CN1982464B - Rice blast gene related with virulence and its use - Google Patents

Abstract

The present invention relates to a gene of Magnaporthe oryzae with high homology with URO-D gene of yeast, human and other biological uroporphyrinogen decarboxylase, named as MgURO-D, the gene encodes 367 amino acids, including 4 Son, 5 exons. The present invention also relates to the expression vector and host containing the gene, the application of the gene in the control of plant diseases (especially rice blast) and the application as a drug target for plant disease control.

Description

A kind of blast fungus gene related to pathogenicity and its application

technical field

The present invention relates to the field of plant disease prevention and control, more specifically, the present invention relates to a gene related to the pathogenicity of rice blast fungus (Magnaporthe grisea) with higher homology with yeast, human and other biological uroporphyrinogen decarboxylase genes , the MgURO-D gene. The present invention also relates to the application of the gene in plant disease control and as the target of medicine for plant disease control.

Background technique

Rice blast, caused by Magnaporthe grisea, is the most serious disease of cultivated rice, causing a loss of 10% to 30% of rice yield every year. In addition to rice, this filamentous fungus can also infect many other species of Poaceae, such as wheat, scorpion and other crops (Ekwamu, 1991; Igarashi et al., 1986). It invades into plant cells through a specialized infection structure, the appressorium. The appressorium is a dome-shaped, melaninized cell wall that develops from germinating conidia induced by a hydrophobic surface (Dean, 1997), which can generate enormous turgor through rapid glycerol accumulation (de Jong , 1997), and invaded the epidermis of rice leaves by virtue of the mechanical pressure generated by the turgor pressure. Infection hyphae grow rapidly in the host leaf cells, and fusiform lesions can be observed within a few days. If rice blast occurs in the seedling stage, it will seriously affect the growth and yield, and what's more, it will cause the death of the seedlings.

Magnaporthe oryzae is a filamentous fungus, the common form is haploid, and the sexual generation belongs to Ascomycota subphylum. Because it is suitable for various classical and modern molecular genetics operations, it can be used as a model species to study fungi and host plants. Interaction. The draft genome and related analysis of Magnaporthe oryzae strain 70-15 has just been reported recently. The whole genome is estimated to be about 40.3Mb, and the sequencing work has reached a coverage rate of more than 7 times, and a sequence of 38.8Mb has been obtained (Dean et al., 2005). Genome sequences are freely available on the Internet, providing researchers in the field with a great opportunity to unravel the pathogenesis of this destructive filamentous fungal pathogen and develop countermeasures.

Compared with traditional transformation methods, Agrobacterium tumefaciens-mediated transformation (ATMT) has many advantages. The method is easy to operate; whether it is through random insertion or by homologous recombination, the transformation efficiency is high; both conidia and hyphae can be used as the initial material for transformation, thus avoiding the tedious protoplast preparation process; In the obtained transformants, the probability of T-DNA single-copy insertion is high, which provides convenience for further research on the correlation between the observed transformant phenotype and the T-DNA marker gene; the insertion of T-DNA on the chromosome is almost Random; the proportion of mutations that cannot be marked by T-DNA is low; the obtained transformant phenotype has a high ratio of co-segregation with T-DNA. Based on the above advantages, the ATMT method is a very valuable transformation tool for systematic mutation research on fungi, whether through site-specific targeted substitution mutation or random insertion mutation (Caroline et al., 2005). Therefore, we adopted this approach.

URO-D (EC 4.1.1.37) is an enzyme that catalyzes the fifth step in the heme synthesis pathway. Heme can participate in many cell functions, and heme is a substance that plays an important role in oxygen-dependent life activities; many proteins related to oxygen binding, oxidative damage defense and electron transfer, etc. Heme is used as a prosthetic group; heme can also regulate gene expression through the activity of specific transcriptional regulators (Laura, 2004). Therefore, heme is a very important substance in living organisms.

