MORTIERELLA ALPINA RECOMBINANT GENE EXPRESSION SYSTEM AND CONSTRUCTION METHOD AND USE THEREOF

摘要

It relates to a Mortierella alpina recombinant gene expression system, to its construction method and application which is constructed by transformation of M. alpina ATCC 32222 uracil auxotroph strain through A. tumefaciens mediate transformation (ATMT) and is based on the existing uracil auxotrophic strain, through genetic engineering methods to obtain a final phenotype complementary strain to achieve the malic enzyme 1 and malic enzyme 2 overexpression strains.

主权项

1. A homologous recombinant Mortierella alpina strain overexpressing a malic enzyme gene, characterized in that the strain is constructed by transforming M. alpina uracil auxotroph strain using Agrobacterium tumefaciens containing malic enzyme gene, and the said Agrobacterium tumefaciens containing malic enzyme gene harbors the plasmid pBIG2-ura5s-malE1 or pBIG2-ura5s-malE2;
The said plasmid pBIG2-ura5s-malE1 is constructed with the following steps: The HPH expressing cassette is PCR amplified from pD4 plasmid and digested with EcoRI and XbaI, followed by insertion into the multiple cloning site (MCS) of pET28a (+) digested with EcoRI and XbaI, to form plasmid pET28a-HPHs; subsequently, the ura5 gene is digested with BspHI and BamHI, and the digested ura5 gene is inserted into pET28a-HPHs digested with NcoI and BamHI, to replace hpt gene to form pET28a-ura5s; the ura5s expressing cassette is obtained by digesting plasmid pET28a-ura5s with EcoRI and XbaI; replace HPH expressing cassette in pBIG2RHPH2 with the resultant ura5s expressing cassette to form transformant plasmid pBIG2-ura5s; the ma/E1 gene segment is digested with BspHI and BamHI, and pET28a-HPHs is digested with NcoI and BamHI, respectively, and ma/E1 gene segment is inserted into NcoI and BamHI site of plasmid pET28a-HPHs by ligation reaction to form plasmid pET28a-malE1; the ma/E1 expressing cassette is obtained by double digesting plasmid pET28a-malE1 with SpeI and XbaI; the malE1 expressing cassette is inserted into pBIG2-ura5s digested with XbaI to form plasmid pBIG2-ura5s-malE1; The said plasmid pBIG2-ura5s-malE2 is constructed with the following steps: the IT noncoding intron DNA segment is achieved by PCR method from M. alpina genome, IT gene segment and plasmid pET28a-HPHs is digested with NcoI and BamHI respectively, then replace hpt gene in the pET28a-HPHS with IT segment by ligation reaction to obtain plasmid pET28a-Its, then pET28a-Its is double digested with SpeI and XbaI, to obtain ITs expression unit; the resultant ITs expression unit is inserted into pBIG2-ura5s digested by XbaI, to form the M. alpina gene manipulation common vector pBIG2-ura5s-ITs; the malE2 gene is double digested with KpnI and XmaI, then conduct ligation with ligase, to form malE2 expression plasmid pBIG2-ura5s-malE2.