CN117947052A - Corn small spot germ ChGCN gene and application thereof - Google Patents
Corn small spot germ ChGCN gene and application thereof Download PDFInfo
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Abstract
The invention is suitable for the technical field of microbial genetic engineering, and provides a corn small spot germ ChGCN gene and application thereof, wherein the DNA sequence of the ChGCN gene is shown as SEQ ID No. 1; the amino acid sequence of the protein encoded by the ChGCN gene is shown as SEQ ID No. 2. The ChGCN gene is applied to the field of prevention and control of corn small spot disease; the conidium and ascospore formation of the corn small spot germ are controlled, and the pathogenic protein ChGcn is deleted, mutated or modified, so that the conidium and ascospore formation is limited, the pathogenicity is reduced, and the method can be used as a target in the design and screening of medicaments for preventing and treating corn small spot diseases.
Description
Technical Field
The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a corn small spot germ ChGCN gene and application thereof.
Background
The stateless name of the corn small spot germ is: bipolaris maydis belonging to the genus Desmodium of the subdivision Desmodii of the genus Desmodii; the sexual morphology is Cochliobolus heterostrophus, belonging to the ascomycotina class of Isosporium. In 1925, the first pathogen was found to be a maize disease. The corn small spot germ belongs to a filamentous fungus, and after hypha grows to a certain stage, conidium grows out from the top end or side of conidium peduncle under the influence of external environment conditions and self factors. Conidia have 3-13 membranes, most of which have 7-9 membranes, black brown, oblong, rounded at both ends, curved in one side in multiple directions, thick in the middle and thin at both sides, 30-115 μm×10-17 μm in size, and umbilical points are depressed within basal cells. Conidia germinate from the cells at both ends to form sprouting tubes, and sometimes intermediate cells germinate into sprouting tubes. The temperature range of conidium formation is 15-33 ℃, and the optimal temperature range is 23-25 ℃; the germination temperature of the conidium ranges from 5 ℃ to 42 ℃ and the optimal temperature ranges from 26 ℃ to 32 ℃. Conidia are relatively resistant to desiccation and survive for at least one year on corn seeds. The conidium can germinate and grow into a sprouting pipe under the moist condition, and hypha is formed through top growth. Conidia are the primary source of infection of the corn small spot germ in the field, and if the corn small spot germ cannot produce conidia, it will not continue to infect and spread.
Under natural conditions, hyphae grow to a certain stage, and under the external environment conditions, the sexual reproduction stage of the corn small spot germ is abnormal and matched, and a single locus is related to sexual reproduction and is named MAT-1 on the genome, and the locus has two different forms, namely MAT-1-1 and MAT-1-2. The sexual reproduction stage of the species leptosphaeria zeylanica is unusual, and the ascus shell can be found occasionally in dead diseased tissues. The ascus shell of the maize small spot germ can be generated by artificial induction under laboratory conditions and is called 'pseudothecium'. The ascus shell takes about one month from formation to maturation, and after the mature ascus shell encounters water, the top end breaks, releasing the ascus and ascospores. The ascus shell is black, spherical, obvious in beak part, long buried in host disease tissue, and mycelium and conidiophore can grow on the surface; inside the bag, an approximately cylindrical asca is generated. The ascus tip is rounded and the base has a handle. Mature asca undergo meiosis and mitosis to form 8 linear haploid ascospores, the ascospores are mutually entangled into a spiral shape in the asca, and each cell can grow into a bud tube during germination, so that hyphae grow. It was found that 80% of the mature asca had 8 ascospores intact and 20% had 4-7 ascospores. The infection and the epidemic of the corn small spot pathogen in the field mainly depend on the spread of the conidium along with the airflow and the rainwater, if the formation of the conidium can be controlled, the infection source can be greatly reduced, the occurrence of the corn small spot disease can be reduced, and the corn yield can be improved.
