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WO2021039659A1 - Procédé de production de plante résistante aux thrips - Google Patents

Procédé de production de plante résistante aux thrips Download PDF

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Publication number
WO2021039659A1
WO2021039659A1 PCT/JP2020/031698 JP2020031698W WO2021039659A1 WO 2021039659 A1 WO2021039659 A1 WO 2021039659A1 JP 2020031698 W JP2020031698 W JP 2020031698W WO 2021039659 A1 WO2021039659 A1 WO 2021039659A1
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mlx56
thrips
plant
family protein
gene
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Japanese (ja)
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浩太郎 今野
光原　一朗
未果 村田
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National Agriculture and Food Research Organization
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National Agriculture and Food Research Organization
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates

Definitions

  • the present invention relates to a method for producing a thrips-resistant plant.
  • insect-resistant crops have been produced by breeding based on crossing between existing varieties, but developing insect-resistant crops by such traditional breeding methods requires enormous time and cost, and it takes a huge amount of time and cost. Not efficient.
  • Bt protein is derived from bacteria, there is a strong sense of resistance when it is used in genetically modified crops, and the use of plant-derived insect-resistant protein is desired.
  • Known plant-derived insect-resistant proteins include cowpea-derived protein inhibitors (Patent Document 1), green bean-derived amylase inhibitors (Non-Patent Document 1), and snowdrop-derived lectins (Patent Document 2).
  • Patent Document 1 cowpea-derived protein inhibitors
  • Non-Patent Document 1 green bean-derived amylase inhibitors
  • Snowdrop-derived lectins Patent Document 2
  • the insect-resistant effects of lectins and digestive enzyme inhibitory proteins are not always sufficient.
  • Patent Document 3 describes that when an MLX56 protein isolated from quail emulsion and having chitin-binding activity but not chitinase activity is fed to a lepidopteran insect larva, the weight gain of the larva is remarkable. It discloses that it suppressed and brought about a high growth inhibitory effect.
  • the LA-b protein contained in quail emulsion which has an amino acid sequence similar to that of the MLX56 protein but has chitinase activity, has a growth inhibitory effect and an insecticidal effect when fed to larvae of Drosophila. It is disclosed that these effects are considered to be due to the chitinase activity of the LA-b protein.
  • Non-Patent Document 2 the MLX56 family protein including MLX56 protein and LA-b is specific to the peritrophic membrane (tube-shaped, usually extremely thin membrane containing chitin as the main component) existing in the digestive tract of many insects. It has been reported that the abnormal thickening of the peritrophic membrane by binding to each other causes digestive dysfunction, resulting in a growth-inhibiting effect.
  • the MLX56 family protein is a repetitive sequence of two chitin-binding domains (Hevein-like domains) and an Extensin domain (Ser-Pro-Pro-Pro) sandwiched between them in the N-terminal region of the mature protein. It is considered that abnormal thickening of the peritrophic membrane occurs due to the binding of the hebein-like domain to chitin of the peritrophic membrane and the swelling of the extensin domain (Non-Patent Document 2).
  • Thrips (Stonefly) is not only a difficult-to-control pest, but also carries a virus, and even a small number of individuals cause great damage to plants, so the development of a more effective control method is desired. Thrips are known to lack the peritrophic membrane (Non-Patent Document 3).
  • An object of the present invention is to provide an efficient method for producing a thrips-tolerant plant.
  • the present inventors have found that the MLX56 family protein has a control effect on thrips, and have completed the present invention.
  • the present invention includes the following.
  • a method for producing a thrips-resistant transformed plant which comprises introducing a MLX56 family protein gene into a plant and testing the thrips resistance of the transformed plant into which the gene has been introduced.
  • thrips is a western flower thrips (Frankliniella occidentalis) or a western flower thrips (Thrips palmi).
  • MLX56 family protein gene Use of the MLX56 family protein gene to confer thrips resistance on plants by gene transfer.
  • Azalea resistance including mating using a transformant of Azalea resistance having an exogenous MLX56 family protein gene as a breeding parent, obtaining offspring plants, and selecting offspring plants having the MLX56 family protein gene. How to breed plants.
  • a method for controlling thrips which comprises feeding thrips a transformed plant resistant to thrips having an exogenous MLX56 family protein gene.
  • a plant having thrips resistance can be efficiently produced.
  • FIG. 1 is a schematic diagram of the structure of the T-DNA region of the binary plasmid vector pEL2 ⁇ :: MLX56.
  • FIG. 2 shows the expression level of the exogenous MLX56 gene (relative expression level compared to the actin expression level) in the transformed tomato plant.
  • FIG. 3 is a photograph showing the results of SDS-PAGE analysis of a protein extract from a transformed tomato plant and its fraction. Left: Negative control, Right: MLX56-73-30 system.
  • T crude protein extract (Total), S: unbound supernatant (Unbound sup), W: washing solution (Wash), U: urea fraction (Urea), E: chitin-binding substance elution fraction (Elute).
  • FIG. 4 shows the survival number of thrips after 2 weeks in the thrips resistance test using transformed tomato plants. A, b, and c in the figure indicate that there is a statistically significant difference between the strains.
  • FIG. 5 is a photograph showing the appearance of the transformed tomato plant after 2 weeks in the thrips resistance test. A: Overall photo of the transformed tomato plant, B: Photo of the leaves of the transformed tomato plant. The state of feeding damage by thrips of tomato plants (A) and the feeding damage marks of leaves (B) were observed.
  • FIG. 6 is a photograph showing mite damage in a transformed tomato plant. A: Control plant, B: MLX56-69, C: MLX56-73.
  • the present invention relates to the control of thrips using the MLX56 family protein. More specifically, the present invention presents a method for producing a Zamiuma-resistant plant by introducing the MLX56 family protein gene into a plant, and a transformation having the exogenous MLX56 family protein gene thus obtained (transgenic). ) Regarding the control method of thistle horse using plants.
  • a gene encoding an MLX56 family protein is referred to as an MLX56 family protein gene.
