CN110283238B - Rice disease-resistant protein RWR1 and application thereof - Google Patents

Abstract

The invention discloses a rice disease-resistant protein RWR1 and application thereof. The invention provides an application of any one of the following substances 1) -3) in regulating and controlling plant disease resistance: 1) the protein RWR 1; 2) a DNA molecule encoding the protein RWR 1; 3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA molecule encoding the protein RWR 1. Experiments prove that when transgenic rice with over-expressed RWR1 gene is invaded by Magnaporthe grisea, compared with wild rice, the growth of Magnaporthe grisea hypha is obviously inhibited, so that the resistance of the transgenic rice to Magnaporthe grisea is obviously improved. Therefore, the RWR1 gene is associated with resistance of rice to Magnaporthe grisea; the RWR1 protein and the coding gene thereof can be used for the cultivation and identification of disease-resistant plant varieties required by agriculture and ecological environment management, and have important function in the field of plant disease-resistant breeding.

Description

Rice disease-resistant protein RWR1 and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a rice disease-resistant protein RWR1 and application thereof.

Background

Rice is the most important grain crop in China, and the rice diseases caused by pathogenic microorganisms seriously affect the yield and the quality of the rice. With the continuous and deep research on the rice-pathogenic bacteria interaction mechanism, the cultivation of rice disease-resistant varieties by using disease-resistant genes has become the most effective, safest and most economical way for rice breeding experts to prevent and treat rice diseases.

Predicting the rice disease resistance related gene: selecting four whole genome sequenced rice of AA gene type wild rice Oryza _ glaberrima, FF gene type wild rice Oryza _ brachhyanth and two cultivated Asia rice (japonica rice Nipponbare and indica rice 9311) to perform comparative genome analysis research by using a comparative bioinformatics method, performing collinear analysis on rice genomes of four varieties by using PanOCT software, determining a collinear region, identifying a disease-resistant related gene, and obtaining a target gene collinear region and a repeated gene Cluster; obtaining homologous protein by combining with OrthMCL homologous protein analysis; dividing a Cluster gene family for the predicted adjacent target genes in the colinear region (the interval between two adjacent target genes is less than 10 genes); combining phylogenetic tree analysis to judge which target genes in the same gene family are homologous tandem repeats and which target genes are heterologous tandem repeats; and finally, analyzing the historical events of the homologous recombination of the target genes in the gene family by using RDP3 software.

Screening rice broad-spectrum disease-resistant genes: based on the results of comparing the biological gene information, a plurality of genes possibly having disease resistance functions are selected for functional verification (comprising 8 published homologous gene genes of functional disease resistance genes) according to the evolution mode of the rice disease resistance genes and the characteristics of the disease resistance genes reported at present.

The gene chip analysis technology is utilized to screen and clone the rice disease-resistant gene in a large scale, and after the rice is transformed by the transgenic technology, the resistance of the transgenic rice plant to rice blast and other germs is identified, so that the effect of the target gene on broad-spectrum disease resistance is determined, and new genes and material resources are provided for cultivating the rice disease resistance with persistence and broad-spectrum.

Disclosure of Invention

An object of the present invention is to provide use of any one of the following 1) to 3).

The invention provides an application of any substance of the following 1) -3) in regulating and controlling plant disease resistance:

1) the protein RWR 1;

2) a DNA molecule encoding the protein RWR 1;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA molecule encoding the protein RWR 1;

the protein RWR1 is (1) or (2) as follows:

(1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

(2) and (b) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table.

In the above application, the DNA molecule is any one of the following 1) to 4):

1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

2) the coding region is a DNA molecule shown as a sequence 3 in a sequence table;

3) DNA molecules which hybridize under stringent conditions with the DNA sequences defined in 1) and which code for proteins having the same function;

4) a DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the DNA sequence defined in 1) and encoding a protein having the same function.

In the application, the disease resistance is rice blast resistance.

In the above application, the plant is a dicotyledonous plant or a monocotyledonous plant;

or the plant is a monocotyledon, in particular a rice.

The application of any substance of 1) to 3) in the cultivation of disease-resistant plants is also within the protection scope of the invention.

In the application, the disease resistance is rice blast resistance;

or, the plant is a dicotyledonous plant or a monocotyledonous plant;

or the plant is a monocotyledon, in particular a rice.

The second purpose of the invention is to provide a method for cultivating disease-resistant transgenic plants.

The invention provides a method for cultivating disease-resistant transgenic plants, which comprises the following steps: improving the expression quantity and/or activity of a DNA molecule of a coding protein RWR1 in a target plant to obtain a transgenic plant, wherein the disease resistance of the transgenic plant is higher than that of the target plant;

the protein RWR1 is (1) or (2) as follows:

(1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

(2) and (b) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table.

In the method, the expression level and/or activity of the DNA molecule encoding the protein RWR1 in the target plant is increased by introducing the DNA molecule encoding the protein RWR1 into the target plant.

In the above method, the disease resistance is rice blast resistance;

in the above method, the plant is a dicotyledonous plant or a monocotyledonous plant;

or the plant is a monocotyledon, in particular a rice.

The disease resistance of the transgenic plant is higher than that of the target plant and is at least one of the following plants:

1) the transgenic plant has fewer necrotic spots than the plant of interest;

2) the number of susceptible stages of the transgenic plant is smaller than that of the target plant;

3) the relative expression quantity of the DNA of MoPot2 of the transgenic plant is lower than that of the target plant;

the transgenic plant is homozygous plant ((/ +)) of transgenic rice.

