阅读说明：本技术 稻瘟病菌MoSpc2基因及其应用 (Magnaporthe grisea MoSpc2 gene and application thereof ) 是由 汤蔚 杨子锋 徐虎啸 李美琴 林碧蓉 陈江峰 吴欢 王敏 于 2021-10-20 设计创作，主要内容包括：本发明公开了一种稻瘟病菌MoSpc2基因及其应用,该基因由SEQ ID No:1所示的核苷酸序列组成,编码如SEQ ID No:2所示氨基酸序列的蛋白。利用潮霉素磷酸转移酶基因HPH替换稻瘟病菌MoSpc2基因后,所得到的稻瘟病菌敲除突变体与野生型稻瘟病菌相比在菌丝生长、附着孢形成以及产孢能力上有明显下降,在应对外界环境因子的应答上有明显的区别；缺失MoSpc2基因在水稻叶片上不能形成明显病斑,说明MoSpc2基因在调控稻瘟病菌致病力方面有显著作用。本发明所提供的MoSpc2基因及其应用在稻瘟病菌致病性方面具有重要作用,为进一步利用该基因防治稻瘟病菌提供了新的方向。(The invention discloses a rice blast bacterium MoSpc2 The gene consists of a nucleotide sequence shown in SEQ ID No. 1 and encodes a protein of an amino acid sequence shown in SEQ ID No. 2. Replacement of Magnaporthe grisea by hygromycin phosphotransferase gene HPH MoSpc2 After the gene is introduced, compared with wild rice blast, the obtained rice blast fungus knockout mutant has obvious reduction in hypha growth, attachment spore formation and spore production capacity, and has obvious difference in response to external environment factors; absence of MoSpc2 The inability of the gene to form obvious lesions on rice leaves indicates MoSpc2 Regulation of geneHas obvious effect on controlling the pathogenicity of rice blast germs. The invention provides MoSpc2 The gene and the application thereof have important effects on the pathogenicity of the rice blast germs and provide a new direction for further utilizing the gene to prevent and control the rice blast germs.)

1. Rice blast bacteriumMoSpc2A gene characterized by: the Magnaporthe griseaMoSpc2The gene has the full-length sequence shown in SEQ ID No. 1.

2. The Pyricularia oryzae according to claim 1MoSpc2A gene characterized by: has the following nucleotide sequence:

(a) has a nucleotide sequence complementary with the nucleotide sequence shown as SEQ ID No. 1; or

(b) Has a nucleotide sequence which has more than 90 percent of identity with the nucleotide sequence shown in SEQ ID No. 1.

3. The Pyricularia oryzae according to claim 1MoSpc2A gene characterized by: the Magnaporthe griseaMoSpc2The cDNA sequence of the gene is shown as SEQ ID No. 2.

4. The rice blast fungus of claim 1MoSpc2The gene-coded rice blast pathogenic protein MoSpc2 is characterized in that: the amino acid sequence of the rice blast pathogenic protein MoSpc2 is shown in SEQ ID No. 3.

5. The rice blast pathogenic protein MoSpc2 according to claim 4, characterized in that: the rice blast pathogenic protein MoSpc2 has an amino acid sequence which has more than 90% of identity with the amino acid sequence shown in SEQ ID No. 2 and has a rice blast pathogenic function.

6. A method for knocking out rice blast fungus as defined in claim 1MoSpc2Genetic Pyricularia oryzae mutantMoSpc2。

7. Magnaporthe grisea of claim 1MoSpc2The gene is in reducing pathogenicity of rice blast bacteriumThe use of (1).

8. Use of the rice blast pathogenic protein MoSpc2 according to claim 4 for reducing the virulence of rice blast.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to rice blast bacteriaMoSpc2Genes and their use.

Background

Rice is an important food crop, and nearly half of the global population takes rice as main grain. Pyricularia oryzae (A)Magnaporthe oryzae) The caused rice blast is a serious fungal disease in world rice production, occurs in each rice area in China, can cause absolute harvest when outbreaks occur to cause disasters, causes 10-30% of rice harvest loss every year, and can sufficiently nourish 6000 million people. At present, the main methods for preventing and controlling rice blast are to lay out disease-resistant varieties and apply chemical agents. Although the effective application of the disease-resistant variety and the nitrogen fertilizer and the effective combination of the bactericide achieve the effect of controlling the rice blast to a certain extent, the disease-resistant variety usually loses the original blast resistance gradually within years after being implemented due to the complex and various rice blast germs in the field; and the single or excessive use of chemical pesticides can easily cause pathogenic bacteria to generate drug resistance, thereby bringing a serious challenge to the effective prevention and control of the rice blast.

