CN112708603B - Application of rice ARE2 gene in regulation of nitrogen metabolism in plants - Google Patents

Abstract

The invention discloses application of a rice ARE2 gene in regulation and control of plant nitrogen metabolism. The invention provides application of ARE2 protein in promoting plant precocity and/or shortening plant growth period. The invention also provides application of the ARE2 gene in breeding transgenic plants with early maturity and/or shortened growth period. The invention also provides an application of the ARE2 protein: regulating and controlling the tolerance of the plant to low nitrogen stress; regulating and controlling the nitrogen metabolism process of plants; affecting the balance between plant nitrogen metabolism and stress response. The inventor researches and discovers that: the rice ARE2 gene is a key factor for regulating and controlling the nitrogen metabolism process of plants; the are2 mutation inhibits the bad shape caused by nitrogen assimilation deficiency, and increases the tolerance of the rice to nitrogen deficiency stress to a certain extent; the over-expression ARE2 gene has the effect of shortening the growth period, promotes the early maturing of rice and is one of effective strategies for rice breeding in high latitude areas; the ARE2 gene encodes a ppGpp hydrolase. Therefore, the full exploitation and scientific application of the ARE2 gene have potential application value in future breeding work.

Description

Application of rice ARE2 gene in regulation of nitrogen metabolism in plants

technical field

The invention belongs to the field of biotechnology and relates to the application of rice ARE2 gene in the regulation of plant nitrogen metabolism.

Background technique

Nitrogen is one of the essential elements for plant growth and development. It is an important part of nucleic acids, proteins, chlorophyll and hormones. It participates in most physiological and biochemical processes in plants and is very important for plant growth and development. People often increase crop yield by applying a large amount of nitrogen fertilizer. Due to the limited ability of crops to use nitrogen, excessive fertilization will not continue to increase yield, but will cause environmental pollution and waste of resources, endanger human health, destroy ecological balance, and produce a "diminishing return" effect. . Faced with population, resource and environmental issues, the Consultative Group on International Agricultural Research (CGIAR) proposed the Second Green Revolution (Second Green Revolution), which aims to increase crop yields by improving nutrient use efficiency. Improving the nitrogen use efficiency (NUE) of crops is one of the important links to achieve sustainable agricultural development. The cultivation and promotion of high nitrogen use varieties can effectively reduce nitrogen fertilizer application and reduce environmental pressure, while meeting the population's demand for food. need.

Plant nitrogen use efficiency is affected by environmental and genetic factors and their interactions. Generally speaking, the main nitrogen source absorbed and utilized by plants is soil inorganic nitrogen. Factors such as temperature, humidity and soil pH affect the uptake of inorganic nitrogen by plants, and plants show preferences for different forms of nitrogen sources in different environments. For example, plants grown in drylands (aerobic soils with high pH) mainly absorb nitrate nitrogen (NO ₃ ⁻ ), while plants grown in paddy fields (reducing soils with low pH) mainly absorb ammonium nitrogen (NH ₄ ^{+ ).} ). With the deepening of research, the key components involved in plant nitrogen metabolism have been gradually isolated and identified, and they constitute the basic nitrogen metabolism network. Plant nitrogen metabolism mainly includes three links: nitrogen uptake and transport, nitrogen assimilation and nitrogen remobilization. Plants absorb soil inorganic nitrogen through nitrate transporter (NRT) and ammonium transporter (AMT) and transport it in the body. Nitrate nitrogen is sequentially processed by nitrate reductase (NR) and nitrite. The action of root reductase (nitrite reductase, NiR) is assimilated to ammonium nitrogen. Ammonium nitrogen is assimilated into organic nitrogen through the glutamine synthetase/glutamine:2-oxoglutarate amidotransferase (GS/GOGAT) cycle, and then various organic compounds are synthesized. After plants enter the reproductive growth and maturity stage, nitrogen is gradually transported in the form of amino acids from senescent tissues to young organs and seeds for reuse and participation in yield formation. Nitrogen in this process mainly comes from leaf proteins. In addition, glutamate dehydrogenase (GDH) can catalyze both the assimilation of ammonium nitrogen and its reverse reaction, which may be an important way for plants to adapt to different nitrogen environments.

Rice is an important food crop, providing the world's population with the staple food and ensuring their basic needs. Genetic, molecular biology and biochemical studies have identified a series of important genetic loci related to nitrogen use efficiency in rice, and scientific utilization of the corresponding superior allelic variation may become an important way to improve rice yield.

Nitrogen assimilation plays an irreplaceable role in plant growth and development, and the loss of function of key genes in this process often leads to severe growth and development defects and even death.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of rice ARE2 gene in the regulation of plant nitrogen metabolism.

The present invention provides the application of ARE2 protein in promoting plant early maturity and/or shortening plant growth period.

The present invention also provides the application of the ARE2 gene in cultivating transgenic plants with early maturity and/or shortened growth period.

The present invention also provides a method for cultivating transgenic plants with early maturity and/or shortened growth period, comprising the steps of: introducing an ARE2 gene into a recipient plant to obtain a transgenic plant with early maturity and/or shortened growth period. Specifically, the ARE2 gene is introduced into the recipient plant through a recombinant vector.

The present invention also provides the application of ARE2 protein, which is as follows (c1) or (c2) or (c3):

(c1) regulating plant tolerance to low nitrogen stress;

(c2) Regulating the nitrogen metabolism process of plants;

(c3) affects the balance between plant nitrogen metabolism and stress response.

The regulation of plant tolerance to low nitrogen stress is that the expression of ARE2 protein is inhibited so that the plant tolerance to low nitrogen stress is increased.

The present invention also provides a method for cultivating a transgenic plant with increased tolerance to low nitrogen stress, comprising the steps of: inhibiting the expression of the ARE2 gene in the target plant to obtain a transgenic plant with increased tolerance to low nitrogen stress.

The present invention also provides a method for cultivating transgenic plants with increased tolerance to low nitrogen stress, comprising the steps of: mutating the ARE2 gene in the target plant as a target gene to obtain tolerance to low nitrogen stress Elevated transgenic plants.

Any of the above-mentioned plants are monocotyledonous or dicotyledonous.

Any of the above-mentioned plants are grasses. Any of the above-mentioned plants are plants of the genus Oryza sativa. Any one of the above-mentioned plants is rice. Any one of the above-mentioned plants is rice japonica. Any one of the above-mentioned plants is rice Nipponbare.

Any of the above-mentioned plants are cruciferous plants. Any of the above plants is an Arabidopsis plant. Any of the above-mentioned plants is Arabidopsis thaliana.

The present invention also protects the use of ARE2 protein as ppGpp hydrolase.

The present invention also protects the ARE2 protein. ARE2 protein was obtained from rice.

The present invention also protects the ARE2 gene. The ARE2 gene was obtained from rice.

Any one of the above ARE2 proteins is the following (a1) or (a2) or (a3) or (a4) or (a5) or (a6) or (a7):

(a1) the protein shown in SEQ ID NO: 3 of the sequence listing;

(a2) a protein derived from rice and having more than 98% identity and the same function as (a1);

(a3) a protein with the same function obtained by substituting and/or deleting and/or adding (a1) one or several amino acid residues;

(a4) a protein encoded by the DNA molecule shown at positions 1581-12019 in SEQ ID NO: 1 of the Sequence Listing;

(a5) a protein encoded by the DNA molecule shown at positions 1301-12512 in SEQ ID NO: 1 of the Sequence Listing;

(a6) a protein encoded by the DNA molecule shown in SEQ ID NO: 1 of the Sequence Listing;

(a7) A protein encoded by the DNA molecule shown in SEQ ID NO: 2 of the Sequence Listing.

Any one of the above-mentioned ARE2 genes is a DNA molecule encoding an ARE2 protein.

Any of the above-mentioned ARE2 genes is the following (b1) or (b2) or (b3) or (b4) or (b5) or (b6):

(b1) a DNA molecule whose cDNA coding region is shown in sequence 2 in the sequence listing;

(b2) The genomic DNA is the DNA molecule shown at positions 1581-12019 in Sequence 1 of the Sequence Listing;

(b3) The genomic DNA is the DNA molecule shown at positions 1301-12512 in Sequence 1 of the Sequence Listing;

(b4) DNA molecules whose genomic DNA is shown in Sequence 1 in the Sequence Listing;

(b5) a DNA molecule derived from rice and having more than 98% identity with (b1) or (b2) or (b3) or (b4) and encoding the ARE2 protein;

(b6) a DNA molecule that hybridizes to (b1) or (b2) or (b3) or (b4) under stringent conditions and encodes the ARE2 protein.

The stringent conditions were hybridization in a solution of 2×SSC, 0.1% SDS at 68°C and washing the membrane twice for 5 min each, and hybridization in a solution of 0.5×SSC, 0.1% SDS at 68°C. And wash the membrane twice, 15min each time.

The recombinant vector containing the ARE2 gene can be constructed using the existing plant expression vector.

When constructing a recombinant vector, any of the enhancing, constitutive, tissue-specific or inducible promoters can be added before the translation initiation nucleotide, either alone or in combination with other plant promoters. In addition, when constructing recombinant vectors, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be in the reading frame of the coding sequence. the same to ensure correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plants, the recombinant vector used can be processed, such as adding genes that express enzymes or light-emitting compounds that can produce color changes in plants, antibiotic markers with resistance, or marker genes for chemical resistance Wait. Considering the safety of transgenic, transformed plants can be directly screened by phenotype without adding any selectable marker gene.

The plant expression vector can specifically be a pCAMBIA1300 binary vector.

The recombinant vector may specifically be a plasmid having the DNA molecule shown in SEQ ID NO: 4 of the sequence listing.

Specifically, the recombinant vector can be a recombinant plasmid obtained by inserting the DNA molecule shown in SEQ ID NO: 4 of the sequence listing into the pCAMBIA1300 binary vector (specifically, it can be inserted between HindIII and EcoRI restriction sites).

The recombinant vector may specifically be a plasmid having the DNA molecule shown in SEQ ID NO: 6 of the sequence listing.

The recombinant vector can specifically be a recombinant plasmid obtained by inserting the DNA molecule shown in SEQ ID NO: 6 of the sequence listing into the pCAMBIA1300 binary vector (specifically, it can be inserted between the HindIII and EcoRI restriction sites).

In order to explore the key regulatory components of the plant nitrogen metabolism pathway and further analyze its genetic regulatory network, the inventors of the present invention carried out chemical mutagenesis of the abc1-1 mutant, and screened a series of abc1-1 suppressor mutants abc1repressors ( are). The are1 mutation partially restores the growth and development defect of abc1-1, indicating that the nitrogen assimilation process in plants is regulated in a complex manner. Further analysis found that the loss of ARE1 gene function has the effect of improving nitrogen use efficiency and increasing yield, and rice yield was negatively correlated with the expression level of ARE1, indicating that ARE1 is a negative regulator of nitrogen metabolism. The present invention conducts systematic and in-depth research on the representative mutant are2 on the basis of screening are series mutants, attempts to clarify the role of the ARE2 gene in the process of nitrogen metabolism, analyzes the molecular regulation mechanism of nitrogen nutrition in rice and other crops, and provides information for rice breeding. and green and efficient agriculture to provide theoretical basis and molecular strategies.

The research results of the inventors of the present invention show that: the rice ARE2 gene is a key factor regulating the nitrogen metabolism process of plants; the are2 mutation inhibits the bad shape caused by nitrogen assimilation defects, and increases the tolerance of rice to nitrogen deficiency stress to a certain extent; overexpression The ARE2 gene has the effect of shortening the growth period and promoting the early maturity of rice, which is one of the effective strategies for rice breeding in high latitudes; the ARE2 gene encodes a ppGpp hydrolase. Therefore, the full exploration and scientific application of ARE2 gene has potential application value in future breeding work.

The experiment of the present invention proves that the present invention isolates and identifies a rice nitrogen metabolism-related gene ARE2, which negatively regulates the nitrogen metabolism process of plants, the are2 mutation part improves the plant's tolerance to nitrogen deficiency stress, and the overexpression of the ARE2 gene promotes rice early maturity. The ARE2 gene encodes a ppGpp hydrolase. These results suggest that ARE2 is an effective locus for nitrogen-efficient breeding in rice. Utilizing the excellent natural variation of ARE2, or the site-directed editing of the ARE2 gene through plant genetic engineering technology, is an effective strategy for cultivating new rice varieties with high nitrogen efficiency.

Description of drawings

Figure 1. Genetic screening of abc1 are2 double mutants.

A is the grain-filling phenotype of wild type (WT), abc1 mutant, abc1 are2-1 mutant, abc1 are2-2 mutant and abc1 are2-1/abc1 are2-2 F ₁ plants. The ruler is 15 cm.