  • the MLX56 family protein according to the present invention is a general term for the natural MLX56 protein contained in mulberry emulsion, its homologue, and their functional mutants and recombinant proteins.
  • An example of the amino acid sequence of the natural MLX56 protein contained in mulberry emulsion is shown in SEQ ID NO: 2, and an example of the MLX56 gene (ORF / CDS sequence) encoding it is shown in SEQ ID NO: 1.
  • the MLX56 family protein according to the present invention may be a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
  • the MLX56 family protein according to the present invention has the amino acid sequence shown in SEQ ID NO: 2, 80% or more, 83% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99. It may be a protein consisting of an amino acid sequence having% or more, 99.5% or more, 99.7% or more, or 100% sequence identity.
  • the MLX56 family proteins according to the present invention also have 1 to 20, 1 to 10, 1 to 9, 1 to 7, 1 to 5, 1 to 3, 1 to 20 in the amino acid sequence shown in SEQ ID NO: 2. It may be a protein consisting of an amino acid sequence containing deletions, substitutions, insertions, and / or additions of two or one amino acid residue.
  • the above MLX56 family protein preferably has a signal peptide (for example, the 1st to 21st amino acid sequences shown in SEQ ID NO: 2) at the N-terminal as a protein encoded by the MLX56 family protein gene.
  • the MLX56 family protein may consist of, for example, the 22nd to 415th sequences of the amino acid sequence shown in SEQ ID NO: 2 in the form of a mature protein in which the signal peptide is cleaved.
  • the MLX56 family protein according to the present invention has thrips control activity.
  • "having a thrips control activity” means that a protein having a signal peptide at the N-terminal exhibits thrips control activity at least in the state of a mature protein.
  • the MLX56 family protein according to the present invention may have chitin-binding activity.
  • a protein having a signal peptide at the N-terminal “has chitin-binding activity” means that it exhibits chitin-binding activity at least in the state of a mature protein.
  • the MLX56 family protein preferably has a characteristic structure of two chitin-binding domains (hebein-like domains) and an extensin domain sandwiched between them in the N-terminal region of the mature protein.
  • the chitin-binding domain (hebein-like domain) is a region showing homology to the amino acid sequence of hebein, which is a rubber latex, and is the 27th to 65th positions in the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO: 9; It corresponds to the 127th to 165th (SEQ ID NO: 11; the second chitin-binding domain on the C-terminal side of the extensin domain) at the N-terminal side (first chitin-binding domain).
  • the MLX56 family protein may have a domain consisting of the amino acid sequence shown in SEQ ID NO: 9 as the N-terminal chitin-binding domain, or 80% or more, 85% or more, 90 with the amino acid sequence shown in SEQ ID NO: 9. It may consist of an amino acid sequence having% or more, 93% or more, 95% or more, or 100% sequence identity, and may have a domain having chitin-binding activity.
  • the MLX56 family protein may have a domain consisting of the amino acid sequence shown in SEQ ID NO: 11 as the C-terminal chitin-binding domain, or 80% or more, 85% or more, 90 with the amino acid sequence shown in SEQ ID NO: 11.
  • the extendin domain in the MLX56 family of proteins is preferably composed of repetitive sequences with Ser-Pro-Pro-Pro-Pro- (Pro) n as the repetitive unit.
  • Ser-Pro-Pro-Pro-Pro- (Pro) n is an integer of 0 or 1 or more, preferably 0 to 10, more preferably 0 to 3, for example, independently for each iteration unit. It can be 0, 1 or 2.
  • the extender domain may include a Ser-Pro-Pro-Pro-Pro repetitive sequence, i.e.
  • (Ser-Pro-Pro-Pro-Pro-Pro) m where m in the equation is, for example, an integer greater than or equal to 2, 3 to 15, more preferably 4 to 13, for example 4, 5, 6, 7, 8, 9 or 10.
  • the integer domain is, for example, Ser-Pro-Pro-Pro-Pro-Ser-Pro-Pro-Pro-Pro-Pro-Pro- (Ser-Pro-Pro-Pro) m'.
  • M'in the equation may be an integer greater than or equal to 1, preferably 2 to 10, more preferably 3 to 8, for example 4, 5, 6, 7 or 8.
  • the extendin domain in the MLX56 family protein corresponds to positions 67 to 118 (SEQ ID NO: 10) in the amino acid sequence shown in SEQ ID NO: 2.
  • the extendin domain can be swellable.
  • the MLX56 family protein may also have a chitinase-like domain, preferably on the C-terminal side of the second chitin binding domain. However, the MLX56 family protein may or may not have chitinase activity, i.e., the chitinase-like domain in the MLX56 family protein may, in some cases, not have chitinase activity.
  • the LA-b protein which is a homologue of the MLX56 protein, typically has chitinase activity.
  • the amino acid sequence of the LA-b protein has about 92.8% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.
  • amino acid changes such as deletion, substitution, insertion, or addition in the MLX56 family protein as compared with the amino acid sequence shown in SEQ ID NO: 2 can retain the functionality of the MLX56 family protein.
  • Substitutions of amino acids compared to the amino acid sequence shown in SEQ ID NO: 2 are preferably, but not limited to, conservative substitutions.
  • Conservative substitutions include, for example, polar uncharged amino acids (serine, threonine, glutamine, aspartic acid, or cysteine), aromatic amino acids (phenylalanine, tyrosine, or tryptophan), acidic amino acids (polar charge; glutamic acid, or aspartic acid), bases.
  • Sexual amino acids (polar charge; lysine, arginine, or histidine), hydrophobic amino acids (alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan), aliphatic amino acids (hydrophobicity; alanin or glycine), branched amino acids Within similar groups of (hydrophobic; valine, leucine, or isoleucine) or hydrophilic amino acids (serine, threonine, aspartic acid, glutamine, tyrosine, tryptophan, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid). It can be a replacement.
  • the MLX56 family protein is deleted in the above signal peptide in the first chitin binding domain, in the second chitin binding domain and / or in the extendin domain as compared to the amino acid sequence shown in SEQ ID NO: 2. , Substitution, insertion, or addition, preferably not including amino acid changes.