Experiments prove that when transgenic rice with over-expressed RWR1 gene is invaded by Magnaporthe grisea, compared with wild rice, the growth of Magnaporthe grisea hypha is obviously inhibited, so that the resistance of the transgenic rice to Magnaporthe grisea is obviously improved. Therefore, the RWR1 gene is associated with resistance of rice to Magnaporthe grisea; the RWR1 protein and the coding gene thereof can be used for the cultivation and identification of disease-resistant plant varieties required by agriculture and ecological environment management, and have important function in the field of plant disease-resistant breeding.

Drawings

FIG. 1 is T₂Generation p1300 RWR1 disease-resistant phenotype of transgenic rice inoculated with Magnaporthe grisea RB 22.

FIG. 2 is T₂Generation p1300, statistics of infection grade of RWR1 transgenic rice after inoculation of Magnaporthe grisea RB 22.

FIG. 3 is T₂Generation p1300: relative value of fungal DNA in leaves of rice after inoculation of rice blast fungus in RWR1 transgenic rice.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

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

The quantitative experiments in the examples below were repeated three times in average and the results averaged.

In The following examples, The rice was of The conventional variety TP309 (hereinafter also referred to as wild-type rice) (described in Wang J, Qu B, Dou S, Li L, Yin D, Pang Z, Zhou Z, Tian M, Liu G, Xie Q, Tang D, Chen X, Zhu L., The E3 ligand OsPUB15 organisms with The receiver-promoter PID2and regulated Plant cell death and origin immunity. BMC Plant biol.2015, 13; 15. publicly available from The institute of genetics and developmental biology).

Example 1 obtaining of RWR1 Gene

Extraction of Rice (Oryza sativa) Oyzabrachyntha (described in Chen J, Huang Q, Gao D, Wang J, Lang Y, Liu T, Li B, Bai Z, Luis Goicoechea J, Liang C, Chen C, Zhang W, Sun S, Liao Y, Zhang X, Yang L, Song C, Wang M, Shi J, Liu G, Liu J, Zhou H, Zhou W, Yu Q, An N, Chen Y, Cai Q, Wang B, Liu B, Min J, Huang Y, Wu H, Li Z, Zhang Y, in Y, Song W, Jiang J, Jacksa, Wing RA, Wang J, Chen M. genome-gene of the lake W, Jiang J, Jackson SA, Wing RA, Wang J, Chen M. genome-gene of the lake of Oryza Hayachu J, Nature PCR was carried out by research institute, Nature DNA research institute, Nature et al, Nature 3. origin, Nature et al, Nature PCR, Nature et al, Nature 3. origin, Nature et al.

RWR1-F：5'ACGGCCAGTGCCAAGCTT GAGAGAGAGGGAGAGATGAA 3'

RWR1-R：5'CGCGCCTCGAGATCCA CCAACGGCGAGTAATAGCAT 3'

The 5273bp PCR product is obtained by amplification.

The PCR product is sent for sequencing, the nucleotide sequence of the PCR product is sequence 3 in a sequence table, the nucleotide sequence comprises a promoter sequence of about 1026bp and a coding region sequence of 4247bp, and a gene shown in 1 st to 5273 th sites of the sequence 3 in the sequence table is named as RWR 1. Sequence 1 in the sequence table is a CDS sequence of the gene, has 3645 basic groups in total, encodes 1214 amino acids, and is named as RWR1 protein (sequence 2).

Example 2 functional verification of RWR1 Gene

1. Obtaining of expression vectors

HindIII and XbaI double-digested original plasmid pCAMBIA1300-flag (described in Wang Z, Li N, Jiang S, Gonzalez N, Huang X, Wang Y, Inz red, Li Y. SCFSAP controls organic size by targeting PPD proteins for digestion in Arabidopsis thaliana. Nat. Commun.2016; 7:11192, publicly available from the institute of genetics and developmental biology; 9.6kb large fragment was recovered to obtain pCAMBIA1300-221-flag vector backbone.

The 5273bp PCR product obtained In example 1 was subjected to an In-fusion reaction with the vector backbone pCAMBIA1300-flag (TAKARA Corp.) to give a binary expression vector pCAMBIA1300:: RWR 1.

RWR1 is a vector obtained by replacing 35S promoter between Hind III and Xba I of pCAMBIA1300-flag plasmid with DNA molecule shown in sequence 3. The DNA molecule shown in the sequence 3 has the RWR1 gene promoter sequence at the 1 st to the 1026 th positions, and the RWR1 gene coding region sequence at the 1027 th and the 5273 th positions.

2. RWR1 gene expression recombinant strain

The recombinant expression vector pCAMBIA1300 of the 1 is that RWR1 transforms agrobacterium EHA105, single clone is selected, and the single clone is shake cultured in LB liquid culture medium containing kanamycin and rifampicin antibiotic at 28 ℃ overnight, thus obtaining transformant.

The transformant is subjected to bacteria liquid PCR identification (RWR1-6500F and Flag-R to obtain about 600bp of positive recombinant bacteria), the positive recombinant bacteria are named as EHA105/p1300:: RWR1, and the positive recombinant bacteria are stored at-70 ℃ for later use.

RWR1-6500F：5′GAGACCTGTCACATGCTCATA 3′

Flag-R：5′CTAGTTAATTAAGACGCGTCCT 3′。

3. Obtaining of RWR1 gene expression rice

The agrobacterium tumefaciens mediated method is adopted for transforming the rice, and the specific method is as follows:

i, preparation of transformation receptor, namely selecting rice TP309 young embryo 14 days after pollination as a receptor infected by agrobacterium.

