【作者】 郭春叶；

【导师】 龚月生； 刘林丽；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2007， 硕士

【摘要】 本试验应用基因重组技术构建葡萄芪合酶基因(stilbene synthase,STS)重组酵母菌,通过半乳糖诱导外源基因表达,验证芪合酶基因在酵母菌中的表达情况,进而为开发一种含有白藜芦醇生物活性成分的酵母微生态制剂提供了重要的试验素材。将从中国野生属葡萄种高抗白粉病的华东葡萄白河-35-1中分离出的芪合酶基因cDNA片段,进行PCR扩增;把纯化回收的目的基因PCR产物定向插入克隆载体pGEM-T Easy中进行测序与序列分析;经DNA测序,克隆片段全长1195 bp,其中编码芪合酶基因的片段长1179 bp(包括起始密码子和终止密码子),共编码392个AA,与Genbank中已有的葡萄芪合酶基因进行同源性比对,其核苷酸同源性达98 %,由此推导出的氨基酸序列与已公布的氨基酸序列同源性为99 %,且位于第164位的半胱氨酸(Cys)酶活中心的碱基未发生突变。将芪合酶基因与克隆载体pGEM-T Easy连接的重组质粒pGEM-T-STS以及酵母表达载体pYES6/CT用BamH I/Xho I双酶切,采用定向克隆的方法将纯化回收的芪合酶基因克隆到大肠杆菌-酵母菌穿梭型载体pYES6/CT上,转化大肠杆菌,提取阳性重组质粒转化酿酒酵母菌(Saccharomyces cerevisiae)菌株INVSc1,经杀稻瘟素(Bsd)抗性筛选出阳性克隆转化子,并进行菌落PCR鉴定;酵母菌落PCR电泳结果显示,在约1200 bp处出现一条亮带,证明芪合酶基因酵母表达载体pYES6/CT-STS构建成功。将含有阳性单克隆的酵母菌用半乳糖诱导表达,收集诱导表达4、8、12、16、20及24 h的酵母菌采用超声波破碎菌体,并进行全蛋白SDS-PAGE电泳分析;SDS-PAGE电泳分析发现与空白菌相比含目的基因的诱导菌有目标带(48 kDa)出现,证明目的基因cDNA片段可以在酵母菌中表达。本试验成功构建了葡萄芪合酶基因酿酒酵母表达载体,并实现了在酿酒酵母菌的大量表达,为下一步利用该菌株生产微生态制剂奠定了基础。更多还原

【Abstract】 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更多还原