B-D are the plant height (B), tiller number (C) and relative chlorophyll content of leaves (D) of wild type, abc1 mutant, abc1 are2-1 mutant and abc1 are2-2 mutant. Values represent mean ± standard deviation, sample size ≥ 15.

** indicates that the difference reached a very significant level in the t test (P<0.01).

Figure 2 shows the phenotypic analysis of are2 single mutants.

A-B are the seedling phenotype (A) and leaf chlorophyll content (B) of wild type (Wild type, WT), are2-1 mutant and are2-2 mutant. The ruler is 2 cm. Values represent mean ± standard deviation, 3 technical replicates. ** indicates that the difference reached a very significant level in the t test (P<0.01).

C is the vegetative phase phenotype of wild type, are2-1 mutant and are2-2 mutant. The ruler is 15 cm.

D-E are the leaf phenotypes of wild type, are2-1 mutants and are2-2 mutants in reproductive growth stages. The ruler is 2 cm.

F-H are the filling stage phenotype (F) and the mature stage phenotype (G and H) of wild type, are2-1 mutant and are2-2 mutant. The ruler is 15 cm.

Figure 3 shows the tolerance analysis of are2 mutants to nitrogen starvation stress.

A-C are the phenotype (A), biomass (B) and root-shoot ratio (C) of wild type (WT) and are2-2 mutants cultured under normal conditions (control) and under nitrogen deficiency conditions for 20 days . The ruler is 2 cm. Values represent mean ± standard deviation, and the sample size is 24. ** indicates that the difference reached a very significant level in the t test (P<0.01).

Figure 4 shows the map-based cloning and genetic verification of the ARE2 gene.

A is the genetic location of the ARE2 gene. In the schematic diagram of the gene structure, black boxes, white boxes and straight lines represent exons, untranslated regions and introns, respectively. are2-1 and are2-2 mutants have base substitutions at the positions shown. M1-M5 markers are Indel 11931, Indel 12655, Indel 12713, SNP12758 and Indel 12928, respectively.

B design primers CDS-1F and CDS-1R flanking are2-1 and are2-2 mutation sites, for wild type (Wildtype, WT), abc1 mutant, abc1are2-2 mutant, abc1 are2-1 mutant Electrophoresis results of the products amplified with the complementary DNA (cDNA) of the are2-1 mutant.

C-E are wild-type, are2-2 mutants, transformants with full-length ARE2 genome sequences in are2-2 mutant backgrounds (COMP) and transformants with FLAG-tagged ARE2 coding sequences in are2-2 mutant backgrounds (ARE2 -FLAG) seedling phenotype (C and D) and leaf chlorophyll content (E). The ruler is 2 cm. Values represent mean ± standard deviation, 3 technical replicates. ** indicates that the difference reached a very significant level in the t test (P<0.01).

F-G are the vegetative growth phase phenotype (F) and the grain filling phase leaf phenotype (G) of the transformants of the wild type, are2-2 mutant and the ARE2 genome full-length sequence of are2-2 mutant background. (F) and (G) scales are 15 cm and 2 cm, respectively.

H is the grain-filling phenotype of wild-type, abc1 mutant, abc1are2-2 mutant and transformants with FLAG-tagged ARE2 coding sequence (ARE2-FLAG/abc1are2-2) in the background of abc1are2-2 mutant. The ruler is 15 cm.

Figure 5 is a phenotypic analysis of ARE2 gene overexpressing plants. A-D are the grain filling stage phenotype (A), panicle phenotype (B), leaf phenotype (C) and leaf chlorophyll content (D) of wild type (WT) and ARE2 gene overexpression plants (Overexpression, OE). ). (A-C) Scale bars are 15 cm, 2 cm, and 2 cm, respectively. (D) Values represent mean ± standard deviation, with a sample size of 24. ** indicates that the difference reached a very significant level in the t test (P<0.01).

Figure 6 is a functional analysis of the ARE2 gene.

A is the phenotype of plants grown on 1/2 MS medium without sucrose for 15 days. The control was normal culture for 15 days, and the dark treatment was normal culture for 7 days and then dark for 8 days. Similar to AtRSH1, ARE2 restores the dark-induced accelerated senescence phenotype of rsh1-1 mutants. The ruler is 1cm.

B is E. coli cultured overnight. Both AtRSH1 and ARE2 restored the slow growth phenotype of the spoT203 strain (CF4943).

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the quantitative tests in the following examples are set to repeat the experiment 2-3 times, and the results are averaged. The BAC plasmid (OSJNBb0050D18) was provided by the Arizona Genomics Institute. Arabidopsis rsh1-1 mutant seeds were provided by researcher Ben Field (Aix Marseille University). E. coli CF4941 and E. coli spoT203 (CF4943) were provided by researcher Michael Cashel (National Institutes of Health). The pCAMBIA1300 binary vector is a commercial vector with a hygromycin resistance gene. Unless otherwise specified, the wild type of rice (indicated by WT) refers to Nipponbare rice, and the wild type of Arabidopsis (indicated by Col-0) refers to the Columbia ecotype Arabidopsis thaliana. The method for detecting chlorophyll content in the examples: Weigh 10-30 mg of the sample ground in liquid nitrogen, add 2 mL of absolute ethanol, mix well, and let stand for 2 hours at 4°C in the dark. The supernatant was aspirated by centrifugation, and the absorbances A _646.8 and A _{663.2 were} measured by a spectrophotometer. C _a =13.95A _{663.2-6.88A 646.8} ; C _b =24.96A _{646.8-7.32A 663.2} ; chlorophyll content (mg/g)=(C _a +C _b )×extract volume/sample fresh weight. The relative chlorophyll content of plant leaves was measured with SPAD chlorophyll meter.

The ARE2 gene in the rice genomic DNA is described in SEQ ID NO: 1 of the Sequence Listing. In Sequence 1 of the sequence listing, from the 5' end to the 3' end direction, the 1-1300th deoxynucleotide is the upstream sequence, the 1301-12512th deoxynucleotide is the exon/intron region, the first Deoxynucleotides 12513-13012 are downstream sequences. In Sequence 1 of the sequence listing, from the 5' end to the 3' end, the 1301-1608 deoxynucleotides are the first exon (wherein the 1301-1580 deoxynucleotides are the 5' untranslated region. , the 1581-1583 deoxynucleotide is the translation initiation site), the 1609-1795 deoxynucleotide is the first intron, and the 1796-2016 deoxynucleotide is the second exon Deoxynucleotides 2017-4294 are the second intron, deoxynucleotides 4295-4480 are the third exon, and deoxynucleotides 4481-4977 are the third intron Intron, deoxynucleotides 4978-5043 are the fourth exon, deoxynucleotides 5044-5165 are the fourth intron, and deoxynucleotides 5166-5258 are the fifth Exons, deoxynucleotides 5259-5358 are the fifth intron, deoxynucleotides 5359-5490 are the sixth exon, and deoxynucleotides 5491-5624 are the sixth Introns, the 5625-5726 deoxynucleotides are the 7th exon, the 5727-5823 deoxynucleotides are the 7th intron, and the 5824-5901 deoxynucleotides are the 7th introns. 8 exons, the 5902-5978 deoxynucleotides are the 8th intron, the 5979-6050 deoxynucleotides are the 9th exon, and the 6051-7015 deoxynucleotides are The 9th intron, the 7016-7123 deoxynucleotides are the 10th exon, the 7124-7198 deoxynucleotides are the 10th intron, and the 7199-7296 deoxynucleotides It is the 11th exon, the 7297-7734 deoxynucleotide is the 11th intron, the 7735-7795 deoxynucleotide is the 12th exon, and the 7796-7888 deoxynucleoside The acid is the 12th intron, the 7889-7951 deoxynucleotide is the 13th exon, the 7952-8058 deoxynucleotide is the 13th intron, and the 8059-8115 deoxynucleus The nucleotide is the 14th exon, the 8116-8192 deoxynucleotide is the 14th intron, the 8193-8294 deoxynucleotide is the 15th exon, and the 8295-8401 deoxynucleotide Nucleotides are intron 15, deoxynucleotides 8402-8560 are exon 16, deoxynucleotides 8561-9140 are intron 16, and deoxynucleotides 9141-9272 Deoxynucleotides are exon 17, deoxynucleotides 9273-9346 are intron 17, deoxynucleotides 9347-9433 are exon 18, and deoxynucleotides 9434-9497 Deoxynucleotides at positions 9498-9569 are in the 18th intron, deoxynucleotides at positions 9498-9569 are in the 19th exon, and deoxynucleotides at positions 9570-10729 are the first 9 introns, the 10730-10816 deoxynucleotides are the 20th exon, the 10817-10882 deoxynucleotides are the 20th intron, and the 10883-11333 deoxynucleotides are The 21st exon, the 11334-11557 deoxynucleotides are the 21st intron, the 11558-11619 deoxynucleotides are the 22nd exon, and the 11620-11776 deoxynucleotides It is the 22nd intron, the 11777-11845 deoxynucleotide is the 23rd exon, the 11846-11926 deoxynucleotide is the 23rd intron, and the 11927-12512 deoxynucleoside The acid is the 24th exon (wherein the 12017-12019 deoxynucleotides are the translation termination site, and the 12020-12512 deoxynucleotides are the 3' untranslated region).

The coding frame of the ARE2 gene in the rice cDNA is shown in sequence 2 of the sequence listing.

The protein encoded by the rice ARE2 gene is shown in sequence 3 of the sequence listing.

The nucleotide sequences of the primers used in the examples are shown in Table 1.

Table 1

Example 1. Genetic screening of abc1 are2 double mutants

Previous studies have found that the rice ABNORMAL CYTOKININ RESPONSE1 (ABC1) gene encodes a key enzyme in the nitrogen assimilation process, namely reduced ferredoxin-dependent glutamate synthase (Fd-GOGAT), and the weak allelic mutation of the gene abc1 -1 led to the decrease of Fd-GOGAT enzyme activity, and the corresponding mutants showed a series of abnormal nitrogen metabolism characteristics, such as reduced plant height, reduced tillering, yellow leaf color and prolonged growth period. This gene is highly conserved in different species, and its loss-of-function mutant has a lethal phenotype at the seedling stage.

The inventors of the present invention performed EMS (Ethylmethylsulfon) chemical mutagenesis on the rice abc1-1 mutant (hereinafter referred to as abc1), and screened to obtain the suppressor mutant abc1 repressors (are) of the abc1 mutant, among which there are two double mutants The phenotypes are similar. The two are crossed, and the phenotype of the progeny (F ₁ ) plant is the same as that of the parent, indicating that they are two allelic variations of the same gene, so the inventors of the present invention named them abc1 are2-1 and abc1 are2 respectively. -2 (A in Figure 1). During the entire growth and development period, the are2 mutation could partially restore the phenotype of the abc1 mutant, including plant height, tillering and leaf color (B, C and D in Figure 1). The above results indicated that the are2 mutation partially inhibited the abnormal nitrogen assimilation phenotype of the abc1 mutant, suggesting that the are2 mutation may enhance the ability of rice to tolerate low nitrogen stress, and ARE2 is a key site involved in nitrogen metabolism in rice.

Example 2. Phenotypic analysis of are2 single mutants

The abc1 are2 double mutant was backcrossed with rice Nipponbare, and the are2 single mutant was isolated. Compared with the wild type, the are2 mutants mainly showed the characteristics of light green leaf color during the whole growth and development process (A, B, C, D, E and F in Figure 2). In addition, the are2 mutants showed higher sensitivity to low temperature due to the decrease in air temperature when rice was from late grain filling to maturity (G and H in Fig. 2). These results suggest that ARE2 is involved in the regulation of nitrogen metabolism in rice, and may affect the balance between nitrogen metabolism and stress response in rice.

Example 3. Tolerance analysis of are2 mutants to nitrogen deficiency stress

1. Preparation of rice nutrient solution

According to Yoshida et al. (1976), the nutrient solution formula was slightly adjusted to prepare mother liquors I-VI. The concentration of mother liquors I-IV and VI is 800 times, and the concentration of mother liquor V is 1000 times. Mother liquor I contains 38.56g/L ammonium sulfate and 52.72g/L magnesium sulfate (or 51.6g/L potassium sulfate and 52.72g/L magnesium sulfate, for the preparation of nitrogen-deficient nutrient solution). Mother liquor II contains 14.8g/L potassium nitrate and 47.92g/L calcium nitrate (or 10.92g/L potassium chloride and 32.42g/L calcium chloride for the preparation of nitrogen-deficient nutrient solution). Mother liquor III contained 19.84 g/L potassium dihydrogen phosphate and 12.72 g/L potassium sulfate. Mother liquor IV contained 2.288 g/L boric acid, 0.064 g/L copper sulfate pentahydrate, 0.176 g/L zinc sulfate heptahydrate, 1.488 g/L manganese chloride tetrahydrate and 0.072 g/L molybdic acid tetrahydrate. Mother liquor V contained 5.57 g/L ferrous sulfate heptahydrate and 7.45 g/L disodium EDTA. Mother liquor VI also contains 40 g/L sodium silicate. Dilute to 0.5× when using, and adjust pH to 5.5.

