酵母高效克隆表达T载体pYES2-T的构建及应用  

Constrcution and Application of Yeast High Throughput Cloning and Expression T Vector pYES2-T

作　　者：曹雪松 曹高燚 武俊杰 詹瑶 丁博 李明 谢晓东 Cao Xuesong;Cao Gaoyi;Wu Junjie;Zhan Yao;Ding Bo;Li Ming;Xie Xiaodong(College of Agronomy&Resources and Environment,International Joint Research Center for Crop Stress Resistance Mechanism and Genetic Improvement,Tianjin,300384)

机构地区：[1]天津农学院农学与资源环境学院,天津市作物抗逆的机理及遗传改良国际联合研究中心,天津300384

出　　处：《基因组学与应用生物学》2020年第6期2607-2613,共7页Genomics and Applied Biology

基　　金：国家自然科学基金(31671611和31801446);天津市高校创新团队和天津市农委项目(TJNWY2017008)共同资助。

摘　　要：为了在酵母中高通量、高效率地对外源基因进行克隆和表达,本研究以酵母表达载体pYES2为基础,构建高效克隆表达T载体。pYES2是酿酒酵母的一种高效表达载体,在其多克隆位点处设计插入XcmⅠ-Intron-XcmⅠ片段,同时将其载体中URA3基因中原有的XcmⅠ酶切位点进行定点突变,构建出pYES2-T酿酒酵母高效表达T载体。利用XcmⅠ酶切载体pYES2-T,使其产生两个突出的T末端,一方面可以通过PCR产物加A的方式直接进行TA克隆;另一方面可在半乳糖诱导启动子的调控下,对TA克隆后的外源基因在酵母中诱导表达。利用该系统高通量地对人工合成耐盐系列基因NLEAs进行筛选,研究结果表明,该系统可以在酿酒酵母中一步式完成片段克隆及耐盐基因的筛选。本研究构建的酵母表达T载体使用方便、效率高、成本低,应用前景广阔。In this study, an efficient expression clone T vector was constructed based on the yeast expression vector p YES2 in order to clone and verify the foreign gene expression in yeast with high throughput and efficiency.The vector pYES2 has high protein expression efficiency in yeast Saccharomyces cerevisiae. Yeast efficient expression vector pYES2-T was constructed through the insertion of XcmⅠ-Intron-XcmⅠ fragment into MCS and the fixed-point mutations of the origin XcmⅠ enzyme cutting site at URA3 gene. Digesting pYES2-T by Xcm Ⅰcan produce two dT ends at the both sides of the vector. Then TA cloning can be carried out directly by adding dA to PCR product of the target gene;On the other hand, the gene expression can be easily to control under galactose inducible promoter after TA cloning of the gene in yeast expression. The synthetic salt tolerant series gene NLEAs were screened by this high-throughput T vector system, indicating that the cloning and salt-tolerant screening can be conducted in one step in Saccharomyces cerevisiae. The yeast expression T vector constructed by this research is easy to use, highly efficient, low cost with wide application prospect.

关 键 词：酿酒酵母 表达T载体 载体构建 

分 类 号：Q78[生物学—分子生物学]