  • the MLX56 family protein is deleted, substituted, in all of the above signal peptides, as well as the two chitin-binding domains and the extensin domain sandwiched therein, as compared to the amino acid sequence shown in SEQ ID NO: 2. Does not include amino acid changes such as insertions or additions.
  • the MLX56 family protein gene according to the present invention encodes the above MLX56 family protein.
  • the MLX56 family protein gene according to the present invention may be a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1.
  • the MLX56 family protein gene according to the present invention contains the nucleotide sequence shown in SEQ ID NO: 1 and 80% or more, 83% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, It may be a gene consisting of a base sequence having 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% sequence identity.
  • the MLX56 family protein gene according to the present invention may be a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence shown in SEQ ID NO: 2 or the 22nd to 415th amino acids of SEQ ID NO: 2. Sequence and 80% or more, 83% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, or 100% It may be a gene encoding a protein consisting of an amino acid sequence having sequence identity.
  • the MLX56 family protein gene according to the present invention also has 1 to 20, 1 to 10, 1 to 9, and 1 to 7 in the amino acid sequence shown in SEQ ID NO: 2 or the 22nd to 415th amino acid sequences of SEQ ID NO: 2.
  • the proteins encoded by these MLX56 family protein genes (MLX56 family proteins) have thrips control activity.
  • the protein encoded by the MLX56 family protein gene may also have chitin-binding activity.
  • the above description of the MLX56 family protein also applies to proteins encoded by the MLX56 family protein gene.
  • the MLX56 family protein genes are, for example, the following a) to c) :.
  • sequence identity (%) with respect to a specific amino acid sequence or base sequence means the sequence identity (%) with respect to the total length of the specific amino acid sequence or base sequence.
  • the MLX56 family protein gene can be prepared by a conventional method. For example, a region containing the full length of the open reading frame (ORF) or protein coding sequence (CDS) of the MLX56 family protein gene using cDNA reverse-transcribed from total mRNA derived from mulberry (for example, Morus alba L.) as a template.
  • mulberry means a plant of the genus Morus unless otherwise specified.
  • mulberries are Mulberry (Morus alba L.), Yamagwa (Morus australis Poir.), Kegwa (Morus cathayana Hemsl.), Ogasawara rugwa (Morus boninensis Koidz.), Hachijogwa (Morus kagayamae Koidz.), Yamabe folia.
  • the introduction of mutations into the MLX56 family protein gene can be performed by a conventional method.
  • a short oligonucleotide in which the desired mutation is introduced into a sequence homologous to the target gene (such as the MLX56 family protein gene consisting of the nucleotide sequence shown in SEQ ID NO: 1) is introduced into a plant cell, and the mismatch repair mechanism of the cell is used.
  • Oligonucleotide-Directed Mutagenesis which induces the desired mutation on the genome, or a short oligo in which the desired mutation is introduced into a sequence homologous to the zinc finger nuclease (ZFN) and the target gene.
  • a site-specific mutagenesis method such as a ZFN-mediated mutagenesis method using nucleotides can be used.
  • the above mutation may be introduced into a target gene via homologous recombination by introducing the MLX56 family protein gene or a nucleic acid fragment containing a mutation site thereof into a plant cell as a template DNA.
  • the isolated MLX56 family protein gene may be modified by an arbitrary mutagenesis method such as the Kunkel method or the Gapped duplex method. Mutation transfer into a gene can also be carried out using, for example, a commercially available site-specific mutagenesis kit.
  • the "gene” in the present specification may be DNA, RNA (mRNA, etc.), a chimeric nucleic acid of DNA and RNA, or an artificial base-containing nucleic acid.
  • the gene may consist of a protein coding sequence (start codon to stop codon; CDS), a 5'untranslated region including a translation start site, a polyadenilation signal, and an RNA degradability control region. 3'Untranslated region including, etc. may be further included.
  • the present invention relates to a method for producing a thrips-resistant transformed plant, which comprises introducing the MLX56 family protein gene into a plant and examining the thrips resistance of the transformed plant into which the gene has been introduced.
  • the MLX56 family protein gene is preferably exogenous to the plant to be introduced.
  • the "foreign" or “foreign MLX56 family protein gene" for an MLX56 family protein gene is whether the MLX56 family protein gene is (i) not naturally present in the genome of the species of the host plant to which the gene is introduced. (ii) Even if the gene is naturally occurring in the species, it does not exist in the genome of the host plant line or individual to which the gene is introduced, or (iii) in the genome of the species, line or individual of the host plant to which the gene is introduced. It means that the MLX56 family protein gene is in a state of being excised or isolated from a naturally occurring site.
  • a plant having an exogenous MLX56 family protein gene is a transformed plant having a heterologous MLX56 family protein gene or having an additional MLX56 family protein gene.
  • the exogenous MLX56 family protein gene can be introduced into plants by genetic engineering.
  • any method used for plant transformation for example, Agrobacterium method, particle gun method, electroporation method, whisker method, polyethylene glycol (PEG).
  • Method, microinjection method, protoplast fusion method and the like can be used. Details of these plant transformation methods can be found in general textbooks such as “Transformation Protocol” (2012) edited by Yutaka Tabei, and Horsch et al., "A Simple and General Method for Transferring Genes into”. Plants. "Science, (1985) 227 (4691): 1229-1231, and Sun et al.," A highly Efficient Transformation Protocol for Micro-Tom, a Model Cultivar for Tomato Functional Genomics. "Plant Cell Physiol., 2006 ) 47, 426-431, etc. can be referred to.
  • a vector in which the MLX56 family protein gene is incorporated in the T-DNA region of a vector suitable for the Agrobacterium method (usually a binary vector) is used as an appropriate Agrobacterium, for example, Agrobacterium.
  • Agrobacterium for example, Agrobacterium.
  • -It may be introduced into Agrobacterium tumefaciens by an electroporation method or the like, and Agrobacterium carrying the vector may be inoculated into plant cells, curls, leaf sections, leaflets and the like to infect them.
