酿酒酵母PMT1与PMT2双基因过表达菌株构建及其复制寿命研究

Construction of protein O-mannosyltransferases 1 and 2 double-gene overexpressed strain and study on replicative lifespan in Saccharomyces Cerevisiae

目的构建酿酒酵母蛋白质O-甘露糖转移酶PMT1与PMT2双基因过表达菌株,研究其对酵母细胞复制性寿命的影响。方法以PCR技术为基础,通过分子克隆的方法构建PMT2基因表达载体;通过同源重组实现PMT1和PMT2双基因在酵母细胞中过表达;实时定量PCR验证过表达菌株中相关基因的转录水平;光学显微镜下分离和计数酵母目细胞产生子细胞的数目,检测酵母细胞的复制性寿命。结果通过克隆技术成功构建PMT2基因过表达载体(pRS305-PMT2-ox),并转化和筛选出PMT1与PMT2双基因过表达菌株(PMT1ox PMT2ox);转录水平分析证明PMT1和PMT2基因表达上调;复制性寿命检测结果显示:与野生型菌株比较,PMT1ox PMT2ox菌株的平均寿命无明显变化。结论过表达PMT1与PMT2基因不影响酵母菌株的平均复制性寿命,提示PMT1与PMT2双基因过表达不参与酵母复制性寿命的调节。

Objective Construction of proteinO - mannosyltransferases 1 （ PMT1 ） and protein - O - mannosyhransferases 2 （ PMT2 ） double - gene overexpressed strain and study of their effects on the replicative lifespan in Saccharomyees Cerevisiae. Methods Vector with PMT2 gene was constructed by cloning technology. PMT1 and PMT2 double - gene overexpression strain, based on the principle of homologous recombination, was constructed by transforming the linearing PMT2 - expression vector into PMT1 overexpres- sion yeast cells. The transcriptional levels of PMT1 and PMT2 in the overexpressed strain were determined by real - time quantitative PCR （RT- PCR）. We counted the replicative life span by separating daughter cells from mother cells using a manual micromanipula- tor equipped with a fiber - optic needle under the microscope. Results We constructed successfully PMT1 - expression vector （ pRS305 - PMT2 - ox）. We screened PMT1 and PMT2 - double - gene overexpression strains on solid auxotrophic medium and veri- fied the transformants by polymerase chain reaction （PCR）. Expression levels of PMT1 and PMT2 were upregulated in the overexpres- sion strain by RT - PCR. This overexpression strain had no difference compared with wide - type cel1＇ s replicative lifespan. Conclu- sion PMT1 and PMT2 double - gene overexpression has not change the replicative lifespan of saccharomyces cerevisiae, which sug-gests that PMT1 and PMT2 double - gene overexpression was involved in regulation of replicative lifespan in yeast.