A Botrytis cinerea gene bcpda1 related to pathogenicity and its application

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: 2019-05-07
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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  • A Botrytis cinerea gene bcpda1 related to pathogenicity and its application
  • A Botrytis cinerea gene bcpda1 related to pathogenicity and its application
  • A Botrytis cinerea gene bcpda1 related to pathogenicity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Correlation analysis of BcPda1 gene

[0024] The open reading frame of BcPda1 gene of Botrytis cinerea consists of 1140 nucleotides, only contains one exon (no intron), and the encoded protein product consists of 379 amino acids. The BcPda1 protein sequence was compared and analyzed (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and it was found that Pda1 widely exists in cellular organisms such as animals, plants, fungi and bacteria. Domain analysis revealed that the BcPda1 protein contains a conserved porphobilinogen deaminase domain (see figure 1 ).

Embodiment 2

[0025] Example 2 Knockout of BcPda1 Gene

[0026] 1) Construction of knockout vector

[0027] Primer Pda1-UP-F(5'- GAATTC CGGCTTCCATTTCTGCTAC-3') and Pda1-UP-R(5'- GGTACC CACAACCACGACCAACCAT-3'), using the genomic DNA of Botrytis cinerea strain B05.10 as a template to amplify the 762bp fragment upstream of the BcPda1 gene, using Pda1-DN-F (5'- TCTAGA TTCACAGGCACATAGGCTACA-3') and Pda1-DN-R(5'- CTGCAG ACTTTGGAATACTCACCTTTATCACT-3') to amplify the downstream 793bp fragment of Botrytis cinerea BcPda1 gene, the reaction system is: 10mmol / L dNTP Mixture, 0.5μL; 10×PCR buffer, 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 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...

Embodiment 3

[0037] Example 3 Genetic Complementation of BcPda1 Gene Deletion Mutants

[0038] Primers C-F (5'-GAATTCCGGCTTCCATTTCTGCTAC-3') and C-R (5'-CTGCAGACTTTGGAATACTCACCTTTATCACT-3') were used to amplify the full-length 2968bp gene of Botrytis cinerea BcPda1 (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 EcoR I and Pst I sites to construct a genetic complementation vector pPda1-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 BcPda1 gene deletion mutant M1 to obtain a genetically complementary strain M1 / Pda1. The primers a and b, c and d used in the mutant verification were selected for PCR amplificat...

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Abstract

The invention discloses a pathogenicity-related botrytis cinerea gene BcPda1 and application thereof and belongs to the technical field of microbiological genetic engineering. The DNA sequence of the gene BcPda1 which is from botrytis cinerea and controls pathogenicity is as indicated in SEQ ID No: 1 and composed of 1140 nucleotides; the amino acid sequence of protein encoded by the gene BcPda1 is as indicated in SEQ ID No: 1 and composed of 379 amino acids; the gene BcPda1 can be applied to the field of plant botrytis-cinerea-resistance genetic engineering; the protein BcPda1 controlling panthogenicity of the botrytis cinerea is subjected to deficiency or mutation or modification, so that pathogenicity of the botrytis cinerea has defects, and the pathogenicity-related botrytis cinerea gene BcPda1 can serve as a target to be applied to design and screening of antifungal medicament.

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): C12N15/60C12N9/88A01N63/04A01P3/00C12R1/645
CPCA01N63/30C12N9/88
Inventor 李桂华曹胜男李乐涛秦庆明张明哲
Owner JILIN UNIV
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