And II, culturing the agrobacterium engineering strain, namely taking a small amount of bacterium liquid from a positive recombinant bacterium agrobacterium EHA105/p1300, RWR1 stock solution (20% glycerol), carrying out streak culture on a YEB solid culture medium (containing 50mg/l kanamycin and 50mg/l rifamycin), and carrying out dark culture at 28 ℃ until a single colony with the diameter of 1mm grows out. A single colony is inoculated on the same culture medium and cultured until the growth period is full. A small amount of the cells were inoculated into 20ml of YEB liquid medium (containing the corresponding antibiotic) and shake-cultured overnight at 28 ℃ under dark conditions at 220 rpm. The next day, the cells were inoculated in 20ml YEB liquid medium containing 100uM acetosyringone at an inoculum size of 2%, cultured to the middle of logarithmic growth, and diluted to slightly turbid with naked eyes (OD600 about 0.1) with more than 3 times of liquid minimal medium for transformation.

And III, infection and co-culture of the agrobacterium rhizogenes, namely putting the bacterial liquid into a culture bottle, adding the pre-cultured callus, slightly shaking and standing for 10 minutes. After drying the callus on sterile filter paper, the callus was placed on a medium supplemented with 100uM acetosyringone and cultured in the dark at 25 ℃ for 3 days.

And IV, screening the resistant callus, namely transferring the co-cultured callus to a screening culture medium containing 25mg/l of hygromycin (hygromycin B) and 500mg/l of cefamycin, and screening for 1-2 times to show that the resistant callus grows out.

Regeneration of resistant plants, transfer of resistant calli to medium containing 25mg/l hygromycin under light conditions as before. Transferring the differentiated resistant buds to a regeneration seedling subculture medium containing 25mg/l hygromycin when the resistant buds grow to 2-3cm, and transferring the resistant buds to a nutrient solution for open culture after the resistant buds grow to a complete plant. After new roots are generated, seedlings are transferred to a greenhouse to obtain T₀Transgenic RWR1 rice.

The same method is adopted to transfer the empty vector pCAMBIA1300-221-flag into the rice TP309 to obtain T₀Transferring the rice with empty carrier.

Mixing the above T₀Seeding with plant to obtain T₁And (5) plant generation.

4. Identification of transgenic RWR1 Rice

Will grow for about two weeks T₁The rice was transferred to RWR1 and leaves of about 3cm were cut and quickly frozen in liquid nitrogen. Extracting total DNA, and performing PCR amplification by using wild rice TP309 as a control to detect the integration of target genes into a genome.

The primers used for PCR detection of RWR1 expression (amplified to obtain a ligated fragment with tag Flag at the end of RWR 13') were:

RWR1-6500F：5′GAGACCTGTCACATGCTCATA 3′

Flag-R：5′CTAGTTAATTAAGACGCGTCCT 3′

recovering PCR amplification product and sequencing to show that RWR1 has integrated into rice genome and the plant integrated into rice genome is marked as positive T₁Transgenic RWR1 rice.

T₁Both the generation-transferred empty vector rice and the wild type rice TP309 did not have RWR1 integration.

5. Obtaining of homozygous RWR1 gene expression rice

Identifying said positive T₁Transforming RWR1 rice single plant to obtain T₂Seeds are replaced, 30-40 seeds are respectively soaked in an aqueous solution containing 25mg/L of hygromycin for germination, and a positive plant is judged according to the growth state of plant roots (the growth of the young roots of a transgenic negative plant is severely inhibited under the action of the hygromycin, the main roots are short, the lateral roots are basically not generated, the young roots stretch to about 1 cm and begin to brown, the transgenic positive material has hygromycin resistance, the growth of the roots is basically not inhibited, and the main roots and the lateral roots are normal), and the transgenic positive material is recorded as T₂Generation p1300 RWR1 transgenic rice, and transplanting experiments are carried out.

T₁Transferring empty carrier rice to harvest and plant to obtain T₂Transferring the rice with empty carrier.

6. Analysis of resistance of RWR1 gene expression rice to rice blast fungus

Before inoculation of the rice, T₂Generation p1300 RWR1 transgenic rice, T₂The generation-transferred empty carrier rice and the wild rice TP309 are cultured under the conventional conditions: light 14 hours, dark 10 hours, temperature 28 ℃.

When the plants grow to the three-leaf one-heart stage (about 20 days), carrying out the inoculation experiment of the Magnaporthe grisea RB22 with the spore liquid concentration of 5X10⁵[ solution ] ml (RB22 described in Kang H, Wang Y, Peng S, Zhang Y, Xiao Y, Wang D, Qu S, Li Z, Yan S, Wang Z, Liu W1, Ning Y, Korniliev P, Leung H, Mezey J, McCouch SR, Wang GL.. Disection of the genetic architecture of the rice resistance to the blast future. mol Plant Patholol.2016, 17(6):959-72., available from the institute of genetics and developmental biology). After the leaves are completely sprayed with moisture and treated in the dark for 24 hours, the disease condition is counted and photographed and recorded after the leaves are cultured for 5 days in the light.

The results show that, compared to wild type rice TP309, homozygous T₂And (3) generation p 1300. the RWR1 transgenic rice has obviously improved resistance to Magnaporthe grisea RB22 after being sprayed with RB22 spore liquid, which shows that RWR1 protein is related to the resistance of rice to Magnaporthe grisea.

The concrete expression is as follows:

1) statistical analysis of plant phenotype after inoculation with RB22

The results are shown in FIG. 1, T is 6 days after spraying₂Generation p1300 leaves of homozygous plants (+/+) and heterozygous plants (+/-) of RWR1 transgenic rice were not substantially infected with Magnaporthe grisea; on the other hand, the leaf of the wild type rice used as a control (the wild type rice isolated in FIG. 1, indicated as-/-) had a large number of necrotic spots.