Corn small spot is a major corn leaf fungal disease, which occurs primarily in warm moist corn producing areas. In the 70 s of the 20 th century, corn with T-type male sterile cytoplasm (T-cms) was grown in large quantities, causing corn small spots to be pandemic in the United states, resulting in 165 billions of kilograms of corn reduced in yield, accounting for 15% of the total yield of United states corn, with about 10 billions of dollars lost in yield, because the losses incurred exceeded the late potato epidemic that occurred in Europe of 1840 and was jarring the world. The corn small spot disease occurs in Jiangsu region of China as early as 20 th century, but only in the rainy year, and is mostly popular in the later stage of corn growth, causing little serious economic loss. However, in the 60 s of the 20 th century, the damage of the small spot disease is more and more serious due to the large-area planting of the maize infectious hybrid, and the small spot disease becomes an important maize leaf disease. By the middle 60 th century, the serious occurrence of corn small spot disease causes serious yield reduction in the river and Hubei partial areas, the yield reduction of the common land parcels reaches more than 20%, and the yield reduction of the serious land parcels reaches 80%, even is out of order. After the 70 th century, the disease-resistant varieties of corns are promoted, and the occurrence of small spot diseases is basically controlled, but the small spot diseases still occur seriously in certain corn producing areas of China due to the large-area singleization of the planting of the disease-resistant varieties and the global warming of the air temperature, so that the serious loss is caused. The research on the formation of conidium and ascospore of the corn small spot germ is conducted, and the key factors of the formation of the conidium of the corn small spot germ are identified, so that the molecular mechanism of the pathogen of the dead nutritional pathogenic fungi of the corn small spot germ is revealed, and the protein which can be used as a target of fungicide action can be possibly found, and a theoretical and technical basis is laid for developing efficient medicaments for preventing and treating the corn small spot disease and other similar diseases.
GCN4 is a gene of unknown function in the species Leptosphaeria maydis. The function of the gene ChGCN of the corn small spot pathogen is analyzed, so that the effect of the gene in the growth and development process of the corn small spot pathogen is evaluated, the potential control target can be identified, and the novel agent for controlling the corn small spot pathogen can be screened.
Disclosure of Invention
The embodiment of the invention aims to provide a corn small spot germ ChGCN gene and application thereof, aiming at solving the problems in the background technology.
The embodiment of the invention is realized in such a way that the DNA sequence of the gene ChGCN of the corn leaf spot germ is shown as SEQ ID No. 1. The DNA sequence is an open reading frame of ChGCN gene, consists of 1701 nucleotides, and contains 1 intron sequence.
Further technical proposal provides the protein coded by ChGCN gene, the amino acid sequence of which is shown as SEQ ID No.2, and the sequence consists of 465 amino acids.
Another object of the embodiment of the invention is the application of the gene ChGCN to the control of the formation of conidia and ascus shells and pathogenicity of the corn small spot pathogen, and the gene ChGCN from the corn small spot pathogen can be applied to the field of prevention and control of corn small spot diseases. Specific:
The protein coded by the gene ChGCN for controlling the formation of conidium and ascosia and pathogenicity from the corn small spot germ is deleted, mutated or modified, so that the formation of conidium and ascosia and pathogenicity are defective, and the gene can be used as a target for designing and screening medicaments for preventing and treating corn small spot disease.
The ChGCN gene of the corn small spot germ provided by the embodiment of the invention proves that the deletion or mutation of the ChGCN gene leads to the obvious reduction of conidium formation of the corn small spot germ, the formation of ascus shells is affected, the pathogenicity is obviously reduced, and the ChGCN gene is a gene necessary for the life cycle of the corn small spot germ. Therefore, screening the compound capable of preventing the expression of the gene and the expression, modification and localization of the protein can effectively control the occurrence of corn small leaf spot, thereby being beneficial to developing a novel bactericide, namely, one important application of the ChGCN4 gene 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 important candidate target sites for the design and screening of agents for preventing and treating corn small spot pathogens.
Drawings
FIG. 1 is a schematic representation of domain prediction of ChGcn4 proteins;
FIG. 2 is a schematic diagram of the knockout strategy of the ChGCN gene of Leptosphaeria maydis (gene replacement by homologous recombination);
FIG. 3 is a PCR-validated electrophoresis of the Leptosphaeria maydis ChGCN gene deletion mutant;
FIG. 4 is a comparison of the culture characteristics of the deletion mutant of the Leptosphaeria maydis ChGCN gene with that of the wild-type strain and the complementation strain Δ ChGCN 4-C;
FIG. 5 is a graph showing the relative yields of the mutant deletion of the ChGCN gene from Leptosphaeria maydis and the conidia of the wild-type strain;
FIG. 6 is a diagram showing the observation of the generation of ascus shells of the deletion mutant and wild strain of the gene ChGCN. Maydis;
FIG. 7 is a graph showing the number of generation of ascus shells of a deletion mutant of the ChGCN gene of Leptosphaeria maydis and a wild-type strain;
FIG. 8 is a diagram showing the pathogenicity analysis of a deletion mutant of the Leptosphaeria maydis ChGCN gene and a wild-type strain;
FIG. 9 is a graph showing the relative infection areas of the mutant and wild-type strains deleted for the ChGCN gene of Leptosphaeria maydis.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Specific implementations of the invention are described in detail below in connection with specific embodiments.