In the course of evolution, the synthetic pathway of heme is very conserved (Wyckoff et al.1996). The synthesis of heme, starting from glycine and succinyl-CoA, involves a total of eight enzymatic reactions (see Accompanying drawing 1), URO-D catalyzes the fifth step reaction in the form of a dimer, and converts uroporphyrinogen III (uroporphyrinogen III) into porphyrinogen III (coproporphyrinogen III) (see accompanying drawing 2). The reaction occurs in the cytoplasm without the participation of cofactors. URO-D catalyzes the sequential removal of four carboxyl groups from the acetic acid side chain of the substrate, while forming the following intermediate products: hepta-carboxyporphyrinogen, hexa-carboxyl Hexa-carboxyporphyrinogen, and penta-carboxyporphyrinogen (Rytka et al., 1984). In humans, one of the most common porphyrias (porphyries): porphyria cutanea tarda (PCT) (Nordmann and Puy, 2002). A significant decrease in URO-D enzyme activity will lead to a large accumulation of water-soluble porphyrin in the human body, which will be activated to generate reactive oxygen species under light conditions, thereby damaging the skin. Decreased activity of this enzyme in mice results in similar symptoms, and homozygous mutations of this gene are lethal in early mouse embryos (Johm D. Phillips et al, 2001). Models for studying human PCT disease have been established in zebrafish and mice (Han Wang, 1998; Johm, Phillips et al, 2001).

The crystal structure of human URO-D has been obtained at a resolution of 1.60 determined on the basis of (see Figure 3). The protein has a molecular weight of 40.8kDa, has only one functional domain (domain), and consists of a (β/α) ₈ -barrel structure with a loop (loop) composed of the carboxy-terminal of the barrel strands (the barrel strands) deep active site cleft. Many conserved residues are clustered in this cleft: Arg37, Arg41, His339 and other residues on the side chain may be related to substrate binding; while Asp86, Tyr164, Ser219 and other residues may be related to substrate binding In addition to binding, it may also participate in catalytic reactions. In solution, URO-D exists as a dimer and tends to form crystals. The dimer assembles and juxtaposes the active site clefts of each monomer, which indicates that the interaction of the two catalytic centers plays an important role in the catalytic function of the enzyme (Frank et al, 1998).

In Saccharomyces cerevisiae, URO-D is encoded by the gene HEM12, which was independently cloned by complementing allelic hem12 and hem6 mutant strains, respectively (Garey et al.1992; Diflumieri et al. .1993). Two haploid mutant strains of Saccharomyces cerevisiae gene HEM12: hem6-1B (point mutation) and BY4742Δhem12-1A (deletion mutation), cannot grow on the medium without heme (hemin), and the colonies form red fluorescence, And for methionine auxotrophy (methionineauxotrophy). KlHEM12, the gene encoding URO-D in another yeast, Kluyveromyceslactis, has also been cloned. This gene can complement two mutants of Saccharomyces cerevisiae: hem6-1B and BY4742Δhem12-1A, and restore their phenotype. The secondary structure prediction shows that the protein conforms to the (β/α) 8-barrel structure reported by human URO-D (Frank G. et al, 1998); Kluyveromyces haploid strain with gene KlHEM12 mutated, in Growth is slow on the medium without adding hemin (hemin), and the colonies form red fluorescence due to the accumulation of porphyrin. Studies have shown that the transcription of this gene is not inhibited by heme or glucose, but slightly induced by non-fermentable carbon sources (Laura N. et al, 2004).

In maize, the heterozygous mutant Les22 of the URO-D gene exhibits a lesion-like phenotype (lesion mimic), with tiny, grayish-white or brown, dot-like necrosis spots appearing on the leaves, and light can promote necrosis. form. The mutation of the gene encoding URO-D leads to the decrease of URO-D activity, the accumulation of its precursor uroporphyrinogen III, and the energy or electrons are transferred to molecular oxygen by light, which becomes active oxygen, thus causing damage to leaf cells ( Hu et al., 1998).

Contents of the invention

The present invention selects a mutant-M230 with significantly reduced pathogenicity from the established population of M. decarboxylases, URO-D) upstream of the start codon 180bp; the gene contains an open reading frame of 1104bp, encoding 367 amino acids, named MgURO-D, containing 4 introns, 5 exons, followed by The URO-D gene comparison of yeast, human and other organisms shows high homology; a fungal expression vector pCMgURO-D for complementation was constructed, which can restore the pathogenicity of M230, which shows that MgURO -D was unexpectedly strongly associated with the virulence of Magnaporthe grisea.

Therefore, an object of the present invention is to provide a kind of gene that is from blast fungus (Magnaporthe grisea) and yeast, human beings etc. biological uroporphyrinogen decarboxylase gene homology is higher, namely MgURO-D gene, its nucleoside The acid sequence is shown in SEQ ID No.1.

