Application of Protein MoUPE3 in Regulating the Virulence of Magnaporthe oryzae

Abstract

The present invention discloses the application of protein MoUPE3 in regulating the pathogenicity of Magnaporthe oryzae. In the present invention, the Magnaporthe oryzae knockout mutant ΔMoUPE3 was obtained by knocking out the gene encoding protein MoUPE3 in Magnaporthe oryzae, and the complemented mutant ΔMoUPE3-com was constructed by complementation. By detecting the colony morphology, sporulation amount, stress resistance, pathogenicity, etc. of the wild type of Magnaporthe oryzae, the Magnaporthe oryzae knockout mutant ΔMoUPE3, and the complemented mutant ΔMoUPE3-com, it was found that the gene encoding protein MoUPE3 of Magnaporthe oryzae can regulate the sporulation amount, stress resistance, and pathogenicity of Magnaporthe oryzae, and can be used as a target for controlling rice blast. The present invention enriches the pathogenicity-related genes of Magnaporthe oryzae, and at the same time provides target genes for the development of effective fungicides, which is helpful for the control of rice blast.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically relates to the application of protein MoUPE3 in regulating the pathogenicity of rice blast fungus. Background Art

[0002] Rice blast, caused by the fungus Magnaporthe oryzae, is a devastating disease in rice production, severely impacting both yield and quality. Blast can occur throughout the rice plant's growth cycle, causing seedling blast, leaf blast, node blast, panicle blast, and grain blast. Among these, panicle blast has the greatest impact on rice yield and quality.

[0003] Currently, the primary control measures for rice blast in rice production involve the use of disease-resistant varieties and chemical pesticides. Advances in molecular pathogen biology have also revealed that many pathogenicity-related genes are closely linked to the pathological processes of plant pathogens. Therefore, identifying genes associated with rice blast pathogenicity could provide new insights into improving rice resistance and preventing and controlling rice blast. Summary of the Invention

[0004] The first object of the present invention is to provide a rice blast fungus protein MoUPE3.

[0005] The second object of the present invention is to provide a gene encoding the protein MoUPE3.

[0006] The third object of the present invention is to provide the use of the protein MoUPE3 in reducing the pathogenicity of rice blast fungus.

[0007] The fourth object of the present invention is to provide the use of the protein MoUPE3 in reducing the spore production of rice blast fungus.

[0008] The fifth object of the present invention is to provide the use of the protein MoUPE3 in reducing the stress resistance of rice blast fungus.

[0009] The sixth object of the present invention is to provide a substance for inhibiting the expression of the rice blast fungus protein MoUPE3 for use in reducing the pathogenicity of the rice blast fungus.

[0010] The seventh object of the present invention is to provide a substance for inhibiting the expression of the rice blast fungus protein MoUPE3 for use in reducing the spore production of rice blast fungus.

[0011] The eighth object of the present invention is to provide a substance for inhibiting the expression of the rice blast fungus protein MoUPE3 for use in reducing the stress resistance of the rice blast fungus.

[0012] The ninth objective of the present invention is to provide a substance for inhibiting the expression of the rice blast fungus protein MoUPE3 for use in preventing and controlling rice blast.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] The present invention identifies a protein, MoUPE3, that regulates the pathogenicity of the blast fungus, Magnaporthe oryzae, whose amino acid sequence is shown in SEQ ID NO. 2. MoUPE3 is an unknown protein that lacks a signal peptide and known structural domains. Prior to this invention, the specific function of MoUPE3 in the blast fungus was unclear.

[0015] The present invention obtains a rice blast fungus knockout mutant ΔMoUPE3 by knocking out the gene encoding protein MoUPE3 in rice blast fungus, and obtains a complemented mutant ΔMoUPE3-com by complementation. By testing the colony morphology, spore production, stress resistance (adversity resistance) and pathogenicity of the wild type of rice blast fungus, the rice blast fungus knockout mutant ΔMoUPE3 and the complemented mutant ΔMoUPE3-com, it was found that the spore production, stress resistance and pathogenicity of the rice blast fungus knockout mutant ΔMoUPE3 were significantly reduced compared with the rice blast fungus wild type; while the spore production, stress resistance and pathogenicity of the complemented mutant ΔMoUPE3-com were restored, indicating that the rice blast fungus protein MoUPE3 regulates the spore production, stress resistance and pathogenicity of rice blast fungus, and can be used as a target for the prevention and control of rice blast. Therefore, the present invention applies to protect the rice blast fungus protein MoUPE3 and its encoding gene, as well as the application of the protein MoUPE3 and its encoding gene.

[0016] The present invention provides a rice blast fungus protein MoUPE3, the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0017] The present invention also provides a gene encoding protein MoUPE3, whose nucleotide sequence is one of the following A, B, and C:

[0018] A. a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2;

[0019] B. the nucleotide sequence shown in SEQ ID NO.1;

[0020] C. Analogs of A or B above obtained by base insertion, deletion, or substitution that still have the function of regulating the pathogenicity of rice blast fungus.

