Gene moupe12 and its application in reducing the pathogenicity of magnaporthe oryzae

Abstract

The application discloses genes MoUPE12 and application thereof in reducing pathogenicity of Magnaporthe oryzae. The application obtains corresponding knockout mutants by constructing a knockout vector of genes MoUPE12 and introducing the knockout vector into Magnaporthe oryzae protoplasts. It is found through determination of conidium germination, appressorium development and pathogenicity to rice of the obtained knockout mutants that the conidium germination and appressorium development of the knockout mutants are slowed down, the glycogen metabolism of the conidia is slowed down, and the pathogenicity to rice is significantly reduced. After the gene MoUPE12 is complemented, the pathogenicity of the complemented mutant restores to the wild type level. That is, inhibition of expression of the gene MoUPE12 can reduce the pathogenicity of Magnaporthe oryzae, and the gene can be used as a target to develop Magnaporthe oryzae prevention and treatment products. The application enriches the pathogenic mechanism of Magnaporthe oryzae and is beneficial to development of Magnaporthe oryzae prevention and treatment products.

Description

Technical Field

[0001] This invention belongs to the technical field of development and manufacturing of biological control agents for plant diseases. More specifically, it relates to genes. MoUPE12 And its application in reducing the pathogenicity of rice blast fungus. Background Technology

[0002] Rice blast fungus ( Magnaporthe oryzae Rice blast, caused by blast disease, is one of the most destructive diseases in rice production, which can seriously affect the yield and quality of rice.

[0003] In the process of controlling rice blast using chemical methods, improper use of chemical agents can easily lead to environmental pollution and pesticide residues, impacting the ecological environment. While biological control methods are environmentally friendly, their effectiveness in controlling rice blast is easily affected by environmental conditions, resulting in unstable efficacy. Therefore, there is still a need to develop green and effective rice blast control products. In-depth research into the pathogenic genes of rice blast fungus and the study of their functions will not only contribute to a comprehensive understanding of the pathogenic molecular mechanisms of rice blast fungus but also provide targets for rice blast control, thereby facilitating the development of rice blast control products. Summary of the Invention

[0004] This invention provides a gene for promoting the development of rice blast control products. MoUPE12 And its application in reducing the pathogenicity of rice blast fungus.

[0005] The first objective of this invention is to provide genes. MoUPE12 Application in reducing the pathogenicity of rice blast fungus.

[0006] A second objective of this invention is to provide a method for inhibiting said gene. MoUPE12 Application of the expressed reagent in reducing the pathogenicity of rice blast fungus.

[0007] A third objective of this invention is to provide a method for inhibiting said gene. MoUPE12 The application of the expressed reagent in the preparation of products that reduce the pathogenicity of rice blast fungus.

[0008] A fourth object of the present invention is to provide a method for inhibiting said gene. MoUPE12 Application of the expressed reagent in the prevention and control of rice blast.

[0009] The fifth object of the present invention is to provide a method for inhibiting said gene. MoUPE12 Application of the expressed reagent in the preparation of rice blast control products.

[0010] A sixth object of the present invention is to provide a method for knocking out said gene. MoUPE12 The application of the reagent in reducing the pathogenicity of rice blast fungus.

[0011] A seventh object of the present invention is to provide a method for knocking out said gene.MoUPE12 The application of the reagent in the preparation of products that reduce the pathogenicity of rice blast fungus.

[0012] An eighth object of the present invention is to provide a method for knocking out said gene. MoUPE12 The application of reagents in the prevention and control of rice blast.

[0013] A ninth object of the present invention is to provide a method for knocking out said gene. MoUPE12 The application of the reagent in the preparation of rice blast control products.

[0014] The above-mentioned objective of this invention is achieved through the following technical solution:

[0015] This invention constructs genes MoUPE12 (or) MoUPE12 A gene knockout vector was created and introduced into the protoplasts of *Strombus haematous*, resulting in the *Strombus haematous* knockout mutant Δ. MoUPE12 The germination of conidia, appressorium development, and pathogenicity of the knockout mutants were measured. The results showed that conidia germination and appressorium development were slowed, glycogen metabolism in the conidia was reduced, and pathogenicity to rice was significantly decreased. In contrast, the gene... MoUPE12 After replacement, the pathogenicity of the replacement mutant returned to the wild-type level. Therefore, this invention seeks protection for the gene. MoUPE12 Application in reducing the pathogenicity of rice blast fungus.

