CN112725351A

CN112725351A - Application of gene OsWRKY43 in resisting bacterial blight of rice

Info

Publication number: CN112725351A
Application number: CN202110066315.6A
Authority: CN
Inventors: 明凤; 钟群; 余江涛; 毛婵娟; 娄玉霞
Original assignee: Shanghai Normal University
Current assignee: Shanghai Normal University
Priority date: 2021-03-23
Filing date: 2021-03-23
Publication date: 2021-04-30
Anticipated expiration: 2041-03-23
Also published as: CN112725351B

Abstract

The application discloses application of a gene OsWRKY43 in resisting bacterial leaf blight of rice, wherein the gene OsWRKY43 is a transcription factor induced by biotic stress in rice, and the gene OsWRKY43 is a WRKY family II type transcription factor; the protein sequence coded by the gene OsWRKY43 is shown in SEQ ID NO. 3.

Description

Application of gene OsWRKY43 in resisting bacterial blight of rice

Technical Field

The invention relates to the field of biological variety cultivation, in particular to application of a gene OsWRKY43 in resisting bacterial blight of rice.

Background

During the production process of rice, a plurality of factors influence the yield and the quality of the rice, wherein the invasion of pathogenic bacteria is a main factor and seriously influences the high and stable yield of the rice. The enhancement of disease resistance is an important guarantee for the yield and quality of rice, and the cultivation of disease-resistant high-yield varieties is always the target of rice breeding. With the maturity of gene editing and transgenic technology, high-quality gene resources become the main factor limiting the breeding of rice molecules. Therefore, the identification of important new genes participating in the rice disease-resistant process can not only perfect the disease-resistant mechanism of rice, but also provide new gene resources and theoretical basis for rice molecular breeding, and has very important practical significance for solving the grain problem.

Bacterial blight of rice is a worldwide bacterial disease, and it is called "three major diseases" of rice together with rice blast and sheath blight. It has wide generation range, fast popularity, great harm and high mutation, and causes great loss to rice production. The pesticide has certain effect on preventing and treating the bacterial leaf blight of rice, but can cause environmental pollution and destroy ecological balance. The method for preventing and treating the bacterial blight of the rice is the most economic, effective and environment-friendly means by utilizing natural disease-resistant genes to cultivate disease-resistant varieties.

Plant transcription factors are pivotal in disease-resistant signaling pathways. When plants sense the signals of invading pathogenic bacteria, after a series of signal transmission events, the transcription factor is activated, and the activated transcription factor and the cis-acting element in the corresponding gene promoter generate DNA-protein specific interaction in the nucleus, thereby activating the transcription expression of genes related to defense response. Among the many plant transcription factors, the disease resistance response is also associated with more research and identification, especially the transcription factor associated with bacterial blight of rice.

Because the resistance of the crop varieties is lost due to the small species genetic variation of the bacterial blight, the bacterial blight resistance resources of the rice are in short supply at present, and the disease resistance of the plants generally comes at the cost of sacrificing the growth quantity, so more resistance-related genes, in particular key genes for regulating and controlling both yield increase and disease resistance, need to be identified and cloned.

Disclosure of Invention

In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a gene fragment capable of resisting bacterial blight of rice. Therefore, the technical personnel in the field are dedicated to developing the gene OsWRKY43 and the application of the protein coded by the gene OsWRKY43 in the disease resistance of rice bacterial blight.

On one hand, the application provides application of a gene OsWRKY43 in resisting bacterial blight of rice, wherein the gene OsWRKY43 is a transcription factor induced by biotic stress in rice, and the gene OsWRKY43 is a WRKY family II type transcription factor.

In certain embodiments, inoculation of a rice plant with a strain of paraquat enables the expression of the gene OsWRKY43 to be upregulated.

In certain embodiments, the mutation of the base T at the 627 position of the coding region of the gene OsWRKY43 to A and the mutation of the corresponding encoded serine to isoleucine can reduce the anti-bacterial leaf blight function of the gene OsWRKY 43.

In certain embodiments, the coding region of the gene OsWRKY43 is obtained by PCR using the primer sequences shown in SEQ ID NO.5 and 6.

In certain embodiments, the protein sequence encoded by the gene OsWRKY43 is set forth in SEQ ID No. 3.

In certain embodiments, the coding sequence of the gene OsWRKY43 is set forth in SEQ ID No. 2.

In certain embodiments, the gene OsWRKY43 has the sequence shown in SEQ ID No. 1.

On the other hand, the application also provides a method for improving the bacterial leaf blight resistance of rice, which comprises introducing a gene OsWRKY43 into the rice, wherein a protein sequence coded by the gene OsWRKY43 is shown as SEQ ID No. 3.

