RU2850542C2 - Application of protoporphyrinogen oxidase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is a method of weed control, characterised in that it involves applying a herbicide containing an effective dose of a protoporphyrinogen oxidase (PPO) inhibitor to a field where at least one transgenic plant is located, wherein said transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and said transgenic plant has increased resistance to PPO inhibitor herbicides compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase, wherein protoporphyrinogen oxidase has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6. Protoporphyrinogen oxidase according to the present invention has high resistance to the PPO inhibitor herbicide. Furthermore, plants containing the polynucleotide sequence encoding protoporphyrinogen oxidase exhibit strong resistance to the PPO inhibitor herbicide and demonstrate highly resistant tolerance to nearly all of the 4-fold field concentrations of oxyfluorfen, saflufenacil and flumioxazin, and 2 times the field concentration of sulfenetrazone.

EFFECT: increased weed control efficiency.

58 cl, 4 dwg, 10 tbl, 3 ex

Description

FIELD OF INVENTION

The present invention relates to the use of protoporphyrinogen oxidase and, in particular, to a method for imparting resistance to a plant to the herbicide inhibitor PPO (from the English PPOtoporphyrinogen oxidase - protoporphyrinogen oxidase) using protoporphyrinogen oxidase originating from prokaryotes, and its use.

PRIOR ART

The porphyrin biosynthetic pathway is used to synthesize chlorophyll and heme, which play important roles in plant metabolism, and is found in the chloroplast. In this pathway, protoporphyrinogen oxidases (PPOs) catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. After protoporphyrin IX is produced, protoporphyrin IX binds to magnesium via magnesium chelatase to synthesize chlorophyll or binds to iron via ferrochelatase to synthesize heme.

Herbicides that act by inhibiting PPO include primarily PPO inhibitor herbicides from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones, triazinones, etc. In plants, PPO inhibitors inhibit the enzymatic activity of PPO, leading to the inhibition of chlorophyll and heme synthesis and the accumulation of the substrate, protoporphyrinogen IX; the accumulated protoporphyrinogen IX is rapidly exported from the chloroplast to the cytoplasm, where protopormyrinogen IX is converted to protoporphyrin IX in a non-enzymatic reaction, during which, in the presence of light and oxygen molecules, highly reactive singlet oxygen ( ¹ O ₂ ) is additionally formed, which damages the cell membrane and quickly leads to the death of plant cells.

Methods for providing plants that are resistant to PPO inhibitor herbicides primarily include the following: 1) detoxifying the herbicide with an enzyme that converts the herbicide or its active metabolite into non-toxic products; 2) overexpressing sensitive PPOs such that the plant produces amounts of the target enzyme that are sufficient for the herbicide, given the rate constants of the enzyme, so as to have a sufficient amount of functional enzyme available despite the presence of its inhibitor; and 3) providing a functional PPO that is less sensitive to the herbicides or their active metabolites but retains the ability to catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. Regarding functional PPOs, while a given functional PPO enzyme may provide useful levels of resistance to some PPO-inhibitor herbicides, the same functional PPO may not be suitable for providing commercial levels of resistance to a superior, more desirable PPO-inhibitor herbicide. For example, PPO-inhibitor herbicides may differ in their weed control spectrum, production costs, and environmental benefits. Accordingly, new methods are needed to confer resistance to PPO-inhibitor herbicides on different crops and crop varieties.

SUMMARY OF THE INVENTION

The aim of the present invention is to propose the use of protoporphyrinogen oxidase. Protoporphyrinogen oxidase originates from prokaryotes, and plants transformed with the polynucleotide sequence encoding protoporphyrinogen oxidase according to the present invention exhibit good resistance to PPO-inhibiting herbicides.

In order to achieve the above-mentioned object, according to the present invention, a method for controlling weeds is provided, comprising applying a herbicide containing an effective dose of a PPO inhibitor to a field where at least one transgenic plant is located, where this transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and this transgenic plant has a reduced degree of plant damage and/or an increased plant yield, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase, where protoporphyrinogen oxidase has at least 88% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

even more preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NO: 1-14;

preferably, the transgenic plant includes monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plant is oat (Avena sativa), wheat (Triticum aestivum), barley (Hordeum vulgare), millet (Setaria italica), corn (Zea mays), sorghum (Sorghum bicolor), Brachypodium distachyon, rice (Oryza sativa), tobacco (Nicotiana tabacum), sunflower (Helianthus annuus), alfalfa (Medicago sativa), soybean (Glycine max), chickpea (Cicer arietinum), peanut (Arachis hypogaea), sugar beet (Beta vulgaris), cucumber (Cucumis sativus), cotton (Gossypium hirsutum), oilseed rape (Brassica napus), potato (Solarium tuberosum), tomato (Solanum lycopersicum) or Arabidopsis thaiiana; even more preferably, the transgenic plant is a glyphosate-resistant plant and the weeds are glyphosate-resistant weeds;

preferably, the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of simple diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;

Even more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine.

Preferably, the polynucleotide sequence of protoporphyrinogen oxidase comprises:

(a) a polynucleotide sequence encoding an amino acid sequence having at least 88% sequence identity to a sequence selected from SEQ ID NOs: 1-14, wherein the polynucleotide sequence does not include SEQ ID NOs: 15-28; or

(b) a polynucleotide sequence as shown in any of SEQ ID NOs: 29-42 or SEQ ID NOs: 62-64.

In addition, the transgenic plant further comprises at least one second polynucleotide encoding a second herbicide-resistant protein that differs from the polynucleotide sequence encoding protoporphyrinogen oxidase.

The second polynucleotide encodes a selectable marker protein, a protein with synthetic activity, a protein with degradative activity, a protein resistant to biotic stress, a protein resistant to abiotic stress, a male sterility protein, a protein that can affect plant yield, and/or a protein that affects plant quality.

In particular, the second polynucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate synthase and/or cytochrome-like protein.

Alternatively, the herbicide containing an effective dose of a PPO inhibitor may further include glyphosate-based herbicides, glufosinate-based herbicides, auxin-like herbicides, graminicides, pre-emergence selective herbicides and/or post-emergence selective herbicides.

In order to achieve the above-mentioned object, according to the present invention, there is further provided a planting combination for controlling weed growth, comprising a PPO inhibitor herbicide and at least one transgenic plant, wherein the herbicide, comprising an effective dose of the PPO inhibitor, is applied to a field, wherein at least one transgenic plant is located, wherein this transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and this transgenic plant has a reduced degree of plant damage and/or an increased plant yield, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase; wherein the protoporphyrinogen oxidase has at least 88% sequence identity with an amino acid sequence, selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

even more preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NO: 1-14;

Preferably, the transgenic plant includes monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plant is oat, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana; even more preferably, the transgenic plant is a glyphosate-resistant plant, and the weeds are glyphosate-resistant weeds.

preferably, the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of simple diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;

Even more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine.

Preferably, the polynucleotide sequence of protoporphyrinogen oxidase comprises:

(a) a polynucleotide sequence encoding an amino acid sequence having at least 88% sequence identity to a sequence selected from SEQ ID NOs: 1-14, wherein the polynucleotide sequence does not include SEQ ID NOs: 15-28; or

(b) a polynucleotide sequence as shown in any of SEQ ID NOs: 29-42 or SEQ ID NOs: 62-64.

In addition, the transgenic plant further comprises at least one second polynucleotide encoding a second herbicide-resistant protein that differs from the polynucleotide sequence encoding protoporphyrinogen oxidase.

The second polynucleotide encodes a selectable marker protein, a protein with synthetic activity, a protein with degradative activity, a protein resistant to biotic stress, a protein resistant to abiotic stress, a male sterility protein, a protein that affects plant yield, and/or a protein that affects plant quality.

In particular, the second polynucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate synthase and/or cytochrome-like protein.

Alternatively, herbicides containing an effective dose of a PPO inhibitor may additionally include glyphosate-based herbicides, glufosinate-based herbicides, auxin-like herbicides, graminicides, pre-emergence selective herbicides and/or post-emergence selective herbicides.

In order to achieve the above-mentioned object, according to the present invention, there is further provided a method for producing a plant that would be resistant to a PPO inhibitor herbicide, wherein the method comprises introducing a polynucleotide sequence encoding protoporphyrinogen oxidase into the genome of the plant, and wherein the herbicide containing an effective dose of the PPO inhibitor is applied to a field where at least the given plant is located, the plant has a reduced degree of plant damage and/or an increased plant yield, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase, wherein the protoporphyrinogen oxidase has at least 88% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

even more preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NO: 1-14;

preferably, the route of administration will include methods of genetic transformation, genome editing or gene mutation;

preferably, the plant includes monocotyledonous plants and dicotyledonous plants; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or Arabidopsis thaliana;

preferably, the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of simple diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;

Even more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine.

In order to achieve the above objective, according to the present invention, a method for cultivating a plant that is resistant to a PPO inhibitor herbicide is provided, comprising:

planting at least one plant propagule, wherein the plant propagule comprises in its genome a polynucleotide encoding protoporphyrinogen oxidase, wherein the protoporphyrinogen oxidase has at least 88% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

allowing the plant propagule to grow into a plant; and

applying a herbicide containing an effective dose of a PPO inhibitor to a field containing at least one plant and harvesting a plant having a reduced level of plant damage and/or an increased plant yield compared to other plants lacking a polynucleotide sequence encoding protoporphyrinogen oxidase;

preferably, the protoporphyrinogen oxidase has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

even more preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NO: 1-14;

preferably, the plant includes monocotyledonous plants and dicotyledonous plants; more preferably, the plant is oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana;

preferably, the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of simple diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;

Even more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine.

In order to achieve the above-mentioned object, according to the present invention, there is further provided a method for protecting a plant from damage caused by a PPO inhibitor herbicide or for imparting resistance to a plant to a PPO inhibitor herbicide, comprising applying a herbicide containing an effective dose of a PPO inhibitor to a field where at least one transgenic plant is located, wherein the transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and the transgenic plant has a reduced degree of plant damage and/or an increased plant yield, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase, wherein the protoporphyrinogen oxidase has at least 88% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

even more preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NO: 1-14;

Preferably, the transgenic plant includes monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plant is oat, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana;

preferably, the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of simple diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;

Even more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine.

In order to achieve the above object, the present invention further provides the use of protoporphyrinogen oxidase for imparting resistance to a plant to a PPO inhibitor herbicide, wherein the protopophyrinogen oxidase has at least 88% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

preferably, the protoporphyrinogen oxidase has at least 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

more preferably, the protoporphyrinogen oxidase has at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;

even more preferably, the protoporphyrinogen oxidase is selected from the amino acid sequences of the group consisting of SEQ ID NO: 1-14;

preferably, the use of protoporphyrinogen oxidase to impart resistance to a plant to a PPO inhibitor herbicide comprises applying a herbicide containing an effective dose of a PPO inhibitor to a field containing at least one transgenic plant, wherein the transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and this transgenic plant has a reduced degree of plant damage and/or an increased plant yield, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase;

preferably, the plant includes monocotyledonous plants and dicotyledonous plants; more preferably, the plant is oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana;

preferably, the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of simple diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones;

Even more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine.

Preferably, the polynucleotide sequence of protoporphyrinogen oxidase comprises:

(a) a polynucleotide sequence encoding an amino acid sequence having at least 88% sequence identity to a sequence selected from SEQ ID NOs: 1-14, wherein the polynucleotide sequence does not include SEQ ID NOs: 15-28; or

(b) a polynucleotide sequence as shown in any of SEQ ID NOs: 29-42 or SEQ ID NOs: 62-64.

As a particular embodiment, the PPO inhibitor herbicide (also known as a PPO inhibitor class herbicide) may be one or more selected from the group consisting of: diphenyl ethers (chlornitrophen, chlormethoxyfen, bifenox, oxyfluorfen, acifluorfen, its salts and esters, fomesafen, lactofen, fluoroglycophenethyl, ethoxyphenethyl, aclonifen, bifenox, ethoxyfen, chlorintrophen and halosaphen); oxadiazolones (oxadiazone and oxadiagril); N-phenylphthalimides (flumioxazin, flumiclorac-pentyl and cinidonethyl); oxazolinones (pentoxazone); phenylpyrazoles (fluazolate and pyraflufenethyl); uracils (benzenediazone, butafenacil, and saflufenacil); thiadiazoles (thidiazimine and fluthiacet); triazolinones (azaphenidine, sulfentrazone, and carfentrazone); triazinones (trifludimoxazine); and others (flufenpyrethyl and pyraclonil), but is not limited to them.

As used in this document, singular terms refer to one or more referenced entities (namely, at least one). For example, "element" refers to one or more elements (components). Furthermore, the term "comprise" or its variations, such as "comprises" or "comprising," should be understood to include a specified element, integer, or step, or group of elements, integers, or steps, but not to exclude any other element, integer, or step, or group of elements, integers, or steps.

As used herein, the term "herbicide tolerant" means the ability of protoporphyrinogen oxidase to retain at least some of its enzymatic activity in the presence of one or more PPO herbicides. Protoporphyrinogen oxidase enzymatic activity can be measured by any means known in the art, such as by an assay in which the production of protoporphyrinogen oxidase product or the consumption of protoporphyrinogen oxidase substrate in the presence of one or more PPO herbicides is measured by fluorescence, high-performance liquid chromatography (HPLC), or mass spectrometry (MS). "Herbicide tolerant" may represent complete or partial insensitivity to a particular herbicide and may be expressed as a percentage (%) of resistance or insensitivity to a particular PPO herbicide.

As used in this document, the term "herbicide tolerance of plants, seeds, plant tissues, or cells" or "herbicide-resistant plants, seeds, plant tissues, or cells" refers to the ability of plants, seeds, plant tissues, or cells to resist the effects of applied herbicides. For example, herbicide-resistant plants can survive or continue to grow in the presence of a herbicide. The tolerance of a plant, seed, plant tissue, or cell to a herbicide can be measured by comparing the plant, seed, plant tissue, or cell to an appropriate control. For example, herbicide tolerance can be measured and assessed by applying the herbicide to a plant (the test plant) that contains a DNA molecule encoding a protein capable of conferring herbicide resistance and then comparing the damage to the two plants, where the tolerance of the test plant to the herbicide is indicated by a reduction in the degree of damage compared to the control plant. A herbicide-resistant plant, seed, plant tissue, or cell exhibits a reduced response to the toxic effects of a herbicide, compared to a control plant, seed, plant tissue, or cell. The term "herbicide-resistant trait" is a transgenic trait that imparts improved herbicide tolerance to a plant compared to a wild-type plant. Plants that could be obtained with the herbicide-resistant trait of the present invention could include, for example, any plant, including agricultural plants such as oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana.

The DNA molecules of the present invention can be synthesized and modified by methods known in the art, either in whole or in part, particularly when it is desirable to provide sequences useful for DNA manipulation (such as restriction endonuclease recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant codon usage frequency or Kozak consensus sequences), or sequences useful for the construction of DNA constructs (such as spacer or linker sequences). The present invention includes DNA molecules, preferably proteins, encoding a protein having at least 88% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity and at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14. The term "percent sequence identity" or "sequence identity %" refers to the percentage of identical amino acids in the protein sequence of a reference or query sequence (or its complementary strand) compared to a test sequence (or its complementary strand) when the two sequences are aligned. Sequence alignment methods are well known in the art and can be performed using mathematical algorithms such as the Myers and Miller (1988) CABIOS 4:11–17 algorithm; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global sequence alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443–453; and the Karlin and Altschul (1990) PPOc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) PPOc. Natl. Acad. Sci. USA 90:5873–5877. Computer implementations of these mathematical algorithms can be used to compare sequences to determine sequence identity. Such implementations include, but are not limited to, CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, CA); the ALIGN program (Version 2.0); and GAP, BESTFIT, BLAST, FAS TA, and TFASTA in the GCG Wisconsin Genetics software package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, CA, USA). Sequence identity, expressed as a percentage, is presented as the fraction of identity multiplied by 100.

In this document, oxyfluorfen refers to 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-trifluoromethylbenzene as a colorless crystalline solid. It is a selective, pre- and post-emergence, contact-acting herbicide and PPO inhibitor with ultra-low dosages based on diphenyl ethers and can be applied as an emulsifiable concentrate. Weeds are controlled primarily by uptake of the herbicide through the coleoptile or mesocotyl. Oxyfluorfen can effectively control weeds in rice, soybeans, corn, cotton, vegetables, grapes, fruit trees, and other agricultural crops. Weeds that can be controlled include, but are not limited to, the monocotyledonous and broadleaf weeds Barnyard grass, Sesbania cannabina, Bromus tectorum, Setaria viridis, Datura stramonium, Agropyron repens, Ambrosia artemisiifolia, Sida spinosa, Abutilon theophrasti, and Brassica kaber.

In the context of this document, an effective dose of oxyfluorfen means an application rate in the range of 180 to 720 g a.i./ha (grams of active ingredient per hectare), including 190 to 700 g a.i./ha, 250 to 650 g a.i./ha, 300 to 600 g a.i./ha or 400 to 500 g a.i./ha.

In the context of this document, saflufenacil refers to N'-[2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydro-1(2H)-pyrimidine)benzoyl]-N-isopropyl-N-methylsulfamide as a light brown expended granular solid. It belongs to the uracil-based PPO inhibitor sterilant herbicides and can be formulated into 70% water-dispersible granules. Saflufenacil is effective against a variety of broadleaf weeds, including those resistant to glyphosate, ALS, and triazines, and is characterized by rapid killing and rapid degradation of residues in soil.

In the context of this document, an effective dose of saflufenacil means an application in the range of 25 to 100 g a.i./ha, including 30 to 95 g a.i./ha, 40 to 90 g a.i./ha, 50 to 85 g a.i./ha or 60 to 80 g a.i./ha.

In this document, flumioxazin refers to 2-[7-fluoro-3,4-dihydro-3-oxo-4-(2-propynyl)-2H-1,4-benzoxazin-6-yl]-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione. It is an N-phenylphthalimide-based PPO inhibitor herbicide that is taken up by seedlings and foliage and is typically applied as a 50% wettable powder and 48% suspension concentrate. Flumioxazin is effective in controlling annual broadleaf weeds and some grassy weeds. It is readily degraded in the environment and is safe for subsequent crops.

In the context of this document, an effective dose of flumioxazin means an application rate in the range from 60 to 240 g a.i./ha, including 70 to 220 g a.i./ha, 85 to 200 g a.i./ha, 90 to 185 g a.i./ha or 100 to 150 g a.i./ha.

In the context of this document, sulfentrazone refers to N-(2,4-dichloro-5-(4-difluoromethyl-4,5-dihydro-3-methyl-5-oxo-1H-1,2,4-triazol-1-yl)phenyl)methanesulfonamide as a tan solid. It is a triazolinone-based PPO inhibitor herbicide in a typical dosage form of 38.9% and 44.5% suspension concentrate. Sulfentrazone can be used to control, among other things, annual broadleaf weeds, grassy weeds and Cyperaceae, such as Ipomoea nil, Amaranthus retroflexus, Chenopodium album, Datura stramonium, Digitaria sanguinalis, Setaria viridis, Xanthium strumarium, Eleusine indica Gaertn, Cyperus rotundus, etc. in fields of corn, sorghum, soybeans, peanuts, etc.

In the context of this document, an effective dose of sulfentrazone means an application rate in the range of 450 to 900 g a.i./ha, including 500 to 850 g a.i./ha, 550 to 700 g a.i./ha, 500 to 685 g a.i./ha or 500 to 650 g a.i./ha.

As used herein, the term "resistance" is heritable and allows a plant to grow and reproduce under conditions where it is effectively treated with a conventional herbicide. As recognized by those skilled in the art, even if a given plant treated with a herbicide experiences a certain degree of injury (such as minor necrosis, decay, chlorosis, or other damage), yield is not significantly reduced, and thus, the plant can still be considered "resistant." In other words, the plant has an increased ability to withstand varying degrees of injury caused by the herbicide, and, in general, injury can be induced in a wild-type plant of the same genotype with the same herbicide dose. The term "resistant" or "tolerance" as used herein is broader than the term "resistance" and includes "tolerance."

In the context of this document, biotic stress-tolerant proteins refer to proteins that are resistant to stress caused by other organisms, including insect-tolerant proteins and disease-tolerant proteins (virus, bacteria, fungi, and nematode).

In the context of this document, abiotic stress-tolerant proteins refer to proteins that are tolerant to stress caused by the external environment, including proteins that are tolerant to herbicides, drought, heat stress, cold, frost, salinity stress, oxidative stress, etc.

In the context of this document, proteins that affect plant quality refer to proteins that affect plant output traits, such as proteins that improve the quality and content of starch, oil, vitamins, etc., and proteins that improve fiber quality.

In addition, an expression cassette containing a polynucleotide sequence encoding protoporphyrinogen oxidase may also be expressed together with at least one protein encoding a herbicide resistance gene in plants. The herbicide resistance protein includes 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate oxidoreductase (GOX), glyphosate-N-acetyltransferase (GAT), glyphosate decarboxylase, glufosinate acetyltransferase (PAT), alpha-ketoglutarate-dependent dioxygenase (AAD), dicamba monooxygenase (DMO), 4-hydroxyphenylpyruvate dioxygenase (HPPD), acetolactate synthase (ALS) and/or cytochrome-like protein (P450), but is not limited to them.

As used herein, "glyphosate" refers to N-phosphonomethyl glycine and its salts, and a "glyphosate-based herbicide" treatment refers to a treatment with any herbicide formulation containing glyphosate. Commercial glyphosate formulations include, but are not limited to, ROUNDUP® (glyphosate as an isopropyl ammonium salt), ROUNDUP®WEATHERMAX (glyphosate as a potassium salt); ROUNDUP®DRY and RIVAL® (glyphosate as an ammonium salt); ROUNDUP®GEOFORCE (glyphosate as a sodium salt); nTOUCHDOWN® (glyphosate as a trimethylsulfonium salt).

In the context of this document, an effective dose of glyphosate means application in an amount ranging from 200 to 1600 g a.i./ha, including 250-1600 g a.i./ha, 300-1600 g a.i./ha, 500-1600 g a.i./ha, 800-1500 g a.i./ha, 1000-1500 g a.i./ha, 800-1500 g a.i./ha.

For the purposes of this document, "glufosinate," also known as phosphinothricin, refers to ammonium 2-amino-4-[hydroxy(methyl)phosphoryl]butyrate, and a treatment with a "glufosinate-based herbicide" refers to a treatment with any herbicide formulation containing glufosinate.

In the context of this document, an effective dose of glufosinate means an application rate in the range of 200 to 800 g a.i./ha, including 200 to 750 g a.i./ha, 250 to 700 g a.i./ha, 300 to 700 g a.i./ha, 350 to 650 g a.i./ha or 400 to 600 g a.i./ha.

In the present invention, auxin-like herbicides mimic or act like natural plant growth regulators called auxins. They affect cell wall plasticity and nucleic acid metabolism, which can lead to uncontrolled cell growth and division. Damage symptoms caused by auxin-like herbicides include epinastic bending and twisting of shoots and petioles, cupping and curling of leaves, and abnormal leaf shape and venation. Auxin-like herbicides include, but are not limited to, a phenoxycarboxylic acid compound, a benzoic acid compound, a pyridinecarboxylic acid compound, a quinolinecarboxylic acid compound, and a benazolineethyl compound. Typically, an auxin-like herbicide is dicamba, 2,4-dichlorophenoxyacetic acid (2,4-D), (4-chloro-2-methylphenoxy)acetic acid (MCPA), and/or 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB).

As used in this document, "dicamba" refers to 3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid and its acids and salts. Its salts include isopropylamine, diglycoamine, dimethylamine, potassium salts, and sodium salts. Commercial dicamba formulations include, but are not limited to, Banvel® (as the DMA salt), Clarity® (as the DGA salt, BASF), VEL-58-CS-11™, and Vanquish® (as the DGA salt, BASF).

2,4-D, as used in the present invention, is a relatively inexpensive and potent broad-spectrum herbicide for broadleaf weeds that has been used to control a wide range of broadleaf weeds for over 65 years in both agricultural and non-agricultural settings. 2,4-D exhibits varying levels of selectivity for different plants (e.g., dicotyledonous plants are more sensitive than grasses). Plants typically metabolize 2,4-D slowly, so different target site activities likely explain the different plant responses to 2,4-D. 2,4-D is metabolized in plants typically through two metabolic steps, namely, hydroxylation followed by conjugation with amino acids or glucose.

Selective pre-emergent herbicides in the present invention include, but are not limited to, acetanilide, acetochlor, acetolactate synthase inhibitor, and dinitroaniline.

Selective post-emergence herbicides in the present invention include, but are not limited to, nicosulfuron, rimsulfuron, and quizalofop-p-ethyl.

The application amount of the herbicide in the present invention varies depending on the soil structure, pH value, organic matter content, farming system and weed size and is determined by checking the appropriate application amount of the herbicide on the herbicide label.

In the context of this document, the term "impart" refers to providing a plant with a characteristic or trait, such as herbicide resistance and/or other desirable traits.

In the context of this document, the term "heterologous" means from another source. In the context of DNA, "heterologous" refers to any foreign, "non-self" DNA, including DNA from another plant of the same species. For example, in the present invention, the soybean HPPD gene, which can be expressed in a soybean plant through transgenic methods, would still be considered "heterologous DNA."

As used herein, the term "nucleic acid" includes a polymer of dioxyribonucleotides or ribonucleotides, either in single- or double-stranded forms, and unless otherwise indicated, encompasses known analogs (e.g., peptide nucleic acids) that have the vital properties of natural nucleotides in that they hybridize with single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

As used in this document, the term "coding" or "encoded," when used in the context of the nucleic acids in question, means that the nucleic acid contains the necessary information to direct the translation of the nucleotide sequence into a specific protein. The information by which a protein is encoded is characterized by the use of codons. A nucleic acid encoding a protein may contain untranslated sequences (e.g., introns) within the translated regions of the nucleic acid or may lack such included untranslated sequences (e.g., as in the case of cDNA (complementary DNA)).

The DNA sequences encoding the protoporphyrinogen oxidase of the present invention are used in providing plants, plant cells and seeds of the present invention that offer improved tolerance to a variety of PPO inhibitor herbicides, compared to similar plants (control plants) that do not contain the DNA sequence encoding the protoporphyrinogen oxidase of the present invention.

