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A Botrytis cinerea gene bcals1 related to pathogenicity and its application

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

Inactive Publication Date: 2019-03-26
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A Botrytis cinerea gene bcals1 related to pathogenicity and its application
  • A Botrytis cinerea gene bcals1 related to pathogenicity and its application
  • A Botrytis cinerea gene bcals1 related to pathogenicity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Correlation analysis of BcAls1 gene

[0024] The open reading frame of the BcAls1 gene of Botrytis cinerea consists of 1918 nucleotides, including 2 exons, the full-length cDNA of the coding region is 1851 nucleotides, and the encoded protein product consists of 616 amino acids. The BcAls1 protein sequence was compared and analyzed (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and it was found that Als1 widely exists in cellular organisms such as animals, plants, fungi and bacteria. Domain analysis revealed that the BcAls1 protein contains a conserved aminolevulinic acid synthase domain (see figure 1 ).

Embodiment 2

[0025] Example 2 Knockout of BcAls1 gene

[0026] 1) Construction of knockout vector

[0027] Using primers Als1-UP-F (5'-GAATTCATGGTGCTCGTATCGTTTGA-3') and Als1-UP-R (5'-GGTACCGCGGGATGATGGATTATTATGT-3') to amplify the upstream of the BcAls1 gene using the genomic DNA of Botrytis cinerea strain B05.10 as a template 603bp fragment, using Als1-DN-F (5'-GGATCCTCCTCGGACCCAATACCAC-3') and Als1-DN-R (5'-AAGCTTCTTTAAGCGAGGTCACGGA-3') to amplify the downstream 911bp fragment of Botrytis cinerea BcAls1 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) 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 products were succ...

Embodiment 3

[0037] Example 3 Genetic Complementation of BcAls1 Gene Deletion Mutants

[0038] Primers C-F (5'-GTCGACTGATGGGAAACCCTGGATG-3') and C-R (5'-GTCGACCTTTAAGCGAGGTCACGGA-3') were used to amplify the full-length 3875bp gene of Botrytis cinerea BcAls1 (including promoter, open reading frame and terminator), and cloned into pMD18-t vector, and then subcloned into the SalI site of pSULF vector (containing chlorimuron-methyl resistance gene), and constructed into genetic complementation vector pAls1-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 BcAls1 gene deletion mutant M1 to obtain a genetically complementary strain M1 / Als1. The primers a and b, c and d used in the mutant verification were selected for PCR amplification, and the results w...

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Abstract

The invention provides a botrytis cinerea gene BcAls1 relative to pathogenicity and application of the botrytis cinerea gene BcAls1, and belongs to the technical field of microbiological genetic engineering. The DNA sequence of the gene BcAls1 sourced from botrytis cinerea and used for controlling pathogenicity is shown in SEQ ID No:1, and comprises 1918 nucleotides. The amino acid sequence of protein coded by the gene BcAls1 is shown in SEQ ID No:2, and comprises 616 amino acids. The gene BcAls1 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 BcAls1 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/10A01H5/00
CPCC12N9/1029C12N15/8282C12Y203/01037
Inventor 秦庆明曹胜男李桂华李乐涛张明哲
Owner JILIN UNIV
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