CN116425842A

CN116425842A - Application of rice blast fungus gene MoHG 1in regulation and control of growth and pathogenicity of rice blast fungus strain

Info

Publication number: CN116425842A
Application number: CN202310226296.8A
Authority: CN
Inventors: 王一; 李成云; 林艾佳; 郭力维; 浦鑫; 吴奇
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2023-03-10
Filing date: 2023-03-10
Publication date: 2023-07-14
Anticipated expiration: 2043-03-10
Also published as: CN116425842B

Abstract

The invention discloses an application of a rice blast fungus gene MoHG 1in regulating and controlling the growth and pathogenicity of a rice blast fungus strain, belonging to the field of plant genetic engineering. The invention introduces the gene knockout fusion fragment into the rice blast fungus protoplast by constructing the gene knockout fusion fragment; the gene MoHG1 is knocked out from rice blast fungus by utilizing a homologous recombination method to obtain a knocked-out mutant delta Mohg1, and a pathogenicity test shows that the deletion of the MoHG1 obviously reduces the virulence of the rice blast fungus and can not form obvious lesions on rice leaves. The pathogenicity of the knockout mutant Δmohg1 is significantly lower than that of the wild type; proved that the rice blast fungus MoHG1 gene is a pathogenic related gene of rice blast fungus; moHG1 was also demonstrated to be necessary for the formation of rice blast attachment cells, maintenance of cell wall integrity and pathogenicity. The rice blast fungus gene MoHG1 and the application thereof provided by the invention have important significance in the prevention and control aspect of rice blast.

Description

Application of rice blast fungus gene MoHG 1in regulation and control of growth and pathogenicity of rice blast fungus strain

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to application of a rice blast fungus gene MoHG 1in regulation and control of growth and pathogenicity of a rice blast fungus strain.

Background

Since Pyricularia oryzae is easy to carry out genetic analysis and has characteristics common to many plant pathogenic fungi in terms of growth and development and infection mechanisms, pyricularia oryzae is also a model organism for researching the growth and development of filamentous fungi and pathogenic mechanisms.

The infection process of the rice blast fungus to the rice mainly comprises the following steps: (1) The conidium spreads along with wind and rain and adheres to the surface of rice leaves; (2) germination of conidia to form a bud tube; (3) differentiation of the shoot tubes to form attachment cells; (4) the attachment cells differentiate to form invasion nails; (5) The invasion spike penetrates the host cells and forms invasion hyphae within the host cells, expanding between the cells. The rice blast fungus mainly infects the tissues of the overground parts of rice by the conidium, when the conidium contacts the epidermis of a host, the conidium germinates to form a germinating pipe, the germinating pipe is specifically differentiated to generate attachment cells, and the attachment cells form invasion nails which penetrate the epidermis of the host and then continuously enter the cells of the host, so that the symptoms of the host plant appear about 5-7 days. The key process of the rice blast fungus to successfully infect the rice is to form a highly specific infection structure, namely an attachment cell, wherein swelling pressure generated after the attachment cell is mature can enable infection nails to penetrate through a rice cuticles layer, so that the rice cells are successfully infected. When Pyricularia oryzae is unable to form intact attachment cells, its pathogenicity is significantly reduced.

In Pyricularia oryzae, the cell wall integrity signal pathway regulates the response of Pyricularia oryzae to cell wall stress through the mitogen-related kinase phosphorylation cascade, affecting Pyricularia oryzae mycelium growth, attachment cell formation and infection capacity. In recent years, more and more researches find that the cell wall integrity signal path is communicated with other signal paths, and the growth and development and infection capacity of the rice blast are regulated together. There is no report on the influence of the MoHG1 gene of Pyricularia oryzae on the pathogenic ability of Pyricularia oryzae in the prior art.

Disclosure of Invention

The invention aims to provide an application of a rice blast fungus gene MoHG 1in regulating and controlling the growth and pathogenicity of rice blast fungus strains.