[0021] Considering that knocking out the gene encoding protein MoUPE3 in rice blast fungus will affect its pathogenicity, spore production and stress resistance, the present invention also applies to the following applications of the protective protein MoUPE3:

[0022] The present invention discloses an application of the protective protein MoUPE3 in reducing the pathogenicity of rice blast fungus.

[0023] The present invention also applies for the use of the protective protein MoUPE3 in reducing the spore production of rice blast fungus.

[0024] The present invention also applies for the use of the protective protein MoUPE3 in reducing the stress resistance of rice blast fungus.

[0025] Specifically, the stress resistance refers to resistance to oxidative stress.

[0026] The present invention also claims the use of the protective protein MoUPE3 in affecting the integrity of the cell wall of rice blast fungus.

[0027] Specifically, the above application is achieved by blocking or inhibiting the expression of protein MoUPE3 in rice blast fungus.

[0028] As an optional embodiment, the expression of protein MoUPE3 can be blocked by knocking out the gene encoding protein MoUPE3 in rice blast fungus, or the expression of protein MoUPE3 in rice blast fungus can be inhibited by RNA interference or the like.

[0029] Specifically, the nucleotide sequence of the gene encoding the protein MoUPE3 is one of the following A, B, and C:

[0030] A. a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2;

[0031] B. The nucleotide sequence shown in SEQ ID NO.1.

[0032] C. Analogs of A or B above obtained by base insertion, deletion, or substitution that still have the function of regulating the pathogenicity of rice blast fungus.

[0033] At the same time, the present invention also applies to protect the use of a substance that inhibits the expression of the rice blast fungus protein MoUPE3 in reducing the pathogenicity of the rice blast fungus.

[0034] The present invention also applies for protection of the use of a substance that inhibits the expression of the rice blast fungus protein MoUPE3 in reducing the spore production of the rice blast fungus.

[0035] The present invention also applies for protection of the use of a substance that inhibits the expression of the rice blast fungus protein MoUPE3 in reducing the stress resistance of the rice blast fungus.

[0036] The present invention also applies for protection of the use of a substance that inhibits the expression of the rice blast fungus protein MoUPE3 in preventing and controlling rice blast.

[0037] Specifically, the rice blast disease is caused by Pyricularia oryzae.

[0038] The present invention also provides a method for preventing and controlling rice blast disease caused by rice blast fungus, which comprises applying an agent capable of inhibiting the rice blast fungus protein MoUPE3 to the aboveground part of rice.

[0039] As an optional embodiment, the agent capable of inhibiting the rice blast fungus protein MoUPE3 may be an antisense RNA or siRNA of the gene encoding the rice blast fungus protein MoUPE3.

[0040] Specifically, the reagent capable of inhibiting the rice blast fungus protein MoUPE3 is the antisense RNA or siRNA of the gene shown in SEQ ID NO.1.

[0041] The present invention has the following beneficial effects:

[0042] The present invention provides novel functions of an uncharacterized protein, MoUPE3, in the rice blast fungus and its encoding gene, MoUPE3. Furthermore, the present invention provides the use of the MoUPE3 protein in reducing the pathogenicity, spore production, and stress resistance of the fungus, as well as the use of substances that inhibit the expression of the MoUPE3 protein in reducing the pathogenicity, spore production, and stress resistance of the fungus and controlling rice blast. This invention enriches the pathogenicity-related genes of the rice blast fungus and provides target genes for the development of effective fungicides, thus facilitating the control of rice blast. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the construction of the rice blast fungus MoUPE3 gene knockout vector.

[0044] Figure 2 The PCR amplification results of the HPH gene of some hygromycin-resistant transformants; where M: DL 2000 Marker; 1: wild type of rice blast fungus; 2-6: hygromycin-resistant transformants ΔMoUPE3-6, ΔMoUPE3-21, ΔMoUPE3-23, ΔMoUPE3-31, and ΔMoUPE3-36.

[0045] Figure 3 The PCR amplification results of the target gene MoUPE3 in some hygromycin-resistant transformants; where M: DL2000 Marker; 1: wild type of rice blast fungus; 2 to 6: transformants ΔMoUPE3-6, ΔMoUPE3-21, ΔMoUPE3-23, ΔMoUPE3-31 and ΔMoUPE3-36 containing hygromycin resistance.

[0046] Figure 4 The following are the Southern blot analysis results of the rice blast knockout positive candidate transformants using the HPH fragment as a probe; 1: rice blast wild type; 2 to 4, candidate positive transformants ΔMoUPE3-21, ΔMoUPE3-23, and ΔMoUPE3-36.

[0047] Figure 5The Southern blot analysis results of the rice blast fungus knockout candidate positive transformants using the MoUPE3 fragment as a probe; 1: rice blast fungus wild type; 2 to 4: candidate positive transformants ΔMoUPE3-21, ΔMoUPE3-23, and ΔMoUPE3-36.