The present invention also relates to the amino acid sequence encoded by SEQ ID No.1, as shown in SEQ ID No.2.

The present invention also relates to expression vectors containing the above-mentioned genes. In a preferred embodiment, the expression vector containing the MgURO-D gene is pCMgURO-D. According to the Budapest Treaty, the Escherichia coli containing the expression vector pCMgURO-D has been deposited on November 29, 2005 in the General Microorganism Collection Center (CGMCC) of China Microbiological Culture Collection Management Committee (CGMCC) in Zhongguancun, Beijing, China, and the preservation number is CGMCC No.1548.

The present invention also relates to microbial hosts containing the above expression vectors. The host can be Escherichia coli or Agrobacterium.

Another object of the present invention is to provide the application of the above gene in the control of plant diseases. The plant disease is especially rice blast caused by Magnaporthe oryzae.

Another object of the present invention is to provide the use of said gene as a drug target for plant disease control. The plant disease is especially rice blast caused by Magnaporthe oryzae.

The present invention also provides a method for treating plant rice blast caused by Magnaporthe oryzae, comprising blocking or inhibiting the expression of MgURO-D gene in Magnaporthe oryzae (for example, using antisense RNA and siRNA of the gene, etc.).

In another aspect of the present invention, there is provided a medicament for blocking or inhibiting the expression of the above-mentioned gene (such as the antisense RNA and siRNA of the gene, etc.) in the preparation of medicines, and the medicine is used to control the disease caused by Magnaporthe oryzae. Plant blast.

This study shows that URO-D is crucial to the pathogenicity of Magnaporthe grisea. When the expression of MgURO-D gene is inhibited due to the insertion of T-DNA into the promoter region, the pathogenicity of the mutant strains is significantly reduced. Lesions are difficult to expand on rice leaves, but after the expression vector pCMgURO-D containing this gene is transferred, the pathogenicity can be restored. It can be seen that after the mutant strain invades rice leaves, it cannot effectively resist the host defense. Inhibition of the growth of infected hyphae.

Description of drawings

Figure 1 shows the synthetic pathway of heme in yeast (http://db.yeastgenome.org/cgi-bin);

Figure 2 shows the decarboxylation reaction catalyzed by URO-D (John D. et al, 2001);

Figure 3 shows the three-dimensional structure of human URO-D dimer (Frank G. et al, 1998);

Figure 4 shows the insertion copy number detection of M230 mutant strain T-DNA (Southern blot analysis, the selected restriction endonucleases are respectively: A.EcoR I; B.Pst I);

Figure 5 shows the insertion site of T-DNA in mutant strain M230;

Figure 6 shows the MgURO-D gene structure (the dark black is the exon, and the gray is the intron);

表示数据源于人体，而空心三角

则表示数据来源于酵母)；Figure 7 shows the amino acid sequence alignment of different biological URO-D proteins (triangular symbols indicate residues closely related to binding substrates or performing catalytic functions, solid triangles

Indicates that the data comes from the human body, while the hollow triangle

means that the data comes from yeast);

Figure 8 shows the URO-D protein phylogenetic tree;

Figure 9 shows the sequence used to construct the expression vector pCMgURO-D;

Figure 10 shows the pCMgURO-D fungal expression vector map;

Figure 11 shows the comparison of the pathogenicity of different strains (the rice blast fungus strains inoculated with the rice variety Nipponbare are, from left to right: water, M230, and the M230 strain transformed into the expression vector pMgURO-D, Y34).

Figure 12 shows the flanking sequence of the T-DNA insertion site in the mutant strain M230 (note: the italic part is the right border sequence of the T-DNA, and the underlined part is the genomic sequence of the mutant strain M230).

Figure 13 shows the comparison of the growth status of mutant strains M230 and Y34 in V8 liquid medium.

Detailed ways

Hereinafter, the present invention will be described in detail with reference to Examples, which are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The scope of the invention is specifically defined by the appended claims.

Example

1. Obtaining of T-DNA insertion mutants of MgURO-D gene:

This mutant was obtained by the Agrobacterium tumefaciens-mediated transformation method (ATMT) (Caroline et al., 2005).

