CN101058812B - Rice SNARE protein gene OsNPSN11 and its application - Google Patents

Abstract

The invention discloses a rice SNARE protein gene OsNPSN11 and an application method thereof, belonging to the field of genetic engineering. The cDNA sequence of the gene is SEQ ID NO.1 and its coded amino acid sequence is SEQ ID NO.2. The gene OsNPSN11 of the present invention is reported for the first time in rice, and mRNA expression analysis shows that the gene is induced by the inoculation of Magnaporthe grisea, and the obtained transgenic plants are subjected to PCR, Southern hybridization and RT-PCR verification to evaluate the disease resistance of rice, and the results show that, Compared with the control, the transgenic plants showed obvious resistance to blast fungus. Therefore, OsNPSN11 can be introduced into plants as a target gene to improve plant disease resistance.

Description

Rice SNARE protein gene OsNPSN11 and its application

technical field

The invention discloses rice SNARE protein gene OsNPSN11 and application thereof, belonging to the field of genetic engineering.

Background technique

Membrane fusion during vesicle transport in eukaryotic cells is mainly mediated by SNARE proteins, whose structures are highly conserved. McNew et al. (2000McNew JA, Parlati F, Fukuda R, Johnston RJ, Paz K, Paumet F, SollnerTH, Rothman JE (2000) Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature, 407(6801): 153-159) The prediction of SNARE protein genes on the whole genome sequences of human, fruit fly, yeast and Arabidopsis found that the number of SNARE protein family members contained in them were 35, 20, 21 and 68 respectively, and the number of SNARE proteins in Arabidopsis was obvious Higher than other species, and the composition is complex. At present, the whole genome sequencing of many eukaryotic organisms has been completed, which provides a lot of information for clarifying the distribution of the SNARE protein family in different eukaryotic genomes and speculating on its function, and is also conducive to the discovery of more new SNARE proteins.

Studies have found that SNARE proteins in plants promote the formation of plant cell plates, can interact with ion channel proteins, are beneficial to the normal growth and development of plants, can improve plant disease resistance and participate in plant gravity. Plant SNARE proteins not only play a role in plant growth and development, but also play an important role in resisting biotic and abiotic stresses (Pratelli et al., 2004 Pratelli R, Sutter JU, Blatt MR (2004) A new catch in the SNARE.Trends Plant Sci 9(4):187-195). Collins et al (2003Collins NC, Thordahl-Christensen H, Lipka V, Baus s, Kombrink E, Qiu J, Hückelhoven R, Stein M, Freialdenhoven A, Somerville SC, Schulze-Lefert P(2003) SNARE-protein-mediated disease resistance at the plant cell wall.Nature 425:973-977) when studying the gene HvSyp121 related to barley non-host resistance, it was found that a large number of vesicles loaded with H ₂ O ₂ gathered in the infected part of the mutant leaves, and it was speculated that the mutant cells The vesicle fails to fuse with the plasma membrane, and the _{H2O2 -} mediated disease resistance signal transduction pathway is blocked, allowing the toxic substances secreted by the pathogen to enter the plant to cause disease (Dangl et al., 2001Dang1 JL, Jones JD(2001) Plant pathogens and integrated defense responses to infection. Nature 411: 826-833; Tsanko et al., 2005 Tsanko SG, Jacques H (2005) Hydrogen peroxide as a signal controlling plant programmed cell death. J Cell Biol 168(1): 17-20). It shows that SNARE proteins in plants are related to the basic disease resistance of plants, that is, non-host resistance.

Contents of the invention

technical problem

The object of the present invention is to disclose the rice SNARE protein gene OsNPSN11 and its application. The gene comes from rice and can be introduced into plants as a target gene to improve plant disease resistance and improve plant varieties.