[0048] Figure 6 The PCR amplification results of the MoUPE3 gene of some bleomycin-resistant transformants; wherein, M: DL 2000 Marker; 1 to 4: bleomycin-resistant transformants ΔMoUPE3-21-com-1, ΔMoUPE3-21-com-2, ΔMoUPE3-21-com-3 and ΔMoUPE3-21-com-4.

[0049] Figure 7 The conidia production statistics of the rice blast fungus knockout mutant ΔMoUPE3 and complement mutant ΔMoUPE3-com were obtained. The data were analyzed by Duncan's new multiple range method (P < 0.05). Different letters indicate significant differences. The data in the figure are mean ± standard deviation.

[0050] Figure 8 Stress resistance analysis of the knockout mutant ΔMoUPE3 and the complemented mutant ΔMoUPE3-com.

[0051] Figure 9 To determine the pathogenicity of the rice blast fungus knockout mutant ΔMoUPE3 and complement mutant ΔMoUPE3-com to rice. DETAILED DESCRIPTION

[0052] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0053] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0054] The test strain used in the following examples was the rice blast fungus (Magnaporthe oryzae) dominant race ZC13 ​​in Guangdong Province; and the test rice was the susceptible indica rice line CO39.

[0055] The cloning vector used was pMD18-T vector, the gene knockout vector was the filamentous fungal expression vector pCT74, and the gene complementation vector was pCTZN (modified by the inventor's laboratory based on the pCT74 plasmid, replacing the SGFP and HPH genes on pCT74 with the bleomycin resistance gene Zeocin).

[0056] Example 1 Construction of a Rice Blast Fungus MoUPE3 Gene Knockout Mutant

[0057] The nucleotide sequence of the rice blast fungus MoUPE3 gene knocked out in this example is shown below (SEQ ID NO.1):

[0058] ACACATTCACAGTCCCAATCACTGCAACTCTTCACAATCTACGCATCAGCAAAAAAACATCTTCCTCAA CATTCACTCGCTCTTCCATTCTCACAACCAACCAACAATCAAA ATGGCCACCCAAGCTTCCAAGGCATCCACCTGCTGCGGCAAGAGTGACGTCTGCGTCTGTG GTAAGTTTCAAGCCTACCTGAAAGCGTCCCACAGCCTTAAAATGACTAC TTAGGTATTCGTCACCATCAAATACTAACCCACAACCCCCACCATCAG CCACTCAAGCAACCTGCAGTTGTGGCAAGCAGTCCGCCCTTCACTGCACGTGCGACAAGGCTTCCACCGAGAACGCCGTCACCGGACCTCGGTGCTCGTGCCGTGCCCGCCCGGCCGGTGAGTGCAACTGCGACCGCGCTGCCGCCGAGAACGTCACCCCCAGCGGAAACGCCTGCGCCTGCGGTGTCAGGCCTGCCG GTATGTTTGACCTTGATTGATCCCCCACCATTAATCCGATGATCCGGCTTTGCTAACATATA ATGGTTTAG ATGCCTGCACTTGTGAGAAGGGAGGAGCCTCGGGCGTCTATGACACTGCCAACGAGACCGACTTCACTACCAAGAAATAA ATGAAGATTATCGGCAAGGGATTTATTTCTGGCGTTATTCTGGGTCTTTTTTCTTCTTCTTTTTT CGTGTACTCTATGACACTCAGTATACCCCATCGCATTGAGCAATGAACTATTACATATTATCATCGTGGATCCCAGA CGCTACAAGGCGAAAAAAATTAAATGCAATTATTTAACCACACTGGGGAACCTTCGACCGAAAGATAAAGATACATT TTGTT

[0059] The underlined parts are introns, and the rest are exons.

[0060] The amino acid sequence of the rice blast fungus protein MoUPE3 is shown below (SEQ ID NO. 2):

[0061] MATQASKASTCCGKSDVCVCATQATCSCGKQSALHCTCDKASTENAVTGPRCSCRARPAGECNCDRAAAENVTPSGNACACGVRPADACTCEKGGASGVYDTANETDFTTKK

[0062] In this example, a gene knockout vector for MoUPE3 in rice blast fungus was constructed and introduced into rice blast fungus protoplasts. The MoUPE3 gene was knocked out from rice blast fungus by homologous recombination to obtain a knockout mutant ΔMoUPE3. Figure 1 shown.

[0063] 1. Amplification of the upstream and downstream homology arms of the MoUPE3 gene

[0064] Sequences of approximately 1000 bp in size were selected upstream and downstream of the MoUPE3 gene (named as homology arm A fragment and homology arm B fragment, respectively), and primers were designed for amplification. The primer sequences are shown in Table 1.