  • the T-DNA region is a region sandwiched between the right border sequence (RB) and the left border sequence (LB) existing in the extrachromosomal vector of Agrobacterium, and is a plant transformation by Agrobacterium.
  • Suitable Agrobacterium includes, but is not limited to, strains such as LBA4404, GV3101, C58, C58C1Rif (R) , EHA101, EHA105, and AGL1.
  • the MLX56 family protein gene in the T-DNA region is integrated into the genome in the plant cell, and transformed plant cells can be obtained.
  • the term "genome” simply means a nuclear genome in principle.
  • sections such as plant leaves may be used, or protoplasts may be prepared and used (Christou P, et al., Bio / technology (1991) 9: 957-962).
  • a gene transfer device for example, PDS-1000 (BIO-RAD), etc.
  • PDS-1000 BIO-RAD
  • the operating conditions can usually be performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.
  • the MLX56 family protein gene is preferably introduced into the plant genome (nuclear genome).
  • the MLX56 family protein gene may be introduced into the chloroplast genome of a plant.
  • a transformation vector or expression cassette suitable for introduction into the chloroplast genome it is preferable to use a transformation vector or expression cassette suitable for introduction into the chloroplast genome.
  • Such transformation vectors or expression cassettes preferably contain the MLX56 family protein gene under the control of promoters and terminators suitable for expression in the chloroplast.
  • Promoters suitable for expression in chloroplasts include, but are not limited to, rrn promoter, psbA promoter, rrn-T7g10 and the like.
  • terminators suitable for expression in chloroplasts include, but are not limited to, psbA terminator, rsp16 terminator, and rbcL terminator.
  • Such a transformation vector or expression cassette preferably contains two homologous sequences derived from the chloroplast genome (eg, the region around trnI-trnA) so as to sandwich the introduction sequence of the MLX56 family protein gene or the like.
  • the gene of interest can be efficiently introduced into the chloroplast genome by homologous recombination with the chloroplast genome based on their homologous sequence in the transformation vector or expression cassette. Numerous combinations of homologous sequences from the chloroplast genome have been reported that can be used for such transformation.
  • the MLX56 family protein gene is introduced into plants in an expressible state.
  • the MLX56 family protein gene is preferably under the control of a plant-functioning promoter (plant expression promoter) in the T-DNA region and in the integrated plant.
  • the MLX56 family protein gene is preferably operably linked to a plant expression promoter (typically downstream of the plant expression promoter).
  • the plant expression promoter is not limited to the following, but may be a constitutive promoter, a tissue-specific promoter, a time-specific promoter, an injury (eg, disease or insect damage) -inducible promoter, or the like. It may be an inducible promoter. Constitutive promoters can constitutively induce systemic gene expression.
  • a tissue-specific promoter or a time-specific promoter can induce gene expression specifically in a specific tissue or time, and in the present invention, it is particularly preferable to be able to induce gene expression in leaves.
  • Injury-inducing promoters can induce gene expression depending on injury stimuli such as insect damage.
  • a constitutive promoter for the purpose of constantly imparting thrips resistance to plants.
  • the "promoter” means a DNA sequence capable of inducing the expression of a gene under its control, and may further include an enhancer sequence or the like.
  • the plant expression promoter used to induce the expression of the MLX56 family protein gene is not limited to the following, but is, for example, the E12 ⁇ promoter (Mitsuhara et al. (1996) Plant Cell Physiol. 37: 49-59). , CaMV35S promoter, ubiquitin promoter, tobacco PR1a promoter, tobacco PI2 promoter, NCR promoter, ADH promoter, NOS promoter, CaMV35Sp / ADH5'-UTR and the like.
  • the E12 ⁇ promoter, CaMV35S promoter, ubiquitin promoter, NCR promoter, ADH promoter, NOS promoter, and CaMV35Sp / ADH5'-UTR are constitutive promoters.
  • the tobacco PR1a promoter is a disease-inducing promoter
  • the tobacco PI2 promoter is a pest-inducing promoter.
  • the E12 ⁇ promoter is the El2 enhancer (5'upstream region of cauliflower mosaic virus 35S promoter -419 to -90 sequence)-(cauliflower mosaic virus 35S promoter -1 to -90 sequence)- ⁇ (tobacco mosaic virus genome). It is a highly expressed promoter having the composition of ⁇ sequence) in the 5'untranslated region of RNA.
  • the promoter for plant expression is a high expression promoter (overexpression promoter).
  • the promoter for plant expression for inducing the expression of the MLX56 family protein gene is preferably not a natural promoter of the MLX56 family protein gene, and may be a heterologous promoter for the MLX56 family protein gene.
  • the plant expression promoter may be used in combination with various terminators (although not limited to, a plant expression terminator is more preferable).
  • the vector or expression cassette used to introduce the MLX56 family protein gene into plants is the expression-stabilizing sequence ASR (anti-silencing region; Kishimoto et al., PLoS One. 2013; 8 (1): e54670. Doi: 10.1371 / journal.pone.0054670) may be included.
  • the MLX56 family protein gene may be introduced into plants with the expression stabilizing sequence ASR. In that case, the MLX56 family protein gene is preferably introduced into the plant under the control of ASR, and the MLX56 family protein gene is preferably operably linked to ASR.
  • the plant into which the MLX56 family protein gene is introduced may be any plant that thrips feeds (thrips parasite).
  • the plant into which the MLX56 family protein gene is introduced may be a monocotyledonous plant or a dicotyledonous plant.
  • Examples of plants into which the MLX56 family protein gene is introduced include, but are not limited to: Gramineae plants (wheat such as wheat and barley, rice, corn, grass, etc.) Legumes (dice, green beans, sardines, adzuki beans, pea, broad beans, chickpeas, etc.) Solanaceae plants (tomatoes, eggplants, peppers, peppers, tobacco, potatoes, etc.) Cucurbitaceae plants (cucumber, melon, pumpkin, watermelon, etc.) Cruciferous plants (cabbage, rapeseed, rapeseed, mustard (Brassica), etc.) Malvaceae plants (cotton, okra, etc.) Amaryllidaceae plants (onions, leeks, garlic, etc.) Rosaceae plants (strawberry, pear, peach, apple, apricot, plum, plum, cherry, rose, etc.) Rutaceae plants (citrus fruits such as
  • the plant into which the MLX56 family protein gene is introduced is not a mulberry plant (mulberry).
