Delta12-fatty acid dehydrogenase gene and application of gene

A fatty acid dehydrogenase and unsaturated fatty acid technology, applied in genetic engineering and biological fields, can solve problems such as difficult separation and difficult biochemical characteristics, and achieve the effects of improving oil quality, low cost and short production cycle

Inactive Publication Date: 2014-06-04
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Especially in recent years with the bifunctional Δ 12 - The discovery of fatty acid dehydrogenase [Edgra B. Cahoon et al, 2001, THE JOURNAL OF BIOLIGICAL CHEMISTRY, 26: 2637-2643.] and the discovery of conjugated oleic acid in health care, people are increasingly aware of unsaturated The importance

Method used

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  • Delta12-fatty acid dehydrogenase gene and application of gene
  • Delta12-fatty acid dehydrogenase gene and application of gene
  • Delta12-fatty acid dehydrogenase gene and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Isolation of Δ from Mortierella chrysogenum M6-22 12 - Nucleotide sequence of fatty acid dehydrogenase

[0024] Genomic DNA of Mortierella flavinum M6-22 was extracted using the Ezup Column Fungal Genomic DNA Extraction Kit (product number: SK8259) of Sangon Bioengineering (Shanghai) Co., Ltd., and 1ul was used as a template for polymerase chain reaction. response, according to the published Δ 12 -Degenerate primers (primer 1 and primer 2) were designed for the amino acid sequence of the histidine conserved region Ⅰ and Ⅲ region of the fatty acid dehydrogenase homologous sequence, and PCR amplification was carried out on a PCR machine (BIOER company) with the above-mentioned extracted DNA template , the primers, components and amplification conditions used in the reaction are as follows:

[0025] Primer 1: FD12CRF1: 5`-CA(T / C)GA(A / G)TG(T / C)GGICA(T / C)CA(A / G)(G / T)CITT-3`

[0026] Primer 2: FD12CRR3: 5`-(A / T)A(A / G)TG(A / G)TGI(A / G)(A / G / C)IAC(A / G)TGIGT-3`

[00...

Embodiment 2

[0048] Example 2: Homology Search of Isolated Nucleotide Sequences

[0049] will infer that Δ 12 - The amino acid sequence of fatty acid dehydrogenase was searched for homology on Genbank, and most of the obtained homologous sequences encoded Δ 12 -Sequences of fatty acid dehydrogenases, a few are hypothetical protein sequences, select Δ that has been functionally verified 12 - Fatty acid dehydrogenase for homology comparison, which is compared with Mucor circinosa ( Mucor circinelloides )Δ 12 - Fatty acid dehydrogenase has the highest homology, with an identity of 58% ( figure 1 ). figure 1 Shows the separation of Δ in Mortierella flavinum M6-22 of the present invention 12 - Fatty acid dehydrogenase and Mucor circinatus Δ 12 -Comparison of amino acid sequence homology of fatty acid dehydrogenase (GenBank: BAB69056.1), MID12 is Mortierella chrysogenum M6-22Δ of the present invention 12 - Fatty acid dehydrogenase, MCD12 for Mucor circinatus Δ 12 - Fatty acid dehydrog...

Embodiment 3

[0050] Example 3: Construction of Saccharomyces cerevisiae recombinant expression vector

[0051] According to the sequence of the coding region shown in SEQ ID NO:1, a pair of gene-specific amplification primers (primer 3 and primer 4) were designed to isolate its potential open reading frame sequence:

[0052] Primer 3: MID12F:5`-ATT AAGCTT ATGCCACCCAACGTAACGTCT-3`

[0053] Primer 4: MID12R:5`-GCA CTCGAG TTAGTTTTTTGAAGAACAAGACGT-3`

[0054] The underlined parts at the 5‵ ends of these two primers respectively contain Hind III and xho Ⅰ Restriction site, the amplification conditions and reaction components used are the same as the PCR reaction using cDNA as template, and the sequencing result of the amplified product shows that it is consistent with the sequence of 1-1194bp shown in SEQ ID NO:1. Then take 25ul of PCR products and 10ulpYES3 / CT for double enzyme digestion, recover the large fragments, and use T4 ligase to ligate at 16 degrees for 4 hours. The ligated p...

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Abstract

The invention discloses a delta12-fatty acid dehydrogenase gene separated from mortierella isabellina M6-22, the nucleotide acid sequence is shown in the SEQIDNO:1, the code is an amino acid sequence shown in the SEQIDNO: 2; by constructing a recombinant carrier and expressing in saccharomyces cerevisiae, the expression product has delta12-fatty acid dehydrogenase function and is capable of catalyzing the oleic acid to convert into linoleic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and relates to a yeast-Mertierella fumigatus ( Mortierella isabellina Δ of clones in ) 12 - Fatty acid dehydrogenase gene, specifically Mortierella chrysogenum Δ 12 - the nucleotide sequence of fatty acid dehydrogenase and its application; the gene is directly or connected with different vectors, transferred into bacteria, yeast, plants or animals, and used to encode Δ 12 - Method and use of fatty acid dehydrogenase for producing unsaturated fatty acids. Background technique [0002] Δ 12 - Fatty acid dehydrogenase (Δ 12 -fatty acid desaturases) is also known as oleate desaturases. Δ 12 - Fatty acid dehydrogenase belongs to a class of membrane integrases, due to the difficulty of isolating and identifying membrane-bound proteins [MaKeon, 1981, Methods in Enzymology, 12141-12147; Wang et al., 1988, Plant Physiology and Biochemistry, 26:777- 792], so it is difficult to ob...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/81C12N1/19C12P7/64C12R1/865C12R1/645
Inventor 张琦李凌彦何仕武胡彬彬魏云林林连兵季秀玲
Owner KUNMING UNIV OF SCI & TECH
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