2. Phenotypic analysis

In order to clarify whether the are2 mutation changes the tolerance of rice to nitrogen deficiency stress, the inventors of the present invention cultivated wild-type and are2-2 mutant plants under normal conditions and nitrogen deficiency conditions, respectively, and measured the above and below ground at about 20 days. Some related physiological indicators. The following two trends were found to be stable: the biomass (indicated by fresh weight) of the are2-2 mutant was significantly lower than that of the wild type under normal conditions, whereas under nitrogen deficient conditions this trait was between the wild type and the are2-2 mutant There was no significant difference; the root-shoot ratio of the are2-2 mutant was significantly higher than that of the wild type under nitrogen deficient conditions (see Figure 3). These results indicated that nitrogen starvation stress inhibited biomass accumulation to a lower degree and promoted root growth to a higher degree in are2 mutants than in wild type, suggesting that are2 mutants have a certain tolerance to nitrogen deficiency stress.

Example 4. Map-based cloning and genetic verification of ARE2 gene

1. Construction of recombinant expression vector

ARE2 genome full-length sequence (genomic DNA, gDNA) genetic complement vector (pARE2::ARE2): using BAC plasmid (OSJNBb0050D18) as a template, with primer pairs ARE2-F/ARE2-1R and ARE2-2F/ARE2-R amplification Two fragments of the ARE2 genomic sequence were obtained. The two fragments were sequentially inserted between the SalI and KpnI restriction sites of the pCAMBIA1300 binary vector, and the recombinant plasmid was obtained after the target fragment was sequenced correctly.

Genetic complementation vector with FLAG tag (pARE2::ARE2-FLAG-NOS): Using rice genomic DNA as a template, the primer pair pARE2-F/pARE2-R was used to amplify the ARE2 promoter (ARE2 native promoter, pARE2, namely Deoxynucleotides 1-1580 in SEQ ID NO: 1) fragment. The ARE2 coding sequence (CDS, ie the 1-2676th deoxynucleotide in sequence 2) was obtained by amplifying the ARE2CDS-F/ARE2CDS-R primer pair with rice complementary DNA (cDNA) as the template. ARE2 promoter, ARE2 coding sequence, FLAG tag sequence and NOS terminator sequence were inserted in sequence between the HindIII and EcoRI restriction sites of the pCAMBIA1300 binary vector, and the recombinant plasmid was obtained after correct sequencing of the target fragment.

2. Genetic mapping and phenotypic analysis of transformed strains

In order to determine the nature of the are2 mutation, the inventors crossed the are2 mutant with the wild type, the first generation (F ₁ ) plants all showed the wild type phenotype, and the second generation (F ₂ ) population appeared trait segregation, and the wild type phenotype and The ratio of plants with mutant phenotypes was close to 3:1, indicating that are2 is a single nuclear gene recessive mutation (see Table 2). The are2-1 mutant was crossed with the indica variety Nanjing 6 to construct an F ₂ mapping population, and individual plants with the are2 mutant phenotype in the population were selected for map-based cloning. Using simple sequence repeats (SSR), insertion-deletion polymorphism (InDel) and single nucleotide polymorphism (SNP) molecular markers, the ARE2 candidate gene was located at No. 3 Within a 103-kb segment of the long arm of the chromosome, this segment contains 11 functionally annotated open reading frames (ORFs). PCR amplification and sequencing of this segment revealed that only one gene (LOC_Os03g22160) was mutated in the are2 mutant. In the are2-1 mutant, the G mutation at the first position of the eighth intron of the gene is A; in the are2-2 mutant, the G mutation at the fifth intron of the gene is A (see A in Figure 4). Both of these mutations caused the splicing disorder of the ARE2 gene transcript, resulting in a partial loss of its function (see Figure 4B).

Table 2 Genetic analysis of are2 mutations

To verify the correctness of the candidate gene, the inventors transferred the full-length genome sequence driven by the promoter of the candidate gene and the coding sequence with the FLAG tag into the are2-2 single mutant for genetic rescue. The transformants exhibited a wild-type phenotype and normal leaf color throughout the growth period (C, D, E, F and G in Figure 4), indicating that the exogenous ARE2 gene restored the phenotype of the are2-2 single mutant. In addition, the transformant with the FLAG-tagged ARE2 coding sequence (ARE2-FLAG) in the are2-2 mutant background was used as the male parent, and the abc1 are2-2 double mutant was used as the female parent for hybridization, and abc1 are2- 2 Transformants with a double mutant background and carrying the ARE2-FLAG transgene exhibited the abc1 mutant phenotype (H in Figure 4), indicating that the exogenous ARE2 gene restored the abc1 are2-2 double mutant phenotype.

The above results indicate that the LOC_Os03g22160 gene is the ARE2 gene.

Example 5. Phenotypic analysis of ARE2 gene overexpressing plants

1. Construction of recombinant expression vector

Overexpression vector (pUbi::ARE2-FLAG-NOS): insert the ubiquitin promoter (pUbi) and the coding sequence of rice ARE2 gene between the HindIII and EcoRI restriction sites of the pCAMBIA1300 binary vector. The exogenous DNA molecules of the 1-2676th deoxynucleotide in sequence 2), the FLAG tag coding sequence and the NOS terminator sequence were sequenced correctly, and a recombinant plasmid was obtained. The exogenous DNA molecule is shown in Sequence 4 of the Sequence Listing. In Sequence 4 of the sequence listing, the 1-1981 position is the ubiquitin promoter, the 1988-4663 position is the coding sequence of the rice ARE2 gene, the 4670-4696 position is the FLAG tag coding sequence, and the 4745-5009 position is the NOS terminator. . In Sequence 4 of the Sequence Listing, positions 1988-4699 form a fusion gene.

2. Preparation of transgenic plants and phenotypic analysis

1. The overexpression vector (pUbi::ARE2-FLAG-NOS) was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

2. The recombinant Agrobacterium obtained in step 1 is taken, and the embryogenic callus of rice Nipponbare is genetically transformed by the Agrobacterium dip method, and then co-culture, screening culture, differentiation culture and rooting culture are sequentially performed to obtain a T0 generation regenerated plant.

3. The T0 generation transgenic plants are selfed to obtain the T1 generation plants, and the transgenic plants are screened from the T1 generation plants (the method of screening the transgenic plants: the genomic DNA of the test plant is used as a template, and two different exons corresponding to the ARE2 gene are used. The primer pair composed of two primers is used for PCR amplification. The exogenously introduced ARE2 gene is cDNA, which is different in size from the amplification product of endogenous genomic DNA. It is judged whether it is a transgenic plant according to whether the amplification product has the expected size).

4. The T1 generation transgenic plants are selfed to obtain T2 generation plants, and the T2 generation plants are subjected to hygromycin resistance identification. If the T2 generation plants obtained by selfing a T1 generation transgenic plant have hygromycin resistance, the T1 generation transgenic plants are hygromycin resistance. The plant is a homozygous transgenic plant, and its selfed progeny is a homozygous transgenic line.

Two homozygous transgenic lines were obtained, the OE1 line and the OE2 line.

5. Phenotypic analysis

Tested plants: rice Nipponbare plants, T3 generation plants of OE1 line, T3 generation plants of OE2 line.

Under parallel conditions, the test plants were cultured normally, and the phenotypes were continuously observed. Compared with the rice Nipponbare plants, the OE1 and OE2 lines mainly showed early maturation characteristics after entering the grain filling stage, which were mainly reflected in earlier leaf senescence and faster grain maturity. As shown in Figure 5, the grains of wild-type plants have not yet begun to mature at this stage, and the overall panicle is cyan, while the grains of OE1 line plants and OE2 line plants begin to mature, and the overall panicle is yellow; compared with wild-type plants, the The OE1 line plants and the OE2 line plants had lighter leaf color and decreased chlorophyll content. The results showed that overexpression of ARE2 gene has the effect of promoting early rice maturity, that is, shortening the growth period of rice.

After rice enters the reproductive growth stage, chilling damage seriously affects rice yield. Therefore, in high latitude regions, excessive growth period is one of the important factors limiting rice yield. The effect of overexpressing ARE2 gene in shortening the growth period can make up for the limitation of the long growth period on the breeding of varieties in high latitudes to a certain extent.

Example 6. Functional analysis of ARE2 gene

1. Construction of recombinant expression vector

1. Construction of Arabidopsis genetic complementation vector

pAtRSH1::AtRSH1-FLAG-NOS vector: insert the AtRSH1 promoter, Arabidopsis AtRSH1 gene coding sequence, FLAG tag coding sequence and NOS terminator sequence between the SalI and EcoRI restriction sites of the pCAMBIA1300 binary vector. Exogenous DNA molecules, sequenced correctly, and recombinant plasmids are obtained. The exogenous DNA molecule is shown in Sequence 5 of the Sequence Listing. In Sequence 5 of the sequence listing, the 1-801 position is the AtRSH1 promoter, the 808-3459 position is the Arabidopsis AtRSH1 gene coding sequence, the 3466-3492 position is the FLAG tag coding sequence, and the 3523-3787 position is the NOS termination. son. In Sequence 5 of the Sequence Listing, positions 808-3495 form a fusion gene.

pAtRSH1::ARE2-FLAG-NOS vector: between the HindIII and EcoRI restriction sites of the pCAMBIA1300 binary vector, insert the AtRSH1 promoter, the coding sequence of the rice ARE2 gene (that is, the 1-2676 deoxynucleoside in sequence 2) acid), the FLAG tag coding sequence and the exogenous DNA molecule of the NOS terminator sequence, the sequencing was correct, and a recombinant plasmid was obtained. The exogenous DNA molecule is shown in Sequence 6 of the Sequence Listing. In Sequence 6 of the sequence listing, the 1-801 position is the AtRSH1 promoter, the 808-3483 position is the rice ARE2 gene coding sequence, the 3490-3516 position is the FLAG tag coding sequence, and the 3565-3829 position is the NOS terminator. In Sequence 6 of the Sequence Listing, positions 808-3519 form a fusion gene.

2. Construction of Escherichia coli Genetic Complementation Vector

pBAD/His A-AtRSH1 vector: insert the coding sequence of the Arabidopsis AtRSH1 gene between the PstI and KpnI restriction sites of the pBAD/His A vector (positions 808-3459 in SEQ ID NO: 5 of the sequence listing, and at the 3' end The stop codon TAA was added to obtain the pBAD/His A-AtRSH1 vector.

pBAD/His A-ARE2 vector: insert the coding sequence of the rice ARE2 gene between the KpnI and HindIII restriction sites of the pBAD/His A vector (as shown in SEQ ID NO: 2 of the sequence listing) to obtain the pBAD/His A-ARE2 vector.

2. Acquisition and identification of transgenic Arabidopsis

In order to analyze the biochemical function of the products encoded by the ARE2 gene, the inventors performed heterologous genetic rescue experiments on the Arabidopsis mutant rsh1-1. The Arabidopsis mutant rsh1-1 is the Arabidopsis ppGpp hydrolase gene loss-of-function mutant rsh1-1. The Arabidopsis mutant rsh1-1 is described in the following literature: Sugliani, M., Abdelkefi, H., Ke, H., Bouveret, E., Robaglia, C., Caffarri, S., and Field, B. (2016 ).An ancient bacterial signalingpathway regulates chloroplast function to influence growth and development in Arabidopsis. Plant Cell 28, 661-679.

1. The pAtRSH1::AtRSH1-FLAG-NOS vector or the pAtRSH1::ARE2-FLAG-NOS vector was introduced into Agrobacterium EHA105 to obtain a recombinant Agrobacterium.

2. The recombinant Agrobacterium obtained in step 1 is taken, and the Arabidopsis mutant rsh1-1 plant is genetically transformed by the flower soaking method, and the seeds are harvested, which are the T0 generation seeds.

3. Cultivate the seeds of the T0 generation into plants, that is, the T0 generation plants; screen the transgenic plants from the T0 generation plants through hygromycin resistance screening.

4. T0 generation transgenic plants were selfed to obtain T1 generation seeds, and the plants grown from T1 generation seeds were T1 generation plants; transgenic plants were screened from T1 generation plants through hygromycin resistance screening.

