宏基因组中不依赖辅酶B12甘油脱水酶基因的筛选和表达  

Screening and Expression of the Gene Encoding Coenzyme B_(12)-Independent Glycerol Dehydratase from Metagenome

作　　者：韦旭钦[1] 陈云来[1] 吴杰群[1] 韦宇拓[1] 杜丽琴[1] 黄日波[1,2] 

机构地区：[1]广西大学生命科学与技术学院广西亚热带农业生物资源保护与利用国家重点实验室,南宁530004 [2]广西科学院国家非粮生物质能源工程技术研究中心,南宁530007

出　　处：《基因组学与应用生物学》2013年第6期729-733,共5页Genomics and Applied Biology

基　　金：国家“863”高科技资助项目(2007AA02Z227)共同资助

摘　　要：通过特定的引物,以土壤宏基因组DNA为模板,采用PCR技术扩增获得3.3 kb的DNA片段,该片段连接于pSE380载体构建重组质粒pSE380-dhaB12用于测序和表达。序列分析表明,该DNA片段编码的氨基酸序列与已报导的丁酸梭菌不依赖辅酶B12甘油脱水酶的相似性达99%。通过IPTG诱导,dhaB12基因在大肠杆菌中表达成功,SDS-PAGE分析表明有88 kD和34 kD两条蛋白质条带,在没有辅酶B12存在的情况下,所表达的酶具有明显的甘油脱水酶活性。With a given primer pair, a DNA segment of 3.3 kb was amplified by polymerase chain reaction （PCR） using a metagenomic DNA of a soil sample as template, and then cloned in vector pSE380, forming recombinant plasmids pSE380-dhaB12 for sequencing and expression. Sequence analysis indicated that the amino acid sequences encoded by this segment was 99% identical to that reported for coenzyme B12-independent glycerol dehydratase of Clostridium butyrieum. The dhaB12 genes were successfully expressed in Escherichia coli by induction of iso- propyl-beta-D-thiogalatopyranoside （IPTG）, and synthesis of two proteins at 88 kD and 34 kD were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. The expressed enzyme showed significant glycerol dehydratase activity in the absence of coenzyme B12.

关 键 词：宏基因组 不依赖辅酶B12 甘油脱水酶 筛选 表达 

分 类 号：Q78[生物学—分子生物学]