MoHG1 (HyphaGrow 1in Magnaporthe oryzae) sequence length in Pyricularia oryzae is 694bp, coding region length is 606bp, 2 exons, 1 intron, and 201 amino acids are encoded, and the gene encoded protein is annotated as putative protein. Predicting and analyzing physicochemical properties, secondary structures, functions, subcellular localization and the like of the protein by utilizing online analysis software, and predicting that the MoHG1 protein belongs to unstable hydrophobin, has 2 transmembrane regions, and has no information related to functions; but the protein secondary structure has 8 alpha helices and 9 random coils; the MoHG1 gene function prediction result shows that the protein can be related to metal ion transmembrane transport; subcellular analysis predicts that the protein may be a protein secreted extracellularly. However, the specific biological function of the Magnaporthe grisea MoHG1 is currently unknown. Experiments show that compared with a wild type, the delta Mohg1 knockout strain has obviously reduced hypha growth, which indicates that MoHG1 regulates hypha growth, but the regulation mechanism is not clear. The Δmohg1 knockout strain is very sensitive to cell wall stress agents (CR, SDS and CFW), suggesting that Mohg1 may be involved in the regulation of the strain cell wall integrity signaling pathway. The pathogenicity of the rice is detected by using a rice blast fungus conidium rice living body spray inoculation method, and the result shows that the infection capability of delta Mohg1 knockout strain to rice is obviously weakened.

The aim of the invention is achieved by the following technical scheme:

the invention provides an application of a rice blast fungus gene MoHG 1in regulating and controlling pathogenicity of rice blast fungus. The amino acid sequence of the rice blast fungus gene MoHG1 is shown in SEQ ID NO: as shown in figure 1, the number of the components,

the nucleotide sequence of the rice blast fungus gene MoHG1 is shown as SEQ ID NO:2, and a DNA sequence shown in SEQ ID NO. 2.

Furthermore, the rice blast fungus gene MoHG1 is applied to regulating the growth and development of rice blast fungus.

Furthermore, the rice blast fungus gene MoHG1 is applied to the regulation and control of the formation of rice blast fungus attached spores.

Furthermore, the application of the rice blast fungus gene MoHG 1in maintaining the integrity of the rice blast fungus cell wall is provided.

The invention provides an application of a rice blast fungus gene MoHG 1in preventing and treating rice blast caused by rice blast fungus.

The invention provides application of a Pyricularia oryzae gene MoHG1 as a drug target for controlling plant diseases, wherein the plant diseases are rice blast caused by Pyricularia oryzae.

The invention provides an application of a rice blast fungus gene MoHG 1in preventing and controlling rice blast caused by rice blast fungus, wherein the prevention and control are realized by blocking or inhibiting the expression of the rice blast fungus gene MoHG1.

Further, the blocking or inhibiting the expression of the rice blast gene MoHG1 is by adopting antisense RNA or siRNA of the rice blast gene MoHG1. Use of an agent that blocks or inhibits expression of the gene MoHG 1in a rice blast fungus (e.g., antisense RNA or siRNA using the gene, etc.) for the preparation of a medicament for controlling plant rice blast caused by the rice blast fungus.

Compared with the prior art, the invention has the following advantages and effects:

the rice blast fungus gene MoHG1 provided by the invention for the first time codes 201 amino acids. The invention introduces the gene knockout fusion fragment into the rice blast fungus protoplast by constructing the gene knockout fusion fragment; the gene MoHG1 is knocked out from rice blast fungus by utilizing a homologous recombination method, and a knocked-out mutant delta MoHG1 is obtained. The pathogenicity test results show that the pathogenicity of the knockout mutant delta MoHG1 is significantly lower than that of the wild type. The test proves that the rice blast fungus MoHG1 gene is the pathogenic related gene of rice blast fungus.

Experiments prove that the spore yield of the delta Mohg1 knockout strain is extremely lower than that of a wild strain YN125, and the results show that MoHG1 has an influence on the spore yield of the rice blast fungus, and MoHG1 influences the colony growth speed of the rice blast fungus; the formation rate of the attachment cells of the delta Mohg1 knockout strain is extremely lower than that of a wild strain YN125, and the result shows that MoHG1 has an influence on the germination of conidia of the rice blast fungi and the formation of the attachment cells; the number of protoplasts released by the delta Mohg1 knockout strain is 27 times that of the wild type strain YN125, and the result shows that the delta Mohg1 knockout strain is sensitive to cell wall degrading enzyme, and MoHG1 regulates the integrity of the cell wall of the rice blast fungus; the rice treated by the delta Mohg1-31 knockout strain and the delta Mohg1-34 knockout strain has lighter disease incidence than the rice treated by the wild strain YN125, which proves that MoHG1 possibly participates in the pathogenic process of the rice blast fungus and is very important for obviously weakening the function of the rice infection capability. The research of the invention is helpful for deeply elucidating pathogenic molecular mechanism of rice blast fungus, and provides a target gene for developing effective bactericides.