5. T1 generation transgenic plants are selfed to obtain T2 generation seeds, and the plants grown from T2 generation seeds are T2 generation plants; T2 plants are identified for hygromycin resistance, if the T2 generation plants obtained by selfing a T1 generation transgenic plant are all With hygromycin resistance, the T1 generation transgenic plant is a homozygous transgenic plant, and its self-crossed progeny is a homozygous transgenic line.

The homozygous transgenic plants obtained by carrying out the above steps with the pAtRSH1::AtRSH1-FLAG-NOS vector are represented by AtRSH1-FLAG. The homozygous transgenic plants obtained by carrying out the above steps with the pAtRSH1::ARE2-FLAG-NOS vector are expressed as ARE2-FLAG.

6. Sow seeds on 1/2 MS solid medium without sucrose (Columbia ecotype Arabidopsis seeds or Arabidopsis mutant rsh1-1 seeds or T2 seeds of AtRSH1-FLAG or T2 seeds of ARE2-FLAG) After culturing for about 1 week, wrap the petri dish with tin foil for dark treatment, and observe the phenotype after culturing for 7-10 days.

The results are shown in Figure 6, A. Under normal conditions, the growth status of each Arabidopsis line was similar. Arabidopsis mutant rsh1-1 plants had a dark-induced accelerated senescence phenotype, while transgenic plants remained green for about a week in the dark, similar to Columbia ecotype Arabidopsis plants. The results showed that the rice ARE2 gene could restore the phenotype of the Arabidopsis rsh1-1 mutant, and the ARE2 gene function was conserved and had the function of the Arabidopsis AtRSH1 gene. AtRSH1 is a ppGpp hydrolase, so ARE2 also has ppGpp hydrolase activity.

3. Acquisition and identification of recombinant Escherichia coli

In order to analyze the biochemical function of the products encoded by the ARE2 gene, the inventors performed heterologous genetic rescue experiments on Escherichia coli spoT203 strain. Escherichia coli spoT203 strain, namely Escherichia coli (p)ppGpp hydrolysis deficient strain spoT203, is described in the following documents: Mechold, U., Cashel, M., Steiner, K., Gentry, D., and Malke, H. (1996) .Functional analysis of a relA/spoT gene homolog from Streptococcusequisimilis.J.Bacteriol.178,1401-1411. The E. coli spoT203 strain (CF4943) is a strain with the spoT203 point mutation in the E. coli CF4941 background, and exhibits a slow-growing phenotype due to (p)ppGpp accumulation in vivo. E. coli CF4941 is a relA ^- genotype strain.

1. The pBAD/His A vector was introduced into Escherichia coli spoT203 strain to obtain recombinant bacteria.

2. The pBAD/His A-AtRSH1 vector was introduced into Escherichia coli spoT203 strain to obtain recombinant bacteria.

3. The pBAD/His A-ARE2 vector was introduced into Escherichia coli spoT203 strain to obtain recombinant bacteria.

4. The test bacteria are inoculated into LB liquid medium and cultivated until the OD ₆₀₀ of the bacterial liquid is the same (about 1.0-1.5), and a certain volume (about 8-10 μL) of bacterial liquid is drawn after equal dilution, and inoculated in a solution containing 0.2% hydrolyzed casein. And 0.5-0.8% arabinose M9glucose minimal solid medium, 37 ℃ overnight culture and observe the bacterial growth state.

The test bacteria are: Escherichia coli CF4941, Escherichia coli spoT203, the recombinant bacteria obtained in step 1, the recombinant bacteria obtained in step 2 or the recombinant bacteria obtained in step 3.

M9 glucose minimal solid medium was prepared according to the method of Xiao et al. (1991) with slight adjustments. Specifically: 11.28 g/L M9 salt (Sigma-Aldrich), 0.2% glucose, 0.1 mM calcium chloride, 1 mM magnesium sulfate, 0.5 μg/mL ferrous sulfate, 1 μg/mL vitamin B ₁ , 1.5% agar powder. Prepare 10 × M9 salt stock solution (pH 7.0), sterilize it with moist heat at 121°C for 20 minutes; prepare 100 × mother solution of other components, and sterilize by suction filtration (vitamin B1 stock solution is stored at ₄ °C, other mother solutions are stored at room temperature). When preparing the medium, dissolve the agar powder with single distilled water, sterilize it with moist heat at 121°C for 20 minutes, and add each mother liquor in proportion. Arabinose is used to induce the expression of foreign genes.

The results are shown in Figure 6, B. The growth rate of the recombinant bacteria introduced with AtRSH1 gene and ARE2 gene was similar to that of E. coli CF4941, that is, both AtRSH1 gene and ARE2 gene could restore the slow growth phenotype of E. coli spoT203. The spoT203 mutation resulted in a decrease in the (p)ppGpp hydrolase activity of SpoT, thus the results indicated that ARE2 has ppGpp hydrolase activity.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention has given particular embodiments, it should be understood that the present invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

sequence listing

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

<120> Application of rice ARE2 gene in the regulation of nitrogen metabolism in plants

