CN113788883B - Rice blast bacterium MoSpc2 gene and application thereof - Google Patents

Abstract

The invention discloses a rice blast fungusMoSpc2The gene consists of a nucleotide sequence shown as SEQ ID No. 1 and codes protein with an amino acid sequence shown as SEQ ID No. 2. Replacing Magnaporthe grisea with hygromycin phosphotransferase gene HPHMoSpc2After the gene, compared with wild rice blast bacteria, the obtained rice blast bacteria knockout mutant has obviously reduced hypha growth, attached spore formation and spore production capacity and obviously different response to external environmental factors; deletion ofMoSpc2The gene can not form obvious disease spots on the rice leaves, which indicates thatMoSpc2The gene has remarkable effect in regulating and controlling the pathogenicity of rice blast bacteria. The invention providesMoSpc2The gene and the application thereof have important functions in the aspect of pathogenicity of the rice blast bacteria, and provide a new direction for further utilizing the gene to prevent and treat the rice blast bacteria.

Description

Rice blast bacterium MoSpc2 gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, in particular to rice blast bacteriaMoSpc2Genes and uses thereof.

Background

Rice is an important grain crop, and nearly half of the population worldwide takes rice as a main grain. Pyricularia oryzaeMagnaporthe oryzae) The rice blast caused by the method is produced by the world riceSerious fungal diseases occur in various rice areas in China, and when the outbreak disaster occurs, the harvest of the rice is stopped, 10% -30% of the harvest losses of the rice are caused every year, and the lost rice is enough for 6000 millions of people to live. At present, the mode of preventing and controlling rice blast mainly comprises the layout of disease-resistant varieties and the application of chemical agents. Although the effective application of disease-resistant varieties and nitrogenous fertilizers and the effective combination of bactericides achieve the effect of controlling rice blast to a certain extent, the disease-resistant varieties gradually lose the original pestilence resistance within a few years due to the complex and various bacterial groups of rice blast in the field; and the single or excessive use of chemical pesticides can easily cause pathogenic bacteria to generate drug resistance, thus providing serious challenges for effective prevention and control of rice blast.

The signal peptide plays a key role in targeting the targeting of the guide protein to the endoplasmic reticulum secretory pathway. The structural characteristics of these signal peptides vary with their output positions and their specificity is usually achieved by interaction with receptors on the target membrane. Signal peptidases play a vital role in the release, maturation and secretion of proteins as the main element of signal peptide processing. Once the signal peptide enzyme fails to cleave the signal peptide, it will cause the secretion substrate to stay intracellular, failing to complete the secretion process. Functional studies on signal peptidases are currently focused mainly on bacteria, yeasts and animals, and there are few studies on fungi and plants. Therefore, the research on the pathogenesis of the signal peptidase can provide an important drug target for the design and screening of the rice blast pathogen targeted drug, and has important theoretical significance and application value for the comprehensive control of the rice blast pathogen.

Disclosure of Invention

The invention aims at solving the problems of serious harm and lack of an effective control method of rice blast in the prior art and provides a rice blast fungusMoSpc2Genes and uses thereof. Pyricularia oryzaeMoSpc2The gene has important effects on hypha vegetative growth, conidium generation, adhesion cell formation and pathogenicity of rice blast germ.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

rice blast fungusMoSpc2The full-length sequence of the gene is shown as SEQ ID No. 1.

Further, the above Pyricularia oryzaeMoSpc2A gene having the nucleotide sequence:

(a) Has a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID No. 1; or (b)

(b) A nucleotide sequence having more than 90% identity with the nucleotide sequence shown in SEQ ID No. 1. Further, the above Pyricularia oryzaeMoSpc2The cDNA sequence of the gene is shown as SEQ ID No. 2.

A rice blast bacterium is prepared from the rice blast bacteriumMoSpc2The amino acid sequence of the pathogenic protein MoSpc2 of the gene-encoded rice blast germ is shown as SEQ ID No. 3.

Furthermore, the pathogenic protein MoSpc2 of the rice blast germ has an amino acid sequence which has more than 90% of identity with the amino acid sequence shown in SEQ ID No. 3 and has the pathogenic function of rice blast.