Drawings

FIG. 1 is a schematic diagram of the construction of a Magnaporthe grisea MoHG1 gene knockout fusion fragment.

FIG. 2 is an amplification electrophoretogram of the fusion flanking fragment upstream and downstream of MoHG1, wherein M: DL2000 DNA markers; lane 1: moHG1 upstream fusion flanking fragment; lane 2: moHG1 downstream fusion flanking fragment; lane 3: negative control ddH2O.

FIG. 3 is PCR identification of ΔMohg1 knockout strain, wherein M: DL2000 DNA markers; lane 1: knockout strain delta Mohg1-31-1; lane 2: knocking out the strain delta Mohg1-31-2; lane 3: knockout strain delta Mohg1-34-1; lane 4: wild strain YN125; lane 5: ddH2O.

FIG. 4 is a colony morphology observation and strain growth rate determination for wild-type strain and ΔMohg1 knockout strain at different time points.

FIG. 5 is a colony morphology observation and strain growth rate determination of wild-type strain and ΔMohg1 knockout strain on different media (CM, MM and OA).

FIG. 6 shows the spore production assay of the Magnaporthe grisea knockout mutant.

FIG. 7 shows measurement of the formation of adherent cells of a Pyricularia oryzae knockout mutant.

FIG. 8 is a measurement of the release of protoplasts by cell wall degrading enzyme.

FIG. 9 is a graph showing a comparison of the areas of spread of lesions on leaves of rice cultivar LTH inoculated with a conidium suspension of three strains YN125, ΔMohg1-31 and ΔMohg 1-34.

FIG. 10 is a comparison of the pathogenicity detection of three strains YN125, ΔMohg1-31 and ΔMohg1-34 inoculated in the leaves of rice cultivar LTH.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.

1 Experimental materials

1.1 test strains and plants

The rice blast fungus is wild strain YN125, and the rice to be tested is Lijiang new-ball black cereal (LTH).

1.2 host bacteria and plasmid vectors

Coli strain DH 5. Alpha; yeast Saccharomyces cerevisiae strain EGY48; plasmid pCX62.

2 Experimental methods

2.1 extraction of wild Pyricularia oryzae YN125 genomic DNA

The DNA of Pyricularia oryzae YN125 genome was extracted by CTAB method.

2.2 Construction of MoHG1 knockout fusion fragment

A schematic diagram of the construction of the Magnaporthe grisea MoHG1 gene knockout fusion fragment is shown in figure 1. A sequence with a length of about 1500bp was selected upstream of the MoHG1 gene (the amino acid and nucleotide sequences are shown as SEQ ID NO:1-2 respectively), a sequence with a length of about 1000bp was selected downstream, and primers were designed (Table 1). The primer sequences are shown in SEQ ID NO: 3-14.

2.2.1 design and Synthesis of primers

TABLE 1 specificity primer for amplification of Pyricularia oryzae MoHG1 gene flanking sequence and verification of knockout transformant

Amplification of 2.2.2HPH

The plasmid pCX62 is used as a template, and PCR amplification is carried out under the action of a primer pair HYG-F/HYG-R to obtain an HPH sequence, and a reaction system and a reaction program are as follows:

the reaction system:

the reaction procedure:

the PCR product gel recovery operation was performed with reference to the instructions in the DNA gel recovery kit.

2.2.3 amplification of MoHG1 upstream fusion flanking fragments

The templates for the following operations were Pyricularia oryzae YN125 genomic DNA.

Two rounds of PCR amplification are respectively carried out by using primer pairs 3-LF1/3-LR1 and 3-LF2/HYG-M-R to obtain an upstream fusion flanking sequence of MoHG1, and a reaction system and a reaction program are as follows:

first round PCR reaction system:

second round PCR reaction system:

the reaction procedure:

the reaction products were separated on a 1% agarose gel and the MoHG1 upstream fusion flanking sequence was recovered.