[0065] Table 1 Primers for amplification of homology arm A and B fragments of MoUPE3 gene

[0066] Primer name Primer sequence 5'-3' (the underlined part is the enzyme cutting site) Restriction site MoUPE3-AF <![CDATA[G ACTAGT GGTCAGTCCGAAACCTCTCA]]> Spe I MoUPE3-AR <![CDATA[GC CAATTG ATAGTCGGATTGCCATAAACGTTCC]]> Mfe MoUPE3-BF <![CDATA[GG GGGCCC CGCAAGCTGTTCTCAGAAGT]]> Apa I MoUPE3-BR <![CDATA[GG GGTACC TGTGCATCCAACCGTGTCAT]]> Kpn I

[0067] The genomic DNA of rice blast fungus was extracted using an OMEGA Fungal DNA Kit; the obtained genomic DNA was used as a template and PCR amplification was performed with primers MoUPE3-AF and MoUPE3-AR to obtain the homology arm A fragment (MoUPE3-A) of the MoUPE3 gene; and the homology arm B fragment (MoUPE3-B) of the MoUPE3 gene was obtained by PCR amplification with primers MoUPE3-BF and MoUPE3-BR.

[0068] The PCR reaction system is shown in the following table:

[0069] Template DNA 1 μL MoUPE3-AF / BF (10 μmol / L) 1 μL MoUPE3-AR / BR (10 μmol / L) 1 μL <![CDATA[10×Ex Taq Buffer(Mg 2+ plus)]]> 5μL dNTP Mixture 4μL Ex Taq DNA Polymerase 0.25 μL <![CDATA[ddH2O]]> 37.75μL Total 50 μL

[0070] PCR reaction conditions were: 94°C for 5 minutes, 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute for 35 cycles, followed by 72°C for 10 minutes. After amplification, the PCR product was cleaned and recovered using the OMEGA Cycle Pure Kit.

[0071] 2. Construction of MoUPE3 gene knockout vector

[0072] Referring to the instructions of the pMD 18-T Vector Cloning Kit (TakaRa), MoUPE3-A and MoUPE3-B were ligated with the T vector to obtain recombinant plasmids pMD18T-MoUPE3-A and pMD18T-MoUPE3-B, respectively.

[0073] Specifically, 1 μL of the pMD18-T vector was added to 4 μL of the PCR product (homologous arm A fragment or homology arm B fragment) and 5 μL of solution I, and the cells were ligated overnight at 16°C. The ligation product was added to 100 μL of E. coli DH5α competent cells and placed on ice for 30 minutes. The cells were heat-shocked in a 42°C water bath for 90 seconds and cooled on ice for 5 minutes. 800 μL of LB liquid medium was added and the cells were incubated at 37°C with shaking at 150 rpm for 45 minutes. The cells were then centrifuged at 4000 rpm for 5 minutes, the supernatant discarded, and the cells were spread on LB solid medium (containing 50 μg / mL Amp). The cells were incubated at 37°C for 8–12 hours. Positive transformants with Amp resistance were selected, and the recombinant plasmid DNA was extracted and sequenced.

[0074] pMD18T-MoUPE3-B and pCT74 vectors were double-digested with Apa I and Kpn I, respectively, to recover fragment B and the pCT74 vector. Fragment B was ligated with pCT74 using T4 DNA ligase and transformed into E. coli DH5α, yielding the recombinant plasmid pCT74-MoUPE3-B. Following the same procedure, pMD18T-MoUPE3-A was double-digested with Spe I and Mfe I, and the recombinant plasmid pCT74-MoUPE3-B was double-digested with Spe I and EcoR I. Fragment A and the recombinant plasmid were recovered. Fragment A was ligated with pCT74-MoUPE3-B using T4 DNA ligase and transformed into E. coli DH5α. Enzyme digestion and identification revealed the gene knockout vector pCT74-MoUPE3-KO.

[0075] 3. Preparation of Rice Blast Protoplasts

[0076] The rice blast fungus was activated on a plate of Jianli medium (5.0 g / L yeast extract, 22.0 g / L anhydrous glucose and 17.0 g / L agar powder) and inverted for about 10 days at 28°C; the mycelial fragments on the Jianli medium were taken with sterile tweezers and transferred to 50 mL of YPS medium (6.0 g / L yeast extract, 6.0 g / L hydrolyzed casein and 10.0 g / L sucrose) and shake-cultured at 28°C and 120 rpm for 2 days; the above culture was filtered through a 200-mesh cell sieve to obtain mycelia containing a small amount of culture solution, which was poured into a sterilized mortar and ground, and an appropriate amount of mycelial fragments was transferred to 200 mL / bottle of YPS medium and shake-cultured at 28°C and 120 rpm for 1 day; the mycelia were filtered through a 200-mesh cell sieve, then rinsed twice with sterile water, and then sterilized with 0.8 mol / L Rinse once with NaCl solution, clamp the mycelium with sterile tweezers and place it in a sterile culture dish, and cover it with 2 to 3 layers of sterile filter paper; transfer the mycelium to a weighed EP tube and weigh it again to obtain the weight of the wet mycelium; add an appropriate amount of 10 mg / mL lytic enzyme solution to the EP tube, with the ratio of enzyme solution to mycelium (volume-to-mass ratio of 10:1), and shake and digest at 80 to 110 rpm at 30°C for about 1 hour; collect the digestion solution by filtering with sterile dust-free paper, place it in a pre-cooled EP tube, and centrifuge it at 3500 rpm at 4°C for 10 minutes; discard the supernatant, and resuspend the precipitate in pre-cooled STC (1.2 mol / L sorbitol, 10 mmol / L Tris-HCl, 50 mmol / L CaCl2, pH 7.5); centrifuge at 5000 rpm for 10 min at 4°C; discard the supernatant, resuspend the precipitate in STC solution, temporarily store on ice, and dilute to the appropriate concentration after counting; calculate the protoplast concentration using a hemocytometer; add an appropriate amount of pre-cooled STC solution and control the final protoplast concentration to 1×10 7-8 pieces / mL.

