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New delta12 desaturase gene derived from Pavlova viridis

A technology of Pavlova viridis and desaturase, which is applied in the field of desaturase gene and its separation, and can solve the problems of sequence structure difference, enzyme activity and function difference, etc.

Active Publication Date: 2015-03-04
江苏桂龙生物技术有限公司
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Problems solved by technology

At present, several delta12 desaturases from different species have been isolated and identified in microalgae, but the homologous enzymes of different species obviously have differences in sequence and structure, which lead to differences in their enzymatic activities and functions

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  • New delta12 desaturase gene derived from Pavlova viridis

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Embodiment Construction

[0018] The isolation process of Pavlova viridis delta12 desaturase gene is as follows:

[0019] (1) Obtain the total mRNA of Pavlova viridis using conventional mRNA extraction and separation means in the art, and use primer OligodT to reverse transcribe it into cDNA by conventional means in the art;

[0020] (2) Using cDNA as a template, design primers P12F and P12R according to the conservation of homologous genes of delta12 desaturase to amplify the gene in the conserved region. The primer sequence is: P12F: 5'-CTGACGGTGGTCATCGCAGG-3'; P12R: 5'-CATGACACAACATCTGAAGA-3'; PCR reaction conditions: 94oC 3 min; 94oC 30 s, 57oC 30 s, 72oC 1 min, 30 cycles; 72oC 10 min; The size of the obtained PCR product is about 449bp;

[0021] (3) The amplification reaction at the 3' end was carried out using cDNA as a template and using the SMART RACE cDNA amplification kit. The amplification primers used were: UPM (10×universal primer mix); des12-3 GGCAGGCTCGGGCATCCTGT. The gradient PCR react...

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Abstract

The invention provides a new delta12 desaturase gene derived from Pavlova viridis. Polyunsaturated fatty acids such as conjugated linoleic acid (CLA), eicosapentaenoic acid (EPA) and the like are necessary important nutrient substances of human bodies. Aiming at microalgae, namely Pavlova viridis which is capable of naturally synthesizing lots of polyunsaturated fatty acids, a new delta12 desaturase gene is cloned by combining conservative region sequence amplification with RACE (rapid-amplification of cDNA ends), thereby providing the basis further knowing and utilizing biosynthesis processes of linoleic acid and conjugated linoleic acid which have great practical significances.

Description

technical field [0001] The application belongs to the technical field of gene cloning and separation, and in particular relates to a desaturase gene related to the synthesis of polyunsaturated fatty acids derived from microalgae and its separation method. Background technique [0002] Polyunsaturated fatty acids are essential nutrients for the human body, and microalgae are an important biological resource because they are rich in polyunsaturated fatty acids. Green Pavlova viridis is rich in polyunsaturated fatty acids, and it is a good experimental material for identifying and cloning polyunsaturated fatty acid-related synthesis genes. [0003] The full name of RACE is rapid-amplification of cDNA ends, which is a commonly used technology for rapid cloning of cDNA ends by PCR. In this experiment, RACE technology was used to obtain the full-length sequence of the target mRNA fragment. [0004] In the biosynthetic pathway of polyunsaturated fatty acids in microalgae, two ma...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/10
Inventor 不公告发明人
Owner 江苏桂龙生物技术有限公司
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