The specific method is the same as the above.

2.2.4 amplification of the products recovered from MoHG1 upstream fusion flanking fragment gel

The PCR product is purified by using a primer pair 3-LF2/HYG-M-R to obtain the recovered product of the MoHG1 upstream fusion flanking fragment gel, and the reaction system and the reaction procedure are as follows:

the reaction system:

the reaction procedure:

the PCR products are purified and collected, and the specific operation steps are carried out according to instructions in a PCR product purification kit.

2.2.5 amplification of fusion flanking fragments downstream of MoHG1

Two rounds of PCR amplification are respectively carried out by using primer pairs 3-RF1/3-RR1 and HYG-M-F/3-RR2 to obtain a downstream fusion flanking sequence of MoHG1, and a reaction system and a reaction program are as follows:

first round PCR:

second round PCR:

the reaction procedure was the same as 2.2.3.

The MoHG1 downstream fusion flanking sequence was recovered from the gel.

2.2.6 amplification of the products recovered from the MoHG1 downstream fusion flanking fragment gel

The PCR product is purified by using a primer pair HYG-M-F/3-RR2 to obtain the recovered product of the MoHG1 downstream fusion flanking fragment gel, and the reaction system and the reaction procedure are as follows:

the reaction system:

the reaction procedure was the same as 2.2.4.

And (3) purifying the PCR product obtained by the method and collecting the target fragment.

2.2.7 detection of upstream and downstream fusion flanking fragments of MoHG1

The purified upstream and downstream fusion flanking fragments of MoHG1 are sent to Kunming department biotechnology Co., ltd for sequencing, and the sequencing result is analyzed by DNAMAN software, and the correct upstream and downstream fusion flanking fragments of MoHG1 are stored in a refrigerator at-20 ℃.

2.3 preparation of Pyricularia oryzae protoplast

Inoculating Pyricularia oryzae into YEG liquid culture medium (glucose 25g, yeast extract 5g, pure water to 1L), and shake culturing at 28deg.C and 120rpm for 3d; the above culture was ground with a sterile grinding bowl, 2mL was pipetted into a 100mL fresh YEG medium and cultured with shaking at 28℃and 120rpm for 2d; the mycelium liquid was filtered through a high-grade filter cloth, the mycelium was washed 3 times with sterile water, 1 time with 20% sucrose solution, and the mycelium was dried with filter paper. After adding an appropriate amount of 20mg/mL lywallzyme solution and ampicillin and streptomycin, and performing enzymolysis for 3 hours at 30 ℃ at 80rpm, the cleavage of protoplast was examined by a hemocytometer every hour. Then filtering with high-grade filter cloth, and collecting the lysate. Centrifuging at 5000rpm at 4deg.C for 10min; carefully remove the supernatant, fully resuspend the pellet with 20mLSTC, centrifuge at 5000rpm for 10min at 4 ℃; the supernatant was carefully removed and the pellet was resuspended in 200uLSTC (1.2M sorbitol, 10mM Tris-HCl, pH 7.5, 50mM CaCl) ₂ ) To give a final protoplast concentration of 1X 10 ⁷ And each mL.

2.4 transformation of Pyricularia oryzae protoplasts

Protoplasts were transformed with 60% PEG, 200. Mu.L protoplasts were incubated with 30. Mu.L of the knocked-out upstream fusion fragment and 30. Mu.L of the downstream fusion fragment in the dark at 28℃for 30min, 1.5mL of PTC solution (750. Mu.L first and 750. Mu.L later) was added, followed by 30min incubation, followed by TB3 liquid medium and overnight resuscitative on a 80r/min shaker at 28℃for 12h. The resuscitated strain is spread on TB3 solid medium containing hygromycin B for 4-5d.

2.5 PCR verification analysis of Pyricularia oryzae knockout mutant

And in the 5d after resuscitating, picking single colony which can normally grow on the culture medium containing hygromycin B TB3, transferring to a PDA culture medium containing hygromycin B for expanding culture of hyphae, extracting hyphae DNA, and carrying out PCR verification on 3-orf-F/3-orf-R, 3-LF1/HYG-M-R, HYG-M-F/3-RR1 by using a primer pair.

