CN116082473A

CN116082473A - Application of protein MoUPE3 in regulation and control of pathogenic force of rice blast fungi

Info

Publication number: CN116082473A
Application number: CN202211183880.1A
Authority: CN
Inventors: 李云锋; 李洁玲; 聂燕芳; 李华平
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-05-09

Abstract

The invention discloses an application of a protein MoUPE3 in regulating and controlling pathogenic force of rice blast fungi. The invention obtains the rice blast fungus knockout mutant delta MoUPE3 by knocking out the gene of the encoding protein MoUPE3 in the rice blast fungus, and obtains the complement mutant delta MoUPE3-com by the complement construction. The bacterial colony morphology, spore yield, stress resistance, pathogenicity and the like of the wild type rice blast fungus, the rice blast fungus knockout mutant delta MoUPE3 and the anaplerotic mutant delta MoUPE3-com are detected, and the encoding gene of the rice blast fungus protein MoUPE3 can regulate and control the spore yield, stress resistance and pathogenicity of the rice blast fungus and can be used as a target point for preventing and controlling rice blast. The invention enriches pathogenic related genes of rice blast fungi, provides target genes for developing effective bactericides, and is beneficial to preventing and controlling rice blast.

Description

Application of protein MoUPE3 in regulation and control of pathogenic force of rice blast fungi

Technical Field

The invention belongs to the technical field of plant genetic engineering. More specifically, the application of the protein MoUPE3 in regulating and controlling the virulence of rice blast fungi is disclosed.

Background

The rice blast caused by the rice blast fungus (Magnaporthe oryzae) is a destructive disease in the rice production, and seriously affects the yield and quality of the rice. The rice blast can occur in the whole growth period of rice, and causes seedling blast, leaf blast, festival blast, neck blast, grain blast and the like; among them, neck blast has the greatest effect on rice yield and quality.

At present, the main measures adopted for preventing and controlling rice blast in rice production are prevention and control by using disease-resistant varieties and chemical pesticides. With the development of pathogenic molecular biology, many pathogenic related genes have also been found to have a close relationship with the pathological processes of plant pathogenic bacteria. Therefore, the gene related to the pathogenic of the rice blast is excavated, and a new idea can be provided for improving the disease resistance of the rice and preventing and controlling the disease of the rice blast.

Disclosure of Invention

The first object of the present invention is to provide a Magnaporthe grisea protein MoUPE3.

A second object of the present invention is to provide a gene encoding the protein MoUPE3.

The third object of the invention is to provide the application of the protein MoUPE3 in reducing the pathogenic force of rice blast bacteria.

The fourth object of the invention is to provide an application of the protein MoUPE3 in reducing the spore yield of rice blast bacteria.

The fifth object of the invention is to provide the application of the protein MoUPE3 in reducing the stress resistance of rice blast bacteria.

A sixth object of the present invention is to provide the use of a substance inhibiting the expression of the blasticidin MoUPE3 protein for reducing the pathogenic force of Pyricularia oryzae.

The seventh object of the present invention is to provide the use of a substance inhibiting the expression of the mopsin MoUPE3 in reducing the spore yield of mopsin.

An eighth object of the present invention is to provide an application of a substance inhibiting the expression of the mopsin MoUPE3 in reducing the stress resistance of mopsis.

The ninth object of the present invention is to provide the use of a substance inhibiting the expression of the mophthora oryzae protein MoUPE3 in controlling rice blast.

The above object of the present invention is achieved by the following technical scheme:

the invention identifies a protein MoUPE3 for regulating and controlling the pathogenicity of rice blast fungus from the rice blast fungus, and the amino acid sequence of the protein MoUPE3 is shown as SEQ ID NO. 2. The protein MoUPE3 is an unknown protein that does not contain a signal peptide and a known domain, and the specific function of the protein MoUPE3 in Pyricularia oryzae has not been known prior to the present invention.

The invention obtains the rice blast fungus knockout mutant delta MoUPE3 by knocking out the gene of the encoding protein MoUPE3 in the rice blast fungus, and obtains the complement mutant delta MoUPE3-com by the complement construction. The bacterial colony morphology, the spore yield, the stress resistance (stress resistance), the pathogenicity and the like of the rice blast wild type, the rice blast knockout mutant delta MoUPE3 and the anaplerosis mutant delta MoUPE3-com are detected, so that the spore yield, the stress resistance and the pathogenicity of the rice blast knockout mutant delta MoUPE3 are obviously reduced compared with those of the rice blast wild type; the spore yield, stress resistance and pathogenicity of the anaplerotic mutant delta MoUPE3-com are recovered, which shows that the rice blast fungus protein MoUPE3 can regulate and control the spore yield, stress resistance and pathogenicity of rice blast fungus and can be used as a target point for preventing and controlling rice blast. Therefore, the application of the invention protects the rice blast fungus protein MoUPE3 and the encoding gene thereof, and the application of the protein MoUPE3 and the encoding gene thereof.