Technical solutions

The application of rice SNARE protein gene OsNPSN11, including:

1) For the extraction of total RNA ^, the rice blast fungus-resistant variety "Hei Ke Zi Jing" (local species of Japonica rice in the Taihu Lake Basin) was selected ^{. 1} ) After the inoculation treatment, the leaves were immediately frozen in liquid nitrogen 24 hours later and stored in a -80°C refrigerator. Take part of the leaves, grind them with a mortar, transfer them into a 1.5mLEP tube filled with Trizol lysate, shake fully, extract the total RNA, and identify the quality of the total RNA by electrophoresis;

2) The cloning of rice SNARE protein gene OsNPSN11 is based on Arabidopsis gene AtNPSN11 (Zheng et al., 2002zheng H, Bednarek SY, Sanderfoot AA, Alonso J, Ecker JR, Raikhel NV (2002) NPSN11 is a cell plate-associated SNARE protein that interacts With the syntax KNOLLE.Plant Physiol 129:530-539) sequence as a probe, search the rice genome sequence and EST sequence database, splicing to get the full-length sequence of 880bp rice SNARE protein gene OsNPSN11, and design primers at both ends:

PI: 5-TGCTTTTGGTTGTTGTGATCC-3,

P2: 5-CTGCTCITGGTTGATTTGTTC-3.

The total RNA obtained in step 1) was reverse-transcribed to synthesize the first strand of cDNA, which was used as a template for PCR amplification with high-fidelity Pfu enzyme. The PCR program was as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 45 seconds, and repeat at 56°C. 1 min at 72°C, 1.5 min at 72°C, 35 cycles, and 5 min at 72°C. Finally, Tag enzyme was incubated at 72°C for 10 min. After adding A at the end, it was cloned into the pGEM-T vector, and the rice SNARE protein gene OsNPSN11 was obtained by entrusting Shanghai Sangong Company to sequence. cDNA sequence SEQ ID NO.1;

3) Construction of plant expression vector According to the cDNA sequence (seeing SEQ ID NO.1) of rice SNARE protein gene OsNPSN11, design primer P3 near the start codon ATG and introduce restriction endonuclease site XbaI, the primer sequence is:

P3: 5-AT TCTAGA TGGATTTGGAGTCGGTCA-3.

Using the PCR amplification product obtained in step 2) as a template, after PCR amplification with P3 and P2 primers, the cDNA of OsNPSN11 was cloned into the intermediate vector pGEM-T, and the XbaI restriction site introduced by primer P3 and pGEM- The SalI restriction site on the T vector was further cloned into the binary expression vector pCAMBIA1301, and sequenced to ensure that the reading frame of the coding region in the expression vector was correct;

4) Acquisition of transgenic plants The expression vector pCAMBIA1301 obtained in step 3) was transferred into Agrobacterium, and further transferred into the rice blast pathogen variety "Su Yunuo", and the obtained transgenic plants were verified by PCR, Southern hybridization and RT-PCR Carry out the disease resistance evaluation of paddy rice afterward, the transgenic T1 generation plant that grows to 3-4 leaf stage is carried out the spore inoculation process of blast fungus, investigate the disease grade number of plant after 7 days and diseased leaf lesion number and lesion length, and The transgenic rice plants with obvious resistance compared with the control are the obtained blast-resistant transgenic plants.

Beneficial effect

1. The present invention discloses rice SNARE protein gene OsNPSN11 and its encoded protein. The rice SNARE protein gene OsNPSN11 was reported for the first time in rice. The mRNA expression analysis showed that the gene was induced by the inoculation of Magnaporthe grisea. The transgenic experiment proved that the overexpression of this gene improved the resistance of rice to Magnaporthe grisea. Therefore, it is expected to be used as a target gene to introduce into plants, improve plant disease resistance, and improve plant varieties.

2. The OsNPSN11 gene of the present invention comes from rice and has optimized codons suitable for the expression of monocotyledonous plants such as rice. Its genetically engineered recipient plants are more suitable for rice, corn except for dicotyledonous plants, such as soybeans, cotton, tobacco, etc. , wheat and other monocotyledonous plants.

