Use of protein moupe11 in reducing the pathogenicity of magnaporthe grisea

Abstract

The application discloses application of protein MoUPE11 in reducing pathogenicity of Magnaporthe oryzae. MoUPE11 The application constructs a knock-out vector of a gene coding the protein MoUPE11, and obtains a knock-out mutant MoUPE11 Pathogenicity analysis shows that, compared with a wild type, the pathogenicity of the knock-out mutant MoUPE11- is significantly reduced, and the pathogenicity of a back-mutant com restores to the wild type level, indicating that the protein MoUPE11 is related to the pathogenicity of Magnaporthe oryzae, and the pathogenicity of Magnaporthe oryzae can be reduced by inhibiting expression of the protein MoUPE11 in Magnaporthe oryzae, so as to reduce the incidence of rice blast, realize prevention and treatment of rice blast, and reduce the adverse effects of rice blast on rice. In addition, the protein MoUPE11 can also be used as a target to develop green prevention and control products for rice blast.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological agent development and preparation of plant diseases. More specifically, it relates to the application of protein MoUPE11 in reducing the pathogenicity of Magnaporthe oryzae. BACKGROUND

[0002] The pathogen of rice blast is Magnaporthe oryzae (Pyricularia oryzae) which can cause 10% to 30% reduction of rice yield every year, threatening the safe production of rice. Magnaporthe oryzae

[0003] The main methods for preventing rice blast are chemical control, biological control and breeding of disease-resistant varieties. Breeding of disease-resistant varieties is the most effective method for preventing rice blast, which is economical and sustainable. However, the breeding cycle of disease-resistant varieties is long, and the mutation rate of Magnaporthe oryzae is fast, which makes it difficult to keep up with the mutation rate of Magnaporthe oryzae. Chemical control has a quick effect, but it can easily lead to drug resistance of the pathogen and environmental pollution and pesticide residues if not used properly. Biological control has the advantage of environmental friendliness, but its control effect is easily affected by environmental factors.

[0004] Taking Magnaporthe oryzae pathogenicity-related genes as targets and reducing the pathogenicity of Magnaporthe oryzae are important methods for preventing rice blast. At present, the molecular mechanism of Magnaporthe oryzae pathogenicity is not yet perfect, and it is necessary to continuously excavate Magnaporthe oryzae pathogenicity-related genes to facilitate the development of rice blast prevention and treatment preparations with multiple targets and low resistance risk. SUMMARY

[0005] In order to perfect the molecular mechanism of Magnaporthe oryzae pathogenicity and enrich the targets for the development of rice blast prevention and treatment preparations, the application provides the application of protein MoUPE11 in reducing the pathogenicity of Magnaporthe oryzae.

[0006] The first object of the application is to provide the application of protein MoUPE11 in reducing the pathogenicity of Magnaporthe oryzae.

[0007] The second object of the application is to provide the application of a reagent for inhibiting the expression of a gene encoding the protein MoUPE11 in reducing the pathogenicity of Magnaporthe oryzae.

[0008] The third object of the application is to provide the application of a reagent for inhibiting the expression of a gene encoding the protein MoUPE11 in preparing a product for reducing the pathogenicity of Magnaporthe oryzae.

[0009] The fourth object of the application is to provide the application of a reagent for knocking out a gene encoding the protein MoUPE11 in reducing the pathogenicity of Magnaporthe oryzae.

[0010] The fifth object of the application is to provide the application of a reagent for knocking out a gene encoding the protein MoUPE11 in preparing a product for reducing the pathogenicity of Magnaporthe oryzae.​

[0011] A sixth object of the present application is to provide the use of an agent inhibiting the expression of a gene encoding the protein MoUPE11 in the prevention and treatment of rice blast.

[0012] A seventh object of the present application is to provide the use of an agent inhibiting the expression of a gene encoding the protein MoUPE11 in the preparation of a product for the prevention and treatment of rice blast.

[0013] An eighth object of the present application is to provide the use of an agent for knocking out a gene encoding the protein MoUPE11 in the prevention and treatment of rice blast.

[0014] A ninth object of the present application is to provide the use of an agent for knocking out a gene encoding the protein MoUPE11 in the preparation of a product for the prevention and treatment of rice blast.

[0015] The above objects of the present application are achieved by the following technical solutions:

[0016] The present application constructs a knockout vector for a gene encoding the protein MoUPE11, and obtains a knockout mutant MoUPE11 and found that, compared with the wild type control, MoUPE11 the conidium germination and appressorium development are slowed down, the glycogen metabolism of the conidium is slowed down, the conidium is more sensitive to oxidative stress and the pathogenicity is significantly reduced, while the phenotype and pathogenicity of the complemented mutant are similar to those of the wild type of Magnaporthe oryzae, indicating that the protein MoUPE11 is related to the pathogenicity of Magnaporthe oryzae. Therefore, the present application claims the application of the protein MoUPE11 in reducing the pathogenicity of Magnaporthe oryzae.

