一种适用于产朊假丝酵母的基因敲除系统及其在gsh1基因敲除中的应用  

A new knocking-out system in Candida utilis and its application on disrupting the gsh1 gene

作　　者：张君丽[1] 卫功元[1] 董红军[2] 朱泰承[2] 李寅[2] 

机构地区：[1]苏州大学医学部基础医学与生物科学学院,江苏苏州215123 [2]中国科学院微生物研究所,北京100101

出　　处：《微生物学通报》2011年第6期795-802,共8页Microbiology China

基　　金：国家自然科学基金项目(No.20906065);江苏省属高校自然科学研究项目(No.09KJB530009)

摘　　要：报道一种适用于产朊假丝酵母Candida utilis的基因敲除系统,利用该敲除系统获得gsh1基因敲除杂合突变株。根据不同种属酵母菌γ-谷氨酰半胱氨酸合成酶(γ-GCS)蛋白质的保守序列,克隆C.utilis SZU 07-01的gsh1基因;以商品化质粒pPICZalpha A为基础,构建gsh1基因的敲除载体pPICZalpha A-kan 3,其中,kan基因的启动子TEF被替换为来自于C.utilis SZU 07-01的GAP启动子(pGAP:kan)。质粒电转化C.utilis,获得gsh1基因敲除杂合突变株C.utilis GSH-6。结合发酵培养得到的数据进行分析,突变株的γ-GCS酶活比出发菌株降低17.5%,GSH合成量降低61%,细胞干重降低18.5%。所构建敲除组件pGAP:kan的成功应用为从分子水平研究C.utilis中谷胱甘肽(GSH)的生理功能提供了一种新借鉴。In this study,we report a novel system of gene knocking-out in C.utilis SZU 07-01 by suc-cessfully disrupting the gene of gsh1.First of all,the gsh1（encoding γ-GCS protein） gene was cloned by genome walking method from C.utilis SZU 07-01 according to γ-GCS protein conservative se-quences among several different yeasts.Then,the disrupting vector,pPICZalpha A-kan 3 was con-structed on the basis of plasmid pPICZalpha A,whose original TEF promoter responding for kananmy-cin resistance gene（kan） transcription was replaced by GAP promoter（pGAP） isolated from C.utilis SZU 07-01.pPICZalpha A-kan 3 was linearized and then transformed into C.utilis,resulting in a gsh1 deleted heterozygotic mutant strain designated as GSH-6.After cultured in the same condition,the mu-tant deficient in glutathione biosynthesis showed decreases of 17.5%,61%,18.5% in γ-GCS activity,glutathione content and dry cell weight,respectively.The disruption element（pGAP：kan） used in this study supplies a new gene genetic manipulation approach to research the physiological function of GSH in C.utilis at a molecular level.

关 键 词：基因敲除 gsh1基因 产朊假丝酵母 谷胱甘肽 γ-GCS酶活 

分 类 号：Q78[生物学—分子生物学]