3. Perform PCR, Southern hybridization and RT-PCR verification on the obtained transgenic plants to evaluate the disease resistance of rice. The transgenic T1 generation plants grown to the 3-4 leaf stage were inoculated with the blast fungus spores. After 7 days, the average disease grade of the investigated plants was 2.5, the average number of diseased spots on the diseased leaves was 4.25, and the average length of the diseased spots was 0.58. mm, while the average disease grade of the control plants was 7 and the average number of diseased spots on the diseased leaves was 44 and the average length of the diseased spots was 5.00mm. The results showed that the transgenic plants showed obvious resistance to blast fungus compared with the control. sex. 4. Utilize the OsNPSN11 gene of the present invention as the target gene to construct a plant expression vector, wherein any promoter such as the cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin promoter or other promoters can be used, and the expression vector can include if necessary An enhancer, whether a transcriptional enhancer or a translational enhancer. To simplify the identification of transformed cells selectable markers can be used including enzymes that are resistant to antibiotics, enzymes that can also recognize compounds by color change (e.g. B-glucuronidase GUS) or luminescence (e.g. luciferase) Classes can also be selected with no markers. As the expression vector used, Ti plasmid, Ri plasmid, plant virus vector and the like can be used. Transformation methods Plants can be transformed by Agrobacterium-mediated method, particle gun method, pollen tube passage method or other methods.

Detailed ways

Example 1

Select the rice variety "Heikezijaponica" (a local japonica rice variety in the Taihu Lake Basin, and a variety resistant to blast fungus). After the rice seedlings grow to the 3-4 leaf stage, inoculate with blast fungus spores, and immediately after 24 hours The leaves were frozen in liquid nitrogen and stored in a -80°C refrigerator. Part of the leaves were taken, crushed with a mortar, and transferred into a 1.5mLEP tube filled with Trizol lysate (TRIzol Reagents, purchased from Invitrogen, USA). After sufficient shaking, total RNA was extracted, and the quality of total RNA was identified by electrophoresis.

Taking the Arabidopsis gene AtNPSN11 (Zheng et al., 2002zheng H, Bednarek SY, Sanderfoot AA, Alonso J, EckerJR, Raikhel NV (2002) NPSN11 is a cell plate-associated SNARE protein that interacts with the syntaxin KNOLLE.Physi9: -539) sequence as a probe, searched the rice genome sequence and EST sequence database, spliced to obtain the full-length sequence of the 880bp rice SNARE protein gene OsNPSN11, and designed primers at both ends (P1: 5-TGCITITGGTTGTTGTGATCC-3, P2: 5-CTGCTCTTGGTTGATITGTTC- 3.) Use the first strand of cDNA synthesized by reverse transcription of total RNA as a template to perform PCR amplification with high-fidelity Pfu enzyme (purchased from Roche). Refolding for 1 min, 72°C extension for 1.5 min, after 35 cycles, 72°C extension for 5 min, and finally Tag enzyme (purchased from Dingguo Company, Beijing) was incubated at 72°C for 10 min, then A was added to the end and cloned into pGEM-T vector (purchased from Promega ), commissioned Shanghai Sangon Company to sequence and obtain the cDNA sequence of the rice SNARE protein gene OsNPSN11.

Analysis of the cDNA sequence SEQ ID NO.1 of the rice SNARE protein gene OsNPSN11 obtained above and its encoded protein SEQ ID NO.2, the protein sequence encoded by the gene is 69% consistent with the protein sequence of the Arabidopsis AtNPSN11, and the C-terminal There are conserved domain Qb-SNARE domain and transmembrane domain.

Example 2

Use primers P1 and P2 designed in Example 1 to carry out semi-quantitative RT-PCR analysis of the expression of rice seedlings at the 3-4 leaf stage after the inoculation process. The results show that the expression of OsNPSN11 is enhanced after 2 hours of inoculation with Magnaporthe grisea spores. Scanning analysis showed that the mRNA expression was 8 times that of the control, and the expression decreased after 24 hours of inoculation treatment, indicating that the expression of OsNPSN11 gene was related to the treatment of Magnaporthe grisea.