<130> GNCYX210431

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13012

<212> DNA

<213> Oryza sativa

<400> 1

aatggttaca cgaggacaaa cattcttccc ccaggaagtc acaaatctca aaaggttttt 60

gacgggtgaa tgcttatttc ttttttctta aacgcaatcc acattttttt tcttcttttg 120

attagcgata ctatttgtgc ttaagatatt taccatgcaa aatggcttgg agagagagaa 180

tgtctggact gaagttaagg attatttgga gagcttaaaa ttctgataag caactgatta 240

gtagccaact tttgacaatc tggaaaagct gggttttcta gtttttgact tcaagttcat 300

tttctagatt ctacaaatac agtttctcag aatctggacc aaaagctaga ctgttttaga 360

tagtttttga ttctgataga agttgcaaca gccaaaagct ctcccaaact cccaaacagg 420

cccttactat caagtgtcat tgcatagtac tttgatagtt gatggatagg ctccgaaaaa 480

acaactgcga gaagagtgtt gcttgcacaa tatgatccat atacaatggc tctgcccatc 540

aggttatttt ccaaccaatg gctcccgtcc ctttgtcacc cctgagatca ctttcttggt 600

tgaggtggtg gctttgatct gacatgttag cataaggtca ctcgccacac caccattgta 660

aatgctctta agcagcctct ctagcgattc cataatggtg agtcagtgac agagcaaacc 720

atgatttgag agaaggagca tatttggaat gctatgatat cttggtttca tacagggttc 780

aaaatttagg tgaagtatct caaatttcca actgttttat atacaaaaca atttattcga 840

tatacgtttt tttctctctt cgaagagaac aaaatacttg catgaatata gtttaggtcc 900

atacatccat acccttttta catgggcaca caatccatgc ttctccccac agcccaactg 960

ttagctagaa gaagagatta catgacatga gcaaaccatc gattctattc ttccgtagtg 1020

ctaccaggat cattgctgtg tgaaaactga aaagccataa cgaaaaattc catagtgctc 1080

acgaccaaga cgtgtgccac taaaaactaa cgagtacacg agcagaacgt gtgtgtgcct 1140

ctcgagtcac gagctgctgt acaccaggta tgcagatggg ggaggaggag agtacgagac 1200

taccggatgt aaaccagaaa aaatccgtaa aaaagccctc caaaatacta ctcctcctac 1260

tgctgctgct gctgcaaaag cgaaagagga ttagctgaaa ccgggcaaaa gggagggcgt 1320

ttgccaccta tttcaccaca cggccgcggc gttttgtgtg gccttcgtct cgtgttatct 1380

cttctcttct tcccaccccc tctctctctc cttctcctcc cttccgcctt catcatcggc 1440

ggcaaacccc ttcttccatc cacttccccc accgaattcg cgcgcgcgcg ccccgtggct 1500

gcggcggagg ggaaggtgag gacggcggcg ggcacggcca cggcctccga gccgccgatg 1560

agattgcgcg ccaccgcgac atgcaacccc cgacgggcgc cgtgtcaggt gatgatcccc 1620

tcccctcgcc tcccctccac cccgtcatct cggcttgctc cccccgcgcg attcggtccg 1680

cggctgctga tgatgatgcg tcgcccgcgg tggatggact agggattggg ggaattgatg 1740

ggggagccct gatgatggct ttgtttgttt gtttggtggt ggtcgcggcg tgcagggtcg 1800

tcgtcgtcgt cgctggagtg cgtgagctcg tgcaggacgt cgtggagggg cggagggagg 1860

ccgtacgaat gcagcgtgct ctcgtgcgcc tggaacgcgc cgcgggcgct cacgggggcg 1920

ctcgccagca ccaccgcgca gtgctcctcc tgcagccacg ccgaggctgg ggcagggtgg 1980

aggcggcggg gccagtcgca gcggagtaac aattcggtgc gttgctgcct gcataattga 2040

ttctgtcatt tacacgtaat gttgattggg attcgccatt ttggcttact ttagagttaa 2100

cggaatggaa aaatggaggt gcttactagt gttttattcc atttcgtagc atcaggtccc 2160

tgagcatcac tgaaactgaa cgatctcatg actaaagcca atatatcata catgttttgt 2220

agtacaagtc aagttggtca atctggctat caggtcatac tgaagttagt tcacttgggg 2280

gaaaaaggct tccatctatc tcatttcatt attgcatata tgctttggct ataaacacta 2340

caagtagcaa gtcctgctga ttctcttcaa gtttggcttg tacatagaca taggtaatgg 2400

ttttctgtag attttgtaag tgttatttga tgccatcaga acatatttcc ttcttagaaa 2460

ataaatggat agtggttttg tacttatata ttttccttgt taagaccttt tatttagaag 2520

ttatggaaat ttgtgatgaa gccttaacca tatgaagttt agttcaccca tggttcctcc 2580

atctattaat ttaattcttc ccaaagtaca ggattggcac catttattcc ctggttaatg 2640

attcagttac tcggctttct ccttgatatt ttttacggct gcaacaggtt gctagtacat 2700

gaaacaaaga agtctaggggg caacattggt actgtgctag agatcgtgaa cggtaaagct 2760

gtgtattgac aacggaatgt ggatttggtt tggtccatct tctttaggaa ctaggatgct 2820

agggaagaag cagggtcaat ttggcgtttg gcaacaaaca ttagtcaatt tggcgtttga 2880

ctaagtttat ttgtctataa acttagtcaa acttaaagca gagtcatctt gagctagcat 2940

gttgtgagcc tgtaaattaa cttgactatg caatgccttg tttgttcctt tattctgtgc 3000

tccttgtagg agtctgacca taatctaata gataaaaatt atatatacaa aaaaaaaagg 3060

tgagcgtgca tgtttaggat agaattgttt ggtacactca tttgtggctt ggatgcgatg 3120

caaatgcgtt gaaatatatt atagacattt ctatgtatat ctttattgta gcgagtatag 3180

gctgttttta gcaagatttt gatggcgatc tagtggaaat gtggaatgac atctgctaaa 3240

atttccatga cttaggttta gcttatatcg taactatttt ccgaatgcca tcagaaggat 3300

tgtcttcacc taacaatcac cattcttcca tatttatctc agctttgttt cctgttaata 3360

ctgagcactc agtatgtaca tgaactaccc tcatgccaaa tagaatggtt ttcccaaacc 3420

atttaggttt catttggttg gaagttgtaa tgtatcacac tggttttgtg acaaagaatt 3480

gagtgtaatg tacgtgggtt acatcagact ttgtgttctc ttggagttct cctgccgtcc 3540

tacttttatt cttaattcat gcatcgggat tagagttttc caaccaaata acaggtaacg 3600

tcttaattgt gcactcaatc atgatctaga ggagaagttg caaaccaaat gccctggttt 3660

aagtgctagt aaatactttc cactttttcc tttatggtcg cttattgacc atgagtcatt 3720

gaaaacatgt ttggccttta atgtaagggg gatttaacca gagtcacctg tttctgatct 3780

acttggctca aaccgatggg gtcaaacctt ggaaaatatc ttataacaaa atactactac 3840

ctccgtttca ggttatagga tgttttgact ttggtcaaag tcaaactgtt tcaagtttga 3900

caaagtttat agaaaaaaat aataacattt tgaacccaag acaaatttat tactatgaaa 3960

atatattcaa ttattgattt aataaaacta atttgatatt ataaatatta ctatatttgt 4020

ctataaactt agtcaaactt aaagtagatt gactttaacc aaagtcaaaa cgtcttataa 4080

cctgaaaacg gagggagtac taacaaatta caattctaag tctgttatga atacagtcaa 4140

atgccatatt gtgtcaaatg tgacatcatt agttttacaa atttgtaagg catcatacca 4200

tcttactttt ttaatggttg ctgtcacgtt cgttatctac atatgaaaca ataattcttt 4260

gcaatgtctt aatactatca acttttttt ccagttactg cacataacct gggcagaagg 4320

catcaacagg gggaagtttg gttatggttc atcagcccat tcttttccca ctggaaattt 4380

tttcaaatct tggtcaactt ccgtggatcc aacatggaga gttttctgct attcatcatc 4440

tgaatcgttc aatcatattt ctccagaaac tttgtgggag gtttgtcact gaattgcaca 4500

attaaacttt tttgctcgtt tttggtacat aattcagttt tgttaaaatt aaatataaga 4560

gttaaagcat ttttaaagga tttatgatta atcatttgtg ggtttgatcc gtgaaacttc 4620

cataaagaag tcaataatca ttttgtgtct ggtagtatag tactacaatc accttcttct 4680

taatgttgta aacacacttc ataattaact agaacatacg gcaatgaaac tcaattcaga 4740

tttggaagat gcggaagtct ccaggttaag cacaatttcc aatagtgctg ccatggtgca 4800

ttttcaaaat gaaaccatta gttgtgactt gttagcataa ttttttgtgcc tagtatgaaa 4860

ttttaaatgt actattcaag tatactgccc tcaaatgcaa ctcatggttg atgttcgttt 4920

gaagaaagag gcaattttgt ttgtggcgct tttaaaattt atgcgattgc atgacaggat 4980

ctcaagccag ctatctcgta ccttcaacct gaagaattga actttgtaca cgatgctctg 5040

aaggtaattg aagtgcaata acattcttcc acgtggtttg tggacaatat tctatacttc 5100

ttttattatt cctgggtagt gcatgcacca ggaggttgta tcatgtacct gattgactct 5160

tgcagttggc atatgaggca cataatgggc agaaacgccg aagtggggaa cctttcatta 5220

ttcatcctgt tgaagttgct cgtatccttg gggagcatgt gagtaatata tcgttaccac 5280

tacggtgatg actttctgcc catataaaca taatgaagtt aaattttctg tcgttccatc 5340

ttctgtggac atatttagga acttgactgg gaatcaatag ctgctggttt attgcatgac 5400

actgttgaag atacagacat ggttactttt gaaagaatag aaaatgagtt tggtgtaacc 5460

gtacgccgta ttgttgaagg ggagacaaag gtacattgtt tcacatttta tattttgcac 5520

atggctgcag tgcatgttta tatttggtat atttctgcaa aatgtttgtg atccaggtag 5580

agggatttta atgctataac agacctattt catttcaaat gcaggtatct aagctaggaa 5640

aacttcagtg caaaaatgag ggtaattcaa aacaggacgt caaggctgaa gatttaagac 5700

agatgtttct tgccatgaca gaagaggttt tttcttttttt tgtttcgtca tgtagactgt 5760

tatttatcga gagctatttg aacaccctac taggttccat gaaaatttaa ataaaaattg 5820

taggttcgtg taatcattgt gaaactggca gacaggctgc ataatatgcg tacactcaca 5880

catatgcctc agcacaagca ggtaaactct tgttccactg aaaataatat ctgtccgtaa 5940

cttagtttct atttttattg taagcactct ctttgcagta tgccattgcc atggagacac 6000

tgcaggtttt tgcaccccta gctaaacttc tcgggatgta tcgaataaag gtatctgatg 6060

ataaatcatc ttgaaatata actgttattt ctgtttattg tatagactaa cttcattact 6120

gcagtgcttt tatgttattg ctgtgtttgc ttacaaacat tttgtactac catgctgttg 6180

tgttttttcc aataaaattt atacttcttt tttaatgata attgttttcg tgtttgcttt 6240

tttgatttta aaaacctgta tttctgctat aatttgacca gtagacttgt ttgtcctgaa 6300

cttattagta atattaagaa acaagtccat tttatcctta aatattgctc aagttcagaa 6360

atctatgcta aaatttggta tttcacaacc gtgcaaaaac aacctccctt gcttaattca 6420

ccaatttttc gttgacttgg ttttctgatt gccaacttta tgctgatgtg gcatacgaat 6480

cagtaacagc ataaagtgat tcagttgtac agattgaaat attcagattt gttgttgttg 6540

atttggatgc aatgtcatca attacacctc cctcggatat tttagaagtc aacttttcac 6600

ctcagaatga gacatccaag ccccaaaaca gttaaaatct aaccatcgtt ttatccactt 6660

tatggtttta atggtggttt tggagagtta aaagaacata atgtacttcc aaagttcagg 6720

ggccaaagaa atcaattccc cttaatctta tgactgtacg agatattttc atagtagtat 6780

tttgtagttc aaggaccaaa ggaaaaaaaa tccagttcaa ggaccaaagg aaaaaaaatc 6840