Signal peptides have a key role in directing proteins to target the secretory pathway of the endoplasmic reticulum. The structural characteristics of these signal peptides vary with their output location and their specificity is usually achieved by interacting with receptors on the target membrane. Signal peptidases, as a major component of signal peptide processing, have a crucial role in the release, maturation and secretion of proteins. Once the signal peptidase fails to cleave the signal peptide, it will cause the secretory substrate to be retained intracellularly and the secretory process cannot be completed. Regarding the functional studies of signal peptidases, the focus is now mainly on bacteria, yeasts and animals, and there are few studies on fungi and plants. Therefore, the research on the pathogenic mechanism of the signal peptidase can provide an important drug target for the design and screening of the rice blast germ targeting drugs, and the research has important theoretical significance and application value on the comprehensive control of the rice blast germ.

Disclosure of Invention

The invention aims to provide a rice blast bacterium aiming at the problems of serious damage and lack of an effective prevention and control method of the existing rice blastMoSpc2Genes and their use. Magnaporthe griseaMoSpc2The gene plays an important role in hypha vegetative growth, conidium generation, attachment cell formation and pathogenicity of rice blast germs.

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

rice blast bacteriumMoSpc2The gene has the full-length sequence shown in SEQ ID No. 1.

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

(a) has a nucleotide sequence complementary with the nucleotide sequence shown as SEQ ID No. 1; or

(b) Has a nucleotide sequence which has more than 90 percent of 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.

The rice blast fungusMoSpc2The amino acid sequence of the gene-coded pathogenic protein MoSpc2 of Magnaporthe grisea is shown in SEQ ID No. 3.

Furthermore, the pathogenic protein MoSpc2 of the rice blast fungus 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 a rice blast pathogenic function.

Rice blast bacterium knockout methodMoSpc2Genetic Pyricularia oryzae mutantMoSpc2。

The above Magnaporthe griseaMoSpc2The application of the gene in reducing the pathogenicity of rice blast bacteria.

The application of the pathogenic protein MoSpc2 of Magnaporthe grisea in reducing pathogenicity of Magnaporthe grisea.

The invention has the advantages that: the invention proves that the physiological functions of the rice blast fungus MoSpc2 gene such as vegetative growth, conidium germination, attachment spore determination, pathogenicity and the like are verifiedMoSpc2The deletion or deletion of the gene results in a decrease in the pathogenicity of Pyricularia oryzae, thus indicating thatMoSpc2The gene is an important gene in the pathogenic process of rice blast germs. The invention providesMoSpc2The gene and the application thereof provide a new direction for the prevention and control of rice blast germs.

Description of the drawings:

FIG. 1 is a drawing ofMoSpc2Schematic diagram of gene knock-out 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 single copy integration of the HPH gene in the mutant genome.

FIG. 3 is a drawing showingMoSpc2Photographs of the colony and aerial hyphae morphology of the knockout mutant on CM, SDC, OMA plate medium.

FIG. 4 is a drawing showingMoSpc2And (3) a colony morphology photo of the knockout mutant under the stress of various external environmental factors.

FIG. 5 is a drawing showingMoSpc2The gene knockout mutant is compared with the wild conidium morphology.

FIG. 6 is a drawing showingMoSpc2Statistics of the number of conidia of knock-out mutants and wild type cultured in SDC medium at 28 ℃ for 8 days.

FIG. 7 is a drawing showingMoSpc2Pathogenicity of gene knockout mutant and wild type on rice leaf

FIG. 8 is a drawing showingMoSpc2Germination of the Acidocella at different times for the knockout mutant and the wild type.

The specific implementation mode is as follows:

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

Test strains and plants:

magnaporthe griseaThe wild type strain Guy11 (of Magnaporthe grisea) isMagnaportheoryzae), the tested rice is rice susceptible rice blast variety Co-39.