[0016] Specifically, the application involves inhibiting genes in the rice blast fungus. MoUPE12 The expression of the gene is achieved; MoUPE12 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0017] Specifically, the protein MoUPE12, whose amino acid sequence is shown in SEQ ID NO.2, is an uncharacterized protein that does not contain a signal peptide, does not contain any known domains, and has secretory function.

[0018] In a specific embodiment of the present invention, the gene MoUPE12 The nucleotide sequence is shown in SEQ ID NO.1.

[0019] More specifically, the gene that inhibits rice blast fungus MoUPE12 The expression of this gene is achieved by interfering with its expression. MoUPE12 The expression of, or through, the gene MoUPE12 The coding region of the gene is mutated or the gene is knocked out. MoUPE12 It was achieved.

[0020] More specifically, the gene that inhibits rice blast fungus MoUPE12The expression is achieved by interfering with gene expression through RNA interference technology. MoUPE12 The expression of, or the manipulation of genes through gene editing systems MoUPE12 Mutate the coding region or knock out the gene through homologous recombination. MoUPE12 It was achieved.

[0021] This invention also provides a method for reducing the pathogenicity of rice blast fungus, the method comprising: inhibiting genes in rice blast fungus. MoUPE12 The expression.

[0022] Specifically, the gene that inhibits rice blast fungus MoUPE12 The expression of this gene is achieved by interfering with its expression. MoUPE12 The expression of, or through, the gene MoUPE12 The coding region of the gene is mutated or the gene is knocked out. MoUPE12 It was achieved.

[0023] More specifically, the gene that inhibits rice blast fungus MoUPE12 The expression is achieved by interfering with gene expression through RNA interference technology. MoUPE12 The expression of, or the manipulation of genes through gene editing systems MoUPE12 Mutate the coding region or knock out the gene through homologous recombination. MoUPE12 It was achieved.

[0024] Given that knocking out genes that suppress rice blast fungus MoUPE12 The expression of this gene can reduce the pathogenicity of rice blast fungus to rice. Therefore, the present invention also seeks protection for the gene. MoUPE12 Application of the expressed reagent in reducing the pathogenicity of rice blast fungus.

[0025] The present invention also seeks protection for the gene that inhibits the gene. MoUPE12 The application of the expressed reagent in the preparation of products that reduce the pathogenicity of rice blast fungus.

[0026] The present invention also seeks protection for the gene that inhibits the gene. MoUPE12 Application of the expressed reagent in the prevention and control of rice blast.

[0027] The present invention also seeks protection for the gene that inhibits the gene. MoUPE12 Application of the expressed reagent in the preparation of rice blast control products.

[0028] Optionally, the repressor gene MoUPE12 The expression reagents are those that target genes using antisense gene technology or RNA interference technology. MoUPE12 Reagents required for expression inhibition.

[0029] Optionally, the reagent is a target gene. MoUPE12 siRNA or dsRNA, which can inhibit the rice blast fungus gene MoUPE12The expression of this method aims to reduce the pathogenicity of rice blast fungus, thereby preventing and controlling rice blast.

[0030] The present invention also claims protection for methods used to knock out said gene. MoUPE12 The application of the reagent in reducing the pathogenicity of rice blast fungus.

[0031] The present invention also claims protection for methods used to knock out said gene. MoUPE12 The application of the reagent in the preparation of products that reduce the pathogenicity of rice blast fungus.

[0032] The present invention also claims protection for methods used to knock out said gene. MoUPE12 Application of the expressed reagent in the prevention and control of rice blast.

[0033] The present invention also claims protection for methods used to knock out said gene. MoUPE12 Application of the expressed reagent in the preparation of rice blast control products.

[0034] Specifically, the gene knockout MoUPE12 The reagent for expression is a gene MoUPE12 Reagents required for functional knockout or knockout of the entire gene.

[0035] Optionally, the actual substance is a gene. MoUPE12 Knockout vector.

[0036] Optionally, the gene MoUPE12 The knockout vector was constructed using the filamentous fungal expression vector pCT74.

[0037] Specifically, the rice blast disease is caused by the rice blast fungus (… Magnaporthe oryzae Caused by ).