Rice blast bacterium knocked outMoSpc2Rice blast bacterial mutant of geneMoSpc2。

Above Pyricularia oryzaeMoSpc2The application of the gene in reducing the pathogenicity of rice blast bacteria.

The application of the pathogenic protein MoSpc2 of the rice blast fungus in reducing the pathogenicity of the rice blast fungus.

The invention has the advantages that: the invention verifies the physiological functions of the rice blast fungus MoSpc2 gene, such as vegetative growth, conidium germination, attached spore measurement, pathogenicity and the likeMoSpc2Knock-out or deletion of the gene results in a decrease in the pathogenicity of Pyricularia oryzae, thus indicatingMoSpc2The gene is an important gene in the pathogenic process of rice blast bacteria. The invention providesMoSpc2The gene and the application thereof provide a new direction for preventing and controlling the rice blast germ.

Description of the drawings:

FIG. 1 is a schematic view ofMoSpc2Schematic diagram of gene knockout mutant construction process.

FIG. 2 shows Southern hybridization for analysis of single copy integration of the HPH gene in mutants. Wherein Guy11 and DMoSpc2Genomic DNA of the mutant was digested with NruI and then hybridized with a probe to verify that the HPH gene was in the processSingle copy integration in the variant genome.

FIG. 3 is a schematic view ofMoSpc2Colony and aerial hypha morphology photograph of the knocked-out mutant on CM, SDC, OMA plate medium.

FIG. 4 is a diagram ofMoSpc2Colony morphology photographs of the knockout mutants under various external environmental factor stresses.

FIG. 5 is a schematic view of a displayMoSpc2The knock-out mutant was compared to the wild-type conidium morphology.

FIG. 6 is a diagram ofMoSpc2Statistical plot of conidia numbers of knock-out mutants versus wild type cultured at 28℃for 8 days in SDC medium.

FIG. 7 is a diagram ofMoSpc2Pathogenic force pattern of gene knockout mutant and wild type on rice leaf

FIG. 8 is a diagram ofMoSpc2Germination of the gene knockout mutant and the wild type attached spore at different times.

The specific embodiment is as follows:

the invention is described in detail below with reference to the drawings and the specific embodiments.

Test strain and plant:

the rice blast bacterial strain is rice blast bacterial wild strain Guy 11%Magnaportheoryzae), the rice to be tested is a rice susceptible rice blast variety Co-39.

(II) preparation of relevant culture medium and solution:

(1) Complete medium (CM ii): 20 times nitrate (50 mL/L), 1000 times trace elements (1 mL/L), 1000 times vitamin solution (1 mL/L), glucose (10 g/L), peptone (2 g/L), acid hydrolyzed casein (1 g/L), yeast extract (1 g/L), and agar powder (20 g/L) added to the solid medium;

1000 x vitamin solution (100 ml): biotin 0.01g,Pyridoxin 0.01g,Thiamine 0.01g,Riboflavin0.01g,PABA (p-aminobenzonic acid) 0.01g,Nicotinic acid 0.01g;

1000 x trace elements (100 ml): znSO (ZnSO) ₄ . 7H ₂ O 2.2g，H ₃ BO ₃ 1.1g，MnCl ₂ . 4H ₂ O 0.5g，FeSO ₄ . 7H ₂ O 0.5g，CoCl ₂ . 6H ₂ O 0.17g，CuSO ₄ . 5H ₂ O 0.16g，Na ₂ MoO ₄ . 5H ₂ O 0.15g ，Na ₄ EDTA 5g；

(2) Protoplast regeneration medium (TB 3): yeast extract (3 g/L), acid hydrolyzed casein (3 g/L), sucrose (200 g/L), and agar powder (12 g/L) added to the solid medium;

(3) Rice straw medium (SDC): corn flour (25 g/L), dried straw (150 g/L), agar powder (20 g/L). Is used for inducing the rice blast bacteria to produce spores.

(4) Oat Medium (OMA): oat (50 g/L), preparing a solid culture medium, and adding agar powder (20 g/L);

example 1 screening of genes

The protein sequence of SPC2 in yeast is obtained by searching NCBI database, and the protein sequence is compared with NCBI functional network by using blastp tool to obtain similar protein (MGG_10832) in Pyricularia oryzae, and the protein is named MoSpc2. The amino acid sequence of pathogenic protein MoSpc2 of Pyricularia oryzae is shown as SEQ ID No. 3. The gene encoding the protein is rice blast fungusMoSpc2The full-length sequence of the gene is shown as SEQ ID No. 1; the cDNA sequence is shown in SEQ ID No. 2.

