Genetic transformation method of haematococcus pluvialis

A genetic transformation method and technology of Haematococcus pluvialis, applied in the field of genetic transformation of Haematococcus pluvialis, can solve the problems of low positive transformation rate, cumbersome operation, low success rate, etc., achieve broad market prospects, avoid cell stress The effect of response and improving the positive rate

Active Publication Date: 2020-11-10
SHENZHEN UNIV
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Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the genetic transformation method of Haematococcus pluvialis in the prior art, such as cumbersome operation, high cost, low positive transformation rate and low success rate, thereby providing a kind of Haematococcus pluvialis Methods of genetic transformation of algae

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  • Genetic transformation method of haematococcus pluvialis
  • Genetic transformation method of haematococcus pluvialis
  • Genetic transformation method of haematococcus pluvialis

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Embodiment 1

[0061] The present embodiment provides a method for genetic transformation of Haematococcus pluvialis, comprising the following steps:

[0062] 1 Linearization of plasmids for transformation

[0063] The plasmid used in this example is pH124. Use the plasmid extraction kit Plasmid Mini Kit II (Omega (Omega Bio-tek), the United States) to extract the pH124 plasmid from Escherichia coli DH5α, linearize the plasmid with the restriction endonuclease Not I, and digest the plasmid with Not I The system is 30 μL, specifically including: 20 μL DNA solution, 2 μL Not I fast cutting enzyme (New England (NEB), USA), 3 μL Cutsmart Buffer (New England (NEB), USA), 5 μL water. The enzyme digestion temperature was 37° C. and the enzyme digestion time was 2 hours to obtain a linearized pH124 plasmid having the DNA sequence shown in SEQ ID NO: 1, and the concentration of the linearized plasmid was about 1.0 μg / μL.

[0064] 2 Genetic transformation of Haematococcus pluvialis

[0065] (1) Col...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a genetic transformation method of haematococcus pluvialis. The method comprises the following step of mediating exogenous genes with a polyethylene glycol solution to enter the protoplast of the haematococcus pluvialis. According to the method, the polyethylene glycol solution is adopted to mediate the exogenous genes to be transferred into the protoplast of the haematococcus pluvialis for the first time; due to the fact that the permeability of cell membranes can be improved through polyethylene glycol, linearized plasmids can be transferred into the protoplast of the haematococcus pluvialis more easily; the method does not cause mechanical damage to cells; and the influence on the conversion of the haematococcus pluvialis due to the cell stress reaction caused by damage is avoided. The method solves the problems of complicated operation, high cost, long period and low conversion rate in genegun and agrobacterium tumefaciens mediated genetic transformation. The method can be widely applied to excellent character improvement, gene editing, expression regulation and the like of the haematococcus cells, and has huge commercial advantages and broad market prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for genetic transformation of Haematococcus pluvialis. Background technique [0002] As an important economic green algae, Haematococcus pluvialis is the main source of natural astaxanthin. Astaxanthin belongs to carotenoids, which can delay aging, promote antibody production and enhance the body's immunity. It is a natural antioxidant; astaxanthin is also effective in the prevention and treatment of oxidative stress diseases such as human diabetes, gout, hypertension and cancer. Obvious effect, so astaxanthin has great market value, and Haematococcus pluvialis has great research significance. [0003] Most of the Haematococcus pluvialis currently used for commercial production of natural astaxanthin are wild-type varieties obtained by screening. The content of astaxanthin in the cells of this wild-type Haematococcus is very limited (less than 2%). T...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12R1/89
CPCC12N15/8206
Inventor 胡章立郭春立王潮岗王会
Owner SHENZHEN UNIV
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