In certain embodiments, wherein the coding sequence of the gene OsWRKY43 is in a vector that enables its expression in rice.

On the other hand, the application also provides application of the protein in resisting bacterial blight of rice, wherein the protein is encoded by a gene OsWRKY43, and the sequence of the protein is shown as SEQ ID No. 3.

On the other hand, the application also provides application of the gene OsWRKY43 in tobacco pathogenic bacteria infection resistance of phytophthora capsici PC35, wherein a protein sequence coded by the gene OsWRKY43 is shown in SEQ ID No. 3.

The transcription factor plays an important role in plant growth and development and stress, and one transcription factor can often regulate and control the expression of a plurality of related genes at the downstream, so that compared with a method for improving the stress resistance of a certain aspect of crops by only introducing a single gene, the method for improving the stress resistance and disease resistance of the crops by using the plant transcription factor through genetic engineering is probably a more effective way. By cloning a transcription factor OsWRKY43 induced by biotic stress in rice and analyzing the gene sequence, OsWRKY43 belongs to WRKY family II type transcription factor and has the closest relationship with OsWRKY1 and OsWRKY 5. The time-space expression mode and the stress response mode are determined, and the result shows that the gene is constitutively expressed in each organ, the expression level is lowest in roots, and the expression level is highest in young ears in the middle development stage. After the rice is treated by IAA, 6-BA, KT and 2,4-D, NAA, the expression level of OsWRKY43 in rice plants treated by 6-BA and NAA is obviously increased. After inoculation of bacterial blight microspecies by taking Nipponbare as a material, the expression level of OsWRKY43 rapidly rises, and OsWRKY43 is also significantly induced by signal hormone SA. In oswrky43-1H10 mutant with Nipponbare as genetic background of rice, after inoculation of small seeds of paraquat, the length of lesion spots of mutant strains is observed to be longer than that of wild type, and the expression quantity of disease-resistant related genes PR1a and PR1b is reduced, which provides gene source and technical support for rice defense reaction mechanism.

The rice gene OsWRKY43 provided by the application can be used for improving plant varieties, such as improving the ability of rice to resist bacterial blight, thereby increasing the yield of rice.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 shows a graph of the results of the analysis and detection of the OsWRKY43 gene organ expression pattern of rice in the present application;

FIG. 2 shows an analysis chart of an expression pattern of a rice gene OsWRKY43 after inoculation of bacterial blight in the present application;

FIG. 3 shows an analysis chart of the expression pattern of the rice gene OsWRKY43 after phytohormone treatment;

FIG. 4 shows an analysis chart of the expression pattern of the rice gene OsWRKY43 after signal hormone treatment;

FIG. 5 shows a schematic representation of the resistance of OsWRKY43 mutant and wild type rice seedling to bacterial blight herein;

FIG. 6 shows the analysis and detection of expression patterns of disease-resistant genes of OsWRKY43 mutant and wild rice seedlings in the application;

FIG. 7 is a graph showing the results of the detection of disease resistance by OsWRKY43 in tobacco.

Detailed Description

The present invention will now be further described with reference to examples, which are intended to be illustrative only, and the present invention may be embodied in many different forms of embodiments, and the scope of the present invention is not limited to the embodiments set forth herein.

Materials, reagents and the like used in the examples of the present application are commercially available unless otherwise specified.

The following description is made in this application with respect to nouns or acronyms: 6-BA: 6-benzylaminopurine; NBA: n-bromoacetamide; 2, 4-D: 2, 4-dichlorophenoxyacetic acid; IAA: indole-3-acetic acid; KT: a kinetin; and SA: salicylic acid; JA: abscisic acid.

The wild rice varieties used in the embodiment of the application are all Nipponbare, and the oswrky43-1H10 mutant takes the Nipponbare as the genetic background of rice and is provided by Western Meijie science and technology Co. Nipponbare (Oryza. Sativa L. spp. japonica, var Nipponbare, AA genome) is the progeny of the cross between "mountain-shaped cells" and "Happy wind", a rice variety, belonging to the subspecies Japonicae. The ear is small, the plant type is vertical, the leaves are wide, the plant grows luxuriantly in high-temperature areas, the grain attachment is thin, the ear weight is poor, the seed is firm and full, the grain is not easy to fall off, and the grains have short awns. Fertilizer resistance: strong stalk, lodging like "jinnanfeng", less leaf scattering under the condition of multiple fertilizers, and suitability for fertile land and high-fertilizer cultivation, plantlet transplanter cultivation, direct seeding cultivation and the like. Resistance to pests: the rice stem borer has weak resistance to stripe disease and flax leaf spot, moderate resistance to other diseases, strong resistance to bacterial leaf blight and resistance to rice stem borer. Is suitable for low-wetland cultivation. The phenomenon of sprouting of the ear rarely occurs. Yield: under the conditions of fertile land and high-fertilizer cultivation, the thousand-grain weight is about 22 g, the abdomen is slightly white, the color is rich, the quality and the appearance are good, and the taste is good.

