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Genetic Transformation Method of Agrobacterium Tumefaciens Mediated by Agrobacterium tumefaciens

A genetic transformation method, technology of Agrobacterium tumefaciens, applied in biochemical equipment and methods, botany equipment and methods, fermentation, etc.

Active Publication Date: 2019-06-21
广西壮族自治区农业科学院生物技术研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there have been no reports on the genetic transformation system of P. solani. Therefore, establishing a genetic transformation system of P. solani and applying it to the research on pathogenicity-related genes of P. solani will help to analyze the pathogenicity of P. solani It is of great significance to promote the molecular biology research of water chestnut stem blight

Method used

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  • Genetic Transformation Method of Agrobacterium Tumefaciens Mediated by Agrobacterium tumefaciens
  • Genetic Transformation Method of Agrobacterium Tumefaciens Mediated by Agrobacterium tumefaciens
  • Genetic Transformation Method of Agrobacterium Tumefaciens Mediated by Agrobacterium tumefaciens

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Experimental program
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Embodiment 1

[0019] Embodiment 1 Obtaining of water chestnut stem blight bacteria T-DNA insertion transformants

[0020] 1> Preparation of conidia of water chestnut stem blight (all operations are carried out under sterile conditions)

[0021] Bacteria spp. on potato-dextrose-agar (PDA) medium ( figure 1 A) After cultivating for 15-20 days, scrape off the conidia of the water chestnut stem blight bacteria with sterilized deionized water, then filter and remove the mycelia with sterile mirror paper, and use the water chestnut stem blight bacteria conidia filtrate obtained Deionized water adjusted to a concentration of 10 5 -10 8 Spores / ml, to obtain the conidia of the water chestnut blight ( figure 1 B) The suspension is ready for use.

[0022] 2> Preparation of carrier-containing Agrobacterium

[0023]Utilize carrying green fluorescent protein gene eGFP and hygromycin resistance screening gene hpth( figure 2 ) is the transformation vector, and the plasmid pEV vector is transformed i...

Embodiment 2

[0028] Example 2 Identification of transformant of water chestnut stem blight

[0029] 1> Fluorescence microscope identification

[0030] The colonies of the transformants of P. chinensis blight were placed under a fluorescence microscope to observe the fluorescence. Fluorescence was not detected in the wild type of P. chinensis, and the transformants transformed with Agrobacterium glowed green under the ultraviolet excitation light of the fluorescence microscope, which proved that the green fluorescent protein GFP of Agrobacterium had been successfully transferred to and expressed in P. chinensis. ( image 3 ).

[0031] 2) PCR verification

[0032] Primers hpth-F: GCGTGGAGAAGTTCCTCATCGAGA (SEQ.ID.No.2) / hpth-R: GAGCCTTGGGCTTCCAGCAA (SEQ. ID.No.3) Carry out PCR amplification on the transformants to preliminarily verify whether a mutant with an insertion is really obtained.

[0033] Composition of PCR reaction solution (25 μL system): Add 2.5 μL 10×PCR Buffer, 2.0 μL 2.5 μm...

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Abstract

The invention discloses a genetic transformation method for agrobacterium tumefaciens-mediated cylindrosporium eleocharidis. The genetic transformation method is characterized in that conidia of the cylindrosporium eleocharidis are used as a transformation receptor material; agrobacterium tumefaciens containing a binary vector is mixed with the conidia of the cylindrosporium eleocharidis and then is co-cultured with the conidia of the cylindrosporium eleocharidis, and screening is carried out by antibiotics to obtain a resistant transformant, so that an efficient genetic transformation system is established for the cylindrosporium eleocharidis. The method is simple to operate, is high in transformation rate and is good in stability; the method is used for constructing a cylindrosporium eleocharidis mutant library; the transformant obtained by genetic transformation of the agrobacterium tumefaciens-mediated cylindrosporium eleocharidis can provide a rich screening bacteria source for screening out cylindrosporium eleocharidis strains with low pathogenicity and pathogenic defects, and also lays the foundation for cloning pathogenic related genes of the cylindrosporium eleocharidis and deeply researching pathogenesis of the cylindrosporium eleocharidis.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to a method for genetic transformation of water chestnut stem blight mediated by Agrobacterium tumefaciens. Background technique [0002] Water chestnut (Eleocharis dulcis) belongs to Cyperaceae (Cyperaceae) Eleocharis (Eleocharis), is a perennial herb, also known as ground chestnut, black taro, horse mang, red arrowhead, horseshoe and so on. It is native to southern my country and India, and has a cultivation history of more than 2,000 years in my country. At present, water chestnuts are widely cultivated in the Yangtze River Basin and the provinces to the south of China, and Guangxi is widely planted in Hepu, Guilin, Hezhou and other regions; it is also cultivated in the United States, North Korea, Vietnam, Australia, Japan and other countries. According to incomplete statistics, the cultivation area of ​​water chestnuts in my country was about 3...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H5/00
CPCC12N15/8205C12N15/8209
Inventor 颜梅新江文欧昆鹏桂杰董伟清高美萍何芳练张尚文
Owner 广西壮族自治区农业科学院生物技术研究所
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