The invention provides a rice blast fungus protein MoUPE3, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention also provides a gene for encoding the protein MoUPE3, and the nucleotide sequence of the gene is one of the following A, B, C:

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

B. a nucleotide sequence shown as SEQ ID NO. 1;

C. analogs of A or B above obtained by base insertion, deletion, or substitution still have the function of regulating the pathogenic ability of Pyricularia oryzae.

In view of the influence on pathogenicity, sporulation quantity, stress resistance and the like of the rice blast fungus after knocking out the gene encoding the protein MoUPE3. Thus, the present invention also applies for the following applications of the protection protein MoUPE 3:

the invention discloses application of a protective protein MoUPE3 in reducing pathogenic force of rice blast fungi.

The invention also discloses application of the protective protein MoUPE3 in reducing the spore yield of the rice blast fungus.

The invention also discloses application of the protective protein MoUPE3 in reducing stress resistance of rice blast bacteria.

Specifically, the stress resistance means resistance to oxidative stress.

The invention also discloses application of the protective protein MoUPE3 in influencing the integrity of the cell wall of the rice blast fungus.

Specifically, the application is realized by blocking or inhibiting the expression of the protein MoUPE3 in the rice blast fungus.

Alternatively, the expression of the protein MoUPE3 may be blocked by knocking out the gene encoding the protein MoUPE3 in Pyricularia oryzae, or the expression of Pyricularia oryzae protein MoUPE3 may be inhibited by RNA interference or the like.

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

B. the nucleotide sequence shown as SEQ ID NO. 1.

Meanwhile, the invention also discloses application of a substance for inhibiting the expression of the rice blast fungus protein MoUPE3 in reducing the pathogenicity of rice blast fungus.

The invention also discloses application of a substance for inhibiting the expression of the Magnaporthe grisea protein MoUPE3 in reducing the spore yield of Magnaporthe grisea.

The invention also discloses application of a substance for inhibiting the expression of the blasticidin MoUPE3 in reducing the stress resistance of the blasticidin.

The invention also discloses application of a substance for inhibiting the expression of the Magnaporthe grisea protein MoUPE3 in preventing and treating rice blast.

Specifically, the rice blast is caused by Pyricularia oryzae.

The invention also provides a method for preventing and controlling rice blast caused by rice blast fungus, which comprises the following steps: an agent capable of inhibiting the blasticidin MoUPE3 is applied to the aerial parts of rice.

Alternatively, the agent capable of inhibiting the mopsin moppe 3 may be an antisense RNA or siRNA of the gene encoding the mopsin moppe 3 protein.

Specifically, the agent capable of inhibiting the blasticidin MoUPE3 is antisense RNA or siRNA of a gene shown as SEQ ID NO. 1.

The invention has the following beneficial effects:

the invention provides uncharacterized protein MoUPE3 in rice blast fungus and a novel function of a coding gene MoUPE3 thereof. On the basis, the invention provides application of the protein MoUPE3 in reducing the pathogenicity, the sporulation quantity and the stress resistance of rice blast fungi, and application of substances for inhibiting the expression of the protein MoUPE3 in reducing the pathogenicity, the sporulation quantity, the stress resistance and preventing and treating rice blast fungi. The invention enriches pathogenic related genes of rice blast fungi, provides target genes for developing effective bactericides, and is beneficial to preventing and controlling rice blast.

Drawings

FIG. 1 is a schematic diagram of the construction of a MoUPE3 gene knockout vector of Pyricularia oryzae.

FIG. 2 shows the result of PCR amplification of a portion of the HPH gene of the hygromycin resistant transformant; wherein M: DL 2000Marker;1: wild type rice blast fungus; 2 to 6: hygromycin resistant transformants ΔMoUPE3-6, ΔMoUPE3-21, ΔMoUPE3-23, ΔMoUPE3-31 and ΔMoUPE3-36.

FIG. 3 shows the result of PCR amplification of a part of the gene MoUPE3 of the order hygromycin resistant transformant; wherein M: DL 2000Marker;1: wild type rice blast fungus; 2 to 6: hygromycin resistant transformants ΔMoUPE3-6, ΔMoUPE3-21, ΔMoUPE3-23, ΔMoUPE3-31 and ΔMoUPE3-36.

FIG. 4 shows the result of Southern blot analysis of a rice blast knockout positive candidate transformant using HPH fragment as a probe; wherein, 1: wild type rice blast fungus; 2-4, candidate positive transformants ΔMoUPE3-21, ΔMoUPE3-23 and ΔMoUPE3-36.