T₂The phenotype of the rice with the generation-transfer empty carrier has no obvious difference with that of wild rice.

2) Investigation and analysis of rice material leaf disease progression

According to the rice blast resistance field identification technical specification (DB51/T714-2007) of rice in Sichuan province, statistics of disease progression is carried out on rice materials, the result is shown in figure 2, and T is carried out 6 days after fungus spraying₂Generation of p1300: RWR1 transgenic RiceThe leaf infection grade of the homozygous plants (+/+) and the heterozygous plants (+/-) is far lower than that of the wild type rice of the control and the wild type rice (-/-) separated from the transgenes.

3) Detecting the relative value of rice blast fungus DNA and rice leaf DNA in rice leaves

After 6 days after RB22 bacterium spraying detection, T is detected₂Generation p1300, the DNA relative expression quantity of rice blast fungus marker gene MoPot2 in RWR1 transgenic rice and wild rice.

The specific method comprises the following steps:

A. DNA extraction from rice leaves

Extraction of total DNA Using the CATB method:

1) taking the rice leaves of each strain about 3cm, grinding the rice leaves into powder in liquid nitrogen, adding 0.3mL of CATB DNA extracting solution, and shaking and mixing uniformly.

2) The slurry was placed in an incubator at 65 ℃ for 30 minutes, 0.3mL of chloroform was added, the tube was closed, the tube was shaken vigorously for 20 seconds, and then centrifuged at 12000g for 5 minutes.

3) About 0.3mL of the upper aqueous phase was placed in a new centrifuge tube, 0.3mL of isopropanol was added, and the tube was left at room temperature for 10 minutes and centrifuged at 12000g for 10 minutes at 4 ℃.

4) The supernatant was discarded, 0.7mL of 75% ethanol was added, vortexed, and centrifuged at 12000g for 1 min at 4 ℃.

5) The supernatant was carefully discarded, and then dried at room temperature or under vacuum for 10 minutes, and dissolved in 0.3mL of double distilled water to obtain DNA.

B. Fluorescent quantitative PCR analysis

1) Reaction system

SYBR Premix 10μL

2) Reaction procedure

95℃，30s；30cycles of(95℃，10s；60℃，20s；72℃，20s)

3) Data analysis

The Ct value is the number of cycles that the fluorescence signal in the PCR tube has undergone to reach a set threshold value.

Δ Ct ═ Ct (gene) -Ct (ubq), the expression level of a gene was measured as the value of 2- Δ Ct.

The genes expressed by the quantitative PCR analysis and the primers used are shown below:

the reference gene is ubqutin, and the Q-PCR primer for amplifying the reference gene is as follows:

UBQ-F:5′-TTCTGGTCCTTCCACTTTCAG-3′

UBQ-R:5′-ACGATTGATTTAACCAGTCCATGA-3′

Q-PCR primers for detecting MoPot2 were:

MoPot2-F:5′-ACGACCCGTCTTTACTTATTTGG-3′

MoPot2-R:5′-AAGTAGCGTTGGTTTTGTTGGAT-3′

the results are shown in FIG. 3, T₂The generation p1300, compared with wild rice TP309 of a control plant, the DNA relative expression of the transgenic rice of RWR1 is obviously reduced by MoPot 2; t is₂Compared with wild rice, the rice with the generation-transferred empty vector has no significant difference in the relative expression quantity of the DNA of MoPot 2.

The above experiments were repeated 3 times, and the results were consistent.

The results prove that the RWR1 gene is related to the resistance of rice to rice blast, and the RWR1 protein and the coding gene thereof can be used for the cultivation and identification of disease-resistant plant varieties in agricultural production and play an important role in the field of plant disease-resistant breeding.

Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

<120> rice disease-resistant protein RWR1 and application thereof

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<170> PatentIn version 3.5

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atggctgagt ttttggttcg gccgctgctg tccacggtgc agaacgcttc cagctatctt 60

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tgttgtgtgt ctgatgaatt tgatgtcgtt agcatcgcaa acaacatatg tgtgagcaca 720

gagagaaatc gtgaaagggc actgcaagat ctgcagaagg aagtaagtgg aaagaagttt 780

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tgccttaagc agggctccag gggcagtgtg gtactaacaa caactcggga tgtcaaagtc 900

gctacaatta tggctaccag tgaagttgaa gtgtataatc ttggtaagct aggagaagtg 960

tatttgaagg aaataatcca aagtaaagca attggtttgc caggaagtga tgagcatttg 1020

gaagttctta ataaaattgt tcagagatgt gatggctctc ctttagctgc aaaatccttt 1080

ggctctgtgt tgtctagcag gagtactgta caagaatgga aggatatatt agccaaaagt 1140

aacatttgca atgaggggga ggacacaatt tttcctatac ttcgtctcag ctatgacgac 1200

ttaccatttg acatgaagca atgctttgct ttctgtgcta tattcccaaa agattatgtg 1260

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tttcaagatg taaagaaatc ttctatatgg accacatgca agatacatga tcttatgcac 1440