[0038] Furthermore, this invention discovered that the rice blast fungus knockout mutant Δ MoUPE12 Conidial germination and appressorium development are slowed, and glycogen metabolism in conidia is reduced. Therefore, inhibiting the aforementioned gene... MoUPE12 Reagents for expression or for knocking out the gene MoUPE12 The application of the reagent in inhibiting the germination of rice blast fungus conidia, appressorium development, and conidium glycogen metabolism is also within the scope of protection of this invention.

[0039] The present invention has the following beneficial effects:

[0040] This invention constructs genes MoUPE12 A knockout vector was introduced into rice blast fungus protoplasts to obtain corresponding knockout mutants. Measurements of conidial germination, appressorium development, and pathogenicity to rice in the obtained knockout mutants revealed that conidial germination and appressorium development were slowed, glycogen metabolism in conidia was reduced, and pathogenicity to rice was significantly decreased. Furthermore, the gene...MoUPE12 After replacement, the pathogenicity of the replacement mutant returned to the wild-type level. This means the suppressor gene... MoUPE12 Expression of this gene can reduce the pathogenicity of rice blast fungus, and this gene can be used as a target to develop rice blast control products. This invention enriches the understanding of the pathogenic mechanism of rice blast fungus and is beneficial to the development of rice blast control products. Attached Figure Description

[0041] Figure 1 Results of secretory function analysis of MoUPE12 protein from rice blast fungus.

[0042] Figure 2 Rice blast fungus MoUPE12 A schematic diagram of the construction of a gene knockout vector.

[0043] Figure 3 For some hygromycin-resistant transformants HPH Gene PCR analysis results; DNA marker: DL 2000 Marker.

[0044] Figure 4 The target gene for some hygromycin-resistant transformants ( MoUPE12 PCR analysis results; DNA marker: DL 2000 Marker.

[0045] Figure 5 For HPH Southern blot analysis results of rice blast fungus knockout transformants with fragments as probes.

[0046] Figure 6 For MoUPE12 Southern blot analysis results of rice blast fungus knockout transformants with fragments as probes.

[0047] Figure 7 The target gene for some bleomycin-resistant transformants ( MoUPE12 PCR analysis results; DNA marker: DL 2000 Marker.

[0048] Figure 8 Δ, a knockout mutant of rice blast fungus MoUPE12 and complement mutant Δ MoUPE12 -com Gene MoUPE12 The results of RT-qPCR analysis are shown; different letters in the figure indicate significant differences. p< 0.05.

[0049] Figure 9 Δ, a knockout mutant of rice blast fungus MoUPE12 and complement mutant Δ MoUPE12 Analysis results of the stress resistance capability of -com.

[0050] Figure 10 Δ, a knockout mutant of rice blast fungus MoUPE12 and complement mutant Δ MoUPE12 Results of observation on conidial germination and glycogen metabolism of -com.

[0051] Figure 11 Δ, a knockout mutant of rice blast fungus MoUPE12 and complement mutant Δ MoUPE12 Statistical results of conidial germination and appressorium formation of -com; Figure p< 0.05.

[0052] Figure 12 Δ, a knockout mutant of rice blast fungus MoUPE12 and complement mutant Δ MoUPE12 -com's results on the pathogenicity of detached rice leaves. Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0054] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0055] The gene described in this invention MoUPE12 The nucleotide sequence of the protein is shown in SEQ ID NO.1. The protein it encodes, MoUPE12, is an uncharacterized protein that does not contain a signal peptide or a known domain. The amino acid sequence of the protein MoUPE12 is shown in SEQ ID NO.2.

[0056] The test strains used in this invention are wild-type rice blast fungus strain ZC13 ​​and sucrase-deficient yeast strain YTK12; the test rice is the susceptible indica rice line CO39; the secretion function verification vector is pSUC2; 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...). GFP and HPH Gene replaced with bleomycin resistance gene Zeocin ).

[0057] Example 1: Detection of the secretory function of protein MoUPE12

[0058] 1. pSUC2-MoUPE12 Construction of recombinant vectors

[0059] The gene sequence encoding the MoUPE12 protein was cloned and ligated into the pSUC2 vector. 10 µL of the ligation product was added to 100 µL of *E. coli* DH5α competent cells and incubated on ice for 30 min. The cells were then heat-shocked in a water bath at 42°C for 90 s, followed by cooling on ice for 5 min. 800 µL of LB broth was added, and the cells were incubated at 37°C with shaking at 180 rpm for 1 h. After centrifugation at 4000 rpm for 5 min, the supernatant was discarded, and 100 µL of the bacterial culture was mixed with the precipitate and spread onto LB solid medium (containing 100 µg / mL Amp). The cells were incubated at 37°C for 8–12 h. Single colonies exhibiting Amp resistance were selected for PCR verification. After successful verification, the colonies were sequenced, and the sequencing results were compared using SnapGene. Correct alignment confirmed the pSUC2 vector. MoUPE12 Recombinant carrier.