FIG. 5 shows the result of Southern blot analysis of rice blast knockout candidate positive transformants using MoUPE3 fragment as a probe; wherein, 1: wild type rice blast fungus; 2 to 4: candidate positive transformants ΔMoUPE3-21, ΔMoUPE3-23 and ΔMoUPE3-36.

FIG. 6 shows the result of PCR amplification of the MoUPE3 gene of a part of bleomycin resistant transformants; wherein M: DL 2000Marker;1 to 4: bleomycin resistant transformants ΔMoUPE3-21-com-1, ΔMoUPE3-21-com-2, ΔMoUPE3-21-com-3 and ΔMoUPE3-21-com-4.

FIG. 7 shows the results of the sporulation statistics of the Pyricularia oryzae knockout mutant ΔMoUPE3 and the back-fill mutant ΔMoUPE3-com; data analysis was performed by Duncan new complex polar difference method (P < 0.05), and differences in letters are significant, and data in figures are mean.+ -. Standard deviation.

FIG. 8 is an analysis of stress resistance of knockout mutant ΔMoUPE3 and of make-up mutant ΔMoUPE3-com.

FIG. 9 shows the pathogenicity determination of Pyricularia oryzae knockout mutant ΔMoUPE3 and anaplerotic mutant ΔMoUPE3-com on rice.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

The test strain used in the following examples was Magnaporthe grisea (Magnaporthe oryzae) ZC13, a dominant minispecies of Guangdong province; the rice tested was a susceptible indica line CO39.

The cloning vector is pMD18-T vector, the gene knockout vector is filamentous fungus expression vector pCT74, and the gene replacement vector is pCTZN (the SGFP and HPH genes on pCT74 are replaced by bleomycin resistance gene Zeocin, which is modified on the basis of pCT74 plasmid by the laboratory of the inventor).

EXAMPLE 1 construction of Magnaporthe grisea MoUPE3 Gene knockout mutant

The nucleotide sequence of the Magnaporthe grisea MoUPE3 gene knocked out in this example is shown as follows (SEQ ID NO. 1):

ACACATTCACAGTCCCAATCACTGCAACTCTTCACAATCTACGCATCAGCAAAAAAACATCTTCCTCAA CATTCACTCGCTCTTCCATATCTCACAACCAACCAACAATCAAAATGGCCACCCAAGCTTCCAAGGCATCCACCTGCTGCGGCAAGAGTGACGTCTGCGTCTGTGGTAAGTTTCAAGCCTACCTGAAAGCGTCCCACAGCCTTAAAATGACTAC TTAGGTATTCGTCACCATCAAATACTAACCCACAACCCCCACCATCAGCCACTCAAGCAACCTGCAGTTGTGGCAAGCAGTCCGCCCTTCACTGCACGTGCGACAAGGCTTCCACCGAGAACGCCGTCACCGGACCTCGGTGCTCGTGCCGTGCCCGCCCGGCCGGTGAGTGCAACTGCGACCGCGCTGCCGCCGAGAACGTCACCCCCAGCGGAAACGCCTGCGCCTGCGGTGTCAGGCCTGCCGGTATGTTTGACCTTGATTGATCCCCCACCATTAATCCGATGATCCGGCTTTGCTAACATATA ATGGTTTAGATGCCTGCACTTGTGAGAAGGGAGGAGCCTCGGGCGTCTATGACACTGCCAACGAGACCGACTTCACTACCAAGAAATAAATGAAGATTATCGGCAAGGGATTTATTTCTGGCGTTATTCTGGGTCTTTTTTCTTCTTCTTTTTT CGTGTACTCTATGACACTCAGTATACCCCATCGCATTGAGCAATGAACTATTACATATTATCATCGTGGATCCCAGA CGCTACAAGGCGAAAAAAATTAAATGCAATTATTTAACCACACTGGGGAACCTTCGACCGAAAGATAAAGATACATT TTGTT

wherein the underlined part is an intron and the rest is an exon.

The amino acid sequence of the Magnaporthe grisea protein MoUPE3 is shown in the following (SEQ ID NO. 2):

MATQASKASTCCGKSDVCVCATQATCSCGKQSALHCTCDKASTENAVTGPRCSCRARPAGECNCDRAAAENVTPSGNACACGVRPADACTCEKGGASGVYDTANETDFTTKK

in the embodiment, a blast fungus MoUPE3 gene knockout vector is constructed, the blast fungus MoUPE3 gene knockout vector is introduced into a blast fungus protoplast, and the MoUPE3 gene is knocked out from the blast fungus by utilizing a homologous recombination method to obtain a knock-out mutant delta MoUPE3. A schematic construction diagram of the Magnaporthe grisea MoUPE3 gene knockout vector is shown in figure 1.