Example 2 Gene knockout

(1) Selecting rice blast fungus wild Guy11 genome as templateMoSpc21000bp fragment upstream of geneMoSpc2F1 (forward direction),MoSpc2Amplification of the F2 (reverse) primerMoSpc2Upper arm segment, selectMoSpc21000bp fragment downstream of the geneMoSpc2F3 (forward direction),MoSpc2F4 (reverse) amplificationMoSpc2Lower arm segment (fig. 1). Then fusing the upper arm, hygromycin phosphotransferase gene HPH (SEQ ID No: 4) and lower arm fragments, and fusing the productsMoSpc2-nest FMoSpc2The post R is amplified in large quantities to give a large number of DNA fusion products. The fusion product was subjected to protoplast transformation to replace the Magnaporthe grisea SPC2 gene.

(2) Protoplast transformation process:

(1) adding 10 mu L of the DNA fusion product into the split-packed wild rice blast fungus protoplast, gently mixing, and standing for 20-25 min (making the protoplast fully contact with DNA);

(2) adding 1 mL PTC, gently mixing, and standing for 20-25 min;

(3) adding a proper amount of TB3 liquid culture medium, gently mixing, sealing the mouth of the tube with a sealing film, and resuscitating in the dark at 28 ℃ in an incubator for 12-16 h.

(4) In the melted TB ₃ Hygromycin (600. Mu.L/200 ml) was added to the solid medium at the desired concentration and poured into resuscitated centrifuge tubes. Mixing, pouring into a sterile 15 cm culture dish, cooling and solidifying the culture medium, pouring into a TB3 culture medium with corresponding concentration hygromycin (1200 mu L/200 ml), covering the surface, cooling and solidifying, sealing the culture dish with a sealing film, and pouring into an illumination incubator at 8 ℃ for culturing 6-10 d;

(5) when the transformant grows out of the culture dish, carefully picking a single colony in the upper culture medium, inoculating the single colony in the CMII solid culture medium plate, and further verifying the transformant;

(3) By means ofMoSpc2Gene inner primerMoSpc2-ko-F andMoSpc2and (3) performing PCR knockout verification on the ko-R, and selecting a non-banded transformant for further verification by taking a negative non-banded transformant as a control. By means ofMoSpc2And (3) performing hygromycin transfer verification by using an internal primer HYG R of the F1 and hygromycin phosphotransferase gene HPH, taking negative non-stripes as a control, and selecting a transformant with the stripes for performing southern blot verification.

(4) Southern hybridization identification: selectingMoSpc2Designing a probe (SEQ ID No: 5) for the sequence of the downstream coding region of the gene, and selecting a restriction endonuclease having No recognition site in the probeNruIGenomic DNA of the candidate transformant and the wild strain Guy11 were digested respectively (FIG. 2). In the hybridization validation plots, a band of the predetermined size 2.07 kb was present in the wild-type lane, while a band of the size 2.60 kb was present in the mutant lane (FIG. 2), consistent with the predictions, indicating that in the transformantMoSpc2The gene has been successfully replaced by the HPH geneInstead, and inserted as a single copy. Identification of Pyricularia oryzae mutantMoSpc2。

(5) Primer sequences used:

SEQ ID NO. 6：MoSpc2-F1：TGGCCATGTTGTTCTTCATC；

SEQ ID NO. 7：MoSpc2-F2：

CATTCATTGTTGACCTCCACTAGCTCCATGTGTACAAGAAAGTTGGTT；

SEQ ID NO. 8：MoSpc2-F3：GCAAAGGAATAGAGTAGATGCCGACCGAAATATCAAATTTCAAGTTA；

SEQ ID NO.9：MoSpc2-F4：AATAGAGCAATACGCAAGAG；

SEQ ID NO. 10：MoSpc2-nest F：ACAGCACATGGTAGTTGCTG；

SEQ ID NO. 11：MoSpc2-nest R：AGCAGCGCTCGGTGAGCATG；

SEQ ID NO. 12：MoSpc2-ko-F：GCAGCTGGTGAGCACATACT；

SEQ ID NO. 13：MoSpc-ko-R：TGTCTGCACCTGTAGCTGAT；

SEQ ID NO. 14：HYG R：CGGTGGTGCAGATGAACTTC。

(6) PCR reaction system used in the experiment:

