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Botrytis cinerea gene BcCpo1 relative to pathogenicity and application of botrytis cinerea gene BcCpo1

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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  • 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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  • Botrytis cinerea gene BcCpo1 relative to pathogenicity and application of botrytis cinerea gene BcCpo1
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  • Botrytis cinerea gene BcCpo1 relative to pathogenicity and application of botrytis cinerea gene BcCpo1

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Experimental program
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Effect test

Embodiment 1B

[0023] Correlation analysis of embodiment 1 BcCpo1 gene

[0024] The open reading frame of the BcCpo1 gene of Botrytis cinerea consists of 1074 nucleotides, including 2 exons, the full-length cDNA of the coding region is 1020 nucleotides, and the encoded protein product consists of 339 amino acids. The BcCpo1 protein sequence was compared and analyzed (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and it was found that Cpo1 widely exists in cellular organisms such as animals, plants, fungi and bacteria. Domain analysis revealed that the BcCpo1 protein contains a conserved coproporphyrinogen III oxidase domain (see figure 1 ).

Embodiment 2B

[0025] The RNA interference of embodiment 2BcCpo1 gene

[0026] 1) Construction of interference carrier

[0027] Using primers Cpo1-Ri-F (5'-GGTACCCTCGAGCGGCAATTGACAAAAT-3') and Cpo1-Ri-R (5'-AGATCTAAGCTTTCAAACAACTTTGCGC-3'), the BcCpo1 gene encoding was amplified using the genomic DNA of Botrytis cinerea strain B05.10 as a template Region 432bp fragment, the reaction system is: 10mmol / LdNTPMixture, 0.5μL; 10×PCRbuffer, 2.5μL; each 1μL of upstream and downstream primers (10μmol / mL); template DNA, 1μL; Ex-Taq, 0.2μL (5U); ddH 2 O, 18.8 μL; amplification program: 94°C pre-denaturation for 3 minutes, then (1) 94°C, denaturation for 30 seconds; (2) 58°C, annealing for 30 seconds; (3) 72°C, extension for 30 seconds; (4) ) cycled 30 times; (5) extended at 72°C for 10 minutes. The above-mentioned DNA amplification products were cloned successively in the opposite direction between XhoI, HindIII and between KpnI and BglII of the pSilent1 vector; then the entire interference region (...

Embodiment 3

[0035] Detection of the expression level of BcCpo1 gene in the embodiment 3 RNA interference mutant

[0036] BcCpo1 protein levels in related strains were detected by western blot. The total protein of the wild-type and mutant strains were extracted respectively, separated by SDS-PAGE electrophoresis, and then transferred to PVDF membrane by electrophoresis. The primary antibody of human Cpo1 protein was used for western blot detection, and the secondary protein conjugated with horseradish peroxidase Color development was performed after anti-hybridization. The primary antibody of yeast Actin protein was used to detect the Actin protein content of Botrytis cinerea-related strains as an internal reference. The results showed that in the RNA interference mutant, the content of BcCpo1 protein was significantly reduced, and its residual level could only reach 18% of that in the wild-type strain (see image 3 ). Therefore, the RNA interference mutant of BcCpo1 can be used for su...

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Abstract

The invention provides a botrytis cinerea gene BcCpo1 relative to pathogenicity and application of the botrytis cinerea gene BcCpo1, and belongs to the technical field of microbiological genetic engineering. The DNA sequence of the gene BcCpo1 sourced from botrytis cinerea and used for controlling pathogenicity is shown in SEQ ID No:1, and comprises 1074 nucleotides. The amino acid sequence of protein coded by the gene BcCpo1 is shown in SEQ ID No:2, and comprises 339 amino acids. The gene BcCpo1 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 BcCpo1 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 Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12R1/645
CPCC12N9/001C12Y103/99022
Inventor 李桂华李乐涛秦庆明张明哲
Owner JILIN UNIV
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