1. Amplification of upstream and downstream homology arms of MoUPE3 Gene

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

TABLE 1 amplification primers for MoUPE3 Gene homology arm A fragment and B fragment

Primer name	Primer sequences 5'-3' (underlined as cleavage sites)	Cleavage site
			MoUPE3-AF	GACTAGTGGTCAGTCCGAAACCTCTCA	Spe I
MoUPE3-AR	GCCAATTGATAGTCGGATTGCCATAAACGTTCC	Mfe I
			MoUPE3-BF	GGGGGCCCCGCAAGCTGTCTCAGAAGT	Apa I
MoUPE3-BR	GGGGTACCTGTGCATCCAACCGTGTCAT	Kpn I

Extracting the rice blast fungus genome DNA by using a fungus DNA extraction kit (OMEGA Fungal DNA Kit); PCR amplification is carried out by using the obtained genome DNA as a template and using primers MoUPE3-AF and MoUPE3-AR to obtain a homologous arm A fragment (MoUPE 3-A) of the MoUPE3 gene; PCR amplification was performed using primers MoUPE3-BF and MoUPE3-BR to obtain the homologous arm B fragment of the MoUPE3 gene (MoUPE 3-B).

The PCR reaction system is shown in the following table:

template DNA	1μL
		MoUPE3-AF/BF(10μmol/L)	1μL
MoUPE3-AR/BR(10μmol/L)	1μL
		10×Ex Taq Buffer(Mg ²⁺ plus)	5μL
dNTP Mixture	4μL
		Ex Taq DNA Polymerase	0.25μL
ddH ₂ O	37.75μL
		Total	50μL

The PCR reaction conditions were: reacting at 94 ℃ for 5min; reacting at 98 ℃ for 10s, reacting at 55 ℃ for 30s, and reacting at 72 ℃ for 1min for 35 cycles; the reaction was carried out at 72℃for 10min. After the amplification, the PCR amplification product was recovered by using OMEGA Cycle Pure Kit kit.

2. Construction of MoUPE3 Gene knockout vector

Referring to the specifications of the pMD18-T Vector Cloning Kit (TakaRa) kit, moUPE3-A and MoUPE3-B were ligated with the T vector, respectively, to obtain recombinant plasmids pMD18T-MoUPE3-A and pMD18T-MoUPE3-B.

The method comprises the following steps: mu.L of pMD18-T vector was taken, and 4. Mu.L of the above-mentioned PCR-recovered product (homology arm A fragment or homology arm B fragment) and 5. Mu.L of solution I were added, respectively, and ligated at 16℃overnight. The ligation product was added to 100. Mu.L of E.coli DH 5. Alpha. Competent cells and left on ice for 30min; heat shock is carried out for 90s in a water bath at the temperature of 42 ℃ and the mixture is cooled for 5min on ice; adding 800 μl of LB liquid medium, and shaking culturing at 37deg.C and 150rpm for 45min; centrifuging at 4000rpm for 5min, discarding supernatant, and coating on LB solid medium (containing 50 μg/mL Amp); culturing at 37 deg.c for 8-12 hr. Positive transformants with Amp resistance are picked up, recombinant plasmid DNA is extracted and sequenced and identified.

The pMD18T-MoUPE3-B and pCT74 vectors were double digested with Apa I and Kpn I, respectively, and the B fragment and pCT74 vectors were recovered. Ligating the B fragment with pCT74 using T4DNA ligase to transform E.coli DH 5. Alpha; obtaining recombinant plasmid pCT74-MoUPE3-B. The same procedure was followed to double-cleave pMD18T-MoUPE3-A with SpeI and Mfe I, double-cleave recombinant plasmid pCT74-MoUPE3-B with SpeI and EcoRI, and recover the A fragment and recombinant plasmid. Ligating the A fragment with pCT74-MoUPE3-B by using T4DNA ligase to transform E.coli DH5 alpha; and obtaining the gene knockout vector pCT74-MoUPE3-KO through enzyme digestion identification.

