CN110305879B

CN110305879B - Maize small leaf spot pathogen ChCDC3 gene and application thereof

Info

Publication number: CN110305879B
Application number: CN201910618994.6A
Authority: CN
Inventors: 张祥辉; 孙玥; 李乐; 王璐; 刘金亮; 潘洪玉
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2019-07-10
Filing date: 2019-07-10
Publication date: 2022-03-29
Anticipated expiration: 2039-07-10
Also published as: CN110305879A

Abstract

The invention provides a corn microsporum ChCDC3 gene and application thereof, belonging to the technical field of microbial genetic engineering, wherein the DNA sequence of the ChCDC3 gene for controlling the colony growth rate, conidiospore formation and ascospore formation from the corn microsporum is shown as SEQ ID No. 1; the amino acid sequence of the protein coded by the ChCDC3 gene is shown as SEQ ID No. 2; the ChCDC3 gene can be applied to the field of plant anti-corn southern leaf blight genetic engineering; the protein ChCDC3 for controlling the colony growth rate and the formation of conidiospores and ascospores of corn microsporum is deleted, mutated or modified, so that the corn microsporum grows slowly, the formation of the conidiospores and the ascospores is limited, the life cycle of the corn microsporum is hindered, and the protein ChCDC3 can be used as a target to be applied to designing and screening of anti-corn microsporum agents, particularly, the protein does not exist in plants, and the protein is safe to the plants.

Description

Maize small leaf spot pathogen ChCDC3 gene and application thereof

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to discovery of a novel gene for controlling pathogenicity of fungi in the field of plant protection and application of a protein coded by the novel gene.

Background

The asexual morphology of the corn microsporum is named as: bipolaris maydis belongs to the genus Deuteromycotina flush, has a morphological name of Cochliobolus heterostrophus, and belongs to Ascomycotina heterospirospora. In 1925, it was first discovered as a pathogen for corn disease. The corn microsporum belongs to filamentous fungi, and when hyphae grow to a certain stage, conidia grow on the top or the side of a conidiophore under the influence of external environmental conditions and self factors. Conidia have 3-13 septa, most of them have 7-9 septa, black brown, oblong shape, blunt two ends, multi-directional side bending, thick middle, thin two sides, size of 30-115 μm × 10-17 μm, and umbilicus point is sunken in basal cell. Conidia germinate to grow germ tubes from cells at two ends, and sometimes middle cells can germinate to grow the germ tubes. The conidium forming temperature range is 15-33 ℃, and the optimal temperature range is 23-25 ℃; the temperature range of conidium germination is 5-42 ℃, and the optimal temperature range is 26-32 ℃. Conidia have a relatively strong resistance to desiccation and can survive for at least one year on corn seeds. The conidium can germinate to grow a germ tube under the humid condition, and hyphae are formed through the top growth.

Under natural conditions, hyphae grow to a certain stage and can enter a sexual reproduction stage under the condition of an external environment, the sexual reproduction stage of the corn microsporum is heterozygosis, a single site is related to sexual reproduction on a genome and is named as MAT-1, and the site has two different forms, namely MAT-1-1 and MAT-1-2. The sexual reproductive stage of maize lentigo is not common and occasionally ascospores are found in deadly diseased tissues. The production of an ascochyta may be induced artificially in laboratory conditions, the ascochyta of maize microsporum being referred to as "pseudothecium". The time from formation to maturation of the ascocarp shell is about one month, and the mature ascocarp shell is ruptured when it is in water to release ascospores and ascospores. The ascocarp shell is black and spherical, the coracoid part is obvious, the ascocarp shell is embedded in host diseased tissue, and mycelium and conidiophores can grow on the surface; the inside of the capsule is provided with a sub-capsule which is approximately cylindrical. The top end of the sub-sac is blunt and round, and the base part is provided with a handle. The mature ascospores are subjected to meiosis and mitosis to form 8 linear haploid ascospores, the ascospores are wound into a spiral shape in the ascospores, and each cell can grow into a germ tube and further grow into hyphae during germination. It was found that 80% of the mature oocysts had 8 intact ascospores and 20% had 4-7 ascospores. The infection and the prevalence of the corn microsporum in the field mainly depend on the propagation of conidia along with airflow and rainwater, if the formation of the conidia can be controlled, the infection source is greatly reduced, the occurrence of the corn microsporum is reduced, and the corn yield is improved.

