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A Botrytis cinerea gene bcudc1 related to pathogenicity and its application

A technology of Botrytis cinerea, 1.bcudc1, applied in the application field of genes and their encoded proteins, can solve the problems of little known molecular mechanisms

Inactive Publication Date: 2019-05-03
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 bcudc1 related to pathogenicity and its application
  • A Botrytis cinerea gene bcudc1 related to pathogenicity and its application
  • A Botrytis cinerea gene bcudc1 related to pathogenicity and its application

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Experimental program
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Embodiment 1B

[0025] Correlation analysis of embodiment 1BcUdc1 gene

[0026] The open reading frame of the BcUdc1 gene of Botrytis cinerea consists of 1323 nucleotides, including 5 exons, the full-length cDNA of the coding region is 1095 nucleotides, and the encoded protein product consists of 364 amino acids. The BcUdc1 protein sequence was compared and analyzed (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and it was found that Udc1 widely exists in cellular organisms such as animals, plants, fungi and bacteria. Phylogenetic tree analysis (http: / / phylogeny.lirmm.fr / phylo_cgi / simple_phylogeny.cgi) showed that the homology between filamentous fungal Udc1 proteins was high, among which BcUdc1 was closest to S. sclerotiorum Udc1; BcUdc1 was closely related to Yeast is a little further away, followed by animals, bacteria, and most distantly related to green algae and plants (see figure 1 ).

Embodiment 2B

[0027] Example 2 Knockout and Genetic Complementation of BcUdc1 Gene

[0028] 1) Construction of knockout vector

[0029] Udc1-UP-F(5'-CTCGAGGCTCCGTGGAAACACCTACA-3)' and Udc1-UP-R(5'-GAATTCAAATAAGTTGACAGGGACGAGA-3') were used to amplify the upstream of BcUdc1 gene using the genomic DNA of Botrytis cinerea strain B05.10 as a template 655bp fragment, use Udc1-DN-F (5'-GGATCCAAACCGCCCAGTTGTCTTCC-3') and Udc1-DN-R (5'-CTGCAGAGAAACCCTCGCCATCAAAC-3') to amplify the downstream 699bp fragment of Botrytis cinerea BcUdc1 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 2O, 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 p...

Embodiment 3

[0041] The role of embodiment 3 BcUdc1 gene in the process of conidia germination of Botrytis cinerea

[0042] The variation of conidia germination of BcUdc1 mutant was determined by slide culture method. PDB (20% potato boiled and filtered, glucose 2%) spore suspension (5×10 4 mL- 1 ) was dropped on the glass slide, incubated at 20°C with moisture, and microscopically observed after 12h and 24h respectively. The experimental results showed that the conidia of the wild-type strain of Botrytis cinerea had germinated within 12 hours, and grew long germ tubes (60-100 μm), while only a small amount of conidia of the BcUdc1 mutant germinated, and the growth was small , even at 24h, it is only about 20 μm, indicating that the BcUdc1 gene plays an important role in the germination of Botrytis cinerea conidia (see Figure 4 ).

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Abstract

The invention provides a botrytis cinerea gene BcUdc1 relative to pathogenicity and application of the botrytis cinerea gene BcUdc1, and belongs to the technical field of microbiological genetic engineering. The DNA sequence of the gene BcUdc1 sourced from botrytis cinerea and used for controlling conidialgermination and pathogenicity is shown in SEQ ID No:1, and comprises 1323 nucleotides. The amino acid sequence of protein coded by the gene BcUdc1 is shown in SEQ ID No:2, and comprises 364 amino acids. The gene BcUdc1 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 BcUdc1 used for controlling the conidialgermination and 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 the germination and pathogenicity of fungal conidia and its encoded 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 w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N9/88C12R1/645
CPCC12N9/88C12Y401/01037
Inventor 秦庆明李乐涛李桂华张明哲
Owner JILIN UNIV
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