[0017] Specifically, the application is achieved by inhibiting the expression of the protein MoUPE11 in Magnaporthe oryzae; the amino acid sequence of the protein MoUPE11 is shown in SEQ ID NO. 1.

[0018] Specifically, the inhibition of the expression of the protein MoUPE11 in Magnaporthe oryzae is achieved by interfering with the expression of a gene encoding the protein MoUPE11, or by mutating the coding region of a gene encoding the protein MoUPE11, or by knocking out a gene encoding the protein MoUPE11.

[0019] More specifically, the inhibition of the expression of the protein MoUPE11 in Magnaporthe oryzae is achieved by interfering with the expression of a gene encoding the protein MoUPE11 by RNA interference technology, or by mutating the coding region of a gene encoding the protein MoUPE11 by a gene editing system, or by knocking out a gene encoding the protein MoUPE11 by homologous recombination.

[0020] The application also provides a method for reducing the pathogenicity of Magnaporthe oryzae, which comprises inhibiting the expression of protein MoUPE11 in Magnaporthe oryzae.

[0021] Specifically, the expression of protein MoUPE11 in Magnaporthe oryzae is inhibited by interfering with the expression of the gene encoding the protein MoUPE11, or by mutating the coding region of the gene encoding the protein MoUPE11, or by knocking out the gene encoding the protein MoUPE11.

[0022] More specifically, the expression of protein MoUPE11 in Magnaporthe oryzae is inhibited by interfering with the expression of the gene encoding the protein MoUPE11 by RNA interference technology, or by mutating the coding region of the gene encoding the protein MoUPE11 by a gene editing system, or by knocking out the gene encoding the protein MoUPE11 by homologous recombination.

[0023] In view of the fact that the pathogenicity of Magnaporthe oryzae can be significantly reduced by knocking out the gene encoding protein MoUPE11 to inhibit the expression of protein MoUPE11 in Magnaporthe oryzae, the prevention and treatment of rice blast is achieved. Therefore, the application also claims the use of a reagent for inhibiting the expression of the gene encoding the protein MoUPE11 in reducing the pathogenicity of Magnaporthe oryzae.

[0024] The application also claims the use of a reagent for inhibiting the expression of the gene encoding the protein MoUPE11 in preparing a product for reducing the pathogenicity of Magnaporthe oryzae.

[0025] The application also claims the use of a reagent for knocking out the gene encoding the protein MoUPE11 in reducing the pathogenicity of Magnaporthe oryzae.

[0026] The application also claims the use of a reagent for knocking out the gene encoding the protein MoUPE11 in preparing a product for reducing the pathogenicity of Magnaporthe oryzae.

[0027] The application also claims the use of a reagent for inhibiting the expression of the gene encoding the protein MoUPE11 in preventing and treating rice blast.

[0028] The application also claims the use of a reagent for inhibiting the expression of the gene encoding the protein MoUPE11 in preparing a product for preventing and treating rice blast.

[0029] The application also claims the use of a reagent for knocking out the gene encoding the protein MoUPE11 in preventing and treating rice blast.

[0030] The application also claims the use of a reagent for knocking out the gene encoding the protein MoUPE11 in preparing a product for preventing and treating rice blast.

[0031] Optionally, the reagent for inhibiting the expression of the gene encoding the protein MoUPE11 is a reagent for knocking out the gene encoding the protein MoUPE11 by using antisense gene technology or RNA interference technology. MoUPE11 The reagent for inhibiting the expression is used.

[0032] Optionally, the reagent is siRNA or dsRNA targeting the gene MoUPE11 .

[0033] Specifically, the reagent for knocking out the gene encoding the protein MoUPE11 is a reagent for functionally knocking out the gene or knocking out the entire gene. MoUPE11

[0034] Optionally, the reagent is a knockout vector for the gene MoUPE11 .

[0035] Optionally, the knockout vector is constructed by using the filamentous fungus expression vector pCT74.

[0036] Specifically, the rice blast is caused by Magnaporthe oryzae. Magnaporthe oryzae .

[0037] In addition, the conidial germination and appressorium development of the Magnaporthe oryzae knockout mutant Δ MoUPE11 are slowed down, and the glycogen metabolism of the conidium is slowed down. Therefore, the application of the reagent for inhibiting the expression of the gene encoding the protein MoUPE11 or the reagent for knocking out the gene encoding the protein MoUPE11 in inhibiting the conidial germination, appressorium development and glycogen metabolism of the conidium of Magnaporthe oryzae is also within the protection scope of the present application.

