Preparation and conversion method of inonotus obliquus protoplast

A technology of Inonotus obliquus and protoplasts, which is applied in the field of genetic engineering, can solve problems such as the lack of reports on the transformation method of Inonotus obliquus, and achieve good quality results

Inactive Publication Date: 2016-09-28
LIAONING UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the transformation method of Inonotus obliquus

Method used

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  • Preparation and conversion method of inonotus obliquus protoplast
  • Preparation and conversion method of inonotus obliquus protoplast
  • Preparation and conversion method of inonotus obliquus protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 A method for preparation and transformation of Inonotus obliquus protoplasts

[0023] (1) Preparation of Inonotus obliquus hyphae membrane

[0024] The hyphae of Inonotus obliquus were inoculated with 0.08% MgSO 4 After 6 days of culture on the PDA solid medium, use an inoculating loop to pick the colony sheet into a 50ml centrifuge tube containing sterile water and glass beads (both glass beads and 50ml centrifuge tube have been sterilized), vortex and shake until Become very small hypha fragments, use a pipette to pipette 1ml hypha fragments to 0.08% MgSO 4 Into the 200ml PDA liquid medium, stand still for 12 days to obtain Inonotus obliquus hypha membrane.

[0025] (2) Preparation of mixed enzyme solution

[0026] Weigh 60mg lysozyme and 30mg collapse enzyme, add 3ml osmotic stabilizer (0.6mol / L KCl solution), after the enzyme is completely dissolved, filter with 0.22μm filter membrane, take the filtrate, and make it with osmotic stabilizer The mixed enzyme soluti...

Embodiment 2

[0041] Example 2 The effect of mixed enzyme solution on the preparation and transformation methods of Inonotus obliquus protoplasts

[0042] The method is the same as that in Example 1, except that the preparation method of the mixed enzyme solution is as follows:

[0043] Weigh 45mg of lysozyme and 30mg of collapse enzyme, add 3ml of osmotic stabilizer (0.6mol / L KCl solution), after the enzyme is completely dissolved, filter with 0.22μm filter membrane, take the filtrate, and make with osmotic stabilizer The mixed enzyme solution with a concentration of 25mg / ml should be placed in a sterile room for use on the same day.

[0044] The preparation, transformation method and release measurement of Inonotus obliquus protoplasts are the same as those in Example 1. After the determination, the concentration of Inonotus obliquus protoplasts prepared by the method of this example is 9×10 6 Pcs / ml. The prepared protoplasts were coated on the regeneration medium, and the regeneration rate was...

Embodiment 3

[0045] Example 3 The effect of mixed enzyme solution on the preparation and transformation of an Inonotus obliquus protoplast

[0046] The method is the same as that in Example 1, except that the preparation method of the mixed enzyme solution is as follows:

[0047] Weigh 75mg of lysozyme and 30mg of collapse enzyme, add 3ml of osmotic stabilizer (0.6mol / L KCl solution), after the enzyme is completely dissolved, filter with 0.22μm filter membrane, take the filtrate, and make with osmotic stabilizer The mixed enzyme solution with a concentration of 35mg / ml should be placed in a sterile room for use on the same day.

[0048] The preparation, transformation method and release measurement of Inonotus obliquus protoplasts are the same as those in Example 1. After determination, the concentration of Inonotus obliquus protoplasts prepared by the method of this example is 2.5×10 7 Pcs / ml. The prepared protoplasts were coated on the regeneration medium, and the regeneration rate was about 6...

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Abstract

The invention discloses a preparation and conversion method of inonotus obliquus protoplast. The preparation method comprises the following steps that a mixed enzyme solution composed of lywallzyme and driselase is adopted for enzymolysis of an inonotus obliquus hypha membrane and filtered through eight layers of lens wiping paper, filtrate collection and centrifugation are carried out, and sediment is obtained and is the inonotus obliquus protoplast. The conversion method comprises the steps that plasmid pAN7-1 is induced by PEG / CaC12 to be converted into inonotus obliquus protoplast, and inonotus obliquus containing plasmid pAN7-1 is obtained. According to the inonotus obliquus protoplast obtained through the method, the yield reaches up to 5*107 / mL, and the regeneration rate is 7%. The preparation and conversion method lays a foundation for carrying out genetic manipulation modification on the inonotus obliquus in the later period, improving the yield of a triterpene compound and a precursor thereof, then improving the medical value of the inonotus obliquus and meeting market requirements.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, relates to fungal biology, and specifically relates to a preparation method and a transformation method of Inonotus obliquus protoplasts. Background technique [0002] Inonotus obliquus (Inonotus obliquus) is a precious medicinal fungus. As early as the 16th century, Inonotus obliquus was widely used by people in Eastern Europe to prevent and treat a variety of intractable diseases, such as gastric cancer, bowel cancer and cardiovascular diseases. The main active component of the medicinal value of Inonotus obliquus is triterpenoids, which have antioxidant, anti-tumor and antibacterial activities. With the increasing depletion of wild resources and the increasing demand for Inonotus obliquus, the application of modern biotechnology to increase the yield of artificially cultivated Inonotus obliquus mycelium triterpenoids is particularly important to meet people's demand for Inonotus obliquus importa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N1/06C12N15/80C12R1/645
CPCC12N1/14C12N1/06C12N15/80
Inventor 刘宏生邓芳博赵健朱俊丰
Owner LIAONING UNIVERSITY
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