Botrytis cinerea gene BcFch1 relative to pathogenicity and application of botrytis cinerea gene BcFch1

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

Inactive Publication Date: 2016-04-13
JILIN UNIV
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  • Abstract
  • Description
  • Claims
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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

Method used

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  • Botrytis cinerea gene BcFch1 relative to pathogenicity and application of botrytis cinerea gene BcFch1
  • Botrytis cinerea gene BcFch1 relative to pathogenicity and application of botrytis cinerea gene BcFch1
  • Botrytis cinerea gene BcFch1 relative to pathogenicity and application of botrytis cinerea gene BcFch1

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Experimental program
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Effect test

Embodiment 1B

[0023] Correlation analysis of embodiment 1 BcFch1 gene

[0024] The open reading frame of BcFch1 gene of Botrytis cinerea consists of 1409 nucleotides, including 3 exons, the full-length cDNA of the coding region is 1308 nucleotides, and the encoded protein product consists of 435 amino acids. The BcFch1 protein sequence was compared and analyzed (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and it was found that Fch1 widely exists in cellular organisms such as animals, plants, fungi and bacteria. Domain analysis revealed that the BcFch1 protein contains a conserved ferrochelatase domain (see figure 1 ).

Embodiment 2B

[0025] The knockout of embodiment 2BcFch1 gene

[0026] 1) Construction of knockout vector

[0027] Using primers Fch1-UP-F (5'-CTCGAGAAGAGCGGCAAGCGTGGGA-3') and Fch1-UP-R (5'-GGTACCGGTACTCGAAAGGCAGACAGA-3'), the upstream of the BcFch1 gene was amplified using the genomic DNA of Botrytis cinerea strain B05.10 as a template 794bp fragment, using Fch1-DN-F (5'-GGATCCGATGTCCTGGATGCAAGAGTGA-3') and Fch1-DN-R (5'-CTGCAGGTGGGTTGGTGTACGATATTCG-3') to amplify the downstream 767bp fragment of Botrytis cinerea BcFch1 gene, the reaction system is: 10mmol / LdNTPMixture, 0.5 μL; 10×PCRbuffer, 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) 58°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 product...

Embodiment 3B

[0037] Genetic Complementation of Example 3 BcFch1 Gene Deletion Mutant

[0038] Primers C-F (5'-GAATTCTTGTTCGCTTCCTCTTCGTC-3') and C-R (5'-CTGCAGGTGGGTTGGTGTACGATATTCG-3') were used to amplify the full-length 3116bp gene of Botrytis cinerea BcFch1 (including promoter, open reading frame and terminator), and cloned into pMD18-t vector, and then subcloned into the pSULF vector (containing chlorimuron-methyl resistance gene) between the EcoRI and PstI sites to construct a genetic complementation vector pFch1-ko-c. The vector was verified by sequencing to confirm that there were no amino acid mutations. Using the Agrobacterium-mediated transformation method as described above, 100 μg / mL chlorimuron-methyl was used for screening, and the complementary fragment was transferred into the genome of the BcFch1 gene deletion mutant M1 to obtain a genetically complementary strain M1 / Fch1. The primers a and b, c and d used in the mutant verification were selected for PCR amplification, a...

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Abstract

The invention provides a botrytis cinerea gene BcFch1 relative to pathogenicity and application of the botrytis cinerea gene BcFch1, and belongs to the technical field of microbiological genetic engineering. The DNA sequence of the gene BcFch1 sourced from botrytis cinerea and used for controlling pathogenicity is shown in SEQ ID No:1, and comprises 1409 nucleotides. The amino acid sequence of protein coded by the gene BcFch1 is shown in SEQ ID No:2, and comprises 435 amino acids. The gene BcFch1 can be applied to the field of gene engineering of plant botrytis cinerea-resisting gray molds. Deletion, mutation or modification is conducted on the protein coded by the gene BcFch1 used for controlling the pathogenicity of botrytis cinerea to ensure that the pathogenicity of protein is flawed, and the flawed protein can be applied to design and screening of antifungal medicaments while serving as a target.

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 the fungus of Ascomycota and is the pathogenic fungus of gray mold. It can infect more than 200 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 ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60
CPCC12N9/88C12Y499/01001
Inventor 李桂华李乐涛秦庆明张明哲
Owner JILIN UNIV
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