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ataagtctgt tatcagaaca tcctaggtat cactttcact catcatacaa agagactgtt 1560

ctcttagatg actttatgag aaaacaatct ccaactctcc ggagtttatt gtttgaacga 1620

tggtttaatt acttcagcac atcacattta tccaagtgca gttctctgcg agcactgaag 1680

ctcctacgat gcagcgaatt cttaccaatc gggcaccttc agcacctaag atatctcaat 1740

atctcatcaa acagttgtat caaaaagctt cctaaagata tatgcatact ctacaatcta 1800

cagactttgg tcctctctta ttgtaaaaat cttgtcgaac ttccaaagga tatgaagtat 1860

atgaaaaatc tgcgacacct ttatacggat ggatgtccaa aattgaagta catgcctccg 1920

gaccttggac agttaacttc cctgcagata ttaacatctt ttgtggtggg agctaggtct 1980

ggttgcagta accttagaga attgcgtacc ttaaaccttt gtggcaggct acagttatgt 2040

ggcctagaaa atgaaaagga agaaaatgcg aaagcagcca atcttcgaaa caaagagaaa 2100

cttacacatt tgtctcttga gtggaatagc aactgccatc ttgaaggaac aaattcccct 2160

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tatacaggca gttcttttcc agcatggata acagaccttg gtgtcctgca aaacttgata 2280

gagctccatt tagagggctg tacaatgtgt ggagaatttc ctcagttcat tcgtttcaag 2340

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ggaagacaag gaacagaaca agcatttcat cagcttgaga aggttgtcat caacatctgt 2460

ccaaagtttc aaacattgtg ctctggtgtg gcatccactg catttccaga actaaaggaa 2520

gtcaagttaa tggatttgga gagctttgag acatgggtgg caatggaagg gaggcaaggt 2580

tacatgccaa catttcctct gcttgaggag gttgaaatca acaagtgccc aaaattgaca 2640

actctacctg aagcaccaaa gctcaagatt ttaaatctaa atgaaaacaa agcgcagctg 2700

tccttgtcat tgcttcaatc cagctatata tcctcattgt ccaagctaag attggaaata 2760

gatgacaaag aaacaaccct gcagctgctt gatcagatcc acgaatcatc tctctcagaa 2820

atggagttaa cacattgcaa cattctcttc cccttgagcc catcacagtc aaaaatgagg 2880

atctgggaat ggcttggaca acttgttgag ctgaaaatcg actcctgcga ttcgctcatc 2940

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tgctataacc taattggccg tcctacccag gtgacaggaa atccaactct cctgccacat 3060

ctcacatcgc tttatgtttc taagtgtgtc aggttgagag agctctttgt tcttccacca 3120

tctatcaaat atattacaat taatgactcc atttgtcttg agagcttctc attcccctcc 3180

tatcatctgc catgcctaga acgtctaagt ttctggaatt gtcgttcagt ggtaacactt 3240

cagaacctac caccatgcct tatgctgtcc attgatgcat gttgggagct tcaatcgctg 3300

tcagggcagc tggatgaact caagcatttg ggcattgtac gctgcaataa actggagtca 3360

ctgaattgct tgggagaatt gccatcactg gaacatcttg accttaagat gtgcaaacgt 3420

ctagcatcgg cgccatgtgg cccaaggagt tactcatctc ttttgagtat tacaatccaa 3480

gactgcccaa gaatgaatat gaagaaggta tatgagtggc tccggccacg gctggatagc 3540

cttgaggaaa gagacctgtc acatgctcat acaagagtaa tttatgaaga gtctaaatgc 3600

ccgacactaa aatcatggaa atatgctatt actcgccgtt ggtga 3645

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<213> Rice (Oryza sativa)

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Met Ala Glu Phe Leu Val Arg Pro Leu Leu Ser Thr Val Gln Asn Ala

1 5 10 15

Ser Ser Tyr Leu Ala Gly Gln Tyr Arg Val Met Glu Gly Met Glu Glu

20 25 30

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

35 40 45

His Asp Ala Gln Asn Arg Thr Lys Gln Ser Gln Val Gly Ala Trp Leu

50 55 60

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

65 70 75 80

Phe Arg Tyr Glu Ala Leu Arg Arg Glu Ala Arg Arg Lys Gly His Gly

85 90 95

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

100 105 110

Met Gly Lys Lys Leu Arg Lys Ile Val Gln Arg Ile Lys Glu Leu Val

115 120 125

Glu Glu Met Asn Ser Phe Gly Leu Val His Arg Gln Glu Thr Pro Arg

130 135 140

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

145 150 155 160

Val Ser Arg Ser Arg Asp Glu Glu Lys Arg Lys Val Val Lys Ile Leu

165 170 175

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

180 185 190

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

195 200 205

Pro Glu Ile Leu Lys His Phe Gln Leu Arg Arg Trp Cys Cys Val Ser

210 215 220

Asp Glu Phe Asp Val Val Ser Ile Ala Asn Asn Ile Cys Val Ser Thr

225 230 235 240

Glu Arg Asn Arg Glu Arg Ala Leu Gln Asp Leu Gln Lys Glu Val Ser

245 250 255

Gly Lys Lys Phe Leu Ile Val Leu Asp Asp Val Trp Asn Arg Asp Ser

260 265 270

Asp Lys Trp Gly Lys Leu Met Thr Cys Leu Lys Gln Gly Ser Arg Gly

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Asp Glu His Leu Glu Val Leu Asn Lys Ile Val Gln Arg Cys Asp Gly

