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Pavlova-viridis-derived delta-6 desaturase new gene

A desaturase and gene technology, applied in the field of desaturase genes and their separation, can solve the problems of different enzyme activities and functions, differences in sequence structure, etc.

Inactive Publication Date: 2014-12-03
广州诺晶生物技术有限公司
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Problems solved by technology

At present, several delta6 desaturases from different species have been isolated and identified in microalgae, but the homologous enzymes of different species obviously have sequence and structural differences, which lead to differences in their enzymatic activities and functions

Method used

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  • Pavlova-viridis-derived delta-6 desaturase new gene

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Embodiment Construction

[0019] The isolation process of Pavlova viridis delta-6 desaturase gene is as follows:

[0020] (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;

[0021] (2) Using cDNA as a template, design primers P6F and P6R according to the conservatism of the delta6 desaturase homologous gene to amplify the gene in the conserved region. The primer sequence is: P6F: 5'-GTTCCTCGCTGTCGTGGTGGA-3'; P3R: P3R: 5'-TGTTGTGACCGCCGCAGAACCA-3'; PCR reaction conditions: 94oC 3 min; 94oC 30 s, 60oC 30 s, 72oC 1 min, 30 cycles; 72oC 10 min;

[0022] (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); des6-3 TTGGATCTCGTTCATCACTTG. The gradient PCR reaction program is: 94oC 3 min; 94oC 30 s, 72oC 3 min,...

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Abstract

The invention relates to a Pavlova-viridis-derived delta-6 desaturase new gene. Polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and dehydroacetic acid (DHA), are important nutrient substances which are necessary for the human body and can not be synthesized by the human body. In view of the microalga-Pavlova viridis capable of naturally synthesizing EPA and DHA, a conserved region sequence amplification-RACE combined method is used for for cloning to obtain the new delta-6 fatty acid desaturase gene. The acquisition of the gene provides references for further recognizing and utilizing the biosynthesis process of the EPA and DHA which have high practical significance.

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. Pavlova viridis is rich in EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), and 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 EPA and DHA in microalgae, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/53
Inventor 不公告发明人
Owner 广州诺晶生物技术有限公司
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