巴斯德毕赤酵母表达系统中甘油阻遏相关基因GR1的分离克隆与鉴定  

Isolation and Identification of GR1 Gene,a Glycerol Repression Associated Gene in Pichia pastoris Expression System

作　　者：姚学勤[1] 薛冲[1] 赵洪亮[1] 何庆[1] 孙博[1] 刘志敏[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2009年第4期462-466,共5页Letters in Biotechnology

摘　　要：目的:分离克隆并鉴定巴斯德毕赤酵母表达系统中甘油阻遏相关基因。方法:PCR扩增LacZ基因,克隆至pPIC9载体,构建pPIC9-LacZ表达载体,经SalⅠ线性化后转化巴斯德毕赤酵母GS115,构建GS115-LacZ模式菌株;用限制酶介导整合(REMI)技术使GS115-LacZ菌体基因组产生随机突变,筛选甘油去阻遏的GS115-LacZΔ菌体;Southern blot鉴定GS115-LacZΔ基因组,用质粒拯救技术和TAIL-PCR克隆未知基因序列并测序。结果:得到甘油去阻遏的GS115-LacZΔ菌体,经Southern blot分析,突变仅发生在1个基因中;通过质粒拯救和TAIL-PCR,分离得到阻遏相关基因GR1,共2863bp,经在线BLAST,发现其编码一种过氧化物酶体自吞噬相关蛋白。结论:分离得到阻遏相关基因GR1,与过氧化物酶体自吞噬相关,提示过氧化物酶体自吞噬相关基因可能对醇氧化酶启动子AOX1的转录活性有影响。Objective： To isolate and identify the genes related to glyerol repression in Pichia pastoris expression system Methods： LacZ gene was amplified via PCR and inserted into pPIC9 plasmid to construct pPIC9-LacZ plasmid.pPIC9-LacZ plasmid was linearied by Sal I and then tansformed into P.pastoris GS115 strain to construct GS115-LacZ strain Subsequently, restriction mediated intergration （REMI） technique was used to produce random mutation in genome of GS115-LacZ. Screening was done for GS115-LacZA with glyecrol derepression. Southern blot was used for analyzing genomic DNA of GS115-LacZA. Plasmid rescue and TAIL-PCR were used for sequence of unknown gene.Results：The GS115-LacZA with glyecrol derepression was constructed successfully, which had mutation in only a gene, as revealed by Southern blot analysis. By plasmid rescue and TAIL-PCR, we isolated the repression related gene GR1 with 2863 be in length. By online BLAST, it was found that it encodes a peroxisomal autophagy-related protein. Conclusion：We so lated successfully the repression related gene GR1 which is a peroxisomal autophagy-related gene, suggesting that perosisonal autophagy-related gene may affect on transcriptional activity of alcohol oxidase 1（AOX1） promoter.

关 键 词：巴斯德毕赤酵母 甘油阻遏 限制酶介导整合技术 TAIL-PCR 

分 类 号：Q78[生物学—分子生物学]