Cre/LoxP重组系统的改造及在树干毕赤酵母中的应用  

Modification of Cre /LoxP recombination system and its application in Pichia stipitis

作　　者：傅静[1,2] 张梁[1,2] 薛卫[3] 石贵阳[1,2] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,无锡214122 [2]江南大学粮食发酵工艺与技术国家工程实验室,无锡214122 [3]南京农业大学信息科技学院,南京210095

出　　处：《生物加工过程》2014年第5期39-45,共7页Chinese Journal of Bioprocess Engineering

基　　金：江苏省自然科学基金(BK2011153;BK2012363);教育部"新世纪优秀人才支持计划"(NCET-11-0665)

摘　　要：为了实现在P.stipitis中进行无痕基因敲除,以Cre/LoxP系统为研究对象,首先通过同源重组构建尿嘧啶营养缺陷型树干毕赤酵母(ura3-);同时通过定点突变pSH47-Hpt质粒的hpt基因和cre基因,将CDS区CTG突变为TTG;最后以乙醛脱氢酶基因为靶基因,验证突变后的Cre/LoxP系统在P.stipitis进行无痕基因敲除的可行性。结果表明:本文在P.stipitis中成功使用潮霉素B抗性标记,经过修饰后的Cre/LoxP敲除系统能够在P.stipitis中无痕敲除目的基因,为后续研究P.stipitis功能基因和改造代谢途径提供了一种试验方法和筛选标记。Heterologous genes rich in CTG could not express in P. stipitis. A modified Cre /LoxP recombination system was developed to knock out the genes in P. stipitis with no trace. Firstly,ura3 was deleted to obtain a uracil auxotroph; Secondly,CTG codons in hpt and cre of pSH47-Hpt were mutagenized to TTG; Finally,the modified Cre /LoxP recombination system was applied to knock out the genes without trace in P. stipitis using acetaldehyde dehydrogenase gene as a target gene. In this study,Hygromycin B resistant marker was referred to,and the modified Cre /LoxP recombination system could knock out the target gene with no trace in P. tsipitis,which provided a method and a new resistance marker for the further study of functional genes and metabolic pathway in P. stipitis.

关 键 词：树干毕赤酵母 基因敲除 Cre/LoxP重组 潮霉素B 

分 类 号：Q812[生物学—生物工程]