[0038] The present application has the following beneficial effects:

[0039] The present application constructs a knockout vector for the gene encoding the protein MoUPE11, and constructs a knockout mutant MoUPE11 The pathogenicity analysis of the knockout mutant shows that the pathogenicity of the knockout mutant MoUPE11 is significantly reduced compared with the wild type, while the pathogenicity of the complementation mutant MoUPE11 com returns to the wild type level, indicating that the protein MoUPE11 is related to the pathogenicity of Magnaporthe oryzae, and the expression of the protein MoUPE11 in Magnaporthe oryzae can be inhibited to reduce the pathogenicity of Magnaporthe oryzae, and thus the incidence of rice blast is reduced, the prevention and control of rice blast are realized, and the adverse effects of rice blast on rice are reduced. In addition, the green prevention and control product of rice blast can also be developed by taking the protein MoUPE11 as a target. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Magnaporthe oryzae​MoUPE11 A schematic diagram of the construction of a gene knockout vector.

[0041] Figure 2 For some hygromycin-resistant transformants HPH Gene PCR analysis results; DNA marker: DL 2000 Marker.

[0042] Figure 3 For some hygromycin-resistant transformants MoUPE11 Gene PCR analysis results; DNA marker: DL 2000 Marker.

[0043] Figure 4 For HPH Southern blot analysis results of rice blast fungus knockout transformants with fragments as probes.

[0044] Figure 5 For some bleomycin-resistant transformants MoUPE11 Gene PCR analysis results; DNA marker: DL2000 Marker.

[0045] Figure 6 Rice blast fungus knockout mutant MoUPE11 and complement mutant MoUPE11 -com Gene MoUPE11 The results of RT-qPCR analysis are shown; different letters in the figure indicate significant differences. p< 0.05.

[0046] MoUPE11 Rice blast fungus knockout mutant Figure 7 and complement mutant MoUPE11 Statistical results of conidial germination and appressorium formation observed in -com; Figure A shows the statistical results of conidial germination rate; Figure B shows the statistical results of appressorium formation rate; Figure p< 0.05.

[0047] MoUPE11 Rice blast fungus knockout mutant Figure 8 and complement mutant MoUPE11 Analysis results of the stress resistance capability of com.

[0048] MoUPE11 Rice blast fungus knockout mutant Figure 9 and complement mutant MoUPE11The results of pathogenicity determination of the com against rice in vitro leaf. DETAILED DESCRIPTION

[0049] The present application will be further described by the following description of the drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the art.

[0050] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0051] The amino acid sequence of the protein MoUPE11 described in the present application is shown below (SEQ ID NO. 1), and the underlined part in the sequence is the signal peptide thereof:

[0052] MoUPE11 IDVRGYVGDNCGGAWVGCANLNPNVCCTPFGGSRASVGFAAIPTNWRIRGQAFTGGGCNSYGGQGDSNGRDFFCLPYTTRGDRTGGSYSFVNRKRAIDETCPAEQPLGRCEATVKPDTIGLADGTEYNITSLSEDQVNELSAIAASGAGVEAVPANFQVLRRSISA.

[0053] The gene encoding the protein MoUPE11, i.e. the gene MQFSALLVTFAAATVSA The nucleotide sequence of the gene encoding the protein MoUPE11, i.e. the gene

[0054] MoUPE11 CTAAGCACTAATTGAGCGGCGCAGCACCTGGAAGTTGGCGGGGACAGCCTCGACACCGGCGCCGGAAG CTTTTGGGAAACCAGTTCAAGTCACGTCAGCATTTCGAGTCATAGAAAGGCACGAGAAAAGTAAGAGAAAGAAAAGAAAAACCCAC CAGCAATGGCGCT CAGCTCGTTAACCTGGTCCTCGGACAGACTCGTGATGTTGTACTCGGTGCCGT CAGCAAGGCCAATGGTGTCGGGCTTGACCGTGGCTTCGCATCTGCCCAGAGGCTGCTCGGCCGGGCAGGTCTCGTC GATGGCGCGCTTGCGGTTGACGAACGAGTACGAGCCGCCCGTGCGATCACCACGCGTCGTGTACGGGAGGCAGAAG AAGTCTCTGCCGTTGGAGTCCCCCTGGCCTCCGTACGAGTTGCAGCCGCCTCCCGTAAAGGCCTGGCCGCGGATAC CTGTACAATTGTAATGAGGTTTTTTGTCTGGTTAAGCTTTGCTCTTGTTTTTTTCCTTTTTTGTGTCTTTGAGAAAAGAAATCATTCGGAAGAAACTCAC GCCAGTTGGTCGGGAT GGCTGCAAAGCCAACGCTAGCCCTCGAGCCGCCAAA CGGTGTACAGCATACGTTGGGGTTGAGGTTTGCGCAACCAACCCAGGCCCCGCCGCAGTTGTCGCCGACGTAGCCC .