Example 1 analysis of the relativity of the ChGCN Gene of Leptosphaeria maydis
The used corn small spot germ strain (Cochliobolus heterostrophus) is collected from field disease corn.
The corn small spot germ ChGCN gene is obtained by comparing GCN4 gene in saccharomycetes in corn small spot germ, and the DNA sequence is shown as SEQ ID No. 1. The open reading frame of the Leptosphaeria maydis ChGCN gene consists of 1701 nucleotides, comprising 1 intron. The encoded protein product consisted of 465 amino acids and domain analysis revealed that ChGcn protein contained a conserved BRLZ domain (see figure 1).
The amino acid sequence of the protein coded by ChGCN gene is shown as SEQ ID No.2, and the sequence consists of 465 amino acids.
Example 2 knockout of the Leptosphaeria maydis ChGCN Gene (the knockout strategy of the Leptosphaeria maydis ChGCN Gene is shown in FIG. 2)
1) Amplification of the maize Micromacula ChGCN Gene upstream and downstream of the hygromycin Gene:
The primer is adopted:
F1：5'-TGGCTCAGTGCCGTCGTGT-3'；
R1：5'-GCTGAAGCCACTTGAGATGA-3'；
The genome DNA of the wild strain C4 of the corn leaf spot germ is used as a template to amplify a 965bp fragment at the upstream of ChGCN genes.
By using primers
F2：5'-CCCAGTGGTCGTTAGGAG-3'；
R2：5'-AGGCGATAATATGAGGAGAA-3'；
Amplifying 939bp fragments of the ChGCN th maize leaf spot germ gene downstream.
The primer is adopted:
M13F：5'-CGCCAGGGTTTTCCCAGTCACGAC-3'；
M13R：5'-AGCGGATAACAATTTCACACAGGA-3'；
the 2549bp hygromycin gene was amplified using vector pUCATPH as a template.
The reaction system is as follows: 10mmol/L dNTP mix, 1. Mu.L; 5 XPCR buffer, 10. Mu.L; 2.5. Mu.L (10. Mu. Mol/mL) of each of the upstream and downstream primers; template DNA,2 μl; phusion polymerase,0.5 μl (5U); ddH 2 O, 31.5. Mu.L;
The amplification procedure was: pre-denaturing at 98 ℃ for 2 minutes, then denaturing at 98 ℃ for 20 seconds; annealing at 65 ℃ for 30 seconds; extending at 72 ℃ for 30 seconds; cycling for 30 times; extension was carried out at 72℃for 10 minutes.
The 3 fragments are jointly transferred into a wild-type strain C4 of the phoma zeylanicum.