1.1 Cultivation of Agrobacterium

Pick a single colony of Agrobacterium tumefaciens strain Agl-1 (Mullins, 2001) containing the plasmid pBHt2 (Mullins, 2001), minimal medium (MM; Hooykaas et al., 1979), 50ug/ml kanamycin, 10ug /ml rifampicin, 250rpm, shake culture at 28°C for 48h; centrifuge at 4000rpm for 5 minutes, discard the supernatant, resuspend in induction medium (IM; Bundock et al., 1995), centrifuge at 4000rpm for 5 minutes, discard the supernatant; IM (containing 300 uM acetosyringone) medium resuspended, 28 ° C, 250 rpm shaking shake bacteria 6h.

1.2 Acquisition of Magnaporthe grisea spores

In this study, the pathogenic strain-Y34 (provided by China Agricultural University) isolated from the field of Yunnan, China was selected as the research object; the mycelium was inoculated into oat medium (OMA; oat 50g) containing 50ug/ml tetracycline and 100ug/ml streptomycin /L) on; 28 ° C dark culture for 5 to 7 days, when the bacterial colony is basically covered with the petri dish, use a triangular scraper to scrape off the surface hyphae, and continue to cultivate for 3 days under continuous light, and the gray spore layer can be seen; The IM liquid medium was scraped, and the spores were collected by Miracloth (CALBIOCHEM ^R , USA) filtration, observed under a microscope, and the spore concentration was adjusted to 1×10 ⁶ /ml by a hemocytometer.

1.3 Co-cultivation of Agrobacterium and Magnaporthe oryzae spores and screening of transformants

Mix equal volumes of Agrobacterium bacterium liquid and Magnaporthe grisea spore liquid induced in IM liquid medium for 6 hours in advance, add AS to make the final concentration reach 500uM, mix well, and then apply 250-350ul/dish evenly on the floor On the IM medium with a microporous filter membrane, culture in the dark at 28°C for 48 hours; after the co-cultivation, scrape the surface of the filter membrane with sterile water to collect transformed spores, add about 2-3ml of water to each dish, and spread evenly to 3-6 On an oat medium (containing 200ug/ml hygromycin, 50ug/ml tetracycline, and 100ug/ml streptomycin), hygromycin was used to screen transformants, and other antibiotics were used to inhibit Agrobacterium. After 3 to 5 days, the expanded colonies were picked and placed on the oat medium containing the same antibiotic.

1.4 Purification and storage of transformants

The transformants obtained by the above method were cultured under continuous light for 7-10 days to produce spores; the spore liquid was dipped with an inoculation needle and streaked on the oat medium. Mycelium oat culture medium, observe the growth after three days, and add sterilized dry filter paper, collect the filter paper with spores and hyphae after 7 to 10 days, put them into a sterilized paper bag, After drying at 37°C for 3-5 days, store in a desiccator for later use.

1.5 Inoculate susceptible rice leaves and detect the pathogenic phenotype of the obtained transformants

The inoculation experiment was carried out in the mesh room. The rice variety Nipponbare (provided by the Institute of Genetics and Development, Chinese Academy of Sciences) susceptible to Magnaporthe oryzae strain Y34 was selected, and inoculated at the two-leaf to three-leaf stage, and it grew for about 14 to 16 days from the time of sowing. In order to obtain the spore liquid for inoculation, inoculate the purified transformants on the oat medium, cultivate in the dark at 28°C for 6 to 8 days, scrape off the surface hyphae with a triangular scraper, light continuously for three days, and sterile water (containing 0.3% Tween-20) to collect spores, filter through Miracloth, examine under a microscope, and use a hemocytometer to adjust the spore concentration to 0.5×10 ⁶ /ml. The inoculation adopts the spray method, and the above-mentioned spore liquid is evenly and finely sprayed on the leaves with a vacuum pump or a perfume bottle. A positive control (Y34 wild type) and a negative control (sterile water containing Tuwen-20) are set for each inoculation. After inoculation, put it in a dark place, keep warm (25-30°C), keep moist (more than 90% relative humidity) for 24 hours, then take it out, put it in a pool, keep warm (18-32°C), keep moist (60-90%). The onset was recorded and analyzed after one week. According to the above method, a mutant with significantly weakened pathogenicity was obtained, code-named M230. After the leaves were inoculated with this strain, there were few lesion spots, and they stopped expanding in the early stage of disease, so the lesion spots were very small. M230 grew normally on solid media such as oat medium, complete medium (CM), and V8 tomato juice (Camplell soup company, the United States. Dilute the centrifuged supernatant with ultrapure water at 4:1), but in V8 tomato juice Shake culture in juice liquid medium, growth amount then significantly declines (see accompanying drawing 13), this shows that this mutant strain reduces to some extent the utilization efficiency of oxygen; Compared with Y34, there is no difference in colony form, conidia form and quantity , there were no significant differences in sporulation formation rate, status, size, melanin precipitation, and turgor pressure.