340 345 350

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

355 360 365

Thr Val Gln Glu Trp Lys Asp Ile Leu Ala Lys Ser Asn Ile Cys Asn

370 375 380

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

385 390 395 400

Leu Pro Phe Asp Met Lys Gln Cys Phe Ala Phe Cys Ala Ile Phe Pro

405 410 415

Lys Asp Tyr Val Ile Asp Val Glu Thr Leu Ile Lys Leu Trp Leu Ala

420 425 430

His Asp Phe Ile Pro Leu Gln Glu Asp Asp Asn Leu Glu Ser Ala Ala

435 440 445

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

450 455 460

Lys Lys Ser Ser Ile Trp Thr Thr Cys Lys Ile His Asp Leu Met His

465 470 475 480

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

485 490 495

Arg Ser Asn Phe Ile Ser Leu Leu Ser Glu His Pro Arg Tyr His Phe

500 505 510

His Ser Ser Tyr Lys Glu Thr Val Leu Leu Asp Asp Phe Met Arg Lys

515 520 525

Gln Ser Pro Thr Leu Arg Ser Leu Leu Phe Glu Arg Trp Phe Asn Tyr

530 535 540

Phe Ser Thr Ser His Leu Ser Lys Cys Ser Ser Leu Arg Ala Leu Lys

545 550 555 560

Leu Leu Arg Cys Ser Glu Phe Leu Pro Ile Gly His Leu Gln His Leu

565 570 575

Arg Tyr Leu Asn Ile Ser Ser Asn Ser Cys Ile Lys Lys Leu Pro Lys

580 585 590

Asp Ile Cys Ile Leu Tyr Asn Leu Gln Thr Leu Val Leu Ser Tyr Cys

595 600 605

Lys Asn Leu Val Glu Leu Pro Lys Asp Met Lys Tyr Met Lys Asn Leu

610 615 620

Arg His Leu Tyr Thr Asp Gly Cys Pro Lys Leu Lys Tyr Met Pro Pro

625 630 635 640

Asp Leu Gly Gln Leu Thr Ser Leu Gln Ile Leu Thr Ser Phe Val Val

645 650 655

Gly Ala Arg Ser Gly Cys Ser Asn Leu Arg Glu Leu Arg Thr Leu Asn

660 665 670

Leu Cys Gly Arg Leu Gln Leu Cys Gly Leu Glu Asn Glu Lys Glu Glu

675 680 685

Asn Ala Lys Ala Ala Asn Leu Arg Asn Lys Glu Lys Leu Thr His Leu

690 695 700

Ser Leu Glu Trp Asn Ser Asn Cys His Leu Glu Gly Thr Asn Ser Pro

705 710 715 720

Tyr Lys Val Leu Asp Ala Leu Lys Pro His His Arg Leu Gln Met Leu

725 730 735

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

740 745 750

Leu Gly Val Leu Gln Asn Leu Ile Glu Leu His Leu Glu Gly Cys Thr

755 760 765

Met Cys Gly Glu Phe Pro Gln Phe Ile Arg Phe Lys Phe Leu Gln Val

770 775 780

Leu Tyr Leu Ser Arg Leu Asp Asn Leu Gln Thr Leu Cys Arg Glu Glu

785 790 795 800

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

805 810 815

Ile Asn Ile Cys Pro Lys Phe Gln Thr Leu Cys Ser Gly Val Ala Ser

820 825 830

Thr Ala Phe Pro Glu Leu Lys Glu Val Lys Leu Met Asp Leu Glu Ser

835 840 845

Phe Glu Thr Trp Val Ala Met Glu Gly Arg Gln Gly Tyr Met Pro Thr

850 855 860

Phe Pro Leu Leu Glu Glu Val Glu Ile Asn Lys Cys Pro Lys Leu Thr

865 870 875 880

Thr Leu Pro Glu Ala Pro Lys Leu Lys Ile Leu Asn Leu Asn Glu Asn

885 890 895

Lys Ala Gln Leu Ser Leu Ser Leu Leu Gln Ser Ser Tyr Ile Ser Ser

900 905 910

Leu Ser Lys Leu Arg Leu Glu Ile Asp Asp Lys Glu Thr Thr Leu Gln

915 920 925

Leu Leu Asp Gln Ile His Glu Ser Ser Leu Ser Glu Met Glu Leu Thr

930 935 940

His Cys Asn Ile Leu Phe Pro Leu Ser Pro Ser Gln Ser Lys Met Arg

945 950 955 960

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

965 970 975

Asp Ser Leu Ile Tyr Trp Pro Glu Glu Glu Phe Leu Cys Leu Val Ser

980 985 990

Leu Lys Lys Leu Thr Ile Lys Glu Cys Tyr Asn Leu Ile Gly Arg Pro

995 1000 1005

Thr Gln Val Thr Gly Asn Pro Thr Leu Leu Pro His Leu Thr Ser

1010 1015 1020

Leu Tyr Val Ser Lys Cys Val Arg Leu Arg Glu Leu Phe Val Leu

1025 1030 1035

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

1040 1045 1050

Glu Ser Phe Ser Phe Pro Ser Tyr His Leu Pro Cys Leu Glu Arg

1055 1060 1065

Leu Ser Phe Trp Asn Cys Arg Ser Val Val Thr Leu Gln Asn Leu

1070 1075 1080

Pro Pro Cys Leu Met Leu Ser Ile Asp Ala Cys Trp Glu Leu Gln

1085 1090 1095

Ser Leu Ser Gly Gln Leu Asp Glu Leu Lys His Leu Gly Ile Val

1100 1105 1110

Arg Cys Asn Lys Leu Glu Ser Leu Asn Cys Leu Gly Glu Leu Pro

1115 1120 1125

Ser Leu Glu His Leu Asp Leu Lys Met Cys Lys Arg Leu Ala Ser

1130 1135 1140

Ala Pro Cys Gly Pro Arg Ser Tyr Ser Ser Leu Leu Ser Ile Thr

1145 1150 1155

Ile Gln Asp Cys Pro Arg Met Asn Met Lys Lys Val Tyr Glu Trp

1160 1165 1170

Leu Arg Pro Arg Leu Asp Ser Leu Glu Glu Arg Asp Leu Ser His

1175 1180 1185

Ala His Thr Arg Val Ile Tyr Glu Glu Ser Lys Cys Pro Thr Leu

1190 1195 1200

Lys Ser Trp Lys Tyr Ala Ile Thr Arg Arg Trp

1205 1210

<210> 3

<211> 5273

<212> DNA

<213> Rice (Oryza sativa)