[0055] The wild-type strain of *Magnapordica oryzae* used in this invention is the dominant race ZC13 ​​in Guangdong Province; the tested rice is the susceptible indica rice line CO39; the cloning vector is pMD18-T vector; the gene knockout vector is the filamentous fungal expression vector pCT74; and the gene complementation vector is pCTZN (derived from the pCT74 plasmid, i.e., the gene on pCT74...). CTGACATCGATGGCCGACACGGTGGCGGCCGCGAAAGTGACGAGAAGTGCTGAGAACTGCAT and GFP Gene replaced with bleomycin resistance gene HPH ).

[0056] Example 1 Zeocin Obtaining knockout mutants and complement mutants

[0057] 1. Experimental Procedure

[0058] Rice blast fungus MoUPE11 A schematic diagram of gene knockout vector construction is shown below. MoUPE11 As shown. By Figure 1 It can be seen that the present invention achieves homologous recombination. Figure 1 Gene replacement MoUPE11 +gfp fragment, to achieve hph Gene knockout.

[0059] (1) Rice blast fungus MoUPE11 Amplification of upstream and downstream homologous arms of genes

[0060] exist MoUPE11 Gene sequences of approximately 1000 bp in length were selected upstream and downstream of the gene as its homologous arms. The upstream homologous arm was named homologous arm A and the downstream homologous arm was named homologous arm B. Amplification primers were designed based on the selected sequences, as shown in Table 1 (SEQ ID NO. 3-6).

[0061] Table 1 MoUPE11 Primers for amplification of gene homologous arms A and B fragments

[0062]

[0063] Genomic DNA was extracted from wild-type strains of *Magnapordica oryzae* using the OMEGA Fungal DNA Kit. Using the obtained genomic DNA as a template, PCR amplification was performed using the primers shown in Table 1 to obtain... MoUPE11 Homologous arm A of the gene ( MoUPE11 -A) and homologous arm B ( MoUPE11 -B).

[0064] The PCR reaction system used for PCR amplification is shown in Table 2:

[0065] Table 2. PCR reaction system used for amplifying upstream and downstream homologous arms

[0066]

[0067] The PCR reaction conditions used for PCR amplification were as follows: 94℃ for 5 min; 98℃ for 10 s, 55℃ for 30 s, 72℃ for 1 min, for a total of 30 cycles; 72℃ for 10 min.

[0068] After the reaction, the PCR amplification products were cleaned and recovered using the OMEGA Cycle Pure Kit.

[0069] (2) MoUPE11 Construction of gene knockout vector

[0070] Referring to the instructions for the pMD18-T Vector Cloning Kit (TakaRa), clean and recover the obtained... MoUPE11 -A and MoUPE11 -B was ligated to the T vector to obtain the recombinant plasmid pMD18T- MoUPE11 -A and pMD18T- MoUPE11 -B. The bonding system used is shown in Table 3, and the reaction conditions were overnight bonding at 16°C.

[0071] Table 3 Connection System

[0072]

[0073] Add 10 µL of the ligation product to 100 µL of E. coli DH5α competent cells and place on ice for 30 min; heat shock in a water bath at 42℃ for 90 s, then cool on ice for 5 min; add 700 µL of LB liquid medium and culture at 37℃ and 180 rpm for 1 h with shaking; centrifuge at 3500 rpm for 5 min, discard the supernatant, and mix 100 µL of the bacterial culture with the precipitate, then spread it on LB solid medium (containing 100 µg / mL Amp); culture at 37℃ for 8–12 h.

[0074] Positive transformants with Amp resistance were selected, and recombinant plasmid DNA was extracted for sequencing and alignment. MoUPE11 I and Apa I respectively analyzed the pMD18T- sequenced and aligned samples. Kpn The A and pCT74 vectors were double-digested and gel-cleaved. The homologous arm A fragment was ligated to pCT74 using T4 DNA ligase, and the resulting compound was transformed into *E. coli* DH5α to obtain the recombinant plasmid pCT74-. MoUPE11 -A. Follow the same procedure, using MoUPE11 I and Xba pMD18T- RI double digestion and sequencing alignment Eco-B and recombinant plasmid pCT74- MoUPE11 -A was then excised and recovered from the gel. The homologous arm B fragment was ligated with pCT74- using T4 DNA ligase. MoUPE11 -A ligation was performed, and the cells were transformed into *E. coli* DH5α. Enzyme digestion confirmed the presence of the gene knockout vector pCT74-. MoUPE11 -KO.