[0060] 2. Preparation, transformation, and screening of yeast competent cells

[0061] The frozen yeast strain YTK12 was activated using YPDA plates. Single colonies were picked and placed in YPDA liquid medium, and cultured at 28°C with shaking for 18 h. Cells were collected by centrifugation at 12000 × g for 1 min, and 50 μL of sterile water was added and gently mixed. 2 μg of pSUC2- MoUPE12 Recombinant vector / pSUC2 blank vector / pSUC2- Avr1b Recombinant vector (as a control), 5 μg carrier DNA, 1.0 mol / L LiAc, and 50% w / v PEG3350 were added to the yeast YTK12 competent cell slurry and mixed thoroughly. After incubation at 30℃ and 220 rpm for 30 min, DMSO solution was added, and the cells were incubated at 42℃ for 15 min. Cells were collected by centrifugation at 5000 ×g for 1 min and resuspended in 400 μL of sterile water. Cells were collected by centrifugation at 5000 ×g for 30 s, resuspended in sterile water, and evenly spread on CMD-W plates. The cells were incubated at 28℃ for 3 days. Single colonies were picked and inoculated onto YPDA, CMD-W, and YPRAA plates and incubated at 28℃ for 3 days to observe the growth of single colonies.

[0062] 3. TTC (triphenyltetrazolium chloride) colorimetric reaction

[0063] The selected single-colony yeast strains were inoculated into YPDA liquid medium and cultured at 28°C and 220 rpm for 24 h with shaking. The cells were collected by centrifugation at 12000 ×g for 1 min. Sterile water was added and the mixture was vortexed. The cells were collected by centrifugation at 12000 ×g for 1 min. This process was repeated once. The cells were resuspended in 750 μL of sterile water, and 250 μL of 10 mmol / L sodium acetate buffer (pH 4.7) and 500 μL of 10% (w / v) sucrose solution were added. The mixture was incubated at 37°C for 10 min and centrifuged at 12000 ×g for 1 min. 100 μL of the supernatant was added to a glass test tube, and 900 μL of 0.1% TTC solution was added. The mixture was incubated at room temperature for 5 min.

[0064] The results of the secretory function analysis of the MoUPE12 protein of rice blast fungus are as follows: Figure 1 As shown. By Figure 1 It can be seen that it contains pSUC2- MoUPE12 The strains containing the recombinant vector could grow on CMD-W and YPRAA plates. Furthermore, the TTC colorimetric reaction results showed that strains containing pSUC2-... Avr1b and pSUC2- MoUPE12 The YTK12 yeast strain could undergo a reduction reaction with TTC to produce an insoluble red precipitate of formazan compound TPF, while the YTK12 yeast strain used as a negative control and the YTK12 yeast strain containing the pSUC2 vector could not react with TTC to produce TPF. These results indicate that the MoUPE12 protein has a secretory function.

[0065] Example 2 Gene MoUPE12 Obtaining knockout mutants and complement mutants

[0066] 1. Rice blast fungus MoUPE12 Amplification of upstream and downstream homologous arms of genes

[0067] Rice blast fungus MoUPE12 A schematic diagram of gene knockout vector construction is shown below. Figure 2 As shown. By Figure 2 It can be seen that the present invention achieves homologous recombination. MoUPE12 Gene replacement hph + gfp Fragments, implementation MoUPE12 Gene knockout.

[0068] exist MoUPE12 Gene sequences of approximately 1000 bp in length were selected upstream and downstream of the gene as homologous arms. The upstream homologous arm was named homologous arm A, and the downstream homologous arm was named homologous arm B. Based on the selected homologous arm sequences, corresponding amplification primers were designed, as shown in Table 1.

