Key enzyme MCAT in synthesis path of Nannochloropsis gaditana fatty acid FAS II

A technology for fatty acid synthesis and Nannochloropsis, which is applied in the field of enzyme proteins, can solve the problems of blocked protein synthesis, lack of related enzymes, and reduced overall cell activity, and achieves the effect of improving the efficiency of oil accumulation

Inactive Publication Date: 2015-05-27
QINGDAO UNIV
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Problems solved by technology

Under nitrogen deficiency stress, the expression of Ng-MCAT gene showed a trend of first increasing and then decreasing. As time went on,

Method used

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  • Key enzyme MCAT in synthesis path of Nannochloropsis gaditana fatty acid FAS II
  • Key enzyme MCAT in synthesis path of Nannochloropsis gaditana fatty acid FAS II
  • Key enzyme MCAT in synthesis path of Nannochloropsis gaditana fatty acid FAS II

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Embodiment 1

[0021]The experiment selected Nannochloropsis gaditana HH-1 from the algal species library of the First Institute of Oceanography, State Oceanic Administration. Competent cells (E.coli) were purchased from Quanshijin Company, Escherichia coli strain DH5α was preserved in our laboratory, Saccharomyces cerevisiae strain and pYES2 expression plasmid were purchased from Invitroen Company; primer synthesis and sequencing were carried out in Shanghai Sangong. For the experimental methods that do not indicate specific conditions in the following examples, conventional conditions can usually be used, such as the conditions described in the "Molecular Cloning Experiment Guide" written by J. Sambrook (Sambrook), or according to the manufacturer's suggestion conditional run. Embodiment 1: Acquisition of full-length cDNA of the key enzyme MCAT encoding protein in the FAS type II fatty acid synthesis pathway of the present invention

[0022] In the present invention, firstly, the whole ge...

Embodiment 2

[0031] Example 2: Bioinformatics analysis of the key enzyme MCAT in the FAS type II fatty acid synthesis pathway

[0032] The open reading frame analysis of Ng-MCAT gene was carried out by NCBI; the theoretical molecular weight and isoelectric point of the protein were predicted by ProtParam software; the TmPred algorithm software was used to analyze and predict the transmembrane region of the protein; the third level of Ng-MCAT protein was predicted by SWIMM-MODEL Structure; finally, compare the determined Ng-MCAT sequence with the MCAT genes of other species known in GenBank with BLAST software, select sequences with higher similarity and use Bioedit software for alignment sorting, and construct a phylogenetic tree; use Bioedit software Amino acid sequence homology analysis was carried out between Ng-MCAT and MCAT of several algae with close evolutionary relationship.

[0033] Using GeneDoc software, the Ng-MCAT amino acid sequence was homologously compared with the MCAT ami...

Embodiment 3

[0034] Embodiment 3: MCAT Saccharomyces cerevisiae expression vector construction

[0035] Construction and main structure of pYES2-MCAT recombinant plasmid Figure 4 shown

[0036] Digest Ng-MCAT and shuttle plasmid pYES2 with BamH I and XbaI, connect with T4 DNA ligase to obtain plasmid pYES2, transform Escherichia coli DH5α, extract a small amount of plasmid DNA and digest it with TBamH I and XbaI, and detect the digested products by electrophoresis , excised a fragment of about 1059bp, indicating that Ng-MCAT was connected to the expression plasmid pYES2. The Ng-MCAT gene was amplified by PCR with the designed primers, and the amplification result was detected by electrophoresis. The amplified fragment of Ng-MCAT was about 1059 bp, which was in line with the expected size.

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Abstract

The invention belongs to the technical field of enzyme proteins, and specifically relates to a key enzyme MCAT in a synthesis path of Nannochloropsis gaditana fatty acid FAS II. MCAT can code protein gene for acquiring full-length cDNA. The enzyme is a key enzyme in the lipid accumulation process through the accumulation effect of MCAT in the synthesis path of high-oil-yield Nannochloropsis gaditana FAS II fatty acid. The expression in saccharomyces cerevisiae discovers that the MCAT can accelerate extension of an aliphatic carbon chain. Meanwhile, the MCAT can also be guided into Nannochloropsis gaditana, rape and arabidopsis through gene engineering, so as to improve the lipid accumulation efficiency better.

Description

technical field [0001] The invention belongs to the technical field of enzyme proteins, and in particular relates to the key enzyme MCAT in the synthesis pathway of Nannochloropsis FAS II fatty acid. Background technique [0002] Marine microalgae have become a hotspot in the research of marine biomass energy because of their high photosynthetic efficiency, strong environmental adaptability, short growth cycle, and no climate restrictions. Nannochloropsis gaditana has a wide ecological distribution. It already has abundant algal strain resources in the global algal species bank. It has a unique evolutionary status. It is an independent genus of single-celled microalgae and has many unique advantages for industrial production: It grows rapidly, has strong stress resistance, accumulates a large amount of TAG, and can accumulate high value-added products such as EPA and carotenoids. Recently, a research team successfully established a transformation system based on homologous ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/81C12P7/64
CPCC12N9/10C12P7/6436
Inventor 王玲赵燕燕王春李妍王杰贾婉军
Owner QINGDAO UNIV
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