Molecular Cloning And E.coli Expression Of Squalene Synthase Gene From Vitis Vinifera

Squalene synthase catalyses the conversion of FPP to squalene,and the synthesis of squalene is the branch point from isoprenoid central metabolism pathway to the synthetic metabolism of plantsterols and triterpenoids which have many functions of health protection and physiology Therefore,people pay much attention to the research of squalene synthase.In this research, a SQS gene was cloned from Vitis vinifera,by using RACE technique.The recombinant plasmid was transformed into E.coli strain BL21 and induced to express.The results were as following:The first, gene cloning of VvSQS. This research extracted total RNA from the leaves of Vitisvinifera with trizol; designed specific primers according tothe conserved domain of other SQS genes from various plants; obtained 957bp conserved sequence of SQS gene from Vitis vinifera; designed specific primers based on this sequence; by RACE amplified the full length cDNA (1595bp) of SQS gene from Vitis vinifera, which was denominated as VvSQS. VvSQS contained a 1242 bp open reading frame(orf )encoding a 413-amino-acid polypeptide of which the estimated molecular weight was 47．3kDa.The second, characterization of VvSQS.Nucleotide-nucleotide BLAST showed that the full length cDNA sequence of VvSQS shared 82%,82%,82% homology with those of Panax notoginseng,Panax ginseng,Glycyrrhiza uralensis respectively;and protein-protein BLAST analysis showed that the deduced amino acid shared 84%, 83%, 84% idedtities and 91%,92%,91% positives with those of Panax notoginseng,Panax ginseng,Glycyrrhiza uralensis respectively.Analyzing conserved domain of the deduced amino acid sequence indicated that VvSQS contained three conserved domains,which was similar to SQS from other plants.Based on the above results it could be inferred that VvSQS had functions of SQS gene.The third,prokaryotic expression of SQS.By inserting the open reading frame (ORF) of myrosinase gene into prokaryotic expressional vector pET-28a(+) to construct recombinant plasmid.The recombinant plasmid was transformed into E.coli strain BL21 and induced by IPTG to express.The result of SDS-PAGE electrophoresis indicated that myrosinase gene had expressed in E.coli and the molecular mass of recombinant protein was about 47kDa.