The fine rice plants used in this application and the bacterial blight of rice (Xanthomonas oryzae pv. oryzae) are commercially available and also available from Shanghai university.

The following examples do not show specific experimental methods, and can be carried out according to conventional methods. Molecular cloning as in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the instructions of the manufacturer.

Example 1 obtaining of Rice Gene OsWRKY43

(1) The rice variety Nipponbare was cultivated in an incubator (SPX-250-GB, Shanghai, China): the growth conditions were photoperiod 16h/8h (L/D), 28 ℃.

(2) DNA extraction: adding about 500mg of fresh rice plant tissue material into 80 μ L of lysis solution, grinding plant tissue with grinding rod, adding 120 μ L of ddH₂And O. Centrifuging at 12000rpm for 15min, transferring the supernatant into a new centrifuge tube, measuring the OD value, and carrying out electrophoresis detection.

(3) Cloning genes: the extracted rice DNA is used as a template, PCR is carried out to obtain the full length of the gene, the sequencing result shows that the full length is 2480bp, and the sequence is shown as SEQ ID NO. 1. PCR is carried out by taking cDNA obtained by reverse transcription as a template through primers shown in SEQ ID NO.5 and 6 to obtain an open reading frame (coding sequence) of 1764bp, wherein the sequence is shown in SEQ ID NO. 2; the coded protein contains 587 amino acid residues, and the amino acid sequence is shown in SEQ ID NO. 3.

Example 2 analysis of different organ expression patterns of OsWRKY43 Gene in Rice

Respectively extracting total RNA in roots, stems, leaves and ears at different periods of rice development, carrying out reverse transcription on the total RNA into cDNA by using a reverse transcription kit, and carrying out Real-time PCR detection by using primers SEQ ID NO.7 and SEQ ID NO.8, as shown in figure 1. The results showed that the gene was expressed in the roots in the lowest amount and in the young ears in the middle of development in the highest amount.

Example 3 analysis of expression Pattern of Rice Gene OsWRKY43 under infection with Paraquat and treatment with phytohormone and Signal hormone

Bacterial strain of Paraquat: dipping bacterial liquid with OD value of 1 in rice seedlings with two weeks for bacterial strain inoculation by using a leaf top shearing method, processing for six time periods of 0h, 2h, 4h, 8h, 12h and 24h to sample leaves, and extracting total RNA in the leaves;

plant hormones: 50 mu M of 6-BA, NAA, 2,4-D, IAA or KT is soaked in the rice seedlings of two weeks for 12 hours, the leaves are sampled at intervals of 2 hours, 4 hours, 8 hours and 12 hours, and the total RNA in the leaves is extracted. Analysis of results was performed using ANOVA (SPSS) analysis.

Signal hormone: 50 mu M of SA, JA and chito-oligosaccharide are respectively soaked in rice seedlings with two weeks, leaves are sampled every 0h, 0.5h, 1h, 4h, 6h, 12h and 24h, and total RNA in the leaves is extracted.

The total RNA obtained above was reverse transcribed into cDNA using a reverse transcription kit in the manner of example 2, and Real-time PCR detection was performed using primers SEQ ID NO.7 and SEQ ID NO. 8. As shown in FIG. 2, the expression level of OsWRKY43 increased rapidly after inoculation of bacterial blight, reached the highest expression level at 2h, and then decreased gradually with the passage of time. In order to study whether OsWRKY43 is regulated by plant hormone, plant hormone treatment is carried out, and the result shows that OsWRKY43 is regulated by 6-BA, NAA, 2,4-D, IAA and KT, and is shown in figure 3. In fig. 3, the letters a, b, c, and d are compared with each other to show whether the difference is significant, a, b, c, and d in each treatment group are different from each other and have significant difference from each other, the response is a significant level of 5%, and the letters are labeled to show that the letters are not different from each other. In order to study whether OsWRKY43 is induced by SAR reaction mechanism, signal-related hormone treatment is carried out, and the results show that OsWRKY43 is significantly induced by SA and the expression level of OsWRKY43 is up-regulated, and are shown in FIG. 4.