cctttatctt atgaatagaa ggggtggttt catactggtg ttttctgtca gtcttagaaa 6900

ggtctcctct gtagttcgtt gcattgtgag aatgtcgatg tgtttcttat gaagcttatc 6960

atagcttctg gtactttgcc tgggtccatt tgaccagttg ctcttattct tgtagtctga 7020

gctagaatat ctgtccttca tgtacatgaa tcctggtgat ttcgctgaac taaagaaaag 7080

agttgaggac ctctacaagg cccatgaaca agaattggaa gaggtacttt gtttttgcta 7140

ttgcactttg atttgcattc tactctgaat gagattcatt ataatttttg aattacaggc 7200

aaatcaaatc ttaggggaaa agattgctga ggatcaattt cttgatcttg ttagtgttga 7260

aacacaagtg cgttcagtct gcaaagaact ctacaggtag agcacacatg atgtcttatt 7320

aattaaaaat aagtattatc ttgaaccttc acttccactt taatactacc cctgcctagt 7380

gatatactgt tggtataaac ttggtttagc ttatttgttt acactactta caaatgcaat 7440

tcatattaga gcacaatgaa tttcctcaca aatttcttta gttatatctt tgtaaaaaag 7500

tggatatttg ttttcactac tttgtcaatt tgcttacaca acatcattaa atagtgtggt 7560

acagttacag actgattgac gttttctgct gcaaaatcct ttttttcttc tcattcacat 7620

tttgcaccta aaaaggtgat caacaattat ttttgcatta ttcattacca agtttgaaat 7680

aggtggcaaa agtgttttct taggcagatt atttatcatt tggtcatatt gcagcattta 7740

taaaactgcg ctgaagtcca agagttcaat aaatgagata aatcaggttg cacaggtatg 7800

attttcctat gtcagataag ttgcctagct ttatcatttt aacgaataac ttattctctg 7860

aaaagtattg tgtttctata ttattcagct gcggatcatc ataaagccaa aatcctgcaa 7920

tggtgtgggg ccattgtgca ctgcacaaca ggtataacat tacaagtgta acataccatc 7980

ttttctgtat gaaattaaaa tttactaatg tgtaaatatg ctcaatccac tgatgcgcaa 8040

gcattcaatt catttcagat ctgctaccat gtccttggtc ttgttcatgg tatctggaca 8100

cctatacctc aagctgtgag ttatctgctt tccttattca ttataatttt tacctctttt 8160

gattttgcca tttctaaaag ccagctgtat aggtgaaaga ttacattgca accccaaagc 8220

ctaatggata ccaaagtcta cacacgacag tgataccatt tctgaatgag agtatgttcc 8280

atttggaagt tcaggtaatg ttggcttagc tgcctgccat ttagctaaat tcatactaca 8340

aactctcaag catgtgctca ttctaatgca agtttcactt tatgttccta ctgtattgca 8400

gataagaaca gaggatatgg atctcatagc agaaagaggc attgctgcgc actacagtgg 8460

aagaggggtt gtttcagggc ctgttcgtcc tggaatatca agtggaagga attcaaatgg 8520

gaaagtgata tgtctgaaca atacaggctt tgctctgagg gtatataatg atgtcaaagc 8580

ttattgcact tcatattaag taccccctct gttctaaaaa ataaggcgtc cttgttttca 8640

ggaaaaatca acatttgaaa actttgactg ataattgatc taaaatattt ttttggtgtc 8700

atgtatgtag taccactaga tttgtatctg aaaatatttt catatcatgt taatttcaaa 8760

gtaccccagt taataatata ttactacccc tgtcctaaaa tgtatgacac cgttgacttt 8820

tcgcataatg tttgaccatt cgtcttaaaa ttttagtgta aatatgaaaa agtataagtt 8880

aatattattt tgatgataaa gcaagtgaca acaaaataaa tgatatttat atcttttttg 8940

aataagacga atggttaaac attatgtaaa aggtcaacgg cgtcatatat tctgggatgg 9000

attataaaag aaatcaatgg tgaaagtata atgttgggat gctgtgaaat aagttagtac 9060

gctttatatt ttagaacgga ggattattta ttatcacatt tccttaagaa agaactccaa 9120

agacttctca tctttgatag attggttggc tcaatgcaat ccgtgaatgg caagaagagt 9180

ttgttggtaa catgagttct agggaatttg ttgatactat cacccgagat cttctgggaa 9240

gccgtgtctt tgtgtttaca ccaaaaggcg aggtgaggtg ctgacttgga ggacatttct 9300

tccatttgag ttgagcgatt ggtttgaata tagttttcat tggcagatta aaaacttgcc 9360

taagggggcc actgtggttg attatgctta cctgattcac acagaaattg ggaacaaaat 9420

ggtagctgca aaggttagtt atggataata gcattatgat catcattgta tttgtgtgct 9480

catgttcttc ctttcaggtg aatggcaatc tagtttcacc aatccatgta cttgcaaatg 9540

ctgaagttgt ggagattata atttatgatg taagtactca tttcatatat tgcatgattt 9600

atttctgttt tatatctttg tgtcaatttg taactttcaa gatatgttta ttgattgagc 9660

catgtctcga cattgatgca catgcccaca acatacaagt tgcaactttg attgcccttt 9720

ttttgtgtgt atatgggtga taacctccgt cccaaaattt tagggatcct ggcattcaaa 9780

atttgtccaa aagtagtagg gattctagag tactactccc tgctaaccac ctcatttaat 9840

gttcacccaa tcttaccccc aaccaccctc gcactaccca cagacccctc aataagaggc 9900

atcatagtct tttcccttta accttaatct ctccaaaaaa atcttaagat cccttatatt 9960

ttggaactga gggagcaact tacacgccag taggtctgaa attttattcc agcatataac 10020

catccgacca tgtggtctgt agcatcatat atcttttttc atttgcaaat gtactccctc 10080

catttcatat tataagtcat ttgatttttt ttcttagtca aagttcttta ggtttgccta 10140

agtttataga aaaagctagc aacatctaca gcactaaatt aatttcatta aatttagcat 10200

gaaatatatt ttaataatat gtttgtttta tattgaaaat gctactatat ttttctacaa 10260

acttaaagaa gtttgactaa gaaaaaagtc aaaaagactt ataataagaa acagatggag 10320

tacgattcag cttgctacag acaaattcat agcagtatga taaccccctt gggggccagg 10380

atcctggccc atacaaagga gccctaaaag ggacagcagc atgacagctc acattgtccc 10440

tgaatcaatt gaaagaccaa aggtgcattt ccagttcata ccgtgggatc agcccttcat 10500

agttcctaat ttcacacttg aaagttacaa cataattctg agggaaatag atggaagtag 10560

atgtgatgtc tctgtgaggc catttgcgat ttttctcagt ttaaggcata ctgttactct 10620

tgctagtgcc ttttatccat caagaggatt ctagagaact attccattgg cattgcttat 10680

attatacctg tgacatccat ttaaactcat ctgccttttc ttgtttcaga aattatctgc 10740

taaatatgca ttccagcgtc accagcagtg gcttcaacat gcgaaaactc gcagtgcaag 10800

acacaaaatt atgaaagtaa gtctaaaagg atttcttaca ttattagatg gttctggcaa 10860

ctaaccgttt gcctttctcc agttcttgcg ggagcaagct gctctttctg ctgctgaaat 10920

aactgccgac gccgttaata actttgttgc cgatcttgaa gatgaaagtg actatgagca 10980

gtcgattcca agctctgaaa ataaggacta tacattcaac tggcagaaga tattgaattc 11040

tgacaaacta tcctttggaa acaagaagag cgactgcttt ttacctgtta aaaatgtctc 11100

tgtgccaaag gttaatggga agcacaacaa aactgtcaaa gaactgggca tcaaaattaa 11160

tggttcaaca tttcgtggtg atagcttcac tgattttatt caccctggtg tttccagcag 11220

taaagaagtt ctccctagtg tggataactg gaaagctggt aaaatttgtg cgtggcacaa 11280

tacagaaggc agctctatcc aatggctctg catagtgtgt gttgatcgaa aaggtttcac 11340

tcctgccatc taattgttgg acaaagctga aaatatttta ttagctttac ctctataatt 11400

tattagtttt aactaataag acgttgcaaa catgtccaca ttttgttcct ttctgcacaa 11460

ctatttacat ctaatttatt tttgggttat gttgttttga cctttacctg caaatattaa 11520

tataatcact tcaaatggct ttcaatattc atcataggta tggttgcgga agtttcatca 11580

gctctaacag cgtgtggaat caccatatgc tcgtgtgtgg taaatggacc aacttgcctc 11640

aataatttag tatcatcctt ttttatgtga taatcaatgc aacattgcat catttaccct 11700

attaagttcc catcttgaat ttgtcagcca cctctttgta ctgtttgtgt ttgatcattt 11760

ctgctataat tcttaggcgg agcgagataa gagaagggga ataggtgtga tgttgttcca 11820

ctttgagggg gcatatgaga atgtggtgtg cttccttaac tgatctgtat tcctgttcta 11880

attgttgcat tgggcactgt acctaaatag aaaattgcaa ttgcaggtca gtgcatgctc 11940

cggtgttgat atgattcttg gggttcttgg ttggtccgtt ggatgcagct gtaatccttt 12000

gggtgttctt gaatgctagc ggacaacagg catcacgctg catcagttca ccatctccag 12060

caaaaaggag agacaatttt gaagcaccaa gaaatcagtc cgctcaatgg ttcaggagca 12120

gtccgatctt ggttgatgat gctgtgcaac ccataacttg tccttcaaag aaggtagagt 12180

gagggtagag caagctggag ccggatattc tggttcgttt ggaagcgaga ggagtgtaca 12240

tagctgagca agctactagt gaactgttag tccagggatt ggttgggtta tagtattata 12300

caaaagcctc catgaatgac caacaatggc agcttctctc ttctattttt tttgtttgcc 12360

attcgtacac tagttatgta tgacatgtgc agctagaatt ttgatctgat gggattccaa 12420

tgccccaaat tttgcccgta ttcgttattt cactcggtga ttatgactat gctgtagctt 12480

cgatttttca atggtataag ttttcttttc ttactttggg gcctaatgaa atgcgttggc 12540

ttaagaacat ctaactggca gataaataat tcagcgaaga gtcctctgtt ttgcagaggt 12600

aaaaaaacag acgcagtcca tactgtacag taactgaaac aagagaatgg cttgtttgct 12660

acagttaact tatatgaaat gcacctggat tgcgactgat ttagaatgtg gacaatgtgg 12720

accaagccaa gccattttct cacgtcttac cgagacgaat cgctcgtgcc aaggactcat 12780

tgacccttga ttcagttgag aaatgggaga tatttctgtt tggcatttga taaaatagtc 12840

tgttgcccgg cgaaaaataa aatatctgtt aatcgacata gtgagaacat tgatgtgttt 12900

attcaactat aacgcactct aataagcgaa aactacgtgt ttcgatcttg tttcatgtgc 12960

tagcgtgtgt gagccactct tcagctttgg tggcgtcgca ggaatacgca gt 13012

<210> 2

<211> 2679

<212> DNA

<213> Oryza sativa

<400> 2

atgcaacccc cgacgggcgc cgtgtcaggg tcgtcgtcgt cgtcgctgga gtgcgtgagc 60

tcgtgcagga cgtcgtggag gggcggaggg aggccgtacg aatgcagcgt gctctcgtgc 120

gcctggaacg cgccgcgggc gctcacgggg gcgctcgcca gcaccaccgc gcagtgctcc 180

tcctgcagcc acgccgaggc tggggcaggg tggaggcggc ggggccagtc gcagcggagt 240

aacaattcgt tactgcacat aacctgggca gaaggcatca acagggggaa gtttggttat 300

ggttcatcag cccattcttt tcccactgga aattttttca aatcttggtc aacttccgtg 360

gatccaacat ggagagtttt ctgctattca tcatctgaat cgttcaatca tatttctcca 420

gaaactttgt gggaggatct caagccagct atctcgtacc ttcaacctga agaattgaac 480

tttgtacacg atgctctgaa gttggcatat gaggcacata atgggcagaa acgccgaagt 540

ggggaacctt tcattattca tcctgttgaa gttgctcgta tccttgggga gcatgaactt 600

gactgggaat caatagctgc tggtttattg catgacactg ttgaagatac agacatggtt 660

acttttgaaa gaatagaaaa tgagtttggt gtaaccgtac gccgtattgt tgaaggggag 720

