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Genetically engineered bacillus amyloliquefaciens effectively preventing gray mold and its preparation method and application

Amyloliquefaciens and genetic engineering technologies are applied in the field of genetically engineered Bacillus amyloliquefaciens for effective control of botrytis cinerea and the field of preparation thereof, and can solve the problems of tomato yield reduction, environmental pollution, crop yield reduction and the like

Active Publication Date: 2021-05-25
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Fungal plant diseases are the main factors restricting the high yield of plants. The prevalence of fungal diseases has caused extremely serious crop yield reduction. Tomatoes are often attacked by fungal diseases such as Botrytis cinerea, leaf mold, root rot, and early blight. leading to a massive reduction in tomato production
At present, the main means of prevention and control of Botrytis cinerea is chemical control, chemical control will pollute the environment, and problems such as pesticide residues are also harmful to human health; the prevention and control of Botrytis cinerea through disease-resistant breeding has been expected to achieve complete control, but At present, no antigenic material of Botrytis cinerea has been found, so it is not feasible to control Botrytis cinerea through disease-resistant breeding; currently, it is the best solution to use some environmentally friendly and harmless microorganisms to prevent and control fungal diseases through biological control. It can not only fill the deficiency of chemical control, but also ensure the cleanliness of crops

Method used

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  • Genetically engineered bacillus amyloliquefaciens effectively preventing gray mold and its preparation method and application
  • Genetically engineered bacillus amyloliquefaciens effectively preventing gray mold and its preparation method and application
  • Genetically engineered bacillus amyloliquefaciens effectively preventing gray mold and its preparation method and application

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specific Embodiment approach 1

[0051] A strain of genetically engineered Bacillus amyloliquefaciens that effectively prevents Botrytis cinerea. On June 29, it was deposited in the China Center for Microorganism Culture Collection (CGMCC), with the preservation number CGMCC No.14366, and the preservation address is No. 3, Yard No. 1, Beichen Road, Chaoyang District, Beijing.

[0052] The genetically engineered Bacillus amyloliquefaciens effectively preventing and treating Botrytis cinerea described in this embodiment, the construction of the recombinant vector is as attached figure 1 As shown, by means of genetic engineering, the chitinase gene ech42 of M. pinkis was amplified and connected to the expression vector pHT43, and the recombinant expression vector pHT43 / ech42 was successfully constructed.

[0053] The genetically engineered Bacillus amyloliquefaciens that effectively prevents Botrytis cinerea described in this embodiment, the target protein electrophoresis test picture is as attached figure 2 ...

specific Embodiment approach 2

[0069] The preparation method of the genetically engineered Bacillus amyloliquefaciens effectively preventing and treating Botrytis cinerea according to specific embodiment 1 comprises the following steps:

[0070] Step A, amplifying the G. pink chitinase gene ech42 through genetic engineering, and connecting the Bacillus amyloliquefaciens expression vector pHT43 to construct the recombinant expression vector pHT43 / ech42,

[0071]Step B. Introduce the recombinant expression vector pHT43 / ech42 described in step 1 into Bacillus amyloliquefaciens by setting electric shock transformation, and the electric shock voltage is 12.5kv / cm or 15kv / cm or 17.5kv / cm.

specific Embodiment approach 3

[0073] According to the preparation method of the genetically engineered Bacillus amyloliquefaciens effectively preventing and treating Botrytis cinerea according to specific embodiment one or two, step A comprises the following steps:

[0074] Step A1, obtaining the C. rosea chitinase gene ech42 by PCR cloning;

[0075] Step A2, linking the chitinase gene ech42 obtained in step A1 with the T carrier;

[0076] Step A3, linking the chitinase gene ech42 obtained in step 2 with the expression vector pHT43.

[0077] The method for preparing the genetically engineered Bacillus amyloliquefaciens that effectively prevents Botrytis cinerea described in this embodiment, in step A1, finds the gene sequence of the C. rosea chitinase ech42 gene (DQ523687) through GenBank, and uses the signal peptide detection software, Detect the signal peptide sequence, and the cloned gene does not contain the enzyme cutting site introduced on the primer, and design primers with primmer 5 software:

[...

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Abstract

The invention relates to genetically engineered bacillus amyloliquefaciens which can effectively prevent gray mold and its preparation method and application, belonging to the field of molecular biology. In the present invention, through genetic engineering, the chitinase gene ech42 of G. pink is amplified, and the expression vector pHT43 is connected to construct the recombinant expression vector pHT43 / ech42. By setting electric shock transformation, the recombinant expression vector pHT43 / When ech42 is introduced into Bacillus amyloliquefaciens, the electric shock voltage is 12.5kv / cm, 15kv / cm, or 17.5kv / cm. In the detection of chitinase activity of the present invention, the enzyme activity of the pHT43-ech42 / BA strain at the 8th hour is about 54.3% higher than that of the wild-type strain BA. In terms of preventing Botrytis cinerea, the disease prevention effect is still 28.9% after spraying for 20 days. % or so, with long-lasting biological control effect.

Description

technical field [0001] The invention belongs to the field of molecular biology; in particular, it relates to a genetically engineered bacillus amyloliquefaciens that can effectively prevent gray mold and its preparation method and application. Background technique [0002] Fungal plant diseases are the main factors restricting the high yield of plants. The prevalence of fungal diseases has caused extremely serious crop yield reduction. Tomatoes are often attacked by fungal diseases such as Botrytis cinerea, leaf mold, root rot, and early blight. leading to a substantial reduction in tomato production. At present, the main means of prevention and control of Botrytis cinerea is chemical control, chemical control will pollute the environment, and problems such as pesticide residues are also harmful to human health; the prevention and control of Botrytis cinerea through disease-resistant breeding has been expected to achieve complete control, but At present, no antigenic materi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75A01N63/22A01P3/00C12R1/07
CPCA01N63/00C12N9/2442C12N15/75C12Y302/01014
Inventor 王傲雪王旭东陈秀玲张淑梅张瑶刘佳音
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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