2. Analysis of T-DNA insertion copy number and insertion site in the pathogenic mutant M230 genome

2.1 Extraction of Genomic DNA of M230 Strain

Inoculate the mycelium of the blast fungus transformant on the oat medium, culture at 28°C for 4-7 days, use V8 tomato juice liquid medium to collect the mycelium, put it in a 50ml centrifuge tube, and set the volume to 15-20ml above V8 medium (containing 75ug/ml hygromycin, 50ug/ml tetracycline, and 100ug/ml streptomycin) was cultured at 28°C with shaking at 250rpm for 2 to 4 days. Then, vacuum-filter the mycelium with Miracloth, rinse twice with sterilized deionized water, suck off the residual liquid of the mycelium, put it in a mortar, add liquid nitrogen and grind it into powder, and the genomic DNA is extracted with reference to the method reported in the literature (Wu Zhihong , 2001). 1% agarose gel electrophoresis was used to detect the quality of DNA extraction, and quantification was carried out according to the brightness comparison with the standard lambda DNA.

2.2 Southern blot analysis to detect the copy number of T-DNA insertion in the mutant

Pipette 1ug of genomic DNA into two tubes, add 10 units of PstI and EcoRI restriction enzymes and 5ul 10X buffer respectively, add sterile redistilled water to a total volume of 50ul, digest at 37°C overnight, at 1% DNA was separated by electrophoresis at 40V on an agarose gel. The transmembrane transfer of DNA, primer labeling and hybridization reactions were all referred to "Molecular Cloning" (Sambrook et al., 1989). A part of the hygromycin resistance gene in T-DNA was selected as a probe, and HPTa and HPTb were used as primers (sequence See Table 1), using pBHt2 plasmid DNA as a template, obtained by PCR amplification reaction, the fragment length is about 800bp, and labeled with α- ³² P-dCTP and Ready To Go DNA Labeling Kit (Promega, USA). Southern blot analysis showed that the pathogenic mutant M230 strain was a single-copy T-DNA insertion (see Figure 4).

Table 1 Primers used in common PCR

2.3 Analysis of the insertion site of T-DNA in the mutant strain M230 genome:

Insertion sites were analyzed using thermal heterogeneous interleaved polymerase chain reaction (TAIL-PCR) combined with sequencing. The primers (see Table 2) and the amplification process required for the TAIL-PCR reaction were carried out according to the method described by EDMulling (2001) et al. The amplified product in the third step was subjected to agarose gel electrophoresis, and the amplified band was recovered with a gel extraction kit (Shenergy Gaming Company, Shanghai), and connected to the pGEM-Teasy vector, and then sequenced (Boya Company, Beijing) ) (see Figure 12 for the sequence). The obtained sequence was compared to the Magnaporthe grisea genome sequencing website ( www.broad.mit.edu/annotation/fungi/magnaporthe/index.html ), and the analysis results showed that in the M230 mutant strain, the T-DNA was inserted in the Annotate the upstream promoter region of gene MG01622.4, which is about 180 bp away from the start codon (see Figure 5). The annotation shows that the gene has URO-D functional domain (Domain), which is -URO-D gene, so it is named MgURO-D.

Table 2 Primers required for TAIL-PCR reaction

3. Correlation analysis of MgURO-D gene:

The gene is a single copy (according to the analysis of the sequencing website), located on chromosome I, with a full length of 1497bp (including introns). Compared with the EST sequence of Magnaporthe oryzae stored on the NCBI website, it is found that several EST sequences are mutually Cohesion can cover the entire coding region, and the distribution of introns and exons is shown in Figure 6. The amino acid sequence of the gene was taken to the NCBI website for Blast p, and it was found that compared with other organisms, especially other fungi, the gene has a high homology, as shown in Table 3. Putting together the URO-D amino acid sequences of various organisms and performing multiple sequence comparisons, it was found that the amino acid residues at the catalytic active site are all conserved, as shown in Figure 7. The phylogenetic tree of URO-D is shown in Figure 8.