(II) preparation of 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), solid medium added with agar powder (20 g/L);

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

1000 × trace elements (100 ml): ZnSO₄. 7H₂O 2.2g，H₃BO₃ 1.1g，MnCl₂. 4H₂O 0.5g，FeSO₄. 7H₂O 0.5g，CoCl₂. 6H₂O 0.17g，CuSO₄. 5H₂O 0.16g，Na₂MoO₄. 5H₂O 0.15g ，Na₄EDTA 5g；

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

(3) straw medium (SDC): corn flour (25 g/L), dry straw (150 g/L) and agar powder (20 g/L). Is used for inducing the rice blast fungus 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 was obtained by NCBI database search, and this was used as a subject to alignment by blastp tool at NCBI official website to obtain a protein (MGG _ 10832) similar to that in Pyricularia oryzae, and this was named MoSpc 2. The amino acid sequence of the pathogenic protein MoSpc2 of rice blast fungus is shown in SEQ ID No. 3. The gene for coding the protein is rice blast bacteriumMoSpc2The gene has the full-length sequence shown in SEQ ID No. 1; the cDNA sequence is shown in SEQ ID No. 2.

Example 2 Gene knockout

(1) Taking the wild type Guy11 genome of rice blast fungus as a template, selectingMoSpc2The upstream 1000bp fragment of the geneMoSpc2-F1 (forward),MoSpc2Amplification of the-F2 (reverse) primerMoSpc2Upper arm segments, selectingMoSpc2A downstream 1000bp fragment of the gene toMoSpc2-F3 (forward),MoSpc2F4 (reverse) amplificationMoSpc2Lower arm segment (fig. 1). Then the upper arm, hygromycin phosphotransferase gene HPH (SEQ ID No: 4) and the lower arm fragment are fused, and the fusion product is expressed asMoSpc2-nest F andMoSpc2-nest R is amplified in large quantities to give large quantities of DNA fusion products. The fusion product was subjected to protoplast transformation in place of the Pyricularia oryzae SPC2 gene.

(2) Protoplast transformation process:

adding 10 mu L of the DNA fusion product into the split-packaged wild rice blast germ protoplast, gently mixing the mixture evenly, and standing the mixture for 20 to 25 min (to ensure that the protoplast is fully contacted with the DNA);

adding 1 mL of PTC, gently mixing uniformly, and standing for 20-25 min;

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

Fourthly, in the well melted TB₃Hygromycin (600. mu.L/200 ml) at the desired concentration was added to the solid medium and poured into resuscitated centrifuge tubes. Mixing, pouring into a sterile 15 cm culture dish, cooling and solidifying, pouring TB3 culture medium with hygromycin (1200 μ L/200 ml) to cover the surface, cooling and solidifying, sealing the culture dish with a sealing film, and pouring into an illumination incubator at 8 deg.C for culturing for 6-10 days;

fifthly, carefully selecting a single colony in the upper layer culture medium to inoculate the single colony in a CMII solid culture medium plate after a transformant grows out from the culture dish, and subsequently further verifying the transformant;

(3) by usingMoSpc2Gene inner primerMoSpc2-ko-F andMoSpc2and (4) performing PCR knockout verification on the-ko-R, and selecting a non-banded transformant by taking a negative non-banded transformant as a control and a positive banded transformant as a control for further verification. By usingMoSpc2And (3) carrying out hygromycin transfer verification on the-F1 and an internal primer HYG R of a hygromycin phosphotransferase gene HPH, and selecting a transformant with a band to carry out southern blot verification by taking a negative non-band as a control.

(4) And (3) Southern hybridization identification: selectingMoSpc2Designing a probe (SEQ ID No: 5) from the sequence of the downstream coding region of the gene, and selecting a restriction endonuclease without a recognition site in the probeNruIThe genomic DNAs of the candidate transformant and the wild type strain Guy11 were digested with enzyme, respectively (FIG. 2). In the hybridization verification map, a band of a predetermined size of 2.07 kb appeared in the wild-type lane, and a band of a size of 2.60 kb appeared in the mutant lane (FIG. 2), in agreement with the prediction, indicating that transformants had been transformedMoSpc2The gene has been successfully replaced by the HPH gene and is a single copy insertion. Identification of Pyricularia oryzae mutantMoSpc2。

(5) The primer sequences used were:

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 for the experiment:

amplification PCR reaction (50. mu.L):template 0.5. mu.L, 2 × reaction mix 25. mu.L, upstream primer 2. mu.L, downstream primer 2. mu.L, ddH₂O25. mu.L. The PCR reaction program is: 94 ℃ for 5min, 94 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 1kb/min, 30 cycles, 72 ℃ for 10min, 16 ℃ for 10 min.

Fusion PCR reaction (25 μ L): the molar ratio of the template upstream fragment to the hygromycin fragment to the downstream fragment is 1: 3: 1 addition, 2.5. mu.L of 10 XPCR buffer, 0.5. mu.L of dNTP mix, 0.25. mu.L of LTaq enzyme, ddH₂021.75 μ L. The PCR reaction program is: 94 ℃ for 3min, 94 ℃ for 35s, 58 ℃ for 5min, 72 ℃ for 5min, 10 cycles, 16 ℃ for 10 min.