Example 4 molecular characterization of OsWRKY43 mutants

Soaking the mutant strain and the wild type seeds in water for 24h to allow the seeds to be fully imbibed, then placing the seeds in a glass culture dish, placing the glass culture dish in a rice constant-temperature culture box for accelerating germination to be white, changing water every day to prevent mildew growth, selecting the seeds with consistent germination, transferring the seeds to a seed rack, placing the seed rack in basic nutrient solution, and culturing in a culture box at 28 ℃.

And respectively extracting total DNA in the mutant and the wild control group, and carrying out PCR detection by using primers SEQ ID NO.9 and SEQ ID NO. 10. The results show that the mutant oswrky43-1H10 (shown as SEQ ID NO. 4) is located at the position 627 of a coding region, TCC is changed into ACC, serine is changed into isoleucine, and homozygous mutants are selected and propagated.

Example 5 analysis of disease resistance of OsWRKY43 mutant

Experimental group of OsWRKY43 mutants: oswrky43-1H10 mutant rice seedlings at heading stage, control group: wild-type rice seedlings in heading stage. The OsWRKY43 mutant and wild type Nipponbare were inoculated with a strain of paraquat in the manner described in example 3, and then phenotypic analysis was performed three weeks later, and the length of lesions was measured and photographed. The method comprises the following specific steps:

(1) soaking the mutant and wild seeds in water for 24h to allow the seeds to fully absorb and expand, placing the seeds in a culture dish, accelerating germination in a constant-temperature incubator at 37 ℃ until the seeds are exposed to the white, and changing water every day to prevent mildew;

(2) uniformly scattering the seeds after germination acceleration on a seedbed, wherein the surface can not accumulate water, and after the seeds are sowed, leveling the seeds by hands to ensure that the seeds are sunk into mud and are not too deep;

(3) after 3-4 weeks of seedling culture, three-leaf seedlings can be transplanted with developed root systems, which can cause root damage and later-stage dysplasia too early, and a small amount of 0.5g urea per seedling can be applied 1-2 weeks after seedling transplantation, and water is generally placed in a greenhouse for 1 day in advance and then watered;

(4) in heading stage, inoculating bacterial strain of Paraquat by using leaf top shearing method, and observing lesion length.

As shown in FIG. 5, the length of the lesion of the OsWRKY43 mutant (1H10) was longer than that of the wild-type (Nip), i.e., the resistance to bacterial blight was weak.

Example 6 modification of expression level of disease-resistance-associated Gene of OsWRKY43 mutant

Plants were grown as in example 5. The OsWRKY43 mutant and two-month-old leaves of Nipponbare strains are selected, the total RNA obtained by the two-month-old leaves is reversely transcribed into cDNA by using a reverse transcription kit, and Real-time PCR detection is carried out by using primers SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO. 14. As shown in FIG. 6, the expression level of the disease-resistant related genes PR1a and PR1b of OsWRKY43 mutant rice is lower than that of the wild type rice.

Example 7 OsWRKY43 heterologous host transient overexpression disease resistance identification

Experimental group (35S: OsWRKY 43): OsWRKY43 was constructed on a 35S: pEarley Gateway overexpression vector (purchased from Shanghai Bioengineering services, Inc.), control group (35S: EV): no OsWRKY43 gene connected 35S, pEarley Gateway over-expression vector. Transforming by agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.), injecting transformed bacteria liquid into tobacco for in vivo transient expression, inoculating phytophthora capsici PC35 tobacco pathogenic bacteria (purchased from Shanghai Bohu Biotechnology Co., Ltd.) after 24h, measuring the lesion area after three days, and taking a picture. The method comprises the following specific steps:

(1) selecting healthy tobacco plants with consistent growth vigor, infecting the tobacco plants after the OsWRKY43 constructed on a 35S pEarley Gateway carrier is transformed into agrobacterium tumefaciens, and repeating the steps for three times by taking an empty carrier as a control group;

(2) selecting infected tobacco leaves after 24 hours, and placing the infected tobacco leaves in a culture dish;

(3) placing phytophthora capsici PC35 tobacco pathogenic bacteria growing in a solid culture medium of V8+ Amp with the same volume and size on the surface of the selected tobacco leaves for inoculation;

(4) the inoculated leaves were cultured at 28 ℃ for three days, and the lesion condition was observed.