Southern leaf blight is a major corn leaf fungal disease, occurring mainly in warm and moist corn producing areas. In the 70's of the 20 th century, due to the massive planting of corn containing T-type male sterile cytoplasm (T-cms), corn macule became pandemic in the United states, causing a 165 hundred million kilograms of corn to be reduced in yield, accounting for 15% of the total corn yield in the United states, with approximately 10 hundred million dollars lost in value, because the losses caused the global surprise over the pandemic of potato late blight occurring in 1840 Europe. Corn southern blight occurs in Jiangsu areas of China as early as 20 th century, but only in rainy years, most of the corn northern blight is popular in the later period of corn growth, and serious economic loss is rarely caused. However, in the 60's of the 20 th century, the harm of northern leaf blight became serious due to the large-area planting of maize susceptible hybrids, and the corn susceptible hybrids became major leaf diseases of maize. In the middle of the 60's of the 20 th century, due to the serious occurrence of corn northern leaf blight, the yield reduction of parts of Hebei and Hubei is serious, the yield reduction of general plots reaches more than 20%, and the yield reduction of serious plots reaches 80%, even the serious plots are completely harvested. After the 70 s in the 20 th century, the occurrence of the southern leaf blight is basically controlled by the popularization of disease-resistant varieties of corns, but because the planting of the disease-resistant varieties is large-area and simple and global warming is realized, in certain corn production areas in China, the southern leaf blight occurs seriously at the time of occurrence, and disastrous loss is caused. The method has the advantages that the conidiospore and ascospore formation of the corn microsporum are deeply researched, the key factor of the sporulation of the corn microsporum is identified, the molecular mechanism of the disease of the necrotrophic pathogenic fungi of the corn microsporum is disclosed, the protein serving as the action target of the fungicide can be found out, and the theoretical and technical foundation is laid for developing efficient medicaments for preventing and treating the corn microsporum and other similar diseases.

CDC3 is a gene of unknown function in maize Microsporum species. By analyzing the functions of the ChCDC3 gene of the maize microsporum, the function of the gene in the growth and development process of the maize microsporum is evaluated, which is beneficial to identifying potential prevention targets and is used for screening novel agents for preventing and controlling the maize microsporum.

Disclosure of Invention

The invention aims to provide a gene for controlling conidiospore and ascospore formation of fungi and a protein coded by the gene.

The gene for controlling the formation of conidium and ascospore provided by the invention is derived from corn microsporum, is named as ChCDC3, and has a DNA sequence shown as SEQ ID No. 1. The DNA sequence is an open reading frame of a ChCDC3 gene and consists of 1403 nucleotides, wherein the DNA sequence comprises 2 intron sequences.

The invention provides a protein coded by a maize plaque bacteria (Cochliobolus heterosporus) ChCDC3 gene, the amino acid sequence of which is shown as SEQ ID No.2 and consists of 432 amino acids.

The ChCDC3 gene from corn microsporum for controlling formation of conidia and ascospores can be applied to the field of plant anti-corn microsporum genetic engineering.

The protein coded by the ChCDC3 gene for controlling the formation of conidia and ascospores from corn microsporum is deleted, mutated or modified, so that the conidia and ascospores are defected, and the protein can be used as a target to be applied to designing and screening of a corn microsporum resistant medicament, particularly, the protein is not contained in a plant, so that the protein is safer to the plant.

The invention proves that the deletion or mutation of the ChCDC3 gene of maize microsporum maydis (Cochliobolus heterosporus) leads to the remarkable reduction of the formation of conidiospore of maize microsporum maydis and can not form normal ascospore, which indicates that the ChCDC3 gene of maize microsporum maydis (Cochliobolus heterosporus) is necessary gene in the life cycle of maize microsporum maydis. At present, the CDC3 gene has not been reported to influence the formation of ascospores, and the research is the first report. Therefore, screening compounds capable of preventing the expression of the gene and the expression, modification and positioning of the protein thereof can effectively control the occurrence of the corn leaf spot, thereby being helpful for developing a novel bactericide, namely an important application of the corn leaf spot pathogen (Cochliobolus heperostrophus) ChCDC3 gene provided by the invention is as follows: the expression of the gene and the expression, modification and positioning of the protein product coded by the gene can be used as an important candidate target site for designing and screening the corn micrococcus disease resistant medicament.