[0077] 4. Transformation of Rice Blast Fungus Protoplasts

[0078] The knockout vector pCT74-MoUPE3-KO was digested with SpeⅠ to obtain the A-HPH-SGFP-B fragment. 5 μg of the A-HPH-SGFP-B fragment was mixed with 200 μL of protoplasts; or the pCTZN-MoUPE3-com fragment after single enzyme digestion was mixed with 200 μL of rice blast knockout mutant protoplasts; ice bath for 20 min; 1 mL of PTC transformation buffer (60% PEG4000, 50 mmol / L CaCl2, 10 mmol / L Tris-HCl, pH 7.5) was added while mixing, and the mixture was placed at room temperature for 20 minutes; the mixture was centrifuged at 5000 rpm at 4°C for 10 minutes; the supernatant was discarded, and the precipitate was resuspended in 1 mL of regeneration liquid culture medium (6.0 g / L yeast extract, 6.0 g / L hydrolyzed casein, and 200.0 g / L sucrose), transferred to a sterile 50 mL Corning tube, and then 3 mL of regeneration liquid culture medium was added. The tube was gently shaken at 100 rpm at 28°C for 16 to 18 hours for recovery culture; 4 mL of the recovered protoplasts were added to 30 mL of regeneration solid culture medium cooled to about 45°C (the regeneration liquid culture medium contained 1.5% agar powder). In experiments involving knockout vector transformation into protoplasts, the regeneration medium contained 200 μg / mL hygromycin; in experiments involving complementation vector transformation into protoplasts, the regeneration medium contained 150 μg / mL bleomycin. Mix well and pour plates. Incubate the solidified plates upside down at 28°C in the dark for approximately 4 days. Pick single colonies of transformants with hygromycin / bleomycin resistance for identification.

[0079] 5. PCR verification analysis of rice blast knockout mutants

[0080] Genomic DNA from the hygromycin-positive transformants was extracted using the OMEGA Fungal DNA Kit instructions and analyzed by PCR. The HPH gene fragment was amplified by PCR using primers HPH-F / HPH-R, and the MoUPE3 gene fragment was amplified by PCR using primers MoUPE3-F / MoUPE3-R. The primer sequences are shown in Table 2 below.

[0081] Table 2 Primers used for PCR verification analysis of rice blast knockout mutants

[0082] Primer name Primer sequence 5'-3' MoUPE3-F ACGTCTGCGTCTGTGGTAAG MoUPE3-R GAAGGTTCCCCAGTGTGGTT HPH-F TGCTGCTCCATACAAGCCAA HPH-R GACATTGGGGAGTTCAGCGA

[0083] The PCR reaction system is shown in the following table:

[0084] Template DNA 0.5μL MoUPE3-F / HPH-F (10 μmol / L) 0.5μL MoUPE3-R / HPH-R (10 μmol / L) 0.5μL 2×TSINGKE Master Mix 12.5μL <![CDATA[ddH2O]]> 11μL Total 25.0μL

[0085] The PCR reaction conditions were as follows: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, for a total of 35 cycles; and 72°C for 10 min to obtain the amplified product.

[0086] In this example, the gene knockout vector was transformed into rice blast fungus protoplasts using homologous recombination method, and 37 candidate positive transformants were obtained. After DNA extraction, PCR verification analysis was performed on the 37 hygromycin-positive transformants using HPH gene-specific primers. The PCR amplification results of the HPH gene of some hygromycin-resistant transformants are shown in Figure 2. Figure 2 As shown in the figure; where M is DL2000Marker; 1 is the wild type of rice blast fungus; 2 to 6 are hygromycin-positive transformants ΔMoUPE3-6, ΔMoUPE3-21, ΔMoUPE3-23, ΔMoUPE3-31 and ΔMoUPE3-36, respectively. Figure 2 It was found that the obtained hygromycin-positive transformants contained the HPH gene.