The genes encoding protoporphyrinogen oxidase according to the present invention are useful for producing plants resistant to PPO-inhibiting herbicides. The genes encoding protoporphyrinogen oxidase according to the present invention are particularly suitable for expression in plants to impart herbicide resistance.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. The terms refer to polymers of amino acids in which one or more amino acid residues are an artificial chemical analog of the corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids. The polypeptides of the invention can be obtained either from the nucleic acid disclosed herein or by standard molecular biology techniques. For example, a truncated protein of the invention can be produced by expressing a recombinant nucleic acid of the invention in an appropriate host cell, or alternatively by a combination of ex vivo methods, such as protease digestion and purification.

The present invention also provides nucleic acid molecules comprising protoporphyrinogen oxidase. In general, the present invention includes any polynucleotide sequence encoding protoporphyrinogen oxidase having one or more conservative amino acid substitutions relative to protoporphyrinogen oxidase. Conservative substitutions providing functionally similar amino acids are well known in the art. Each of the following five groups contains amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (I), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).

Accordingly, sequences that have a protoporphyrinogen oxidase inhibitor herbicide resistance activity and hybridize with genes encoding protoporphyrinogen oxidase of the present invention under stringent conditions are included in the present invention. Exemplary sequences have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NOs: 29-42 and SEQ ID NOs: 62-64 of the invention. Genes encoding protoporphyrinogen oxidase of the present invention do not include SEQ ID NOs: 15-28.

Any conventional method of nucleic acid hybridization and amplification can be used to identify the presence of the PPO gene according to the present invention. A nucleic acid molecule or fragment thereof can specifically hybridize with other nucleic acid molecules under certain conditions. In the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, then these two nucleic acid molecules can be considered to be specifically hybridized with each other. If two nucleic acid molecules exhibit complete complementarity, then one nucleic acid molecule of the two is said to be "complementary" to the other nucleic acid molecule. In the present invention, when each nucleotide of a nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule, then these two nucleic acid molecules are said to exhibit "complete complementarity." If two nucleic acid molecules can hybridize to each other with sufficient stability to allow annealing and binding under at least conventional "low stringency" conditions, then the two nucleic acid molecules are said to be "minimally complementary." Similarly, if two nucleic acid molecules can hybridize to each other with sufficient stability to allow annealing and binding under conventional "high stringency" conditions, then the two nucleic acid molecules are said to be "complementary." A deviation from perfect complementarity is permissible, provided that this deviation does not completely prevent the two molecules from forming a double-stranded structure. To ensure that a nucleic acid molecule functions as a primer or probe, it is only necessary to ensure that the molecule has sufficient sequence complementarity to allow the formation of a stable double-stranded structure under the conditions of a particular solvent and salt concentration.

In the present invention, a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize with the complementary strand of a suitable nucleic acid molecule under high stringency conditions. Suitable stringency conditions that cause DNA hybridization are well known to those skilled in the art; for example, suitable stringency conditions can be achieved by treatment with 6.0× sodium chloride/sodium citrate (SSC) under conditions of approximately 45°C and then washing with 2.0×SSC under conditions of 50°C. For example, the salt concentration in the washing step can be selected from the following conditions: from a low stringency condition of approximately 2.0×SSC and 50°C to a high stringency condition of approximately 0.2×SSC and 50°C. Furthermore, the temperature conditions in the washing step can increase from low stringency conditions of room temperature (approximately 22°C) to high stringency conditions of approximately 65°C. Both the temperature conditions and the salt concentration can be varied, and it is also possible for one of the two conditions to remain constant while the other is changed. Preferably, the stringent conditions in the present invention can be achieved by specific hybridization with the genes encoding the protoporphyrinogen oxidase of the present invention in 6×SSC, 0.5% SDS solution at 65°C and then washing the membrane each time with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

As used herein, the term "hybridizing" or "specifically hybridizing" refers to the binding, duplexing, or hybridization of a molecule only to a specific nucleotide sequence under stringent conditions when that sequence is in a DNA or RNA (e.g., total cellular) complex mixture.

Due to genetic codon degeneracy, a variety of different DNA sequences can encode the same amino acid sequence. Obtaining these alternative DNA sequences encoding the same or substantially the same protein is within the skill of one skilled in the art. These different DNA sequences are included within the scope of the present invention. "Substantially the same" sequence refers to a sequence with an amino acid substitution, deletion, addition, or insertion that does not substantially affect herbicide resistance activity and includes a fragment that retains herbicide resistance activity.

The term "functional activity" or "activity" in the present invention means that the protein/enzyme used in the present invention (alone or in combination with other proteins) has the ability to degrade a herbicide or reduce the activity of a herbicide. A plant producing a protein according to the present invention preferably produces an "effective amount" of the protein such that when the plant is treated with a herbicide, the expression level of the protein is sufficient to confer on the plant complete or partial resistance to the herbicide (unless otherwise specified, in total amount). The herbicide can be used in an amount that would normally kill the target plant or in a normal field amount and concentration. Preferably, the plant cell or plant according to the present invention is protected from growth inhibition or damage caused by herbicide treatment. The transformed plant and plant cell according to the present invention are preferably resistant to PPO inhibitor herbicides, i.e., the transformed plant and plant cell can grow in the presence of an effective dose of PPO inhibitor herbicides.

The gene and protein in the present invention not only comprise a particular exemplary sequence, but also comprise a portion and/or fragment (including an internal deletion and/or a terminal deletion, compared to the full-length protein), a variant, a mutant, a protein variant, a substitution (a protein having replaced amino acids), a chimera, and a fusion protein that retain the activity characteristic of a particular exemplary protein.

The term "variant" as used herein is intended to denote substantially similar sequences. In the case of polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within a reference polynucleotide and/or a substitution of one or more nucleotides at one or more sites in a herbicide resistance gene. As used herein, the term "reference polynucleotide or polypeptide" includes a herbicide resistance polynucleotide sequence or amino acid sequence, respectively. As used herein, the term "native polynucleotide or polypeptide" includes a naturally occurring polynucleotide sequence or amino acid sequence, respectively. In the case of nucleic acid molecules, conservative variants include those polynucleotide sequences that, due to the degeneracy of the genetic code, encode one of the protoporphyrinogen oxidases of the present invention. Naturally occurring allele variants such as these can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques, as described below. Variants of nucleic acid molecules also include nucleic acid molecules of synthetic origin, such as nucleic acid molecules generated, for example, by site-directed mutagenesis, but which still encode the nucleotide sequence of the protoporphyrinogen oxidase of the invention. Typically, variants of a particular nucleic acid molecule of the invention will have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the sequence of the particular nucleic acid molecule, as determined by sequence alignment programs and parameters.

The term "protein variant" in the present invention is intended to mean a protein derived from a reference protein by deletion or addition of one or more amino acids at one or more internal sites in protoporphyrinogen oxidase and/or substitution of one or more amino acids at one or more sites in protoporphyrinogen oxidase. Protein variants encompassed by the present invention are biologically active, i.e., they continue to possess the desired biological activity of the protoporphyrinogen oxidase of the invention, namely, protoporphyrinogen oxidase activity and/or herbicide resistance, as described herein. Such variants may result, for example, from genetic polymorphism or human manipulation. Biologically active variants of protoporphyrinogen oxidase according to the invention will have at least about 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity across the amino acid sequence for protoporphyrinogen oxidase, as determined by sequence alignment programs and parameters. A biologically active variant of a protein according to the invention may differ from that protein by only 1-15 amino acid residues, only 1-10, such as 6-10, only 5, only 4, 3, 2, or even 1 amino acid residue.

In some examples, nucleotide sequences encoding protoporphyrinogen oxidases of the present invention or protoporphyrinogen oxidase variants thereof that retain protoporphyrinogen oxidase activity can be combined with any combination of nucleotide sequences of interest to create plants with a desired trait. The term "trait" refers to a phenotype derived from a specific sequence or groups of sequences. For example, amino acids/polynucleotides encoding the protoporphyrinogen oxidase of the invention, or a variant thereof that retains protoporphyrinogen oxidase activity, may be combined with any other nucleotides encoding polypeptides that impart a desirable trait, including, but not limited to, disease, insect, and herbicide resistance, heat and drought tolerance, reduced crop maturity time, improved industrial processing, such as with respect to the conversion of starch or biomass into fermentable sugars, and improved agronomic quality, such as high oil content and high protein content.

It is well known to those skilled in the art that the benefit of a combination of two or more modes of action in improving the spectrum of weeds being controlled and/or controlling more naturally tolerant or resistant weed species may also extend to chemicals that have been engineered to be herbicide tolerant in crops by artificial means (either transgenic or non-transgenic) in addition to PPO-resistant crops. In fact, traits encoding the following resistances can be combined individually or in multiple combinations to provide the ability to effectively control or prevent weed-to-herbicide switching: glyphosate resistance (such as EPSPS, GOX, and GAT from resistant plants or bacteria), glufosinate resistance (such as PAT and Bar), acetolactate synthase inhibitor (ALS) herbicide resistance (such as imidazolinones, sulfonylureas, triazolopyrimidines, sulfonated aniline, pyrimidinylthiobenzoic acids, and other chemical resistance genes such as AHAS, CsrI, and SurA), phenoxyauxin herbicide resistance (such as aryloxyalkanoate dioxygenase-12 (AAD-12)), dicamba herbicide resistance (such as dicamba monooxygenase (DMO)), bromoxynil resistance (such as Bxp), phytoene desaturase inhibitor (PDS) resistance, photosystem II inhibitor herbicide resistance (such as psbA), photosystem I inhibitor herbicide resistance, 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide resistance (such as PPO-1), phenylurea herbicide resistance (such as CYP76B1), and CYP-degrading enzyme resistance dichloromethoxybenzoic acid.

Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grassy weed species. However, repeated glyphosate applications in glyphosate-resistant crops and non-agricultural applications have led (and will continue) to weed switching to naturally more resistant species, or glyphosate-resistant biotypes. Most herbicide resistance management programs recommend the use of effective doses of tank-mixed "partner" herbicides as a means of delaying the emergence of resistant weeds. These "partner" herbicides provide control of the same species but have different modes of action. Combining the gene encoding protoporphyrinogen oxidase of the present invention with a glyphosate resistance trait (and/or resistance traits to another herbicide) can achieve control of a glyphosate-resistant weed species (a broadleaf weed species controlled by one or more PPO inhibitor herbicides) in glyphosate-resistant crops by providing selective application of glyphosate and PPO inhibitor herbicides (such as oxyfluorfen, saflufenacil and flumioxazin) in the same crop. Applications of these herbicides can be carried out simultaneously in a tank mix containing two or more herbicides with different modes of action, or can be carried out separately in a single herbicide formulation in sequential applications (e.g., pre-planting or pre- or post-emergence) (with an application time interval ranging from 2 hours to 3 months); or, alternatively, applications of these herbicides may be carried out by using a combination of any number of herbicides representing each category of applicable compounds at any time (from 7 months after planting of the crop until the time the crop is harvested (or the interval before harvest in the case of a single herbicide, whichever is the shortest)).

Flexibility in broadleaf weed control is important, in terms of application timing, the application rate of a single herbicide, and the ability to control resistant or tolerant weeds. The application range of glyphosate, combined with the glyphosate resistance gene/protoporphyrinogen oxidase-encoding gene of the present invention, in agricultural crops can range from 250 to 2,500 g a.i./ha. The application range of one or more PPO inhibitor herbicides can range from 10 to 1,000 g a.i./ha. The optimal timing combination for these applications depends on specific conditions, species, and the environment.

The herbicide composition (e.g., an ester, acid, or salt-forming compound, or a soluble concentrate, emulsifiable concentrate, or soluble liquid) and the tank mix additive (e.g., an adjuvant or compatibility agent) can significantly affect the weed control achieved using a given herbicide or a combination of one or more herbicides. Any chemical combination of any of the above herbicides is within the scope of the present invention.

Furthermore, the gene encoding protoporphyrinogen oxidase of the present invention, alone or in combination with other herbicide-resistant crop traits, can be combined with one or more other input traits (e.g., insect resistance, fungal resistance, or stress tolerance, etc.) or output traits (e.g., increased yield, improved oil content, improved fiber quality, etc.). Thus, the present invention can be used to provide ready-made solutions in agriculture for improving the qualities of crops with the ability to flexibly and cost-effectively control any number of agricultural pests.

These combined combinations can be created by any method, including, but not limited to, crossing plants using any conventional or TopCross methodology or genetic transformation. If the sequences are combined by genetic transformation of plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant containing one or more desired traits can be used as a target for the introduction of additional traits by subsequent transformation. Traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences are to be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or different promoters. In some cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This transformation cassette can be combined with any combination of other suppression or overexpression cassettes to create the desired combination of traits in a plant. Furthermore, it should be recognized that polynucleotide sequences can be combined at the desired position in the genome using site-specific recombination.

The gene encoding protoporphyrinogen oxidase according to the present invention has a higher resistance to PPO inhibitor herbicides, which is an important basis for the possibility of obtaining herbicide-resistant crops and a selectable marker trait.

The term "expression cassette" as used herein means a nucleic acid molecule capable of directing the expression of a specific polynucleotide sequence in an appropriate host cell, comprising a promoter effectively linked to the polynucleotide sequence of interest (namely, a polynucleotide encoding the protoporphyrinogen oxidase of the present invention or a protein variant thereof that retains protoporphyrinogen oxidase activity, alone or in combination with one or more additional nucleic acid molecules encoding polypeptides that impart the desired features), which is effectively linked to termination signals. The coding region typically encodes the protein of interest, but may also encode a functional RNA of interest, such as antisense RNA or untranslated RNA, in either the sense or antisense direction. An expression cassette containing a polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette produced in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host; that is, the specific DNA sequence of the expression cassette does not naturally occur in the host cell and must be introduced into a new host cell through a transformation event. Expression of the polynucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to a specific external stimulus. Additionally, the promoter may also be specific for a particular tissue, organ, or developmental stage.

The present invention encompasses the transformation of plants with expression cassettes capable of expressing a polynucleotide of interest (namely, a polynucleotide encoding the protoporphyrinogen oxidase of the present invention or a variant thereof that retains protoporphyrinogen oxidase activity alone or in combination with one or more additional nucleic acid molecules encoding polypeptides that confers the desired traits). The expression cassette will include, in the 5'-3' direction of transcription, a transcription and translation initiation region (namely, a promoter) and an open reading frame of the polynucleotide. The expression cassette may optionally contain a transcription and translation termination region (namely, a termination region) that is functional in plants. In some embodiments, the expression cassette contains a selectable marker gene to ensure the selection of stable transformants. The expression constructs of the invention may also contain a leader sequence and/or a sequence that allows inducible expression of the polynucleotide of interest.

The regulatory sequences of the expression construct are effectively linked to the polynucleotide of interest. Regulatory sequences in the present invention include, but are not limited to, a promoter, transit peptide, terminator, enhancer, leader sequence, intron, and other regulatory sequences operably linked to the gene encoding protoporphyrinogen oxidase.

The promoter is a plant-expressible promoter. "Plant-expressible promoter" refers to a promoter that directs expression of the coding sequence associated with it in a plant cell. A plant-expressible promoter may be a constitutive promoter. Examples of promoters directing constitutive expression in plants include, but are not limited to, the 35S promoter from cauliflower mosaic virus, the maize Ubi promoters, the rice GOS2 gene promoters, and the like. Alternatively, a plant-expressible promoter may be a tissue-specific promoter, meaning the promoter directs expression of the coding sequence in multiple tissues, such as green tissues, at a level higher than in other plant tissues (measurable by conventional RNA assays), such as the PEP carboxylase (phosphoenolpyruvate carboxylase) promoter. Alternatively, the plant-expressed promoter may be a wound-inducible promoter. A wound-inducible promoter, or a promoter directing a wound-inducible expression profile, means that when a plant suffers wounding, whether due to mechanical injury or insect gnawing, expression of the coding sequence under the regulation of the promoter is significantly enhanced compared to normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, the promoters of the potato and tomato protease inhibitor genes (pin I and pin II) and the maize protease inhibitor gene (MPI).

A transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a specific organelle or cellular compartment. In the case of a receptor protein, the transit peptide can be heterologous, for example, targeting the chloroplast using the chloroplast transit peptide encoding sequence, targeting the endoplasmic reticulum using the "KDEL" retention sequence, or targeting the vacuole using the barley phytolectin gene CTP.

A leader sequence includes, but is not limited to, the following: the leader sequence of small RNA viruses, such as the EMCV leader (5' noncoding region of encephalomyocarditis virus); the leader sequence of potato virus group Y, such as the MDMV leader; the human immunoglobulin heavy chain binding protein (BiP); the untranslated leader sequence of the alfalfa mosaic virus coat protein mRNA (RNA4 of AMV); and the leader sequence of tobacco mosaic virus (TMV).

Enhancer includes but is not limited to the following: cauliflower mosaic virus (CaMV) enhancer, figwort mosaic virus (FMV) enhancer, carnation etched ring virus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, mirabilis mosaic virus (MMV) enhancer, cestrum yellow leaf curl virus (CmYLCV) enhancer, cotton leaf curl Multan virus (CLCuMV) enhancer, Commelina yellow leaf spot virus (CoYMV) enhancer. commelina yellow mottle virus) and the enhancer of peanut chlorotic streak caulimovirus (PCLSV).

For monocot use, an intron includes, but is not limited to, the maize hsp70 intron, the maize ubiquitin intron, the Adh 1 intron, the sucrose synthase intron, or the rice Act1 intron. For dicot use, an intron includes, but is not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "superubiquitin" intron.

The terminator may be a suitable polyadenylation signal sequence that functions in a plant, including, but not limited to, a polyadenylation signal sequence derived from the nopaline synthetase gene of Agrobacterium tumefaciens (NOS), a polyadenylation signal sequence derived from the protease inhibitor II (pinII) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a polyadenylation signal sequence derived from the α-tubulin gene.

The phrase "effective linkage" as used herein refers to the linkage of nucleic acid sequences that enables one of the sequences to provide the function required for the sequence linked to it. "Efficient linkage" as used herein may involve linking a promoter to a sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter. When the sequence of interest encodes a protein and expression of the protein is desired, "effective linkage" means that the promoter is linked to the sequence such that the resulting transcript is effectively translated. If the linkage of the promoter to the coding sequence is a transcript fusion, and expression of the encoded protein is desired, the linkage is such that the first translation initiation codon in the resulting transcript is the initiation codon in the coding sequence. Alternatively, if the binding of the promoter to the coding sequence is a translational fusion and expression of the encoded protein is to be achieved, the binding is such that the first translation initiation codon contained in the 5' untranslated sequence is linked to the promoter such that the relationship of the resulting translation product to the translation open reading frame encoding the desired protein is in-frame. Nucleic acid sequences that can be "effectively linked" include, but are not limited to, sequences that provide gene expression functions (namely, gene expression elements such as promoters, 5'-untranslated regions, introns, protein coding regions, 3'-untranslated regions, polyadenylation sites, and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (namely, T-DNA border sequences, site-specific recombinase recognition sites, and integrase recognition sites), sequences that provide selection functions (namely, antibiotic resistance markers and biosynthetic genes), sequences that provide marker evaluation functions, sequences that assist in the manipulation of sequences, in vitro or in vivo (namely, polylinker sequences and site-specific recombination sequences), and sequences that provide replication functions (namely, bacterial origins of replication, autonomously replicating sequences, and centromeric sequences).

The genome of a plant, plant tissue, or plant cell as used herein refers to any genetic material within a plant, plant tissue, or plant cell, and includes nuclear, plastid, and mitochondrial genomes.

As used herein, the term "plant part" or "plant tissue" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant fragments, and plant cells that are intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, twigs, fruits, stones, ears, cobs, glumes, pedicels, roots, root tips, anthers, and the like.

The mutant PPO protein of the present invention can be applied to various types of plants. The dicotyledonous plant includes, but is not limited to, alfalfa, beans, cauliflower, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, peas, pepper, zucchini, radish, oilseed rape, spinach, soybean, pumpkin, tomato, Arabidopsis thaliana, peanut, or watermelon; preferably, the dicotyledonous plant is cucumber, soybean, Arabidopsis thaliana, tobacco, cotton, or oilseed rape. The monocotyledonous plant includes, but is not limited to, corn, rice, sorghum, wheat, barley, rye, millet, sugarcane, oats, or lawn grass. Preferably, the monocotyledonous plant is corn, rice, sorghum, wheat, barley, millet, sugarcane, or oats.

As used herein, the term "plant transformation" means that once the herbicide-resistant or tolerant nucleic acid molecules encoding the protoporphyrinogen oxidase of the present invention, alone or in combination with one or more additional nucleic acid molecules encoding polypeptides that confer the desired traits, have been cloned into an expression system, they are transformed into a plant cell. The receptors and target expression cassettes of the present invention can be introduced into a plant cell by a variety of routes recognized in the art. The term "introduction" in the context of a polynucleotide, such as a nucleotide construct of interest, is intended to mean providing the polynucleotide to the plant in such a way that the polynucleotide gains access to the interior of the plant cell. When more than one polynucleotide is to be introduced, these polynucleotides can be assembled as part of a single nucleotide construct or as separate nucleotide constructs and can be located on the same or different transformation vectors. Accordingly, these polynucleotides can be introduced into the host cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol. The methods of the invention are independent of the specific method of introducing one or more polynucleotides into a plant, except that the polynucleotide(s) gain access to the interior of at least one plant cell. Methods for introducing one or more polynucleotides into plants are known in the art, including, but not limited to, transient transformation methods, stable transformation methods, and virus-mediated methods or genome editing techniques.

The term "stable transformation" means that an exogenous gene is introduced into the plant genome and stably integrated into the plant or its genome of any subsequent generations, such that the exogenous gene is stably inherited.

The term "transient transformation" means that a nucleic acid molecule or protein is introduced into a plant cell to perform a function, but it is not integrated into the plant genome, so that the exogenous gene cannot be stably inherited.

The term "genome editing technology" refers to techniques used to modify the genome that can precisely manipulate genomic sequences to perform operations such as site-directed gene mutations, insertions, and deletions. Currently, genome editing technologies mainly include homing endonucleases (HE), zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR).

Numerous transformation vectors available for plant transformation are known to those skilled in the art, and the genes related to this invention can be used in conjunction with any such vectors. The choice of vector will depend on the preferred transformation technique and the target species for transformation. For some target species, different antibiotic or herbicide selectable markers may be preferred. Selectable markers routinely used in transformation include the nptII gene (which was published by Bevan et al., Nature 304:184-187 (1983)), which confers resistance to kanamycin and related antibiotics or herbicides; the pat and bar genes, which confer resistance to the herbicide glufosinate (also called phosphinothricin; see White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theon. Appl. Genet. 79: 625-631 (1990) and U.S. Pat. Nos. 5,561,236 and 5,276,268); the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol. Cell. Biol. 4: 2929-2931); the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)); the EPSPS gene, which confers glyphosate resistance (U.S. Pat. Nos. 4,940,935 and 5,188,642); the glyphosate N-acetyltransferase (GAT) gene, which also confers glyphosate resistance (Castle et al. (2004) Science, 304:1151-1154; U.S. Patent Application Publication Nos. 20070004912, 20050246798, and 20050060767); and the mannose-6-phosphate isomerase gene, which confers the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).

Plant regeneration methods are also well known in this field. For example, Ti plasmid vectors have been used to deliver foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and bombarding microparticles.

In the present invention, weeds refer to plants that compete with cultivated transgenic plants on agricultural land.

The term "control" and/or "prevention" in the present invention refers to at least the direct application (e.g., by spraying) of an effective dose of a PPO inhibitor herbicide to agricultural land in such a way as to minimize weed development and/or stop weed growth. At the same time, the cultivated transgenic plants should be morphologically normal and can be cultivated in traditional methods of consumption and/or product production; and preferably, compared to non-transgenic wild-type plants, the cultivated plants have reduced plant damage and/or increased plant yield. Reduced plant damage includes, but is not limited to, improved stem resistance and/or increased grain weight, etc. The "control" and/or "prevention" effect exerted by protoporphyrinogen oxidase on weeds may exist independently and will not be reduced and/or lost due to the presence of other substances that can "control" and/or "prevent" weeds. In particular, if any tissue of a transgenic plant (containing a gene encoding protoporphyrinogen oxidase of the present invention) has and/or produces protoporphyrinogen oxidase and/or another substance that can control weeds simultaneously and/or separately, then the presence of the other substance will neither affect the "control" and/or "prevention" effect exerted by protoporphyrinogen oxidase on weeds, nor lead to the "control" and/or "prevention" effect, completely and/or partially achieved by the other substance, independent of protoporphyrinogen oxidase.

"Plant propagule" as used herein includes, but is not limited to, plant sexual propagules and plant vegetative propagules. Plant sexual propagules include, but are not limited to, plant seeds; and plant vegetative propagules refer to vegetative organs or specific tissue of a plant that can form a new plant ex vivo. Vegetative organs or specific tissue include, but are not limited to, roots, stems, and leaves, such as: plants with roots as vegetative propagules, including strawberries, sweet potatoes, etc.; plants with stems as vegetative propagules, including sugarcane, potatoes (tubers), etc.; and plants with leaves as vegetative propagules, including aloe, begonia, etc.

The present invention can impart a novel herbicide resistance trait to a plant without any observed negative effects on phenotypes (including yield). The plant in the present invention can tolerate, for example, 2×, 3×, 4×, or 5× the total application level of at least one tested herbicide. Improving these tolerance levels is within the scope of the present invention. For example, predictive optimization and further development can be carried out using various techniques known in the art to increase the expression level of a given gene.

According to the present invention, there is provided the use of protoporphyrinogen oxidase, which has the following advantages:

1. Tolerance to a wide range of herbicides. The present invention discloses for the first time that protoporphyrinogen oxidase PPO1-PPO14 can exhibit higher tolerance to PPO-inhibiting herbicides, thus offering potential for widespread use in plants.

2. Strong herbicide resistance. The protoporphyrinogen oxidase PPO1-PPO14 disclosed by the present invention exhibits strong resistance to PPO inhibitor herbicides, and almost all of the protoporphyrinogen oxidases PPO1-PPO14 exhibit high resistance to four times the field concentration of oxyfluorfen, saflufenacil, and flumioxazine, and two times the field concentration of sulfentrazone.

3. Low impact on field conditions. Plant resistance to herbicides is directly correlated with crop yield. Plants with high resistance will not be affected by herbicides, which will not affect crop yield. However, the yield of plants with moderate and low resistance will be significantly lower than that of plants with high resistance.