  • Plant cells, callus, leaf sections, or leaflets into which the MLX56 family protein gene has been introduced are cultured in a selective medium according to, for example, a conventionally known plant tissue culture method, and the surviving callus is subjected to a redifferentiation medium (appropriate concentration).
  • plant hormones including auxin, cytokinin, gibberellin, absidic acid, ethylene, or brushnolide
  • plants transformed by the introduction of the MLX56 family protein gene can be regenerated.
  • a transformed (transgenic) plant into which the MLX56 family protein gene has been introduced can be produced as described above.
  • Whether or not the MLX56 family protein gene has been reliably introduced into the plant may be confirmed by using a nucleic acid amplification method such as PCR method, a Southern hybridization method, a Northern hybridization method, a Western blotting method, or the like.
  • a nucleic acid amplification method such as PCR method, a Southern hybridization method, a Northern hybridization method, a Western blotting method, or the like.
  • genomic DNA extracted from the leaves of transformed plants or cDNA reverse-transcribed from total RNA may be amplified by PCR or the like using a primer specific to the MLX56 family protein gene.
  • the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a clear band to the plant.
  • the introduction of the MLX56 family protein gene can be confirmed. It is also possible to bind the amplification product to a solid phase such as a microplate and detect the amplification product by fluorescence, enzymatic reaction or the like.
  • thrips resistance can be imparted to the plant.
  • the present invention also provides a method for conferring thrips resistance on a plant, which comprises introducing the MLX56 family protein gene into the plant described above.
  • the present invention also provides the use of the MLX56 family protein gene to confer thrips resistance on plants by gene transfer.
  • the transformed plant into which the MLX56 family protein gene has been introduced After introducing the MLX56 family protein gene into the plant, to determine whether the transformed plant into which the MLX56 family protein gene has been introduced has thrips resistance and, in some cases, to determine the degree of thrips resistance acquired. It is preferable to test the thrips resistance of the transformed plant. Azamiuma resistance in transformed plants into which the MLX56 family protein gene has been introduced is enhanced (preferably statistically significantly) compared to comparable control plants except that they have not been introduced with the MLX56 family protein gene. If so, the test plant (transformed plant) is shown to be resistant to thistle horse.
  • Azamiuma resistance can be tested using a plant after the introduction of the MLX56 family protein gene, for example, a transformed plant cell or a transformed plant obtained by the gene transfer, or a part thereof.
  • a plant into which the MLX56 family protein gene has been introduced and regenerated or a progeny plant thereof is maintained for a certain period of time (for example, 2 weeks) in the presence of the azalea under conditions in which the azalea cannot escape.
  • the level of thrips feeding damage to the test plant was observed and improved (preferably statistically significantly) compared to the thrips feeding damage level in the control plant. If so, it can be determined that the test plant (transformed plant) has thrips resistance.
  • the feeding damage level may be judged from the appearance when there is a large difference, but may be judged by comparing the feeding damage areas in the leaves, for example.
  • the surviving number of thrips is counted and compared to the surviving number of thrips in control plants.
  • the test plant can reduce the survival number of thrips that feed on it, i.e. it is determined to be thrips resistant. be able to. For example, 20 thrips were released in a closed space containing plants and maintained at 25 ⁇ 1 ° C for 2 weeks under the conditions of 14 hours light period / 10 hours dark period, and then thrips (larvae, pupae, adults) in the test plants. If the surviving number of (including) is reduced by 10% or more, preferably 20% or more, as compared with the surviving number of thrips in the control plant, it can be judged that the test plant has thrips resistance.
  • the thrips resistance of the transformed plant obtained by gene transfer can be tested, and the transformed plant having thrips resistance can be selected.
  • Thrips means an insect belonging to the order Thrips (Thysanoptera).
  • Thrips Thysanoptera
  • Thripidae are Thripidae, Thripidae (Aeolothripidae), Thripidae (Merothripidae), Thripidae (Phlaeothripidae), Thripidae (Fauriellidae), Thripidae (Fauriellidae), Thripidae (Fauriellidae) It may be a family (Uzelothripidae), but is not limited to these.
  • Thrips include, for example, Thrips, Anaphothrips, Chirothrips, Frankliniella, Fulmekiola, Hydatothrips, and Syltoslips.
  • Sericothrips Taeniothrips, Yoshinothrips, Helionothrips, Arrhenothrips, Ecacanthothrips, Ecacanthothrips
  • the genus Haplothrips the genus Holothrips, the genus Liothrips, the genus Litotetothrips, the genus Mychiothrips, the genus Oidanothrips, the genus Pentagonothrips.
  • Ponticulothrips Psalidothrips, Bactrothrips, or Gastrothrips, but is not limited to these.
  • thrips examples include Thrips flavus, Thrips tabaci, Thrips nigropilosus, Thrips coloratus, Thrips coloratus, Thrips thrips, Thrips thrips, Thrips thrips, Thrips thrips, Thrips thrips, Thrips thrips, Thrips thrips, Thrips thrips ), Thrips thrips (Thrips setosus), Thrips alliorum, Thrips thrips (Anaphothrips obscurus), Thrips palmi Karny (Chirothrips manicatus), Thrips palmi Karny (Chirothrips manicatus), Thrips palmi Karny ), Kahonkahana thrips (Frankliniella tenuicornis), Satoukibichibia thrips (Fulmekiola serrata), Haraobia thrips (Hydatoth
  • Transformed plants (P generation) into which the MLX56 family protein gene has been introduced and confirmed to have thrips resistance are self-mated, seeds are collected, and next-generation plants (F1 plants or later generation plants) are cultivated. You may.
