Isochrysis sphaerica delta 4-fatty acid desaturase gene and cloning method thereof

A technology of isochrysis and desaturase, applied in botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as low yield and high extraction cost

Active Publication Date: 2013-10-30
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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Problems solved by technology

It is known that certain microorganisms such as fungi and marine microalgae can synthesize PUFAs from scratch and are abundant in the body. At present, commercial PUFAs are only successfully prepared from these microorganisms by fermentation and culture in very few species. High extraction costs and low yields need to be overcome. the main bottleneck of

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  • Isochrysis sphaerica delta 4-fatty acid desaturase gene and cloning method thereof
  • Isochrysis sphaerica delta 4-fatty acid desaturase gene and cloning method thereof
  • Isochrysis sphaerica delta 4-fatty acid desaturase gene and cloning method thereof

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[0036]1. the cloning method of Isochrysis sphaerica (Isochrysis sphaerica) Δ4-fatty acid desaturase gene isfad4, it comprises the steps:

[0037] The reported amino acid sequences of Δ4-fatty acid desaturase were compared, and degenerate primers IsF1 and IsR1 were designed in the conserved regions LQHMGGH and QIEHHMAP according to the comparison results; the total genomic DNA extracted from Isochrysis sphaerica was used as a template , using IsF1 / IsR1 as primers, using the Touch-Down PCR method to amplify the gene fragment corresponding to the conserved interval, clone it into the pMD18-T vector and sequence it; PCR (Adaptor-PCR) and thermal asymmetric staggered PCR (TAIL-PCR) amplified the flanking sequences of the 5' and 3' ends of the isfad4 gene fragment of Isochrysis sphaerica; The fragments were assembled to obtain the full-length sequence of the isfad4 structural gene of Isochrysis sphaerica (ie, the full-length sequence of the Isochrysis sphaerica Δ4-fatty acid desatur...

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Abstract

The invention relates to a delta 4-fatty acid desaturase gene originated from marine microalgae isochrysis sphaerica and a cloning method thereof. The full-length delta 4-fatty acid desaturase gene isfad 4 is obtained by the following steps of: taking the isochrysis sphaerica as a material; designing degenerate primers and amplifying a gene core sequence; and amplifying the full-length delta 4-fatty acid desaturase gene isfad 4 by using a genome walking method. Heterologous expression of the isfad 4 in pichia pastoris proves the 4-fatty acid desaturase function of IsFAD 4.

Description

technical field [0001] The invention relates to a Δ4-fatty acid desaturase gene derived from marine microalga Isochrysis sphaerica and a cloning method thereof. Background technique [0002] Polyunsaturated fatty acids (PUFAs), such as Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA), are the main components of the membrane lipid structure of the human central nervous system. Components are essential to the development of the human body, especially the brain and nervous system of infants, and play an important role in medicine and nutritional health care. The efficiency of the human body to synthesize EPA and DHA is extremely low, and it is extremely important to supplement enough EPA and DHA in daily life to maintain good health. At present, such fatty acids in the market are mainly derived from deep-sea fish oil. The problem of environmental pollution and the limitation of fish resources make it necessary for people to find long-lasting, stable and safe alternat...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/10C12R1/89
Inventor 江木兰万霞龚阳敏梁焯张银波郭兵张燕
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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