A recombinant Mortierella alpina overexpressing ω-3 desaturase derived from Phytophthora parasitica, its construction method and application

A technology of Mortierella alpina and Phytophthora parasitica applied in the field of genetic engineering

Active Publication Date: 2020-03-10
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, Xue et al. isolated three ω-3 desaturases—PaD 17, PsD 17, and PrD 17—from Pythium melon and fruit, Phytophthora soybean, and sudden death of oak trees. All omega-6 polyunsaturated fatty acids with carbons have catalytic ability, but they prefer to catalyze AA with 20 carbons, and the conversion rates reach 56%, 47% and 36% respectively

Method used

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  • A recombinant Mortierella alpina overexpressing ω-3 desaturase derived from Phytophthora parasitica, its construction method and application
  • A recombinant Mortierella alpina overexpressing ω-3 desaturase derived from Phytophthora parasitica, its construction method and application
  • A recombinant Mortierella alpina overexpressing ω-3 desaturase derived from Phytophthora parasitica, its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Enzyme cleavage reaction

[0051] According to the codon usage preference of Mortierella alpina, the nucleotide sequence of the ω-3 desaturase gene (Genbank accession No: XM_008906963) derived from the natural Phytophthora parasitica was codon-optimized, and the optimized gene was artificially synthesized Sequence oPpFADS17, as shown in SEQ No.1 and 2. Then it was connected with PUC57 vector to obtain PUC57-oPpFADS17.

[0052] Under the condition of 37°C, first digest the plasmid PUC57-oPpFADS17 and the vector pBIG2-ura5s-ITs fragment overnight with the restriction endonuclease Hind III. The Hind III digestion system (100 μL) is: 2 μL Hind III-HF, 30 μL plasmid or Carrier, 10 μL Cutsmart Buffer, 58 μL deionized water, incubated at 37°C for 12h.

[0053] Among them, the vector pBIG2-ura5s-Its is directly obtained according to the Chinese patent application CN201310524221.4.

[0054]The HPH expression unit was obtained from the pD4 plasmid by PCR, and the HP...

Embodiment 2

[0057] Example 2: Ligation reaction

[0058] The digested and purified ω-3 desaturase gene fragment oPpFADS17 was ligated with the vector pBIG2-ura5s-ITs with T4 ligase, and ligated at 4°C for 12 hours to obtain the recombinant expression vector pBIG2-ura5s-oPpFADS17. The ligation system is (10 μL): 2 μL of the digested fragment of the target gene, 3 μL of the digested fragment of the vector, 1 μL of ligase buffer, 1 μL of T4 ligase, 3 μL of sterile water, and ligate at 4°C for 12 hours.

[0059] The ligation product was transformed into Escherichia coli TOP10 competent cells, and the transformation method was as follows:

[0060] (1) Take 100 μL of competent cells in a sterile state, add 1-2 μL of the ligation product, and mix by blowing and aspiration.

[0061] ⑵ Transfer the mixed competent cells into the pre-cooled electroporation cuvette to avoid the generation of air bubbles.

[0062] ⑶Put the electric cup into the Bio-Rad electroporation instrument, adjust to the appr...

Embodiment 3

[0068] Example 3: Agrobacterium tumefaciens-mediated transformation of Mortierella alpina

[0069] On the basis of the existing domestic and foreign literature reports on the transformation method of Agrobacterium tumefaciens, appropriate optimization adjustments were made, as follows:

[0070] (1) Agrobacterium tumefaciens C58C1 containing the plasmid pBIG2-ura5s-oPpFADS17 stored at -80°C was streaked on a YEP solid medium plate containing 100 μg / mL rifampicin and 100 μg / mL kanamycin. Incubate at 28°C for 48 hours in the dark.

[0071] (2) Pick a single clone and inoculate it into 20 mL of liquid YEP medium containing 100 μg / mL rifampicin and 100 μg / mL kanamycin at 28°C, 200 rpm, and incubate in the dark for 24-48 hours.

[0072] (3) Centrifuge at 4000×g for 5 minutes to collect the bacteria, and discard the supernatant. Add 5mL IM medium to resuspend the bacteria, centrifuge at 4000×g for 5min, and discard the supernatant. Add 2mL IM medium to resuspend the bacteria.

[...

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Abstract

The invention provides restructured mortierella alpina strain MA-oPpFADS17-4 with heterologous experession from omega-3 desaturase genes of phytophthora parasitica, and provides a method for establishing the restructured mortierella alpina. The mortierella alpina uracil heterotrophia type strain is used as materials and produces a constant-temperature high-yield EPA restructured strain through the genetic operation technology of agrobaterium tumefaciens mediate. Production of EPA reaches 1197.3 mg / L and accounts for 31.5% of TFA content, and conversion rate for AA is 77.6%. The restructured mortierella alpina strain is of great significance for basic theory research and product development of the oil-producing fungus, mortierella alpina.

Description

【Technical field】 [0001] The invention relates to the field of genetic engineering, in particular to a strain of Mortierella alpina overexpressing the ω-3 desaturase oPpFADS17 gene derived from Phytophthora parasitica, and also relates to its construction method and application. 【Background technique】 [0002] Mortierella alpina is an oleaginous fungus whose lipid accumulation can reach 50% of the dry cell weight. It is an important model organism for basic research on lipid biochemistry. Mortierella alpina has been used in the industrial production of arachidonic acid (Arachidonic acid, AA, C20:4), and the edible oil it produces has passed the safety assessment of the US Food and Drug Administration (FDA). In addition to synthesizing AA, Mortierella alpina also has a certain ability to synthesize eicosapentanoic acid (EPA, C20:5). EPA belongs to omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), which has important physiological functions, such as: promoting the de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N15/80C12P7/64C12R1/645
CPCC12N9/0071C12P7/6427C12Y114/19
Inventor 陈海琴陈永泉陈卫梅甜甜顾震南张灏
Owner JIANGNAN UNIV
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