Table 3 Homology comparison between MgURO-D protein and URO-D protein from other biological sources

Immediately after the reaction catalyzed by URO-D, coproporphyrinogenoxidase catalyzes the sixth step reaction of the heme synthesis pathway. A comparative analysis of the EST library (website: http://cogeme.ex.ac.uk/transcript.html) established under different conditions found that the mycelia grown on rice cell wall as the only carbon source medium were used as materials In the constructed EST library, the EST number of the gene encoding porphyrinogen oxidase was significantly higher than the EST number of the gene in the EST library of seven other materials (for example, the hyphae grown on basic or complete medium were EST library established by materials, etc.). It can be seen that the heme synthesis pathway is very active in the blast fungus that invades rice leaves, and this pathway is crucial for the survival and expansion of the blast fungus in the host.

4. Complementation experiment of mutant M230

4.1 Construction of MgURO-D gene fungal expression vector

The plasmid used to construct the expression vector was pSULFgfp (Ane Sesma, 2004), and the MgURO-D gene fragment was amplified by PCR (see Table 1 for primers). In addition to including the entire coding region, the amplified product also includes 1.1 Kb upstream of the start codon and 0.5 Kb downstream of the stop codon, a total of 3.2 Kb. The sequence is shown in Figure 9. There are XbaI and EcoRI restriction enzyme sites (indicated in italics and underlined) at the 5' ends of primers MgURO-DF and MgURO-DR, respectively. Oligonucleotide primers were synthesized by Shanghai Boya Biological Co., Ltd., using Y34 genomic DNA as a template, in the presence of 1x amplification buffer, 0.2uM/L amplification primers, 0.2mM/L dNTPS, and 0.5 units of high-fidelity DNA polymerase (Stratagene company) 50uL amplification system for PCR amplification reaction. Amplification conditions are pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 50 seconds, annealing at 60°C for 50 seconds, extension at 72°C for 3 minutes and 30 seconds, 30 cycles, and extension at 72°C for 8 minutes, and the amplification product is run on 1% agarose Gel electrophoresis, strip gel recovery as described above, ligated with pGEM-T easy carrier overnight at 16°C, took 2ul ligation products and transformed E. Pick white colony on LB culture medium (containing 100ug/ml ampicillin) solid culture medium, through plasmid extraction, restriction endonuclease analysis, after confirming that the connection is successful, this transformant is handed over to biological company (Sanbo polygala, Beijing ) sequencing, SEQ ID No.1. Digest the plasmid of the transformant with XbaI and EcoRI restriction endonuclease, and connect with the large fragment of the pSULFgfp binary vector digested with XbaI and EcoRI restriction endonuclease, and transform Escherichia coli DH10B competent cells by electroporation. Colonies were picked on LB solid medium with 50ug/ml kanamycin, extracted from the plasmid, and determined to be transformants after enzyme digestion analysis, named pCMgURO-D, and the map is shown in Figure 10. The vector is transformed into A. tumefaciens (A. Tumefaciens) Agl-1 strain by electroporation, screened by kanamycin, and the plasmid analysis proves that its structure is completely correct, and then it can be used for transformation of Magnaporthe grisea.

4.2 Complementary transformation of mutant strain M230

The method of transforming Magnaporthe grisea mutant M230 strain with the Agrobacterium AGL-1 strain containing the vector pCMgURO-D is as described above, the difference is that the selection medium after co-cultivation uses DCM (Sweigard), and the antibiotic is replaced by herbicide Chlorimuron-methyl (100ug/ml) (Dedi Agrochemical Products Marketing Company, Beijing), after the transformant grows, it is transferred to the medium containing the same herbicide and screened again, and then transferred to oat culture (200ug/ml hygromycin; 100ug/ml streptomycin, 50ug/ml tetracycline), sporulation was produced under continuous light, and after purification by streaking, they were collected by filter paper and stored dry for later use.