[0087] In this example, MoUPE3 gene-specific primers were further used to perform PCR verification analysis on the five positive transformants amplified to the HPH gene by PCR. The results were as follows: Figure 3 shown by Figure 3 It can be seen that the MoUPE3 gene was not amplified in any of the five transformants, indicating that these five transformants were positive transformants.

[0088] 6. Southern blot analysis of rice blast knockout mutants

[0089] Primers MoUPE3-F / MoUPE3-R were used to amplify the target gene probe, and HPH-F / HPH-R were used to amplify the HPH gene probe.

[0090] The PCR amplification system of DNA probe is shown in the following table:

[0091]

[0092]

[0093] The PCR reaction conditions were as follows: 94°C for 5 min; 98°C for 10 s, 55°C for 30 s, and 72°C for 1 min, for a total of 35 cycles; and 72°C for 10 min to obtain the amplified product.

[0094] Southern blot hybridization was performed according to the instructions of DIG High Prime DNA Labeling and Detection Starter Kit I (Roche LOT 28309220).

[0095] In this example, three of the five positive transformants (containing the HPH gene but not the MoUPE3 target gene) were selected for Southern blot analysis. The Southern blot analysis results of the positive candidate transformants for the blast fungus knockout using the HPH fragment as a probe are shown in Figure 2. Figure 4 The Southern blot analysis results of the rice blast fungus knockout candidate positive transformants using the MoUPE3 fragment as a probe are shown in FIG. Figure 5 As shown; where 1: wild type of rice blast fungus; 2 to 4: candidate positive transformants ΔMoUPE3-21, ΔMoUPE3-23 and ΔMoUPE3-36. Figure 4 and Figure 5 The results show that when HPH was used as a probe for hybridization, single copy bands appeared in all three transformants ( Figure 4 When the target gene MoUPE3 was used as a probe for hybridization, no hybridization bands were found in the three transformants ( Figure 5 ); The above results further showed that these three transformants were positive transformants, namely, rice blast fungus MoUPE3 gene knockout mutants.

[0096] Example 2 Construction of a Mutant Complementing the MoUPE3 Gene of Rice Blast Fungus

[0097] The invention constructs a gene complementation vector and introduces the vector into ΔMoUPE3 protoplasts; and uses a random insertion method to complement the gene into the knockout mutant to obtain the complementation mutant ΔMoUPE3-com.

[0098] 1. Amplification of MoUPE3 complementation fragment

[0099] A promoter sequence with a length of 1500 bp was selected upstream of the MoUPE3 gene, and a terminator sequence with a length of 500 bp was selected downstream. Primers were designed for amplification. The primer sequences are shown in Table 3.

[0100] Table 3 Primers for amplification of the complementation fragment of MoUPE3 gene

[0101]

[0102] The genomic DNA of rice blast fungus was extracted using an OMEGA Fungal DNA Kit. The obtained genomic DNA was used as a template and PCR amplified with primers Com-MoUPE3-F and Com-MoUPE3-R to obtain the complemented fragment of the MoUPE3 gene (MoUPE3-com).

[0103] The PCR reaction system is shown in the following table:

[0104] Template DNA 1 μL Com-MoUPE3-F (10 μmol / L) 1 μL Com-MoUPE3-R (10 μmol / L) 1 μL <![CDATA[10×Ex Taq Buffer(Mg 2+ plus)]]> 5μL dNTP Mixture 4μL Ex Taq DNA Polymerase 0.25 μL <![CDATA[ddH2O]]> 37.75μL Total 50 μL

[0105] PCR reaction conditions were: 94°C for 5 minutes, 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 4 minutes for 35 cycles, followed by 72°C for 10 minutes. After amplification, the PCR product was cleaned and recovered using the OMEGA Cycle Pure Kit.

[0106] 2. Construction of MoUPE3 gene complementation vector

[0107] MoUPE3-com and the pCTZN vector were double-digested with Spe I and Not I, respectively, to recover the MoUPE3-com fragment and the pCTZN vector. The MoUPE3-com fragment was ligated with pCTZN using T4 DNA ligase and transformed into Escherichia coli DH5α to obtain the recombinant plasmid pCTZN-MoUPE3-com. After enzyme digestion and identification, the gene-complementing vector pCTZN-MoUPE3-com was obtained.

[0108] 3. Preparation of protoplasts of rice blast knockout mutants

[0109] The preparation method of the protoplasts of the blast fungus knockout mutant is the same as that of the blast fungus protoplasts in Example 1.

[0110] 4. Transformation of protoplasts of rice blast knockout mutants

[0111] The transformation of protoplasts of the rice blast fungus knockout mutant was the same as in Example 1.

[0112] 5. PCR verification analysis of MoUPE3 complementation mutants

[0113] The genomic DNA of the bleomycin-positive transformants was extracted according to the instructions of the OMEGA Fungal DNA Kit and subjected to PCR verification analysis. The gene fragment MoUPE3 was amplified by PCR using primers MoUPE3-F / MoUPE3-R.