acaaaggtat ctaagctagg aaaacttcag tgcaaaaatg agggtaattc aaaacaggac 780

gtcaaggctg aagatttaag acagatgttt cttgccatga cagaagaggt tcgtgtaatc 840

attgtgaaac tggcagacag gctgcataat atgcgtacac tcacacatat gcctcagcac 900

aagcagtatg ccattgccat ggagacactg caggtttttg cacccctagc taaacttctc 960

gggatgtatc gaataaagtc tgagctagaa tatctgtcct tcatgtacat gaatcctggt 1020

gatttcgctg aactaaagaa aagagttgag gacctctaca aggcccatga acaagaattg 1080

gaagaggcaa atcaaatctt aggggaaaag attgctgagg atcaatttct tgatcttgtt 1140

agtgttgaaa cacaagtgcg ttcagtctgc aaagaactct acagcattta taaaactgcg 1200

ctgaagtcca agagttcaat aaatgagata aatcaggttg cacagctgcg gatcatcata 1260

aagccaaaat cctgcaatgg tgtggggcca ttgtgcactg cacaacagat ctgctaccat 1320

gtccttggtc ttgttcatgg tatctggaca cctatacctc aagctgtgaa agattacatt 1380

gcaaccccaa agcctaatgg ataccaaagt ctacacacga cagtgatacc atttctgaat 1440

gagagtatgt tccatttgga agttcagata agaacagagg atatggatct catagcagaa 1500

agaggcattg ctgcgcacta cagtggaaga ggggttgttt cagggcctgt tcgtcctgga 1560

atatcaagtg gaaggaattc aaatgggaaa gtgatatgtc tgaacaatac aggctttgct 1620

ctgaggattg gttggctcaa tgcaatccgt gaatggcaag aagagtttgt tggtaacatg 1680

agttctaggg aatttgttga tactatcacc cgagatcttc tgggaagccg tgtctttgtg 1740

tttacaccaa aaggcgagat taaaaacttg cctaaggggg ccactgtggt tgattatgct 1800

tacctgattc acacagaaat tgggaacaaa atggtagctg caaaggtgaa tggcaatcta 1860

gtttcaccaa tccatgtact tgcaaatgct gaagttgtgg agattataat ttatgataaa 1920

ttatctgcta aatatgcatt ccagcgtcac cagcagtggc ttcaacatgc gaaaactcgc 1980

agtgcaagac acaaaattat gaaattcttg cgggagcaag ctgctctttc tgctgctgaa 2040

ataactgccg acgccgttaa taactttgtt gccgatcttg aagatgaaag tgactatgag 2100

cagtcgattc caagctctga aaataaggac tatacattca actggcagaa gatattgaat 2160

tctgacaaac tatcctttgg aaacaagaag agcgactgct ttttacctgt taaaaatgtc 2220

tctgtgccaa aggttaatgg gaagcacaac aaaactgtca aagaactggg catcaaaatt 2280

aatggttcaa catttcgtgg tgatagcttc actgatttta ttcaccctgg tgtttccagc 2340

agtaaagaag ttctccctag tgtggataac tggaaagctg gtaaaatttg tgcgtggcac 2400

aatacagaag gcagctctat ccaatggctc tgcatagtgt gtgttgatcg aaaaggtatg 2460

gttgcggaag tttcatcagc tctaacagcg tgtggaatca ccatatgctc gtgtgtggcg 2520

gagcgagata agagaagggg aataggtgtg atgttgttcc actttgaggg ggcatatgag 2580

aatgtggtca gtgcatgctc cggtgttgat atgattcttg gggttcttgg ttggtccgtt 2640

ggatgcagct gtaatccttt gggtgttctt gaatgctag 2679

<210> 3

<211> 892

<212> PRT

<213> Oryza sativa

<400> 3

Met Gln Pro Pro Thr Gly Ala Val Ser Gly Ser Ser Ser Ser Ser Leu

1 5 10 15

Glu Cys Val Ser Ser Cys Arg Thr Ser Trp Arg Gly Gly Gly Arg Pro

20 25 30

Tyr Glu Cys Ser Val Leu Ser Cys Ala Trp Asn Ala Pro Arg Ala Leu

35 40 45

Thr Gly Ala Leu Ala Ser Thr Thr Ala Gln Cys Ser Ser Cys Ser His

50 55 60

Ala Glu Ala Gly Ala Gly Trp Arg Arg Arg Gly Gln Ser Gln Arg Ser

65 70 75 80

Asn Asn Ser Leu Leu His Ile Thr Trp Ala Glu Gly Ile Asn Arg Gly

85 90 95

Lys Phe Gly Tyr Gly Ser Ser Ala His Ser Phe Pro Thr Gly Asn Phe

100 105 110

Phe Lys Ser Trp Ser Thr Ser Val Asp Pro Thr Trp Arg Val Phe Cys

115 120 125

Tyr Ser Ser Ser Glu Ser Phe Asn His Ile Ser Pro Glu Thr Leu Trp

130 135 140

Glu Asp Leu Lys Pro Ala Ile Ser Tyr Leu Gln Pro Glu Glu Leu Asn

145 150 155 160

Phe Val His Asp Ala Leu Lys Leu Ala Tyr Glu Ala His Asn Gly Gln

165 170 175

Lys Arg Arg Ser Gly Glu Pro Phe Ile Ile His Pro Val Glu Val Ala

180 185 190

Arg Ile Leu Gly Glu His Glu Leu Asp Trp Glu Ser Ile Ala Ala Gly

195 200 205

Leu Leu His Asp Thr Val Glu Asp Thr Asp Met Val Thr Phe Glu Arg

210 215 220

Ile Glu Asn Glu Phe Gly Val Thr Val Arg Arg Ile Val Glu Gly Glu

225 230 235 240

Thr Lys Val Ser Lys Leu Gly Lys Leu Gln Cys Lys Asn Glu Gly Asn

245 250 255

Ser Lys Gln Asp Val Lys Ala Glu Asp Leu Arg Gln Met Phe Leu Ala

260 265 270

Met Thr Glu Glu Val Arg Val Ile Ile Val Lys Leu Ala Asp Arg Leu

275 280 285

His Asn Met Arg Thr Leu Thr His Met Pro Gln His Lys Gln Tyr Ala

290 295 300

Ile Ala Met Glu Thr Leu Gln Val Phe Ala Pro Leu Ala Lys Leu Leu

305 310 315 320

Gly Met Tyr Arg Ile Lys Ser Glu Leu Glu Tyr Leu Ser Phe Met Tyr

325 330 335

Met Asn Pro Gly Asp Phe Ala Glu Leu Lys Lys Arg Val Glu Asp Leu

340 345 350

Tyr Lys Ala His Glu Gln Glu Leu Glu Glu Ala Asn Gln Ile Leu Gly

355 360 365

Glu Lys Ile Ala Glu Asp Gln Phe Leu Asp Leu Val Ser Val Glu Thr

370 375 380

Gln Val Arg Ser Val Cys Lys Glu Leu Tyr Ser Ile Tyr Lys Thr Ala

385 390 395 400

Leu Lys Ser Lys Ser Ser Ile Asn Glu Ile Asn Gln Val Ala Gln Leu

405 410 415

Arg Ile Ile Ile Lys Pro Lys Ser Cys Asn Gly Val Gly Pro Leu Cys

420 425 430

Thr Ala Gln Gln Ile Cys Tyr His Val Leu Gly Leu Val His Gly Ile

435 440 445

Trp Thr Pro Ile Pro Gln Ala Val Lys Asp Tyr Ile Ala Thr Pro Lys

450 455 460

Pro Asn Gly Tyr Gln Ser Leu His Thr Thr Val Ile Pro Phe Leu Asn

465 470 475 480

Glu Ser Met Phe His Leu Glu Val Gln Ile Arg Thr Glu Asp Met Asp

485 490 495

Leu Ile Ala Glu Arg Gly Ile Ala Ala His Tyr Ser Gly Arg Gly Val

500 505 510

Val Ser Gly Pro Val Arg Pro Gly Ile Ser Ser Gly Arg Asn Ser Asn

515 520 525

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

530 535 540

Trp Leu Asn Ala Ile Arg Glu Trp Gln Glu Glu Phe Val Gly Asn Met

545 550 555 560

Ser Ser Arg Glu Phe Val Asp Thr Ile Thr Arg Asp Leu Leu Gly Ser

565 570 575

Arg Val Phe Val Phe Thr Pro Lys Gly Glu Ile Lys Asn Leu Pro Lys

580 585 590

Gly Ala Thr Val Val Asp Tyr Ala Tyr Leu Ile His Thr Glu Ile Gly

595 600 605

Asn Lys Met Val Ala Ala Lys Val Asn Gly Asn Leu Val Ser Pro Ile

610 615 620

His Val Leu Ala Asn Ala Glu Val Val Glu Ile Ile Ile Tyr Asp Lys

625 630 635 640

Leu Ser Ala Lys Tyr Ala Phe Gln Arg His Gln Gln Trp Leu Gln His

645 650 655

Ala Lys Thr Arg Ser Ala Arg His Lys Ile Met Lys Phe Leu Arg Glu

660 665 670

Gln Ala Ala Leu Ser Ala Ala Glu Ile Thr Ala Asp Ala Val Asn Asn

675 680 685

Phe Val Ala Asp Leu Glu Asp Glu Ser Asp Tyr Glu Gln Ser Ile Pro

690 695 700

Ser Ser Glu Asn Lys Asp Tyr Thr Phe Asn Trp Gln Lys Ile Leu Asn

705 710 715 720

Ser Asp Lys Leu Ser Phe Gly Asn Lys Lys Ser Asp Cys Phe Leu Pro

725 730 735

Val Lys Asn Val Ser Val Pro Lys Val Asn Gly Lys His Asn Lys Thr

740 745 750

Val Lys Glu Leu Gly Ile Lys Ile Asn Gly Ser Thr Phe Arg Gly Asp

755 760 765

Ser Phe Thr Asp Phe Ile His Pro Gly Val Ser Ser Ser Lys Glu Val

770 775 780

Leu Pro Ser Val Asp Asn Trp Lys Ala Gly Lys Ile Cys Ala Trp His

785 790 795 800

Asn Thr Glu Gly Ser Ser Ile Gln Trp Leu Cys Ile Val Cys Val Asp

805 810 815

Arg Lys Gly Met Val Ala Glu Val Ser Ser Ala Leu Thr Ala Cys Gly

820 825 830

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

835 840 845

Gly Val Met Leu Phe His Phe Glu Gly Ala Tyr Glu Asn Val Val Ser

850 855 860

Ala Cys Ser Gly Val Asp Met Ile Leu Gly Val Leu Gly Trp Ser Val

865 870 875 880

Gly Cys Ser Cys Asn Pro Leu Gly Val Leu Glu Cys

885 890

<210> 4

<211> 5009

<212> DNA

<213> Artificial Sequence

<400> 4

tgcagcgtga cccggtcgtg cccctctcta gagataatga gcattgcatg tctaagttat 60

aaaaaattac cacatatttt ttttgtcaca cttgtttgaa gtgcagttta tctatcttta 120

tacatatatt taaactttac tctacgaata atataatcta tagtactaca ataatatcag 180

tgttttagag aatcatataa atgaacagtt agacatggtc taaaggacaa ttgagtattt 240

tgacaacagg actctacagt tttatctttt tagtgtgcat gtgttctcct ttttttttgc 300

aaatagcttc acctatataa tacttcatcc attttattag tacatccatt tagggtttag 360

ggttaatggt ttttatagac taatttttttt agtacatcta ttttattcta ttttagcctc 420

taaattaaga aaactaaaac tctattttag ttttttttatt taataattta gatataaaat 480

agaataaaat aaagtgacta aaaattaaac aaataccctt taagaaatta aaaaaactaa 540

ggaaacattt ttcttgtttc gagtagataa tgccagcctg ttaaacgccg tcgacgagtc 600

taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag cagacggcac 660

ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg ttggacttgc 720

tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg gcacggcagg 780

cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc caccgctcct 840

tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttcccc 900

aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa tccacccgtc 960

ggcacctccg cttcaaggta cgccgctcgt cctcccccccc cccccctctc taccttctct 1020

agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc atgtttgtgt 1080

tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg cgacctgtac 1140

gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc ctgggatggc 1200

tctagccgtt ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt gcatagggtt 1260

tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt 1320

tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc gttctagatc 1380

ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc tgtatgtgtg 1440

tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg atctaggata 1500

ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt tgttcgcttg 1560

gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg agtagaatac 1620

tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg tcatacatct 1680

tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat acatgttgat 1740

gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 1800

cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 1860

acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 1920

ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 1980

taagcttatg caacccccga cgggcgccgt gtcagggtcg tcgtcgtcgt cgctggagtg 2040

cgtgagctcg tgcaggacgt cgtggagggg cggagggagg ccgtacgaat gcagcgtgct 2100

ctcgtgcgcc tggaacgcgc cgcgggcgct cacgggggcg ctcgccagca ccaccgcgca 2160

gtgctcctcc tgcagccacg ccgaggctgg ggcagggtgg aggcggcggg gccagtcgca 2220

gcggagtaac aattcgttac tgcacataac ctgggcagaa ggcatcaaca gggggaagtt 2280

tggttatggt tcatcagccc attcttttcc cactggaaat tttttcaaat cttggtcaac 2340

ttccgtggat ccaacatgga gagttttctg ctattcatca tctgaatcgt tcaatcatat 2400

ttctccagaa actttgtggg aggatctcaa gccagctatc tcgtaccttc aacctgaaga 2460

attgaacttt gtacacgatg ctctgaagtt ggcatatgag gcacataatg ggcagaaacg 2520

ccgaagtggg gaacctttca ttattcatcc tgttgaagtt gctcgtatcc ttggggagca 2580

tgaacttgac tgggaatcaa tagctgctgg tttattgcat gacactgttg aagatacaga 2640

catggttact tttgaaagaa tagaaaatga gtttggtgta accgtacgcc gtattgttga 2700

aggggagaca aaggtatcta agctaggaaa acttcagtgc aaaaatgagg gtaattcaaa 2760

acaggacgtc aaggctgaag atttaagaca gatgtttctt gccatgacag aagaggttcg 2820

tgtaatcatt gtgaaactgg cagacaggct gcataatatg cgtacactca cacatatgcc 2880

tcagcacaag cagtatgcca ttgccatgga gacactgcag gtttttgcac ccctagctaa 2940

acttctcggg atgtatcgaa taaagtctga gctagaatat ctgtccttca tgtacatgaa 3000

tcctggtgat ttcgctgaac taaagaaaag agttgaggac ctctacaagg cccatgaaca 3060

agaattggaa gaggcaaatc aaatcttagg ggaaaagatt gctgaggatc aatttcttga 3120

tcttgttagt gttgaaacac aagtgcgttc agtctgcaaa gaactctaca gcatttataa 3180

aactgcgctg aagtccaaga gttcaataaa tgagataaat caggttgcac agctgcggat 3240

catcataaag ccaaaatcct gcaatggtgt ggggccattg tgcactgcac aacagatctg 3300

ctaccatgtc cttggtcttg ttcatggtat ctggacacct atacctcaag ctgtgaaaga 3360

ttacattgca accccaaagc ctaatggata ccaaagtcta cacacgacag tgataccatt 3420

tctgaatgag agtatgttcc atttggaagt tcagataaga acagaggata tggatctcat 3480

agcagaaaga ggcattgctg cgcactacag tggaagaggg gttgtttcag ggcctgttcg 3540

tcctggaata tcaagtggaa ggaattcaaa tgggaaagtg atatgtctga acaatacagg 3600

ctttgctctg aggattggtt ggctcaatgc aatccgtgaa tggcaagaag agtttgttgg 3660

taacatgagt tctagggaat ttgttgatac tatcacccga gatcttctgg gaagccgtgt 3720

ctttgtgttt acaccaaaag gcgagattaa aaacttgcct aagggggcca ctgtggttga 3780

ttatgcttac ctgattcaca cagaaattgg gaacaaaatg gtagctgcaa aggtgaatgg 3840

caatctagtt tcaccaatcc atgtacttgc aaatgctgaa gttgtggaga ttataattta 3900

tgataaatta tctgctaaat atgcattcca gcgtcaccag cagtggcttc aacatgcgaa 3960

aactcgcagt gcaagacaca aaattatgaa attcttgcgg gagcaagctg ctctttctgc 4020

tgctgaaata actgccgacg ccgttaataa ctttgttgcc gatcttgaag atgaaagtga 4080

ctatgagcag tcgattccaa gctctgaaaa taaggactat acattcaact ggcagaagat 4140

attgaattct gacaaactat cctttggaaa caagaagagc gactgctttt tacctgttaa 4200

aaatgtctct gtgccaaagg ttaatgggaa gcacaacaaa actgtcaaag aactgggcat 4260

caaaattaat ggttcaacat ttcgtggtga tagcttcact gattttattc accctggtgt 4320

ttccagcagt aaagaagttc tccctagtgt ggataactgg aaagctggta aaatttgtgc 4380

gtggcacaat acagaaggca gctctatcca atggctctgc atagtgtgtg ttgatcgaaa 4440

aggtatggtt gcggaagttt catcagctct aacagcgtgt ggaatcacca tatgctcgtg 4500

tgtggcggag cgagataaga gaaggggaat aggtgtgatg ttgttccact ttgagggggc 4560

atatgagaat gtggtcagtg catgctccgg tgttgatatg attcttgggg ttcttggttg 4620

gtccgttgga tgcagctgta atcctttggg tgttcttgaa tgccccgggg attacaagga 4680

tgacgacgat aagtgctaag ctagcggatc cactagttct agaggatccc cgggtaccga 4740

gctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg 4800

ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa 4860

ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat 4920

tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc 4980

gcgcggtgtc atctatgtta ctagatcgg 5009

<210> 5

<211> 3787

<212> DNA

<213> Artificial Sequence

<400> 5

ttttgaatga gcttattaaa ttatatataa agtatataca accaatatgc atattttctt 60

gtttatattc acctaataat aaccaatgaa ataataaaat acaaatgtat attattttcc 120

ttttggtcac aaatgtattt tcttttccct tcttatatta tatacacaat aaaataaaat 180

aaaaatatga aattatcctt tttttgtgtg gacattttat catttaagaa aatgaaatta 240

ataattctaa attactatct tattacaaat gaaaattgta aattaattaa atatttacaa 300

gcatccatat aattttggtc attcaatata attcctaaat actaaaaact attgactttt 360

tttttttttt tttttttttt tttgtaaacc ctaaaaacta ttgactaaaa aaaagtattt 420

taacagaaaa agaagagaat atataatttt aatgtttgac cacaaaggga aaaaaatatc 480

tcaaagtttt ttctttcttt ggtatatcgg acggtatttg tgtggccagg gagggctacc 540

tgagactcca tatctgtctc tcttcttctt cttcttcctc ttcttcctct gcttcttctt 600

cttcacatca ctgtgtgcgt ctctctatct ctctattcta tcatttcgaa atcctctcta 660

tccgaatttg aactcgtctc ttcttcgtaa agagttctta atctccgtct ctgtgatttc 720

ggaatctgga ggttttgaat ttcttgttcg tatcaatcaa atggtggatc gatttagtga 780

gattgcttta cctcattagc agtcgacatg acttctgctt cttccatgtc cgtgtctgtg 840

gaatgtgtga acatatgtaa tctaacgaaa ggagatggga atgcaagaag cgattgcagt 900

gctctctcct gtgcttggaa agctccaaga gcgttaactg ggtttctagc tagcactgct 960

catccacctg tgtgttctgt gtattcatgt ggcagaaatg gaagaaagag cagaatgaaa 1020

gcttgcgcct ggcagaggta tgaatatgaa gtaggctttt ctgaggctcc ttactttgta 1080

aatgtgagaa atatcttgaa gtccagatta tcttgtggtg gtcataaaag atgggaactg 1140

tattgcgtat cagctgaatc ttcttctggt gcatccagtg atgttaccgt cgaaacattg 1200

tgggaggacc ttttcccatc aatatcttat ctaccccgta aagaattaga atttgttcaa 1260

aagggcctta agttagcgtt tgaggcacat catggtcaaa agagacgtag tggggaacca 1320

ttcattatac atcccgttgc agttgctcgt atccttgggg aacttgaatt ggattgggag 1380

tctattgttg ctggattact acatgacaca gtcgaggata caaatttcat tacttttgaa 1440

aagatagaag aagagtttgg tgcaactgtg cgtcacatcg tagaagggga gaccaaggtg 1500

tcaaaactgg gaaagttaaa gtgtaaaacg gaaagtgaaa caatacaaga tgtaaaagca 1560

gatgatttgc ggcagatgtt tctggcgatg acagacgagg tccgcgtcat tattgtcaaa 1620

ctagctgacc ggttgcataa tatgcgaact ctctgccaca tgcctcccca taagcagtcc 1680

agcattgcag gggagacttt gcaggtcttt gctcctttag caaaattatt gggaatgtat 1740

tcaataaagt ctgaactgga aaatctgtct ttcatgtacg taagtgctga ggattatgat 1800

agagtcacta gcaggattgc taacctctac aaagagcatg aaaaagaact cactgaggca 1860

aacagaattt tggtgaaaaa gattgaagat gatcagtttc tggaccttgt gactgtgaat 1920

actgatgttc gatctgtttg caaggaaact tacagcatct acaaagctgc tctcaaatcg 1980

aaaggatcaa ttaatgatta caaccagatt gctcagcagt tacggattgt tgtaaagcca 2040

aaaccatctg taggggtcgg gcctttgtgc agtccacaac agatatgcta tcacgttctg 2100

gggcttgttc atgagatctg gaaacctatt ccacgaacag taaaagatta cattgcaacc 2160

ccgaagccca atggatacca gagcctccat actactgtga ttccattctt gtatgagagt 2220

atgtttcgac tggaggttca gatcagaacc gaggagatgg acttgattgc tgaaaggggc 2280

attgctgttt actacaatgg caagtctcta tctactggat tagttggaaa cgcggttcct 2340

ttaggtagaa attcaagggg gaagacgggt tgcctcaaca atgcagattt tgcactcagg 2400

gttgggtggc taaatgcaat aagggaatgg caagaggagt ttgtgggtaa catgagctct 2460

agagaatttg tggataccat tacgagggat cttttaggta gtcgtgtgtt tgtattcaca 2520

cctaaaggag agataaagaa cctcccgaaa ggggccaccg ttgttgacta cgcttatctg 2580

attcacaccg aaatcggaaa caagatggta gcagcaaagg tcaatggtaa tcttgtttcc 2640

ccaactcacg ttcttgagaa tgctgaggtc gtggagatag tcacctacaa cgccctctca 2700

agtaaatctg ctttccaaag acataaacag tggttgcaac atgccaaaac aaggagtgca 2760

agacacaaga ttatgaggtt cctaagggag caagctgcac aatgtgctgc cgaaattacc 2820

caggatcaag tgaatgactt tgtggcggac tctgatagtg atgtggaaga tctcacagaa 2880

gattcaagaa agagcctaca atggtgggag aaaatcctcg tcaatgttaa gcaattccag 2940

tcacaagaca aaagtagaga tacaacaccc gctcctcaaa acggaagcgt ttgggcccca 3000

aaggtgaatg gaaaacacaa caaagccata aagaactcga gttctgatga gccagagttc 3060

ctcctacctg gagatggaat tgccaggatt ttacctgcta atatccctgc ttataaggaa 3120

gtgttgcccg gcttagacag ttggcgagac agtaaaattg ccacatggca tcatctcgaa 3180

ggtcagtcca tcgaatggtt atgtgtagta tccatggatc gcaaaggcat aatcgcagag 3240

gttacaacag tcctcgcagc tgaaggcatt gcattatgtt cttgcgtggc cgagattgac 3300

agaggaagag gattagcagt aatgttattt caaatagaag caaacattga aagtttggtt 3360

agtgtttgcg ctaaggtgga tttagtcttg ggtgtgttgg gatggtccag tggttgtagc 3420

tggccaagat caacagagaa tgctcaagtt cttgagtgtc ccggggatta caaggatgac 3480

gacgataagt gctaagctag cggatccccg ggtaccgagc tcgaatttcc ccgatcgttc 3540

aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat 3600

catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt 3660

atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga 3720

aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact 3780

agatcgg 3787

<210> 6

<211> 3829

<212> DNA

<213> Artificial Sequence

<400> 6

ttttgaatga gcttattaaa ttatatataa agtatataca accaatatgc atattttctt 60

gtttatattc acctaataat aaccaatgaa ataataaaat acaaatgtat attattttcc 120

ttttggtcac aaatgtattt tcttttccct tcttatatta tatacacaat aaaataaaat 180

aaaaatatga aattatcctt tttttgtgtg gacattttat catttaagaa aatgaaatta 240

ataattctaa attactatct tattacaaat gaaaattgta aattaattaa atatttacaa 300

gcatccatat aattttggtc attcaatata attcctaaat actaaaaact attgactttt 360

tttttttttt tttttttttt tttgtaaacc ctaaaaacta ttgactaaaa aaaagtattt 420

taacagaaaa agaagagaat atataatttt aatgtttgac cacaaaggga aaaaaatatc 480

tcaaagtttt ttctttcttt ggtatatcgg acggtatttg tgtggccagg gagggctacc 540

tgagactcca tatctgtctc tcttcttctt cttcttcctc ttcttcctct gcttcttctt 600

cttcacatca ctgtgtgcgt ctctctatct ctctattcta tcatttcgaa atcctctcta 660

tccgaatttg aactcgtctc ttcttcgtaa agagttctta atctccgtct ctgtgatttc 720

ggaatctgga ggttttgaat ttcttgttcg tatcaatcaa atggtggatc gatttagtga 780

gattgcttta cctcattagc aaagcttatg caacccccga cgggcgccgt gtcagggtcg 840

tcgtcgtcgt cgctggagtg cgtgagctcg tgcaggacgt cgtggagggg cggagggagg 900

ccgtacgaat gcagcgtgct ctcgtgcgcc tggaacgcgc cgcgggcgct cacgggggcg 960

ctcgccagca ccaccgcgca gtgctcctcc tgcagccacg ccgaggctgg ggcagggtgg 1020

aggcggcggg gccagtcgca gcggagtaac aattcgttac tgcacataac ctgggcagaa 1080

ggcatcaaca gggggaagtt tggttatggt tcatcagccc attcttttcc cactggaaat 1140

tttttcaaat cttggtcaac ttccgtggat ccaacatgga gagttttctg ctattcatca 1200

tctgaatcgt tcaatcatat ttctccagaa actttgtggg aggatctcaa gccagctatc 1260

tcgtaccttc aacctgaaga attgaacttt gtacacgatg ctctgaagtt ggcatatgag 1320

gcacataatg ggcagaaacg ccgaagtggg gaacctttca ttattcatcc tgttgaagtt 1380

gctcgtatcc ttggggagca tgaacttgac tgggaatcaa tagctgctgg tttattgcat 1440

gacactgttg aagatacaga catggttact tttgaaagaa tagaaaatga gtttggtgta 1500

accgtacgcc gtattgttga aggggagaca aaggtatcta agctaggaaa acttcagtgc 1560

aaaaatgagg gtaattcaaa acaggacgtc aaggctgaag atttaagaca gatgtttctt 1620

gccatgacag aagaggttcg tgtaatcatt gtgaaactgg cagacaggct gcataatatg 1680

cgtacactca cacatatgcc tcagcacaag cagtatgcca ttgccatgga gacactgcag 1740

gtttttgcac ccctagctaa acttctcggg atgtatcgaa taaagtctga gctagaatat 1800

ctgtccttca tgtacatgaa tcctggtgat ttcgctgaac taaagaaaag agttgaggac 1860

ctctacaagg cccatgaaca agaattggaa gaggcaaatc aaatcttagg ggaaaagatt 1920

gctgaggatc aatttcttga tcttgttagt gttgaaacac aagtgcgttc agtctgcaaa 1980

gaactctaca gcatttataa aactgcgctg aagtccaaga gttcaataaa tgagataaat 2040

caggttgcac agctgcggat catcataaag ccaaaatcct gcaatggtgt ggggccattg 2100

tgcactgcac aacagatctg ctaccatgtc cttggtcttg ttcatggtat ctggacacct 2160

atacctcaag ctgtgaaaga ttacattgca accccaaagc ctaatggata ccaaagtcta 2220

cacacgacag tgataccatt tctgaatgag agtatgttcc atttggaagt tcagataaga 2280

acagaggata tggatctcat agcagaaaga ggcattgctg cgcactacag tggaagaggg 2340

gttgtttcag ggcctgttcg tcctggaata tcaagtggaa ggaattcaaa tgggaaagtg 2400

atatgtctga acaatacagg ctttgctctg aggattggtt ggctcaatgc aatccgtgaa 2460

tggcaagaag agtttgttgg taacatgagt tctagggaat ttgttgatac tatcacccga 2520

gatcttctgg gaagccgtgt ctttgtgttt acaccaaaag gcgagattaa aaacttgcct 2580

aagggggcca ctgtggttga ttatgcttac ctgattcaca cagaaattgg gaacaaaatg 2640

gtagctgcaa aggtgaatgg caatctagtt tcaccaatcc atgtacttgc aaatgctgaa 2700

gttgtggaga ttataattta tgataaatta tctgctaaat atgcattcca gcgtcaccag 2760

cagtggcttc aacatgcgaa aactcgcagt gcaagacaca aaattatgaa attcttgcgg 2820

gagcaagctg ctctttctgc tgctgaaata actgccgacg ccgttaataa ctttgttgcc 2880

gatcttgaag atgaaagtga ctatgagcag tcgattccaa gctctgaaaa taaggactat 2940

acattcaact ggcagaagat attgaattct gacaaactat cctttggaaa caagaagagc 3000

gactgctttt tacctgttaa aaatgtctct gtgccaaagg ttaatgggaa gcacaacaaa 3060

actgtcaaag aactgggcat caaaattaat ggttcaacat ttcgtggtga tagcttcact 3120

gattttattc accctggtgt ttccagcagt aaagaagttc tccctagtgt ggataactgg 3180

aaagctggta aaatttgtgc gtggcacaat acagaaggca gctctatcca atggctctgc 3240

atagtgtgtg ttgatcgaaa aggtatggtt gcggaagttt catcagctct aacagcgtgt 3300

ggaatcacca tatgctcgtg tgtggcggag cgagataaga gaaggggaat aggtgtgatg 3360

ttgttccact ttgagggggc atatgagaat gtggtcagtg catgctccgg tgttgatatg 3420

attcttgggg ttcttggttg gtccgttgga tgcagctgta atcctttggg tgttcttgaa 3480

tgccccgggg attacaagga tgacgacgat aagtgctaag ctagcggatc cactagttct 3540

agaggatccc cgggtaccga gctcgaattt ccccgatcgt tcaaacattt ggcaataaag 3600

tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa 3660

ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt 3720

tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc 3780

aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatcgg 3829

Claims (5)

1. The application of ARE2 protein is as follows (c1) or (c2) or (c3): (c1) regulating plant tolerance to low nitrogen stress; (c2) Regulating the nitrogen metabolism process of plants; (c3) affect the balance between plant nitrogen metabolism and stress response; The ARE2 protein is the protein shown in SEQ ID NO: 3 of the sequence listing; the plant is rice. 2. a method for cultivating a transgenic plant with increased tolerance to low nitrogen stress, comprising the steps of: suppressing the expression of the gene encoding an ARE2 protein in a target plant to obtain a transgenic plant with increased tolerance to low nitrogen stress; The ARE2 protein is the protein shown in SEQ ID NO: 3 of the sequence listing; the plant is rice. 3. The method of claim 2, wherein the gene encoding the ARE2 protein is the following (b1) or (b2) or (b3) or (b4): (b1) a DNA molecule whose cDNA coding region is shown in sequence 2 in the sequence listing; (b2) The genomic DNA is the DNA molecule shown at positions 1581-12019 in Sequence 1 of the Sequence Listing; (b3) The genomic DNA is the DNA molecule shown at positions 1301-12512 in Sequence 1 of the Sequence Listing; (b4) The genomic DNA is a DNA molecule shown in SEQ ID NO: 1 in the sequence listing. 4. a method for cultivating a transgenic plant with increased tolerance to low-nitrogen stress, comprising the steps of: mutating the gene encoding the ARE2 protein in the target plant as a target gene to obtain an increased tolerance to low-nitrogen stress. Transgenic plant; the ARE2 protein is the protein shown in SEQ ID NO: 3 of the sequence listing; the plant is rice. 5. The method of claim 4, wherein the gene encoding the ARE2 protein is the following (b1) or (b2) or (b3) or (b4): (b1) a DNA molecule whose cDNA coding region is shown in sequence 2 in the sequence listing; (b2) The genomic DNA is the DNA molecule shown at positions 1581-12019 in Sequence 1 of the Sequence Listing; (b3) The genomic DNA is the DNA molecule shown at positions 1301-12512 in Sequence 1 of the Sequence Listing; (b4) The genomic DNA is a DNA molecule shown in SEQ ID NO: 1 in the sequence listing.

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