The technical solution of the present invention is further illustrated in detail below by means of graphic materials and examples.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 is a schematic structural diagram of the recombinant expression vector DBN11375 containing the nucleotide sequence of PPO1A for Arabidopsis thaliana according to the present invention;

Fig. 2 is a schematic structural diagram of the control recombinant expression vector DBN12337N according to the present invention;

Fig. 3 is a schematic structural diagram of the recombinant expression vector DBN11375 containing the nucleotide sequence of PPO1B for maize according to the present invention;

Fig. 4 is a schematic structural diagram of the control recombinant expression vector DBN12354N according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The technical solutions regarding the use of protoporphyrinogen oxidases according to the present invention will be further illustrated by means of the following examples.

Example 1. Acquisition and testing of transgenic Arabidopsis thaliana plants

1. Acquisition of genes encoding protoporphyrinogen oxidases

The amino acid sequences of the microbial protoporphyrinogen oxidases PPO1, PPO2, PPO3, PPO4, PPO5, PPO6, PPO7, PPO8, PPO9, PPO10, PPO11, PPO12, PPO13 and PPO14 are set out as SEQ ID NOs: 1-14 in the SEQUENCE LISTING; the nucleotide sequences of PPO1-PPO14 encoding the corresponding protoporphyrinogen oxidases PPO1-PPO14 are set out as SEQ ID NOs: 15-28 in the SEQUENCE LISTING; The nucleotide sequences of PPO1A-PPO14A encoding the corresponding protoporphyrinogen oxidases PPO1-PPO14, which were obtained based on the normal codon preference of Arabidopsis thalianalcow, are set forth as SEQ ID NOs: 29-42 in the SEQUENCE LISTING; and the nucleotide sequences of PPO1B, PPO6B and PPO12B encoding the corresponding protoporphyrinogen oxidases PPO1, PPO6 and PPO12, which were obtained based on the normal codon preference of maize, are set forth as SEQ ID NOs: 62-64 in the SEQUENCE LISTING.

The amino acid sequence of the Escherichia coli protoporphyrinogen oxidase PPO-EC is set forth as SEQ ID NO: 43 in the SEQUENCE LISTING; the nucleotide sequence of PPO-EC encoding the corresponding Escherichia coli protoporphyrinogen oxidase PPO-EC is set forth as SEQ ID NO: 44 in the SEQUENCE LISTING; and the nucleotide sequence of PPO-ECA encoding the corresponding Escherichia coli protoporphyrinogen oxidase PPO-EC, which was obtained based on the common codon preference of Arabidopsis thaliana/soybean, is set forth as SEQ ID NO: 45 in the SEQUENCE LISTING.

The amino acid sequence of the Arabidopsis protoporphyrinogen oxidase PPO-AT is set forth as SEQ ID NO: 46 in the SEQUENCE LISTING; the nucleotide sequence of the PPO-AT encoding the corresponding Arabidopsis protoporphyrinogen oxidase PPO-AT is set forth as SEQ ID NO: 47 in the SEQUENCE LISTING; and the nucleotide sequence of the PPO-ATA encoding the corresponding Arabidopsis protoporphyrinogen oxidase PPO-AT, which was obtained based on the normal codon preference of Arabidopsis thalianalcow, is set forth as SEQ ID NO: 48 in the SEQUENCE LISTING.

The amino acid sequence of the Arsenophonus protoporphyrinogen oxidase PPO-AP is set forth as SEQ ID NO: 65 in the SEQUENCE LISTING; the nucleotide sequence of the PPO-AP encoding the corresponding Arsenophonus protoporphyrinogen oxidase PPO-AP is set forth as SEQ ID NO: 66 in the SEQUENCE LISTING; the nucleotide sequence of the PPO-APA encoding the corresponding Arsenophonus protoporphyrinogen oxidase PPO-AP, which was obtained based on the normal codon preference of Arabidopsis thalianalcow, is set forth as SEQ ID NO: 67 in the SEQUENCE LISTING; and the nucleotide sequence of PPO-APB encoding the corresponding Arsenophonus protoporphyrinogen oxidase PPO-AP, which was obtained based on the normal maize codon preference, is set forth as SEQ ID NO: 68 in the SEQUENCE LISTING.

2. Synthesis of the above mentioned nucleotide sequences

The 5'- and 3'-ends of the nucleotide sequences PPO1A-PPO14A, the nucleotide sequence PPO-ECA, the nucleotide sequence PPO-ATA and the nucleotide sequence PPO-APA (SEQ ID NO: 29-42, SEQ ID NO: 45, SEQ ID NO: 48 and SEQ ID NO: 67), respectively, were linked with universal primer-adapter 1:

Universal 5'-end adapter primer 1: 5'-taagaaggagatatacatatg-3', as set forth in SEQ ID NO: 49 in the SEQUENCE LISTING;

Universal 3'-end adapter primer 1: 5'-gtggtggtggtggtgctcgag-3', as set forth in SEQ ID NO: 50 in the SEQUENCE LISTING.

3. Construction of recombinant expression vectors containing the nucleotide sequences PPO1A-PPO14A, the nucleotide sequence PPO-ECA, the nucleotide sequence PPO-ATA and the nucleotide sequence PPO-APA for Arabidopsis thaliana

The plant expression vector DBNBC-01 was double-digested using Spe I and Asc I restriction enzymes to linearize the plant expression vector. The digestion product was purified to obtain the linearized backbone of the DBNBC-01 expression vector (vector backbone: pCAMB1A2301 (available from CAMBIA)), which was then recombined with the nucleotide sequence of PP01A (SEQ ID NO: 29) linked to the universal primer-adapter 1 according to the procedure in the instructions of the Takara In-Fusion Seamless Linking Kit (Clontech, CA, USA, Cat. No: 121416) to construct the recombinant expression vector DBN12337 with the schematic structure as shown in Fig. 1 (Spec: spectinomycin resistance gene; RB: right border; eFMV: figwort mosaic virus 34S enhancer (SEQ ID NO: 51); prBrCBP: eukaryotic oilseed rape elongation factor-1α gene promoter (Tsf 1) (SEQ ID NO: 52); spAtCTP2: Arabidopsis thaliana chloroplast transit peptide (SEQ ID NO: 53); EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 54); tPsE9: pea RbcS gene terminator (SEQ ID NO: 55); prAtUbi10: Arabidopsis thaliana ubiquitin 10 gene promoter (SEQ ID NO: 56); spAtCLPI: transit peptide Arabidopsis thaliana white or light green chloroplasts (SEQ ID NO: 57); PP01A: nucleotide sequence of PP01A (SEQ ID NO: 29); tNos: nopaline synthase gene terminator (SEQ ID NO: 58); pr35S: cauliflower mosaic virus 35S promoter (SEQ ID NO: 59); cPAT: phosphinothricin acetyltransferase gene (SEQ ID NO: 60); t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 61); LB: left border).

_T1 competent Escherichia coli cells were transformed with the recombinant expression vector DBN12337 by using the heat shock method under the following heat shock conditions: 50 μl of _T1 competent Escherichia coli cells and 10 μl of plasmid DNA (recombinant expression vector DBN12337) were exposed to a water bath at 42°C for 30 seconds, cultured with shaking at 37°C for 1 hour (using a shaker at 100 rpm for shaking), and then cultured under the condition of 37°C on a solid LB plate (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 mg/L agar; adjusted to pH 7.5 with NaOH) containing 50 mg/L spectinomycin for 12 h; white bacterial colonies were selected and cultured at 37°C overnight in LB liquid culture medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 50 mg/L spectinomycin; pH was adjusted to 7.5 with NaOH). Plasmids in the cells were isolated using an alkali-based method: the bacterial solution was centrifuged at 12,000 rpm. for 1 min, the supernatant was removed and the sedimented thallus was suspended for 1 min, the supernatant was removed and the sedimented thallus was suspended with 100 μl of pre-cooled on ice solution I (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid) and 50 mM glucose with pH 8.0); 200 μl of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)), mixed by inverting the tube 4 times and placed on ice for 3-5 min; 150 μl of ice-cooled solution III (3 M potassium acetate, 5 M acetic acid) were added, immediately mixed homogeneously and placed on ice for 5-10 min; The mixture was centrifuged at 4°C and 12,000 rpm for 5 min, 2 times the volume of anhydrous ethanol was added to the supernatant, mixed uniformly, and then placed at room temperature for 5 min; the mixture was centrifuged at 4°C and 12,000 rpm for 5 min, the supernatant was discarded, and the pellet was washed with ethanol at 70% (v/v) and then air-dried; 30 μl of TE (10 mM Tris-HCl and 1 mM EDTA, pH 8.0) containing RNase (20 μg/ml) was added to dissolve the pellet; the resulting product was exposed to a water bath at 37°C for 30 min to digest RNA; and stored at -20°C for use. The isolated plasmids were identified by sequencing. The results showed that the nucleotide sequence between the SpeI and AscI sites in the recombinant expression vector DBN12337 was the nucleotide sequence as set forth in SEQ ID NO: 29 of the SEQUENCE LISTING, namely, the nucleotide sequence of PPO1A.

According to the above method for constructing the recombinant expression vector DBN12337, the nucleotide sequences of PPO2A-PPO14A, the nucleotide sequence of PPO-ECA, the nucleotide sequence of PPO-ATA and the nucleotide sequence of PPO-APA, which were linked to the universal primer-adapter 1, respectively, were subjected to a recombination reaction with the linearized backbone of the expression vector DBNBC-01 to sequentially construct the recombinant expression vectors DBN12338-DBN12353. Sequencing confirmed that the above nucleotide sequences were correctly inserted into the recombinant expression vectors DBN12338-DBN12353.

According to the method for constructing the recombinant expression vector DBN12337 as described above, the recombinant expression vector DBN12337N was constructed as a control, and its structure is shown in Fig. 2 (Spec: spectinomycin resistance gene; RB: right border; eFMV: figwort mosaic virus 34S enhancer (SEQ ID NO: 51); prBrCBP: oilseed rape elongation factor 1a eukaryotic gene promoter (Tsf1) (SEQ ID NO: 52); spAtCTP2: Arabidopsis thaliana chloroplast transit peptide (SEQ ID NO: 53); EPSPS: 5-enolpyruvateshikimate-3-phosphate synthase gene (SEQ ID NO: 54); tPsE9: pea RbcS gene terminator (SEQ ID NO: 55); pr35S: cauliflower mosaic virus 35S promoter (SEQ ID NO: 59); cPAT: phosphinothricin acetyltransferase gene (SEQ ID NO: 60); t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 61); LB: left border).

4. Transformation of Agrobacterium with recombinant expression vectors for Arabidopsis thaliana

Agrobacterium GV3101 was transformed with the recombinant expression vectors DBN12337 to DBN12350, DBN12352, DBN12353 and the above-mentioned control recombinant expression vector DBN12337N, which were correctly constructed, respectively, using the liquid nitrogen-based method under the following transformation conditions: 100 μl of Agrobacterium GV3101 and 3 μl of plasmid DNA (recombinant expression vectors DBN12337 to DBN12350, DBN12352, DBN12353 and DBN12337N) were placed in liquid nitrogen for 10 minutes and immersed in warm water at 37°C for 10 minutes; The transformed Agrobacterium GV3101 were inoculated into a tube with LB, cultured at 28°C and 200 rpm for 2 hours, and spread onto a solid LB plate containing 50 mg/L rifampicin and 50 mg/L spectinomycin until positive single clones grew, and the single clones were selected for cultivation, and their plasmids were isolated. The isolated plasmids were identified by sequencing. The results showed that the structures of the recombinant expression vectors DBN12337 - DBN12350, DBN12352, DBN12353, and DBN12337N were absolutely correct.

5. Generation of transgenic Arabidopsis thaliana plants Wild-type Arabidopsis thaliana seeds were suspended in 0.1% (w/v) agarose solution. The suspended seeds were stored at 4°C for 2 days until dormancy was satisfied to ensure synchronous seed germination. Vermiculite was mixed with horse manure-containing soil, the mixture was watered until moist, and water was allowed to drain from the soil mixture for 24 hours. Pretreated seeds were germinated in the soil mixture and covered with a moisture blanket for 7 days. Seeds were germinated, and plants were grown in a greenhouse under long-wavelength sunlight (16-h light/8-h dark) at a constant temperature (22°C) and constant humidity (40-50%) with a light intensity of 120-150 μmol/ ^m2 s ^-1 . Plants were initially watered with Hoagland's nutrient solution and then with deionized water, thus keeping the soil moist but preventing water penetration.

Arabidopsis thaliana was transformed using the floral dip method. One or more 15-30 ml precultures of LB culture solution containing spectinomycin (50 mg/L) and rifampin (10 mg/L) were inoculated with selected Agrobacterium colonies. The precultures were incubated at 28°C and 220 rpm with shaking at a constant speed overnight. Each preculture was used to inoculate two 500 ml cultures of YEP culture solution containing spectinomycin (50 mg/L) and rifampin (10 mg/L), and the cultures were incubated at 28°C with continuous shaking overnight. Centrifugation at approximately 4000 rpm was performed at room temperature for 20 min to pellet the cells, and the resulting supernatant was discarded. The cell pellet was carefully resuspended in 500 mL of osmotic medium containing 1/2× MS/vitamin B5 salt, 10% (w/v) sucrose, 0.044 μM benzylaminopurine (10 μL/L (1 mg/mL stock solution in DMSO (dimethyl sulfoxide)) and 300 μL/L Silvet L-77. Arabidopsis thaliana plants aged approximately 1 month were immersed in the osmotic culture medium containing the resuspended cells for 15 seconds to ensure immersion of the very last inflorescence. Arabidopsis thaliana plants were then laid laterally and covered, and kept humid in the dark for 24 h. Arabidopsis thaliana plants were normally cultured under a 16 h light/8 h dark photoperiod at 22°C. The seeds were collected after approximately 4 weeks.

Freshly collected seeds (PPO1A-PPO14A nucleotide sequences, PPO-ATA nucleotide sequence, PPO-APA nucleotide sequence, and DBN12337N control vector) were dried at room temperature for 7 days. Seeds were sown on 26.5 cm × 51 cm germination disks, and 200 mg of _T1 seeds (approximately 10,000 seeds) were allowed per disk, where the seeds were previously suspended in distilled water and stored at 4°C for 2 days to satisfy the dormancy requirement and ensure synchronous seed germination.

Vermiculite was mixed with soil containing horse manure, watered until moist, and allowed to drain by gravity. Pre-treated seeds were sown evenly in the soil mixture using a pipette and covered with a moistening blanket for 4-5 days. The blanket was removed one day before glufosinate (used for PAT gene selection during co-transformation) was applied by spraying after germination to select the initial transformant.

T ₁ plants were sprayed with a 0.2% solution of Liberty herbicide (200 g a.i./L glufosinate) using a DeVilbiss compressed air nozzle at a spray volume of 10 ml/disc (703 L/ha) at 7 days after planting (DAP) and 11 DAP (cotyledon stage and 2-4 leaf stages, respectively) to provide an effective rate of glufosinate of 280 g a.i./ha per application. Surviving plants (actively growing plants) were identified 4-7 days after the final spray and transplanted into 7 cm × 7 cm square pots prepared from horse manure-vermiculite soil (3-5 plants/disc). The transplanted plants were covered with a humidifying blanket for 3-4 days and placed in a culture chamber at 22°C or directly transferred to the greenhouse as described above. The blanket was then removed, and at least 1 day before the analysis of the ability of the PPO1A-PPO14A nucleotide sequences, the PPO-ATA nucleotide sequence, the PPO-APA nucleotide sequence, and the control vector to provide tolerance to PPO-inhibiting herbicides, the plants were placed in a greenhouse (22±5°C, 50±30% RH (relative humidity), 14 h light: 10 h dark, at least 500 µE/m2 s ^{-1 natural} plus supplemental light).

6. Identification of resistance of transgenic Arabidopsis thaliana plants to herbicide

Transformed _T1 Arabidopsis thaliana plants were initially selected using a glufosinate-based herbicide. Arabidopsis thaliana (PPO1A) plants into which the nucleotide sequence PPO1A was introduced, T ₁ Arabidopsis thaliana (PPO2A) plants into which the nucleotide sequence PPO2A was introduced, Arabidopsis thaliana (PPO3A) plants into which the nucleotide sequence PPO3A was introduced, T ₁ Arabidopsis thaliana (PPO4A) plants into which the nucleotide sequence PPO4A was introduced, T ₁ Arabidopsis thaliana (PPO5A) plants into which the nucleotide sequence PPO5A was introduced, T ₁ Arabidopsis thaliana (PPO6A) plants into which the nucleotide sequence PPO6A was introduced, T ₁ Arabidopsis thaliana (PPO7A) plants into which the nucleotide sequence PPO7A was introduced, T ₁ Arabidopsis thaliana (PPO8A), into which the nucleotide sequence PPO8A was introduced, T ₁ Arabidopsis thaliana plants (PPOEA), into which the nucleotide sequence PPOEA was introduced, T ₁ Arabidopsis thaliana plants (PPO10A), into which the nucleotide sequence PPO10A was introduced, T ₁ Arabidopsis thaliana plants (PPO11A), into which the nucleotide sequence PPO11A was introduced, T ₁ Arabidopsis thaliana plants (PPO12A), into which the nucleotide sequence PPO12A was introduced, T ₁ Arabidopsis thaliana plants (PPO13A), into which the nucleotide sequence PPO13A was introduced, T ₁ Arabidopsis thaliana plants (PPO14A), into which the nucleotide sequence PPO14A was introduced, T ₁ Arabidopsis thaliana plants (PPO-APA) plants inserted with the nucleotide sequence of PPO-APA, T ₁ Arabidopsis thaliana plants (control vector) inserted with the control vector, and wild-type Arabidopsis thaliana plants (CK) (24 plants per genotype) (18 days after sowing) were sprayed with oxyfluorfen at three different concentrations (180 g a.i./ha (single field concentration, 1×), 720 g a.i./ha (fourfold field concentration, 4×), and 0 g a.i./ha (water, 0×), saflufenacil at three concentrations (25 g a.i./ha (single field concentration, 1×), 100 g a.i./ha (fourfold field concentration, 4×), and 0 g a.i./ha (water, 0×)), flumioxazin at three concentrations (60 g a.i./ha (single field concentration, 1×), 240 g a.i./ha (four times the field concentration, 4×), and 0 g a.i./ha (water, 0×)), and sulfentrazone at three concentrations (450 g a.i./ha (single field concentration, 1×), 900 g a.i./ha (two times the field concentration, 2×), and 0 g a.i./ha (water, 0×)) to determine the herbicide tolerance of Arabidopsis thaliana. The injury level of each plant caused by the herbicide was assessed according to the average injury level (%) of plants (average injury level (%) of plants = injured leaf area/total leaf area × 100%) at 7 days after spraying (7 DAT), and namely, the pesticide damage score: a score of 0 means that the plant growth status is substantially consistent with the growth statuses of plants sprayed with a blank solvent (water); a score of 1 means that the average plant damage levels are less than 10%; a score of 2 means that the average plant damage levels are greater than 10%; and a score of 3 means that the average plant damage levels are 100%. Plants with growth status falling within scores of 0 and 1 are classified as highly resistant plants; plants with growth status falling within score of 2 are classified as low-resistant plants; and plants with growth status falling within score of 3 are classified as non-resistant plants. The experimental results are shown in Tables 1-4.

In the case of Arabidopsis thaliana, 180 g a.i./ha of oxyfluorfen herbicide is an effective dose, discriminating between susceptible and intermediately resistant plants. The results in Table 1 show that, compared with the control vector and CK, all genotypes PPO1A-PPO14A demonstrated highly resistant resistance to oxyfluorfen at different concentrations, while both genotypes PPO-APA and PPO-ATA demonstrated largely no resistance.

In the case of Arabidopsis thaliana, 25 g a.i./ha of saflufenacil-based herbicide is an effective dose that discriminates between susceptible and intermediate-resistant plants. The results in Table 2 show that, compared with the control vector and CK, (1) all genotypes PPO1A-PPO14A exhibited highly resistant saflufenacil at a single field concentration, while both genotypes PPO-APA and PPO-ATA did not exhibit resistance; (2) All genotypes PPO1A-PPO7A and PPO9A-PPO14A showed highly resistant resistance to saflufenacil at four times the field concentration (only 2 plants in genotype PPO8A showed moderate or low resistant resistance, and all other 22 plants showed highly resistant resistance), while both genotypes PPO-APA and PPO-ATA did not show resistance.

In the case of Arabidopsis thaliana, 60 g a.i./ha of flumioxazin herbicide is an effective dose, discriminating between susceptible and intermediately resistant plants. The results in Table 3 show that, compared with the control vector and CK, all genotypes PPO1A-PPO14A demonstrated highly resistant flumioxazin at different concentrations, while both genotypes PPO-APA and PPO-ATA demonstrated mostly no resistance.

In the case of Arabidopsis thaliana, 450 g a.i./ha of sulfentrazone herbicide is an effective dose, discriminating between susceptible and intermediately resistant plants. The results in Table 4 show that, compared with the control vector and CK, all genotypes PPO1A-PPO14A demonstrated highly resistant resistance to sulfentrazone at different concentrations, while both genotypes PPO-APA and PPO-ATA demonstrated no resistance.

Example 2: Production and testing of transgenic soybean plants

1. Transformation of Agrobacterium with recombinant expression vectors

The recombinant expression vectors DBN12337, DBN12342, DBN12348, DBN12351 and DBN12353 (containing the nucleotide sequences of PPO1A, the nucleotide sequence of PPO6A, the nucleotide sequence of PPO12A, the nucleotide sequence of PPO-ECA and the nucleotide sequence of PPO-APA, respectively) and the control recombinant expression vector DBN12337N, as described in point 3 of Example 1, were transformed into Agrobacterium LBA4404 (Invitrogen, Chicago, USA, CAT: 18313-015), respectively, using a liquid nitrogen-based method, under the following transformation conditions: 100 μl of Agrobacterium LBA4404 and 3 μl of plasmid DNA (recombinant expression vector) were placed in liquid nitrogen for 10 minutes and kept in a warm water bath at 37°C for 10 minutes; the transformed Agrobacterium LBA4404 were inoculated into a tube with LB, cultured at 28°C and 200 rpm for 2 hours and then spread on a plate with LB containing 50 mg/L rafampicin and 50 mg/L spectinomycin until positive single clones grew, and single clones were selected for cultivation, and plasmids were isolated from them. The isolated plasmids were identified by sequencing. The results showed that the structures of the recombinant expression vectors DBN12337, DBN12342, DBN12348, DBN12351, DBN12353 and the control recombinant expression vector DBN12337N were absolutely correct.

2. Obtaining transgenic soybean plants

According to the conventional method of infection with Agrobacterium, the cotyledonary node tissue of the sterilely cultivated soybean variety Zhonghuang13 was co-cultured with Agrobacterium as described in paragraph 1 of this Example, so as to introduce T-DNA (containing the sequence of the 34S enhancer of figwort mosaic virus, the sequence of the promoter of the eukaryotic gene of oilseed rape elongation factor 1α (Tsf1), the sequence of the chloroplast transit peptide of Arabidopsis thaliana, the gene of 5-enolpyruvylshikimate-3-phosphate synthase, the sequence of the terminator of the pea RbcS gene, the sequence of the promoter of the gene of ubiquitin 10 of Arabidopsis thaliana, the transit peptide of white or pale green chloroplasts of Arabidopsis thaliana, the nucleotide sequence of PPO1A, the nucleotide sequence of PPO6A, nucleotide sequence of PPO12A, nucleotide sequence of PPO-ECA, nucleotide sequence of PPO-APA, nopaline synthetase gene terminator sequence, 35S cauliflower mosaic virus promoter sequence, phosphinothricin-N-acetyl-transferase gene and 35S cauliflower mosaic virus terminator sequence) of recombinant expression vectors DBN12337, DBN12342, DBN12348, DBN12351, DBN12353 and control recombinant expression vector DBN12337N into soybean chromosomes, thereby obtaining, respectively, soybean plants into which the nucleotide sequence of PPO1A was introduced, soybean plants into which the nucleotide sequence of PPO6A was introduced, soybean plants into which the nucleotide sequence of PPO12A, soybean plants into which the nucleotide sequence of PPO-ECA was introduced, soybean plants into which the nucleotide sequence of PPO-APA was introduced, and soybean plants into which the control vector DBN12337N was introduced.

For Agrobacterium-mediated soybean transformation, briefly, mature soybean seeds were germinated in soybean germination medium (3.1 g/L B5 salt, vitamin B5, 20 g/L sucrose and 8 g/L agar, pH 5.6) and then cultured under the conditions of 25±1°C; and photoperiod (light/dark) of 16 h/8 h. After 4-6 days of germination, sterile soybean seedlings swollen at bright green cotyledonary nodes were selected, hypocotyledonous parts were cut off 3-4 mm below the cotyledonary nodes, cotyledons were cut longitudinally, and apical buds, lateral buds and embryonic roots were removed. A wound was created at the level of the cotyledon node using the back of a scalpel, and the wounded cotyledon node tissues were brought into contact with the Agrobacterium suspension, where the Agrobacterium could transfer the PPO1A nucleotide sequence, the PPO6A nucleotide sequence, the PPO12A nucleotide sequence, the PPO-ECA nucleotide sequence, or the PPO-APA nucleotide sequence, respectively, into the wounded cotyledon node tissues (stage 1: infection stage). In this stage, the cotyledon tissues were preferably immersed in Agrobacterium suspension ( _OD660 0.5-0.8, infection culture medium (2.15 g/L MS salt, vitamin B5, 20 g/L sucrose, 10 g/L glucose, 40 mg/L acetosyringone (AS), 4 g/L 2-morpholineethanesulfonic acid (MES) and 2 mg/L zeatin (ZT), pH 5.3)) to initiate inoculation. The cotyledon tissues were co-cultured with Agrobacterium for a period of time (3 days) (stage 2: co-culture stage). Preferably, after the infection stage, the cotyledon tissues were cultured on solid medium (4.3 g/L MS salts, vitamin B5, 20 g/L sucrose, 10 g/L glucose, 4 g/L MES, 2 mg/L ZT, 8 g/L agar, pH 5.6). Following this co-cultivation step, an optional "recovery" step may take place, in which a recovery culture medium (3.1 g/L B5 salt, vitamin B5, 1 g/L MES, 30 g/L sucrose, 2 mg/L ZT, 8 g/L agar, 150 mg/L cephalosporin, 100 mg/L glutamic acid and 100 mg/L aspartic acid, pH 5.6) was used with the addition of at least one antibiotic (150-250 mg/L cephalosporin) to inhibit the growth of Agrobacterium, and without the addition of a selective agent for the plant transformant (step 3: recovery step). Preferably, the tissue blocks regenerated from the cotyledonary nodes were cultured in a solid medium containing an antibiotic and not containing a selective agent, so as to eliminate Agrobacterium and to provide a recovery period for the infected cells. Then, the tissue blocks regenerated from the cotyledon nodes were cultured in a culture medium containing a selective agent (glyphosate), and growing transformed calli were selected (step 4: selection step). Preferably, the tissue blocks regenerated from the cotyledon nodes were cultured in a solid culture medium for screening (3.1 g/L B5 salt, vitamin B5, 1 g/L MES, 30 g/L sucrose, 1 mg/L 6-benzyladenine (6-BAP), 8 g/L agar, 150 mg/L cephalosporin, 100 mg/L glutamic acid, 100 mg/L aspartic acid, and 0.25 mol/L N-(phosphonomethyl)glycine, pH 5.6) containing a selective agent, thereby obtaining selective growth of transformed cells. Plants were then regenerated from the transformed cells (stage 5: regeneration stage). Preferably, tissue blocks regenerated from cotyledon nodes grown in a culture medium containing a selective agent were cultured in solid culture media (B5 differentiation culture medium and B5 rooting culture medium) for plant regeneration.