4.3 PCR detection of transformants

Get 2～4ng of rice blast fungus transformant genomic DNA as amplification template, in containing 1X amplification buffer, 0.2uM/L amplification primers MgURO-DF2 and MgURO-DR2 (in MgURO-D gene interior, respectively located in T- On both sides of the DNA insertion site, the sequence is shown in Table 1), 0.2mM/LdNTPs, 0.5 unit TaqDNA polymerase 20ul amplification system for PCR amplification reaction. The amplification conditions were: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 50 seconds, annealing at 60°C for 50 seconds, extension at 72°C for 5 minutes, and 30 cycles. Take 6ul of the amplification reaction mixture and run it on a 1% agarose gel. There are two expected amplified fragments, 4Kb and 1.7Kb in size, respectively. Electrophoresis results show that nearly 50% (10/21) of the transformants are in line with expectations, that is, both bands are amplified, and the rest only have 4Kb bands. Since the herbicide chlorimuron-methyl-resistant gene ILV1 was cloned from Magnaporthe grisea, homologous recombination may occur after pCMgURO-D enters Magnaporthe grisea, although it can still resist chlorimuron-methyl herbicide, it is used for The complementary gene fragment is lost, therefore, only the 4Kb gene fragment containing T-DNA can be amplified.

4.4 Inoculation of susceptible rice leaves, detection of pathogenicity

From the ten transformants whose PCR results obtained above were in line with expectations, 5 were randomly selected, cultured, sporulated, and used for pathogenicity detection, and the method was the same as above. The results showed that the pathogenicity of the five transformants was similar to that of the wild-type Y34 (see Figure 11), indicating that the complementation was successful. It can be concluded that the reason why the pathogenicity of M230 is significantly reduced is that T-DNA is inserted in the upstream promoter region of MgURO-D gene, which affects its expression.

It can be seen from this example that the inactivation of the gene claimed in the present invention directly leads to the decrease of the pathogenicity of Magnaporthe oryzae. Therefore, the gene claimed in the present invention can be used for plant disease control, especially rice blast caused by blast fungus. In addition, the genes claimed in the present invention can be used as targets for the development of drugs for plant disease control. Those skilled in the art can develop medicines for preventing and treating plant diseases, especially rice blast, according to the teaching and inspiration of this manual.