Example 3:MoSpc2deletion of genes affecting vegetative growth of Pyricularia oryzae

The wild type Guy11, the mutantMoSpc2Inoculating into CM II, SDC and OMA culture medium plates, culturing at 28 deg.C in dark for 7 d, measuring colony growth diameter, and taking pictures for storage. The experimental results showed that the colony growth diameter of the mutant was smaller than that of the wild type in all three media (fig. 3), especially in SDC media, indicating a significant reduction in SDC mediaMoSpc2The deletion of the gene affects the vegetative growth of rice blast fungi.

Example 4: MoSpc2the deletion of the gene influences the response of the rice blast fungus to the external environment stress

Wild type strain Guy11, mutant ΔMoSpc2They were inoculated in CM II medium containing 0.5 mM diamine (Diamide, causing oxidative stress), 2 mM DTT (dithiothreitol, causing stress to the endoplasmic reticulum), and 400. mu.g/mL CFW (cell wall inhibitor calcium fluorescent white, causing stress to the cell wall) and cultured in the dark at 28 ℃ for 7 d, respectively. The results of the experiment showed that the mutants grew significantly slower on CM II plates containing diamine and DTT and significantly faster on CM II plates containing CFW (FIG. 4). The above results show that it is possible to obtain,MoSpc2the deletion of the gene influences the response of the rice blast fungus to the external environment stress.

Example 5:MoSpc2the gene participates in the asexual propagation process of regulating and controlling rice blast bacteria

The wild type Guy11, the mutantMoSpc2Is inoculated toAnd (3) scraping off aerial hyphae on the surface of the culture medium after culturing the medium in the dark at the temperature of 28 ℃ for 5 d in the center of the SDC culture medium flat plate, placing the medium in a greenhouse at the temperature of 28 ℃, inducing 3 d sporulation under the regulation and control of a black light lamp, and quantitatively counting the sporulation yield of the corresponding strain in the SDC culture medium. The experimental results show that the mutant isMoSpc2The produced partial spores were morphologically abnormal, and conidia of different strains were stained with CFW and further observed for morphology (fig. 5): from microscopic observation, the conidia of the wild strain are basically three-cell two-septum, while the mutant has three spore morphologies: there are no septa, one and two, where the spore fraction of a single septum is 64.6% and the size of the conidia is also reduced relative to the wild type. On the other hand, the mutant is Δ compared with the wild type Guy11MoSpc2Also the sporulation yield of (a) decreased significantly (fig. 6). The above results illustrateMoSpc2The gene is important for the asexual propagation process of rice blast germs.

Example 6: MoSpc2the gene participates in the regulation of the pathogenic process of rice blast bacteria

Collecting the wild type Guy11, the mutantMoSpc2Conidia suspension (1X 10)⁵one/mL) of the cells were inoculated on three-week-old rice leaves, respectively, and the rice spray test was performed, and the lesion spots on the leaves were observed after 6 days. The results show that on rice leaves, the wild type Guy11 can produce typical rice blast disease spots, and the mutant ΔMoSpc2No lesion was produced (fig. 7). In conclusion, the results show that,MoSpc2the pathogenicity of the rice blast bacterium is obviously reduced by the deletion of the gene, which shows thatMoSpc2The gene plays an important role in the pathogenic process of rice blast germs.

Example 7: MoSpc2deletion of the Gene delays maturation of the adherent cells

Respectively collecting the wild type Guy11 and the mutantMoSpc2Conidia of the strain induce the germination of the anchorage cell under hydrophobic condition. The germination and formation of anchorage cells of different strains at 4 h, 8 h and 16 h are respectively observed by a microscope. It was found (FIG. 8) that conidia of the wild type Guy11 strain had germinated substantially at 4 h, and that the mutant ΔMoSpc2The conidiophores of (1) have delayed germination, and the germination time is 16 hThe germination rate is only 82%. According to the statistical result, the mutant is shownMoSpc2The formation rate of the attached cells of the conidia is similar to the result of the germination rate, the conidia of the wild type already basically form the attached cells at 8 h, but the formation rate of the attached cells of the mutant at 16 h is only 61%. The above results show thatMoSpc2Deletion of the gene delays development of the adherent cells.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> Magnaporthe oryzae MoSpc2 gene and application thereof

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<170> PatentIn version 3.3

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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