The resistant tissues that were screened were transferred to B5 differentiation culture medium (3.1 g/L B5 salt, vitamin B5, 1 g/L MES, 30 g/L sucrose, 1 mg/L ZT, 8 g/L agar, 150 mg/L cephalosporin, 50 mg/L glutamic acid, 50 mg/L aspartic acid, 1 mg/L gibberellin, 1 mg/L auxin, and 0.25 mol/L N-(phosphonomethyl)glycine, pH 5.6) and cultured at 25°C for differentiation. The differentiated seedlings were transferred to B5 rooting medium (3.1 g/L B5 salt, vitamin B5, 1 g/L MES, 30 g/L sucrose, 8 g/L agar, 150 mg/L cephalosporin, and 1 mg/L indole-3-butyric acid (IBA)), cultured in the rooting medium at 25°C until they reached a height of about 10 cm, and then transferred to the greenhouse until fruiting. In the greenhouse, the plants were cultured at 26°C for 16 hours and then cultured at 20°C for 8 hours per day.

3. Testing transgenic soybean plants using TaqMan

Approximately 100 mg of leaves from soybean plants inserted with the PPO1A nucleotide sequence, soybean plants inserted with the PPO6A nucleotide sequence, soybean plants inserted with the PPO12A nucleotide sequence, soybean plants inserted with the PPO-ECA nucleotide sequence, soybean plants inserted with the PPO-APA nucleotide sequence, and soybean plants inserted with the DBN12337N control vector were collected as samples, and their genomic DNA was isolated using the DNeasy Plant Maxi kit from Qiagen, and the EPSPS gene copy number was detected using the Taqman fluorescence-based quantitative PCR method to determine the PPO gene copy number. Meanwhile, wild-type soybean plants were used as a control and were detected and analyzed according to the above-mentioned method. Three replicates were set for these experiments, and the average value was calculated.

The specific method for determining the number of copies of the EPSPS gene was as follows:

Step 11: 100 mg of leaves of soybean plants into which the nucleotide sequence of PPO1A was introduced, soybean plants into which the nucleotide sequence of PPO6A was introduced, soybean plants into which the nucleotide sequence of PPO12A was introduced, soybean plants into which the nucleotide sequence of PPO-ECA was introduced, soybean plants into which the nucleotide sequence of PPO-APA was introduced, soybean plants into which the control vector DBN12337N was introduced, and wild-type soybean plants were taken and ground into a homogenate using liquid nitrogen in a mortar, and three replicates were made for each sample;

Step 12: Genomic DNA of the above mentioned samples was isolated using the DNeasy Plant Mini kit from Qiagen, following the specific method as described in the product manual;

Step 13: Genomic DNA concentrations of the above samples were determined using NanoDrop 2000 (Thermo Scientific);

Step 14: The genomic DNA concentrations of the above samples were adjusted to the same value in the range of 80 to 100 ng/µL;

Step 15: The copy number of samples was identified using a fluorescence-based quantitative PCR method using a Taqman probe, where samples for which the copy number was known and identified were taken as standards, wild-type soybean plant samples were taken as controls, and three replicates were taken for each sample and averaged; the primer and probe sequences for the fluorescence-based quantitative PCR were as follows:

The following primers and probe were used to detect the EPSPS gene sequence:

primer 1: ctggaaggcgaggacgtcatcaata, as set forth in SEQ ID NO: 69 of the SEQUENCE LISTING;

primer 2: tggcggcattgccgaaatcgag, as set forth in SEQ ID NO: 70 of the SEQUENCE LISTING;

Probe 1: atgcaggcgatgggcgcccgcatccgta, as set forth in SEQ ID NO: 71 of the SEQUENCE LISTING;

PCR reaction system:

JumpStart™ Taq ReadyMix™ (Sigma) 10 µl

50× primer/probe mix 1 µl

genomic DNA 3 µl

water (ddH ₂ O) 6 µl

50× Primer/Probe Mix contained 45 µL of each primer at 1 mM, 50 µL of probe at 100 µM, and 860 µL of 1× TE buffer and was stored at 4°C in an amber glass tube.

PCR reaction conditions:

Data were analyzed using SDS2.3 software (Applied Biosystems).

By analyzing the results of the EPSPS gene copy number experiment, it was further demonstrated that all of the PPO1A nucleotide sequence, the PPO6A nucleotide sequence, the PPO12A nucleotide sequence, the PPO-ECA nucleotide sequence, the PPO-APA nucleotide sequence, and the DBN12337N control vector were inserted into the chromosome of the detected soybean plants, and all of the soybean plants into which the PPO1A nucleotide sequence was introduced, the soybean plants into which the PPO6A nucleotide sequence was introduced, the soybean plants into which the PPO12A nucleotide sequence was introduced, the soybean plants into which the PPO-ECA nucleotide sequence was introduced, the soybean plants into which the PPO-APA nucleotide sequence was introduced, and the soybean plants into which the DBN12337N control vector was introduced resulted in the production of single-copy transgenic soybean plants.

4. Identification of herbicide resistance in transgenic soybean plants

Soybean plants (PPO1A) introduced with the nucleotide sequence of PPO1A, soybean plants (PPO6A) introduced with the nucleotide sequence of PPO6A, soybean plants (PPO12A) introduced with the nucleotide sequence of PPO12A, soybean plants (PPO-ECA) introduced with the nucleotide sequence of PPO-ECA, soybean plants (PPO-APA) introduced with the nucleotide sequence of PPO-APA, soybean plants (control vector) introduced with the control vector, and wild-type soybean plants (CK) (with 16 plants included in each genotype) (18 days after sowing) were taken and sprayed with saflufenacil at three concentrations (50 g a.i./ha (two-fold field concentration, 2×), 100 g a.i./ha (four-fold field concentration, 4×) and 0 g a.i./ha (water, 0×)), oxyfluorfen at three concentrations (360 g a.i./ha (two times the field concentration, 2×), 720 g a.i./ha (four times the field concentration, 4×) and 0 g a.i./ha (water, 0×), and flumioxazin at three concentrations (120 g a.i./ha (two times the field concentration, 2×), 240 g a.i./ha (four times the field concentration, 4×) and 0 g a.i./ha (water, 0×) to determine the tolerance of soybean plants to the herbicides. According to the method as described above in 6. Example 1, after 7 days of spraying (7 DAT), the injury level of each plant caused by the herbicide was assessed according to the average damage levels (%) of plants. The experimental results are shown in Tables 5-7.

The results in Table 5 show that (1) compared with the control vector and CK, the genotypes PPO1A, PPO6A, PPO12A, and PPO-ECA could show different degrees of resistance to saflufenacil, while PPO-APA basically did not show resistance; (2) when treated with saflufenacil at twice the field concentration, the injury levels in the genotypes PPO1A, PPO6A, and PPO12A were scored as 0, while about 44% of the plants in the genotype PPO-ECA were scored as 1; and (3) all the genotypes PPO1A, PPO6A, and PPO12A showed highly resistant resistance to saflufenacil at four times the field concentration, while about 31% of the plants in the genotype PPO-ECA showed moderate or low resistant resistance.

The results in Table 6 show that, compared with the control vector and SC, (1) all genotypes of PPO1A, PPO6A, PPO12A, and PPO-ECA exhibited highly resistant resistance to oxyfluorfen at twice the field concentration, while 50% of plants in the PPO-APA genotype did not exhibit resistance; and (2) all genotypes of PPO1A, PPO6A, PPO12A, and PPO-ECA exhibited highly resistant resistance to oxyfluorfen at four times the field concentration, while the PPO-APA genotype did not exhibit resistance.

The results in Table 7 show that, compared with the control vector and CK, all genotypes PPO1A, PPO6A, PPO12A and PPO-ECA showed highly resistant to flumioxazin at different concentrations, while the genotype PPO-APA did not show resistance to flumioxazin.

Example 3. Production and testing of transgenic corn plants

1. Construction of recombinant maize expression vectors containing PPO genes

The 5' and 3' ends of the nucleotide sequence of PPO1B, the nucleotide sequence of PPO6B and the nucleotide sequence of PPO12B and the nucleotide sequence of PPO-APB, as described in point 1 of Example 1, respectively, were linked with the following universal primer-adapter 2:

Universal 5'-end adapter primer 2: 5'-ccaagcggccaagctta-3', as set forth in SEQ ID NO: 72 in the SEQUENCE LISTING;

Universal 3' adapter primer 2: 5'-tgtttgaacgatcggcgcgcc-3', as set forth in SEQ ID NO: 73 in the SEQUENCE LISTING.

The plant expression vector DBNBC-02 was double digested using Spe I and Asc I restriction enzymes to linearize the plant expression vector. The digestion product was purified to obtain the linearized backbone of the DBNBC-02 expression vector (vector backbone: pCAMB1A2301 (available from CAMBIA)), which was then recombined with the nucleotide sequence of PPO1B linked to the universal primer-adapter 2 according to the procedure in the instructions of the Takara In-Fusion Seamless Linking Kit (Clontech, CA, USA, CAT: 121416) to construct the recombinant expression vector BN12354 with the vector structure as shown in Fig. 3. (Spec: spectinomycin resistance gene; RB: right border; PPOsAct1: rice actin 1 promoter (SEQ ID NO: 74); cPAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO: 60); t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 61); pr35S-06: cauliflower mosaic virus 35S promoter (SEQ ID NO: 75); iZmHSP70: Zea mays 70 kDa heat shock protein intron (SEQ ID NO: 76); spAtCLPI: Arabidopsis thaliana white or light green chloroplast transit peptide (SEQ ID NO: 57); PPO1B: nucleotide sequence of PPO1B (SEQ ID NO: 62); tNos: nopaline synthase gene terminator (SEQ ID NO: 58); prZmUbi: Zea mays ubiquitin 1 gene promoter (SEQ ID NO: 77); PMI: phosphomannose isomerase gene (SEQ ID NO: 78); tNos: nopaline synthase gene terminator (SEQ ID NO: 58); LB: left border).

Competent _T1 Escherichia coli cells were transformed according to the heat shock method described in Section 3 of Example 1, and plasmids in the cells were isolated using the alkaline method. The isolated plasmid was identified by sequencing. The results indicated that the recombinant expression vector DBN12354 contained the nucleotide sequence set forth in SEQ ID NO: 62 in the SEQUENCE LISTING, namely, the nucleotide sequence of PPO1B.

According to the above method for constructing the recombinant expression vector DBN12354, the nucleotide sequence of PPO6B, the nucleotide sequence of PPO12B, and the nucleotide sequence of PPO-APB, which were linked to the universal primer-adapter 2, respectively, were subjected to a recombination reaction with the linearized backbone of the expression vector DBNBC-02, to sequentially construct the recombinant expression vectors DBN12355 - DBN12357. Sequencing confirmed that the nucleotide sequence of PPO6B, the nucleotide sequence of PPO12B, and the nucleotide sequence of PPO-APB were correctly inserted into the recombinant expression vectors DBN12355 - DBN12357.

According to the method for constructing the recombinant expression vector DBN12354 as described above, the recombinant expression vector DBN12354N was constructed as a control, and its structure is shown in Fig. 4 (Spec: spectinomycin resistance gene; RB: right border; prOsAct1: rice actin 1 promoter (SEQ ID NO: 74); cPAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO: 60); t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 61); prZmUbi: Zea mays ubiquitin 1 gene promoter (SEQ ID NO: 77); PMI: phosphomannose isomerase gene (SEQ ID NO: 78); tNos: nopaline synthase gene terminator (SEQ ID NO: 58); LB: left border).

2. Transformation of Agrobacterium with recombinant expression vectors

Agrobacterium LBA4404 (Invitrogen, Chicago, USA, CAT: 18313-015) were respectively transformed with the recombinant expression vectors DBN12354 to DBN12357, which were correctly constructed, and the above-mentioned control recombinant expression vector DBN12354N using the liquid nitrogen-based method under the following transformation conditions: 100 μl of Agrobacterium LBA4404 and 3 μl of plasmid DNA (recombinant expression vector) were placed in liquid nitrogen for 10 minutes and kept in a warm water bath at 37°C for 10 minutes; the transformed Agrobacterium LBA4404 were inoculated into a LB tube, cultured under the conditions of 28°C and 200 rpm. for 2 hours and then spread on LB plate containing 50 mg/L rifampicin and 50 mg/L spectinomycin until positive single clones grew, and the single clones were selected for cultivation, and their plasmids were isolated. The isolated plasmids were identified by sequencing. The results showed that the structures of the recombinant expression vectors DBN12354 - DBN12357 and the control vector DBN12354N were absolutely correct.

3. Obtaining transgenic corn plants

According to a commonly used Agrobacterium infection method, young embryos of the sterilely cultivated maize variety Zong31 (Z31) were co-cultured with Agrobacterium as described in paragraph 2 of this Example so as to introduce T-DNA (containing a rice actin 1 promoter sequence; a phosphinothricin N-acetyltransferase gene; a 35S terminator sequence of cauliflower mosaic virus; a 35S promoter sequence of cauliflower mosaic virus; a 70 kDa heat shock protein intron sequence of Zea mays; a white or light green chloroplast transit peptide of Arabidopsis thaliana; a nucleotide sequence of PPO1B, a nucleotide sequence of PPO6B; a nucleotide sequence of PPO12B; a nucleotide sequence of PPO-APB; a nopaline synthetase gene terminator sequence; a promoter sequence Zea mays ubiquitin 1 gene; phosphomannose isomerase gene; and nopaline synthase gene terminator sequence) of the recombinant expression vectors DBN12354 to DBN12357 and the control recombinant expression vector DBN12354N, as constructed in item 1 of this Example, into maize chromosomes, respectively, to obtain maize plants into which the nucleotide sequence of PPO1B is introduced, maize plants into which the nucleotide sequence of PPO6B is introduced, maize plants into which the nucleotide sequence of PPO12B is introduced, maize plants into which the nucleotide sequence of PPO-APB is introduced, and maize plants into which the control vector DBN12354N is introduced; meanwhile, the wild-type maize plants were used as a control.

For Agrobacterium-mediated transformation of maize, briefly, immature young embryos were separated from maize and brought into contact with an Agrobacterium suspension, where the Agrobacterium can transfer the PPO1B nucleotide sequence, the PPO6B nucleotide sequence and the PPO12B nucleotide sequence, or the PPO-APB nucleotide sequence, into at least one cell of one of the young embryos (stage 1: infection stage). At this stage, the young embryos were preferably immersed in a suspension of Agrobacterium ( _OD660 is 0.4-0.6, infection medium (4.3 g/L MS salts, vitamin N6, 300 mg/L casein, 68.5 g/L sucrose, 36 g/L glucose, 40 mg/L AS (acetosyringone), 1 mg/L 2,4-D; pH 5.3)) to initiate inoculation. The young embryos were co-cultured with Agrobacterium for a period of time (3 days) (stage 2: co-culture stage). Preferably, after the infection step, the young embryos were cultured on a solid medium (4.3 g/L MS salts, MS vitamins, 300 mg/L casein, 20 g/L sucrose, 10 g/L glucose, 100 mg/L AS, 1 mg/L 2,4-D g/L, 8 g/L agar, pH 5.8). After the co-culture step, there may be an optional "recovery" step, in which a recovery medium (4.3 g/L MS salts, MS vitamins, 300 mg/L casein, 30 g/L sucrose, 1 mg/L 2,4-D, 3 g/L plant gel, pH 5.8) with the addition of at least one antibiotic (cephamycin) to inhibit the growth of Agrobacterium was used, and without the addition of any selective agent for plant transformants (step 3: recovery step). Preferably, the young embryos were cultured in a solid culture medium containing an antibiotic and lacking a selective agent, so as to eliminate Agrobacterium and provide a recovery period for the infected cells. Then, the inoculated young embryos were cultured on a medium containing a selective agent (mannose), and growing transformed calli were selected (step 4: selection step). Preferably, the young embryos were cultured in a solid selective medium (4.3 g/L MS salts, MS vitamins, 300 mg/L casein, 30 g/L sucrose, 12.5 g/L mannose, 1 mg/L 2,4-D, 3 g/L plant gel; pH 5.8) containing a selective agent, to obtain selective growth of the transformed cells. Then, the plants were regenerated from the calli (step 5: regeneration step). Preferably, calli growing on the medium containing the selective agent were cultured in a solid medium (MS differentiation medium and MS rooting medium) to regenerate plants.

The resistant calli obtained as a result of screening were transferred to MS differentiation medium (4.3 g/L MS B5 salts, MS vitamins, 300 mg/L casein, 30 g/L sucrose, 2 mg/L 6-benzyladenine, 5 mg/L mannose, 3 g/L plant gel; pH 5.8) and cultured at 25°C for differentiation. Differentiated seedlings were transferred to MS rooting medium (2.15 g/L MS salts, MS vitamins, 300 mg/L casein, 30 g/L sucrose, 1 mg/L indole-3-acetic acid, 3 g/L plant gel; pH 5.8) and cultured at 25°C. When the plants reached a height of approximately 10 cm, they were transferred to a greenhouse and cultivated until fruiting. In the greenhouse, the plants were grown at 28°C for 16 hours and then at 20°C for 8 hours per day.

4. Testing transgenic maize plants using TaqMan

According to the method described in paragraph 3 of Example 2, to test transgenic soybean plants using TaqMan, maize plants into which the nucleotide sequence of PPO1B was introduced, maize plants into which the nucleotide sequence of PPO6B was introduced, maize plants into which the nucleotide sequence of PPO12B was introduced, maize plants into which the nucleotide sequence of PPO-APB was introduced, and maize plants into which the control vector DBN12354N was introduced were detected and analyzed. The copy number of the PMI gene was detected by quantitative fluorescence-based PCR with a Taqman probe, thereby determining the copy number of the PPO gene. Meanwhile, wild-type maize plants were used as a control and were detected and analyzed according to the above-mentioned method. Three replicates were set for these experiments, and the average value was calculated.

The following primers and probe were used to detect the PMI gene sequence:

Primer 3: gctgtaagagcttactgaaaaaattaaca, as set forth in SEQ ID NO: 79 of the SEQUENCE LISTING;

Primer 4: cgatctgcaggtcgacgg, as set forth in SEQ ID NO: 80 of the SEQUENCE LISTING;

Probe 2: tctcttgctaagctgggagctcgat.ee, as set forth in SEQ ID NO: 81 in the SEQUENCE LISTING.

In addition, by analyzing the results of the PMI gene copy number experiment, it was confirmed that all of the PPO1B nucleotide sequence, the PPO6B nucleotide sequence, the PPO12B nucleotide sequence, the PPO-APB nucleotide sequence, and the DBN12354N control vector were inserted into the chromosomes of the detected maize plants; and all of the maize plants into which the PPO1B nucleotide sequence was introduced, the maize plants into which the PPO6B nucleotide sequence was introduced, the maize plants into which the PPO12B nucleotide sequence was introduced, the maize plants into which the PPO-APB nucleotide sequence was introduced, and the maize plants into which the DBN12354N control vector was introduced resulted in single-copy transgenic maize plants.

5. Identification of herbicide resistance in transgenic maize plants

Maize plants (PPO1B) introduced with the nucleotide sequence of PPO1B, maize plants (PPO6B) introduced with the nucleotide sequence of PPO6B, maize plants (PPO12B) introduced with the nucleotide sequence of PPO12B, maize plants (PPO-APB) introduced with the nucleotide sequence of PPO-APB, maize plants (control vector) introduced with the control vector, and wild-type maize plants (CK) (with 16 plants included in each genotype) (18 days after sowing) were selected and sprayed with saflufenacil at three concentrations (50 g a.i./ha (two-fold field concentration, 2×), 100 g a.i./ha (four-fold field concentration, 4×), and 0 g a.i./ha (water, 0×)). oxyfluorfen at three concentrations (360 g a.i./ha (two times the field concentration, 2×), 720 g a.i./ha (four times the field concentration, 4×) and 0 g a.i./ha (water, 0×), and flumioxazin at three concentrations (120 g a.i./ha (two times the field concentration, 2×), 240 g a.i./ha (four times the field concentration, 4×) and 0 g a.i./ha (water, 0×)), to determine the tolerance of maize plants to the herbicides. According to the method as described above in 6. Example 1, after 7 days of spraying (7 DAT), the injury level of each plant caused by the herbicide was assessed according to the average injury level (%) of plants (average injury level (%) of plants = area of injured leaves/total leaf area × 100%). The experimental results are shown in Tables 8–10.

The results in Table 8 show that, compared with the control vector and CK, (1) all genotypes PPO1B, PPO6B, and PPO12B showed highly resistant saflufenacil resistance at twice the field concentration, while approximately 56% of plants in the PPO-APB genotype showed no resistance; (2) all genotypes PPO1B, PPO6B, and PPO12B showed highly resistant saflufenacil resistance at four times the field concentration, while the PPO-APB genotype mostly showed no resistance.

The results in Table 9 show that, compared with the control vector and CK, (1) all genotypes PPO1B, PPO6B, and PPO12B exhibited highly resistant resistance to oxyfluorfen at two-fold field concentration, while approximately 50% of plants in the PPO-APB genotype did not exhibit resistance; (2) all genotypes PPO1B, PPO6B, and PPO12B exhibited highly resistant resistance to oxyfluorfen at four-fold field concentration, while the PPO-APB genotype did not exhibit resistance.

The results in Table 10 show that, compared with the control vector and CK, all genotypes of PPO1B, PPO6B and PPO12B showed highly resistant resistance to flumioxazin at different concentrations, while the PPO-APB genotype generally did not show resistance to flumioxazin.

To summarize the above, with respect to plants, the protoporphyrinogen oxidases PPO1-PPO14 of the present invention can provide good tolerance to PPO inhibitor herbicides in Arabidopsis thaliana plants; in particular, PPO1, PPO6 and PPO12 can provide good tolerance to PPO inhibitor herbicides in Arabidopsis thaliana, soybean and corn plants. Thus, the protoporphyrinogen oxidases PPO1-PPO14 can provide good tolerance to plants. With respect to herbicides, the present invention discloses for the first time that the protoporphyrinogen oxidases PPO1-PPO14 can provide higher tolerance to PPO inhibitor herbicides in plants, to such an extent that these plants can tolerate at least four times the field concentration of oxyfluorfen, saflufenacil or flumioxazin and two times the field concentration of sulfentrazone. Thus, the present invention has prospects for wide application in plants.

Finally, it should be noted that all of the above Examples are used only to illustrate embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with respect to preferred Examples, those skilled in the art will understand that these embodiments of the present invention can be modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

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SEQUENCE LIST

<110> BEIJING DABEINUNG BIOTECHNOLOGY CO., LTD.