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sequence listing

<110>Institute of Microbiology, Chinese Academy of Sciences

<120> A Magnaporthe grisea gene related to pathogenicity and its application

<130>IB056407

<160>2

<170>PatentIn version 3.1

<210>1

<211>1104

<212>DNA

<213>Magnaporthe grisea

<400>1

atgggtcata ctttcgagcc actcaaaaac gacctggtta tcaggactgc ctggggccaa 60

aaagtcgaga gaccaccaat atggatcatg agacaggctg gccggtactt gcccgagtat 120

catgaggcca agggcaacag agactttttc gagtgctgtc gcgacccaga ggtggcctcc 180

accctgactc tacagcccat cgaccgcttc gatggcctgc ttgacgcctc catcatcttc 240

tccgacattc ttgtcatccc ccaggccatg ggcatggtcg ttgagatggt cgacaagaag 300

ggcccgcact tcccaaaccc ccttcagtcg cccgacgatg gccagtacgc cgaggtgctc 360

gcgcgggatg tggacgtctc caaggagctg gactacgtct acaaggccat caccctgacc 420

cgtaagaagc ttcagggccg agtgccgctc attggcttct gcggtgctcc ctggaccctg 480

ttctgctaca tggtcgaggg tggtggcacc aagctcttca tccagagcaa gaaatggata 540

ttcaggcatc ccgaggagag caagaagatg ctgcaaaaga tcgccgagct ctgcgtcgag 600

tatctcgccc tgcaggttca ggccggagca cagcttgtcc aagtctttga ctcttgggca 660

ggagagctct ctcctgcatc cttcgagcag ttttctgctc cttacctgcg ctatatttcg 720

gaaaacctcc cgaaaaggct caaggagctg agtctggatc ccgtcccaat gattgttttc 780

cccaagggtg cctggttcgc cctggaggat tgctgcgcca tgggttacaa cgtcgttggg 840

ctcgattggc tctacagtcc tgccaaggcc aaccaggtcc gaggagatcg ggaaatcgtc 900

ctgcagggca acgccgaccc tggagtgctc tacggcacta aggatgccat tacagctgct 960

gtcaaggaca tggttgaagg atttgactgg aaggagaaga agaagggctg gatagtaaac 1020

ctgggccacg gaattacacc gctcgtcaac ccggacgacc tgaagtttta tctgcaggag 1080

atccacagac aaaccaaaga ctag 1104

<210>2

<211>367

<212>PRT

<213>Magnaporthe grisea

<400>2

Met Gly His Thr Phe Glu Pro Leu Lys Asn Asp Leu Val Ile Arg Thr

1 5 10 15

Ala Trp Gly Gln Lys Val Glu Arg Pro Pro Ile Trp Ile Met Arg Gln

20 25 30

Ala Gly Arg Tyr Leu Pro Glu Tyr His Glu Ala Lys Gly Asn Arg Asp

35 40 45

Phe Phe Glu Cys Cys Arg Asp Pro Glu Val Ala Ser Thr Leu Thr Leu

50 55 60

Gln Pro Ile Asp Arg Phe Asp Gly Leu Leu Asp Ala Ser Ile Ile Phe

65 70 75 80

Ser Asp Ile Leu Val Ile Pro Gln Ala Met Gly Met Val Val Glu Met

85 90 95

Val Asp Lys Lys Gly Pro His Phe Pro Asn Pro Leu Gln Ser Pro Asp

100 105 110

Asp Gly Gln Tyr Ala Glu Val Leu Ala Arg Asp Val Asp Val Ser Lys

115 120 125

Glu Leu Asp Tyr Val Tyr Lys Ala Ile Thr Leu Thr Arg Lys Lys Leu

130 135 140

Gln Gly Arg Val Pro Leu Ile Gly Phe Cys Gly Ala Pro Trp Thr Leu

145 150 155 160

Phe Cys Tyr Met Val Glu Gly Gly Gly Thr Lys Leu Phe Ile Gln Ser

165 170 175

Lys Lys Trp Ile Phe Arg His Pro Glu Glu Ser Lys Lys Met Leu Gln

180 185 190

Lys Ile Ala Glu Leu Cys Val Glu Tyr Leu Ala Leu Gln Val Gln Ala

195 200 205

Gly Ala Gln Leu Val Gln Val Phe Asp Ser Trp Ala Gly Glu Leu Ser

210 215 220

Pro Ala Ser Phe Glu Gln Phe Ser Ala Pro Tyr Leu Arg Tyr Ile Ser

225 230 235 240

Glu Asn Leu Pro Lys Arg Leu Lys Glu Leu Ser Leu Asp Pro Val Pro

245 250 255

Met Ile Val Phe Pro Lys Gly Ala Trp Phe Ala Leu Glu Asp Cys Cys

260 265 270

Ala Met Gly Tyr Asn Val Val Gly Leu Asp Trp Leu Tyr Ser Pro Ala

275 280 285

Lys Ala Asn Gln Val Arg Gly Asp Arg Glu Ile Val Leu Gln Gly Asn

290 295 300

Ala Asp Pro Gly Val Leu Tyr Gly Thr Lys Asp Ala Ile Thr Ala Ala

305 310 315 320

Val Lys Asp Met Val Glu Gly Phe Asp Trp Lys Glu Lys Lys Lys Gly

325 330 335

Trp Ile Val Asn Leu Gly His Gly Ile Thr Pro Leu Val Asn Pro Asp

340 345 350

Asp Leu Lys Phe Tyr Leu Gln Glu Ile His Arg Gln Thr Lys Asp

355 360 365

Claims (9)

1. A gene from blast fungus (Magnaporthe grisea), its nucleotide sequence is as shown in SEQ ID No.1. 2. The amino acid sequence encoded by SEQ ID No.1, as shown in SEQ ID No.2. 3. An expression vector containing the gene according to claim 1. 4. The expression vector according to claim 3, which is pCMgURO-D. 5. A microbial host comprising an expression vector according to claim 3 or 4. 6. The application of the gene according to claim 1 in the control of rice blast caused by Magnaporthe oryzae. 7. Use of the gene as claimed in claim 1 as a target of a drug for controlling a plant disease which is rice blast caused by Magnaporthe oryzae. 8. A method for treating plant blast disease caused by Magnaporthe oryzae, comprising blocking or inhibiting the expression of MgURO-D gene in Magnaporthe oryzae. 9. the application of the medicament of blocking or inhibiting the expression of the gene described in claim 1 in the preparation of medicine, the medicament is the antisense RNA or siRNA of the gene described in claim 1, and the medicine is used to control the cause plant blast.

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