[0069] Table 1 MoUPE12Primers for amplification of gene homologous arms A and B fragments

[0070]

[0071] Genomic DNA was extracted from the wild-type ZC13 ​​strain of *Magnaporthe oryzae* using the OMEGA Fungal DNA Kit. Using the obtained genomic DNA as a template, the reaction system was prepared according to the instructions of the PCR amplification reagents. PCR amplification was performed using the primers shown in Table 1 to obtain the desired results. MoUPE12 Homologous arm A of the gene ( MoUPE12 -A) and homologous arm B ( MoUPE12 -B).

[0072] After PCR amplification, the PCR amplification products were cleaned and recovered using the OMEGA Cycle Pure Kit.

[0073] 2. MoUPE12 Construction of gene knockout vector

[0074] Referring to the instructions of the pMD18-T Vector Cloning Kit (TakaRa), the PCR amplification and recovery results were... MoUPE12 -A and MoUPE12 -B was ligated to the T vector, and the ligation product was transformed into *E. coli* DH5α competent cells. Positive transformants with Amp resistance were selected, and recombinant plasmid DNA was extracted and sequenced to obtain the recombinant plasmid pMD18T-. MoUPE12 -A and pMD18T- MoUPE12 -B.

[0075] use Apa I and Kpn I respectively targeted the recombinant plasmid pMD18T- MoUPE12 The A and pCT74 vectors were double-digested and gel-cleaved. The homologous arm A fragment was ligated to pCT74 using T4 DNA ligase. The resulting cells were transformed into *E. coli* DH5α competent cells. Positive transformants resistant to Amp were selected, and recombinant plasmid DNA was extracted and sequenced to obtain the recombinant plasmid pCT74-. MoUPE12 -A. Similarly, using Xba I and Eco RI double enzyme digestion of recombinant plasmid pMD18T- MoUPE12 -B and recombinant plasmid pCT74- MoUPE12 -A was then excised and recovered from the gel. The homologous arm B fragment was ligated with pCT74- using T4 DNA ligase. MoUPE12 -A is used to connect and transform E. coli DH5α competent cells.

[0076] Enzyme digestion verification yielded MoUPE12 Gene knockout vector pCT74- MoUPE12 -KO.

[0077] 3. MoUPE12 Amplification of gene complement fragments

[0078] exist MoUPE12 A 1500 bp promoter sequence was selected upstream of the gene, and a 500 bp terminator sequence was selected downstream. Based on the selected sequences, corresponding amplification primers were designed, as shown in Table 2.

[0079] Table 2 MoUPE12 Amplification primers for gene complement fragments

[0080]

[0081] Using the extracted wild-type genomic DNA of rice blast fungus as a template, primer Com- MoUPE12 -F and Com- MoUPE12 PCR amplification was performed using the -R method. After amplification, the amplification products were cleaned and recovered using the OMEGA Cycle Pure Kit. MoUPE12 Gene complement fragment ( MoUPE12 -com).

[0082] 4. MoUPE12 Construction of gene complementation vector

[0083] use Kpn I and Apa I respectively to MoUPE12 The -com and pCTZN vectors were double-digested and then gel-cleaved. The digested DNA was ligated using T4 DNA ligase. MoUPE12 The -com fragment was ligated with pCTZN and transformed into E. coli DH5α competent cells. The recombinant plasmid was extracted and verified by enzyme digestion. MoUPE12 Gene complementation vector pCTZN- MoUPE12 -com.

[0084] 5. Preparation of rice blast fungus protoplasts

[0085] Wild-type strain of *Stromboma oryzae* was activated on Jianli medium (containing 5.0 g / L yeast extract, 22.0 g / L anhydrous glucose, and 17.0 g / L agar powder) plates and cultured upside down at 28°C for 10 days. Mycelia were scraped and transferred to 50 mL YPS liquid medium (containing 6.0 g / L yeast extract, 6.0 g / L hydrolyzed casein, and 10.0 g / L sucrose) and cultured at 28°C with shaking at 120 rpm for 2 days. Mycelia were collected using a 200-mesh cell sieve, crushed, and transferred to 200 mL YPS liquid medium and cultured at 28°C with shaking at 120 rpm for 1 day. Mycelia were collected by filtration through a 200-mesh cell sieve, washed twice with ddH2O, and then washed once with sterile 0.7 mol / L NaCl solution. Add 10 mL of enzyme solution (containing 15 mg / mL lysozyme and 15 mg / mL wall-breaking enzyme) to each 1 g of mycelium and incubate at 30 °C and 80 rpm for 1.5 h with shaking. Filter the enzymatic digest using four layers of sterile paper, wash with pre-cooled 0.7 mol / L NaCl solution and collect. Centrifuge at 4 °C and 3500 rpm for 10 min. Resuspend the precipitate in pre-cooled STC solution (containing 1.2 mol / L sorbitol, 10 mmol / L Tris-HCl, 50 mmol / L CaCl2, pH 7.5). Repeat once, then add an appropriate amount of pre-cooled STC solution to control the final protoplast concentration at 1 × 10⁻⁶. 7 ~1×10 8 Quantity / mL, aliquoted and stored at -80℃.