<120> APPLICATION OF PROTOPORPHYRIN GEN OXIDASE

<130> FP1210437P

<160> 81

<170> SIPOSequenceListing 1.0

<210> 1

<211> 179

<212> PRT

<213> Ewingella americana

<220>

<223> Amino acid sequence of PPO1

<400> 1

Met Lys Ala Leu Val Leu Tyr Ser Thr Arg Asp Gly Gln Thr His Ala

1 5 10 15

Ile Ala Ser Tyr Ile Ala Asn Cys Met Lys Glu Lys Tyr Glu Cys Asp

20 25 30

Val Ile Asp Leu Leu His Ala Gln His Val Thr Leu Ser Arg Tyr Asp

35 40 45

Lys Val Leu Val Gly Ala Ser Ile Arg Tyr Gly His Phe Asn Ala Val

50 55 60

Leu Asp Lys Phe Val Lys Gln Asn Ile Gln Gln Leu Asn Ser Met Pro

65 70 75 80

Ser Ala Phe Phe Ala Val Asn Leu Thr Ala Arg Lys Pro Glu Lys Arg

85 90 95

Thr Pro Gln Thr Asn Ser Tyr Val Arg Lys Phe Leu Leu Gly Thr Pro

100 105 110

Trp Lys Pro Ala Met Cys Gly Val Phe Ala Gly Ala Leu Arg Tyr Pro

115 120 125

Arg Tyr Arg Trp Val Asp Lys Val Met Ile Gln Leu Ile Met Arg Met

130 135 140

Thr Gly Gly Glu Thr Asp Thr Ser Lys Glu Val Glu Tyr Thr Asp Trp

145 150 155 160

Glu Gln Val Lys Lys Phe Ala Glu Asp Phe Gly Lys Leu Ser Tyr Lys

165 170 175

Lys Thr Leu

<210> 2

<211> 174

<212> PRT

<213> Vibrio sp. HDW18

<220>

<223> Amino acid sequence of PPO2

<400> 2

Met Arg Ala Leu Leu Leu Tyr Ser Ser Gln Glu Gly Gln Thr Arg Lys

1 5 10 15

Ile Ile Gln Arg Ile Ala Ala Gln Met Pro Glu Tyr Thr Cys Glu Val

20 25 30

Gln Asp Leu His Gln Ser Ile Asp Ile Asp Trp Ala Glu Tyr Asp Lys

35 40 45

Val Leu Ile Gly Ala Ser Ile Arg Tyr Gly Arg Leu Asn Pro Ala Leu

50 55 60

Tyr Arg Phe Ile Glu His His Leu Val Gly Leu Thr Ser Arg Lys Ala

65 70 75 80

Ala Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Glu Gln Gln Gly Lys

85 90 95

Asp Thr Pro Gln Gly Ser Ala Tyr Ile Gln Thr Phe Leu Lys Lys Ser

100 105 110

Ala Trp Gln Pro Glu Arg Ile Ala Val Phe Ala Gly Ala Leu Tyr Tyr

115 120 125

Pro Arg Tyr Arg Trp Phe Asp Lys Met Met Ile Arg Leu Ile Met Thr

130 135 140

Leu Thr Gly Gly Glu Thr Asp Thr Ser Gln Glu Val Glu Tyr Thr Asn

145 150 155 160

Trp Asp Lys Val Val Lys Phe Ala Glu Gln Phe Arg Asn Trp

165 170

<210> 3

<211> 176

<212> PRT

<213> Proteus hauseri

<220>

<223> Amino acid sequence of PPO3

<400> 3

Met Ser Ala Leu Leu Leu Tyr Cys Ser Thr Asp Gly Gln Thr Lys Lys

1 5 10 15

Ile Met Thr Gln Ile Ala Asn Glu Leu Arg Gln His Gly Tyr Glu Cys

20 25 30

Asp Val Arg Asp Leu Thr Ser Val Gln Gln Asn Leu Asn Leu Ser Ala

35 40 45

Tyr Asn Lys Val Leu Val Gly Ala Ser Ile Arg Tyr Gly Tyr Phe Asn

50 55 60

Lys Ala Leu Asp Lys Phe Ile Thr Arg His Leu Ser Gln Leu Asn Asn

65 70 75 80

Met Pro Ser Ala Phe Phe Gly Val Asn Leu Thr Ala Arg Lys Glu Glu

85 90 95

Lys Ser Thr Pro Glu Thr Asn Ser Tyr Ile Cys Lys Phe Leu Asp Lys

100 105 110

Thr Pro Trp Lys Pro Thr Leu Thr Gly Val Phe Ala Gly Ala Leu Phe

115 120 125

Tyr Pro Arg Tyr Lys Trp Ile Asp Arg Val Met Ile Gln Leu Ile Met

130 135 140

Lys Met Thr Lys Gly Glu Thr Asp Pro Thr Lys Glu Ile Glu Tyr Thr

145 150 155 160

Asp Trp Asn Lys Val Ser Glu Phe Ala Thr Asn Phe Ala Lys Ile Gly

165 170 175

<210> 4

<211> 171

<212> PRT

<213> Rodentibacter genomosp.2

<220>

<223> Amino acid sequence of PPO4

<400> 4

Met Lys Thr Leu Ile Leu Tyr Ser Ser His Asp Gly Gln Thr Lys Lys

1 5 10 15

Ile Ser Glu Phe Leu Thr Ala Leu Leu Asn Gly Glu Val Leu Val Asp

20 25 30

Ala Leu Arg Glu Asp Tyr Asp Ile Glu Thr Phe Asp Gln Val Ile Ile

35 40 45

Gly Ala Ser Ile Arg Tyr Gly His Phe Ser Lys Thr Leu Asp Arg Phe

50 55 60

Val Val Arg Asn Arg Val His Leu Glu Gln Lys Arg Ala Val Phe Phe

65 70 75 80

Ser Val Asn Leu Thr Ala Arg Lys Glu Gly Lys Asp Asn Pro Glu Thr

85 90 95

Asn Val Tyr Val Arg Lys Phe Leu Gln Arg Thr Lys Trp Gln Pro Lys

100 105 110

Leu Ala Ala Val Phe Ala Gly Ala Leu Leu Tyr Pro Arg Tyr Lys Trp

115 120 125

Phe Asp Arg Val Met Ile Arg Phe Ile Met Lys Ile Thr Gly Gly Glu

130 135 140

Thr Asp Ile Thr Lys Glu Ile Glu Tyr Thr Asp Trp Glu Lys Val Arg

145 150 155 160

Glu Phe Ala Gln Arg Ile Asn Arg Leu Glu Lys

165 170

<210> 5

<211> 176

<212> PRT

<213> Mannheimia pernigra

<220>

<223> Amino acid sequence of PPO5

<400> 5

Met Lys Thr Leu Ile Leu Tyr Phe Thr Thr Asp Gly Gln Thr Lys Lys

1 5 10 15

Ile Ala Glu Arg Leu Ala Asp Val Ile Thr His Glu Val Glu Leu Ile

20 25 30

Ser Leu Lys Asp Gln Ala Val Asp Phe Ala Gln Lys Met Ala Asn Val

35 40 45

Asp Gln Val Val Ile Gly Ala Ser Ile Arg Tyr Gly His Phe Ser Pro

50 55 60

Leu Val Tyr Gln Phe Ile Glu Gln His Tyr Ala Met Leu Asn Glu Lys

65 70 75 80

Lys Thr Ala Phe Tyr Gly Val Asn Leu Thr Ala Arg Lys Ala Asn Arg

85 90 95

Asn Thr Pro Thr Thr Asn Thr Tyr Ile Arg Asn Phe Leu Ala Lys Val

100 105 110

Lys Trp Gln Pro Asn Tyr Val Glu Val Ile Ala Gly Ala Leu Phe Tyr

115 120 125

Pro Arg Tyr Arg Phe Ile Asp Arg Met Met Ile Arg Phe Ile Met Lys

130 135 140

Val Thr Lys Gly Glu Thr Asp Val Ser Lys Glu Tyr Glu Tyr Thr Asn

145 150 155 160

Trp Gln Gln Val Glu Glu Phe Gly Lys Lys Leu Ser Asp Gln Leu Ile

165 170 175

<210> 6

<211> 231

<212> PRT

<213> Leptomonas pyrrhocoris

<220>

<223> amino acid sequence of PPO6

<400> 6

Met Ser Ser Pro Asn Gly Lys Tyr Leu Met Leu Tyr Ser Thr Thr Asp

1 5 10 15

Gly His Thr Lys Thr Ile Met Asp Thr Met Ala Lys His Ile Met Glu

20 25 30

Glu Ala Lys Val Gln Cys Asp Val Val Asp Met Arg Asp Gly Asp Lys

35 40 45

Tyr Glu Leu Ala Ala Tyr Glu Lys Val Met Leu Gly Ala Ser Ile Arg

50 55 60

Tyr Gly Phe Phe Ser Arg Thr Leu His Thr Tyr Thr Thr His His Val

65 70 75 80

Asp Glu Leu Asn Ser Met Pro Ser Ala Phe Phe Gly Val Asn Leu Thr

85 90 95

Ala Arg Lys Thr Ser Lys Asn Thr Ala Met Thr Asn Ala Tyr Thr Arg

100 105 110

Lys Phe Leu Asp Gln Ser Met Trp Val Pro Gln Leu Ser Gly Val Phe

115 120 125

Ala Gly Ala Leu Trp Tyr Pro Arg Tyr Asn Phe Phe Asp Arg Val Met

130 135 140

Ile Gln Phe Ile Met Lys Val Thr Gly Gly Glu Thr Asn Thr Thr Lys

145 150 155 160

Glu Ile Val Tyr Thr Asp Trp Asp Ala Val His Lys Phe Ala Thr Asp

165 170 175

Phe Val Gln Leu Pro Ala Thr Ala Ile Pro Arg Ser Lys Pro Ala Thr

180 185 190

Ser Val Pro Pro Ala Ser Val Ala Asn Tyr Asp Asn Gly Ala Arg Val

195 200 205

Ala Leu Val Val Val Gly Ile Ser Ala Ala Ile Ile Phe Gly Arg Arg

210 215 220

Leu Ile Leu Ala Lys Arg Phe

225 230

<210> 7

<211> 174

<212> PRT

<213> Aeromonas aquatica

<220>

<223> Amino acid sequence of PPO7

<400> 7

Met Asp Lys Ile Leu Val Leu Tyr Ser Ser Gln Asp Gly Gln Thr Arg

1 5 10 15

Lys Ile Val Asp Ala Met Leu Glu Glu Ile Ser Gly Cys Asp Val Val

20 25 30

Ile Gln Asp Leu His Thr Leu Pro Lys Cys Asn Leu Ser Lys Tyr Ala

35 40 45

Lys Val Leu Ile Gly Ala Ser Ile Arg Tyr Gly Asn Phe His Pro Ser

50 55 60

Leu Leu Ser Phe Ile Asn Ala His His Glu Gln Leu Glu Val Ala Asn

65 70 75 80

Ala Ala Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Pro Glu Lys Gln

85 90 95

Thr Pro Gln Thr Asn Ala Tyr Met Lys Lys Phe Leu Arg Leu Ser Pro

100 105 110

Trp Lys Pro Lys Thr Leu Gly Val Phe Ala Gly Ala Leu Gln Tyr Ser

115 120 125

Arg Tyr Asn Trp Trp Gln Thr Arg Ile Ile Gln Leu Ile Met Lys Met

130 135 140

Thr Gly Gly Ser Thr Asp Thr Ser Gln Asp Leu Glu Phe Thr Asp Trp

145 150 155 160

Val Lys Val Arg Ser Phe Ala Arg Glu Phe Trp Ser Lys Arg

165 170

<210> 8

<211> 191

<212> PRT

<213> Psychromonas sp. SA13A

<220>

<223> Amino acid sequence of PPO8

<400> 8

Met Ser Asn Glu Thr Gln Ala Lys Gln Ala Glu Thr Lys Gln Thr Glu

1 5 10 15

Ser Ser Val Val Ser Lys Thr Leu Val Leu Tyr Ser Ser Val Asp Gly

20 25 30

Gln Thr Leu Lys Ile Ile Asn Arg Ile Lys Gln Thr Leu Thr Gly Glu

35 40 45

Val Ile Val Leu Asn Val Asp Asp Asn Pro Thr Ile Asp Phe Ser Leu

50 55 60

Tyr Gln Lys Val Leu Ile Gly Ala Ser Ile Arg Tyr Gly Asn Phe Arg

65 70 75 80

Pro Asn Ile Ile Lys Phe Val Asn Gln His Lys Thr Gln Leu Asp Ala

85 90 95

Val Cys Asn Ala Phe Phe Val Val Cys Leu Thr Ala Arg Lys Pro Glu

100 105 110

Lys Ala Val Pro Glu Asn Asn Ala Tyr Met Lys Lys Phe Asp Gln Leu

115 120 125

Ser Gln Trp Gln Pro Gln Phe Lys Gly Val Phe Ala Gly Ala Leu Leu

130 135 140

Tyr Ser Arg Tyr Asn Trp Trp Gln Thr Leu Leu Ile Gln Leu Ile Met

145 150 155 160

Lys Met Thr Gly Gly Ser Thr Asp Lys Thr Gln Asp Ile Glu Leu Thr

165 170 175

Asp Trp Glu Lys Val Asp Ser Phe Ala Gln Asp Phe Ala Ala Arg

180 185 190

<210> 9

<211> 174

<212> PRT

<213> Photobacterium aquae

<220>

<223> Amino acid sequence of PPO9

<400> 9

Met Glu Lys Val Leu Leu Leu His Ser Ser Ser Asp Gly Gln Thr Val

1 5 10 15

Lys Ile Leu Arg His Ile Glu Thr Thr Leu Gly Gly Asp Tyr Gln Cys

20 25 30

Glu Leu Val Asp Leu Asn Thr Leu Pro Thr Val Asp Phe Ala Asp Tyr

35 40 45

Asp Arg Val Leu Val Gly Ala Ser Ile Arg Tyr Gly His Leu Asn Lys

50 55 60

Lys Leu Tyr Gln Phe Ile Glu Arg Tyr Arg Asp Glu Leu Asp Gln Asn

65 70 75 80

Lys Val Gly Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Glu Gly Lys

85 90 95

Asn Thr Pro Glu Thr Ser Val Tyr Met Lys Thr Phe Leu Lys Lys Ser

100 105 110

Pro Trp Cys Pro Lys Leu Gln Glu Val Phe Ala Gly Ala Leu Arg Tyr

115 120 125

Pro Arg Tyr Gly Trp Phe Asp Arg Val Met Ile Gln Phe Ile Met Arg

130 135 140

Ile Thr Gly Gly Glu Thr Asp Thr Ser Lys Glu Val Glu Tyr Thr Asp

145 150 155 160

Trp Gln Lys Val Glu Ala Phe Ala Ala Arg Phe Lys His Tyr

165 170

<210> 10

<211> 174

<212> PRT

<213> Vibrio mimicus

<220>

<223> Amino acid sequence of PPO10

<400> 10

Met Lys Ala Leu Phe Leu Tyr Ser Ser Gln Glu Gly Gln Thr Arg Lys

1 5 10 15

Ile Ile Glu Arg Ile Ala Gln Gln Met Pro Glu Tyr Asp Cys Asp Ile

20 25 30

Gln Asp Leu His Gln Val Ser Asp Ile Ala Leu Ala Asp Tyr Asp Lys

35 40 45

Ile Leu Ile Gly Ala Ser Ile Arg Tyr Gly Arg Leu Asn Asp Lys Leu

50 55 60

Tyr Gln Phe Ile Gln Arg Tyr Leu Pro Asp Leu Arg Ser Asn Lys Ala

65 70 75 80

Ala Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Glu Glu Gln Gly Lys

85 90 95

Asp Thr Pro Glu Gly Ser Val Tyr Ile Gln Thr Phe Leu Lys Lys Ser

100 105 110

Pro Trp Gln Pro Glu Arg Ile Ala Val Phe Ala Gly Ala Leu Tyr Tyr

115 120 125

Pro Arg Tyr Arg Trp Phe Asp Lys Met Met Ile Arg Leu Ile Met Thr

130 135 140

Leu Thr Gly Gly Glu Thr Asp Thr Ser Lys Glu Val Glu Tyr Thr Asn

145 150 155 160

Trp Asn Lys Val Ser Gln Phe Ala Glu Gln Phe Arg Asn Trp

165 170

<210> 11

<211> 175

<212> PRT

<213> Vibrio

<220>

<223> Amino acid sequence of PPO11 (unclassified vibrio)