2) Transformation of corn small spot germ:
2.1, spore-forming culture of the corn small spot germ:
Taking a small amount of conidium of a corn small spot germ C4 strain from a refrigerator at the temperature of minus 80 ℃, and dripping the conidium into a CMX culture medium (each liter of the CMX culture medium comprises 10mL of 0.1g/mL of calcium nitrate tetrahydrate solution, 10mL of solution B,0.5mL of microelement solution, 1g of yeast extract, 0.5g of enzymolysis casein, 0.5g of acidolysis casein, 10g of xylose and 20g of agar powder, wherein each liter of solution B comprises 20g of potassium dihydrogen phosphate, 25g of magnesium sulfate heptahydrate and 15g of sodium chloride; each liter of the trace element solution comprises 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 and 948.2mg of ferric chloride hexahydrate, and the trace element solution is cultured for 1 week at 24 ℃, and is cultured by using CM liquid culture medium (10 mL of 0.1g/mL of calcium nitrate tetrahydrate solution, 10mL of solution B,0.5mL of trace element solution, 1g of yeast extract, 0.5g of enzymolysis casein, 0.5g of acidolysis casein, 10g of glucose and 20g of agar powder, wherein each liter of solution B comprises 20g of potassium dihydrogen phosphate, 25g of magnesium sulfate heptahydrate and 15g of sodium chloride, each liter of trace element solution comprises 57.2mg of boric acid, 393mg of copper sulfate pentahydrate, 13.1mg of potassium sulfate tetrahydrate, 60.4mg of manganese sulfate monohydrate, 36.8mg of ammonium molybdate tetrahydrate, 5.49g of zinc sulfate tetrahydrate, 948.2 g of ferric sulfate hexahydrate and 53 mg of ferric chloride, and the spore concentration of spore is collected by a microscope for observation, and the spore concentration is adjusted by taking a microscope, and observing the spore concentration of the spore by using a microscope.
2.2, Conversion of corn small spot bacteria:
1mL of spore suspension was aspirated into 100mLCM liquid medium, shaking culture (150 rpm) was performed at 24℃for 12-18h, mycelia were collected by centrifugation and digested in 80mL of an enzymatic hydrolysate (3.27 g of sodium chloride, 0.8g of crashing enzyme) for 2h, and protoplasts were collected. Protoplasts were washed 3 times with 10mLSTC solutions and finally dissolved in 500. Mu. LSTC solution (21.86 g sorbitol, 1Mtris-HCL1mL,0.735g calcium chloride dihydrate per 100: 100mLSTC solution). 25mL of the prepared PCR fragment was thoroughly mixed with 100. Mu.L of the protoplast solution, and 1mLPEG solutions (30 g of polyethylene glycol, 0.5mL of 1M tris-HCL, 0.37g of calcium chloride dihydrate were added per 50: 50mLPEG solutions) were added. Finally, diluted with 1mLSTC solution and mixed with regeneration medium, cultured overnight at 30℃and 10mL of water agar containing 150. Mu.g/mL hygromycin was added to each dish, and after 3d of culture at 30℃extended colonies were picked up on CMX medium containing the same antibiotic.
2.3, Verification of deletion mutants:
Three pairs of primers were selected and used to screen transformants by PCR amplification. The amplification results were consistent with the following, determined as ChGCN gene deletion mutants: primer U (5'-ACCCTTGACCTGGTGGCA-3') on the genome outside the upstream homology arm was paired with primer NLC37 (5'-GGATGCCTCCGCTCGAAGTA-3') of hygromycin resistance gene to amplify a recombinant fragment of the expected size (2.3 kb); primer D (5'-CTTTGTGCCTGTAAGGGT-3') on the genome outside the downstream homology arm was paired with primer NLC38 (5'-CGTTGCAAGACCTGCCTGAA-3') of the hygromycin resistance gene to amplify a recombinant fragment of the expected size (2.9 kb); the coding region primers F (5'-CCGCCACCATACCCACTCT-3') and R (5'-GAACCTTGGTCTCCTGCTC-3') have no amplified band (the wild strain can be amplified to 0.84kb fragment) (see FIG. 3, wherein F/R, U/NLC37 and D/NLC38 are the primers used, WT is wild strain of Leptosphaeria maydis, 1 is the amplification result of the upstream sequence of ChGCN gene plus part of hygromycin sequence, 2 is the amplification result of the ChGCN gene, 3 is the amplification result of the downstream sequence of ChGCN gene plus part of hygromycin sequence). As a result, 2 ChGCN gene deletion mutants were selected from the transformants for subsequent functional analysis.
EXAMPLE 3 genetic complementation of the Leptosphaeria maydis ChGCN gene deletion mutant
The primer is adopted:
C-F1：5'-TGGCTCAGTGCCGTCGTGT-3'；
C-R1：5'-CACTGGAACAACTGGCATGAGGCGATAATATGAGGAGAA-3'；
amplifying the full length 3733bp (comprising upstream and downstream sequences) of the gene ChGCN of the leptospora maydis;
The primer is adopted:
C-F2：5'-CAGGTACACTTGTTTAGATTCGGGTCCTTGATTAG-3'；
C-R2：5'-GATGGCATAGTTGCTCGT-3'；
The downstream sequence 851bp of the downstream gene ChGCN of the leptospora maydis is amplified.