  • Next-generation plants may have the MLX56 family protein gene heterozygous or homozygous in the nuclear genome.
  • next-generation plants may have some chloroplasts transformed with the MLX56 family protein gene (heteroplasmy), all or most. All chloroplasts may be transformed with the MLX56 family protein gene (homoplasmy).
  • the MLX56 family protein genes, whether heterozygous or homozygous, can result in thrips resistance.
  • Transformed plants carrying the MLX56 family protein gene can be propagated by sexual reproduction (autologous or allogeneic) or asexual reproduction (vegetative reproduction, clonal reproduction, etc.).
  • sexual reproduction autologous or allogeneic
  • asexual reproduction vegetative reproduction, cloning
  • a plant directly obtained by introducing the MLX56 family protein gene using a transformation technique.
  • a plant produced by (cultivation, etc.) is called a progeny plant.
  • Offspring plants that inherit the exogenous MLX56 family protein gene from P generation plants are also included in the range of transformed plants that carry the MLX56 family protein gene, and the exogenous MLX56 family protein gene introduced into P generation plants is , It is also called an exogenous MLX56 family protein gene in offspring plants that inherit it.
  • the present invention comprises mating using a lizard-resistant transformed plant having an exogenous MLX56 family protein gene as a breeding parent to obtain a progeny plant and selecting a progeny plant having the MLX56 family protein gene. Breeding methods for resistant plants are also provided.
  • "Mating using a transformed plant according to the present invention as a breeding parent” means that the transformed plant according to the present invention is used for the purpose of introducing an exogenous MLX56 family protein gene in a transformed plant resistant to horse mackerel into a progeny plant. It means that plants are crossed with each other (self-mating), or the transformed plant according to the present invention is crossed with a plant of the same species, a different strain or a closely related species. Mating may be performed once or repeatedly.
  • the transformed plant according to the present invention is crossed with a plant of the same species or a closely related species (repeated parent), the progeny plant is mated with the repetitive parent (backcross), and the progeny plant is further mated with the repetitive parent. May be repeated (continuous backcrossing).
  • the transformed plant according to the present invention may be crossed with a plant of the same species or a closely related species, and the progeny plant thereof may be crossed with another plant of the same species or a closely related species.
  • Selection of progeny plants having the MLX56 family protein gene can be performed by detecting the MLX56 family protein gene or its gene product (mRNA or MLX56 family protein) in the plant.
  • progeny plants having the MLX56 family protein gene it is also preferable to test thrips resistance of progeny plants having the MLX56 family protein gene and select progeny plants having thrips resistance.
  • By obtaining offspring plants in this way and selecting offspring plants having the MLX56 family protein gene it is possible to breed plants having the Azamiuma resistance conferred by the MLX56 family protein gene.
  • the present invention also provides a method for controlling thrips, which comprises feeding thrips a transformed plant resistant to thrips having an exogenous MLX56 family protein gene.
  • the present invention also relates to a method for controlling thrips, which comprises cultivating a thrips-resistant transformed plant having a exogenous MLX56 family protein gene in a thrips distribution area.
  • the thrips-resistant transformed plant having the exogenous MLX56 family protein gene according to the present invention can also be used as a counter plant for thrips control.
  • the present invention also provides a thrips control agent containing a thrips-resistant transformed plant having an exogenous MLX56 family protein gene or an MLX56 family protein isolated from the transformed plant.
  • the thrips control agent containing the plant body or seed of the transformed plant according to the present invention can be used as a counter plant for controlling thrips by reducing the population of thrips.
  • the thrips control agent containing the transformed plant according to the present invention for example, a part of the plant body (leaves, stems, etc.) or the MLX56 family protein isolated from the plant, can be thrips by a method such as sowing on farmland. It can be used to reduce the abundance of thrips.
  • the MLX56 family protein may be extracted from the transformed plant according to the present invention.
  • the MLX56 family protein expressed and accumulated in the transformed plant according to the present invention is sugar chain-modified.
  • the thrips control activity of the MLX56 family protein according to the present invention is preferably 10% or more, more preferably 10% or more, in terms of the number of thrips inhabiting the thrips by feeding the protein, as compared with the control group not given the MLX56 family protein. Can result in a reduction of 20% or more.
  • the term "plant” refers to various growth stages and parts of a plant, such as plant body (whole plant), stem, leaf, root, flower, bud, fruit (flesh, pericarp), shoot, seed, tissue, cell, And crows etc. are basically included.
  • the transformed plant according to the present invention may be a transformed plant cell, a transformed plant, or a part thereof (leaves, etc.).
  • the term "plant” in the present invention may refer to a plant, a part of a plant, or a cell or tissue depending on the context, but those skilled in the art can easily understand the meaning. ..
  • the present invention by introducing the MLX56 family protein gene into a plant, it is possible to efficiently enhance the thrips resistance of the plant as compared with the conventional breeding method.
  • Example 1 Construction of MLX56 expression plasmid vector and introduction into Agrobacterium
  • the binary vector pEL2 ⁇ -MCS (Ohtsubo, N., et al., (1999) Plant Cell) Physiol. 40: 808-817) was used.
  • pEL2 ⁇ -MCS contains a multicloning site (MCS) downstream of the E12 ⁇ promoter (Mitsuhara et al. (1996) Plant Cell Physiol. 37: 49-59) and contains a kanamycin resistance gene as a selectable marker gene.
  • the petiole of the mulberry variety Shinichinose (Morus alba L, cv. Shin-Ichinose) (produced in Tsukuba City, Ibaraki Prefecture) was cut, the emulsion secreted from the wound was recovered, and the total RNA was isolated from the emulsion.
  • CDNA was synthesized from total RNA (1 ⁇ g) using oligo-dT-Adapter primers (Takara Bio Inc., Kyoto, Japan) and TaKaRa One Step RNA PCR Kit (AMV) (Takara Bio Inc.).
  • nucleic acid amplification is performed by PCR using primers MLX56 ORF51 (5'-aattTCTAGAatgaagtttagaactcttttaatc-3'; SEQ ID NO: 3) and MLX56 ORF31 (5'-tataGAGCTCttacattcgagcaacttccga-3'; SEQ ID NO: 4).