The result is shown in fig. 7, after the gene OsWRKY43 is transiently overexpressed in tobacco leaves, the lesion area is obviously smaller than that of a control group, and the gene OsWRKY43 can improve the disease resistance of plants; however, after the rice bacterial blight is infected by the rice, a host can generate a series of immune reactions, wherein the disease resistance mechanism of the effective protein in the rice is similar to the disease resistance mechanism of the phytophthora capsici serving as a fungus in the embodiment with a specific effector activating the plant immune system, so the experiment also proves the disease resistance effect of the OsWRKY43 gene on the rice bacterial blight.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

<110> Shanghai university of Master

Application of gene OsWRKY43 in resisting bacterial blight of rice

<130> CN015-20011PICN

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 2480

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsWRKY43 gene sequence

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atggagtcct acgtgggtgt caaggggaag aatgtcgtcg ggggcggcga cgtcggcagg 60

gagatgccgg tggcgccacc gtcgtcgtcg tcggcggcgg tagggatggt ggagttcccg 120

gcggcggcgg cggggctggg gtacgccggg atgacggcga aggaggccgg gggtggttac 180

caggagagga gggtggtggt cggcgagatg gacttcttca agacggcgga gaagcgtggg 240

gagaggaagg agccgccgcc ggcgacggcg acggcggcgg cgagtggaca cgccggtgcc 300

tcgccggatg acctcagcct caacaaggac gacctcacca tcaatgtaac ggatcagttg 360

atgcgtgatg gtttgagaga tttttttttc ttggttgatg cgtggtttaa tttcgtgatc 420

ttgttgcgtc gcagatggga ttgctcgtcg gccggaggag gaacagcggc agcgaggaat 480

ccatcgtcga cgatggcggc gtctcctcca acgacgagga gcaccgcgag gccaaagctg 540

cggtgagcaa acagcagcaa aatcaatagg atctctctgt gtttataaat tcgcttttga 600

atttctgaat gcgaggagga gagatcggtc tagtgaccgt ttgccgtttt cttcatcatc 660

agaaattcta gttagtgtaa accggagaac aacaaaaatt gttgttgcca tgatctgatc 720

ataattttct ctcggaattt atgatcagct ggcagtaacc aaagctgaga ttgggcggct 780

gagtgaggag aacaagaggc tcaagaacat gctgagcaac gtgaccacga aatacaactc 840

tctccagatg cagttcgtca cactgatgca gcagaggaga tcagtcctag ccgctccgat 900

ccaccagcaa gaggtaatta agaaaaaaga acaaatcata attattacca tgcaagattc 960

aagatcgctc atggcagcaa tgctcaagat cgctcatgtg gtgtaattca tcgacgattt 1020

gtatgatcgg cagctgcttg atccggagaa gaaggagcaa gaggggagcc agcagcagca 1080

gcagcagctg atcccgaggc agttcatcag cctcggctcc gcgtcgctgc agcccgacgt 1140

cgaggcgccg cactccgtcg tcgtcgtcgg cggcgatgtc tgcgcgccgt cgtcgagcaa 1200

ccccgacgcg gcggtgccgg cgatgatgcc gttgccgcat ttcgaccacc acaaccacca 1260

ccaccccatc catggtggaa gggagagggg gagcagcccg gcggaggccg accaccaccg 1320

ccaccatcag caagagcagc cgccgccgcc gccgcagcag cagcagcagt tgccgccgag 1380

ctggcttccc gccgacaagg tgcccaggtt cctccccggc aaggggcccg agcccgtccc 1440

ggaggccgcc accatgcgca aggcccgcgt ctccgttcga gctcgctccg atgcacccat 1500

ggtgtgtgtg tgtgtgtgct cgatcatgtt ccatatgcag ctagtgttct tcttcttcgc 1560

atggatgttt gatatggtga tgtgcagatc agcgatgggt gccaatggag gaagtacggg 1620

cagaagatgg cgaagggaaa cccttgcccg cgcgcgtact accggtgcac catggccgcc 1680

ggctgcccgg tgcggaagca ggtgtgtgtg caagatcgcc ggagcgtacg agcgtgtttg 1740

atttgctgtg tcgtcgtcgt cgtcaatggc gcgttcgctg atgtgcggcc atggttttgc 1800

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accacccgct gccgccggcg gcgatggcga tggcgtcgac cacggcggcg gcggcgtcca 1920

tgctgctgtc gggctccatg ccgagcgcgg acggcagcct catggccggg tccaacttcc 1980

tggcgcgcgc cgtgctgccg tgctcgtcca ccgtcgccac catctcggcg tcggcgccgt 2040

tcccgacggt cacgctcgac ctcacgcaga cggcgccgcc gccgccgccg gcgtcgtcga 2100

cgcagccgca gccgccgcgc ccggagcccg cgcagctcca ggcggcgctc gccgaggccg 2160

cgcggccggt ggcgctgccc cagctgttcg ggcagaagct ctacgaccag tccaagctct 2220

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cgctgacgtc gtacataggc agtagaagcg gcagcggcgg cgccggcgcc ggcgggagca 2400

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taggagagca aaacagctaa 2480

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<223> OsWRKY43 coding region sequence