[0075] (3) MoUPE11 Amplification of gene complement fragments

[0076] exist MoUPE11 A promoter sequence of 1500 bp was selected upstream of the gene, and a terminator sequence of 500 bp was selected downstream. Amplification primers were designed based on the selected sequences, as shown in Table 4 (SEQ ID NO. 7-8).

[0077] Table 4 MoUPE11 Amplification primers for gene complement fragments

[0078]

[0079] Using the above genomic DNA as a template, primer Com- MoUPE11 MoUPE11 -F and Com- MoUPE11 -R is used for PCR amplification to obtain... MoUPE11 Gene complement fragment ( MoUPE11 -com).

[0080] The PCR reaction system used for PCR amplification is shown in Table 5:

[0081] Table 5 Amplification MoUPE11 PCR reaction system used for gene complementation fragment

[0082]

[0083] The PCR reaction conditions used for PCR amplification were as follows: 94℃ for 5 min; 98℃ for 10 s, 55℃ for 30 s, 72℃ for 4 min, for a total of 30 cycles; and 72℃ for 10 min.

[0084] After the reaction, the PCR amplification products were cleaned and recovered using the OMEGA Cycle Pure Kit.

[0085] (4) MoUPE11 Construction of gene complementation vector

[0086] use Xba I and Eco RI respectively MoUPE11 The -com and pCTZN vectors were double-digested and then gel-cleaved and recovered. The DNA was ligated using T4 DNA ligase.MoUPE11 The -com fragment was ligated with pCTZN, and the mixture was transformed into E. coli DH5α to obtain the recombinant plasmid pCTZN- MoUPE11 -com. The gene complementation vector pCTZN- was obtained after enzyme digestion verification. MoUPE11 -com.

[0087] (5) Preparation of rice blast fungus protoplasts

[0088] Activated *Magnaporphyra yezoensis* strains were inoculated onto Jianli solid medium (yeast extract 5.0 g / L, anhydrous glucose 22.0 g / L, and agar powder 17.0 g / L) and cultured at 28°C for approximately 10 days. Mycelia were then collected using sterile forceps and transferred to 50 mL of YPS liquid medium (yeast extract 6.0 g / L, hydrolyzed casein 6.0 g / L, and sucrose 10.0 g / L) and cultured at 28°C with shaking at 120 rpm for 2 days. Mycelia were collected through a 200-mesh sieve, thoroughly ground, and transferred to 200 mL of YPS medium, and cultured for another day under the same conditions. Mycelia were then collected by filtration through a sieve and washed twice with sterile ddH2O and once with 0.7 mol / L NaCl solution. After drying, weigh 0.3–0.5 g of wet mycelium and add 10 mL of enzyme solution (containing 15 mg / mL lysozyme and 15 mg / mL wall-breaking enzyme) per 1 g of mycelium for enzymatic hydrolysis. Hydrolyze at 30°C and 80–110 rpm for approximately 1.5 h with shaking, observing the degree of hydrolysis under a microscope during this period. Filter the hydrolysate through four layers of clean paper. Wash the filtrate with pre-cooled 0.7 mol / L NaCl and collect it. Centrifuge at 4°C and 3500 rpm for 10 min. Resuspend the precipitate in 10 mL of pre-cooled STC solution (1.2 mol / L sorbitol, 10 mmol / L Tris-HCl, 50 mmol / L CaCl2, pH 7.5), centrifuge again, and resuspend in 1 mL of STC. Count the protoplasts using a hemocytometer and adjust the protoplast concentration to 1 × 10⁻⁶. 7 ~1×10 8 Quantity / mL, aliquoted and stored at -80℃.

[0089] (6) Transformation of rice blast fungus protoplasts

[0090] use Xba I linearized knockout vector pCT74- MoUPE11 -KO, take 200 μL of rice blast fungus protoplasts and add 3-5 μg of the above linearized plasmid; or add the linearized complementation vector pCTZN- MoUPE11The -com plasmid was mixed with the protoplasts of the rice blast fungus knockout mutant; after incubating on ice for 20 min, 2 mL of pre-chilled PTC solution (60% PEG4000, 50 mmol / L CaCl2, 10 mmol / L Tris-HCl, pH 7.5) was added and mixed, and the mixture was incubated on ice for another 15 min; 20 mL of pre-chilled STC solution was added, and the mixture was centrifuged at 4000 rpm for 15 min at 4 °C. The pellet was resuspended in 4 mL of liquid regeneration medium (6.0 g / L yeast extract, 6.0 g / L hydrolyzed casein, and 200.0 g / L sucrose) and thawed at 28 °C and 100 rpm for 16–18 h; 30 mL of solid regeneration medium containing 200 μg / mL hygromycin was added to the centrifuge tube and cooled to about 45 °C; in the experiment of transforming protoplasts with the supplemental vector, the regeneration medium contained 150 µg / mL bleomycin, and after mixing, the mixture was poured into plates and incubated upside down in the dark at 28 °C for 4–5 days. Select resistant transformants and transfer them to Jianli medium containing the same concentration of hygromycin / bleomycin. Incubate at 28°C for 2–3 days for secondary verification to obtain stable transformed strains.

