赤霞珠白藜芦醇合酶基因的克隆及酿酒酵母表达载体的构建

为克隆酿酒葡萄品种赤霞珠的白藜芦醇合酶基因并构建其酿酒酵母表达载体,利用改良CTAB法提取赤霞珠葡萄叶片组织中的DNA,根据文献报道的序列(Genbank登录号AF128861)设计引物扩增得到白藜芦醇合酶基因全长序列。将全长序列克隆至克隆载体后,通过重叠延伸PCR扩增到RS基因cDNA片段。然后分别将RS基因全序列和cDNA片段连接至真核表达载体pGBKT7,经克隆和测序分析后,构建两种真核表达载体pGBKT7/RSDNA和pGBKT7/RScDNA,用SD-trp选择培养基筛选出阳性克隆,菌落PCR验证得到阳性转化子。

Cloning of Resveratrol Synthase Gene from Cabernet Sauvignon and Construction of Yeast Expression Vector

The aim of this study is to clone resveratrol synthase gene from vinifera cultivar Cabernet Sauvignon and to construct its yeast expression vector. The DNA of Cabernet Sauvignon from leaf tissue was extracted with modified CTAB method.Then the full length gene sequence was obtained by PCR with the primers designed from previously reported RS gene sequence(Genbank accession number is AF128861). After the full length gene sequence was cloned into cloning vector, we acquired the RS gene full length cDNA sequence with overlap extension PCR method.Then the full length gene and cDNA were cloned into pGBKT7, a shuttle type plasmid vector of yeast, and transformed Saccharomyces cerevisiae AH109, respectively. Two kinds of yeast expression vector, pGBKT7/RSDNA and pGBKT7/RScDNA, were confirmed to be constructed using sequencing analysis. The recombinant strains were selected with selective SD trp media and identified by colony PCR.