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atggagtcct acgtgggtgt caaggggaag aatgtcgtcg ggggcggcga cgtcggcagg 60

gagatgccgg tggcgccacc gtcgtcgtcg tcggcggcgg tagggatggt ggagttcccg 120

gcggcggcgg cggggctggg gtacgccggg atgacggcga aggaggccgg gggtggttac 180

caggagagga gggtggtggt cggcgagatg gacttcttca agacggcgga gaagcgtggg 240

gagaggaagg agccgccgcc ggcgacggcg acggcggcgg cgagtggaca cgccggtgcc 300

tcgccggatg acctcagcct caacaaggac gacctcacca tcaatatggg attgctcgtc 360

ggccggagga ggaacagcgg cagcgaggaa tccatcgtcg acgatggcgg cgtctcctcc 420

aacgacgagg agcaccgcga ggccaaagct gcgctggcag taaccaaagc tgagattggg 480

cggctgagtg aggagaacaa gaggctcaag aacatgctga gcaacgtgac cacgaaatac 540

aactctctcc agatgcagtt cgtcacactg atgcagcaga ggagatcagt cctagccgct 600

ccgatccacc agcaagagct gcttgatccg gagaagaagg agcaagaggg gagccagcag 660

cagcagcagc agctgatccc gaggcagttc atcagcctcg gctccgcgtc gctgcagccc 720

gacgtcgagg cgccgcactc cgtcgtcgtc gtcggcggcg atgtctgcgc gccgtcgtcg 780

agcaaccccg acgcggcggt gccggcgatg atgccgttgc cgcatttcga ccaccacaac 840

caccaccacc ccatccatgg tggaagggag agggggagca gcccggcgga ggccgaccac 900

caccgccacc atcagcaaga gcagccgccg ccgccgccgc agcagcagca gcagttgccg 960

ccgagctggc ttcccgccga caaggtgccc aggttcctcc ccggcaaggg gcccgagccc 1020

gtcccggagg ccgccaccat gcgcaaggcc cgcgtctccg ttcgagctcg ctccgatgca 1080

cccatggtcc agaggtgcgc ggaggacagg acggtgctga taacgacgta cgaggggaac 1140

cacaaccacc cgctgccgcc ggcggcgatg gcgatggcgt cgaccacggc ggcggcggcg 1200

tccatgctgc tgtcgggctc catgccgagc gcggacggca gcctcatggc cgggtccaac 1260

ttcctggcgc gcgccgtgct gccgtgctcg tccaccgtcg ccaccatctc ggcgtcggcg 1320

ccgttcccga cggtcacgct cgacctcacg cagacggcgc cgccgccgcc gccggcgtcg 1380

tcgacgcagc cgcagccgcc gcgcccggag cccgcgcagc tccaggcggc gctcgccgag 1440

gccgcgcggc cggtggcgct gccccagctg ttcgggcaga agctctacga ccagtccaag 1500

ctctccgccg tgcaggccgt ggccggcacc aagggctccg acggcggcgc gctcgccgac 1560

acggtgaacg ccgcgacggc cgcgatcgcg tcggacccaa acttcaccgc cgtgctcgcc 1620

gccgcgctga cgtcgtacat aggcagtaga agcggcagcg gcggcgccgg cgccggcggg 1680

agcagcggca cggtgcagcc gctgatgagc ggcggcggcg atagctgtag tagagacgac 1740

aagataggag agcaaaacag ctaa 1764

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsWRKY43 encoding protein sequence

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Met Glu Ser Tyr Val Gly Val Lys Gly Lys Asn Val Val Gly Gly Gly