Drawings

FIG. 1 is a schematic diagram of domain prediction of ChCDC3 protein

Wherein: finding a conserved CDC-Septin functional structural domain;

FIG. 2 is a schematic diagram of the knockout strategy of ChCDC3 gene (gene replacement by homologous recombination) of maize Microplaque pathogen

Wherein: ChC4 is a wild strain of corn microsporum; Δ Chcdc3 is a deletion mutant of Chcdc3 gene; primers F1/R1 and F2/R2 are respectively used for amplifying the upstream and downstream sequences of the ChCDC3 gene and are used as homologous arms for knockout; primers F/R, U/NLC37 and NLC38/D are used for verifying the mutant;

FIG. 3 is the PCR-verified electrophoresis chart of the deletion mutant of the ChCDC3 gene of maize Microplaque pathogen

Wherein: F/R, U/NLC37 and D/NLC38 are used as primers; 1 is a corn microsporum wild strain, 2 and 3 are ChCDC3 gene deletion mutants; (1) is the result of partial ChCDC3 gene amplification; (2) adding a part of hygromycin sequence to the upstream sequence of the ChCDC3 gene for amplification; (3) adding a part of hygromycin sequence to a downstream sequence of the ChCDC3 gene for amplification;

FIG. 4 is a photograph comparing the culture characteristics of deletion mutant of maize Microplaque pathogen ChCDC3 gene with wild type strain and complementary strain Δ ChCDC 3-C;

wherein: the culture medium is CMX, the culture is carried out at 24 ℃, and the observation and the photographing are carried out 7 days after the inoculation; WT is a wild type strain of Microsporum maydis, and the numbering of other strains is as described above.

FIG. 5 shows the colony diameters of deletion mutants of the maize Microplaque pathogen ChCDC3 gene, wild type strains and complementary strains delta ChCDC3-C after 7 days of culture;

wherein: the culture medium is CMX, cultured at 24 ℃, and measured 7 days after inoculation; WT is a wild type strain of Microsporum maydis, and the numbering of other strains is as described above.

FIG. 6 is a microcosmic contrast picture of conidium growth of deletion mutant, wild strain and complementary strain delta ChCDC3-C of maize small spot pathogen ChCDC3 gene

Wherein: the spores are corresponding strains, inoculated on a CMX culture medium and cultured for 7-9d to produce spores, spore suspension is prepared, dripped on a glass slide, and observed and photographed under a microscope.

FIG. 7 shows the relative yields of conidia of deletion mutant of ChCDC3 gene and wild strain and complementary strain Δ ChCDC 3-C;

wherein: the spores are used as corresponding strains, inoculated on a CMX culture medium and cultured for 9d to produce spores, spore suspension is prepared, and the spore concentration is calculated by using a blood cell plate counter.

FIG. 8 shows the conidium germination rates of deletion mutants of ChCDC3 gene, wild type strains and complementary strains delta ChCDC 3-C.

Wherein: the spores are produced by inoculating corresponding strains on a CMX culture medium and culturing for 9d, spore suspension is prepared and dripped on a glass slide, moisture preservation culture is carried out for 6h at 24 ℃, and the spore germination rate is measured.

FIG. 9 shows the numbers of the deletion mutant of ChCDC3 gene and the wild strain and the complementary strain Δ ChCDC3-C produced capsulets.

Wherein: the deletion mutant of the ChCDC3 gene, a wild strain, a complementary strain delta ChCDC3-C and a strain CB7 of the corn small leaf spot are hybridized, and the number of the subcapsular shells is measured after the deletion mutant is cultured for 21 days on a Sach culture medium at 25 ℃.

FIG. 10 is the microscopic observation picture of deletion mutant of ChCDC3 gene, wild strain and complementary strain delta ChCDC3-C ascospore.

Wherein: the deletion mutant of the ChCDC3 gene, a wild strain and a complementary strain delta ChCDC3-C are hybridized with a strain CB7 of the corn small leaf spot, the ascospores are obtained after the strain is downloaded into Sach culture medium at 25 ℃ and cultured for 21 days, and the observation and the photographing are carried out under a microscope.

