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Agrobacterium tumefaciens-mediated genetic transformation method of corynespora cassiicola

A genetic transformation method, the technology of Agrobacterium tumefaciens, is applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials. Problems such as inability to convert

Inactive Publication Date: 2019-04-05
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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Problems solved by technology

However, the conditions required for genetic transformation of different fungi are different, the transformation parameters are different, and the influencing factors are different
So far, there is no report on the genetic transformation of Corynespora polybasicum mediated by Agrobacterium tumefaciens, and the transformation cannot be carried out smoothly by referring to the transformation methods of other filamentous fungi

Method used

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  • Agrobacterium tumefaciens-mediated genetic transformation method of corynespora cassiicola
  • Agrobacterium tumefaciens-mediated genetic transformation method of corynespora cassiicola
  • Agrobacterium tumefaciens-mediated genetic transformation method of corynespora cassiicola

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Embodiment 1

[0039] Agrobacterium tumefaciens-mediated multi-main Corynesium genetic transformation test, the specific steps are as follows:

[0040] (1) Cc- Sensitivity determination of GX strains to hygromycin. Agrobacterium tumefaciens mediated C. Cc- In the genetic transformation system of GX strain, choosing the appropriate concentration of hygromycin is very important for the selection of mutants. The colony preserved on the slant was transferred to a PDA plate and activated in the dark at 25°C. Then, use a sterilized hole puncher to punch a bacterium cake with a diameter of 5 mm from the edge of the colony, and then place the bacterium cake in the center of a new PDA plate in turn, inoculate a piece of bacterium cake on each plate, and place the inoculated plate at a constant temperature Incubate in the dark at 25°C for 7 days in an incubator. Then use a sterilized hole puncher to punch a 5mm-diameter bacterium cake from the edge of the colony, and place the bacterium cake in ...

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Abstract

The invention provides an agrobacterium tumefaciens-mediated genetic transformation method of corynespora cassiicola. The method comprises the following steps: (1) determining the sensitivity of the corynespora cassiicola to hygromycin; (2) screening the concentration of antibiotics inhibiting the normal growth of agrobacterium; (3) preparing suspension liquid of conidia of the corynespora cassiicola; (4) preparing an agrobacterium tumefaciens bacteria solution containing a binary vector; (5) co-culturing agrobacterium tumefaciens with the conidia of the corynespora cassiicola; (6) screening transformants; and (7) performing molecular identification on the transformants, and performing insertion of a copy number and genetic stability analysis. The method provided by the invention can be used for preparing protoplast without needing removal of the fungal cell wall, and is simple to operate and high in conversion efficiency; and the obtained transformants are large in single copy ratio and can be stably inherited. The invention provides the stable and efficient agrobacterium tumefaciens-mediated genetic transformation method of the corynespora cassiicola, thus providing a potential possibility for obtaining pathogenic mutants, and laying a foundation for in-depth research on pathogenic related genes and pathogenic mechanisms.

Description

technical field [0001] The invention relates to a method for genetic transformation of Corynesium polybasicum mediated by Agrobacterium tumefaciens, belonging to the field of genetic engineering. Background technique [0002] Corynespora polyarcha is an important plant pathogen with a wide range of hosts. The pathogen often damages the leaves of plants to form target-like lesions, and can also infect the flowers, fruits, stems and roots of the host. However, the genetic mechanism of Corynespora polymata pathogenicity is still unclear. In view of the economic importance of Corynespora polymata leaf spot on crops, new strategies should be developed to provide new methods for in-depth study of pathogenicity-related diseases at the molecular level. Genes and pathogenic mechanisms are imperative. [0003] Fungal transformation technology destroys genes in a random or targeted manner to produce mutants, which provides mutant materials for in-depth study of fungal pathogenicity-r...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12R1/645
CPCC12N15/80
Inventor 李俊香古勤生
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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