1 5 10 15

Asp Val Gly Arg Glu Met Pro Val Ala Pro Pro Ser Ser Ser Ser Ala

20 25 30

Ala Val Gly Met Val Glu Phe Pro Ala Ala Ala Ala Gly Leu Gly Tyr

35 40 45

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

50 55 60

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

65 70 75 80

Glu Arg Lys Glu Pro Pro Pro Ala Thr Ala Thr Ala Ala Ala Ser Gly

85 90 95

His Ala Gly Ala Ser Pro Asp Asp Leu Ser Leu Asn Lys Asp Asp Leu

100 105 110

Thr Ile Asn Met Gly Leu Leu Val Gly Arg Arg Arg Asn Ser Gly Ser

115 120 125

Glu Glu Ser Ile Val Asp Asp Gly Gly Val Ser Ser Asn Asp Glu Glu

130 135 140

His Arg Glu Ala Lys Ala Ala Leu Ala Val Thr Lys Ala Glu Ile Gly

145 150 155 160

Arg Leu Ser Glu Glu Asn Lys Arg Leu Lys Asn Met Leu Ser Asn Val

165 170 175

Thr Thr Lys Tyr Asn Ser Leu Gln Met Gln Phe Val Thr Leu Met Gln

180 185 190

Gln Arg Arg Ser Val Leu Ala Ala Pro Ile His Gln Gln Glu Leu Leu

195 200 205

Asp Pro Glu Lys Lys Glu Gln Glu Gly Ser Gln Gln Gln Gln Gln Gln

210 215 220

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

225 230 235 240

Asp Val Glu Ala Pro His Ser Val Val Val Val Gly Gly Asp Val Cys

245 250 255

Ala Pro Ser Ser Ser Asn Pro Asp Ala Ala Val Pro Ala Met Met Pro

260 265 270

Leu Pro His Phe Asp His His Asn His His His Pro Ile His Gly Gly

275 280 285

Arg Glu Arg Gly Ser Ser Pro Ala Glu Ala Asp His His Arg His His

290 295 300

Gln Gln Glu Gln Pro Pro Pro Pro Pro Gln Gln Gln Gln Gln Leu Pro

305 310 315 320

Pro Ser Trp Leu Pro Ala Asp Lys Val Pro Arg Phe Leu Pro Gly Lys

325 330 335

Gly Pro Glu Pro Val Pro Glu Ala Ala Thr Met Arg Lys Ala Arg Val

340 345 350

Ser Val Arg Ala Arg Ser Asp Ala Pro Met Val Gln Arg Cys Ala Glu

355 360 365

Asp Arg Thr Val Leu Ile Thr Thr Tyr Glu Gly Asn His Asn His Pro

370 375 380

Leu Pro Pro Ala Ala Met Ala Met Ala Ser Thr Thr Ala Ala Ala Ala

385 390 395 400

Ser Met Leu Leu Ser Gly Ser Met Pro Ser Ala Asp Gly Ser Leu Met

405 410 415

Ala Gly Ser Asn Phe Leu Ala Arg Ala Val Leu Pro Cys Ser Ser Thr

420 425 430

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

435 440 445

Leu Thr Gln Thr Ala Pro Pro Pro Pro Pro Ala Ser Ser Thr Gln Pro

450 455 460

Gln Pro Pro Arg Pro Glu Pro Ala Gln Leu Gln Ala Ala Leu Ala Glu

465 470 475 480

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

485 490 495

Asp Gln Ser Lys Leu Ser Ala Val Gln Ala Val Ala Gly Thr Lys Gly

500 505 510

Ser Asp Gly Gly Ala Leu Ala Asp Thr Val Asn Ala Ala Thr Ala Ala

515 520 525

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

530 535 540

Ser Tyr Ile Gly Ser Arg Ser Gly Ser Gly Gly Ala Gly Ala Gly Gly

545 550 555 560

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

565 570 575

Ser Arg Asp Asp Lys Ile Gly Glu Gln Asn Ser

580 585

<210> 4

<211> 1764

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsWRKY43 coding region mutant sequence