Detailed Description

In order to better describe the invention, the following examples are further illustrated, and the methods in the following examples, unless otherwise specified, are conventional.

The corn northern leaf blight strain (Cochliobolus heterostemus) used in the present invention was collected from field diseased corn.

Example 1 correlation analysis of the ChCDC3 Gene

The ChCDC3 gene of maize microsporum is obtained by the group through comparing CDC genes in yeast in maize microsporum. The open reading frame of the maize alternaria minitans ChCDC3 gene consists of 1403 nucleotides and comprises 2 introns. The encoded protein product consists of 432 amino acids, and the structural domain analysis shows that the protein encoded by the maize lentigines ChCDC3 comprises a conserved CDC-Septin functional domain (see figure 1).

Example 2 knockout of the ChCDC3 Gene of maize Microplaque Virus

1) Amplification of upstream and downstream of ChCDC3 gene and hygromycin gene

Primers F1(5'-T C G T G C C T C C T C A T T T A A C C-3') and R1(5'-TCCTGTGTGAAATTGTTATCCGCTGGAGAAGTAGCACCGTTGGA-3') are adopted, genome DNA of a maize plaque pathogen wild strain C4 is used as a template to amplify an upstream 942p fragment of a ChCDC3 gene, F2(5'-GTCGTGACTGGGAAAACCCTGGCGGCTATCGGTAAACGGGACAA-3') and R2(5'-CCCGCTCATGATACTCTTCG-3') are adopted to amplify a downstream 805bp fragment of a maize plaque pathogen ChCDC3 gene, primers M13F (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and M13R (5'-AGCGGATAACAATTTCACACAGGA-3') are adopted, and vector pUCTATPH is used as a template to amplify a 2549bp hygromycin gene. The reaction system is as follows: 10mmol/L dNTP mix, 1. mu.L; 5 XPCR buffer, 10 uL; 2.5. mu.L (10. mu. mol/mL) of each of the upstream and downstream primers; template DNA, 2. mu.L; phusion polymerase, 0.5. mu.L (5U); ddH₂O, 31.5 μ L; the amplification procedure was: pre-denaturation at 98 ℃ for 2 min, followed by (1) denaturation at 98 ℃ for 20 sec; (2) annealing at 65 ℃ for 30 seconds; (3) extension at 72 ℃ for 30 seconds; (4) circulating for 30 times; (5) extension at 72 ℃ for 10 min. The 3 fragments are co-transferred into a maize Microsporum maydis wild type strain C4.

2) Corn microsporum transformation

a. Sporulation culture of corn microsporum

A small amount of conidia of the strain maize microsporum C4 was taken from a refrigerator at-80 ℃ and added dropwise to a CMX medium [ per liter of CMX medium contains: 10mL of 0.1g/mL calcium nitrate tetrahydrate solution, 10mL of solution B, 0.5mL of trace element solution, 1g of yeast extract, 0.5g of enzymatic casein, 0.5g of acidolysis casein, 10g of xylose and 20g of agar powder. (solution B per liter: 20g potassium dihydrogen phosphate, 25g magnesium sulfate heptahydrate, 15g sodium chloride) (microelement per liter: 57.2mg boric acid, 393mg copper sulfate pentahydrate, 13.1mg potassium iodide, 60.4mg manganese sulfate monohydrate, 36.8mg ammonium molybdate tetrahydrate, 5.49g zinc sulfate monohydrate, 948.2mg ferric chloride hexahydrate.) was incubated at 24 ℃ for 1 week with CM broth [ CMX medium per liter: 10mL of 0.1g/mL calcium nitrate tetrahydrate solution, 10mL of solution B, 0.5mL of trace element solution, 1g of yeast extract, 0.5g of enzymatic casein, 0.5g of acidolysis casein, 10g of glucose and 20g of agar powder. (solution B per liter: 20g of potassium dihydrogen phosphate, 25g of magnesium sulfate heptahydrate, 15g of sodium chloride) (microelement solution per liter: 57.2mg of boric acid, 393mg of copper sulfate pentahydrate, 13.1mg of potassium iodide, 60.4mg of manganese sulfate monohydrate, 36.8mg of ammonium molybdate tetrahydrate, 5.49g of zinc sulfate monohydrate, 948.2mg of ferric chloride hexahydrate.) spores were scraped, collected, observed microscopically, and the concentration of spores was adjusted to 1X 10 by a hemocytometer⁶/mL。