Experimental example 3

According to the full-length sequence of OsNPSN11 obtained according to Example 1 (see SEQ ID NO.1), primer P3 was designed near the start codon ATG and a restriction endonuclease site XbaI was introduced (P3: 5-AT TCTAGA TGGATTTGGAGTCGGTCA- 3), using the amplification product obtained in Example 1 as a template, after PCR amplification with P3 and P2 primers, the cDNA of OsNPSN11 was cloned into the intermediate vector pGEM-T, and the XbaI restriction site introduced by primer P3 and The SalI restriction site on the pGEM-T vector was further cloned into the binary expression vector pCAMBIA1301, sequenced and identified to ensure that the reading frame of the coding region in the expression vector was correct, and then it was transformed into Agrobacterium, and further transformed into a rice blast-susceptible strain of rice " Su Yunuo" (a local variety of japonica rice in the Taihu Lake Basin), the obtained transgenic plants were verified by PCR, Southern hybridization and RT-PCR to evaluate the disease resistance of rice. The transgenic T1 generation plants grown to the 3-4 leaf stage were inoculated with the blast fungus spores. After 7 days, the average disease grade of the investigated plants was 2.5, the average number of diseased spots on the diseased leaves was 4.25, and the average length of the diseased spots was 0.58. mm, while the average disease grade of the control plants was 7 and the average number of diseased spots on the diseased leaves was 44 and the average length of the diseased spots was 5.00mm. The results showed that the transgenic plants showed obvious resistance to blast fungus compared with the control. sex.

In summary, the OsOSNPSN11 gene provided by the inventors is a new gene isolated in rice for the first time and reported for the first time in rice. Its function is related to rice disease resistance and can be introduced into plants as a target gene to improve plant disease resistance. for plant variety improvement. The OsNPSN11 gene of the present invention can be used as the target gene to construct a plant expression vector, wherein any promoter such as cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin promoter or other promoters can be used, and the expression vector can include enhanced An enhancer, whether a transcriptional enhancer or a translational enhancer. To simplify the identification of transformed cells selectable markers can be used including enzymes that are resistant to antibiotics and compounds that can be identified by a color change (e.g. B-glucuronidase GUS) or luminescence (e.g. luciferase) Classes can also be selected with no markers. As the expression vector used, Ti plasmid, Ri plasmid, plant virus vector and the like can be used. Transformation methods Plants can be transformed by Agrobacterium-mediated method, particle gun method, pollen tube passage method or other methods.

The sequences and symbols involved in the present invention are listed as follows:

(1) Information on SEQ ID NO.1

(i) Sequential features:

(A) Length: 880bp

(B) Type: Nucleotide

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: Nucleotide

(iii) Sequence description: SEQ ID NO.1

(2) Information of SEQ ID NO.2

(i) Sequential features:

(A) Length: 261aa

(B) Type: amino acid

(C) chain: single chain

(D) Topology: linear

(ii) Molecule type: protein

(iii) Sequence description: SEQ ID NO.2

sequence listing

<110> Nanjing Agricultural University

<120> Rice SNARE protein gene OsNPSN11 and its application

<130> instruction manual

<140>00

<141>2007-03-10

<160>5

<170>PatentIn version 3.1

<210>SEQ ID NO.1

<211>880

<212>DNA

<213>Oryza sativa (rice)

<221>5'UTR

<222>(1)..(60)

<221> CDS

<222>(61)..(843)

<221>3'UTR

<222>(847)..(880)