[0086] The protoplasts of the rice blast fungus knockout mutant were prepared according to the above-described preparation steps for rice blast fungus protoplasts.

[0087] 6. Transformation of rice blast fungus protoplasts

[0088] use Kpn I linearized knockout vector pCT74- MoUPE12-KO, add 3-5 μg of linearized knockout vector to 200 μL of wild-type rice blast fungus protoplasts, and incubate on ice for 20 min; add pre-chilled PTC solution (60% PEG4000, 50 mmol / L CaCl2, 10 mmol / L Tris-HCl, pH 7.5) and incubate on ice for 15 min; add pre-chilled STC solution, centrifuge at 4℃ and 4000 rpm for 15 min, resuspend the pellet in liquid regeneration medium (6.0 g / L yeast extract, 6.0 g / L hydrolyzed casein and 200.0 g / L sucrose), and revive at 28℃ and 100 rpm for 16-18 h with shaking; incubate in solid regeneration medium (containing 200 μg / mL hygromycin) at 28℃ in the dark for 4-5 days; pick hygromycin-resistant transformants and transfer them to Jianli medium (containing 200 μg / mL hygromycin), and incubate at 28℃ for 2-3 days to obtain stable transformed strains. In the experiment of transforming protoplasts of rice blast fungus knockout mutants with replenishment vectors, bleomycin was used as the antibiotic to screen for resistant transformants.

[0089] 7. PCR Validation Analysis of Rice Blast Genus Knockout Mutant

[0090] Genomic DNA was extracted from the obtained hygromycin-resistant transformants (candidate positive transformants) using the OMEGA Fungal DNA Kit, and PCR verification analysis was performed using the primers shown in Table 3.

[0091] Table 3 Primers used for PCR validation analysis of knockout mutants

[0092]

[0093] This invention utilizes homologous recombination to construct... MoUPE12 Gene knockout vectors were transformed into rice blast fungus protoplasts, yielding 25 candidate positive transformants. Genomic DNA was extracted from these candidate positive transformants and used... HPH Gene-specific primers were used for PCR validation analysis. Some hygromycin-resistant transformants... HPH Gene PCR analysis results as follows Figure 3 As shown. By Figure 3 It can be seen that the transformant can be amplified to HPH The gene was then subjected to further PCR verification analysis.

[0094] The target gene of some hygromycin-resistant transformants ( MoUPE12 PCR analysis results are as follows Figure 4 As shown. By Figure 4 It can be seen that, when it can be amplified to HPH The gene could not be amplified in any of the transformants. MoUPE12The gene sequence indicates that the transformant is a knockout transformant of rice blast fungus, and further analysis will be performed using Southern blot.

[0095] 8. Southern blot analysis of knockout mutants of rice blast fungus

[0096] Use primers MoUPE12 -F / MoUPE12 -R amplifies the target gene probe, using HPH -F / HPH -R amplification HPH Gene probes were used to perform Southern blot hybridization on the resistant transformants that had been validated by PCR, according to the instructions of DIG High Prime DNA Labeling and Detection Starter Kit I (Roche LOT28309220).

[0097] by HPH Southern blot analysis results of rice blast fungus knockout transformants with the fragment as a probe are as follows: Figure 5 As shown. By Figure 5 It can be seen that all the rice blast fungus knockout transformants tested showed single-copy bands.

[0098] by MoUPE12 Southern blot analysis results of rice blast fungus knockout transformants with the fragment as a probe are as follows: Figure 6 As shown. By Figure 6 It can be seen that, with HPH The fragments are three rice blast fungus knockout transformants with single-copy bands on the probe, in order to MoUPE12 The absence of hybridization bands in both the fragments and probes indicates that the present invention has successfully obtained the rice blast fungus. MoUPE12 Gene knockout mutant Δ MoUPE12 .

