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Method for transforming gene of crypthecodinium cohnii producing docosahexaenoic acid (DHA)

A technology of gene transformation and Cryptodinoflagellate, applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems such as the lack of gene transformation methods of Cryptodinoflagellate, the inability to carry out research and productivity improvement work, etc. To achieve the effect of improving the yield of DHA

Active Publication Date: 2016-06-08
KUNMING ZAONENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is still a lack of effective gene transformation methods for Cryptidinosa, making it impossible to carry out related research and work on improving yields.

Method used

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  • Method for transforming gene of crypthecodinium cohnii producing docosahexaenoic acid (DHA)
  • Method for transforming gene of crypthecodinium cohnii producing docosahexaenoic acid (DHA)
  • Method for transforming gene of crypthecodinium cohnii producing docosahexaenoic acid (DHA)

Examples

Experimental program
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Effect test

Embodiment 1

[0040] A method for gene transformation of Cryptodinium ATCC30556, comprising the steps of:

[0041] (1) Determination of the action concentration of hygromycin B, a sensitive antibiotic of Cryptodinoflagellates ATCC30556:

[0042] The determination of action concentration of hygromycin B, a sensitive antibiotic of Cryptodinofinnoflagellate ATCC30556, is realized through antibiotic concentration gradient;

[0043] The steps are: setting a concentration gradient for hygromycin B in the C9N2 medium, 10mg / L-50mg / L. Cryptidinium was shaken and cultured at 25°C and 180rpm for 48 hours, and 50 μl of cell suspension was evenly spread on 5 gradient plates, and cultured upside down at 25°C for 2 weeks (no anti-antibiotic plate culture was used as a positive control); the final determination of Cryptidinium The sensitive concentration of ATCC30556 to hygromycin B is 40mg / L.

[0044] (2) Preparation of competent cells of Cryptodinoflagellate ATCC30556 and transformation of fluorescentl...

Embodiment 2

[0059] This embodiment is basically the same as Embodiment 1, except that the electric shock conversion setting voltage is 1.6 kV.

Embodiment 3

[0061] This embodiment is basically the same as Embodiment 1, except that the electric shock conversion setting voltage is 1.8kV.

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Abstract

The invention discloses an antibiotic namely hygromycin B that is sensitive to crypthecodinium cohnii producing DHA and a method using hygromycin B as the resistance screening marker to transform the gene of crypthecodinium cohnii. The method comprises the following steps: (1) determining the action concentration of hygromycin B; (2) establishing a gene transformation system; (3) preparing an exogenous gene section; (4) converting crypthecodinium cohnii by the exogenous gene section. According to the method, an exogenous gene can be introduced into cells of crypthecodinium cohnii, and at the same time, hygromycin B is taken as the screening marker to obtain an engineering strain of crypthecodinium cohnii with an exogenous gene. An important technological base is provided for the genetic engineering of crypthecodinium cohnii.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and in particular relates to a method for genetically transforming Cryptidinosa for producing docosahexaenoic acid. Background technique [0002] Docosahexaenoic Acid (DHA) is one of the important substances for the development and growth of the human brain. It is an important component of the brain and retina. The content in the human cerebral cortex is as high as 20%, and the proportion in the eye retina The largest, accounting for about 50%. Therefore, it plays an important role in the development of the brain and retina of fetuses and infants, as well as the maintenance of adult brain functions. In addition, DHA also has a positive effect on diseases of the cardiovascular system such as coronary heart disease, stroke, hypertension, nervous system diseases such as neurological disorders, Alzheimer's disease and depression. As an essential fatty acid, DHA has more significant effects ...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12R1/89
Inventor 刘璐刁进进陈磊张卫文
Owner KUNMING ZAONENG BIOTECH CO LTD
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