[0091] (8) PCR verification analysis of rice blast fungus knockout mutant

[0092] Genomic DNA was extracted from the hygromycin-positive transformants according to the instructions of the OMEGA Fungal DNA Kit, and then extracted using primers. HPH -F / HPH -R and MoUPE11 -F / MoUPE11 -R to proceed HPH Gene fragments and MoUPE11 PCR validation analysis of gene fragments. Primers used for PCR validation analysis of knockout mutants are shown in Table 6 (SEQ ID NO. 9-12).

[0093] Table 6 Primers used for PCR validation analysis of knockout mutants

[0094]

[0095] The PCR reaction system used for PCR validation analysis is shown in Table 7:

[0096] Table 7 PCR reaction system used for PCR validation analysis

[0097]

[0098] The PCR reaction conditions were as follows: 98℃ for 3 min; 98℃ for 10 s, 60℃ for 10 s, 72℃ for 10 s, for a total of 30 cycles; 72℃ for 10 min to obtain the amplification product.

[0099] (9) PCR verification analysis of the complemented mutant of rice blast fungus

[0100] Genomic DNA was extracted from the bleomycin-positive transformants according to the instructions of the OMEGA Fungal DNA Kit, and primers were used. MoUPE11 -F / MoUPE11 -R performs gene fragmentation MoUPE11 The PCR validation analysis used the same PCR reaction system and conditions as the knockout mutant PCR validation analysis.

[0101] (10) Southern blot analysis of rice blast fungus knockout mutant

[0102] Use primers HPH -F / HPH -R amplification HPH Gene probes were used according to the instructions of the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche LOT28309220). HPH The gene probe was subjected to Southern blot hybridization. The PCR reaction system used to amplify the gene probe is shown in Table 8.

[0103] Table 8 PCR reaction system used for amplifying gene probes

[0104]

[0105] The PCR reaction conditions were as follows: 94℃ for 5 min; 98℃ for 10 s, 55℃ for 30 s, 72℃ for 1 min, for a total of 35 cycles; and 72℃ for 10 min to obtain the amplification product.

[0106] (11) RT-qPCR validation analysis of rice blast fungus knockout and complement mutants

[0107] The obtained rice blast fungus was extracted using a column-based fungal total RNA extraction and purification kit (Shanghai Sangon Biotech Co., Ltd.). MoUPE11 Genomic RNA from knockout mutants and complement mutants was used; the RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit (Takara). MoActin The gene was used as an internal reference gene and measured using RT-qPCR. MoUPE11 In knockout mutants and complement mutants MoUPE11 The relative expression levels of the target gene. Primers used for RT-qPCR validation analysis are shown in Table 9 (SEQ ID NO. 13-16).

[0108] Table 9 Primers used for RT-qPCR validation analysis

[0109]

[0110] The qPCR reaction system used for RT-qPCR validation analysis is shown in Table 10.

[0111] Table 10. qPCR reaction system used for RT-qPCR validation analysis

[0112]

[0113] The qPCR reaction conditions were as follows: Step 1: 95℃, 30 s; Step 2: 95℃, 5 s; 60℃, 30 s, 40 cycles; Step 3: 95℃, 10 s; 65℃, 5 s; 95℃, 5 s. Each experiment was repeated 3 times. The method calculates the relative expression level.

[0114] 2. Experimental Results

[0115] This invention utilizes homologous recombination to knock out the pCT74- MoUPE11 -KO transformation of *Magnaporum oryzae* protoplasts yielded 45 candidate hygromycin-resistant transformants. (Partial list of hygromycin-resistant transformants follows.) HPH Gene PCR analysis results as follows Figure 2 As shown. By Figure 2 It can be seen that all three transformants tested amplified [the virus / transformation]. HPH Genes. Further analysis of the three transformants tested... MoUPE11 The gene was validated by PCR, and the results are as follows: Figure 3 As shown. By Figure 3 It can be seen that none of the three transformants tested amplified to [the desired amplification level]. MoUPE11 The genes further confirm that these three transformants are positive transformants.

[0116] Based on the PCR validation analysis results, this invention detected positive transformants in 3 positive transformants (including...) HPH Genes without MoUPE11 Two genes (target genes) were selected for Southern blot analysis, among which, HPH Southern blot analysis results of *Magnaporum oryzae* knockout transformants with the fragment as a probe are as follows: Figure 4 As shown. By Figure 4 It can be seen that, with HPH Hybridization was performed using the fragment as a probe, and single-copy bands appeared in both transformants. These results indicate that the present invention successfully obtained a *Blastomyces oryzae* knockout mutant. MoUPE11 .

