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Pathogenicity-related botrytis cinerea gene BcEXO70 and application thereof

A technology of Botrytis cinerea and genes, applied in the application field of genes and their encoded proteins, can solve the problems of little known molecular mechanisms

Inactive Publication Date: 2019-06-11
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

To a large extent, Botrytis cinerea achieves the above goals by changing its own metabolic pathways and secreting related effectors (such as toxins), but the genes, proteins and metabolites involved in the corresponding process and the molecular mechanism of their regulation are still unknown. know little

Method used

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  • Pathogenicity-related botrytis cinerea gene BcEXO70 and application thereof
  • Pathogenicity-related botrytis cinerea gene BcEXO70 and application thereof
  • Pathogenicity-related botrytis cinerea gene BcEXO70 and application thereof

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Experimental program
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Effect test

Embodiment 1B

[0027] Correlation analysis of embodiment 1BcEXO70 gene

[0028] The open reading frame of BcEXO70 gene of Botrytis cinerea consists of 2067 nucleotides, including 3 exons, the full-length cDNA of the coding region is 1965 nucleotides, and the encoded protein product consists of 654 amino acids. Domain analysis found that BcExo70 protein contains a conserved Exo70 domain (see figure 1 ).

Embodiment 2B

[0029] The knockout of embodiment 2BcEXO70 gene

[0030] 1) Construction of knockout vector

[0031] Using primers EXO70-UP-F (5'-CTCGAGTGTGGGAAATGTGGGATG-3') and EXO70-UP-R (5'-GAATTCGCACGCTAGACCTAATGC-3'), the upstream of the BcEXO70 gene was amplified using the genomic DNA of Botrytis cinerea strain B05.10 as a template 791bp fragment, using EXO70-DN-F (5'-TCTAGACCTGTGGGTGAGACGAGA-3') and EXO70-DN-R (5'-AAGCTTAATGCGAAATGCGAAACT-3') to amplify the downstream 751bp fragment of Botrytis cinerea BcEXO70 gene, the reaction system is: 10mmol / L dNTP Mixture, 0.5 μL; 10×PCR buffer, 2.5 μL; each 1 μL of upstream and downstream primers (10 μmol / mL); template DNA, 1 μL; Ex-Taq, 0.2 μL (5U); ddH 2 O, 18.8 μL; amplification program: 94°C pre-denaturation for 3 minutes, then (1) 94°C, denaturation for 50 seconds; (2) 56°C, annealing for 50 seconds; (3) 72°C, extension for 60 seconds; (4) ) cycled 30 times; (5) extended at 72°C for 10 minutes. The above two DNA amplification products ...

Embodiment 3B

[0045] The effect of embodiment 3BcEXO70 gene in the hyphal growth process of Botrytis cinerea

[0046] The plate culture method was used to evaluate the variation of BcEXO70 mutant's mycelial growth and other related phenotypes. Use a sterile puncher to punch out the cakes of the strains to be tested, inoculate them in the center of the PDA medium, and cultivate in the dark at 20°C. After three days, it was observed that the colony morphology of mutants was substantially normal, but the growth rate was significantly slowed down (see Figure 4 ). The colony sizes of the two independently obtained mutant strains were very close, and both were significantly smaller than the wild-type colonies. The colony diameter of each bacterial strain is measured and calculated, and it is found that the colony size of the mutant has only about 62% of the wild type (see Figure 5 ). The above results indicated that the BcEXO70 gene has the ability to maintain the normal growth of hyphae. ...

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Abstract

The invention discloses a pathogenicity-related botrytis cinerea gene BcEXO70 and application thereof, and belongs to the technical field of microbial genetic engineering. The pathogenicity-controlledgene BcEXO70 from botrytis cinerea has a DNA sequence as shown in SEQ ID No:1 and consists of 2067 nucleotides. An amino acid sequence of a protein coded by the BcEXO70 gene is shown as SEQ ID No:2,and the protein is composed of 654 amino acids. The BcEXO70 gene can be applied to the field of plant botrytis cinerea resistance genetic engineering; the gene BcEXO70 for controlling the pathogenicity of the botrytis cinerea is knocked out, so that the pathogenicity of the gene BcEXO70 is defective, and the gene BcEXO70 can be applied to reducing the pathogenicity of the botrytis cinerea.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and specifically relates to the application of a gene for controlling fungal pathogenicity and its coded protein in the field of plant protection. Background technique [0002] Botrytis cinerea, also known as Botrytis cinerea, belongs to Ascomycota fungus and is the pathogenic fungus of gray mold. It can infect more than 1400 kinds of plants, including almost all vegetables and fruit trees. The host can be infected from the seedling stage, fruit-bearing stage to storage stage, and all parts of the plant can be infected by Botrytis cinerea. The typical symptoms of the disease on the leaves are "V"-shaped lesions, and the flowers are mainly rotten and Adjust wilting, the fruit is mainly manifested as rot and shedding. The occurrence and spread of the disease are closely related to the humidity and temperature of the environment, and it will be serious when the relative humidi...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/90C12N15/31C07K14/37
Inventor 李桂华加娜尔孙姣孙晨浩秦庆明张明哲佰娜尔古丽·居马汗迪丽拜尔·麦麦提
Owner JILIN UNIV
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