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Rainbow trout fatty acid elongase gene, recombinant expression vector, application

A technology for expression vector and gene, applied in the field of increasing the content of 11,14,17-cis-eicosatrienoic acid in silkworm pupa tissue, and can solve the problems of increasing the content of unsaturated fatty acid in silkworm tissue and the like

Active Publication Date: 2018-11-02
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of silkworm transgenic technology to express exogenous fatty acid desaturase genes in silkworms to increase the content of a specific unsaturated fatty acid in silkworm tissues

Method used

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  • Rainbow trout fatty acid elongase gene, recombinant expression vector, application
  • Rainbow trout fatty acid elongase gene, recombinant expression vector, application
  • Rainbow trout fatty acid elongase gene, recombinant expression vector, application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Preparation of rainbow trout fatty acid elongase gene OmElo.

[0032] Source gene: fatty acid elongase gene (OmElo) sequence of rainbow trout (NCBI accession number: NP_001118108.1).

[0033] According to the codon preference of the coding gene expression sequence in the silkworm genome sequence data, the source gene was optimized and designed, and the rainbow trout fatty acid elongase gene OmElo was obtained after optimization. The nucleotide sequence is shown in SEQ ID No.1, named OmElo Gene.

[0034] The amino acid sequence of the protein encoded by the OmElo gene is shown in SEQ ID No.2.

Embodiment 2

[0036] The silkworm transgenic recombinant vector pBac[A3-EGFP+IE1-OmElo-SV40] was constructed.

[0037] Such as figure 2 As shown, the intermediate vector obtained by inserting the OmElo gene into the multiple cloning site of the starting vector pSL1180, and then inserting the complete expression cassette IE1-OmElo-SV40 containing the OmElo gene into the final vector pBac[A3-EGFP] for recombinant expression carrier.

[0038] Specific implementation operations:

[0039] Step S1, preparation of vector pSL1180[Ser1-OmElo-SV40]

[0040] The OmElo gene was connected to the pUC57 vector plasmid; the pUC57 vector plasmid was digested by BamHI / NotⅠ double enzymes, and the OmElo gene fragment was recovered (the recovery operation was carried out according to the instructions of the TAKARA gel recovery (small amount) kit), and the recovered fragment was digested with the same enzyme The backbone fragment of the pSL1180[Ser1-pGH-SV40] vector was ligated according to the instructions...

Embodiment 3

[0058] Breeding of silkworm transgenic lines with rainbow trout fatty acid elongase gene (OmElo).

[0059] Using the commercially polyvalent silkworm line 305 as the raw material, the parental silkworm eggs were reared on normal mulberry leaves and mated with moths to lay eggs.

[0060] Take 10nL~15nL of the recombinant vector pBac[A3-EGFP+IE1-OmELo-Sv40] prepared in Example 1 with a concentration of 400ng / μL and the mixture of the helper plasmid pHA3PIG to form a mixture, and inject the mixture into 400 capsules Silkworm eggs laid by female moths of silkworm strain 305 for 2 hours to 6 hours were sealed with non-toxic glue and placed in an environment of 25° C. and 85% relative humidity to accelerate hatching. After hatching, 98 G0 generation ant silkworms were obtained from the pBac[A3-EGFP+IE1-OmElo-SV40] vector.

[0061] The obtained ant silkworms are fed with mulberry leaves until moths become moths, and the obtained silkworms are backcrossed or selfed. Obtain 33 moth c...

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Abstract

The invention discloses a rainbow trout fatty acid elongase gene named as an OmElo gene. The nucleotide sequence of the rainbow trout fatty acid elongase gene is SEQ ID No.1. The OmElo gene is the novel elongase gene obtained after a source gene is optimized and designed according to the preference of encoding gene expression sequential codons in silkworm genome sequential data. The amino acid sequence of the protein encoded by guidance of the OmElo gene is SEQ ID No.2. The invention further provides a recombinant expression vector comprising the OmElo gene and a construction method thereof. A promoter of an expression cassette of the recombinant expression vector is the BmNPV very-early promoter IE1. The recombinant expression vector comprising OmElo gene can be applied to obtaining a silkworm transgenic strain with the high content of cis-11,14,17-epoxyeicosatrienoic acid. The invention further provides a method for improving the content of the cis-11,14,17-epoxyeicosatrienoic acid in silkworm tissue, the rainbow trout fatty acid elongase OmElo gene can be guided into a silkworm embryo, and screening is performed to obtain the target silkworm transgenic strain.

Description

technical field [0001] The invention relates to a rainbow trout fatty acid elongase gene and its encoded protein, a recombinant vector, and a method for increasing the content of 11,14,17-cis-eicosatrienoic acid in silkworm chrysalis tissues, belonging to the fields of genetic engineering and transgenic animal breeding. Background technique [0002] Fatty acids can be divided into saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs) according to the degree of unsaturation. In organisms, many metabolic processes are carried out on or related to membranes, and unsaturated fatty acids are important components of cell membrane phospholipids. Since the freezing point of fatty acids decreases with increasing unsaturation, the higher the content of unsaturated fatty acids in membrane lipids, the lower its phase transition temperature and the greater its fluidity. Furthermore, various functions of the cell membrane, such as cell recognition, tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N15/66C12N15/85A01K67/04
Inventor 沈以红于新波黄先智
Owner SOUTHWEST UNIV
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