解脂酵母中POX3基因的敲除研究  

Deletion of POX3 gene from Yarrowia lipolytica

作　　者：师慧敏[1] 王芳[1] 黄琳[1] 刘常金[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,山东威海246209

出　　处：《食品工业科技》2010年第11期232-234,238,共4页Science and Technology of Food Industry

摘　　要：对耶氏酵母(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POX3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POX3基因的ORF两侧序列同源的长引物,以pUG6为模板构建带有loxP-KanMX-loxP系统的敲除组件,之后转化耶氏酵母,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。最后在YPD培养基中传代丢失pSH65质粒,获得POX3缺陷型菌株。由于在解脂酵母中表达生产γ-癸内酯直接牵扯到若干个需要抑制的基因,单独缺失其中的一个基因理论上可能实现不了产量的极大值。本研究为进一步构建组合敲除准备了条件。The POX3 gene could encode acyl- coenzyme A oxidase （ acyl- CoA） in Yarrowia lipolytica and get involved in biosynthesis of γ- decalactone. POX3 knockout was mediated with a loxP - KanMX- loxP cassette centered in a PCR fragment employing 80bp primer pairs in which 60 nucleotides fully match the POX3 open- reading frame.After transformed the linear disruption cassette into Yarrowia lipolytica cells, selected transformants were checked by PCR for correct integration and deletion of the target sequence.The KanMX marker was efficiently removed by transforming pSH65 plasmid and inducing the Cre expression.pSH65 was then lost after continually incubated in YPD.At last,the knockout mutant of POX3 gene was generated.The research provides a basis for combination knockouts of other genes that inhibit γ-decalactone production.

关 键 词：耶氏酵母 POX3基因 基因敲除 CRE/LOXP系统 Γ-癸内酯 

分 类 号：Q789[生物学—分子生物学]