Method for breeding AA-high-producing strains by utilizing protoplast fusion and combining with fusant screening through fluorescence staining

A technology of protoplast fusion and fluorescent staining, applied in biochemical equipment and methods, microbial determination/inspection, fungi, etc., can solve problems such as heavy workload and cumbersome operation, and achieve the effect of reducing workload

Inactive Publication Date: 2016-10-26
INST OF PLASMA PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For fusion sub-screening methods and techniques, auxotrophic markers or resistance markers are often used, which is cumbersome and requires a lot of work.

Method used

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  • Method for breeding AA-high-producing strains by utilizing protoplast fusion and combining with fusant screening through fluorescence staining
  • Method for breeding AA-high-producing strains by utilizing protoplast fusion and combining with fusant screening through fluorescence staining
  • Method for breeding AA-high-producing strains by utilizing protoplast fusion and combining with fusant screening through fluorescence staining

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] (1) Cultivation and collection of two parental strains

[0046] G12 and F24 were inoculated with PDA solid medium and cultured for 6 days, the spores were scraped off, and the spore suspension was prepared with sterile water and diluted to 10 8 spores / ml, take 1ml of the spore suspension and put it into a 250ml Erlenmeyer flask with a liquid volume of 50ml PDA liquid medium, and shake it at 28°C for 16 hours at 150 r / min to obtain two parental strains;

[0047] (2) Preparation of protoplasts from two parental strains

[0048] Wash and centrifuge the mycelium obtained in step (1) twice with sterile water, then wash once with a 0.8mol / NaCl solution and centrifuge, take 1mg of the wet mycelium obtained by centrifugation and add 5ml of mixed enzymatic hydrolysis solution, mix The enzymatic hydrolysis solution includes 10-20 mg / ml helicase, 5-10 mg / ml cellulase, 0.2-0.8 mg / ml lysozyme and 0.2-0.5 mg / ml chitinase. The body and the enzymatic hydrolysis solution were fully m...

Embodiment 2

[0056] (1) Cultivation and collection of two parental strains

[0057] Two strains of Mortierella alpina G12 and F24 were respectively inoculated on PDA solid medium for 6 days, the spores were scraped off, and the spore suspension was prepared with sterile water and diluted to 10 8 spores / ml, take 1ml of the spore suspension and put it into a 250ml Erlenmeyer flask with a liquid volume of 50ml, and shake it at 28°C for 16 hours at 150 r / min to obtain two parental strains.

[0058] (2) Preparation of protoplasts from two parental strains

[0059] Wash and centrifuge the mycelium obtained in step (1) twice with sterile water, then wash once with a 0.8mol / L NaCl solution and centrifuge, take 1mg of the wet mycelium obtained by centrifugation and add 5ml of mixed enzymatic hydrolysis solution, The mixed enzymatic solution includes 10-20 mg / ml helicase, 5-10 mg / ml cellulase, 0.2-0.8 mg / ml lysozyme and 0.2-0.5 mg / ml chitinase. Shake gently to fully mix the centrifuged bacteria a...

Embodiment 3

[0067] (1) Cultivation and collection of two parental strains

[0068] Two strains of Mortierella alpina G12 and F24 were respectively inoculated on PDA solid medium for 6 days, the spores were scraped off, and the spore suspension was prepared with sterile water and diluted to 10 8 spores / ml, take 1ml of the spore suspension and put it into a 250ml Erlenmeyer flask with a liquid volume of 50ml PDA liquid medium, and shake at 28°C for 16 hours at 150 r / min to obtain two parental strains.

[0069] (2) Preparation of protoplasts from two parental strains

[0070] Wash and centrifuge the mycelium obtained in step (1) twice with sterile water, then wash once with a 0.8mol / L NaCl solution and centrifuge, take 1mg of the wet mycelium obtained by centrifugation and add 5ml of mixed enzymatic hydrolysis solution, The mixed enzymatic solution includes 10-20 mg / ml helicase, 5-10 mg / ml cellulase, 0.2-0.8 mg / ml lysozyme and 0.2-0.5 mg / ml chitinase. . Gently shake to mix the centrifuge...

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Abstract

The invention provides a method for breeding AA-high-producing strains by utilizing protoplast fusion and combining with fusant screening through fluorescence staining. In order to obtain mortierella alpina strains with high yield of arachidonic acid, the invention provides the method adopting the following scheme: two mortierella alpina strains with different superior characteristics are subjected to fusion treatment by adopting a protoplast fusion method, and fusants are screened by combining with the fluorescence staining method. The method provided by the invention comprises the following specific steps: culturing two parental strains, preparing protoplasts of the two parental strains, carrying out fluorescence staining on the protoplasts of the two parental strains, fusing the protoplasts of the two parental strains, and screening and verifying the fusants. The method provided by the invention is easy to operate, the process is easy to realize, and the breeding success probability is high. The method can be used for improving the yielding level of arachidonic acid producing strains, optimizing the fat components of the arachidonic acid product, and improving the characters of the strains.

Description

Technical field: [0001] The invention relates to a method for preparing Mortierella alpina fusogens and screening them with fluorescent dyeing. Background technique: [0002] Arachidonic acid (AA) is a kind of 20-carbon unsaturated fatty acid, which plays an important role in many physiological processes of the human body. It has the functions of esterifying cholesterol, inhibiting platelet aggregation, reducing blood viscosity, regulating white blood cell function, improving immunity, and promoting brain development of infants and young children. The Ministry of Health officially approved in 1994 that AA can be added to infant formula. In 1999, AA was officially listed as a new type of nutritional fortifier. In recent years, it has been widely used in health food, medicine, cosmetics and other fields. [0003] A small amount of natural AA exists in animal liver and adrenal gland, fish oil and microorganisms (protozoa, amoeba, microalgae and fungi). Because the content is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/04C12N1/14C12Q1/04C12P7/64
CPCC12N1/14C12N15/04C12P7/6427C12Q1/04
Inventor 潘淼孙立洁陈祥松袁丽霞吴金勇凌瑞姚建铭
Owner INST OF PLASMA PHYSICS CHINESE ACAD OF SCI
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