<400> 3

gagagagagg gagagatgaa tgcataaaaa gagaaaagtt ttagtgggac tcacattaag 60

aatggtgcac tatggagctt gtatcctatg tgtggctttg tcctacgtga catctctttt 120

tttatgagag agtggcctgc atagtgtatg tccgtggtac ttttttagtt atggaggcct 180

ctctatgtca ccatccttga gcaggtacca ctgttttcta tgtaaaattt ggtacctctt 240

gttaccttag gtactagaag gtaccaaatt ttaagtttta ctctcatccc tcctttttta 300

tcttaaggta ccggtatctc gcggtaccaa atcatttatg atcgttggat caaacagtgc 360

acatcctatt tagctagatc caatggtgag aaacgatttg gtatctcgag gtactggtac 420

ctcgaggtat aaaaggaagg ataagagtaa aactcaccaa attttacata taaaacagtg 480

gtacctcttg taccttctta aggatggaaa aaaaactctt ttaacaacat atattgtcta 540

aaaaatacca gcaggatgct tgcatcggat cgttggtaac gaaaaattac ggggcattta 600

actttttgtc actcttaaaa ttggttaata ataaatttat cactcattat atatgatata 660

tacgctctga tatgttaggt ccaatgataa atatgttaat tattgccggt tatgtacgcc 720

gtgtgtccag agcatctcat taaagtagga atcactctat cttctctccc acatcgatcg 780

accacctctc tctcttccat ctccatccac agcatcccct cggagattag ggcctccgtt 840

ctctgatcga tctagatcga tcccccacaa catctagatc gggtctcgac gaaggttagt 900

gccgctagct agctgcctgc gatatcatat catttcagtt ctgcaatgtg gagtaattaa 960

ccgtgttccc tctcccatcc atgatgcaga gaagagaacg aagcatcatc ttcagaggga 1020

gcaacgatgg ctgagttttt ggttcggccg ctgctgtcca cggtgcagaa cgcttccagc 1080

tatcttgcag gccagtacag ggtgatggaa ggcatggagg agcagcgcaa agctctggag 1140

cgcatgcttc cactcatcct caccgtcatc cacgacgcac agaacagaac caaacaatcc 1200

caagtaggcg cttggctgca agagctcaag aaggtgtcct acgaggcgac cgacgtgttc 1260

gacgagttca gatacgaggc gctccggcgc gaagccagga ggaaagggca cggcgctgta 1320

agcctcttct cctctcgtaa cccaatcgtg tttcgctaca ggatgggcaa gaagctgcgg 1380

aagatcgtgc agagaatcaa ggaacttgtc gaggagatga attcctttgg gctcgtacac 1440

cggcaggaaa caccgaggca gtcgaggcaa actgattcag tgatgcttga ttttgagaag 1500

gatattgtta gcagatccag agatgaggag aagaggaagg ttgtcaagat attggtggat 1560

gaagctagcg acagggagct cacagtcctt cctgttgttg gaatgggtgg tcttggcaag 1620

actacatttg cacagctcat ctacaatgac cctgaaatcc tgaagcattt tcagcttcgc 1680

aggtggtgtt gtgtgtctga tgaatttgat gtcgttagca tcgcaaacaa catatgtgtg 1740

agcacagaga gaaatcgtga aagggcactg caagatctgc agaaggaagt aagtggaaag 1800

aagtttctga tagtgttgga tgatgtgtgg aatagggatt ctgacaagtg gggaaagtta 1860

atgacctgcc ttaagcaggg ctccaggggc agtgtggtac taacaacaac tcgggatgtc 1920

aaagtcgcta caattatggc taccagtgaa gttgaagtgt ataatcttgg taagctagga 1980

gaagtgtatt tgaaggaaat aatccaaagt aaagcaattg gtttgccagg aagtgatgag 2040

catttggaag ttcttaataa aattgttcag agatgtgatg gctctccttt agctgcaaaa 2100

tcctttggct ctgtgttgtc tagcaggagt actgtacaag aatggaagga tatattagcc 2160

aaaagtaaca tttgcaatga gggggaggac acaatttttc ctatacttcg tctcagctat 2220

gacgacttac catttgacat gaagcaatgc tttgctttct gtgctatatt cccaaaagat 2280

tatgtgattg atgtggagac tttgattaag ctatggttgg cacatgactt cataccatta 2340

caagaggatg acaatctaga atcggcagcc gaagatatct tcaaggagct agtttggagg 2400

tcattttttc aagatgtaaa gaaatcttct atatggacca catgcaagat acatgatctt 2460

atgcacgaca ttgctcaatc tgttatggga aaagaatgtg tcagcatagc tggaaggtcc 2520

aattttataa gtctgttatc agaacatcct aggtatcact ttcactcatc atacaaagag 2580

actgttctct tagatgactt tatgagaaaa caatctccaa ctctccggag tttattgttt 2640

gaacgatggt ttaattactt cagcacatca catttatcca agtgcagttc tctgcgagca 2700

ctgaagctcc tacgatgcag cgaattctta ccaatcgggc accttcagca cctaagatat 2760

ctcaatatct catcaaacag ttgtatcaaa aagcttccta aagatatatg catactctac 2820

aatctacaga ctttggtcct ctcttattgt aaaaatcttg tcgaacttcc aaaggatatg 2880

aagtatatga aaaatctgcg acacctttat acggatggat gtccaaaatt gaagtacatg 2940

cctccggacc ttggacagtt aacttccctg cagatattaa catcttttgt ggtgggagct 3000

aggtctggtt gcagtaacct tagagaattg cgtaccttaa acctttgtgg caggctacag 3060

ttatgtggcc tagaaaatga aaaggaagaa aatgcgaaag cagccaatct tcgaaacaaa 3120

gagaaactta cacatttgtc tcttgagtgg aatagcaact gccatcttga aggaacaaat 3180

tccccttata aggttcttga tgctcttaaa cctcatcaca ggctgcagat gcttaaggta 3240

atttcctata caggcagttc ttttccagca tggataacag accttggtgt cctgcaaaac 3300

