Research On The Construction Of Stilbene Synthase (STS) Genetic Yeast Expression Vector And Its Expression In Yeast

With the technology of gene recombination, the recombinant yeast of STS gene was constructed. It may provide important experiment materials for developing a yeast micro-ecological agent containing resveratrol.A cDNA fragment of STS gene derived from Vitis pesudoreticulata Baihe-35-1 was amplified by PCR. And then the recycling target gene was inserted into the clone vector pGEM-T Easy and carried on sequence analysis. After sequencing, the length of the cloned fragment was 1195 bp and the fragment encoding stilbene synthase gene was 1179 bp (including the start codon and termination codon) which encode 392 AA. The identity of the gene with the grape STS gene registered in Genbank was 98 % and the derived protein was 99 %. And the enzyme activity center-cysteine (Cys) which was the 164th AA did not mutate at all.Both pGEM-T-STS and pYES6/CT were digestioned by BamH I and Xho I . With the directional cloning method the reclaimed and purified STS gene was cloned into pYES6/CT, a shuttle type plasmid vector of E.coli-yeast and transformed into E.coli. The positive vector was transformed into Saccharomyces cerevisiae INVSc1 strains. The positive colones were screened out with Bsd and further identified by the colony PCR. The yeast colony PCR results showed that a 1200 bp band proved stilbene synthase gene yeast expression vector was constructed successfully.The yeast containing positive clones were induced with galactose. 4, 8, 12, 16, 20 and 24-hour collection of ultrasonic broken yeast cells was collected and analyzed by SDS-PAGE. By SDS-PAGE analysis the target protein band (48 kDa) appeared in the induced yeast containing the target gene, which identified the target gene cDNA fragment can express in Saccharomyces cerevisiae.This experiment successfully constructed Saccharomyces cerevisiae grape stilbene synthase gene expression vector, and realized a lot of target protein expression in Saccharomyces cerevisiae. The results layed the foundation for the forward experiment of producing the yeast micro-ecological agent containing resveratrol...