The reaction system:

reaction conditions:

2.6 phenotypic observations of Magnaporthe grisea knockout mutants

2.6.1 measurement of Pyricularia oryzae colony morphology observation and growth rate

Wild strain YN125 and two knockout strains DeltaMohg 1-31 and DeltaMohg 1-34 are respectively inoculated on CM, MM and OA solid culture media, and are subjected to inverted culture at 28 ℃ in the dark for 9d. The colony morphology on the 7d CM medium and each of 3, 6, and 9d media was observed and the colony diameter was measured.

2.6.2 spore-forming culture of Pyricularia oryzae and measurement of spore-forming amount

(1) YN125, deltaMohg 1-31 and DeltaMohg 1-34 mycelium blocks were inoculated onto OA, cultured in the dark at 28℃for 4d, and cultured in the light for 10d, respectively.

(2) Conidia were washed off by adding 5mL of sterile water to the plates, counted on a hemocytometer and counted for sporulation. The calculation formula of the sporulation quantity is as follows:

total spore count in 1mL conidium fluid = total spore count in large cell in hemocytometer plate x 10 ⁴ And each.

Determination of the formation Rate of Pyricularia oryzae attachment cells of 2.6.3

(1) Preparation of conidium solutions of YN125, deltaMohg 1-31 and DeltaMohg 1-34, and adjustment of the spore concentration to 1X 10 ⁵ And each mL.

(2) And (3) dripping 20 mu L of spore suspension with adjusted concentration on the hydrophobic rupture disc, observing germination conditions of the conidia at 2h,4h,6h and 8h respectively, and calculating germination rate of the conidia and formation rate of attached cells. The calculation formula of the germination rate and the formation rate of the attachment cells of the conidium is as follows:

germination rate (%) = (number of germinated conidia/(total number of conidia examined) ×100)

Attachment cell formation rate (%) = (number of conidium attachment cells/total number of conidium examined) ×100

2.6.4 cell wall integrity stress assay

Protoplast preparation referring to the calumniate method, the preparation time was 150min, and the prepared protoplasts were counted under a microscope with a hemocytometer. The calculation formula of the number of protoplasts is as follows:

total number of protoplasts in 1mL lywallzyme solution = number of large total protoplasts in hemocytometer plate x 10 ⁴ And each.

Identification of pathogenicity

The concentration of the preparation is 1 multiplied by 10 ⁵ individual/mL of different suspensions of Pyricularia oryzae spores.

(1) Spraying spores on rice plants uniformly by adopting a Spray method, bagging and moisturizing, culturing in the dark at 28 ℃ for 24 hours, moving to a greenhouse and moisturizing for 6 days, counting disease indexes, observing the disease condition of the rice plants, and grading according to the disease standard of the LTH monogenic line. The disease index is calculated as follows:

disease index=100×Σ (leaf number of each stage×representative value of each stage)/(total leaf number of investigation×representative value of highest stage)

3 results and analysis

3.1 obtaining upstream and downstream fusion flanking fragments of Pyricularia oryzae MoHG1

The first round of amplification was performed using the extracted YN125 genomic DNA as a template, using MoHG1 upstream specific primer 3-LF1/3-LR1 and downstream specific primer 3-RF1/3-RR1 (FIG. 1). After the first round of product glue obtained by amplification is recovered, the upstream fragment and the downstream fragment are respectively subjected to PCR fusion amplification with the HPH fragment by using primer pairs 3-LF2/HYG-M-R and HYG-M-F/3-RR2, and after agarose gel electrophoresis detection, the actual values of the upstream fusion flanking fragment and the downstream fusion flanking fragment of the target gene are found to be consistent with theoretical values, wherein the theoretical values are 1957bp and 1793bp (figure 2). And (3) respectively cutting glue to recover an upstream fusion flanking fragment and a downstream fusion flanking fragment of MoHG1, respectively sending the two target fragments to Kunming biotechnology Co-Ltd for sequencing, and the sequencing result shows that the HPH fragment is successfully fused with the upstream flanking fragment and the downstream flanking fragment of the target gene respectively.