3. Preparation of Pyricularia oryzae protoplast

Activating Pyricularia oryzae on a plate of a Murray medium (yeast extract 5.0g/L, anhydrous glucose 22.0g/L and agar powder 17.0 g/L), and culturing at 28deg.C for about 10 days; mycelium fragments on the medium were removed with forceps using sterilized forceps, transferred to 50mL of YPS medium (yeast extract 6.0g/L, hydrolyzed casein 6.0g/L and sucrose 10.0 g/L), and shake-cultured at 28℃for 2d at 120 rpm; filtering the above culture with 200 mesh cell sieve to obtain mycelium containing small amount of culture solution, and pouring into sterilized containerGrinding in a mortar, transferring proper amount of mycelium fragments into YPS culture medium with 200 mL/bottle, and shake culturing at 28deg.C at 120rpm for 1d; filtering mycelium with 200 mesh cell sieve, washing with sterile water for 2 times, washing with sterilized 0.8mol/L NaCl solution for 1 time, clamping mycelium into a sterilized culture dish with sterilized forceps, and covering 2-3 layers of sterilized filter paper; transferring the mycelia into a weighed EP tube, and weighing again to obtain the weight of wet mycelia; adding a proper amount of 10mg/mL of lywallzyme liquid into an EP tube, and carrying out oscillation digestion for about 1h at the temperature of 30 ℃ at 80-110 rpm according to the ratio of the enzyme liquid to hypha (volume mass ratio of 10:1); filtering and collecting the digestion solution by using sterilized dust-free paper, placing the digestion solution in a precooled EP tube, and centrifuging at 3500rpm for 10min at 4 ℃; the supernatant was discarded and the pellet was resuspended in precooled STC (1.2 mol/L sorbitol, 10mmol/L Tris-HCl,50mmol/L CaCl) ₂ pH 7.5); centrifuging at 5000rpm at 4deg.C for 10min; discarding the supernatant, re-suspending the precipitate in STC solution, temporarily preserving on ice, and diluting to a proper concentration after counting; calculating the protoplast concentration with a hemocytometer; adding proper amount of pre-cooled STC solution to control the final concentration of protoplast to 1×10 ^7-8 And each mL.

4. Transformation of Pyricularia oryzae protoplast

The knock-out vector pCT74-MoUPE3-KO was subjected to single cleavage with SpeI to obtain an A-HPH-SGFP-B fragment. Mu.g of the A-HPH-SGFP-B fragment was mixed with 200. Mu.L of protoplast; or uniformly mixing the pCTZN-MoUPE3-com fragment after single enzyme digestion with 200 mu L of the Pyricularia oryzae knockout mutant protoplast; ice bath for 20min; 1mL of PTC conversion buffer (60% PEG4000, 50mmol/L CaCl) was added dropwise ₂ 10mmol/L Tris-HCl, pH 7.5), adding while mixing, and standing at room temperature for 20min; centrifuging at 5000rpm at 4deg.C for 10min; discarding the supernatant, re-suspending the precipitate with 1mL of regenerated liquid culture medium (yeast extract 6.0g/L, hydrolyzed casein 6.0g/L and sucrose 200.0 g/L), transferring to a sterilized 50mL corning tube, adding 3mL of regenerated liquid culture medium, and carrying out shaking recovery culture at 28 ℃ for 16-18 h at 100 rpm; 4mL of the resuscitated protoplast was added to 30mL of a regenerated solid medium (1.5% agar powder in a regenerated liquid medium) cooled to about 45 ℃. In experiments in which the knockout vector transformed protoplasts, the regeneration medium contained 200. Mu.g/mL hygromycin; at the backIn the experiment of transforming protoplast by supplementing carrier, the regeneration medium contains 150 mug/mL bleomycin, the plates are evenly mixed and poured, and the solidified plates are inversely cultured for about 4 days under the darkness at 28 ℃; single colonies of hygromycin/bleomycin resistant transformants were picked for identification.

5. PCR verification analysis of Pyricularia oryzae knockout mutant

Genomic DNA of the hygromycin positive transformant was extracted and analyzed by PCR according to the instructions of the fungus DNA extraction kit (OMEGA Fungal DNA Kit). PCR amplification of the HPH gene fragment is performed by using primers HPH-F/HPH-R; PCR amplification analysis of the MoUPE3 gene fragment was performed using the primers MoUPE3-F/MoUPE3-R, the primer sequences are shown in Table 2 below.

TABLE 2 primers for PCR-validated analysis of Pyricularia oryzae knockout mutants

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

The PCR reaction system is shown in the following table:

template DNA	0.5μL
		MoUPE3-F/HPH-F(10μmol/L)	0.5μL
MoUPE3-R/HPH-R(10μmol/L)	0.5μL
		2×TSINGKE Master Mix	12.5μL
ddH ₂ O	11μL
		Total	25.0μL

The PCR reaction conditions were: reacting at 94 ℃ for 5min;94 ℃ for 30s,55 ℃ for 30s,72 ℃ for 1min, and 35 cycles are performed; and (3) reacting for 10min at 72 ℃ to obtain an amplification product.