<400>1

tgcttttggt tgttgtgatc cctgtggggc tcgggttccg gccggtgtag gggaggggag 60

atg gat ttg gag tcg gtc aac ccg gag ctc gcc gag atc gac ggc cag 108

Met Asp Leu Glu Ser Val Asn Pro Glu Leu Ala Glu Ile Asp Gly Gln

1 5 10 15

atc ggc gac atc ctc cgc gca ttg caa aat ggg ttc cag aag ctg gat 156

Ile Gly Asp Ile Leu Arg Ala Leu Gln Asn Gly Phe Gln Lys Leu Asp

20 25 30

aag atc aag gat gcc aat cga cgg agc agg caa ctc gaa gag ctc act 204

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

35 40 45

gat aag atg cgg gat tgc aag agg ctt atc aag gac ttt gag cga gtt 252

Asp Lys Met Arg Asp Cys Lys Arg Leu Ile Lys Asp Phe Glu Arg Val

50 55 60

gtc aaa gat atg gca gga agt acc gat cct gag act gct agg atg ctt 300

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

65 70 75 80

cat gat agg aaa cag tca atg atc aaa gaa ttg aac tcc tat gtt gct 348

His Asp Arg Lys Gln Ser Met Ile Lys Glu Leu Asn Ser Tyr Val Ala

85 90 95

ttg aag aaa caa tat gca agt gaa aat aag cga gtt gat ctt ttt gat 396

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

100 105 110

ggc cca agt gtt gaa gat ggc ttt ggt gaa gaa aat gtc ctg tta gca 444

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

115 120 125

tca aat atg aca aac caa cag tta atg gat caa gga aac caa cta atg 492

Ser Asn Met Thr Asn Gln Gln Leu Met Asp Gln Gly Asn Gln Leu Met

130 135 140

gat gag act gat caa gct att gca aga tct aaa cag acc gtc caa gag 540

Asp Glu Thr Asp Gln Ala Ile Ala Arg Ser Lys Gln Thr Val Gln Glu

145 150 155 160

acc atc aat gta ggt aca gaa act gca gct gct ctc aaa tca cag aca 588

Thr Ile Ash Val Gly Thr Glu Thr Ala Ala Ala Leu Lys Ser Gln Thr

165 170 175

gag caa atg agc aga att gtt aat gaa ctg gat tcc att cat ttc tcc 636

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

180 185 190

att aaa aag gca tca caa atg gtg aaa gaa att ggt agg cag gtt gca 684

Ile Lys Lys Ala Ser Gln Met Val Lys Glu Ile Gly Arg Gln Val Ala

195 200 205

act gat cgc tgc atc atg gcc ttg ctt ttt ctc att gtt gct gga gtc 732

Thr Asp Arg Cys Ile Met Ala Leu Leu Phe Leu Ile Val Ala Gly Val

210 215 220

ata gca ata ata gtc gtt aag att gta aac cca cag aac aag act atc 780

Ile Ala Ile Ile Val Val Lys Ile Val Asn Pro Gln Asn Lys Thr Ile

225 230 235 240

cga gac att cct ggt ctc gct cca cca gtt agc aga agg cta ttg agt 828

Arg Asp Ile Pro Gly Leu Ala Pro Pro Val Ser Arg Arg Leu Leu Ser

245 250 255

att gta gaa gac atc tgaacttcat acatgtgaac aaatcaacca agagcag 880

Ile Val Glu Asp Ile

260

<210>SEQ ID NO.2

<211>261

<212>PRT

<213>Oryza sativa (rice)

<400>2

Met Asp Leu Glu Ser Val Asn Pro Glu Leu Ala Glu Ile Asp Gly Gln

1 5 10 15

Ile Gly Asp Ile Leu Arg Ala Leu Gln Asn Gly Phe Gln Lys Leu Asp

20 25 30

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

35 40 45

Asp Lys Met Arg Asp Cys Lys Arg Leu Ile Lys Asp Phe Glu Arg Val

50 55 60

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

65 70 75 80

His Asp Arg Lys Gln Ser Met Ile Lys Glu Leu Asn Ser Tyr Val Ala

85 90 95

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

100 105 110

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

115 120 125

Ser Asn Met Thr Asn Gln Gln Leu Met Asp Gln Gly Asn Gln Leu Met

130 135 140

Asp Glu Thr Asp Gln Ala Ile Ala Arg Ser Lys Gln Thr Val Gln Glu

145 150 155 160

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

165 170 175

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

180 185 190

Ile Lys Lys Ala Ser Gln Met Val Lys Glu Ile Gly Arg Gln Val Ala

195 200 205

Thr Asp Arg Cys Ile Met Ala Leu Leu Phe Leu Ile Val Ala Gly Val

210 215 220

Ile Ala Ile Ile Val Val Lys Ile Val Asn Pro Gln Asn Lys Thr Ile

225 230 235 240

Arg Asp Ile Pro Gly Leu Ala Pro Pro Val Ser Arg Arg Leu Leu Ser

245 250 255

Ile Val Glu Asp Ile

260

<210>SEQ ID NO.3

<211>21

<212>DNA

<213> Synthetic

<221> Primer P1

<222>(1)..(21)