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

[0100] Genomic DNA was extracted from the bleomycin-resistant transformants using the OMEGA Fungal DNA Kit, and primers were used to extract the DNA. MoUPE12 -F / MoUPE12 -R is used for PCR validation analysis.

[0101] This invention utilizes a random insertion method to insert the gene complementation vector pCTZN- MoUPE12 -com transforms rice blast fungus knockout mutant Δ MoUPE12 (Δ) MoUPE12Seven bleomycin-resistant transformants were obtained from protoplasts (-22) of the sample. Genomic DNA was extracted from these bleomycin-resistant transformants and analyzed using primers. MoUPE12 -F / MoUPE12 -R was used for PCR verification analysis. The target gene in some bleomycin-resistant transformants ( MoUPE12 PCR analysis results are as follows Figure 7 As shown. By Figure 7 It can be seen that the present invention has obtained the rice blast fungus. MoUPE12 Gene complementation mutant.

[0102] 10. RT-qPCR validation analysis of knockout mutants and complement mutants of rice blast fungus

[0103] Genomic RNA was extracted from hygromycin / bleomycin-resistant transformants that had been validated by PCR using a column-based fungal total RNA extraction and purification kit (Shanghai Sangon Biotech Co., Ltd.). The obtained RNA was reverse transcribed into cDNA according to the instructions of a reverse transcription kit (Takara). MoActin The gene was used as an internal reference gene, and the concentration of the gene in the transformants was measured using RT-qPCR. MoUPE12 The relative expression levels of genes. Primers used for RT-qPCR validation analysis are shown in Table 4.

[0104] Table 4 Primers used for RT-qPCR validation analysis

[0105]

[0106] The qPCR reaction system used for RT-qPCR validation analysis is shown in Table 5.

[0107] Table 5. qPCR reaction system used for RT-qPCR validation analysis

[0108]

[0109] 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.

[0110] Rice blast fungus knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 -com Gene MoUPE12 The RT-qPCR analysis results are as follows Figure 8 As shown. By Figure 8 It can be seen that, compared with the wild-type ZC13 ​​strain, the gene knockout mutant obtained has a higher gene density. MoUPE12The relative expression level was significantly reduced, while the complement mutant recovered to the wild-type level.

[0111] Example 3 Gene MoUPE12 Observational analysis of knockout mutants and complement mutants

[0112] 1. Colony morphology observation and growth rate determination

[0113] Wild-type ZC13 ​​strain of rice blast fungus and knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 -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.

[0114] Observations showed that the knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 The colony morphology and growth rate of -com were not significantly different from those of the wild type of rice blast fungus.

[0115] 2. Analysis of stress resistance

[0116] Wild-type ZC13 ​​strain of rice blast fungus and knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 -com were inoculated onto YDA medium containing 0.8 mol / L sodium chloride (NaCl), 0.01% SDS (sodium dodecyl sulfate), 0.8 mol / L Sorbitol (sorbitol), 0.02 mol / L H2O2, 0.3 g / L CR (Congo red), and 0.08 g / L CFW (fluorescent whitening agent), respectively. After incubation at 28℃ with the medium inverted for 10 days, the colony growth of different strains was observed.

[0117] Rice blast fungus knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 The results of the stress resistance analysis of -com are as follows: Figure 9 As shown. By Figure 9 It can be seen that, compared with the wild-type strain, MoUPE12 There were no significant differences in sensitivity to NaCl, Sorbitol, SDS, H2O2, CR, and CFW.

[0118] 3. Observation on the production and germination of conidia, appressorium formation, and glycogen metabolism of conidia.

[0119] Wild-type ZC13 ​​strain and knockout mutant Δ of rice blast fungus were scraped off separately. MoUPE12 and complement mutant Δ MoUPE12-com mycelium was added to ddH2O, and 500 μL of mycelial solution was transferred to tomato oat medium (40 g of raw oats, boiled in ddH2O for 1 h, filtered, and then 150 mL of tomato juice, 0.06 g of calcium carbonate and 2.5%–3% agar powder were added, and the volume was adjusted to 1 L with ddH2O and water). The medium was incubated upside down at 28℃ for 4–7 days. Mycelium was scraped and added to ddH2O, filtered through 4 layers of dust-free paper, and 20 µL of spore solution was transferred to a hydrophobic glass slide and incubated in the dark at 28℃. The germination of conidia was observed at different time points.