[0114] The PCR reaction system is shown in the following table:

[0115] Template DNA 0.5μL MoUPE3-F (10 μmol / L) 0.5μL MoUPE3-R (10 μmol / L) 0.5μL 2×TSINGKE Master Mix 12.5μL <![CDATA[ddH2O]]> 11μL Total 25.0μL

[0116] The PCR reaction conditions were as follows: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, for a total of 35 cycles; and 72°C for 10 min to obtain the amplified product.

[0117] In this example, the gene complementation vector pCTZN-MoUPE3-com was transformed into protoplasts of the rice blast fungus knockout mutant ΔMoUPE3 (ΔMoUPE3-21) using a random insertion method, and five positive transformants with bleomycin resistance were obtained. After extracting genomic DNA from the positive transformants, PCR analysis was performed on these positive transformants using MoUPE3 gene-specific primers. The results are shown in Figure 2. Figure 6 As shown, there are 4 positive transformants that can amplify the target gene fragment, indicating that these 4 transformants contain the MoUPE3 gene, confirming that these 4 transformants are positive transformants.

[0118] Example 3 Phenotypic Observation of Rice Blast Fungus MoUPE3 Knockout Mutants and Complemented Mutants

[0119] 1. Colony morphology observation and growth rate determination

[0120] The wild type of rice blast fungus, the MoUPE3 gene knockout mutant ΔMoUPE3 and the complemented mutant ΔMoUPE3-com were inoculated on Miri medium, cultured at 28℃ in the dark, and the colony morphology was observed every day. The colony diameter was measured on the 10th day.

[0121] The results of colony morphology observation and growth rate determination showed that the colony morphology and growth rate of ΔMoUPE3 were not significantly different from those of the wild-type strain of Magnaporthe grisea.

[0122] 2. Observation of conidia production

[0123] The activated wild-type, knockout mutant ΔMoUPE3, and complemented mutant ΔMoUPE3-com colonies of rice blast fungus were moistened with sterile water (2 mL to 3 mL per dish), and the mycelia of rice blast fungus were crushed with a sterilized spoon. The mycelia were transferred to a tomato oatmeal medium (40 g of raw oats, boiled with double-distilled water for 1 h, filtered, and then 150 mL of tomato juice, 0.06 g of calcium carbonate, and 2.5% to 3% agar powder were added to the medium, and the volume was adjusted to 1 L with double-distilled water). 500 μL of mycelia were added to each dish, and the mycelia were evenly spread on the plate with a glass rod; the culture was illuminated at 28 ° C for 24 h and inverted for 10 d; 5 mL of sterile water was transferred to the tomato oatmeal medium with a pipette, and the colonies were scraped with a spoon; the spore liquid was collected by filtration with a sterilized 200-mesh cell sieve or 4 layers of dust-free paper for spore production analysis.

[0124] The blast fungus knockout mutant ΔMoUPE3 and the complement mutant ΔMoUPE3-com were inoculated into tomato oat culture medium and cultured for 10 days before the spore production was analyzed. Figure 7 As shown. Figure 7 It can be seen that compared with the wild type of rice blast fungus, the spore production of the knockout mutant ΔMoUPE3 was significantly reduced, while the spore production of the complemented mutant ΔMoUPE3-com was restored to the wild type level, indicating that knocking out the gene MoUPE3 will significantly reduce the spore production of the resulting rice blast fungus knockout mutant.

[0125] Example 4 Analysis of Stress Resistance of Rice Blast Fungus MoUPE3 Knockout Mutants and Complemented Mutants

[0126] 1. Oxidative stress analysis

[0127] The wild type, knockout mutant ΔMoUPE3 (ΔMoUPE3-21 and ΔMoUPE3-36) and complemented mutant ΔMoUPE3-com (ΔMoUPE3-com-4) of rice blast fungus were inoculated on a culture medium containing 20 mmol / L H2O2, respectively. After inverted culture in a 28°C incubator for 10 days, the colony growth of the knockout mutant ΔMoUPE3, complemented mutant ΔMoUPE3-com and wild type strain was observed, and their colony growth inhibition rate was determined.

[0128] 2. Cell wall integrity analysis

[0129] The wild type, knockout mutant ΔMoUPE3 (ΔMoUPE3-21 and ΔMoUPE3-36) and complement mutant ΔMoUPE3-com (ΔMoUPE3-com-4) of rice blast fungus were inoculated on a culture medium containing 0.01% SDS (sodium dodecyl sulfate), 0.2 g / LCR (Congo red) and 0.05 g / mL CFW (fluorescent whitening agent), respectively. After inverted culture in a 28°C incubator for 10 days, the colony growth of the knockout mutant ΔMoUPE3, complement mutant ΔMoUPE3-com and wild type strain was observed, and their colony growth inhibition rate was determined.