In the embodiment, 37 candidate positive transformants are obtained by transforming the gene knockout vector into the Pyricularia oryzae protoplast by using a homologous recombination method. After DNA extraction, PCR validation analysis was performed on 37 hygromycin positive transformants using HPH gene specific primers. The results of PCR amplification of a portion of the hygromycin resistant transformant HPH gene are shown in FIG. 2; wherein M is DL 2000Marker;1 is a wild type rice blast fungus; 2-6 are hygromycin positive transformants delta MoUPE3-6, delta MoUPE3-21, delta MoUPE3-23, delta MoUPE3-31 and delta MoUPE3-36 in sequence. As can be seen from FIG. 2, the obtained hygromycin positive transformant contains the HPH gene.

In this example, the PCR verification analysis of MoUPE3 was further performed on the 5 positive transformants amplified to HPH gene by the PCR using MoUPE3 gene-specific primers, and the results are shown in FIG. 3; as can be seen from FIG. 3, none of the 5 transformants amplified the MoUPE3 gene, indicating that these 5 transformants were positive transformants.

6. Southern blot analysis of Pyricularia oryzae knockout mutant

The primer MoUPE3-F/MoUPE3-R is used for amplifying the target gene probe, and HPH-F/HPH-R is used for amplifying the HPH gene probe.

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

the PCR reaction conditions were: reacting at 94 ℃ for 5min; reacting at 98 ℃ for 10s, reacting at 55 ℃ for 30s, and reacting at 72 ℃ for 1min for 35 cycles; and (3) reacting for 10min at 72 ℃ to obtain an amplification product.

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

In this example, 3 of the above 5 positive transformants (containing HPH gene and not containing MoUPE3 target gene) were selected for Southern blot analysis. Southern blot analysis results of the rice blast knockout positive candidate transformants using the HPH fragment as a probe are shown in FIG. 4; the Southern blot analysis result of the rice blast knockout candidate positive transformant using the MoUPE3 fragment as a probe is shown in FIG. 5; wherein, 1: wild type rice blast fungus; 2 to 4: candidate positive transformants ΔMoUPE3-21, ΔMoUPE3-23 and ΔMoUPE3-36. As can be seen from the results shown in FIGS. 4 and 5, when hybridization was performed using HPH as a probe, single copy bands appeared in each of 3 transformants (FIG. 4). When hybridization was performed using the target gene MoUPE3 as a probe, none of the 3 transformants had a hybridization band (FIG. 5); the results further show that the 3 transformants are positive transformants, namely the Magnaporthe grisea MoUPE3 gene knockout mutant.

EXAMPLE 2 construction of Magnaporthe grisea MoUPE3 Gene anaplerotic mutant

The invention introduces the gene complement vector into the delta MoUPE3 protoplast by constructing the gene complement vector; the gene is complemented into the knockout mutant by a random insertion method to obtain a complemented mutant delta MoUPE3-com.

1. Amplification of MoUPE3 anaplerotic fragment

A promoter sequence with the length of 1500bp is selected at the upstream of the MoUPE3 gene, a terminator sequence with the length of 500bp is selected at the downstream of the MoUPE3 gene, and primers are designed for amplification, wherein the primer sequences are shown in Table 3.

TABLE 3 amplification primers for MoUPE3 Gene anaplerotic fragments

Extracting the rice blast fungus genome DNA by using a fungus DNA extraction kit (OMEGA Fungal DNA Kit); PCR amplification was performed using the obtained genomic DNA as a template and the primers Com-MoUPE3-F and Com-MoUPE3-R to obtain the copy piece of the MoUPE3 gene (MoUPE 3-Com).

The PCR reaction system is shown in the following table:

template DNA	1μL
		Com-MoUPE3-F(10μmol/L)	1μL
Com-MoUPE3-R(10μmol/L)	1μL
		10×Ex Taq Buffer(Mg ²⁺ plus)	5μL
dNTP Mixture	4μL
		Ex Taq DNA Polymerase	0.25μL
ddH ₂ O	37.75μL
		Total	50μL

The PCR reaction conditions were: reacting at 94 ℃ for 5min; reacting at 98 ℃ for 10s, reacting at 55 ℃ for 30s, and reacting at 72 ℃ for 4min for 35 cycles; the reaction was carried out at 72℃for 10min. After the amplification, the PCR amplification product was recovered by using OMEGA Cycle Pure Kit kit.

2. Construction of MoUPE3 Gene repair vector

The MoUPE3-com and pCTZN vectors were digested with SpeI and NotI, respectively, and the MoUPE3-com fragment and pCTZN vector were recovered. Ligating the MoUPE3-com fragment with pCTZN using T4DNA ligase to transform E.coli DH 5. Alpha; the recombinant plasmid pCTZN-MoUPE3-com was obtained. And obtaining a gene compensation vector pCTZN-MoUPE3-com through enzyme digestion identification.