amplification PCR reaction System (50. Mu.L): template 0.5. Mu.L, 2x reaction mix 25. Mu.L, upstream primer 2. Mu.L, downstream primer 2. Mu.L, ddH ₂ O25. Mu.L. The PCR reaction procedure was: 94℃for 5min,94℃for 30s,55℃for 30s,72℃for 1kb/min,30 cycles, 72℃for 10min,16℃for 10min.

Fusion PCR reaction System (25. Mu.L): template upstream fragment, hygromycin fragment and downstream fragment molar ratio 1:3:1, 10x PCR buffer 2.5. Mu.L, dNTP mix 0.5. Mu.L, LTaq enzyme 0.25. Mu.L, ddH ₂ 0.21.75 μl. The PCR reaction procedure was: 94℃for 3min,94℃for 35s,58℃for 5min,72℃for 5min,10 cycles, 16℃for 10min.

Example 3:MoSpc2the deletion of the Gene affects the vegetative growth of Pyricularia oryzae

Fatting wild Guy11 and mutantMoSpc2Inoculating into CM II, SDC and OMA three different culture medium plates respectively at 28deg.CCulturing in darkness 7 d, measuring colony growth diameter after culturing, and photographing and preserving. The experimental results show that the colony growth diameter of the mutant is smaller than that of the wild type in all three media (FIG. 3), especially in SDC media, showing that significant reduction occursMoSpc2The deletion of the gene affects the vegetative growth of Pyricularia oryzae.

Example 4: MoSpc2gene deletion affects the response of Pyricularia oryzae to external environmental stresses

The wild strain Guy11 and the mutant are fattedMoSpc2Inoculated in CM II medium containing 0.5 mM Diamide (causing oxidative stress), 2 mM DTT (dithiothreitol, causing stress on endoplasmic reticulum) and 400. Mu.g/mL CFW (cell wall inhibitor calcium fluorescent white, causing stress on cell wall) and cultured in darkness at 28℃for 7 d, respectively. The experimental results showed that the mutants grew significantly slower on CM ii plates containing Diamide and DTT and grew significantly faster on CM ii plates containing CFW (fig. 4). The above-mentioned results show that,MoSpc2the deletion of the gene affects the response of rice blast bacteria to external environmental stress.

Example 5:MoSpc2gene participating in regulating and controlling asexual propagation process of rice blast germ

Fatting wild Guy11 and mutantMoSpc2Inoculating to the center of SDC culture medium plate, dark culturing at 28deg.C for 5 d, scraping off aerial hyphae on the surface of the culture medium, placing in a greenhouse at 28deg.C, inducing 3 d to produce spores under the regulation of black light lamp, and quantitatively counting the yield of conidium of corresponding strain in SDC culture medium. Experimental results show that the mutantMoSpc2Part of the spores produced were abnormal in morphology, and conidia of different strains were stained with CFW, and the morphology was further observed (fig. 5): the conidia of the wild-type strain were found to be essentially three-cell two-diaphragm from microscopic observation, while the mutants had three spore forms: without the membrane, one and two, the spore proportion of the individual membranes was 64.6% and the size of the conidia was also reduced relative to the wild type. On the other hand, the mutant fates compared with the wild type Guy11MoSpc2The spore yield of (a) was also significantly reduced (fig. 6). Description of the resultsMoSpc2GeneIs important to the asexual propagation process of rice blast germ.

Example 6: MoSpc2gene involved in regulating pathogenic process of Pyricularia oryzae

Collecting wild Guy11 and mutant fatterMoSpc2Conidium suspension (1X 10) ⁵ Each mL), and the rice leaves are inoculated to three weeks old rice leaves for rice spray experiments, and leaf lesions are observed after 6 d. The results show that on rice leaves, wild Guy11 can produce typical rice blast lesions, while the mutant fatesMoSpc2No lesions were generated (fig. 7). In conclusion, the results show that,MoSpc2the deletion of the gene significantly reduced the pathogenicity of Pyricularia oryzae, indicating thatMoSpc2The gene plays an important role in the pathogenic process of rice blast bacteria.