<400> 11

Met Gln Lys Ala Leu Phe Leu Tyr Ser Ser Arg Glu Gly Gln Thr Lys

1 5 10 15

Lys Ile Leu Asn Tyr Ile Lys Glu Glu Leu Ser Glu Phe Asp Cys Glu

20 25 30

Leu Ala Asp Leu His Gln Ile Glu Asp Ile Asp Leu Ala Leu Tyr Asp

35 40 45

Arg Val Leu Ile Gly Ala Ser Ile Arg Tyr Gly His Leu Asn Lys Lys

50 55 60

Leu Tyr Gln Phe Ile Glu Lys His Ala Glu Gln Leu Ala Leu Ala Lys

65 70 75 80

Ala Ala Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Glu Ala Gln Gly

85 90 95

Lys Asp Thr Pro Glu Gly Ser Val Tyr Ile Lys Thr Phe Leu Ser Lys

100 105 110

Ser Ala Trp Gln Pro Lys Leu Ile Gly Val Phe Ala Gly Ala Leu Tyr

115 120 125

Tyr Pro Arg Tyr His Phe Phe Asp Lys Val Met Ile Arg Phe Ile Met

130 135 140

Ser Leu Thr Gly Gly Glu Thr Asp Thr Ser Lys Glu Val Glu Tyr Thr

145 150 155 160

Asn Trp Gln Lys Val Ser Leu Phe Ser Asp Asn Phe Lys Lys Met

165 170 175

<210> 12

<211> 176

<212> PRT

<213> Vibrio hepatarius

<220>

<223> Amino acid sequence of PPO12

<400> 12

Met Lys Lys Ala Leu Phe Leu Tyr Ser Ser Arg Glu Gly Gln Thr Lys

1 5 10 15

Lys Ile Leu Asn Tyr Ile Glu Gly Glu Leu Ser Asn Phe Glu Cys Glu

20 25 30

Leu Val Asp Leu His Thr Leu Asp Ala Ile Asp Phe Ser Val Tyr Asp

35 40 45

Arg Val Leu Ile Gly Ala Ser Ile Arg Tyr Gly His Leu Asn Lys Lys

50 55 60

Leu Tyr Gln Phe Ile Glu Asn Asn Arg Gln Gln Leu Glu Asn His Thr

65 70 75 80

Lys Val Ala Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Glu Asp Gln

85 90 95

Gly Lys Asp Thr Pro Glu Gly Ser Val Tyr Ile Gln Lys Phe Leu Lys

100 105 110

Lys Ser Pro Trp Gln Pro Lys Leu Ile Gly Val Phe Ala Gly Ala Leu

115 120 125

Tyr Tyr Pro Arg Tyr Gly Phe Phe Asp Lys Met Met Ile Lys Leu Ile

130 135 140

Met Thr Leu Thr Asp Gly Glu Thr Asp Thr Thr Lys Glu Val Glu Tyr

145 150 155 160

Thr Asn Trp Lys Lys Val Ser Leu Phe Ala Lys Lys Phe Ala Glu Leu

165 170 175

<210> 13

<211> 175

<212> PRT

<213> Vibrio owensii

<220>

<223> Amino acid sequence of PPO13

<400> 13

Met Ala Lys Ala Leu Phe Leu Tyr Ser Ser Arg Glu Gly Gln Thr Lys

1 5 10 15

Lys Ile Leu His Tyr Ile Asp Glu Lys Leu Thr Gly Phe Glu Cys Glu

20 25 30

Leu Val Asp Leu His Asn Val Glu Thr Ile Asp Phe Ser Gln Tyr Asp

35 40 45

Arg Val Leu Ile Gly Ala Ser Ile Arg Tyr Gly His Leu Asn Lys Lys

50 55 60

Leu Tyr Gln Phe Ile Asp Arg Asn Leu Asn Gln Leu Glu Gln Ser Lys

65 70 75 80

Val Ala Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Glu Asp Gln Gly

85 90 95

Lys Asp Thr Pro Glu Gly Ser Ala Tyr Ile Arg Lys Phe Leu Ile Lys

100 105 110

Ser Pro Trp Lys Pro Thr Leu Ile Gly Val Phe Ala Gly Ala Leu Tyr

115 120 125

Tyr Pro Arg Tyr Gly Trp Phe Asp Lys Thr Met Ile Lys Phe Ile Met

130 135 140

Ser Met Thr Gly Gly Glu Thr Asp Thr Thr Lys Glu Val Glu Tyr Thr

145 150 155 160

Asn Trp Glu Lys Val Ala Leu Phe Ala Asp Lys Phe Lys Glu Leu

165 170 175

<210> 14

<211> 177

<212> PRT

<213> Vibrio xiamenensis

<220>

<223> Amino acid sequence of PPO14

<400> 14

Met Ser Asn Lys Ala Leu Leu Leu Tyr Ser Thr Arg Glu Gly Gln Thr

1 5 10 15

Lys Lys Ile Leu Gln Tyr Ile Asp Lys Glu Leu His Asp Tyr Gln Cys

20 25 30

Asp Tyr Val Asp Leu His Thr Met Pro Ile Glu Ile Asp Phe Ser Gly

35 40 45

Tyr Asp Lys Val Leu Val Gly Ala Ser Ile Arg Tyr Gly His Leu Asn

50 55 60

Lys Lys Leu Tyr Arg Phe Ile Glu Arg Tyr Lys Ala Glu Leu Lys Asp

65 70 75 80

Asn Asn Ala Ala Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Glu Gln

85 90 95

Gln Gly Lys Asp Thr Pro Glu Gly Ser Ala Tyr Ile Gln Thr Phe Leu

100 105 110

Lys Lys Ser Pro Trp Gln Pro Ala Leu Ile Gly Val Phe Ala Gly Ala

115 120 125

Leu Arg Tyr Pro Lys Tyr Arg Trp Phe Asp Lys Met Met Ile Arg Leu

130 135 140

Ile Met Thr Met Thr Gly Gly Glu Thr Asp Thr Ser Lys Glu Val Glu

145 150 155 160

Tyr Thr Asn Trp Lys Lys Val Ser Leu Phe Val Glu Asn Trp Gln Lys

165 170 175

Gln

<210> 15

<211> 537

<212> DNA

<213> Ewingella americana

<220>

<223> Nucleotide sequence of PPO1

<400> 15

gagcgttttt ttgtacgata acttgccaaa atcctcggca aacttcttaa cttgctccca 60

atcggtgtat tccacttctt tactggtatc cgtttcgcca ccggtcatgc gcataataag 120

ttgaatcatg actttatcta cccaccgata gcgcggataa cgcagcgcgc cagcgaatac 180

accgcacatg gcaggtttcc atggtgtccc cagtaggaac ttgcgtacat aggagttggt 240

ctgaggcgtg cgcttctcag gcttacgcgc agtaaggttc accgcaaaga acgccgacgg 300

catgctatta agctgttgga tattctgttt tacaaattta tccagtacgg cattgaagtg 360

gccgtagcgg attgacgcgc ccaccagcac cttatcgtag cgagaaagcg tcacatgctg 420

cgcgtgcagc aggtcaatca catcgcactc atacttctct ttcatgcaat ttgctatata 480

agaagcaatg gcatgtgttt gtccatcacg cgttgaatag agcaccaatg ctttcat 537

<210> 16

<211> 522

<212> DNA

<213> Vibrio sp. HDW18

<220>

<223> Nucleotide sequence of PPO2

<400> 16

gtgagagcgt tattactgta ttcaagccaa gaagggcaaa cgcgtaaaat tatccaacgt 60

atcgccgcac agatgccaga atatacttgt gaggtgcaag acttacatca aagcatcgat 120

attgactggg cggaatacga caaagtactg attggggcgt caattcgcta tggtcgactt 180

aatcccgcct tgtaccgttt tattgagcat cacctagtgg gattgaccag ccgcaaagcc 240

gccttctttt gtgtcaattt aaccgctaga aaagaacagc aaggaaaaga tactccacag 300

ggcagtgcct atatccaaac ttttctgaaa aagtcggctt ggcagccaga gcgaatcgca 360

gtatttgctg gcgcgcttta ttatccacgt taccgctggt tcgataaaat gatgatccgc 420

ctgatcatga ccttaactgg cggcgagacc gatacgagtc aagaagtcga gtataccaac 480

tgggataaag tcgttaaatt tgctgaacaa ttccgcaatt gg 522

<210> 17

<211> 528

<212> DNA

<213> Proteus hauseri

<220>

<223> Nucleotide sequence of PPO3

<400> 17

atgagcgcgc tattgcttta ttgtagtact gatggtcaaa caaaaaaaat aatgactcaa 60

attgcgaatg aattaaggca gcatggctat gaatgtgatg tgagagattt aacttctgtt 120

cagcagaact taaatctatc tgcttataat aaggtattgg tcggtgcttc tattcgttat 180

ggttacttta ataaagcctt ggataaattt ataactcgac atctttctca actcaataat 240

atgccttcag cattctttgg cgttaatctt acagcacgaa aagaagaaaa gagtacgcca 300

gagactaatt cttatatatg caagttttta gataagacac cttggaagcc aacattaaca 360

ggtgtatttg ctggtgcgct tttttaccct cgctataaat ggattgatag agtgatgatc 420

caacttatta tgaaaatgac aaagggtgaa agacccaa caaaagaaat tgaatatacg 480

gattggaata aagttagtga atttgctact aattttgcca aaataggc 528

<210> 18

<211> 513

<212> DNA

<213> Rodentibacter genomosp.2

<220>

<223> Nucleotide sequence of PPO4

<400> 18

atgaaaacat taattcttta ttcaagccat gatgggcaaa caaaaaaaat ctcggaattt 60

ttaaccgcac ttttaaatgg tgaagtgctg gtcgatgctt tacgagaaga ttatgatatt 120

gaaacctttg atcaggttat tattggcgct tccatacgtt atggtcattt tagtaaaacc 180

ctcgatcgtt ttgttgttcg taaccgtgtg catttggagc aaaaaagagc ggtatttttt 240

agcgttaatt taactgctcg taaagagggc aaggataacc ccgaaactaa tgtatatgta 300

cgcaaatttt tacaacgcac aaaatggcag cctaaattgg ctgcagtgtt tgccggagca 360

ctattatatc cacgctataa atggtttgat cgtgtgatga tacgattcat tatgaaaatc 420

actggtggtg agacagatat aacaaaagag atcgaatata cggattggga aaaagtaagg 480

gaatttgccc aacggattaa caggttggag aaa 513

<210> 19

<211> 528

<212> DNA

<213> Mannheimia pernigra

<220>

<223> Nucleotide sequence of PPO5

<400> 19

aattaactga tcgcttaatt ttttgccaaa ctcttccacc tgttgccaat tcgtatattc 60

atactcttta ctgacgtctg tttcgccttt agttactttc ataataaagc gaatcatcat 120

acggtcaatg aaacgatagc gtgggtagaa caacgcccca gcaatcactt caacatagtt 180

tggttgccat ttcactttag ccaaaaagtt acgtatataa gtattagtgg taggtgtgtt 240

acgatttgct ttgcgagcgg tcagatttac gccataaaat gccgttttct tttcatttaa 300

catagcataa tgctgttcga taaattgata aaccaacgga ctaaaatgtc cataacgaat 360

ggaggcacca atcacaactt gatcaacatt tgccattttt tgggcaaaat cgaccgcttg 420

atccttaagg gagatcagct ccacttcgtg tgtgatcaca tccgctagtc gctcagcaat 480

ctttttcgtt tgaccatcgg tcgtaaaata gagaattaag gttttcat 528

<210> 20

<211> 693

<212> DNA

<213> Leptomonas pyrrhocoris

<220>

<223> Nucleotide sequence of PPO6

<400> 20

atgtcttcac cgaacggaaa atacctgatg ttgtactcga caacagacgg ccacaccaag 60

acgatcatgg acacgatggc gaagcacatt atggaagagg cgaaggtgca atgcgatgtg 120

gttgatatga gggacgggga caagtacgaa ctcgccgcgt acgaaaaggt catgctcgga 180

gcttcgatcc ggtacggatt cttcagcagg acgctgcaca cctacaccac acatcacgtt 240

gacgagctga actcgatgcc ctccgcgttc ttcggtgtca accttaccgc gaggaagaca 300

agcaaaaaca cagcgatgac aaatgcgtac acgcgcaaat ttctggacca atcgatgtgg 360

gtgccacaat tatcgggggt ctttgctggc gctctatggt atccgcggta taacttcttt 420

gatcgagtga tgattcaatt tatcatgaaa gtaaccggcg gtgagacaaa cacaacaaaa 480

gaaattgtct acacggattg ggatgcggtt cataaatttg ctaccgactt cgtacagcta 540

ccggcgacgg ctattcctcg ctccaagccc gcgacctcag tgccgccagc gagtgttgca 600

aactacgata atggtgctcg tgtggcgctt gtcgtcgttg gcatttcagc agccattatc 660

ttcggtcgcc gcctcattct cgcgaaacgg ttt 693

<210> 21

<211> 522

<212> DNA

<213> Aeromonas aquatica

<220>

<223> Nucleotide sequence of PPO7

<400> 21

atggacaaga ttttggtgct ttattcttcc caggacgggc aaacacggaa aattgtggat 60

gccatgctgg aggaaatatc aggatgcgac gtggtgatac aggatctgca taccctgccc 120

aagtgcaatc tcagcaagta tgccaaggta ttgatcggtg cctccattcg ctatggcaac 180

ttccacccca gcctgctcag cttcatcaat gctcaccatg aacagctgga agtggccaat 240

gccgcattct tctgcgtcaa cctgaccgcc cgcaagccag agaagcagac gccgcaaacc 300

aatgcctata tgaagaagtt cctgcgcctc tctccctgga agccgaaaac cctgggggtc 360

ttcgccggtg cgctgcaata ctcccgctat aactggtggc agacccgcat cattcagctg 420

atcatgaaga tgaccggcgg cagcacggac accagccagg acctcgagtt taccgattgg 480

gtaaaggtac gcagttttgc gcgagaattc tggtcaaaaa ga 522

<210> 22

<211> 573

<212> DNA

<213> Psychromonas sp. SA13A

<220>

<223> Nucleotide sequence of PPO8

<400> 22

acgtgctgca aagtcctgag caaaagaatc aactttttcc caatcggtta attctatgtc 60

ctgtgtttta tcggtactgc cgcccgtcat tttcataatc aactgaatta acagggtttg 120

ccaccaatta taacgtgaat acaatagagc tccggcaaac acccctttga actgtggttg 180

ccattgtgaa agctgatcaa attttttcat ataggcatta ttttctggca ctgctttttc 240

tggcttacgt gcagttaaac aaaccacaaa aaaagcatta catactgcat ctaattgcgt 300

tttatgctga ttaacaaact taattatatt gggacgaaag ttaccataac gaatagatgc 360

accgattaag actttttggt acaaagaaaa atcaatggtc ggattatcat caacattcaa 420

tacaatcact tcgccagtaa gcgtttgctt aatacgatta attattttta atgtttgtcc 480

atcgacagag gaataaagta ctaaagtttt gcttactaca ctactttcag tttgcttggt 540

ttcagcttgc ttagcttgag tttcattaga cat 573

<210> 23

<211> 522

<212> DNA

<213> Photobacterium aquae

<220>

<223> Nucleotide sequence of PPO9

<400> 23

atagtgctta aaccgggctg caaaggcctc tactttctgc caatctgtat attcgacttc 60

tttactagta tctgtttccc cgccagtgat acgcataatg aattgaatca tgacacgatc 120

aaaccatcca taccgcggat agcgtaatgc cccagcaaat acttcctgca actttggaca 180

ccatggagac ttcttcaaga acgttttcat atacacgctt gtctccggcg tattcttacc 240

ttccttacgt gccgtaagat tcacgcagaa aaaacccacc ttattctggt caagctcatc 300

tcggtacctc tcgataaact gatagagctt tttatttaga tggccgtaac gaatagaggc 360

tccgaccaat acccgatcat aatccgcaaa atccaccgta ggtaatgtgt tcaaatcaac 420

caattcacac tggtagtcac cacctagcgt cgtttcgata tgacgtaata ttttgacagt 480

ctggccatca ctgcttgaat gcagcaacaa aactttttcc at 522

<210> 24

<211> 522

<212> DNA

<213> Vibrio mimicus

<220>

<223> Nucleotide sequence of PPO10

<400> 24

ccaattgcgg aattgttcgg caaattgaga gactttattc cagttagtgt actccacttc 60

tttactggta tcggtctctc caccagttag cgtcatgatt agacgtatca tcattttatc 120

gaaccaacgg tagcgcgggt aatacagcgc cccagcaaat acggcgatac gctctggctg 180

ccaaggcgac tttttcagga aggtttgaat gtagacactc ccttccggcg tatcttttcc 240

ttgttcttct tttcttgcgg taaggttaac gcaaaagaag gcggctttat tactgcgtag 300

atcgggaaga taacgctgga tgaactggta gagtttgtcg ttgaggcggc cataacgaat 360

cgatgcacca atcaagattt tgtcgtaatc agctagcgct atgtcactta cctgatgcag 420

atcttggata tcacaatcgt attccggcat ctgctgcgcg atgcgttcga taattttccg 480

cgtctgccct tcttggcttg aatacaaaaa tagcgctttc ac 522

<210> 25

<211> 525

<212> DNA

<213> Vibrio

<220>

<223> Nucleotide sequence of PPO11 (unclassified vibrio)