[0117] This invention utilizes a random insertion method to insert the gene complementation vector pCTZN- MoUPE11 -com transforms rice blast fungus knockout mutant MoUPE11 ( MoUPE11 Bleomycin-resistant transformants were obtained from protoplasts containing -13 (-13). The target gene of some of the bleomycin-resistant transformants ( MoUPE11 PCR analysis results are as follows Figure 5 As shown. By Figure 5 It can be seen that the target gene fragment was amplified in 4 candidate positive transformants, indicating that these 4 transformants contain... MoUPE11 Gene analysis confirmed that these four transformants were positive transformants.

[0118] Rice blast fungus knockout mutant MoUPE11 and complement mutant MoUPE11 -com Gene MoUPE11 The RT-qPCR analysis results are as follows Figure 6 As shown. By Figure 6 It can be seen that the present invention has successfully obtained rice blast fungus. MoUPE11 Knockout mutants and complement mutants.

[0119] Example 2 MoUPE11 Observational analysis of knockout mutants and complement mutants

[0120] 1. Colony morphology observation and growth rate determination

[0121] Wild-type and knockout mutant of rice blast fungus MoUPE11 and complement mutant MoUPE11 -com were inoculated onto Jianli medium and cultured at 28°C in the dark. The colony morphology was observed daily, and the colony diameter was measured on day 10.

[0122] Observation revealed that the knockout mutant MoUPE11 and complement mutant MoUPE11 The colony morphology and growth rate of -com were not significantly different from those of the wild-type rice blast fungus.

[0123] 2. Observation on the production and germination of conidia, appressorium formation, and glycogen metabolism of conidia.

[0124] Moisten the activated rice blast fungus (wild type, knockout mutant) with 2-3 mL of sterile water. MoUPE11 and complement mutant MoUPE11On the surface of the *Bacillus oryzae* colonies, crush the mycelia with a sterilized spoon. Transfer the mycelial solution to tomato-oat medium (40 g raw oats, boiled in double-distilled water for 1 h, filtered, then mixed with 150 mL tomato juice, 0.06 g calcium carbonate, and 2.5%–3% agar powder, and diluted to 1 L with double-distilled water) using a pipette, approximately 500 µL of mycelial solution per plate. Spread the mycelial solution evenly onto the plate using a glass rod. Incubate at 28°C for 24 h under light, then invert for 4–7 days. Add 5 mL of sterile water to the tomato-oat medium using a pipette, and scrape the colonies with a spoon. Collect the spore solution by filtering through a sterile 200-mesh cell sieve or four layers of dust-free paper. Adjust the spore concentration to 5 × 10⁻⁶. 4 1 spore / mL; 20 µL of spore solution was pipetted onto a hydrophobic glass slide; kept moist and incubated in the dark at 28℃. Samples were taken at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h and 24 h, and photographs were taken to observe the germination of conidia. The germination rate of conidia was counted.

[0125] Adjust the concentration of the collected spore solution to 5 × 10⁻⁶. 4 Specimen density / mL; 20 µL of spore solution was pipetted onto a hydrophobic glass slide; the slide was kept moist in a culture dish and cultured in the dark at 28°C. Samples were taken at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h and 24 h for glycogen staining. Glycogen staining was performed using KI / I2 solution (60 mg / mL KI, 10 mg / mL I2). After staining for 1 min, the glycogen metabolism process of the conidia was observed by photographing with a regular optical microscope.

[0126] Observations on conidial glycogen metabolism showed that in wild-type strains and complemented mutants of *Oryza sativa*... MoUPE11 In -com, glycogen begins to be transported from conidia to newly formed appressoria at 6 h; by 12 h, the glycogen in conidia has been largely transferred to appressoria; and by 24 h, most of the glycogen in appressoria has been decomposed; while in Δ MoUPE11 During this period, glycogen in conidia begins to be transported from conidia to newly formed appressoria at 8–10 h. Glycogen staining can still be observed in conidia and appressoria at 24 h, i.e., Δ MoUPE11 Glycogen metabolism is slowed down in conidia.

[0127] Conidial germination and appressorium formation were observed and statistically analyzed at 4 h and 12 h, respectively. The results are as follows: Figure 7 As shown; Figure 7 In the table, A represents the statistical results of conidial germination rate, and B represents the statistical results of appressorium formation rate. Figure 7 It can be seen that Δ MoUPE11The conidial germination rate at 4 h and the appressorium formation rate at 12 h were significantly lower than those of the wild type and the complement mutant.

[0128] The above results indicate that MoUPE11 It affects the development and maturation of appressorium.