[0130] 3. Hyperosmotic stress analysis

[0131] The wild type, knockout mutant ΔMoUPE3 (ΔMoUPE3-21 and ΔMoUPE3-36) and complemented mutant ΔMoUPE3-com (ΔMoUPE3-com-4) of rice blast fungus were inoculated on a culture medium containing 0.8 mol / L NaCl and 0.8 mol / L Sorbitol, respectively. After inverted culture in an incubator at 28°C for 10 days, the colony growth of the knockout mutant ΔMoUPE3, complemented mutant ΔMoUPE3-com and wild type strain was observed, and their colony growth inhibition rate was determined.

[0132] The results of stress resistance analysis of the knockout mutant ΔMoUPE3 and the complement mutant ΔMoUPE3-com are shown in Figure 2. Figure 8 As shown. Figure 8 It can be seen that compared with the wild type, the knockout mutant ΔMoUPE3 has enhanced sensitivity to 20mmol / L H2O2, 0.2g / L CR and 0.05g / LCFW, and has no significant difference in sensitivity to 0.8mol / L NaCl, 0.8mol / L Sorbitol and 0.01% SDS.

[0133] Cell wall integrity plays a crucial role in pathogen resistance to host cell immune defenses. ΔMoUPE3 enhances sensitivity to cell wall inhibitors CFW and CR, suggesting that knockout of the MoUPE3 gene compromises cell wall integrity in rice blast fungi.

[0134] Example 5 Pathogenicity Analysis of Rice Blast Fungus MoUPE3 Gene Knockout Mutants and Complemented Mutants

[0135] Conidia of wild type, knockout mutants ΔMoUPE3 (ΔMoUPE3-21 and ΔMoUPE3-36) and complement mutant ΔMoUPE3-com (ΔMoUPE3-com-4) of rice blast fungus were collected (concentration of 5×10 4 The 5-dose (0.1% dapoxetine / mL, containing 0.05% Tween-20) was sprayed on the living rice leaves at 28°C with 12h light / 12h dark and moisturizing. After 5 days, the disease condition of the rice leaves was investigated and the pathogenicity analysis was performed.

[0136] The results of pathogenicity analysis of MoUPE3 gene knockout mutants and complementation mutants of rice blast fungus are shown in Figure 2. Figure 9 As shown by Figure 9 It can be seen that compared with the wild type of rice blast fungus, ΔMoUPE3 has fewer lesions on rice leaves, and the pathogenicity of ΔMoUPE3-com has returned to the wild type level, indicating that the knockout of the MoUPE3 gene will lead to a decrease in the pathogenicity of rice blast fungus.

[0137] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of protein MoUPE3 in reducing the pathogenicity of Magnaporthe oryzae, characterized in that, The amino acid sequence of the protein is as shown in SEQ ID NO.2; the application is achieved by knocking out the gene encoding protein MoUPE3 in Magnaporthe oryzae to block the expression of protein MoUPE3, or by RNA interference to inhibit the expression of protein MoUPE3 in Magnaporthe oryzae.

2. Use of protein MoUPE3 in reducing the sporulation amount of Magnaporthe oryzae, characterized in that, The amino acid sequence of the protein is as shown in SEQ ID NO.2; the application is achieved by knocking out the gene encoding protein MoUPE3 in Magnaporthe oryzae to block the expression of protein MoUPE3, or by RNA interference to inhibit the expression of protein MoUPE3 in Magnaporthe oryzae.

3. Application of protein MoUPE3 in reducing stress resistance of Magnaporthe oryzae, characterized in that, The amino acid sequence of the protein is as shown in SEQ ID NO.2; the application is achieved by knocking out the gene encoding protein MoUPE3 in Magnaporthe oryzae to block the expression of protein MoUPE3, or by RNA interference to inhibit the expression of protein MoUPE3 in Magnaporthe oryzae; the stress resistance is antioxidant stress.

4. The application according to any one of claims 1 to 3, characterized in that, The nucleotide sequence of the gene encoding protein MoUPE3 is as shown in SEQ ID NO.

1.

5. Application of a knockout vector of the gene encoding the protein MoUPE3 in reducing the pathogenicity of Magnaporthe oryzae, characterized in that, The nucleotide sequence of the gene is as shown in SEQ ID NO.

1.

6. Use of a knockout vector of the gene encoding the protein MoUPE3 in reducing the sporulation amount of Magnaporthe oryzae, characterized in that, The nucleotide sequence of the gene is as shown in SEQ ID NO.

1.

7. Use of a knockout vector of the gene encoding the protein MoUPE3 in reducing the stress resistance of Magnaporthe oryzae, characterized in that, The nucleotide sequence of the gene is as shown in SEQ ID NO.1; the stress resistance is antioxidant stress.

8. Application of a knockout vector of the gene encoding the protein MoUPE3 in controlling rice blast, characterized in that, The nucleotide sequence of the gene is as shown in SEQ ID NO.1.

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