<400> 25

cattttttta aagttatcac taaaaagaga tactttttgc cagtttgtgt actcaacttc 60

ttttgaggtg tcagtttcac cgccagtcaa tgacatgatg aaacgaatca tcaccttatc 120

gaaaaagtga taacgtggat agtaaagagc accagcgaaa actccgatta gcttgggttg 180

ccatgctgat ttactgagaa aggttttaat atagacactt ccttctggag tatctttacc 240

ctgcgcttct tttcttgcag ttaagttgac acaaaagaac gccgctttag cgagtgctaa 300

ttgctcggca tgtttttcta taaattgata aagcttttta tttaaatgtc cgtagcgaat 360

agaagcacca attaagactc gatcgtaaag agctaaatca atatcctcaa tttgatgtaa 420

atcggcaagt tcgcaatcaa actcagaaag ttcctctttt atatagttaa gaattttctt 480

ggtttgacct tcacgggaag aatagagaaa aagagctttt tgcac 525

<210> 26

<211> 528

<212> DNA

<213> Vibrio hepatarius

<220>

<223> Nucleotide sequence of PPO12

<400> 26

gtgaaaaaag ctttatttct gtattcttca cgcgaaggtc aaaccaaaaa gattcttaac 60

tatatagagg gtgagcttag caacttcgaa tgtgagctgg ttgacctaca tacattagac 120

gcaatagatt tcagcgttta tgacagagtg ctgattgggg cttcgatccg atacggacat 180

ttaaataaga aactctatca gtttatcgag aacaatcgac aacaactgga aaatcacact 240

aaggtggcat ttttctgcgt taacttgacc gcgcgtaagg aagaccaagg taaagatacc 300

cctgaaggca gcgtttatat tcagaagttt ttaaagaaat cgccttggca acctaagtta 360

atcggggtat tcgccggggc tttatattac ccgcgctatg gtttcttcga caaaatgatg 420

attaaactga ttatgacgct gaccgatggt gaaacggata ctactaaaga agtcgagtac 480

accaattgga aaaaggtctc tcttttcgcg aaaaaattcg ctgagttg 528

<210> 27

<211> 528

<212> DNA

<213> Vibrio owensii

<220>

<223> Nucleotide sequence of PPO13

<400> 27

gtggcaaaag cattatttct atattcctcc cgtgaagggc agactaaaaa aattctccac 60

tatatagatg agaagctaac aggcttcgaa tgtgagctgg tggatttaca taatgttgag 120

acgattgatt ttagccaata cgacagggta ttgattggag cgtctattcg ttatggtcat 180

cttaataaga agctgtacca gtttattgat cgaaacctca atcagcttga acaaagtaaa 240

gtggcatttt tctgcgtaaa cctaaccgct cgtaaagaag accaaggaaa ggatacgcca 300

gagggcagcg cttacattcg taagttccta attaagtcac cttggaaacc aaccttaatc 360

ggtgtttttg ctggtgctct ttattaccca cgttacggat ggttcgacaa aacgatgatt 420

aagttcatta tgtcgatgac gggtggcgaa accgatacga ccaaagaagt ggaatacacc 480

aattgggaaa aagtggctct tttcgcagat aaattcaaag aactgtag 528

<210> 28

<211> 531

<212> DNA

<213> Vibrio xiamenensis

<220>

<223> Nucleotide sequence of PPO14

<400> 28

ttgagtaaca aagcgctatt gctttattca acgcgagaag ggcaaaccaa gaaaattctc 60

caatacatag ataaagagct gcacgactat cagtgcgatt acgttgactt acataccatg 120

ccaatagaga tagatttctc tggttatgac aaagtgctgg tggggggcttc tattcgttat 180

gggcatctca acaaaaaact gtatcggttt attgagcgtt ataaagccga attgaaagac 240

aacaacgcgg cgttcttttg cgttaattta acggcgcgca aagaacagca aggaaaagat 300

accccagagg gcagcgctta tattcaaacc tttcttaaga agtccccatg gcaaccagca 360

ttgattggtg tgtttgctgg ggcattgcgc tatccgaagt accgttggtt tgacaaaatg 420

atgattcgtt tgatcatgac catgacaggt ggtgaaacgg ataccagtaa agaagtcgaa 480

tatacaaact ggaaaaaagt ctctcttttt gtcgaaaatt ggcaaaaaca g 531

<210> 29

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO1A

<400> 29

atgaaggctt tggtactcta cagcacaaga gacggtcaga cacacgcgat cgcgtcgtac 60

attgctaatt gcatgaaaga gaagtatgag tgtgacgtta ttgatcttct tcatgctcag 120

catgttacat tatcacggta tgataaggtt cttgtaggag cttctatacg atatggacat 180

ttcaatgctg tgttggacaa atttgtgaag caaaacatcc aacaactaaa ctcaatgcct 240

tctgctttct ttgctgttaa tttgactgcc aggaaacctg agaaacgtac tcctcagacc 300

aattcttatg ttaggaagtt tcttctgggg acaccatgga aaccagcaat gtgtggcgtg 360

tttgcaggag cattgaggta tccacgttac agatgggttg ataaagtgat gattcagctc 420

ataatgagaa tgactggtgg tgaaacagat acaagcaaag aagttgagta tactgattgg 480

gaacaagtca aaaagttcgc agaagatttt ggaaagctaa gttacaagaa aaccctctga 540

<210> 30

<211> 525

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO2A

<400> 30

atgagagcct tgcttctcta cagcagtcaa gaagggcaga caagaaaaat cattcagaga 60

atagcagctc aaatgcctga gtacacttgt gaagttcaag atttgcatca atcaatagat 120

attgattggg ccgagtatga caaagtttta attggtgcat ccatccggta tggacgtttg 180

aaccctgcac tttacaggtt cattgagcat cacctcgttg gtttgacctc aaggaaggct 240

gctttcttct gcgtaaatct taccgcaagg aaagaacaac aagggaaaga cactccacag 300

ggctctgcat atattcaaac atttttgaag aagagtgctt ggcaacccga aaggattgct 360

gtttttgctg gtgcccttta ttatccaaga tacagatggt ttgataagat gatgatccga 420

ctcatcatga cgttaactgg aggagaaact gatacatctc aggaggtgga atatacaaac 480

tgggataaag tcgtgaagtt tgcagagcag tttcggaatt ggtga 525

<210> 31

<211> 531

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO3A

<400> 31

atgagtgccc tgttgttgta ttgttcaact gatggtcaaa ccaagaagat aatgacacag 60

atagctaatg aactgagaca acatggttat gaatgtgatg tgcgggatct tacttctgtt 120

caacaaaatc tcaatctttc agcttacaac aaagttttgg taggagcttc cataagatat 180

ggatacttca ataaagcact tgacaagttc atcacgaggc acctctctca gttaaacaac 240

atgccttctg ctttctttgg tgtcaatttg actgcaagaa aagaagagaa gagcactccc 300

gaaacaaaca gttacatctg caagtttcta gacaaaacac catggaaacc aaccttaact 360

ggagtcttcg ccggggcgct tttttatcct cgttacaaat ggattgatcg agtgatgatt 420

cagcttatca tgaagatgac taaaggagaa accgatccga caaaagagat tgagtataca 480

gattggaaca aggtttcgga gtttgctacg aattttgcaa agattggctg a 531

<210> 32

<211> 516

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO4A

<400> 32

atgaaaaccc tgattcttta cagcagtcat gatggacaga ccaagaaaat ctcagagttc 60

cttacggcgt tgttgaatgg cgaagttcta gttgatgctc tacgtgaaga ttatgatatt 120

gaaacatttg accaagttat aatcggagct tccataaggt atggtcactt ttctaagact 180

ttagacagat ttgtggtgag aaacagggta catctggaac aaaaacgagc agtattcttc 240

tctgttaact taactgcccg aaaagaaggt aaagacaatc ctgaaactaa tgtctatgtc 300

agaaagtttc tccagagaac aaaatggcaa ccaaaacttg ctgccgtctt cgcaggtgca 360

ctcttgtacc cgagatataa gtggtttgat cgtgtgatga ttcggtttat catgaagatt 420

actgggggag agacggatat aacaaaggag attgagtaca cagattggga aaaggttagg 480

gagttcgctc agagaatcaa ccgccttgag aaatga 516

<210> 33

<211> 531

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO5A

<400> 33

atgaagactc ttatcctcta cttcacaaca gacggccaaa cgaaaaagat tgccgagaga 60

ctggctgatg tgattactca tgaagtggag ctaatctcct tgaaagatca agctgttgat 120

tttgctcaga agatggctaa tgttgaccaa gtagtcattg gagcgagtat aaggtatggt 180

cacttcagcc ctcttgttta tcagtttatt gaacaacatt atgcaatgtt gaatgagaag 240

aaaaccgctt tttatggagt gaatctcaca gcaaggaaag caaatcgaaa caccccaacc 300

acaaatactt acatccggaa cttcttagca aaggtgaaat ggcaacctaa ctatgttgag 360

gtcatagctg gtgccttgtt ttacccgcgt tacagattca ttgatagaat gatgattcgc 420

ttcatcatga aagtaacgaa aggagaaact gatgtttcaa aagaatatga atacacaaac 480

tggcagcaag ttgaagagtt tgggaagaag ctttctgatc agttaatatg a 531

<210> 34

<211> 696

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO6A

<400> 34

atgtcttctc ctaatgggaa gtatttgatg ctttacagta ccaccgacgg tcacacaaaa 60

accattatgg atacaatggc taaacatata atggaagaag caaaagtaca atgtgatgtt 120

gtggacatga gagatggaga caaatacgag ttagctgctt atgagaaggt gatgctagga 180

gcttctatta gatatggatt cttcagccgc actcttcata catataccac tcatcatgtt 240

gatgaactta actcaatgcc ttccgctttc tttggagtca atctcactgc tcgtaaaact 300

tcaaagaaca ctgcaatgac taatgcatac acaagaaagt ttctggatca atccatgtgg 360

gttcctcaat tatctggcgt ttttgctggt gctttgtggt atcctaggta caatttcttt 420

gacagggtta tgattcagtt catcatgaag gtaactggtg gtgaaacaaa cacaacgaaa 480

gagatagttt acactgattg ggatgctgtt cacaaatttg caacagattt tgtgcagcta 540

ccagccacgg cgataccccg gagcaagccg gcgacgtcgg tcccaccagc tagtgtggcc 600

aactatgata atggggcacg agtagcgctt gtggttgtgg gaatctcagc agccatcatc 660

tttggccgtc gattgattct cgcaaagaga ttctga 696

<210> 35

<211> 525

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO7A

<400> 35

atggataaga tattagtcct gtacagtagt caagatgggc agacgaggaa gattgttgat 60

gcaatgcttg aagaaatctc tggttgtgat gttgtgattc aagacttgca cacactaccg 120

aaatgcaacc tatccaaata tgcgaaagtg ttgattggtg cttccataag gtatggaaac 180

ttccatcctt ctcttctcag cttcatcaat gctcatcatg aacaacttga ggttgctaat 240

gcggccttct tctgtgtaaa tctcactgcc cgaaaacctg agaagcaaac tccccagaca 300

aacgcttaca tgaagaagtt tttacgctta tcaccatgga aaccaaagac ccttggagtg 360

tttgcaggag ctttgcaata ttcaagatac aattggtggc aaacccgtat cattcagctg 420

ataatgaaaa tgactggtgg ctcaacagac acatctcagg atttggagtt tactgattgg 480

gttaaagtcc ggtcgtttgc aagagaattt tggagcaaga gatga 525

<210> 36

<211> 576

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO8A

<400> 36

atgtcaaatg agacacaagc caaacaagct gaaacgaaac agactgagtc ttctgtcgta 60

agcaaaacgc tggtgttgta ctcgtccgtc gatggtcaaa ccttaaagat cattaatcgt 120

attaagcaga ccctgacagg agaagttatt gttttgaatg ttgatgataa tccaactatc 180

gattttagcc tttatcagaa ggtcttgatt ggagcatcaa tccggtatgg gaactttaga 240

ccgaacatca taaagtttgt taatcagcat aaaacacaac ttgacgcggt gtgcaacgct 300

ttcttcgtgg tttgtctcac agcaagaaaa ccagagaaag ctgttcctga aaacaatgca 360

tacatgaaga aatttgatca gctaagtcaa tggcaacctc agttcaaggg agtattcgcg 420

ggtgctttat tgtattcacg atacaactgg tggcagacac ttctcataca actaataatg 480

aagatgactg gtggcagtac tgacaaaact caagatattg agcttaccga ttgggaaaag 540

gtggattctt tcgcacaaga ctttgctgcc aggtga 576

<210> 37

<211> 525

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO9A

<400> 37

atggagaaag ttcttctact gcattcttcc tctgatggtc agacagtgaa gattctcaga 60

catattgaaa caacattagg tggtgattac caatgtgagc ttgtggattt gaatactctg 120

ccgactgttg attttgctga ttatgaccgt gtcttagtag gcgcgagtat acgctatggg 180

cacctcaaca agaaactcta ccagtttata gagagataca gagatgaatt ggaccaaaac 240

aaagtaggtt tcttctgcgt gaatcttact gcaaggaaag aaggaaagaa tacgcctgag 300

actagcgttt acatgaagac ctttttgaag aagtcgccat ggtgtccaaa attgcaagaa 360

gttttcgctg gagctcttcg ttatcctcgg tatggatggt ttgatagggt catgattcag 420

ttcatcatga gaatcacggg gggagaaacc gacacatcaa aagaggttga gtatactgat 480

tggcaaaagg ttgaagcttt tgcagcccga ttcaaacatt actga 525

<210> 38

<211> 525

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO10A

<400> 38

atgaaagctc tctttctgta ctcgtctcaa gaaggacaga caaggaagat tatagagaga 60

atcgcccagc aaatgccgga gtatgattgt gatattcaag atcttcatca agtaagcgac 120

attgcattag ctgattatga caagatccta ataggggcca gtatccgata tggaagattg 180

aatgataaac tatatcaatt tattcagaga tatcttcccg atctccgctc caacaaagca 240

gctttcttct gcgtcaatct tacggcgaga aaggaagaac aaggaaaaga caccccagaa 300

ggttctgttt acatccagac tttcttgaag aaatcaccat ggcaacctga gaggattgca 360

gtgtttgctg gtgctcttta ctatcctcgt tacagatggt ttgataagat gatgattcgg 420

ttaataatga ctttgaccgg cggtgaaact gatacatcaa aagaagttga gtacacaaat 480

tggaacaagg tttctcagtt tgcggagcag ttccgtaact ggtga 525

<210> 39

<211> 528

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO11A

<400> 39

atgcaaaaag ctcttttcct ctacagttca agagaaggac aaacaaagaa aatactcaac 60

tacatcaagg aagaactatc agagtttgat tgtgagcttg ctgatcttca tcagattgaa 120

gatattgatt tagccttgta tgatcgagta ttaataggcg caagcatccg gtatggtcac 180

ttgaacaaaa agctttatca gtttattgag aagcatgctg agcaacttgc gctggcaaaa 240

gcagctttct tctgcgtgaa tctcactgca aggaaagagg ctcaaggaaa agatactccg 300

gaaggttctg tttacataaa gacgtttctg tccaagtctg cttggcaacc aaagttgatt 360

ggagtttttg ctggtgccct atactatcct cgttaccatt tctttgacaa agtgatgatt 420

agattcatca tgtctttaac cgggggagaa actgacacat caaaagaagt tgagtataca 480

aattggcaga aagtctcgtt gtttagtgac aatttcaaga agatgtga 528

<210> 40

<211> 531

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO12A

<400> 40

atgaagaaag ctcttttcct gtacagctcg agagaaggcc agacaaaaaa gattctcaac 60

tacatagaag gagagttatc aaattttgaa tgtgaattgg ttgaccttca cacgcttgat 120

gctattgatt tctccgtcta tgatcgggtg ctcattggtg cttctatacg atatggtcat 180

ttgaacaaga agctttatca gtttatagag aacaacagac aacaactaga gaatcatacc 240

aaagttgcct tcttctgcgt caatcttact gcaaggaaag aagatcaagg aaaagacact 300

cctgagggga gtgtttacat ccagaaattt ctcaagaaat caccatggca accgaagttg 360

attggagttt tcgcgggggc attatactac cctcgttatg gattctttga taagatgatg 420

atcaaactca tcatgacttt aactgatggt gaaaccgaca caacaaagga agtggagtat 480

acaaattgga aaaaagtatc tttgtttgca aagaagtttg ctgagctgtg a 531

<210> 41

<211> 528

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO13A

<400> 41

atggctaaag ctcttttcct gtactcatct agagaaggac aaacaaagaa gattcttcat 60

tacatagacg agaagttaac tgggtttgag tgtgaactcg ttgatttgca caatgtcgaa 120

actattgatt tttctcagta tgatagagtc ttaattggag cgtccatacg atatggtcat 180

ctcaacaaga aattatatca gtttatcgac cgtaacctga accagctaga gcaaagcaag 240

gtagctttct tctgcgtgaa tctcaccgca cggaaagagg atcaaggaaa agatactcct 300

gaaggctcag cttacataag gaagtttctt atcaaatcgc cttggaaacc gacattgatt 360

ggtgttttcg caggtgcact ttactatcca agatatgggt ggtttgataa aacaatgatc 420

aaattcatca tgagtatgac tggtggagaa accgacacga ccaaggaagt tgagtacaca 480

aattgggaaa aagtggcctt gtttgctgat aagttcaagg agctatga 528

<210> 42

<211> 534

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO14A

<400> 42

atgtctaaca aggccttgct gctctatagt acaagagaag gtcaaactaa aaagattctt 60

cagtacatag acaaagagct tcatgattac caatgtgatt atgtcgatct tcacacaatg 120

cctatagaga ttgatttctc tggttatgat aaagttttgg taggggcgtc catccggtat 180

ggacatttaa acaaaaaact ttacagattc atcgagaggt acaaagcaga gttgaaggat 240

aataatgctg ctttcttctg cgtcaatcta accgcaagga aagaacaaca aggaaaagat 300

actcctgaag gatcagctta tattcagact tttctcaaga agtctccatg gcagccagca 360

ttgataggtg tttttgctgg agctttacgt tacccgaagt atagatggtt tgacaagatg 420

atgattcgac taatcatgac tatgacgggc ggtgaaaccg acacatcaaa ggaagtggag 480

tatacaaatt ggaagaaagt gagcctcttt gttgaaaact ggcaaaagca gtga 534

<210> 43

<211> 181

<212> PRT

<213> Escherichia coli

<220>

<223> Amino acid sequence of PPO-EC

<400> 43

Met Lys Thr Leu Ile Leu Phe Ser Thr Arg Asp Gly Gln Thr Arg Glu

1 5 10 15

Ile Ala Ser Tyr Leu Ala Ser Glu Leu Lys Glu Leu Gly Ile Gln Ala

20 25 30

Asp Val Ala Asn Val His Arg Ile Glu Glu Pro Gln Trp Glu Asn Tyr

35 40 45

Asp Arg Val Val Ile Gly Ala Ser Ile Arg Tyr Gly His Tyr His Ser

50 55 60

Ala Phe Gln Glu Phe Val Lys Lys His Ala Thr Arg Leu Asn Ser Met

65 70 75 80

Pro Ser Ala Phe Tyr Ser Val Asn Leu Val Ala Arg Lys Pro Glu Lys

85 90 95

Arg Thr Pro Gln Thr Asn Ser Tyr Ala Arg Lys Phe Leu Met Asn Ser

100 105 110

Gln Trp Arg Pro Asp Arg Cys Ala Val Ile Ala Gly Ala Leu Arg Tyr

115 120 125

Pro Arg Tyr Arg Trp Tyr Asp Arg Phe Met Ile Lys Leu Ile Met Lys

130 135 140

Met Ser Gly Gly Glu Thr Asp Thr Arg Lys Glu Val Val Tyr Thr Asp

145 150 155 160

Trp Glu Gln Val Ala Asn Phe Ala Arg Glu Ile Ala His Leu Thr Asp

165 170 175

Lys Pro Thr Leu Lys

180

<210> 44

<211> 546

<212> DNA

<213> Escherichia coli

<220>

<223> Nucleotide sequence of PPO-EC

<400> 44

gtgaaaacat taattctttt ctcaacaagg gacggacaaa cgcgcgagat tgcctcctac 60

ctggcttcgg aactgaaaga actggggatc caggcggatg tcgccaatgt gcaccgcatt 120

gaagaaccac agtgggaaaa ctatgaccgt gtggtcattg gtgcttctat tcgctatggt 180

cactaccatt cagcgttcca ggaatttgtc aaaaaacatg cgacgcggct gaattcgatg 240

ccgagcgcct tttactccgt gaatctggtg gcgcgcaaac cggagaagcg tactccacag 300

accaacagct acgcgcgcaa gtttctgatg aactcgcaat ggcgtcccga tcgctgcgcg 360

gtcattgccg gggcgctgcg ttacccacgt tatcgctggt acgaccgttt tatgatcaag 420

ctgattatga agatgtcagg cggtgaaacg gatacgcgca aagaagttgt ctataccgat 480

tgggagcagg tggcgaattt cgcccgagaa atcgcccatt taaccgacaa accgacgctg 540

aaataa 546

<210> 45

<211> 546

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO-ECA

<400> 45

atgaaaacac ttatcttgtt ctcaactaga gatggacaga caagagagat tgcttcttac 60

ttggcttcag aacttaagga gttggggtatt caagctgatg ttgctaatgt tcatagaatt 120

gaagagcctc agtgggaaaa ctatgataga gttgttattg gagcttctat tagatatggt 180

cattaccatt cagcttttca agagttcgtt aagaaacatg ctactagact taactctatg 240

ccatcagctt tttactctgt taacttggtt gctagaaagc ctgagaaaag aactccacaa 300

acaaactctt acgctagaaa gttccttatg aactcacagt ggagacctga tagatgcgct 360

gttattgctg gtgctcttag atatccaaga tacagatggt acgatagatt catgatcaag 420

ttgattatga aaatgtctgg aggtgaaact gatacaagaa aggaggttgt ttacacagat 480

tgggaacagg ttgctaattt cgctagagag attgctcatc ttactgataa gccaacattg 540

aaatga 546

<210> 46

<211> 537

<212> PRT

<213> Arabidopsis thaliana

<220>

<223> Amino acid sequence of PPO-AT

<400> 46

Met Glu Leu Ser Leu Leu Arg Pro Thr Thr Gln Ser Leu Leu Pro Ser

1 5 10 15

Phe Ser Lys Pro Asn Leu Arg Leu Asn Val Tyr Lys Pro Leu Arg Leu

20 25 30

Arg Cys Ser Val Ala Gly Gly Pro Thr Val Gly Ser Ser Lys Ile Glu

35 40 45

Gly Gly Gly Gly Thr Thr Ile Thr Thr Asp Cys Val Ile Val Gly Gly

50 55 60

Gly Ile Ser Gly Leu Cys Ile Ala Gln Ala Leu Ala Thr Lys His Pro

65 70 75 80

Asp Ala Ala Pro Asn Leu Ile Val Thr Glu Ala Lys Asp Arg Val Gly

85 90 95

Gly Asn Ile Ile Thr Arg Glu Glu Asn Gly Phe Leu Trp Glu Glu Gly

100 105 110

Pro Asn Ser Phe Gln Pro Ser Asp Pro Met Leu Thr Met Val Val Asp

115 120 125

Ser Gly Leu Lys Asp Asp Leu Val Leu Gly Asp Pro Thr Ala Pro Arg

130 135 140

Phe Val Leu Trp Asn Gly Lys Leu Arg Pro Val Pro Ser Lys Leu Thr

145 150 155 160

Asp Leu Pro Phe Phe Asp Leu Met Ser Ile Gly Gly Lys Ile Arg Ala

165 170 175

Gly Phe Gly Ala Leu Gly Ile Arg Pro Ser Pro Pro Gly Arg Glu Glu

180 185 190

Ser Val Glu Glu Phe Val Arg Arg Asn Leu Gly Asp Glu Val Phe Glu

195 200 205

Arg Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala Gly Asp Pro Ser

210 215 220

Lys Leu Ser Met Lys Ala Ala Phe Gly Lys Val Trp Lys Leu Glu Gln

225 230 235 240

Asn Gly Gly Ser Ile Ile Gly Gly Thr Phe Lys Ala Ile Gln Glu Arg

245 250 255

Lys Asn Ala Pro Lys Ala Glu Arg Asp Pro Arg Leu Pro Lys Pro Gln

260 265 270

Gly Gln Thr Val Gly Ser Phe Arg Lys Gly Leu Arg Met Leu Pro Glu

275 280 285

Ala Ile Ser Ala Arg Leu Gly Ser Lys Val Lys Leu Ser Trp Lys Leu

290 295 300

Ser Gly Ile Thr Lys Leu Glu Ser Gly Gly Tyr Asn Leu Thr Tyr Glu

305 310 315 320

Thr Pro Asp Gly Leu Val Ser Val Gln Ser Lys Ser Val Val Met Thr

325 330 335

Val Pro Ser His Val Ala Ser Gly Leu Leu Arg Pro Leu Ser Glu Ser

340 345 350

Ala Ala Asn Ala Leu Ser Lys Leu Tyr Tyr Pro Pro Val Ala Ala Val

355 360 365

Ser Ile Ser Tyr Pro Lys Glu Ala Ile Arg Thr Glu Cys Leu Ile Asp

370 375 380

Gly Glu Leu Lys Gly Phe Gly Gln Leu His Pro Arg Thr Gln Gly Val

385 390 395 400

Glu Thr Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala

405 410 415

Pro Pro Gly Arg Ile Leu Leu Leu Asn Tyr Ile Gly Gly Ser Thr Asn

420 425 430

Thr Gly Ile Leu Ser Lys Ser Glu Gly Glu Leu Val Glu Ala Val Asp

435 440 445

Arg Asp Leu Arg Lys Met Leu Ile Lys Pro Asn Ser Thr Asp Pro Leu

450 455 460

Lys Leu Gly Val Arg Val Trp Pro Gln Ala Ile Pro Gln Phe Leu Val

465 470 475 480

Gly His Phe Asp Ile Leu Asp Thr Ala Lys Ser Ser Leu Thr Ser Ser

485 490 495

Gly Tyr Glu Gly Leu Phe Leu Gly Gly Asn Tyr Val Ala Gly Val Ala

500 505 510

Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu Thr Ala Ile Glu Val Asn

515 520 525

Asn Phe Met Ser Arg Tyr Ala Tyr Lys

530 535

<210> 47

<211> 1614

<212> DNA

<213> Arabidopsis thaliana

<220>

<223> Nucleotide sequence of PPO-AT

<400> 47

atggagttat ctcttctccg tccgacgact caatcgcttc ttccgtcgtt ttcgaagccc 60

aatctccgat taaatgttta taagcctctt agactccgtt gttcagtggc cggtggacca 120

accgtcggat cttcaaaaat cgaaggcgga ggaggcacca ccatcacgac ggattgtgtg 180

attgtcggcg gaggtattag tggtctttgc atcgctcagg cgcttgctac taagcatcct 240

gatgctgctc cgaatttaat tgtgaccgag gctaaggatc gtgttggagg caacattatc 300

actcgtgaag agaatggttt tctctgggaa gaaggtccca atagttttca accgtctgat 360

cctatgctca ctatggtggt agatagtggt ttgaaggatg atttggtgtt gggagatcct 420

actgcgccaa ggtttgtgtt gtggaatggg aaattgaggc cggttccatc gaagctaaca 480

gacttaccgt tctttgattt gatgagtatt ggtgggaaga ttagagctgg ttttggtgca 540

cttggcattc gaccgtcacc tccaggtcgt gaagaatctg tggagggagtt tgtacggcgt 600

aacctcggtg atgaggtttt tgagcgcctg attgaaccgt tttgttcagg tgtttatgct 660

ggtgatcctt caaaactgag catgaaagca gcgtttggga aggtttggaa actagagcaa 720

aatggtggaa gcataatagg tggtactttt aaggcaattc aggagaggaa aaacgctccc 780

aaggcagaac gagacccgcg cctgccaaaa ccacaggggcc aaacagttgg ttctttcagg 840

aagggacttc gaatgttgcc agaagcaata tctgcaagat taggtagcaa agttaagttg 900

tcttggaagc tctcaggtat cactaagctg gagagcggag gatacaactt aacatatgag 960

actccagatg gtttagtttc cgtgcagagc aaaagtgttg taatgacggt gccatctcat 1020

gttgcaagtg gtctcttgcg ccctctttct gaatctgctg caaatgcact ctcaaaacta 1080

tattacccac cagttgcagc agtatctatc tcgtacccga aagaagcaat ccgaacagaa 1140

tgtttgatag atggtgaact aaagggtttt gggcaattgc atccacgcac gcaaggagtt 1200

gaaacattag gaactatcta cagctcctca ctctttccaa atcgcgcacc gcccggaaga 1260

attttgctgt tgaactacat tggcgggtct acaaacaccg gaattctgtc caagtctgaa 1320

ggtgagttag tggaagcagt tgacagagat ttgaggaaaa tgctaattaa gcctaattcg 1380

accgatccac ttaaattagg agttagggta tggcctcaag ccattcctca gtttctagtt 1440

ggtcactttg atatccttga cacggctaaa tcatctctaa cgtcttcggg ctacgaaggg 1500

ctatttttgg gtggcaatta cgtcgctggt gtagccttag gccggtgtgt agaaggcgca 1560

tatgaaaccg cgattgaggt caacaacttc atgtcacggt acgcttacaa gtaa 1614

<210> 48

<211> 1614

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO-ATA

<400> 48

atggagttgt ctcttttaag accaacaaca caatctttgc ttccttcgtt ctctaaaccc 60

aatctcagat tgaatgtcta caaaccacta cgattgcgat gctctgtggc tggtggtcct 120

actgttggaa gttcaaagat agaaggaggc ggtggtacaa ccatcactac agattgtgtg 180

attgtaggtg gtggcatttc gggtctttgc attgctcaag ctctcgctac aaaacatcct 240

gacgctgcgc ccaacttaat agtgacggaa gcaaaagaca gagttggtgg gaatattatc 300

acaagagaag agaatgggtt tttatgggaa gaaggaccga acagctttca accgtcagat 360

ccaatgctta caatggtggt tgattctgga ctgaaagatg atctcgtact tggtgatccg 420

acggccccaa ggtttgtttt gtggaatgga aaattgcgac ctgttccaag caaattaaca 480

gatttacctt tcttcgatct catgagtata ggtgggaaga tcagggccgg ttttggtgca 540

ctgggaatcc gtccttctcc tcctggaaga gaggagcg tagaagagtt tgtgcgtagg 600

aacctgggag atgaagtttt tgagaggttg attgagccat tttgctcagg cgtttatgca 660

ggtgacccga gcaagctgtc tatgaaggct gctttcggca aagtttggaa gctcgagcag 720

aatggtggat ctattattgg aggaactttc aaggccattc aagaaagaaa gaatgctcct 780

aaagcagaga gggaccctag acttcccaag cctcaaggac aaaccgtcgg ctctttcagg 840

aaaggattaa gaatgttgcc agaggctatc agtgcgcgcc ttggaagcaa ggttaaactt 900

tcatggaaac tatctgggat aaccaaactt gaatcaggag gttacaattt aacttatgaa 960

actccggatg gacttgtttc tgtacagagt aagtcagttg tgatgacggt tccttcacat 1020

gtcgcttctg gtctgttgcg tccattatcc gaatctgccg ctaatgcatt atccaaactc 1080

tactatcctc ccgtggcagc agtcagtatc tcttatccaa aagaagctat tagaacagaa 1140

tgtcttattg atggtgagct taagggattt ggacagcttc accctcgaac tcaggtgtg 1200

gaaacactgg ggactattta tagttcctcc ttgtttccta accgtgcacc acctgggaga 1260

attcttcttc taaactacat tggagggtca acaaacaccg gtatcctatc gaagagtgag 1320

ggagaattag ttgaagctgt cgacagagac ctaaggaaga tgctgataaa gccaaattca 1380

accgaccccc tcaagctcgg agttcgcgtg tggcctcagg caattccgca attcctggta 1440

ggacattttg atatattgga tactgccaaa tcttctctta cttcatcggg ctacgaaggt 1500

ttgtttcttg gcggaaacta tgttgctgga gttgccttgg ggcgctgtgt agaggtgct 1560

tatgagactg caatagaggt gaacaatttc atgtctagat atgcttacaa gtga 1614

<210> 49

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Universal 5'-End Adapter Primer 1

<400> 49

taagaaggag atatacatat g 21

<210> 50

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Universal 3'-End Adapter Primer 1

<400> 50

gtggtggtgg tggtgctcga g 21

<210> 51

<211> 542

<212> DNA

Figwort mosaic virus

<220>

<223> 34S enhancer

<400> 51

aattctcagt ccaaagcctc aacaaggtca gggtacagag tctccaaacc attagccaaa 60

agctacagga gatcaatgaa gaatcttcaa tcaaagtaaa ctactgttcc agcacatgca 120

tcatggtcag taagtttcag aaaaagacat ccaccgaaga cttaaagtta gtgggcatct 180

ttgaaagtaa tcttgtcaac atcgagcagc tggcttgtgg ggaccagaca aaaaaggaat 240

ggtgcagaat tgttaggcgc acctaccaaa agcatctttg cctttattgc aaagataaag 300

cagattcctc tagtacaagt ggggaacaaa ataacgtgga aaagagctgt cctgacagcc 360

cactcactaa tgcgtatgac gaacgcagtg acgaccacaa aagaattagc ttgagctcag 420

gatttagcag cattccagat tgggttcaat caacaaggta cgagccatat cactttattc 480

aaattggtat cgccaaaacc aagaaggaac tcccatcctc aaaggtttgt aaggaagaat 540

tc 542

<210> 52

<211> 1534

<212> DNA

<213> Tsf1, Brassica napus

<220>

<223> promoter of the eukaryotic gene elongation factor-1α of oilseed rape

<400> 52

gattatgaca ttgctcgtgg aatgggacag ttatggtatt tttttgtaat aaattgtttc 60

cattgtcatg agattttgag gttaatctat gagacattga atcacttagc attagggatt 120

aagtagtcac aaatcgcatt caagaagctg aagaacacgt tatggtctaa tggttgtgtc 180

tctttattag aaaatgttgg tcagtagcta tatgcactgt ttctgtaaaa ccatgttggt 240

gttgtgttta tttcaagaca catgttgagt ccgttgattc agagcttttg tcttcgaaca 300

caatctagag agcaaatttg ggttcaattt ggatatcaat atgggttcga ttcagataga 360

acaataccct ttgatgtcgg gtttcgattt ggttgagatt catttttatc gggtttggtt 420

cgattttcga attcggttta ttcgccccct catagcatct acattctgca gattaatgta 480

caagttatgg aaaaaaaaat gtggttttcg aattcggttt agtagctaaa cgttgcttgc 540

agtgtagtta tgggaattat gaaacacgac cgaaggtatc aattagaaga acgggtcaac 600

gggtaagtat tgagaaatta ccggagggta aaaataaaca gtattctttt tttttcttaa 660

cgaccgacca aggttaaaaa aagaaaggag gacgagatac aggggcatga ctgtaattgt 720

acataagatc tgatctttaa accctaggtt tccttcgcat cagcaactat aaataattct 780

gagtgccact cttcttcatt cctagatctt tcgccttatc gctttagctg aggtaagcct 840

ttctatacgc atagacgctc tcttttctct tctctcgatc ttcgttgaaa cggtcctcga 900

tacgcatagg atcggttaga atcgttaatc tatcgtctta gatcttcttg attgttgaat 960

tgagcttcta ggatgtattg tatcatgtga tggatagttg attggatctc tttgagtgaa 1020

ctagctagct ttcgatgcgt gtgatttcag tataacagga tccgatgaat tatagctcgc 1080

ttacaattaa tctctgcaga tttattgttt aatcttggat ttgatgctcg ttgttgatag 1140

aggatcgttt atagaactta ttgattctgg aattgagctt gtgtgatgta ttgtatcatg 1200

tgatcgatag ctgatggatc tatttgagtg aactagcgta cgatcttaag atgagtgtgt 1260

attgtgaact gatgattcga gatcagcaaa acaagatctg atgatatctt cgtcttgtat 1320

gcatcttgaa tttcatgatt ttttattaat tatagctcgc ttagctcaaa ggatagagca 1380

ccacaaaatt ttattgtggt agaaatcggt tcgattccga tagcagctta ctgtgatgaa 1440

tgattttgag atttggtatt tgatatatgt ctactgtgtt gaatgatcgt ttatgcattg 1500

tttaatcgct gcagatttgc attgacaagt agcc 1534

<210> 53

<211> 228

<212> DNA

<213> Arabidopsis thaliana

<220>

<223> chloroplast transit peptide

<400> 53

atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60

tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca 120

cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180

tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc 228

<210> 54

<211> 1368

<212> DNA

<213> Artificial sequence

<220>

<223> 5-enolpyruvylshikimate-3-phosphate synthase gene

<400> 54

atgcttcacg gtgcaagcag ccgtccagca actgctcgta agtcctctgg tctttctgga 60

accgtccgta ttccaggtga caagtctatc tcccacaggt ccttcatgtt tggaggtctc 120

gctagcggtg aaactcgtat caccggtctt ttggaaggtg aagatgttat caacactggt 180

aaggctatgc aagctatggg tgccagaatc cgtaaggaag gtgatacttg gatcattgat 240

ggtgttggta acggtggact ccttgctcct gaggctcctc tcgatttcgg taacgctgca 300

actggttgcc gtttgactat gggtcttgtt ggtgtttacg atttcgatag cactttcatt 360

ggtgacgctt ctctcactaa gcgtccaatg ggtcgtgtgt tgaacccact tcgcgaaatg 420

ggtgtgcagg tgaagtctga agacggtgat cgtcttccag ttaccttgcg tggaccaaag 480

actccaacgc caatcaccta cagggtacct atggcttccg ctcaagtgaa gtccgctgtt 540

ctgcttgctg gtctcaacac cccaggtatc accactgtta tcgagccaat catgactcgt 600

gaccacactg aaaagatgct tcaaggtttt ggtgctaacc ttaccgttga gactgatgct 660

gacggtgtgc gtaccatccg tcttgaaggt cgtggtaagc tcaccggtca agtgattgat 720

gttccaggtg atccatcctc tactgctttc ccattggttg ctgccttgct tgttccaggt 780

tccgacgtca ccatccttaa cgttttgatg aacccaaccc gtactggtct catcttgact 840

ctgcaggaaa tgggtgccga catcgaagtg atcaacccac gtcttgctgg tggagaagac 900

gtggctgact tgcgtgttcg ttcttctact ttgaagggtg ttactgttcc agaagaccgt 960

gctccttcta tgatcgacga gtatccaatt ctcgctgttg cagctgcatt cgctgaaggt 1020

gctaccgtta tgaacggttt ggaagaactc cgtgttaagg aaagcgaccg tctttctgct 1080

gtcgcaaacg gtctcaagct caacggtgtt gattgcgatg aaggtgagac ttctctcgtc 1140

gtgcgtggtc gtcctgacgg taagggtctc ggtaacgctt ctggagcagc tgtcgctacc 1200

cacctcgatc accgtatcgc tatgagcttc ctcgttatgg gtctcgtttc tgaaaaccct 1260

gttactgttg atgatgctac tatgatcgct actagcttcc cagagttcat ggatttgatg 1320

gctggtcttg gagctaagat cgaactctcc gacactaagg ctgcttga 1368

<210> 55

<211> 643

<212> DNA

<213> Pisum sativum)

<220>

<223> pea RbcS gene terminator

<400> 55

agctttcgtt cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat 60

tgcgcacaca ccagaatcct actgagtttg agtattatgg cattgggaaa actgtttttc 120

ttgtaccatt tgttgtgctt gtaatttact gtgtttttta ttcggttttc gctatcgaac 180

tgtgaaatgg aaatggatgg agaagagtta atgaatgata tggtcctttt gttcattctc 240

aaattaatat tatttgtttt ttctcttatt tgttgtgtgt tgaatttgaa attataagag 300

atatgcaaac attttgtttt gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag 360