[0129] 3. Analysis of stress resistance

[0130] (1) Oxidative stress analysis

[0131] Wild-type and knockout mutant of rice blast fungus MoUPE11 and complement mutant MoUPE11 -com were inoculated onto Jianli medium containing 20 mmol / L H2O2, and incubated upside down in an incubator at 28℃ for 10 days. The colony growth of different strains was then observed.

[0132] (2) Cell wall integrity analysis

[0133] Wild-type and knockout mutant of rice blast fungus MoUPE11 and complement mutant MoUPE11 -com were inoculated onto Jianli medium containing 0.01% SDS (sodium dodecyl sulfate), 0.3 g / L CR (Congo red), and 0.08 g / L CFW (fluorescent whitening agent), respectively. After incubation at 28°C with the medium inverted for 10 days, the colony growth of different strains was observed.

[0134] (3) Analysis of high osmotic pressure stress

[0135] Wild-type and knockout mutant of rice blast fungus MoUPE11 and complement mutant MoUPE11 -com were inoculated onto Jianli medium containing 0.8 mol / L NaCl and 0.8 mol / L Sorbitol, respectively, and incubated upside down in an incubator at 28°C for 10 days. The colony growth of different strains was then observed.

[0136] Rice blast fungus knockout mutant MoUPE11 and complement mutant MoUPE11- The results of the stress resistance analysis of com are as follows: Figure 8 As shown. By Figure 8 It can be seen that, compared with the wild type of rice blast fungus, MoUPE11The sensitivity to 20 mmol / L H2O2 was enhanced, while there was no significant difference in sensitivity to 0.8 mol / L NaCl, 0.8 mol / L Sorbitol, 0.01% SDS, 0.08 g / L CFW and 0.3 g / L CR.

[0137] Example 3 MoUPE11 Pathogenicity analysis of knockout and complement mutants

[0138] Place moistened filter paper in a petri dish, and cut the fourth leaf of rice (CO-39) and spread it on the filter paper; collect wild-type and knockout mutant rice blast fungi. MoUPE11 and complement mutant MoUPE11 -com's conidial solution (conidial solution concentration is 1×10) 5 The above 10 μL conidial solution (containing 0.05% Tween 20) was added dropwise to rice leaves and placed in a dark artificial climate chamber at 25℃ and 90% humidity for 24 h. After culturing under 12 h light / 12 h dark conditions for 4–5 days, the disease incidence of the rice leaves was observed and photographed.

[0139] Rice blast fungus knockout mutant MoUPE11 and complement mutant MoUPE11 -com's results on the pathogenicity test of detached rice leaves are as follows: Figure 9 As shown. By Figure 9 It can be seen that, compared with the wild type of rice blast fungus and MoUPE11 Compared to -com, Δ MoUPE11 It will not form gray necrotic spots, and the lesion area is relatively small.

[0140] The above results indicate that Δ MoUPE11 The reduced pathogenicity to detached rice leaves indicates that... MoUPE11 Genes are related to the pathogenicity of rice blast fungus, and can be used as targets to develop rice blast control agents.

[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of protein MoUPE11 in reducing the pathogenicity of rice blast fungus, characterized in that, The application is achieved by inhibiting the expression of the protein MoUPE11 in rice blast fungus; the amino acid sequence of the protein MoUPE11 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, Inhibiting the expression of the protein MoUPE11 in rice blast fungus is achieved by interfering with the expression of the gene encoding MoUPE11, or by mutating the coding region of the gene encoding MoUPE11, or by knocking out the gene encoding MoUPE11.

3. The use of a reagent that inhibits the expression of the gene encoding the protein MoUPE11 described in claim 1 in reducing the pathogenicity of rice blast fungus.

4. The use of a reagent for inhibiting the expression of the gene encoding the protein MoUPE11 described in claim 1 in the preparation of a product that reduces the pathogenicity of rice blast fungus.

5. The use of a reagent for knocking out the gene encoding the protein MoUPE11 of claim 1 in reducing the pathogenicity of rice blast fungus.

6. The use of a reagent for knocking out the gene encoding the protein MoUPE11 of claim 1 in the preparation of a product that reduces the pathogenicity of rice blast fungus.

7. The application of a reagent for inhibiting the expression of the gene encoding the protein MoUPE11 described in claim 1 in the prevention and control of rice blast.

8. The use of a reagent for inhibiting the expression of the gene encoding the protein MoUPE11 described in claim 1 in the preparation of rice blast control products.

9. The use of a reagent for knocking out the gene encoding the protein MoUPE11 of claim 1 in the prevention and control of rice blast.

10. The use of a reagent for knocking out the gene encoding the protein MoUPE11 of claim 1 in the preparation of rice blast control products.

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