耶氏酵母菌POX5基因的敲除  

Deletion of POX5 Gene from Yarrowia lipolytica

作　　者：黄琳[1] 刘常金[1] 王芳[1] 师慧敏[1] 张治洲[1,2] 

机构地区：[1]天津科技大学食品工程与生物技术学院,天津300457 [2]哈尔滨工业大学(威海)科学与工业技术研究院

出　　处：《现代食品科技》2010年第4期331-336,共6页Modern Food Science and Technology

基　　金：新世纪优秀人才计划(2006);哈工大人才引进启动资金(hitwh200904)资助项目;上海爱普香料有限公司资金合作项目

摘　　要：对于耶氏酵母(Yarrowia lipolytica)中编码与γ–癸内酯合成相关的乙酰辅酶A氧化酶基因(POX1-POX5)中的POX5基因用Cre/loxP同源重组系统进行敲除。设计含有60个核苷酸与POX5基因的ORF两侧序列同源的长引物,以pUG6为模板构建带有Cre/loxP系统的敲除组件,之后转化耶氏酵母YL(Yarrowia lipolytica),通过PCR确定阳性克隆子。将质粒pSH65转入阳性克隆子,半乳糖诱导表达Cre酶以切除KanMX基因并经过PCR确认。最后在YPD培养基中连续传代培养丢失pSH65质粒,成功获得POX5缺陷型菌株。The POX5 gene in Yarrowia lipolytica could encode acyl-coenzyme A oxidase （acyl-CoA） which was involved in biosynthesis of γ-decalactone. The gene POX5 disruption was mediated with a loxP-KanMX-loxP cassette centered in a PCR fragment employing 80bp primer pairs which contained 60 nucleotides and fully match the upstream or downstream of the POX5 open-reading frame. After transformation of the linear disruption cassette with a Kan marker into Yarrowia lipolytica cells, transformants were selected by PCR for correct integration of the cassette and concurrent deletion of the genomic target sequence. After correctly integrating the transformants into the genome, the Kan marker was efficiently removed by transforming pSH65 plasmid and inducing the Cre expression. The expression of the Cre resulted in the removal of the marker gene, 1eaving behind a single loxP site at the chromosomal locus. The pSH65 plasmid was then lost after continually incubated in YPD, giving the deletion mutant of POX5 gene.

关 键 词：耶氏酵母 POX5基因 基因敲除 CRE/LOXP系统 γ–癸内酯 

分 类 号：Q78[生物学—分子生物学]