ttaatatgag gagtaaaaca cttgtagttg taccattatg cttattcact aggcaacaaa 420

tatattttca gacctagaaa agctgcaaat gttactgaat acaagtatgt cctcttgtgt 480

tttagacatt tatgaacttt cctttatgta attttccaga atccttgtca gattctaatc 540

attgctttat aattatagtt atactcatgg atttgtagtt gagtatgaaa atatttttta 600

atgcatttta tgacttgcca attgattgac aacatgcatc aat 643

<210> 56

<211> 1322

<212> DNA

<213> Arabidopsis thaliana

<220>

<223> ubiquitin 10 gene promoter

<400> 56

gtcgacctgc aggtcaacgg atcaggatat tcttgtttaa gatgttgaac tctatggagg 60

tttgtatgaa ctgatgatct aggaccggat aagttccctt cttcatagcg aacttattca 120

aagaatgttt tgtgtatcat tcttgttaca ttgttattaa tgaaaaaata ttattggtca 180

ttggactgaa cacgagtgtt aaatatggac caggccccaa ataagatcca ttgatatatg 240

aattaaataa caagaataaa tcgagtcacc aaaccacttg ccttttttaa cgagacttgt 300

tcaccaactt gatacaaaag tcattatcct atgcaaatca ataatcatac aaaaatatcc 360

aataacacta aaaaattaaa agaaatggat aatttcacaa tatgttatac gataaagaag 420

ttacttttcc aagaaattca ctgattttat aagcccactt gcattagata aatggcaaaa 480

aaaaacaaaa aggaaaagaa ataaagcacg aagaattcta gaaaatacga aatacgcttc 540

aatgcagtgg gacccacggt tcaattattg ccaattttca gctccaccgt atatttaaaa 600

aataaaacga taatgctaaa aaaatataaa tcgtaacgat cgttaaatct caacggctgg 660

atcttatgac gaccgttaga aattgtggtt gtcgacgagt cagtaataaa cggcgtcaaa 720

gtggttgcag ccggcacaca cgagtcgtgt ttatcaactc aaagcacaaa tacttttcct 780

caacctaaaa ataaggcaat tagccaaaaa caactttgcg tgtaaacaac gctcaataca 840

cgtgtcattt tattattagc tattgcttca ccgccttagc tttctcgtga cctagtcgtc 900

ctcgtctttt cttcttcttc ttctataaaa caatacccaa agcttcttct tcacaattca 960

gatttcaatt tctcaaaatc ttaaaaactt tctctcaatt ctctctaccg tgatcaaggt 1020

aaatttctgt gttccttatt ctctcaaaat cttcgatttt gttttcgttc gatcccaatt 1080

tcgtatatgt tctttggttt agattctgtt aatcttagat cgaagacgat tttctgggtt 1140

tgatcgttag atatcatctt aattctcgat tagggtttca taaatatcat ccgatttgtt 1200

caaataattt gagttttgtc gaataattac tcttcgattt gtgatttcta tctagatctg 1260

gtgttagttt ctagtttgtg cgatcgaatt tgtcgattaa tctgagtttt tctgattaac 1320

ag 1322

<210> 57

<211> 234

<212> DNA

<213> Arabidopsis thaliana

<220>

<223> chloroplast transit peptide of white or light green color

<400> 57

atggctacag ctactacaac tgctactgct gctttttcag gagttgtttc tgttggtaca 60

gaaactagaa gaatctattc attctctcat cttcaacctt cagctgcttt tcctgctaag 120

ccatcttcat tcaaatcact taagttgaag cagtctgcta gacttacaag aagattggat 180

catagaccat ttgttgttag atgtgaggct tcttcatcta acggaagact tact 234

<210> 58

<211> 253

<212> DNA

<213> Agrobacterium tumefaciens

<220>

<223> nopaline synthase gene terminator

<400> 58

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgttactag atc 253

<210> 59

<211> 530

<212> DNA

<213> cauliflower mosaic virus

<220>

<223> 35S Promoter

<400> 59

ccatggagtc aaagattcaa atagaggacc taacagaact cgccgtaaag actggcgaac 60

agttcataca gagtctctta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg 120

agcacgacac gcttgtctac tccaaaaata tcaaagatac agtctcagaa gaccaaaggg 180

caattgagac ttttcaacaa agggtaatat ccggaaacct cctcggattc cattgcccag 240

ctatctgtca ctttattgtg aagatagtgg aaaaggaagg tggctcctac aaatgccatc 300

attgcgataa aggaaaggcc atcgttgaag atgcctctgc cgacagtggt cccaaagatg 360

gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 420

aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 480

cgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggaca 530

<210> 60

<211> 552

<212> DNA

<213> Streptomyces viridochromogenes

<220>

<223> phosphinothricin-N-acetyltransferase gene

<400> 60

atgtctccgg agaggagacc agttgagatt aggccagcta cagcagctga tatggccgcg 60

gtttgtgata tcgttaacca ttacattgag acgtctacag tgaactttag gacagagcca 120

caaacaccac aagagtggat tgatgatcta gagaggttgc aagatagata cccttggttg 180

gttgctgagg ttgaggtgt tgtggctggt attgcttacg ctgggccctg gaaggctagg 240

aacgcttacg attggacagt tgagagtact gtttacgtgt cacataggca tcaaaggttg 300

ggcctaggat ccacattgta cacacatttg cttaagtcta tggaggcgca aggttttaag 360

tctgtggttg ctgttatagg ccttccaaac gatccatctg ttaggttgca tgaggctttg 420

ggatacacag cccggggtac attgcgcgca gctggataca agcatggtgg atggcatgat 480

gttggttttt ggcaaaggga ttttgagttg ccagctcctc caaggccagt taggccagtt 540

acccagatct ga 552

<210> 61

<211> 195

<212> DNA

<213> cauliflower mosaic virus

<220>

<223> 35S terminator

<400> 61

ctgaaatcac cagtctctct ctacaaatct atctctctct aataataatgt gtgagtagtt 60

cccagataag ggaattaggg ttcttatagg gtttcgctca tgtgttgagc atataagaaa 120

cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 180

accaaaatcc agtgg 195

<210> 62

<211> 540

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO1B

<400> 62

atgaaagcgt tggttcttta cagtacaaga gatggtcaga cccacgccat tgcgagctat 60

atcgcaaact gcatgaagga gaagtatgaa tgtgacgtca ttgatctctt gcacgcgcag 120

catgtcaccc ttagccgcta cgacaaggtc cttgttgggg catcgatacg ctatggccat 180

ttcaacgccg tgctggacaa atttgttaag cagaatatac aacagctgaa ctccatgcct 240

tctgcattct tcgcggtaaa tctgaccgct cgtaagccag aaaaaaggac accacaaaca 300

aattcatacg tcagaaagtt cctcctagga acgccgtgga agcccgccat gtgcggtgtt 360

tttgctggcg ccttgcgata ccctcggtat cggtgggtgg acaaggtgat gatccaatta 420

atcatgagga tgacgggggg cgagactgat acttctaaag aagtggagta cactgattgg 480

gagcaggtga aaaaatttgc tgaggacttc ggaaagctct cctacaagaa gacgctgtga 540

<210> 63

<211> 696

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO6B

<400> 63

atgtcaagtc ccaacgggaa gtaccttatg ctgtatagca cgactgatgg acacaccaag 60

acaataatgg acaccatggc gaagcacatt atggaagagg ccaaagttca gtgcgacgtc 120

gtagacatga gggacgggga caagtatgaa cttgcggctt acgagaaggt catgctcggc 180

gcctccataa ggtacggctt cttcagccgc accttgcata catatactac ccatcatgtt 240

gatgagctta actccatgcc atcggcattc tttggtgtta atttaacggc gcggaaaact 300

agcaagaaca cagccatgac caatgcttat acacggaaat tcctggatca atctatgtgg 360

gtgccacagc tgagtggggt cttcgccggt gcactctggt acccccgcta caactttttt 420

gatcgagtga tgatccagtt catcatgaag gtgacgggcg gcgaaacaaa tactacaaaa 480

gagatcgtct acacggactg ggatgcagtg cacaagtttg ctactgactt cgtccaactc 540

cctgccaccg ccattccaag atctaaacct gctacgtcgg tgccgccggc gtcagtagca 600

aactatgata atggtgctcg tgttgcgctg gtggtggttg gaatttctgc agccatcatc 660

ttcggccgca ggctaatttt ggccaagaga ttttga 696

<210> 64

<211> 531

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO12B

<400> 64

atgaagaagg cgctgttttt atattcaagt cgtgaggggcc aaacaaaaaa aatattgaac 60

tacattgaag gtgaattgtc caattttgag tgcgagctcg tcgacctcca cacgctggac 120

gccattgatt tctcggtcta tgacagggtt ttgattggag caagcatacg gtacggccac 180

ctaaacaaga aactttatca attcattgag aataaccgcc agcagctgga aaatcatacc 240

aaggtggcct tcttctgtgt aaatcttact gcaaggaagg aagatcaagg caaggacacc 300

cctgaaggga gcgtctacat ccagaagttc ctgaaaaaat ctccctggca gccaaaactt 360

atcggggtgt tcgctggcgc gctctactac ccgagatatg gtttttttga caagatgatg 420

atcaagctca tcatgacgct gacagatgga gagactgata ctacaaagga ggtggagtac 480

accaactgga agaaagtttc cctcttcgcc aagaagtttg ctgagctttg a 531

<210> 65

<211> 175

<212> PRT

<213> Arsenophonus

<220>

<223> Amino acid sequence of PPO-AP

<400> 65

Met Ser Tyr Leu Leu Leu Tyr Ser Gly Lys Asn Gly Gln Thr Lys Lys

1 5 10 15

Ile Ile Leu Lys Ile Thr Glu Tyr Leu Arg Lys Glu Gly Lys Gln Cys

20 25 30

Asp Val Arg Asp Leu Asn Met Asp Lys Asn Phe Asn Leu Ser Val Tyr

35 40 45

Gln Lys Val Leu Val Gly Ala Ser Ile Arg Tyr Gly His Phe Asn Pro

50 55 60

Ala Val Leu Asn Phe Ala Gln Asn His Gln Lys Glu Leu Asn Thr Ile

65 70 75 80

Gln Ser Ala Phe Phe Gly Val Asn Leu Met Ala Arg Lys Gln Gly Lys

85 90 95

Asp Thr Pro Glu Thr Asn Val Tyr Val Arg Lys Phe Leu Ala Lys Ile

100 105 110

Pro Trp Gln Pro Thr Ile Thr Asn Val Phe Ser Gly Ala Leu Arg Tyr

115 120 125

Pro Gln Tyr Lys Trp Leu Asp Arg Ile Met Ile Gln Phe Ile Met Lys

130 135 140

Ile Thr Gly Gly Glu Thr Asn Lys Asn Lys Glu Val Glu Tyr Thr Asp

145 150 155 160

Trp Glu Lys Val Glu Tyr Phe Ala Arg Glu Phe Asn Ala Leu Lys

165 170 175

<210> 66

<211> 528

<212> DNA

<213> Arsenophonus

<220>

<223> Nucleotide sequence of PPO-AP

<400> 66

atgagctatt tgctacttta ttctggtaaa aatggtcaga ctaaaaaaat aatacttaaa 60

attactgaat atttacgtaa agaaggtaag caatgtgatg ttagagattt aaatatggat 120

aaaaatttta atctttctgt ctaccaaaaa gtattagtcg gtgcatcaat ccgttatggt 180

cattttaatc ctgcagtttt gaattttgcg caaaatcacc aaaaagagtt gaatactatc 240

caaagtgcat tttttggcgt taatttaatg gccagaaaac agggaaaaga tacacctgaa 300

acaaatgttt atgtacgtaa atttttagct aaaattccgt ggcaaccaac tattactaat 360

gtgttttcgg gagcgctacg ttatccccaa tataaatggt tagatcgtat tatgatccaa 420

tttattatga aaataacagg cggagaaacg aataaaaata aagaggttga atatactgat 480

tgggaaaaag tcgaatactt tgcgcgtgaa tttaatgcgc ttaaataa 528

<210> 67

<211> 528

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO-APA

<400> 67

atgtcttacc tgctgttata ttcagggaaa aatggacaaa ccaagaagat cattctgaaa 60

ataacagagt atcttcgaaa agaaggaaag caatgtgatg ttagagattt gaacatggat 120

aagaacttca acctttctgt atatcagaag gtgctcgttg gtgcctcgat aagatatggt 180

catttcaatc ccgcagtcct caattttgct cagaatcacc aaaaggagtt aaacactatt 240

caatcagctt tctttggcgt gaatttgatg gctcgtaaac aaggaaaaga tactcctgag 300

acaaatgtgt atgtgaggaa gtttcttgcc aaaattccat ggcaaccaac aatcaccaat 360

gttttcagtg gagcgttgag gtatcctcag tacaaatggc ttgaccggat aatgattcag 420

tttatcatga agattactgg tggtgaaaca aacaaaaaca aggaagttga atatactgat 480

tgggaaaagg tagagtactt tgcaagagag tttaatgctt tgaaatga 528

<210> 68

<211> 528

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleotide sequence of PPO-APB

<400> 68

atgtcctacc tcttgctgta ttcggggaaa aatggtcaaa ccaagaagat catcctcaaa 60

ataactgaat atcttcgtaa agaaggaaag cagtgcgacg tcagagatct taacatggac 120

aagaatttta atttaagtgt gtaccagaag gttcttgtcg gagcttcaat tcgctacggc 180

cacttcaacc ccgccgtgct gaacttcgcg cagaatcatc aaaaagagct gaacacaatt 240

caatctgcct tctttggcgt caacctgatg gcgaggaagc aagggaagga tacaccagaa 300

accaatgttt atgttcggaa gtttttggct aaaattccat ggcagccgac gatcaccaac 360

gtatttagcg gtgcactcag atatcctcag tacaagtggc tggataggat aatgatccag 420

ttcatcatga agattactgg cggcgagaca aataagaaca aggaggtgga gtacacggac 480

tggggagaaag tggaatactt cgcccgcgag ttcaatgcac taaaatga 528

<210> 69

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 1 for detection of the EPSPS gene

<400> 69

ctggaaggcg aggacgtcat caata 25

<210> 70

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 2 for detection of the EPSPS gene

<400> 70

tggcggcatt gccgaaatcg ag 22

<210> 71

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Probe 1 for detection of EPSPS gene

<400> 71

atgcaggcga tgggcgcccg catccgta 28

<210> 72

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Universal 5'-End Adapter Primer 2

<400> 72

ccaagcggcc aagctta 17

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Universal 3' End Adapter Primer 2

<400> 73

tgtttgaacg atcggcgcgc from 21

<210> 74

<211> 1411

<212> DNA

<213> Oryza sativa L.

<220>

<223> rice actin 1 promoter

<400> 74

tactcgaggt cattcatatg cttgagaaga gagtcgggat agtccaaaat aaaacaaagg 60

taagattacc tggtcaaaag tgaaaacatc agttaaaagg tggtataaag taaaatatcg 120

gtaataaaag gtggcccaaa gtgaaattta ctcttttcta ctattataaa aattgaggat 180

gtttttgtcg gtactttgat acgtcatttt tgtatgaatt ggtttttaag tttattcgct 240

tttggaaatg catatctgta tttgagtcgg gttttaagtt cgtttgcttt tgtaaataca 300

gagggatttg tataagaaat atctttagaa aaacccatat gctaatttga cataattttt 360

gagaaaaata tatattcagg cgaattctca caatgaacaa taataagatt aaaatagctt 420

tcccccgttg cagcgcatgg gtattttttc tagtaaaaat aaaagataaa cttagactca 480

aaacatttac aaaaacaacc cctaaagttc ctaaagccca aagtgctatc cacgatccat 540

agcaagccca gcccaaccca acccaaccca acccacccca gtccagccaa ctggacaata 600

gtctccacac ccccccacta tcaccgtgag ttgtccgcac gcaccgcacg tctcgcagcc 660

aaaaaaaaaa agaaagaaaa aaaagaaaaa gaaaaaacag caggtgggtc cgggtcgtgg 720

gggccggaaa cgcgaggagg atcgcgagcc agcgacgagg ccggccctcc ctccgcttcc 780

aaagaaacgc cccccatcgc cactatatac ataccccccc ctctcctccc atccccccaa 840

ccctaccacc accaccacca ccacctccac ctcctccccc ctcgctgccg gacgacgagc 900

tcctcccccc tccccctccg ccgccgccgc gccggtaacc accccgcccc tctcctcttt 960

ctttctccgt ttttttttcc gtctcggtct cgatctttgg ccttggtagt ttgggtgggc 1020

gagaggcggc ttcgtgcgcg cccagatcgg tgcgcgggag gggcgggatc tcgcggctgg 1080

ggctctcgcc ggcgtggatc cggcccggat ctcgcgggga atggggctct cggatgtaga 1140

tctgcgatcc gccgttgttg ggggagatga tggggggttt aaaatttccg ccgtgctaaa 1200

caagatcagg aagaggggaa aagggcacta tggtttatat ttttatatat ttctgctgct 1260

tcgtcaggct tagatgtgct agatctttct ttcttctttt tgtgggtaga atttgaatcc 1320

ctcagcattg ttcatcggta gtttttcttt tcatgatttg tgacaaatgc agcctcgtgc 1380

ggagcttttt tgtaggtaga agtgatcaac c 1411

<210> 75

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<223> 35S promoter of cauliflower mosaic virus

<400> 75

ggtccgattg agacttttca acaaagggta atatccggaa acctcctcgg attccatgc 60

ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc 120

catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa 180

gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca 240

aagcaagtgg attgatgtga tggtccgatt gagacttttc aacaaagggt aatatccgga 300

aacctcctcg gattccatg cccagctatc tgtcacttta ttgtgaagat agtggaaaag 360

gaaggtggct cctacaaatg ccatcattgc gataaaggaa aggccatcgt tgaagatgcc 420

tctgccgaca gtggtcccaa agatggaccc caccaccacga ggagcatcgt ggaaaaagaa 480

gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg 540

gatgacgcac aatcccacta tccttcgcaa gacccttcct ctatataagg aagttcattt 600

catttggaga ggacacgctg acaagctgac tctagcaga 639

<210> 76

<211> 806

<212> DNA

<213> Zea mays

<220>

<223> 70 kDa heat shock protein intron Zea mays

<400> 76

accgtcttcg gtacgcgctc actccgccct ctgcctttgt tactgccacg tttctctgaa 60

tgctctcttg tgtggtgatt gctgagagtg gtttagctgg atctagaatt acactctgaa 120

atcgtgttct gcctgtgctg attacttgcc gtcctttgta gcagcaaaat atagggacat 180

ggtagtacga aacgaagata gaacctacac agcaatacga gaaatgtgta atttggtgct 240

tagcggtatt tatttaagca catgttggtg ttatagggca cttggattca gaagtttgct 300

gttaatttag gcacaggctt catactacat gggtcaatag tatagggatt catattatag 360

gcgatactat aataatttgt tcgtctgcag agcttattat ttgccaaaat tagatattcc 420

tattctgttt ttgtttgtgt gctgttaaat tgttaacgcc tgaaggaata aatataaatg 480

acgaaatttt gatgtttatc tctgctcctt tattgtgacc ataagtcaag atcagatgca 540

cttgttttaa atattgttgt ctgaagaaat aagtactgac agtattttga tgcattgatc 600

tgcttgtttg ttgtaacaaa atttaaaaat aaagagtttc ctttttgttg ctctccttac 660

ctcctgatgg tatctagtat ctaccaactg acactatatt gcttctcttt acatacgtat 720

cttgctcgat gccttctccc tagtgttgac cagtgttact cacatagtct ttgctcattt 780

cattgtaatg cagataccaa gcggcc 806

<210> 77

<211> 1993

<212> DNA

<213> Zea mays

<220>

<223> ubiquitin 1 gene promoter Zea mays

<400> 77

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cggggggattc ctttcccacc 840

gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900

ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960

cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020

ttctctagat cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt 1080

ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140

ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200

gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260

agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320

atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380

tagatcggag tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440

tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500

aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560

cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620

gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680

acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740

gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800

tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860

tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920

catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980

gttacttctg cag 1993

<210> 78

<211> 1176

<212> DNA

<213> Escherichia coli

<220>

<223> phosphomannose isomerase gene

<400> 78

atgcaaaaac tcattaactc agtgcaaaac tatgcctggg gcagcaaaac ggcgttgact 60

gaactttatg gtatggaaaa tccgtccagc cagccgatgg ccgagctgtg gatgggcgca 120

catccgaaaa gcagttcacg agtgcagaat gccgccggag atatcgtttc actgcgtgat 180

gtgattgaga gtgataaatc gactctgctc ggagaggccg ttgccaaacg ctttggcgaa 240

ctgcctttcc tgttcaaagt attatgcgca gcacagccac tctccattca ggttcatcca 300

aacaaacaca attctgaaat cggttttgcc aaagaaaatg ccgcaggtat cccgatggat 360

gccgccgagc gtaactataa agatcctaac cacaagccgg agctggtttt tgcgctgacg 420

cctttccttg cgatgaacgc gtttcgtgaa ttttccgaga ttgtctccct actccagccg 480

gtcgcaggtg cacatccggc gattgctcac tttttacaac agcctgatgc cgaacgttta 540

agcgaactgt tcgccagcct gttgaatatg cagggtgaag aaaaatcccg cgcgctggcg 600

attttaaaat cggccctcga tagccagcag ggtgaaccgt ggcaaacgat tcgtttaatt 660

tctgaatttt acccggaaga cagcggtctg ttctccccgc tattgctgaa tgtggtgaaa 720

ttgaaccctg gcgaagcgat gttcctgttc gctgaaacac cgcacgctta cctgcaaggc 780

gtggcgctgg aagtgatggc aaactccgat aacgtgctgc gtgcgggtct gacgcctaaa 840

tacattgata ttccggaact ggttgccaat gtgaaattcg aagccaaacc ggctaaccag 900

ttgttgaccc agccggtgaa acaaggtgca gaactggact tcccgattcc agtggatgat 960

tttgccttct cgctgcatga ccttagtgat aaagaaacca ccattagcca gcagagtgcc 1020

gccattttgt tctgcgtcga aggcgatgca acgttgtgga aaggttctca gcagttacag 1080

cttaaaccgg gtgaatcagc gtttattgcc gccaacgaat caccggtgac tgtcaaaggc 1140

cacggccgtt tagcgcgtgt ttacaacaag ctgtaa 1176

<210> 79

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 3 for detection of the PMI gene sequence

<400> 79

gctgtaagag cttactgaaa aaattaaca 29

<210> 80

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 4 for detection of the PMI gene sequence

<400> 80

cgatctgcag gtcgacgg 18

<210> 81

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Probe 2 for detection of PMI gene sequence

<400> 81

tctcttgcta agctgggagc tcgatcc 27

<---

Claims (64)

1. A method for controlling weeds, characterized in that it includes applying a herbicide containing an effective dose of a protoporphyrinogen oxidase (PPO) inhibitor to a field where at least one transgenic plant is located, wherein said transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and said transgenic plant has increased resistance to PPO inhibitor herbicides, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase, wherein protoporphyrinogen oxidase has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6. 2. The method for controlling weeds according to claim 1, wherein the protoporphyrinogen oxidase has at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6. 3. The method for controlling weeds according to claim 1 or 2, wherein the protoporphyrinogen oxidase is the amino acid sequence of SEQ ID NO: 6. 4. The method for controlling weeds according to any one of claims 1-3, wherein said transgenic plant comprises monocotyledonous plants and dicotyledonous plants. 5. The method of controlling weeds according to claim 4, wherein the transgenic plant is oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, Cicer arietinum, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or Arabidopsis thaliana. 6. The method of controlling weeds according to claim 4 or 5, wherein the transgenic plant is a glyphosate-resistant plant and the weeds are glyphosate-resistant weeds. 7. The method for controlling weeds according to any one of claims 1-6, wherein the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones. 8. The method of controlling weeds according to claim 7, wherein the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin. 9. A method for controlling weeds according to any one of paragraphs 1-8, characterized in that the polynucleotide sequence of protoporphyrinogen oxidase includes: (a) a polynucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6, wherein the polynucleotide sequence does not include SEQ ID NO: 20; or (b) the polynucleotide sequence presented in SEQ ID NO: 34 or SEQ ID NO: 63. 10. A method for controlling weeds according to any one of claims 1-9, characterized in that the transgenic plant additionally contains at least one second polynucleotide encoding a second herbicide-resistant protein that differs from the polynucleotide sequence encoding protoporphyrinogen oxidase. 11. The method for controlling weeds according to claim 10, characterized in that the second polynucleotide encodes a selectable marker protein, a protein with synthetic activity, a protein with degradative activity, a protein resistant to biotic stress, a protein resistant to abiotic stress, a male sterility protein, a protein that affects plant yield, and/or a protein that affects plant quality. 12. The method for controlling weeds according to claim 11, wherein the second polynucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate synthase and/or cytochrome-like protein. 13. The method for controlling weeds according to any one of claims 1-12, characterized in that the herbicide containing an effective dose of a PPO inhibitor further comprises a glyphosate-based herbicide, a glufosinate-based herbicide, an auxin-like herbicide, a graminicide, a pre-emergence selective herbicide and/or a post-emergence selective herbicide. 14. A planting combination for controlling weed growth comprising a PPO inhibitor herbicide and at least one transgenic plant, wherein the herbicide comprising an effective dose of the PPO inhibitor is applied to a field where said at least one transgenic plant is located, wherein said transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and said transgenic plant has increased resistance to PPO inhibitor herbicides, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase; wherein protoporphyrinogen oxidase has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6. 15. A planting combination for controlling weed growth according to claim 14, wherein the protoporphyrinogen oxidase has at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6. 16. A planting combination for controlling weed growth according to claim 14 or 15, wherein the protoporphyrinogen oxidase is the amino acid sequence of SEQ ID NO: 6. 17. A planting combination for controlling weed growth according to any one of claims 14-16, wherein the transgenic plant comprises monocotyledonous plants and dicotyledonous plants. 18. The planting combination for controlling weed growth according to claim 17, wherein the transgenic plant is oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, Cicer arietinum, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato or Arabidopsis thaliana. 19. A planting combination for controlling weed growth according to claim 17 or 18, wherein the transgenic plant is a glyphosate-resistant plant and the weeds are glyphosate-resistant weeds. 20. A planting combination for controlling weed growth according to any one of claims 14-19, wherein the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones. 21. The planting combination for controlling weed growth according to claim 20, wherein the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin. 22. A planting combination for controlling weed growth according to any of paragraphs 14-21, characterized in that the polynucleotide sequence of protoporphyrinogen oxidase includes: (a) a polynucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6, wherein the polynucleotide sequence does not include SEQ ID NO: 20; or (b) a polynucleotide sequence as shown in SEQ ID NO: 34 or SEQ ID NO: 63. 23. A planting combination for controlling weed growth according to any one of claims 14-22, characterized in that the transgenic plant further comprises at least one second polynucleotide encoding a second herbicide-resistant protein that differs from the polynucleotide sequence encoding protoporphyrinogen oxidase. 24. A planting combination for controlling weed growth according to claim 23, characterized in that the second polynucleotide encodes a selectable marker protein, a protein with synthetic activity, a protein with degradative activity, a protein resistant to biotic stress, a protein resistant to abiotic stress, a male sterility protein, a protein that affects plant yield, and/or a protein that affects plant quality. 25. A planting combination for controlling weed growth according to claim 24, characterized in that the second polynucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate synthase and/or cytochrome-like protein. 26. A planting combination for controlling weed growth according to any one of claims 14-25, characterized in that the herbicide containing an effective dose of a PPO inhibitor further comprises a glyphosate-based herbicide, a glufosinate-based herbicide, an auxin-like herbicide, a graminicide, a pre-emergence selective herbicide and/or a post-emergence selective herbicide. 27. A method for producing a plant that is resistant to a PPO inhibitor herbicide, characterized in that said method includes introducing a polynucleotide sequence encoding protoporphyrinogen oxidase into the genome of said plant, and when a herbicide containing an effective dose of a PPO inhibitor is applied to a field where at least said plant is located, said plant has increased resistance to PPO inhibitor herbicides, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase, wherein protoporphyrinogen oxidase has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6. 28. A method for producing a plant that is resistant to a PPO inhibitor herbicide according to claim 27, wherein the protoporphyrinogen oxidase has at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6. 29. A method for producing a plant that is resistant to a PPO inhibitor herbicide according to claim 27 or 28, wherein the protoporphyrinogen oxidase is the amino acid sequence of SEQ ID NO: 6. 30. A method for producing a plant that is resistant to a PPO inhibitor herbicide according to any one of claims 27-29, wherein the method of introduction includes methods of genetic transformation, genome editing or gene mutation. 31. A method for producing a plant that is resistant to a PPO inhibitor herbicide according to any one of claims 27-30, wherein the plant comprises monocotyledonous plants and dicotyledonous plants. 32. The method of producing a plant that is resistant to the PPO inhibitor herbicide of claim 31, wherein the transgenic plant is oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, Cicer arietinum, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana. 33. A method for producing a plant that is resistant to a PPO inhibitor herbicide, claims 27-32, wherein the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones. 34. The method for producing a plant that is resistant to a PPO inhibitor herbicide according to claim 33, wherein the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine. 35. A method for cultivating a plant that is resistant to a PPO inhibitor herbicide, characterized in that it comprises: planting at least one plant propagule, wherein said plant propagule comprises in its genome a polynucleotide encoding protoporphyrinogen oxidase, wherein said protoporphyrinogen oxidase has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6; allowing the plant propagule to grow into a plant; and applying a herbicide containing an effective dose of a PPO inhibitor to a field containing at least said plant, and harvesting said plant having increased resistance to PPO inhibitor herbicides, compared to other plants lacking a polynucleotide sequence encoding protoporphyrinogen oxidase. 36. A method for cultivating a plant that is resistant to a PPO inhibitor herbicide according to claim 35, wherein the protoporphyrinogen oxidase has at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6. 37. A method for cultivating a plant that is resistant to a PPO inhibitor herbicide according to claim 35 or 36, wherein the protoporphyrinogen oxidase is SEQ ID NO: 6. 38. A method for cultivating a plant that is resistant to a PPO inhibitor herbicide according to any one of claims 35-37, wherein the plant comprises monocotyledonous plants and dicotyledonous plants. 39. The method of cultivating a plant that is resistant to the PPO inhibitor herbicide of claim 38, wherein the plant is oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, Cicer arietinum, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana. 40. A method for cultivating a plant that is resistant to a PPO inhibitor herbicide according to any one of claims 35-39, wherein the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones. 41. A method for cultivating a plant that is resistant to a PPO inhibitor herbicide according to claim 40, wherein the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine. 42. A method for protecting a plant from damage caused by a PPO inhibitor herbicide, characterized in that it includes applying a herbicide containing an effective dose of a PPO inhibitor to a field where at least one transgenic plant is located, wherein said transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and said transgenic plant has increased resistance to PPO inhibitor herbicides, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase, wherein protoporphyrinogen oxidase has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6. 43. A method for imparting resistance to a PPO inhibitor herbicide to a plant, characterized in that it comprises applying a herbicide containing an effective dose of a PPO inhibitor to a field where at least one transgenic plant is located, wherein said transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and said transgenic plant has increased resistance to PPO inhibitor herbicides, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase, wherein protoporphyrinogen oxidase has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6. 44. The method according to claim 42 or 43, wherein the protoporphyrinogen oxidase has at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6. 45. The method according to any one of claims 42-44, wherein the protoporphyrinogen oxidase is SEQ ID NO: 6. 46. The method according to any one of claims 42-45, wherein the transgenic plant comprises monocotyledonous plants and dicotyledonous plants. 47. The method of claim 46, wherein the transgenic plant is oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, Cicer arietinum, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana. 48. The method according to any one of claims 42-47, wherein the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones. 49. The method of claim 48, wherein the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine. 50. Use of protoporphyrinogen oxidase for imparting resistance to a plant to a PPO inhibitor herbicide, wherein the protopophyrinogen oxidase has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6. 51. The use of protoporphyrinogen oxidase for imparting resistance to a plant to a PPO inhibitor herbicide according to claim 50, wherein the protoporphyrinogen oxidase has at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. 52. Use of protoporphyrinogen oxidase for imparting resistance to a plant to a PPO inhibitor herbicide according to claim 50 or 51, wherein the protoporphyrinogen oxidase is SEQ ID NO: 6. 53. The use of protoporphyrinogen oxidase to impart resistance to a plant to a PPO inhibitor herbicide according to any one of claims 50-52, wherein the use of protoporphyrinogen oxidase to impart resistance to a plant to a PPO inhibitor herbicide comprises applying a herbicide containing an effective dose of a PPO inhibitor to a field where at least one transgenic plant is located, wherein said transgenic plant contains in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, and said transgenic plant has increased resistance to PPO inhibitor herbicides, compared to other plants without a polynucleotide sequence encoding protoporphyrinogen oxidase. 54. The use of protoporphyrinogen oxidase to impart resistance to a plant to a PPO inhibitor herbicide according to any one of claims 50-53, wherein the plant comprises monocotyledonous plants and dicotyledonous plants. 55. The use of protoporphyrinogen oxidase to impart resistance to a plant to a PPO inhibitor herbicide according to claim 54, wherein the plant is oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, Cicer arietinum, peanut, sugar beet, cucumber, cotton, oilseed rape, potato, tomato, or Arabidopsis thaliana. 56. The use of protoporphyrinogen oxidase to impart resistance to a plant to a PPO inhibitor herbicide according to any one of claims 50-55, wherein the PPO inhibitor herbicide comprises a PPO inhibitor herbicide from the class of diphenyl ethers, oxadiazolones, N-phenylphthalimides, oxazolinones, phenylpyrazoles, uracils, thiadiazoles, triazolinones and/or triazinones. 57. The use of protoporphyrinogen oxidase to impart resistance to a plant to a PPO inhibitor herbicide according to claim 56, wherein the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazine. 58. The use of protoporphyrinogen oxidase to impart resistance to a plant to the PPO inhibitor herbicide according to claim 50, characterized in that the polynucleotide sequence of the protoporphyrinogen oxidase comprises: (a) a polynucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6, wherein the polynucleotide sequence does not include SEQ ID NO: 20; or (b) a polynucleotide sequence as shown in SEQ ID NO: 34 or SEQ ID NO: 63.