3. Preparation of Pyricularia oryzae knockout mutant protoplast

The preparation method of the Pyricularia oryzae knockout mutant protoplast is the same as that of Pyricularia oryzae protoplast in example 1.

4. Transformation of Pyricularia oryzae knockout mutant protoplast

Transformation of Pyricularia oryzae knockout mutant protoplasts was as in example 1.

5. PCR validation analysis of MoUPE3 anaplerotic mutants

Genomic DNA of the obtained bleomycin positive transformant was extracted according to the instructions of the fungal DNA extraction kit method (OMEGA Fungal DNA Kit), and PCR verification analysis was performed. PCR amplification of the gene fragment MoUPE3 was performed with the primer MoUPE3-F/MoUPE 3-R.

The PCR reaction system is shown in the following table:

template DNA	0.5μL
		MoUPE3-F(10μmol/L)	0.5μL
MoUPE3-R(10μmol/L)	0.5μL
		2×TSINGKE Master Mix	12.5μL
ddH ₂ O	11μL
		Total	25.0μL

In this example, 5 bleomycin-resistant positive transformants were obtained by transforming protoplasts of the Pyricularia oryzae knockout mutant ΔMoUPE3 (ΔMoUPE3-21) with the gene-complementing vector pCTZN-MoUPE3-com by random insertion. After extracting genomic DNA of positive transformants, PCR verification analysis was performed on these positive transformants using MoUPE3 gene-specific primers, and as a result, as shown in FIG. 6, 4 positive transformants were amplified to the target gene fragment, indicating that these 4 transformants contained MoUPE3 gene, confirming that these 4 transformants were positive transformants.

Example 3 phenotypic observations of Magnaporthe grisea MoUPE3 Gene knockout mutant and anaplerotic mutant

1. Colony morphology observation and growth rate measurement

The wild type Magnaporthe grisea, the Magnaporthe grisea MoUPE3 gene knockout mutant delta MoUPE3 and the anaplerotic mutant delta MoUPE3-com are respectively inoculated on a Murray medium, cultured under the dark condition of 28 ℃, the colony morphology is observed every day, and the colony diameter is measured at the 10 d.

Colony morphology observation and growth rate measurement results show that compared with the wild type of the rice blast fungus, the colony morphology and growth rate of delta MoUPE3 are not obviously different from those of the wild type strain of the rice blast fungus.

2. Observation of conidium production

Wetting the activated rice blast wild type, knockout mutant delta MoUPE3 and anaplerotic mutant delta MoUPE3-com colony surfaces with sterile water (2-3 mL/dish) respectively, mashing rice blast hypha with a sterilized small spoon, transferring the hypha liquid to a tomato oat culture medium (raw oat 40g, boiling with double distilled water for 1h, filtering, adding 150mL tomato juice, 0.06g calcium carbonate and 2.5% -3% agar powder, fixing volume to 1L with double distilled water, adding 500 mu L hypha liquid to each dish, and uniformly coating the hypha liquid on a flat plate with a glass rod; illumination is carried out for 24 hours at the temperature of 28 ℃, and inverted culture is carried out for 10 days; transferring 5mL of sterile water to a tomato oat culture medium by a liquid transferring gun, and scraping colonies by a spoon; spore liquid was collected by filtration through a sterilized 200 mesh cell sieve or 4 layers of dust free paper, and analyzed for spore yield.

The pestilence knockout mutant delta MoUPE3 and the anaplerotic mutant delta MoUPE3-com are respectively inoculated to tomato oat culture medium for 10d, and then the spore yield analysis is carried out. The results of the spore yield statistics of the Pyricularia oryzae knockout mutant ΔMoUPE3 and the anaplerotic mutant ΔMoUPE3-com are shown in FIG. 7. As can be seen from fig. 7, the knockout mutant Δmoupe3 showed significantly reduced spore yield compared to the wild type of rice blast, while the complementation mutant Δmoupe3-com showed that the knockout gene moupe3 significantly reduced spore yield of the resulting rice blast knockout mutant.

Example 4 analysis of stress resistance of Magnaporthe grisea MoUPE3 Gene knockout mutant and anaplerotic mutant

1. Oxidative stress analysis

The wild type, knockout mutant ΔMoUPE3 (ΔMoUPE3-21 and ΔMoUPE 3-36) and the back-fill mutant ΔMoUPE3-com (ΔMoUPE 3-com-4) of Pyricularia oryzae were inoculated to a strain containing 20mmol/L H, respectively ₂ O ₂ After culturing in an incubator at 28℃for 10 days in an inverted manner, colony growth conditions of the knockout mutant ΔMoUPE3, the complementation mutant ΔMoUPE3-com and the wild strain were observed, and colony growth inhibition rates were measured.

