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A Chinese Mortierella hirsutella sinensis transformed bacterial strain expressing green fluorescent protein and its preparation method

A technology of green fluorescent protein and Trichosporum sinensis, which is applied in the field of genetic engineering, can solve the problems such as no transformants have been seen, and achieve the effects of stable fluorescence characteristics, simple preparation process, and simple materials

Active Publication Date: 2020-10-30
成都图径生物科技有限公司
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Problems solved by technology

[0005] However, Hirsutella sinensis is a very special entomogenous fungus, so far, no transformant of Hirsutella sinensis containing EGFP fluorescent marker has been seen

Method used

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  • A Chinese Mortierella hirsutella sinensis transformed bacterial strain expressing green fluorescent protein and its preparation method
  • A Chinese Mortierella hirsutella sinensis transformed bacterial strain expressing green fluorescent protein and its preparation method
  • A Chinese Mortierella hirsutella sinensis transformed bacterial strain expressing green fluorescent protein and its preparation method

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Embodiment 1

[0048] Embodiment 1 The present invention prepares the method for the transformation bacterial strain of Chinese Mortierella Hirsutella sinensis and the bacterial strain obtained

[0049] 1. Preparation method

[0050] (1) Preparation of Chinese Mortierella Hirsutella sinensis blastospore suspension

[0051] The hyphae of Hirsutella sinensis were broken up with a dispersing head and then inserted into the sporulation liquid medium, cultured at 18°C ​​and 130r / min for 14 days, and then collected by filtering the mycelium with a layer of nylon membrane (150 mesh) to obtain blastospores. The IM medium was resuspended three times and counted on the hemocytometer, and the concentration was adjusted at 10 4 ~10 5 A / mL spare. The morphology and structure of blastospores are shown in the appendix figure 1 .

[0052] (2) Preparation of Agrobacterium tumefaciens containing recombinant plasmid pRF-AETH

[0053] A single colony of Agrobacterium tumefaciens AGL-1 was picked, inoculat...

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Abstract

The invention discloses a method for preparing a Hirsutella sinensis transforming strain expressing green fluorescent protein. The method comprises: (1) preparing a Hirsutella sinensis blastospore suspension; (2) preparing Agrobacterium tumefaciens containing recombinant plasmid pRF-AETH; (3) preparing an Agrobacterium tumefaciens liquid; (4) mixing the Hirsutella sinensis blastospore suspension and the Agrobacterium tumefaciens liquid, and co-culturing; and (5) carrying out selective culture, identifying, and obtaining the Hirsutella sinensis capable of stably expressing green fluorescent protein. The invention further provides a Hirsutella sinensis transforming strain. According to the present invention, with the method, the Hirsutella sinensis transforming strain capable of stably expressing egfp gene can be obtained, can provide the convenience for the molecular biology research of the Hirsutella sinensis functional gene, can monitor the growth and reproduction and the development and differentiation of the Hirsutella sinensis in host insects, can provide the direct evidence for the clarification of the interaction between the Hirsutella sinensis and the host, and has excellent application prospects.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a transformed bacterial strain of Hirsutella sinensis expressing green fluorescent protein and a preparation method thereof. Background technique [0002] Cordyceps sinensis is a compound with good health care and medicinal effects formed by the entomogenous fungus Hirsutellasinensis parasitizing on the larvae of the Lepidoptera bat moth in a specific ecological environment (Zhang YJ et al., 2012 ; Paterson R R., 2008). Because of its unique health care and medicinal effects, the market demand has surged, and the collection of natural resources has been frenzied, causing the resources to be on the verge of exhaustion. Therefore, artificial cultivation of Cordyceps sinensis has become an important way for people to seek to supplement the dwindling sources of wild Cordyceps sinensis (Holliday J et al., 2008). However, the mechanism by which the fungus enters the larvae of the b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12N15/65C12N1/15C12R1/645
CPCC12N15/65C12N15/80C12N2800/10
Inventor 蓝贤清苏文赵成向金鹏方呈祥
Owner 成都图径生物科技有限公司
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