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Agrobacterium tumefaciens mediated Corynespora cassiicola efficient genetic transformation method

A genetic transformation method, Agrobacterium-mediated technology, applied in the field of Agrobacterium-mediated high-efficiency genetic transformation of Corynespora polybasicum, can solve the problems of low transformation efficiency and achieve high transformation efficiency and simple operation

Pending Publication Date: 2019-09-20
上海润庄农业科技有限公司
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Problems solved by technology

[0005] The present invention proposes an Agrobacterium-mediated high-efficiency genetic transformation method of Corynespora multiprimum, which solves the problem of low transformation efficiency of the PEG-mediated protoplast genetic transformation method in the prior art

Method used

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Effect test

Embodiment 1

[0029] A high-efficiency genetic transformation method for Corynespora polymains mediated by Agrobacterium, comprising:

[0030] (1) the preparation step of many main Corynesporum spore suspensions;

[0031] Corynespora multiprimum (HG20101029-1) was cultured on a PDA plate (diameter 9cm) at 25°C for 10 days, with 12 hours of light and 12 hours of darkness alternately every day; (b) 5 mL of ddH was added to each plate 2 O, wash the spores gently with a brush, and then filter them with three layers of sterile gauze; (c) collect the spores by centrifugation at room temperature at 7000rpm for 5min, and use ddH 2 O Resuspend the spores and adjust the spore concentration of the spore suspension to 2 × 10 5 CFU / mL.

[0032] (2) Transform Agrobacterium AGL1 competent cells with binary vector pCAMBIA1300; screen and identify positive Agrobacterium transformants.

[0033] (3) Activate the above-mentioned positive Agrobacterium transformants on LB plates containing 100 μg / mL rifampic...

Embodiment 2

[0038] A high-efficiency genetic transformation method for Corynespora polymains mediated by Agrobacterium, comprising:

[0039] (1) the preparation step of many main Corynesporum spore suspensions;

[0040] Corynespora multiprimum (HG20101029-1) was cultured on a PDA plate (diameter 9cm) at 25°C for 10 days, with 12 hours of light and 12 hours of darkness alternately every day; (b) 5 mL of ddH was added to each plate 2 O, wash the spores gently with a brush, and then filter them with three layers of sterile gauze; (c) collect the spores by centrifugation at room temperature at 7000rpm for 5min, and use ddH 2 O Resuspend the spores and adjust the spore concentration of the spore suspension to 2 × 10 5 CFU / mL.

[0041] (2) Transform Agrobacterium AGL1 competent cells with binary vector pCAMBIA1300; screen and identify positive Agrobacterium transformants.

[0042] (3) Activate the above-mentioned positive Agrobacterium transformants on LB plates containing 100 μg / mL rifampic...

Embodiment 3

[0047] A high-efficiency genetic transformation method for Corynespora polymains mediated by Agrobacterium, comprising:

[0048] (1) the preparation step of many main Corynesporum spore suspensions;

[0049] Corynespora multiprimum (HG20101029-1) was cultured on a PDA plate (diameter 9cm) at 25°C for 10 days, with 12 hours of light and 12 hours of darkness alternately every day; (b) 5 mL of ddH was added to each plate 2 O, wash the spores gently with a brush, and then filter them with three layers of sterile gauze; (c) collect the spores by centrifugation at room temperature at 7000rpm for 5min, and use ddH 2 O Resuspend the spores and adjust the spore concentration of the spore suspension to 2 × 10 5 CFU / mL.

[0050] (2) Transform Agrobacterium AGL1 competent cells with binary vector pCAMBIA1300; screen and identify positive Agrobacterium transformants.

[0051] (3) Activate the above-mentioned positive Agrobacterium transformants on LB plates containing 100 μg / mL rifampic...

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Abstract

The invention provides an Agrobacterium tumefaciens mediated Corynespora cassiicola efficient genetic transformation method. The method includes the steps of firstly, preparing a Corynespora cassiicola spore suspension; secondly, using binary vector pCAMBIA1300 to convert Agrobacterium tumefaciens AGL1 competent cells; screening and identifying positive Agrobacterium tumefaciens transformants; activating the Agrobacterium tumefaciens transformants, and transferring single colony into an LB liquid culture medium containing rifampicin and kanamycin to perform culture; fourthly, preparing a positive Agrobacterium tumefaciens transformant bacterium solution; fifthly, mixing the Corynespora cassiicola spore suspension with the positive Agrobacterium tumefaciens transformant bacterium solution, incubating, and culturing in an IM culture medium containing acetosyringone; sixthly, performing resistance screening on Corynespora cassiicola positive transformants. The method is high in conversion efficiency and simple inn operation.

Description

technical field [0001] The invention relates to the field of genetic transformation, in particular to an Agrobacterium-mediated high-efficiency genetic transformation method of Corynespora multiprimum. Background technique [0002] Corynespora cassiicola (Berk. & Curt.) Wei. is a filamentous fungus of the subphylum Corynespora, which can infect more than 500 kinds of vegetables and economic crops such as cucumbers, tomatoes, tobacco, rubber, and cotton , causing leaf spot disease, the annual occurrence area exceeds 667,000 hectares, and the loss exceeds 5 billion. The identification, cloning and functional analysis of pathogenicity-related genes will help to reveal the molecular mechanism of the pathogenic bacteria and the effective prevention and control of the disease. At present, PEG-mediated protoplast genetic transformation can be used to study the gene function of pathogens. [0003] However, on the one hand, the PEG-mediated protoplast genetic transformation method ...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12R1/645
CPCC12N15/80
Inventor 高士刚戴富明曾蓉徐丽慧王良军杨晓峰
Owner 上海润庄农业科技有限公司
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