3.2 screening of Pyricularia oryzae knockout mutants

3.2.1 obtaining of knockout transformants

Through the PEG-protoplast transformation method, 41 grown putative transformants are picked up on a recovery medium containing hygromycin B for 5-7d after transformation to an OA medium containing hygromycin B, and after continuous culture for 5d, DNA is extracted for knocking out strain identification.

3.2.2 molecular characterization of the knockout Strain

PCR amplification screening was performed by extracting genomic DNA of 41 putative transformants using the target gene MoHG1 specific primer 3-orf-F/3-orf-R as a template. The detection results show that the No. 31 and No. 34 transformants do not amplify the target fragment (694 bp). After the two transformants are subjected to single spore selection and separation culture, the number 31 transformant obtains 2 single spores, and the number 34 transformant obtains 1 single spore. The obtained single spore cultured DNA was used as a template, and MoHG1 specific primers 3-orf-F/3-orf-R, primers 3-LF1/HYG-M-R (MoHG 1 upstream forward primer and HPH reverse primer) and HYG-M-F/3-RR1 (HPH forward primer and MoHG1 downstream reverse primer) were used as templates, respectively, and the results showed that the target gene specific bands were not amplified in the three single spore DNA templates, but the target gene upstream, downstream and HPH gene fusion fragments (2029 bp and 1860bp, FIG. 3) were obtained, and the delta Mohg1 knockout strain was obtained. The knocked-out strain was stored in filter paper for subsequent testing.

3.3 analysis of colony morphology and growth Rate of Pyricularia oryzae knockout mutants

Two DeltaMohg 1 knockout strains, deltaMohg 1-31 and DeltaMohg 1-34, were obtained by gene knockout, and colony diameters of three strains YN125, deltaMohg 1-31 and DeltaMohg 1-34 on CM solid medium were measured at 3d,6d and 9d after inoculation. The results showed that the Δmohg1 knockout strain grew at a significantly lower rate than the wild-type strain (fig. 4). The knocked-out strain and the wild-type strain were inoculated on CM, MM and OA media, respectively, colony morphology was observed on day 7 after inoculation, and colony diameter was measured. The results showed that the diameter of the colonies of the knocked-out strain was significantly lower on the three media than that of the colonies of the wild-type strain (fig. 5). These results demonstrate that MoHG1 affects the colony growth rate of blasticidia.

3.4 spore production of Magnaporthe grisea knockout mutant

Conidia of three strains YN125, deltaMohg 1-31 and DeltaMohg 1-34 cultured under the same spore-producing condition are washed off, and the spore-producing amounts of the respective strains are calculated by counting with an optical microscope and compared. Results discovery

The average number of conidia of DeltaMohg 1-31 is (1.20.+ -. 0.20). Times.10 ⁵ ·mL ^-1 The average number of conidia of DeltaMohg 1-34 is (1.50.+ -. 0.17). Times.10 ⁵ mL ^-1 And the average conidium number of YN125 is (2.2.+ -. 0.1). Times.10 ⁵ mL ^-1 After single-factor analysis of variance, the spore yield of the two delta Mohg1 knockout strains was found to be extremely lower than that of the wild strain YN125 (FIG. 6), and the result shows that MoHG1 has an effect on the spore yield of Pyricularia oryzae.

3.5 Adhesives formation of Magnaporthe grisea knockout mutants

mu.L of the prepared suspension droplets of YN125, deltaMohg 1-31 and DeltaMohg 1-34 spores were sucked and cultured on a hydrophobic glass slide, and after 8 hours, the strain attachment cell formation condition was observed, the average attachment cell formation rate of DeltaMohg 1-31 was 18.20+ -0.01%, the average attachment cell formation rate of DeltaMohg 1-34 was about 19.71+ -0.23%, and the average attachment cell formation rate of YN125 was about 29.58+ -0.36%, and after single factor variance analysis, it was found that the attachment cell formation rate of the two DeltaMohg 1 knockout strains was extremely lower than that of the wild strain YN125 (see FIG. 7). Experimental results show that MoHG1 has an effect on the germination of conidia of Pyricularia oryzae and the formation of attachment cells.