<400> 4

atggagtcct acgtgggtgt caaggggaag aatgtcgtcg ggggcggcga cgtcggcagg 60

gagatgccgg tggcgccacc gtcgtcgtcg tcggcggcgg tagggatggt ggagttcccg 120

gcggcggcgg cggggctggg gtacgccggg atgacggcga aggaggccgg gggtggttac 180

caggagagga gggtggtggt cggcgagatg gacttcttca agacggcgga gaagcgtggg 240

gagaggaagg agccgccgcc ggcgacggcg acggcggcgg cgagtggaca cgccggtgcc 300

tcgccggatg acctcagcct caacaaggac gacctcacca tcaatatggg attgctcgtc 360

ggccggagga ggaacagcgg cagcgaggaa tccatcgtcg acgatggcgg cgtctcctcc 420

aacgacgagg agcaccgcga ggccaaagct gcgctggcag taaccaaagc tgagattggg 480

cggctgagtg aggagaacaa gaggctcaag aacatgctga gcaacgtgac cacgaaatac 540

aactctctcc agatgcagtt cgtcacactg atgcagcaga ggagatcagt cctagccgct 600

ccgatccacc agcaagagct gcttgaaccg gagaagaagg agcaagaggg gagccagcag 660

cagcagcagc agctgatccc gaggcagttc atcagcctcg gctccgcgtc gctgcagccc 720

gacgtcgagg cgccgcactc cgtcgtcgtc gtcggcggcg atgtctgcgc gccgtcgtcg 780

agcaaccccg acgcggcggt gccggcgatg atgccgttgc cgcatttcga ccaccacaac 840

caccaccacc ccatccatgg tggaagggag agggggagca gcccggcgga ggccgaccac 900

caccgccacc atcagcaaga gcagccgccg ccgccgccgc agcagcagca gcagttgccg 960

ccgagctggc ttcccgccga caaggtgccc aggttcctcc ccggcaaggg gcccgagccc 1020

gtcccggagg ccgccaccat gcgcaaggcc cgcgtctccg ttcgagctcg ctccgatgca 1080

cccatggtcc agaggtgcgc ggaggacagg acggtgctga taacgacgta cgaggggaac 1140

cacaaccacc cgctgccgcc ggcggcgatg gcgatggcgt cgaccacggc ggcggcggcg 1200

tccatgctgc tgtcgggctc catgccgagc gcggacggca gcctcatggc cgggtccaac 1260

ttcctggcgc gcgccgtgct gccgtgctcg tccaccgtcg ccaccatctc ggcgtcggcg 1320

ccgttcccga cggtcacgct cgacctcacg cagacggcgc cgccgccgcc gccggcgtcg 1380

tcgacgcagc cgcagccgcc gcgcccggag cccgcgcagc tccaggcggc gctcgccgag 1440

gccgcgcggc cggtggcgct gccccagctg ttcgggcaga agctctacga ccagtccaag 1500

ctctccgccg tgcaggccgt ggccggcacc aagggctccg acggcggcgc gctcgccgac 1560

acggtgaacg ccgcgacggc cgcgatcgcg tcggacccaa acttcaccgc cgtgctcgcc 1620

gccgcgctga cgtcgtacat aggcagtaga agcggcagcg gcggcgccgg cgccggcggg 1680

agcagcggca cggtgcagcc gctgatgagc ggcggcggcg atagctgtag tagagacgac 1740

aagataggag agcaaaacag ctaa 1764

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsWRKY43 coding sequence forward primer

<400> 5

atggagtcct acgtgggtgt caagg 25

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsWRKY43 coding sequence reverse primer

<400> 6

ttagctgttt tgctctccta tcttg 25

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> detection of Forward primer 1 by PCR

<400> 7

cgatccacca gcaagagc 18

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PCR detection of reverse primer 1

<400> 8

gaactgcctc gggatcag 18

<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PCR detection of Forward primer 2

<400> 9

gcaagagctg cttgaaccgg agaagaagga g 31

<210> 10

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PCR detection of reverse primer 2

<400> 10

ctccttcttc tccggttcaa gcagctcttg c 31

<210> 11

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsPR1a Forward primer

<400> 11

acctcggcgt cttcatca 18

<210> 12

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsPR1a reverse primer

<400> 12

gagattggcc gacgaagtt 19

<210> 13

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsPR1b Forward primer

<400> 13

ggtgacctgg gacacgag 18

<210> 14

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> OsPR1b reverse primer

<400> 14

gttgctggag tggatcagg 19

Claims

1. Application of gene OsWRKY43 in resisting bacterial blight of rice, wherein the gene OsWRKY43 is a transcription factor induced by biotic stress in rice, and the gene OsWRKY43 is a WRKY family II type transcription factor.

2. The use as claimed in claim 1, wherein inoculation of the rice plant with a strain of paraquat enables up-regulation of the expression of the gene OsWRKY 43.

3. The use as claimed in claim 3, wherein the base T at 627 site of the coding region of the gene OsWRKY43 is mutated into A, and the corresponding encoded serine is mutated into isoleucine, so that the anti-bacterial leaf blight function of the gene OsWRKY43 is reduced.

4. The use as claimed in claim 3, wherein the coding region of the gene OsWRKY43 is obtained by PCR using primer sequences shown in SEQ ID No.5 and 6.

5. The use as claimed in claim 4, wherein the gene OsWRKY43 has a protein sequence shown in SEQ ID NO. 3.

6. The use as claimed in claim 5, wherein the coding sequence of the gene OsWRKY43 is shown in SEQ ID No. 2.

7. The use as claimed in claim 6, wherein the gene OsWRKY43 has a sequence shown in SEQ ID No. 1.

8. A method for improving the bacterial leaf blight resistance of rice comprises introducing a gene OsWRKY43 into the rice, wherein a protein sequence coded by the gene OsWRKY43 is shown in SEQ ID No. 3.

9. The method as claimed in claim 8, wherein the coding sequence of the gene OsWRKY43 is located in a vector capable of expressing it in rice.

10. The gene OsWRKY43 is applied to tobacco resistance to phytophthora capsici PC35 tobacco pathogenic bacteria infection, wherein a protein sequence coded by the gene OsWRKY43 is shown in SEQ ID No. 3.