Agrobacterium-mediated Leptosphaeria biglobosa genetic transformation method

A genetic transformation method, Agrobacterium-mediated technology, applied in biochemical equipment and methods, introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of cumbersome operation, low transformation efficiency, and unstable transformants. Achieve the effect of genetic stability, low copy and high transformation efficiency

Active Publication Date: 2019-09-03
INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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Problems solved by technology

At present, the widely used fungal transformation methods include plasmid co-transformation, electric shock transformation, gene gun, restriction endonuclease-mediated transformation (REMI) and Agrobacterium-mediated transformation, but plasmid co-transformation, electric shock transformation, gene gun method, restriction Transformation methods such as endonuclease-mediated transformation are prone to

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  • Agrobacterium-mediated Leptosphaeria biglobosa genetic transformation method
  • Agrobacterium-mediated Leptosphaeria biglobosa genetic transformation method
  • Agrobacterium-mediated Leptosphaeria biglobosa genetic transformation method

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Embodiment 1

[0035] Medium used in this embodiment:

[0036] PDA medium: 200g of potatoes, 20g of glucose, 15g of agar powder, add water to make up to 1000mL, sterilize by high pressure steam at 121°C for 20min.

[0037] LB medium: yeast powder 5g, tryptone 10g, sodium chloride 10g, agar powder 15g, add water to 1000mL, sterilize by high pressure steam at 121°C for 20min.

[0038] CM co-cultivation medium: K 2 HPO 4 3.44g, KH 2 PO 4 l.45g, NaCl0.15g, MgSO 4 ·7H 2 O0.5g, (NH 4 )2SO 4 0.5gCaCl 2 ·H 2 O0.067g, FeSO 4 ·7H 2 O0.0025g, Glucose1.8g, MES7.8g, glycerol 5mL, agar powder 15g, distilled water to 1000mL, pH value 5.8.

[0039] IM medium: based on CM medium, add acetosyringone with a final concentration of 200 μg / mL in CM medium.

[0040] SM screening medium: Based on PDA medium, cefotaxime sodium with a final concentration of 100 mg / mL and hygromycin B with a final concentration of 50 mg / mL were added to the PDA medium.

[0041] Method steps:

[0042] Agrobacterium LBA44...

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Abstract

The invention provides an Agrobacterium-mediated leptosphaeria biglobosa genetic transformation method, which belongs to the technical field of genetic transformation of plant pathogenic fungi. The method comprises the following steps: 1) inoculating agrobacterium containing plasmid pCHs-GFP in a LB liquid medium for culture, and then transferring the agrobacterium into a CM liquid medium containing 150-250 [mu]g/mL acetosyringone to obtain agrobacterium bacterial fluid; 2) collecting the conidiospore of the leptosphaeria biglobosa, and mixing the conidiospore with sterile water to prepare a conidiospore suspension having a concentration of (0.5-9)*10<6>/mL; and 3) mixing the agrobacteriumbacteria liquid with the conidiospore suspension, co-cultivating the material for 3-4 days, transferring the material to a screening medium, and screening and culturing the material for 7-15 days to obtain a transformant; The method has high transformation efficiency, low copy and genetic stability.

Description

technical field [0001] The invention belongs to the technical field of genetic transformation of plant pathogenic fungi, and in particular relates to a method for genetic transformation of Rapeseed blackleg fungus mediated by Agrobacterium. Background technique [0002] Rapeseed blackleg (Phoma stem canker) is currently one of the important factors restricting the healthy development of rapeseed industry in many countries. Studies have shown that in many rapeseed producing areas where black shank is endemic in the world, the pathogenic compound species Leptosphaeria maculans and Leptosphaeria biglobosa co-exist and infect host crops alone or together. L.maculans can cause rot at the base of rapeseed stems and cause severe yields loss. The spread and evolution of rapeseed black shank in various countries in the world showed that L.biglobosa was gradually replaced by L.maculans. In 1999, the pathogenic bacteria L.biglobosa was isolated and obtained for the first time in my c...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65
CPCC12N15/65C12N15/8205
Inventor 宋培玲皇甫海燕李子钦吴晶史志丹燕孟娇郭晨皇甫九茹贾晓清郝丽芬魏晓军郭建靖杨永青
Owner INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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