Example 7: MoSpc2deletion of the Gene delays maturation of the attachment cell

The wild Guy11 and the mutant fatter are respectively collectedMoSpc2Conidia of the strain induce germination of its attachment cells under hydrophobic conditions. The germination and formation of adherent cells at 4 h, 8 h and 16 h, respectively, was observed by microscopy for the different strains. It was found experimentally (FIG. 8) that the conidia of the wild-type Guy11 strain had germinated substantially at 4 h, whereas the mutant +MoSpc2The germination of the conidia is delayed, and the germination rate is only 82% at 16 h. Based on the statistical results, the mutants were shownMoSpc2The rate of attachment cell formation of the conidia was similar to that of germination, with the wild-type conidia already forming substantially attachment cells at 8 h, but the mutant had only 61% attachment cell formation at 16 h. The above results indicate thatMoSpc2Deletion of the gene delays development of the attachment cell.

SEQUENCE LISTING

<110> Fujian university of agriculture and forestry

<120> Magnaporthe grisea MoSpc2 gene and application thereof

<130> 14

<160> 14

<170> PatentIn version 3.3

<210> 1

<211> 1142

<212> DNA

<213> SEQ ID No.1

<400> 1

gcttactcca atccgaccac gtgttcgagg tttcgacagc aaaccaaagg acaaaaccat 60

cttccactcg cgacatccaa aattataaca tcaacaaccc aaaacaaaaa ccaactttct 120

tgtacacaat ggcggctcaa gagaagatat cggtctataa tgcagctggt gagcacatac 180

tgccttgttt cctttacaat tacttcatct tcgacttggc aagttcgtcc tccttgtttg 240

cctatgctga ctgccaacgt acgtacgcac tttcaagacc tcaagaacac atccgatgat 300

gccataccca attacctcaa ctcgctcaag ttcaagcagt cacacacgct cacggatgtg 360

cgcctgacgc ttggctattc ggcctttgcc atatcggccg cctgtttctt ctgggactac 420

aagttgggat ttgacagcac caagtactac acggccgccg ccgtcgctct atatgccatc 480

ttgaatggcg ctctcaccct ctggtctttc tttgtcgaga agaacatcgt ctacgttggc 540

acagcgccaa gcggagaaaa ggttcgttcc ttggtctcga gcatatcacc aacctggatt 600

tgtacaaagc gtcgtggcca ctactaacac gccagctgtc actcagatca ccatagcgtc 660

tagcgtcaac aagtacgacc ctacctacag gcttgccatc acaacagtcc ccaagggcgc 720

ttccaagggc cagagcatcg aggtctctcg cccttttgcc gagtggtttg actcagttgg 780

tcacttcatc gctgcacctt tccagacgat gctggctact tcagtgccgg cgatagccaa 840

ggtggattcc aaaaggcttg ccaacatccc cgccgctcag gcagagacct cggcacccaa 900

ggaagatggt tacagtgccg agatgttgga cgctattttg tcctcgtccg gatcagctac 960

aggtgcagac accaccggct cagtatcagc caagaagggt ggaaagcgcc gcaaggcttg 1020

aaaatatcaa atttcaagtt aaactaggga gactcggtag catggaattt tgtacactag 1080

actggtgttt tcatcacgta ggccgggatt ggttccagaa ttgggtgatc tattgaagtt 1140

tt 1142

<210> 2

<211> 699

<212> DNA

<213> SEQ ID No.2

<400> 2

atggcggctc aagagaagat atcggtctat aatgcagctg acctcaagaa cacatccgat 60

gatgccatac ccaattacct caactcgctc aagttcaagc agtcacacac gctcacggat 120

gtgcgcctga cgcttggcta ttcggccttt gccatatcgg ccgcctgttt cttctgggac 180

tacaagttgg gatttgacag caccaagtac tacacggccg ccgccgtcgc tctatatgcc 240

atcttgaatg gcgctctcac cctctggtct ttctttgtcg agaagaacat cgtctacgtt 300

ggcacagcgc caagcggaga aaagatcacc atagcgtcta gcgtcaacaa gtacgaccct 360

acctacaggc ttgccatcac aacagtcccc aagggcgctt ccaagggcca gagcatcgag 420

gtctctcgcc cttttgccga gtggtttgac tcagttggtc acttcatcgc tgcacctttc 480