  • MLX56 ORF51 5'-aattTCTAGAatgaagtttagaactctttttaatc-3'; SEQ ID NO: 3
  • MLX56 ORF31 5'-tataGAGCTCttacattcgagcaacttccga-3'; SEQ ID NO: 4
  • the obtained amplified fragment and the binary vector pEL2 ⁇ -MCS were cleaved with Xba1 and Sac1, and the cleaved ends were ligated with DNA ligase and introduced into competent cells of Escherichia coli JM109 strain.
  • E. coli colonies carrying the recombinant plasmid were selected based on kanamycin resistance, the plasmid was isolated, the inserts in the plasmid were sequenced, and the MLX56 gene ORF sequence (SEQ ID NO: 1) under the control of the E12 ⁇ promoter in the vector. Selected clones that were correctly inserted.
  • the MLX56 expression binary plasmid vector thus obtained was named pEL2 ⁇ :: MLX56.
  • the MLX56 gene containing the nucleotide sequence shown in SEQ ID NO: 1 encodes the MLX56 protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
  • a schematic diagram of the structure of the T-DNA region of pEL2 ⁇ :: MLX56 is shown in FIG.
  • Example 2 Preparation of transgenic tomatoes The transformation in this example was obtained from the National Bio-Resource Center (NITE (NBRC); Japan), an independent administrative agency. Tomatoes (Solanum lycopersicum; variety name Micro-Tom) were used.
  • NITE National Bio-Resource Center
  • Micro-Tom Micro-Tom
  • the seeds are placed in a seeding medium and placed in a culture room at 25 ° C. for 7 to 10 days under 16-hour light period / 8-hour dark period conditions. It was cultured, germinated and grown. The cotyledon was cut out using a sterilized scalpel, the petiole and the tip were removed, and the cotyledon was divided into two parts perpendicular to the middle rib (cotyledon section) and subjected to subsequent transformation.
  • the cotyledon slices were picked up from the bacterial cell suspension with tweezers, placed on a sterile Kim towel to absorb the bacterial solution, placed in a coexisting medium, and co-cultured with Agrobacterium at 25 ° C in the dark. went.
  • the cotyledon slices were transplanted into a callus-inducing medium containing an antibiotic and cultured at 25 ° C. under 16-hour light period / 8-hour dark period conditions.
  • the medium was changed every 7 to 14 days, and the callus formed from the cotyledon section was transplanted to shoot-forming medium 1 in about 3 weeks, and the culture was continued for another 10 to 14 days, and then transplanted to new shoot-forming medium 1 again. Incubated for 10-14 days. Then, it was transplanted to shoot-forming medium 2, the medium was changed every 10 to 14 days, and when adventitious buds grew, it was transplanted to rooting medium. Plants whose rooting was observed by culturing in a rooting medium were extracted from the medium, transplanted to culture soil, and grown in a culture room. In this way, the transformed tomato plant could be regenerated.
  • the medium composition used is shown in Table 1 below.
  • Example 3 Confirmation of expression of transgene The confirmation of expression of the transgene (MLX56 gene) in the transformed tomato plant is carried out according to the method described in Kawazu et al. (2012) Arthropod-Plant Interactions, 6: 221-230. It was conducted. Specifically, first, about 100 mg of upper-developed leaves were cut from the plant individuals regenerated according to Example 2, and frozen in liquid nitrogen. Frozen leaf samples were crushed using a mortar and pestle cooled with liquid nitrogen, and then total RNA was extracted. Extraction of total RNA was performed using the TRIzol (R) reagent (Thermo Fisher Scientific) and according to the manufacturer's instructions. After extraction, the RNA-containing pellet obtained by isopropanol precipitation was dissolved in TE buffer.
  • RNA was obtained total RNA as a template.
  • a reverse transcription reaction was carried out using the iScript TM cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions, and complementary DNA (cDNA) was synthesized (first-strand DNA).
  • cDNA complementary DNA
  • qRT-PCR and fluorescence detection were performed using a CFX96 real-time PCR analysis system (Bio-Rad) using first-stranded DNA as a template.
  • MLX56RT51 5'-CCAAGTCCACCTCCACCAAGTC-3'(SEQ ID NO: 5) and MLX56RT31: 5'-TTTCCGAGGGCTCTTCCACATC-3' (SEQ ID NO: 6), and premix iQ TM SuperMix (Bio) -Rad) was used.
  • QRT-PCR and fluorescence detection using tomato actin gene sequence detection primers (LeActinRT51: 5'-CCAGGTATTGCTGATAGAATGAG-3'(SEQ ID NO: 7) and LeActinRT31: 5'-GAGCCTCCAATCCAGACAC-3' (SEQ ID NO: 8)) as internal standards.
  • the relative value of the MLX56 gene measurement to the actin gene measurement was calculated, thereby expressing the amount of MLX56 mRNA in the leaf sample.
  • MLX56-69 Two lines of transformed tomatoes expressing the introduced MLX56 gene particularly strongly were obtained and named MLX56-69 and MLX56-73.
  • MLX56-69 the expression level of MLX56 mRNA was about 3 times that of the tomato actin gene
  • MLX56-73 the expression level of MLX56 mRNA was about 5 times that of the tomato actin gene (Fig. 2). ..
  • the expression level of the MLX56 gene was also confirmed in the plurality of next-generation plant individuals acquired in Example 4 described later.
  • Example 4 Detection of MLX56 protein
  • the accumulation of MLX56 protein in transformed tomato plants was confirmed by utilizing the chitin binding property of MLX56 protein.
  • the transformed tomato MLX56-73 obtained in Example 3 was self-mated to obtain a next-generation plant.
  • an extract solution (20 mM Tris-HCl (pH 9.5) and protease inhibitors cOmplete, Mini (Roche).
  • Addition in the amount according to the above; the same applies hereinafter in this example) 800 ⁇ L was added and ground with a dairy pot and a dairy stick.