b. Corn microsporum transformation

1mL of spore suspension is sucked into 100mLCM liquid medium, shaking culture (150rpm) is carried out for 12-18h at 24 ℃, hyphae are collected by centrifugation and are subjected to enzymolysis for 2h in 80mL of enzymolysis liquid (3.27g of sodium chloride, 0.8g of collapse enzyme), and protoplasts are collected. The protoplasts were washed 3 times with 10ml of LSTC solution and finally dissolved in 500. mu.L of LSTC solution (21.86 g sorbitol, 1Mtris-HCL1mL, 0.735g calcium chloride dihydrate per 100ml of LSTC solution). 25mL of the prepared PCR fragment was mixed well with 100. mu.L of the protoplast solution, and 1mL of PEG solution (each 50mL of PEG solution contains 30g of polyethylene glycol, 0.5mL of 1Mtris-HCL0.5mL of calcium chloride dihydrate) was added thereto. Finally, the cells were diluted with 1mLSTC solution, mixed with regeneration medium, incubated overnight at 30 ℃ and 10mL of water agar containing 150. mu.g/mL hygromycin was added to each dish, and after incubation for 3 days at 30 ℃ the expanded colonies were picked up on CMX medium containing the same antibiotics.

3) Validation of deletion mutants

Three pairs of primers were selected for screening of the transformants by PCR amplification. The amplification result is consistent with the following results and is determined as a deletion mutant of the ChCDC3 gene: the primer U (5'-CGACAAGAGCGAGTTGAACA-3') on the genome outside the upstream homology arm paired with primer NLC37(5'-GGATGCCTCCGCTCGAAGTA-3') of the hygromycin resistance gene amplified a recombinant fragment of the expected size (2.4 kb); the primer D (5'-GGGTGCCATTTGTTGCTATT-3') on the genome outside the downstream homology arm can be paired with primer NLC38(5'-CGTTGCAAGACCTGCCTGAA-3') of the hygromycin resistance gene to amplify a recombinant fragment of the expected size (3.0 kb); while the coding region primers F (5'-ATTGTTGACAACCGCATTCA-3') and R (5'-CTCCATCTTCTGGAGCTTGG-3') had no amplified band (the wild type strain amplified a 0.6kb fragment) (see FIG. 3). As a result, 2 strains of ChCDC3 gene deletion mutants were selected from the transformants for subsequent functional analysis.

Example 3 genetic complementation of deletion mutants of the ChCDC3 Gene

The full length 2755bp (including upstream and downstream sequences) of the maize alternaria miniata ChCDC3 gene is amplified by adopting primers C-F1(5'-TCGTGCCTCCTCATTTAACC-3') and C-R1(5'-CACTGGAACAACTGGCATGCACTCCTCGCTCCGAACTAC-3'), and the downstream sequence 827bp of the maize alternaria miniata ChCDC3 gene is amplified by adopting primers C-F2(5'-CAGGTACACTTGTTTAGAGGTCTCAGATATGGCACCCATGA-3') and C-R2 (5'-TTGTAGACGACGATGCCGTA-3'). Then, nptII gene was amplified using vector pII 99 as a template and DW69(5'-CATGCCAGTTGTTCCAGTG-3') and DW70(5'-ACCTCTAAACAAGTGTACCTG-3') as primers. And transferring the three complementary fragments into a ChCDC3 gene deletion mutant genome, and screening a genetic complementary strain delta ChCDC3-C by taking geneticin as a screening marker. And selecting a primer F/R for PCR verification.

Example 4 Effect of ChCDC3 Gene in the hyphal growth of maize Microsporum maydis

The mutants of ChCDC3 were evaluated for phenotypic variation associated with hyphal growth by plating. Taking 10 μ L of strain CMX spore suspension (1 × 10)⁶mL^-1) Inoculating in the center of solid CMX medium, culturing at 24 deg.C, 16h light, and 8h dark. Seven days later, the colony morphology of the mutant is obviously different from that of the wild type strain and the complementary strain, the colony of the mutant is raised around, the growth speed is obviously slowed down, and the color of the thallus is lightened, which indicates that the ChCDC3 is a gene necessary for the hypha extension growth of the corn leaf spot germ (see figures 4 and 5).