<400>3

tgcttttggt tgttgtgatc c 21

<210>SEQ ID NO.4

<211>21

<212>DNA

<213> Synthetic

<221> Primer P2

<222>(1)..(21)

<400>4

ctgctcttgg ttgatttgtt c 21

<210>SEQ ID NO.5

<211>26

<212> DNA

<213> Synthetic

<221> Primer P3

<222>(1)..(26)

<400>5

attctagatg gatttggagt cggtca 26

Claims (1)

1. The rice blast resistance function application of rice SNARE protein gene OsNPSN11, the specific steps are: 1) Extraction of total RNA The rice blast fungus-resistant variety "Hei Ke Zi Japonica" was selected, and when the rice seedlings grew to the 3-4 leaf stage, they were inoculated with 5× ¹⁰⁴ /ml of blast fungus spores, and the leaves were frozen in liquid nitrogen immediately after 24 hours. Store in a -80°C refrigerator; take part of the leaves, grind them with a mortar, transfer them into a 1.5mL EP tube filled with Trizol lysate, shake fully, extract total RNA, and identify the quality of total RNA by electrophoresis; 2) Cloning of rice SNARE protein gene OsNPSN11 Using the Arabidopsis gene AtNPSN11 sequence as a probe, searched the rice genome sequence and EST sequence database, spliced to obtain the full-length sequence of the 880bp rice SNARE protein gene OsNPSN11, and designed primers at both ends: P1: 5-TGCTTTTGGTTGTTGTGATCC-3, P2: 5-CTGCTCTTGGTTGATTTGTTC-3; The total RNA obtained in step 1) was reverse-transcribed to synthesize the first strand of cDNA, which was used as a template for PCR amplification with high-fidelity Pfu enzyme. The PCR program was as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 45 seconds, and repeat at 56°C. 1 min at 72°C, 1.5 min at 72°C, 35 cycles, and 5 min at 72°C, and finally Tag enzyme was incubated at 72°C for 10 min, then A was added to the end and cloned into the pGEM-T vector, sequenced to obtain the cDNA sequence SEQ ID NO. 1; 3) Construction of plant expression vectors According to the cDNA sequence SEQ ID NO.1 of rice SNARE protein gene OsNPSN11, primer P3 was designed near the start codon ATG and a restriction endonuclease site XbaI was introduced. The primer sequence is: P3: 5-AT TCTAGA TGGATTTGGAGTCGGTCA-3, Using the PCR amplification product obtained in step 2) as a template, after PCR amplification with P3 and P2 primers, the cDNA of OsNPSN11 was cloned into the intermediate vector pGEM-T, and the XbaI restriction site introduced by primer P3 and pGEM- The SalI restriction site on the T vector was further cloned into the binary expression vector pCAMBIA1301, and sequenced to ensure that the reading frame of the coding region in the expression vector was correct; 4) Obtaining of transgenic plants The expression vector pCAMBIA1301 obtained in step 3) was transferred into Agrobacterium, and further transferred into the rice blast pathogen variety "Su Yunuo", and the obtained transgenic plants were subjected to PCR, Southern hybridization and RT-PCR verification to carry out rice disease resistance To evaluate the resistance, the transgenic T1 generation plants grown to the 3-4 leaf stage were inoculated with the spores of Magnaporthe grisea, and the disease grade of the plants and the number and length of diseased spots on the diseased leaves were investigated after 7 days. Compared with the control group, they had obvious resistance. Sexual transgenic rice plants are the obtained blast-resistant transgenic plants.