  • the obtained grinding solution was centrifuged at 12,000 rpm for 5 minutes to remove insoluble matter, and the supernatant was collected to prepare a crude protein extract (Total).
  • transformed tomatoes variants of microtoms having the firefly luciferin gene under the control of the same promoter E12 ⁇ (Ueda et al., (2016) Journal of Plant Interactions, Vol.14, No.1, 73-78) 200 mg of the upper-developed leaves of Tomato was collected, and a crude protein extract was obtained in the same manner. Unless otherwise specified, leaf samples and extracts were treated in an ice-cooled state.
  • Example 5 Thrips resistance test The thrips resistance of the next-generation plant individuals of the transformed tomato strains MLX56-69 and MLX56-73 obtained in Examples 2 and 3 was examined. As a control, a transformed tomato (cultivar Microtom) having the firefly luciferin gene under the control of the promoter E12 ⁇ , which was the same as that used in Example 4, was used. Fifteen individuals were tested for each transformed tomato strain.
  • FIG. 5 shows a representative photograph of the appearance of the transformed tomato after the test period of 2 weeks.
  • the control plants were significantly damaged by thrips, but the MLX56-69 and MLX56-73 strains were clearly less damaged. It was shown that by introducing the MLX56 gene into plants, the resistance of thrips in plants can be significantly increased and the feeding damage caused by thrips can be reduced.
  • Example 6 Tick resistance test (comparative example) The tick resistance of transformed tomato strains MLX56-69 and MLX56-73, as well as the same control transformed tomato plants used in Example 5, was examined.
  • FIG. 6 shows a representative photograph of the appearance of the transformed tomato 26 days after inoculation.
  • MLX56-69, MLX56-73, and control plants were all found to be severely harmed by spider mites. Transduction of the MLX56 gene into plants has been shown to result in mite resistance.
  • Example 7 Conferring resistance to Thrips palmi Karny by transient expression of MLX56 gene in cucumber
  • transient expression of MLX56 gene in cucumber was induced to thrips thrips. We examined whether or not resistance could be imparted to cucumbers.
  • the plasmid vectors include pBE2113-GUS (control vector; Mitsuhara et al. 1996 Plant Cell Physiol. 37: 49-59) and pEL2 ⁇ -MCS (Ohtsubo, N. et al.,) Which is a modified vector of pBE2113-GUS. (1999)
  • Each plasmid vector was introduced into the Agrobacterium tumefaciens C58C1 strain by the electroporation method to prepare two types of Agrobacterium. Culturing of Agrobacterium, induction of infectivity, infection to plants, and transient expression were basically carried out according to the method described in Kawazu et al., Plant Biotechnology 29, 495-499 (2012).
  • the two types of Agrobacterium produced were cultured overnight, and after collecting each of them, the acetosyringone-containing buffer solution described in Kawazu et al., Plant Biotechnology 29, 495-499 (2012).
  • Suspension in (10 mM MES (pH 5.6), 10 mM MgCl 2 , 20 ⁇ M acetosyringone) at OD 600 0.2 and allowing to stand at room temperature for 3 hours induces infectivity of Agrobacterium and agro A bacterial suspension was prepared.
  • the unfolded 3rd leaf of cucumber (variety name: ZQ-7, growth stage: 5 weeks old) was cut out, and after cutting out a leaf piece with a diameter of 1.5 cm, the surface was washed and immersed in the above Agrobacterium suspension. ..
  • the Agrobacterium suspension was infiltrated into the intercellular spaces of the leaf pieces by lowering the atmospheric pressure of the suspension and the soaked leaf pieces and then returning the pressure to normal pressure. Then, the leaf pieces were taken out, the surface Agrobacterium suspension was absorbed by a paper towel to remove it, and then air-dried at room temperature to evaporate the excess water in the intercellular spaces.
  • the above leaf pieces were placed one by one in a manger cell, and one adult female Thrips palmi was encapsulated, and then bred at 25 ° C in the light period for 16 hours and the dark period for 8 hours. Eggs laid after 3 days were visualized by trypan blue staining, and the number of eggs laid per leaf was counted. The results are shown in Table 2.
  • the group using pBE2113-GUS-introduced leaf pieces is called the control group
  • the group using pEl2Omega :: MLX56-introduced leaf pieces is called the MLX56 group.
  • a plant having thrips resistance can be produced.
  • SEQ ID NO: 1 MLX56 ORF
  • SEQ ID NO: 2 MLX56 protein
  • SEQ ID NO: 3 Primer SEQ ID NO: 4: Primer SEQ ID NO: 5: Primer SEQ ID NO: 6: Primer SEQ ID NO: 7: Primer SEQ ID NO: 8: Primer SEQ ID NO: 9: First chitin binding domain
  • SEQ ID NO: 10 Extending Domain
  • SEQ ID NO: 11 Second Chitin Binding Domain

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Abstract

La présente invention concerne un procédé de production d'une plante transformée résistante aux thrips, le procédé consistant à : introduire un gène d'une protéine de la famille des MLX56 dans une plante ; et mesurer la résistance aux thrips de la plante transformée dans laquelle le gène a été introduit.
PCT/JP2020/031698 2019-08-23 2020-08-21 Procédé de production de plante résistante aux thrips Ceased WO2021039659A1 (fr)

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Citations (3)

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BRPI0808895A2 (pt) * 2007-03-02 2014-09-02 Nat Inst Of Aglobiological Sciences Proteína de resistência a insetos derivadas de uma planta, gene de resistência a insetos, vetor recombinante, célula hospedeira, c~elula de planta, transformante, método para produzir um transformante, proteína e agente de resistência a insetos

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JP2007000031A (ja) * 2005-06-22 2007-01-11 Matsushita Electric Works Ltd 生物誘引システム及びこれを用いて行う生物誘引方法
JP2011528018A (ja) * 2008-07-17 2011-11-10 ビーエーエスエフ ソシエタス・ヨーロピア 害虫駆除用のアゾリン−２−イルアミノ化合物
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