2. Cell wall integrity analysis

The wild type, knockout mutant ΔMoUPE3 (ΔMoUPE3-21 and ΔMoUPE 3-36) and anaplerotic mutant ΔMoUPE3-com (ΔMoUPE 3-com-4) of Pyricularia oryzae were inoculated on medium containing 0.01% SDS (sodium dodecyl sulfate), 0.2g/L CR (Congo red) and 0.05g/mL CFW (fluorescent whitening agent), respectively, and after culturing in an incubator for 10 days at 28℃with inversion, colony growth conditions of the knockout mutant ΔMoUPE3, anaplerotic mutant ΔMoUPE3-com and wild strain were observed, and colony growth inhibition rates were measured.

3. High osmotic stress analysis

The wild type, knockout mutant ΔMoUPE3 (ΔMoUPE3-21 and ΔMoUPE 3-36) and complementation mutant ΔMoUPE3-com (ΔMoUPE 3-com-4) of Pyricularia oryzae were inoculated into medium containing 0.8mol/L NaCl and 0.8mol/L Sorbitol (Sorbitol), respectively, and after culturing in an incubator at 28℃for 10 days in an inverted manner, colony growth conditions of the knockout mutant ΔMoUPE3, the complementation mutant ΔMoUPE3-com and the wild type strain were observed, and colony growth inhibition rates were measured.

The results of the stress resistance analysis of the knockout mutant ΔMoUPE3 and the make-up mutant ΔMoUPE3-com are shown in FIG. 8. As can be seen from FIG. 8, the knockout mutant ΔMoUPE3 pair compared to the wild type20mmol/L H ₂ O ₂ Sensitivity enhancement of 0.2g/L CR and 0.05g/L CFW was not significantly different for 0.8mol/L NaCl, 0.8mol/L Sorbitol and 0.01% SDS.

The integrity of the cell wall plays an important role in the immune defensive response of pathogenic bacteria against host cells. CFW and CR are cell wall inhibitors, and Δmoupe3 has increased sensitivity to them, indicating that knockout of the MoUPE3 gene affects the cell wall integrity of rice blast.

EXAMPLE 5 pathogenic analysis of Magnaporthe grisea MoUPE3 Gene knockout mutant and anaplerotic mutant

Conidium solutions (with the concentration of 5 multiplied by 10) of wild type Magnaporthe grisea, knockout mutant delta MoUPE3 (delta MoUPE3-21 and delta MoUPE 3-36) and anaplerosis mutant delta MoUPE3-com (delta MoUPE 3-com-4) of Magnaporthe grisea are respectively taken ⁴ And 0.05% Tween-20) is sprayed on living rice leaves, the living rice leaves are irradiated at 28 ℃ for 12 hours or are dark for 12 hours and are moisturized, and the pathogenicity analysis is carried out after 5 days by investigating the disease condition of the rice leaves.

As shown in FIG. 9, the results of the pathogenicity analysis of the Magnaporthe grisea MoUPE3 gene knockout mutant and the anaplerotic mutant are shown, and as shown in FIG. 9, the number of lesions on rice leaves of ΔMoUPE3 is smaller than that of the Magnaporthe grisea wild type, and the pathogenicity of ΔMoUPE3-com is restored to the wild type level, which indicates that the knockout of the MoUPE3 gene leads to the decrease of the pathogenicity of the Magnaporthe grisea.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A protein MoUPE3 for regulating and controlling the pathogenicity of rice blast fungi is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

2. A gene encoding the protein MoUPE3 of claim 1, wherein the nucleotide sequence of the gene is one of the following A, B, C:

B. a nucleotide sequence shown as SEQ ID NO. 1;

3. Use of the protein MoUPE3 according to claim 1 for reducing pathogenic force of rice blast bacteria.

4. The use of the protein MoUPE3 according to claim 1 for reducing the spore yield of rice blast bacteria.

5. Use of the protein MoUPE3 according to claim 1 for reducing stress resistance of rice blast bacteria.

6. The use according to claims 3 to 5, characterized in that it is achieved by blocking or inhibiting the expression of the protein MoUPE3 in rice blast.

7. Use of a substance inhibiting expression of the blasticidin MoUPE3 protein of claim 1 for reducing pathogenic effects of blasticidin.

8. Use of a substance inhibiting expression of the blasticidin MoUPE3 protein of claim 1 for reducing spore production of blasticidin.

9. Use of a substance inhibiting expression of the blasticidin MoUPE3 of claim 1 for reducing stress resistance of a rice blast.

10. Use of a substance inhibiting expression of the mophthora oryzae protein MoUPE3 of claim 1 for controlling rice blast.