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A kind of synthetic fatty acid desaturase coding gene and its application

A fatty acid and transgenic cell line technology, applied to the artificially synthesized fatty acid desaturase coding gene and its application field, can solve the problems of nematode less, expensive, time-consuming limited source of seafood and biological enrichment, etc., to achieve a reduction ratio Effect

Active Publication Date: 2018-05-25
南宁壮博生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far the only feasible way to increase omega-3 content in carnivores is by feeding supplemented omega-3 fatty acid diets (such as feeding flaxseed, fishmeal or other seafood), but this is not sustainable because not only is it expensive , time-consuming and limited seafood sources and potential contamination from bioaccumulation remain concerns
The ω-3PUFA dehydrogenase encoded by the fat-1 gene of the nematode (Caenorhabditis elegans) dehydrogenates the ω-6PUFAs of 16-20 carbons in the organism to generate the corresponding ω-3PUFA. Spychalla, kang, Lai, etc. are prepared by Transgenic Arabidopsis thaliana, transgenic mice and transgenic pigs have verified its function, but there are few reports on the function of fat-1 gene in nematode (Caenorhabditis briggsae) of the same species and different genus

Method used

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  • A kind of synthetic fatty acid desaturase coding gene and its application
  • A kind of synthetic fatty acid desaturase coding gene and its application
  • A kind of synthetic fatty acid desaturase coding gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Codon optimization and total gene synthesis of nematode FAT-1 fatty acid desaturase gene

[0048] According to the complete CDS sequence of the wild-type nematode (Caenorhabditis briggsae) FAT-1 fatty acid desaturase gene—cbrfat-1 (as shown in sequence 3 in the sequence listing), a gene suitable for expression in mammalian cells was obtained after design and repeated verification Optimized FAT-1 fatty acid desaturase gene sequence, that is, transforming the wild-type nematode (Caenorhabditisbriggsae) FAT-1 fatty acid desaturase gene sequence into a codon-optimized sequence preferred by mammals (high frequency usage), thereby improving the nematode FAT-1 Expression levels of fatty acid desaturases in mammalian cell culture settings.

[0049] According to the above method, an optimized nematode FAT-1 fatty acid desaturase coding gene designed according to mammalian codon preference was finally obtained, which was named hsfat-1 gene, and its gene sequence was No...

Embodiment 2

[0051] Embodiment 2, hsfat-1, the construction of cgfat-1 gene mammalian stable expression cell line

[0052] 1. Construction of recombinant expression vectors pcDNA3.1(+)-hsfat-1 and pcDNA3.1(+)-cgfat-1

[0053] 1. Construction of recombinant expression vector pcDNA3.1(+)-hsfat-1

[0054] The artificially synthesized DNA fragment in Example 1 (position 911-2137 of Sequence 1) was double-digested with restriction endonucleases HindIII and EcoR I, which was larger than the backbone of the plasmid pcDNA3.1(+) after the same double-digestion The fragments were ligated, and the ligated product was transformed into Escherichia coli DH5α competent cells. Obtain several single colonies and inoculate them with corresponding antibiotics (Amp + ) in LB medium, cultured overnight at 37°C with vigorous shaking, the plasmid was extracted, and the recombinant plasmid was identified by endonuclease Hind III and EcoR I. The identification results are as follows: figure 1 As shown in middl...

Embodiment 3

[0068] Example 3, hsfat-1, cg-fat-1 gene stable mammalian expression cell line fatty acid extraction and determination

[0069] The hsfat-1 gene mammalian stable expression cell line (experimental group), cg-fat-1 gene mammalian stable expression cell line (positive control group, WT) and pcDNA3.1(+) empty cell line constructed from Example 2 Five vector-positive clones (negative control group, CK1) were randomly selected and inoculated in 60 mm culture dishes containing 4 ml of cell culture medium (about 4×10 5 cells / dish) when the cell confluency reaches 90%, digest with 0.1% trypsin and transfer to a 100mm culture dish to continue culturing to expand the cell volume, add arachidonic acid with a final concentration of 100μM, and cultivate for 48 hours until the cell confluence 100%, the fatty acid is extracted, the fatty acid is subjected to methyl esterification reaction, and the content of fatty acid methyl ester is measured by GC-MS method. The content of fatty acid methy...

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Abstract

The invention discloses an artificially synthesized fatty acid desaturase encoding gene and its application. The gene provided by the present invention is specifically any one of the following a)-d): a) the coding sequence is the nucleic acid molecule shown at position 932-2131 of sequence 1; b) shown in position 932-2131 of sequence 1 A nucleic acid molecule; c) a nucleic acid molecule that hybridizes to the nucleic acid molecule defined in a) or b) under stringent conditions, and encodes a protein with the same amino acid sequence as the nucleic acid molecule shown in position 932-2131 of SEQ ID NO: 1; d) is a nucleic acid molecule that is identical to a ) or b) or c) The defined nucleic acid molecule has more than 90% homology, and encodes a protein with the same amino acid sequence as the nucleic acid molecule shown in position 932-2131 of SEQ ID NO: 1. Experiments show that, in pig fetal fibroblasts, the optimized nematode FAT-1 fatty acid desaturase gene of the present invention has a stronger ability to convert omega-6 fatty acid than the wild-type nematode FAT-1 fatty acid desaturase gene. The ability to convert to omega-3 fatty acids can significantly reduce the omega-6 / omega-3 ratio.

Description

technical field [0001] The invention relates to an artificially synthesized fatty acid desaturase gene and its application, in particular to an artificially synthesized fatty acid desaturase gene and its application in converting fatty acid Omega-6 into Omega-3. Background technique [0002] Omega-3 polyunsaturated fatty acids are widely accepted by the public for their many health benefits, so the demand for these fatty acids is increasing year by year. [0003] China is a big producer and consumer of pork. According to data from the China Health and Nutrition Survey, pork alone accounts for more than 70% of Chinese meat consumption. The 8 main fatty acids in lard are myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and arachidic acid. Among them, unsaturated fatty acids account for 58%, and unsaturated fatty acids, especially Omega-6 and Omega-3, are especially beneficial to human health. However, the ratio of Omega-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/85C12N5/10C12P7/64
Inventor 牟玉莲程英任红艳樊俊华黄雷吴添文阮进学李奎
Owner 南宁壮博生物科技有限公司
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