[0120] Adjust the concentration of the collected spore solution to 5 × 10⁻⁶. 4 spores / mL; pipette 20 µL of spore solution onto a hydrophobic slide and incubate in the dark at 28°C. Samples were taken at different time points, stained with KI / I2 solution (60 mg / mL KI, 10 mg / mL I2), and observed under a microscope.

[0121] Rice blast fungus knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 The results of conidial germination and glycogen metabolism observations at -com are as follows: Figure 10 As shown. By Figure 10 It can be seen that the wild-type ZC13 ​​strain and Δ MoUPE12 Glycogen from conidia begins to be transported to newly formed appressoria at 6 hours, and by 12 hours, the glycogen in conidia has been largely transferred to appressoria. In Δ MoUPE12 In the process, glycogen in conidia begins to be transported from conidia to newly formed appressoria at 8–10 h, and glycogen staining can still be observed in conidia and appressoria at 24 h. These results indicate that knocking out... MoUPE12 This leads to a delay in the transfer of glycogen from the conidia of rice blast fungus to the appressorium, i.e., a slowdown in glycogen metabolism in its conidia.

[0122] Rice blast fungus knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 Statistical results of conidial germination and appressorium formation of -com are as follows: Figure 11 As shown. By Figure 11 It can be seen that Δ MoUPE12 The conidial germination rate at 4 h and the appressorium formation rate at 12 h were both significantly lower than those of the wild-type ZC13 ​​strain and Δ MoUPE12 -com. The above results indicate that knocking out MoUPE12 This leads to a slowdown in the germination of conidia and the development of appressorium of rice blast fungus, thus affecting the development and maturation of appressorium.

[0123] Example 4 Gene MoUPE12 Pathogenicity analysis of knockout and complement mutants

[0124] 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 the wild-type ZC13 ​​strain of rice blast fungus and the knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 -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 cultured in an artificial climate chamber at 25℃ and 90% humidity (12 h light / 12 h dark) for 4-5 days. The disease incidence of the rice leaves was then observed and photographed.

[0125] Rice blast fungus knockout mutant Δ MoUPE12 and complement mutant Δ MoUPE12 -com's results on the pathogenicity test of detached rice leaves are as follows: Figure 12 As shown. By Figure 12 It can be seen that, along with the wild-type ZC13 ​​strain of rice blast fungus and MoUPE12 Compared to -com, Δ MoUPE12 The pathogenicity was significantly reduced, and the lesion area was significantly smaller, indicating that Δ MoUPE12 The reduced pathogenicity to detached rice leaves further illustrates... MoUPE12 Genes can affect the pathogenicity of rice blast fungus.

[0126] 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. Genes MoUPE12 Its application in reducing the pathogenicity of rice blast fungus is characterized by, The application involves inhibiting genes in the rice blast fungus. MoUPE12 The expression of the gene is achieved; MoUPE12 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The gene that inhibits rice blast fungus MoUPE12 The expression of this gene is achieved by interfering with its expression. MoUPE12 The expression of, or through, the gene MoUPE12 The coding region of the gene is mutated or the gene is knocked out. MoUPE12 It was achieved.

3. Suppressing the gene described in claim 1 MoUPE12 Application of the expressed reagent in reducing the pathogenicity of rice blast fungus.

4. Inhibition of the gene described in claim 1 MoUPE12 The application of the expressed reagent in the preparation of products that reduce the pathogenicity of rice blast fungus.

5. Inhibition of the gene described in claim 1 MoUPE12 Application of the expressed reagent in the prevention and control of rice blast.

6. Inhibition of the gene described in claim 1 MoUPE12 Application of the expressed reagent in the preparation of rice blast control products.

7. A device for knocking out the gene described in claim 1 MoUPE12 The application of the reagent in reducing the pathogenicity of rice blast fungus.

8. A device for knocking out the gene described in claim 1 MoUPE12 The application of the reagent in the preparation of products that reduce the pathogenicity of rice blast fungus.

9. A device for knocking out the gene described in claim 1 MoUPE12 The application of reagents in the prevention and control of rice blast.

10. A tool for knocking out the gene described in claim 1 MoUPE12 The application of the reagent in the preparation of rice blast control products.

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