ttgatagagc tccatttaga gggctgtaca atgtgtggag aatttcctca gttcattcgt 3360

ttcaagtttc ttcaggttct ttatctgagt agacttgata acttgcaaac cctatgtcgc 3420

gaggaaggaa gacaaggaac agaacaagca tttcatcagc ttgagaaggt tgtcatcaac 3480

atctgtccaa agtttcaaac attgtgctct ggtgtggcat ccactgcatt tccagaacta 3540

aaggaagtca agttaatgga tttggagagc tttgagacat gggtggcaat ggaagggagg 3600

caaggttaca tgccaacatt tcctctgctt gaggaggttg aaatcaacaa gtgcccaaaa 3660

ttgacaactc tacctgaagc accaaagctc aagattttaa atctaaatga aaacaaagcg 3720

cagctgtcct tgtcattgct tcaatccagc tatatatcct cattgtccaa gctaagattg 3780

gaaatagatg acaaagaaac aaccctgcag ctgcttgatc agatccacga atcatctctc 3840

tcagaaatgg agttaacaca ttgcaacatt ctcttcccct tgagcccatc acagtcaaaa 3900

atgaggatct gggaatggct tggacaactt gttgagctga aaatcgactc ctgcgattcg 3960

ctcatctact ggccagaaga agagttccta tgcttggtat ccctgaagaa attgaccatc 4020

aaggagtgct ataacctaat tggccgtcct acccaggtga caggaaatcc aactctcctg 4080

ccacatctca catcgcttta tgtttctaag tgtgtcaggt tgagagagct ctttgttctt 4140

ccaccatcta tcaaatatat tacaattaat gactccattt gtcttgagag cttctcattc 4200

ccctcctatc atctgccatg cctagaacgt ctaagtttct ggaattgtcg ttcagtggta 4260

acacttcaga acctaccacc atgccttatg ctgtccattg atgcatgttg ggagcttcaa 4320

tcgctgtcag ggcagctgga tgaactcaag catttgggca ttgtacgctg caataaactg 4380

gagtcactga attgcttggg agaattgcca tcactggaac atcttgacct taagatgtgc 4440

aaacgtctag catcggcgcc atgtggccca aggagttact catctctttt gagtattaca 4500

atccaagact gcccaagaat gaatatgaag aaggtatatg agtggctccg gccacggctg 4560

gatagccttg aggaaagaga cctgtcacat gctcatacaa gagtaattta tgaaggtaca 4620

ctccattgct agcattttcc ttctttttcc gtttttcgtt tcttttacat agatgaatag 4680

attttatgtt cctgtgccag tgtatatctg cactcaaagt tccaatcatc aggccctgtg 4740

ttttataact gaataagata gaaattgtgt ttcttggtta aagagattct agtctaacct 4800

tgatcgaaca aaggttcgtt ccaccaaaac cataggagct ccatcactgt ttattgacaa 4860

gtttgtgtct acctgtgagg tgtgctatgt gactgcattt gtagcttcct ataatattca 4920

acataattta gtcggtgtcg ataccatgca tgaaatgagc atttttttta tcctcgagaa 4980

actattataa ggaagcactg ttttttatat aaaatttggt actttctagt atctaatata 5040

ctaacagtgg tacctcttag taccttctca aggatcgtaa tatcgctcgc atggaatagt 5100

tcctttccct ctgtactcca ttttattttt tctctttttc tgcagtttat ttcgattgtg 5160

agatattatc taaaaaaaat tcggttgtga gactgacatt tatttttcat gctacagagt 5220

ctaaatgccc gacactaaaa tcatggaaat atgctattac tcgccgttgg tga 5273

Claims (4)

1. The application of any substance of the following 1) to 3) in regulating and controlling rice blast resistance of rice:

1) the protein RWR 1;

2) a DNA molecule encoding the protein RWR 1;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA molecule encoding the protein RWR 1;

the protein RWR1 is a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table.

2. Use according to claim 1, characterized in that:

the DNA molecule is any one of the following 1) to 2):

1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

2) the coding region is a DNA molecule shown as a sequence 3 in a sequence table.

3. The application of any substance of the following 1) to 3) in cultivating rice blast resistance rice;

1) the protein RWR 1;

2) a DNA molecule encoding the protein RWR 1;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a DNA molecule encoding the protein RWR 1;

the protein RWR1 is a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table.

4. A method for cultivating rice blast resistant transgenic rice comprises the following steps: improving the expression quantity of a DNA molecule of a coding protein RWR1 in target rice to obtain transgenic rice, wherein the rice blast resistance of the transgenic rice is higher than that of the target rice;

the protein RWR1 is a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

the method for improving the expression quantity of the DNA molecule of the encoding protein RWR1 in the target rice is to introduce the DNA molecule of the encoding protein RWR1 shown in the sequence 3 into the target rice.

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