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Method for genetic transformation of volvariella volvacea by using electric shock method

A genetic transformation method, the technology of straw mushrooms, applied in the direction of biochemical equipment and methods, methods based on microorganisms, using vectors to introduce foreign genetic materials, etc., to achieve the effect of simple process, strong operability and low cost

Inactive Publication Date: 2016-09-07
SHANGHAI ACAD OF AGRI SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The electroshock genetic transformation method has the advantages of convenient operation, simple process and low input cost. It has been successfully applied to many edible fungi. However, there are no relevant reports on the application of the electroshock genetic transformation method to straw mushrooms at home and abroad.

Method used

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  • Method for genetic transformation of volvariella volvacea by using electric shock method
  • Method for genetic transformation of volvariella volvacea by using electric shock method
  • Method for genetic transformation of volvariella volvacea by using electric shock method

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Experimental program
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Effect test

Embodiment 1

[0032] 1) Determination of the sensitivity of the straw mushroom strain V23 to hygromycin: prepare PDA plates containing 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5ug / ml hygromycin, and treat each concentration 3 times repeat. Afterwards, on the ultra-clean bench, insert a square-shaped strain with a side length of 5 mm on each plate, and use the PDA plate without hygromycin as a control. After inoculation, they were cultured in a constant temperature incubator at 32°C, observed and recorded every day. Hygromycin has a great influence on the growth rate of straw mushroom V23. The hyphae grow extremely slowly at 15ug / ml, stop growing at 20ug / ml, and do not grow at 22.5ug / ml. Therefore, hygromycin was chosen as the screening reagent for resistance of straw mushrooms, and the optimal screening concentration of V23 on the PDA plate was determined to be 20ug / ml.

[0033] 2) Cultivation of V23 mycelia of variegated mushrooms: the strain was inoculated in 100 mL of liquid PDA medium ...

Embodiment 2

[0039] 1) Construction Using SacI and KpnI in the multi-cloning site on the vector pCAMBIA1300, the antifreeze gene AFP in carrots is constructed into the binary vector pCAMBIA1300 to form a recombinant vector pCAMBIA1300::AFP, and the vector construction map is as follows image 3 shown.

[0040] 2) The bacteria were inoculated in 100 mL of liquid PDA medium, and cultured statically at 32° C. for 2 days.

[0041] 3) The configuration method of the enzymolysis solution is the same as in Example 1. The cultured mycelium was filtered with two layers of sterilized gauze, and then washed three times with sterile water. Blot the water with sterile paper, suspend in an appropriate amount of prepared enzyme solution, 32°C, 100rpm shaking enzymolysis; after 5 hours of enzymolysis, centrifuge at 2000rpm for 10min. Centrifuge and wash twice with osmotic pressure stabilizer, add 500 μL electric shock buffer to make mycelia liquid, and finally count with hemocytometer. There were 5 rep...

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Abstract

The invention relates to a method for genetic transformation of volvariella volvacea by using an electric shock method. The method comprises the following steps: determining the minimum tolerance concentration of a volvariella volvacea mycelium to hygromycin, culturing the volvariella volvacea mycelium in a PDA (Potato Dextrose Agar) culture medium, degrading the cell wall of a volvariella volvacea mycelium cell by utilizing a lywallzyme and collecting the volvariella volvacea mycelium; putting a constructed exogenous recombinant carrier plasmid and the volvariella volvacea mycelium into an electric shock cup, finishing the electric shock process under the action of different applied electric fields, coating the volvariella volvacea mycelium after electric shock in a regeneration culture medium, performing resistance screening on the culture medium containing the hygromycin and molecularly detecting whether a normally-growing monoclone is a positive clone or not. The invention provides a novel method for volvariella volvacea molecular biology research, opens up a convenient and economic way for the establishment of a volvariella volvacea bioreactor simultaneously and provides a technical support and a guarantee for effectively solving the difficulties in the production and the storage of volvariella volvacea.

Description

technical field [0001] The invention belongs to the field of biological engineering of straw mushrooms, and in particular relates to a genetic transformation method of straw mushrooms by electric shock method. Background technique [0002] Straw mushroom (Volvariella volvacea) is an important common edible fungus. Its cultivation originated in my country and has a long history of cultivation. It is known as "Chinese mushroom" in the world. Straw mushroom is a saprophytic fungus native to tropical and subtropical high-temperature and rainy regions. It mostly grows on fiber-rich substrates, such as straw and banana leaves. Belongs to Basidiomycetes, Agaricaceae, Amanitaceae, and Straw Mushroom. Straw mushrooms are mainly cultivated in Asia, such as China's southeast coastal provinces and Southeast Asian countries: Singapore, Malaysia, etc. Straw mushrooms have rich economic value: (1) high nutritional value, straw mushrooms contain a variety of essential amino acids - serine...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/645
CPCC12N15/80
Inventor 吕贝贝唐雪明蒋玮吴潇武国干王金斌李鹏白蓝刘华于海龙王荣谈孙斌张静芳
Owner SHANGHAI ACAD OF AGRI SCI
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