Example 5 Effect of the ChCDC3 Gene on the production of conidia of maize Microsporum maydis

Respectively inoculating the maize microsporum wild strain C4, the ChCDC3 gene deletion mutant and the complementary strain on a solid CMX culture medium, flushing conidia with 5mL of sterile water after nine days of growth, collecting spore suspension, counting the spores by using a blood counting plate, and observing the spore morphology under a microscope. Compared with the wild strain, the spore yield of the strain with the deletion mutant of the ChCDC3 gene is only 6 percent of that of the wild strain, and the conidium yield is obviously reduced (see figure 7). It was found by microscopic observation that the conidia of the mutant strain with the deletion of the ChCDC3 gene are irregular in shape and have reduced spore membranes, the conidia of the wild strain and the complementary strain generally contain 5-7 membranes, while the conidia of the mutant strain with the deletion of the ChCDC3 gene have only 1-2 membranes (see FIG. 6). Conidium suspensions of the wild strain, the ChCDC3 gene deletion mutant and the complementary strain are respectively dripped on a glass slide, moisture-preserving culture is carried out for 6h at 25 ℃, and the conidium germination rate is observed under a microscope. The result shows that the conidiophoresis rate of the ChCDC3 gene deletion mutant is only 70% of that of the wild strain (see figure 8). These results show that the ChCDC3 gene of maize microsporum has important effects on the formation, morphology and germination rate of maize microsporum.

Example 6 Effect of the ChCDC3 Gene on the production of ascospores of Microsporum maydis

The wild type strain C4 of maize maculate germ, the gene deletion mutant of ChCDC3 and the complementary strain are respectively cultured with maize maculate germ CB7 (strain C4 contains MAT1-1 mating type gene, CB7 contains MAT1-2 mating type gene). After culturing at 25 ℃ for 21 days, the production of the ascospores and ascoshells was observed. According to observation, the number of the ascocarp of the deletion mutant of the ChCDC3 gene is only about 12% of that of the wild strain and the complementary strain, and the production amount of the ascocarp is obviously reduced (see figure 9). In addition, the wild type strain usually produces 7-8 filamentous ascospores in the ascospores, while the ChCDC3 gene deletion mutant cannot form normal ascospores, and the ascospores are in a bubble shape and are easy to break (see FIG. 10). Therefore, the gene ChCDC3 of the maize lentinus edodes has important effect on the generation of the ascospores and the ascospores.

Sequence listing

The invention name is as follows: maize small leaf spot pathogen ChCDC3 gene and application thereof

Sequence of SEQ ID No.1

(i) Sequence characteristics: (A) length: 1403 bp; (B) type (2): a nucleotide; (C) chain property: single strand