cagacgatgc tggctacttc agtgccggcg atagccaagg tggattccaa aaggcttgcc 540

aacatccccg ccgctcaggc agagacctcg gcacccaagg aagatggtta cagtgccgag 600

atgttggacg ctattttgtc ctcgtccgga tcagctacag gtgcagacac caccggctca 660

gtatcagcca agaagggtgg aaagcgccgc aaggcttga 699

<210> 3

<211> 232

<212> PRT

<213> SEQ ID No.3

<400> 3

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

1 5 10 15

Asn Thr Ser Asp Asp Ala Ile Pro Asn Tyr Leu Asn Ser Leu Lys Phe

20 25 30

Lys Gln Ser His Thr Leu Thr Asp Val Arg Leu Thr Leu Gly Tyr Ser

35 40 45

Ala Phe Ala Ile Ser Ala Ala Cys Phe Phe Trp Asp Tyr Lys Leu Gly

50 55 60

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

65 70 75 80

Ile Leu Asn Gly Ala Leu Thr Leu Trp Ser Phe Phe Val Glu Lys Asn

85 90 95

Ile Val Tyr Val Gly Thr Ala Pro Ser Gly Glu Lys Ile Thr Ile Ala

100 105 110

Ser Ser Val Asn Lys Tyr Asp Pro Thr Tyr Arg Leu Ala Ile Thr Thr

115 120 125

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

130 135 140

Phe Ala Glu Trp Phe Asp Ser Val Gly His Phe Ile Ala Ala Pro Phe

145 150 155 160

Gln Thr Met Leu Ala Thr Ser Val Pro Ala Ile Ala Lys Val Asp Ser

165 170 175

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

180 185 190

Lys Glu Asp Gly Tyr Ser Ala Glu Met Leu Asp Ala Ile Leu Ser Ser

195 200 205

Ser Gly Ser Ala Thr Gly Ala Asp Thr Thr Gly Ser Val Ser Ala Lys

210 215 220

Lys Gly Gly Lys Arg Arg Lys Ala

225 230

<210> 4

<211> 1368

<212> DNA

<213> SEQ ID No.4

<400> 4

tggagctagt ggaggtcaac aatgaatgcc tattttggtt tagtcgtcca ggcggtgagc 60

acaaaatttg tgtcgtttga caagatggtt catttaggca actggtcaga tcagccccac 120

ttgtagcagt agcggcggcg ctcgaagtgt gactcttatt agcagacagg aacgaggaca 180

ttattatcat ctgctgcttg gtgcacgata acttggtgcg tttgtcaagc aaggtaagtg 240

gacgacccgg tcataccttc ttaagttcgc ccttcctccc tttatttcag attcaatctg 300

acttacctat tctacccaag catccaaatg aaaaagcctg aactcaccgc gacgtctgtc 360

gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 420

gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 480

agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 540

ctcccgattc cggaagtgct tgacattggg gagttcagcg agagcctgac ctattgcatc 600

tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 660

ctccagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 720

gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 780

tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 840

gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 900

cggcacctcg tgcatgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 960

acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 1020

atcctcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 1080

aggcatccgg agcttgcagg atcgccgcgc ctccgggcgt atatgctccg cattggtctt 1140

gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 1200

cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 1260

agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1320

cgccccagca ctcgtccgag ggcaaaggaa tagagtagat gccgaccg 1368

<210> 5

<211> 964

<212> DNA

<213> SEQ ID No.5

<400> 5

aaatatcaaa tttcaagtta aactagggag actcggtagc atggaatttt gtacactaga 60

ctggtgtttt catcacgtag gccgggattg gttccagaat tgggtgatct attgaagttt 120

tgctgctaca ttattgccaa caggggttcc tgcaagcctt ggagtgttta ccataatctt 180

