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Novel application of delta12-fatty acid desaturase gene

A fatty acid dehydrogenase and gene technology, applied in the fields of genetic engineering and biology, can solve problems such as the inability of output and quality to meet market demand, and achieve the effects of low cost, high feasibility and short production cycle

Inactive Publication Date: 2015-09-09
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the production of some PUFAs has been industrialized, and many others are being developed and utili

Method used

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  • Novel application of delta12-fatty acid desaturase gene
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  • Novel application of delta12-fatty acid desaturase gene

Examples

Experimental program
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Effect test

Embodiment 1

[0015] Example 1: Known and potential Δ for some species of reference yeast 12 - Fatty acid dehydrogenase gene sequence, design specific primers (primer 1 and primer 2), and use cDNA as a template to carry out PCR reaction, the primer sequence is as follows:

[0016] Primer 1: RKD12F:5`-ATAAGCTTATGGCCGCTACCCTCCGCCAGC-3` (SEQ ID NO: 1)

[0017] Primer 2: RKD12R:5`-ATGAATTCCTAGAGTCCCTCGACGCCCGAGT-3` (SEQ ID NO: 2)

[0018] First, use the eukaryotic cell total RNA extraction kit (Promega company product) kit to extract the total RNA of the cell, use the reverse transcription kit (fermentas company product) to reverse transcribe and synthesize the first cDNA, and then use it as a template for PCR Reaction, PCR amplification system (25 μL) is composed as follows:

[0019] 10×Ex Taq Buffer 2.5 μL

[0020] dNTP (2.5 μmol / L) 2 μL

[0021] Template 3 μL

[0022] P1 (10 μmol / L) 2 μL

[0023] R1 (10 μmol / L) 2 μL

[0024] Ex Taq DNA polymerase (5U / μL) 2 μL

[0025] Sterile ddH...

Embodiment 2

[0027] Embodiment 2: Construction of Saccharomyces cerevisiae recombinant expression vector

[0028] According to the coding region sequence of the sequence obtained in Example 1, a pair of gene-specific amplification primers (primer 3 and primer 4) were designed to isolate its potential open reading frame sequence:

[0029] Primer 3: RKD12F:5`-ATA AGCTT ATGGCCGCTACCCTCCGCCAGC-3`

[0030] Primer 4: RKD12R:5`-ATG AATTC CTAGAGTCCCTCGACGCCCGAGT-3`

[0031] The underlined parts at the 5‵ ends of these two primers respectively contain Eco RI and Hind Ⅲ Restriction site, the amplification conditions and reaction components used are the same as the PCR reaction using cDNA as a template, and the sequencing results of the amplified product show that it is consistent with the sequence of 1-1341bp shown in GenBank accession number KJ502671.1; Then take 25 μl of PCR products and 10ul pYES3 / CT (Invitrogen) for double enzyme digestion, electrophoresis to recover the large fragments,...

Embodiment 3

[0032] Example 3: Saccharomyces cerevisiae recombinant expression vector pY3RKD12 transforms Saccharomyces cerevisiae cells

[0033] Pick a single colony of Saccharomyces cerevisiae strain INVSC1 in 5ml YEPD liquid medium, cultivate it overnight on a shaker at 30°C, transfer it to 100ml of YEPD liquid medium according to the inoculum size of 1%, shake it at 30°C until the OD600 is between 1.3- Between 1.5; 4°C, 4000g centrifugation for 5 minutes to collect the pelleted cells, and use 100ml pre-cooled sterile ddH 2 O Wash the cells, centrifuge at 4°C and 4000g for 5 minutes to collect the pelleted cells, and then use 50ml pre-cooled sterile ddH 2 O Wash the cells, centrifuge at 4°C and 4000g for 5min to collect the precipitated cells, wash the cells with 20ml of pre-cooled 1M sorbitol, centrifuge at 4°C and 4000g for 5min to collect the precipitated cells, suspend the cells with 0.5ml of pre-cooled 1M sorbitol, each 80μl Dispense into 1.5ml pre-cooled centrifuge tubes, take 2...

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Abstract

The invention discloses novel application of a delta12-fatty acid desaturase gene and discloses novel application to encoding of a sequence of a delta12-fatty acid desaturase gene separated from rhodosporidium toruloides YM25235, namely application to production of polyunsaturated fatty acids. A gene encoding product has the activity of delta15-fatty acid desaturase and can catalyze linoleic acid to be converted into alpha-linolenic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and relates to a Δ 12 -Application of fatty acid dehydrogenase gene sequence (GenBank accession number: KJ502671.1) in the production of α-linolenic acid. Background technique [0002] Polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids with two or more double bonds and 18-22 carbon atoms. Many studies have shown that, such as arachidonic acid AA (Arachidonic acid ARA, 20:4n-6), eicosapentaenoic acid EPA (Eicosapentaenoic acid, C20:5n-3) and docosahexaenoic acid DHA (Docosahexaenoic acid acid, 22:6n-3) and other long-chain polyunsaturated fatty acids are important components of cell membranes, and are also precursors of hormone-like (Hormone-like) biologically active substances in animals and humans. Anti-tumor and anti-inflammation, promote cell growth, prevent cardiovascular and cerebrovascular diseases, benefit the formation of brain retina, immune reg...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/81C12P7/64
Inventor 张琦杨晓霞季秀玲魏云林林连兵
Owner KUNMING UNIV OF SCI & TECH
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