(ii) Molecular type: DNA

(iii) Description of the sequence: SEQ ID No.1

1 ATGGACCAGA CATTCAACGG GTCCAACCCT GCGGTCCACA CCATCACGGC TAAGAACCCC 61 AAGGCCGCTG CGGCCATGGC CAACGACATG AACATTGTCC GCAGAAAGCT TACCGGCTAT 121 GTTGGTTTCG CCAACCTGCC CAACCAGTGG CACCGCAAGA GTGTGCGCAA GGGATTCAAC 181 TTCAACGTCA TGGTTGTTGG TACGTGTGAC CTGCCAGCAT GCCGTGTCGA ATCTAACTAA 241 CGTGTCGTCC CAGGCGAGTC TGGACTCGGA AAGTCTACCC TTGTAAACAC ACTGTTCAAC 301 ACGTCGCTGT ACCCGCCCAA GGAACGCCAG CCCCCGAGTC TGGACATTTC CAAGACTGTA 361 TCGATCCAGT CCATCAGCGC CGACATTGAG GAGAACGGCG TTCGTCTGAG GTTAACCGTT 421 GTCGATACAC CTGGCTTTGG CGACTTCATC AACAACGATG AGTCCTGGGA CCCCATTGTC 481 AAGAACATTG AGCAACGGTT CGACGCATAC CTGGATGCCG AGAACAAGGT CAACCGCATG 541 AACATTGTTG ACAACCGCAT TCACGCCGTC GTCTACTTTA TTCAGCCTAC GGGCCACTCT 601 CTGAAGCCCA TTGACATTGA GGTGATGAAG AAGCTCCACA CCAAGGTCAA CCTGATTCCC 661 GTCATTGCCA AGGCCGACAC CGTTACGGAC GATGAGATTG ACAACTACAA GAAGAGGGTA 721 AGTAGCTTTA TTTTGCGAAG TGGTTGGGGC TATGTTGACT TGTCCAGATT CTTGCCGACA 781 TTGCGTACCA CAAGATCCAG ATCTTCGAAG GACCCCGGTA TGAGCTTGAC GACGAGGAGA 841 CGATTGCCGA GAACCAGGAG ATCATGGCCA AGGTTCCCTT TGCTGTTGTC GGCTCCAACA 901 CCGAGGTGAC GACGGTTGAC GGCCGCAAGG TCCGCGGACG CGCACTCCCC TGGGGTGTGG 961 TCGAGGTTGA CAACGAGGAG CACTGCGACT TTGTCAAGCT CCGACAGATG CTCATCCGCA 1021 CCCACATGGA GGAGCTCAAG GAAAACACCA ACAATGTCTT GTACGAGAAC TACCGCTCGG 1081 ACAAGCTTGC ACAGATGGGC ATCCAACAGG ACTCGAGCGT GTTCAAGGAG GTCAACCCTG 1141 CTGTCAAGCA AGAGGAGGAG CGCTCGCTGC ACGAGGCCAA GCTCCAGAAG ATGGAGATGG 1201 AGATGAAGAT GGTGTTCCAG CAAAAGGTGC AGGAAAAAGA AAGCAAGCTG CGCCAGAGCG 1261 AGGAAGAGCT GTACGCCCGT CACAAGGAGA TGAAGGACCA GCTGGACCGA CAACGACAGG 1321 AGCTCGAAGA GAAGAAGGCA CGCATCGAGT CTGGCCGGCC GATCGAAGAG AAGGGCAAGC 1381 GAAAGGGCTT CTCGCTCCGC TAA

Sequence of SEQ ID No.2

(i) Sequence characteristics: (A) length: 432 amino acids; (B) type (2): an amino acid; (C) chain property: single strand

(ii) Molecular type: polypeptides

(iii) Description of the sequence: SEQ ID No.2

1 MDQTFNGSNP AVHTITAKNP KAAAAMANDM NIVRRKLTGY VGFANLPNQW HRKSVRKGFN 61 FNVMVVGESG LGKSTLVNTL FNTSLYPPKE RQPPSLDISK TVSIQSISAD IEENGVRLRL 121 TVVDTPGFGD FINNDESWDP IVKNIEQRFD AYLDAENKVN RMNIVDNRIH AVVYFIQPTG 181 HSLKPIDIEV MKKLHTKVNL IPVIAKADTV TDDEIDNYKK RILADIAYHK IQIFEGPRYE 241 LDDEETIAEN QEIMAKVPFA VVGSNTEVTT VDGRKVRGRA LPWGVVEVDN EEHCDFVKLR 301 QMLIRTHMEE LKENTNNVLY ENYRSDKLAQ MGIQQDSSVF KEVNPAVKQE EERSLHEAKL 361 QKMEMEMKMV FQQKVQEKES KLRQSEEELY ARHKEMKDQL DRQRQELEEK KARIESGRPI 421 EEKGKRKGFS LR

Claims

1. Corn leaf spot bacteria (A)Cochliobolus heterostrophus）ChCDC3The application of the gene in regulating formation of conidia and ascospores of maize microsporum is disclosed, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. Corn leaf spot bacteria (A)Cochliobolus heterostrophus）ChCDC3The application of the gene as a target in designing and screening the anti-corn maculopathy agent, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

3. Corn leaf spot bacteria (A)Cochliobolus heterostrophus）ChCDC3The application of the protein encoded by the gene in the defect of formation of conidia and ascospores of maize microsporum is disclosed, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 2.