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Genetic transformation method of lentinus edodes

A technology of genetic transformation and Lentinus edodes, applied in the field of Lentinus edodes genetic transformation, can solve the problems of low regeneration rate, unsuitable for monokaryon, complicated preparation process of Lentinus edodes protoplasts, etc., and achieve the effect of improving transformation efficiency and

Pending Publication Date: 2018-08-31
SHANGHAI ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The preparation process of Lentinus edodes protoplasts is complicated and the regeneration rate is low
Spores and fruiting bodies are not suitable for monokaryon transformation, and some laboratories do not have the conditions for fruiting

Method used

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  • Genetic transformation method of lentinus edodes
  • Genetic transformation method of lentinus edodes
  • Genetic transformation method of lentinus edodes

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Embodiment 1

[0035] In this embodiment, taking the transformation of pYN6981 plasmid as an example, the method for genetic transformation of shiitake mushrooms provided in this embodiment is described, which includes the following steps:

[0036] (1) Place the round mycelium block on the PDA plate. After the mycelium is overgrown, it is crushed and inoculated in the PDB liquid medium with a homogenizer under sterile conditions, and cultured at 25°C for 5 days to obtain a liquid strains.

[0037] (2) The culture (i.e. the liquid strain) is again pulverized under aseptic conditions with a homogenizer, and inoculated in 100mL PDB liquid with a 10% inoculum size (the secondary crushing is to remove the solid PDA medium in the liquid bacteria) ; 25 ℃ static culture for 4 days, or shake occasionally during the period.

[0038] (3) Collect mycelium by filtering with a funnel with non-woven fabric; wash twice with 0.6 M mannitol solution, remove mannitol by centrifugation at 6000 rpm for 10 min, ...

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Abstract

The invention discloses a genetic transformation method of lentinus edodes and relates to the technical field of molecular genetic transformation of the lentinus edodes. The method takes an incompletely enzymolyzed lentinus edodes hypha fragment as a transformation receptor for the first time and adopts an electric shock transformation method; electric shock transformation is conducted under the condition of 1800-2200kV / cm voltage, 20-30MFD capacitance and 380-420 omega resistance; a complicated lentinus edodes protoplast preparation step is removed; the transformation efficiency and a transformant regeneration rate can be effectively improved and increased; and the method is also suitable for monokaryon transformation.

Description

technical field [0001] The invention relates to the technical field of molecular genetic transformation of shiitake mushrooms, in particular to a method for genetic transformation of shiitake mushrooms. Background technique [0002] At present, the existing mushroom genetic transformation recipients are usually protoplasts, spores or fruiting bodies. The preparation process of Lentinus edodes protoplasts is complicated and the regeneration rate is low. Spores and fruiting bodies are not suitable for monokaryon transformation, and some laboratories do not have the conditions for fruiting. [0003] In view of this, the present invention is proposed. Contents of the invention [0004] The purpose of the present invention is to provide a method for genetic transformation of shiitake mushrooms. The method is combined with the electroshock transformation method, using incompletely enzymolyzed shiitake mushroom mycelium fragments as transformation receptors, which can effective...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80
CPCC12N15/80
Inventor 刘建雨尚晓冬张美彦章炉军张丹宋春艳李巧珍徐珍杨慧
Owner SHANGHAI ACAD OF AGRI SCI
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