The nptII gene was then amplified using vector pII 99 as template and DW69 (5'-CATGCCAGTTGTTCCAGTG-3') and DW70 (5'-ACCTCTAAACAAGTGTACCTG-3') as primers. Three complementary fragments are transferred into ChGCN gene deletion mutant genome, and genetic complementary strain delta ChGCN-C is selected by taking geneticin as a screening mark. And (5) selecting a primer F/R for PCR verification.
Example 4 action of the Leptosphaeria maydis ChGCN Gene in the mycelium growth Process of Leptosphaeria maydis
The mutation of the phenotype related to hypha growth of ChGCN4 mutant was evaluated by plate culture. 10. Mu.L of the strain CMX spore suspension (1X 10 6mL-1) to be tested was inoculated in the center of the solid CMX medium, cultured at 24℃for 16h of light and 8h of darkness. After seven days, the colony morphology of the mutant is obviously different from that of the wild type and the complementary strain, and the mutant hyphae are obviously whitened, which shows that ChGCN4 is a gene necessary for the normal development of the hyphae of the alternaria alternate (see figure 4).
Example 5 Effect of the Leptosphaeria maydis ChGCN Gene on the production of conidia of Leptosphaeria maydis
The wild-type strains C4 and ChGCN gene deletion mutants of the Spot-corn germ were inoculated onto solid CMX medium, respectively, and after nine days of growth, conidia were rinsed with 5mL of sterile water, the spore suspension was collected, spores were counted using a hemocytometer, and the spore morphology was observed under a microscope. The ChGCN gene deletion mutant strain was found to not produce conidia by comparison with the wild-type strain (see fig. 5). These results indicate that the gene ChGCN of Leptosphaeria maydis has an important role in the formation of conidia of Leptosphaeria maydis.
Example 6 Effect of the Leptosphaeria maydis ChGCN Gene on the production of the ascomycetes Leptosphaeria maydis
The wild-type strain C4 and ChGCN gene deletion mutants of the Spot-corn pathogen were cultivated in the opposite direction to the Spot-corn pathogen CB7 (strain C4 contains MAT1-1 mating type gene, CB7 contains MAT1-2 mating type gene). After culturing at 25℃for 21d, the ascal shell production was observed. Through observation, chGCN4 gene deletion mutants have significantly improved white ascal number and reduced black ascal production (see fig. 6 and 7). Therefore, the ChGCN gene of the corn leaf spot germ ChGCN plays an important role in the generation of ascus shells.
Example 7 Effect of the Leptosphaeria maydis ChGCN Gene on the pathogenicity of Leptosphaeria maydis
The wild strain C4 and ChGCN gene deletion mutant of the corn leaf spot germ are inoculated on a solid CMX culture medium respectively, after growing for 9d, conidium is washed by Tween water to prepare a spore suspension with the concentration of 5 multiplied by 10 4/mL, and inoculated on corn leaves cultured for two weeks, and each leaf is inoculated with 2mL of the spore suspension. Three days later, the onset was observed. Experimental results show that compared with the wild type strain, the area of the lesion caused by ChGCN gene deletion mutant is obviously reduced, and the pathogenicity is obviously reduced (see FIG. 8 and FIG. 9).
The gene ChGCN for controlling the formation of conidium and ascosion and pathogenicity from the corn small spot germ can be applied to the field of plant corn small spot disease resistance genetic engineering.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (4)
1. The gene ChGCN of the leptosphaeria zeylanica, which is characterized in that the DNA sequence of the ChGCN gene is shown as SEQ ID No. 1.
2. The gene ChGCN of leptosphaeria zea according to claim 1, wherein the protein coded by ChGCN gene has an amino acid sequence shown in SEQ ID No. 2.
3. The application of the gene ChGCN of the corn small spot pathogen 3834 based on the gene ChGCN of the corn small spot pathogen according to any one of the above claims 1 and 2, characterized in that the gene ChGCN is applied to the prevention and control of corn small spot disease.
4. The use of the gene ChGCN of leptosphaeria zeae according to claim 3, wherein the gene ChGCN is used as a target in the design and screening of agents for controlling leptosphaeria zeae.
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