tcttcaggcc gcgagcttcg cccagctgct ccgcgcactc atggctgatt gtcgaacttc 240

cctgactggg tcgccacagg cagtagctag gacacgagaa atctgagcct tgtactggcg 300

caggagctgc tcatctatct tctctgccac ctcctggagc agttgtaaac tgagccttcg 360

acagagggcc ggattgccag gattcaccgt aagtgcagcc gcatcagtcg gcgttgcctg 420

ttgatcatga gactgttcca agcccgaggt caccatcgat atggctcctg ctacaacggc 480

ctttagttga tcctttacag catcggactt gttggctagg atttgccgaa gaacttgaag 540

agccgcctcg acatcagcca atcgtttggg cgatgacaca ctagcaatta gtatccgcgt 600

tattcgatct gtgtcctccg cgtatactgt aaattccatg tgtttcagaa catggactac 660

ggcgatggcg ttgacaactc cttgaggcct gtcttgcgtt atggggaacg actgctctag 720

tttcggcttc aacacggaga tgtaaaccca ctgctcagac aggggcctca gaatcgcgtg 780

gttttgcaca ctcagttggc ttttggcggc cagtagccct tccaggcgtg gagccatttg 840

cttcccatgc tcaccgagcg ctgctggaag ggatgcgagg atcttcacta gaccttctac 900

gtccctgtca cgccttgcca ccgctccagc agcgaatccc agagctcttg cgtattgctc 960

tatt 964

<210> 6

<211> 20

<212> DNA

<213> SEQ ID NO. 6：MoSpc2-F1

<400> 6

tggccatgtt gttcttcatc 20

<210> 7

<211> 48

<212> DNA

<213> SEQ ID NO. 7：MoSpc2-F2

<400> 7

cattcattgt tgacctccac tagctccatg tgtacaagaa agttggtt 48

<210> 8

<211> 47

<212> DNA

<213> SEQ ID NO. 8：MoSpc2-F3

<400> 8

gcaaaggaat agagtagatg ccgaccgaaa tatcaaattt caagtta 47

<210> 9

<211> 20

<212> DNA

<213> SEQ ID NO.9：MoSpc2-F4

<400> 9

aatagagcaa tacgcaagag 20

<210> 10

<211> 20

<212> DNA

<213> SEQ ID NO. 10：MoSpc2-nest F

<400> 10

acagcacatg gtagttgctg 20

<210> 11

<211> 20

<212> DNA

<213> SEQ ID NO. 11：MoSpc2-nest R

<400> 11

agcagcgctc ggtgagcatg 20

<210> 12

<211> 20

<212> DNA

<213> SEQ ID NO. 12：MoSpc2-ko-F

<400> 12

gcagctggtg agcacatact 20

<210> 13

<211> 20

<212> DNA

<213> SEQ ID NO. 13：MoSpc-ko-R

<400> 13

tgtctgcacc tgtagctgat 20

<210> 14

<211> 20

<212> DNA

<213> SEQ ID NO. 14：HYG R

<400> 14

cggtggtgca gatgaacttc 20

Claims (1)

1. The application of the rice blast bacterium mutant delta MoSpc2 for knocking out the rice blast bacterium MoSpc2 gene in reducing the pathogenicity of the rice blast bacterium is characterized in that: the full-length sequence of the rice blast fungus MoSpc2 gene is shown as SEQ ID No. 1, the cDNA sequence is shown as SEQ ID No. 2, and the amino acid sequence of the encoded rice blast fungus pathogenic protein MoSpc2 is shown as SEQ ID No. 3;

the construction method of the rice blast fungus mutant delta MoSpc2 comprises the following steps: amplifying a primer MoSpc2-F1/MoSpc2-F2 to obtain a MoSpc2 upper arm fragment, amplifying a primer MoSpc2-F3/MoSpc2-F4 to obtain a MoSpc2 lower arm fragment, then fusing the upper arm fragment, hygromycin phosphotransferase gene HPH and the lower arm fragment, amplifying a large amount of fusion products of the fusion products by using a primer MoSpc2-nestF/MoSpc2-nestR to obtain a large amount of DNA fusion products, carrying out genetic transformation to obtain transformants, and screening and identifying;

the sequences of the primers MoSpc2-F1/MoSpc2-F2 are shown in SEQ ID No. 6-7;

the sequences of the primers MoSpc2-F3/MoSpc2-F4 are shown in SEQ ID No. 8-9;

the sequence of the primer MoSpc2-nestF/MoSpc2-nestR is shown in SEQ ID No. 10-11;

the HPH sequence of the hygromycin phosphotransferase gene is shown as SEQ ID No. 4.