3.6 cell wall integrity stress assay

The release of protoplasts was detected using cell wall degrading enzymes. After 150min of treatment, the experimental results show that

The average protoplast number of DeltaMohg 1-31 was about (14.43.+ -. 0.81). Times.10 ⁶ ·mL ^-1 The average protoplast number of DeltaMohg 1-34 was about (14.77.+ -. 3.20). Times.10 ⁶ ·mL ^-1 The average number of protoplasts of YN125 was about (0.53.+ -. 0.37). Times.10 ⁶ ·mL ^-1 Analysis found that the two Δmohg1 knockout strains released 27 times the number of protoplasts, respectively, than the wild-type strain YN125 (fig. 8), indicating that the two Δmohg1 knockout strains are sensitive to cell wall degrading enzymes. These results indicate that MoHG1 regulates the integrity of the cell wall of blasticidia.

3.7 DeltaMohg 1 knockout Strain pathogenicity detection

The conidium suspensions of three strains YN125, deltaMohg 1-31 and DeltaMohg 1-34 were inoculated onto the leaves of rice variety LTH, and the onset of the rice leaves was observed at 7 d. From the experimental results, it can be seen that: at 7d, the degree of susceptibility of rice leaves treated with ΔMohg1-31 and ΔMohg1-34 was significantly less than that of rice leaves treated with wild-type strain YN 125. According to the grading standard of the severity of the rice diseases, the rice leaves treated by the wild strain YN125 have 5-level lesions, the lesions are obviously expanded, and the expansion area is larger; although both ΔMohg1-31 and ΔMohg1-34 treated rice leaves exhibited disease, they exhibited disease resistance greater than YN125 strain, with stage 4 lesions at the highest on the leaves, and the spread of lesions on the treated leaves was significantly smaller than that of the wild-type strain (FIG. 9). The statistical results of the disease indexes show that the disease index of the rice leaves treated by the delta Mohg1-31 knockout strain is 65.63 +/-2.74, the disease index of the rice leaves treated by the delta Mohg1-34 knockout strain is 64.44 +/-2.95, and the disease index of the rice leaves treated by YN125 is 82.67 +/-7.57, and the disease indexes of the two delta Mohg1 knockout strains are extremely lower than that of the wild strain YN125 after single-factor analysis of variance (figure 10). Therefore, the rice treated with the delta Mohg1-31 knockout strain and the delta Mohg1-34 knockout strain is lighter than the rice treated with the wild-type strain YN125, indicating that MoHG1 may be involved in the pathogenic process of Pyricularia oryzae.

Claims

1. The application of the rice blast fungus gene MoHG 1in inhibiting the growth of rice blast fungus and reducing the pathogenicity of rice blast fungus is characterized in that: the amino acid sequence of the encoding gene MoHG1 of the rice blast fungus is shown as SEQ ID NO: 1.

2. The use according to claim 1, characterized in that: the nucleotide sequence of the rice blast fungus gene MoHG1 is shown in SEQ ID NO:2, and a DNA sequence shown in SEQ ID NO. 2.

3. The use according to claim 1, characterized in that: the application is at least one of the following (1) - (3): (1) The application of the rice blast fungus gene MoHG 1in regulating the growth and development of rice blast fungus;

(2) The application of the rice blast fungus gene MoHG 1in reducing the formation of rice blast fungus attached spores;

(3) The application of the rice blast fungus gene MoHG 1in maintaining the integrity of rice blast fungus cell walls.

4. The use of the Pyricularia oryzae gene MoHG1 according to claim 1 for controlling Pyricularia oryzae.

5. The use of the Pyricularia oryzae gene MoHG1 according to claim 4 for controlling Pyricularia oryzae, wherein: the control is realized by blocking or inhibiting the expression of the rice blast fungus gene MoHG1.

6. The use of the Pyricularia oryzae gene MoHG1 according to claim 5 for controlling Pyricularia oryzae, wherein the rice blast is caused by Pyricularia oryzae: the blocking or inhibiting the expression of the rice blast fungus gene MoHG1 adopts antisense RNA or siRNA of the rice blast fungus gene MoHG1.

7. Use of the blasticidin gene MoHG1 as a target for a drug for plant disease control as claimed in claim 1, characterized by: the plant disease is rice blast caused by rice blast fungus.