厌氧菌株Clostridium sp.WGC702的筛选及其β-葡萄糖苷酶基因的克隆  

Screening of anaerobic bacteria strains WGC702 from biogas digester and cloning its β-glucosidase gene

作　　者：申佩弘[1,2,3] 蒋承建[1,2,3] 杨霞[1,2,3] 沈建涛[1,2,3] 武波[2,3,4] 

机构地区：[1]广西大学生命科学与技术学院 [2]微生物及植物遗传工程教育部重点实验室,南宁530005 [3]广西亚热带生物资源保护利用重点实验室 [4]广西民族大学化学与生态工程学院

出　　处：《工业微生物》2011年第1期21-25,共5页Industrial Microbiology

摘　　要：从沼气池中筛选一株产β-葡萄糖苷酶的厌氧菌菌株WGC702,基于16S rDNA序列及生理特性将其确定为梭菌属,命名为Clostridium sp.WGC702。用BamHI不完全酶切WGC702的总DNA,回收2～10kb范围内的DNA片段,连接到pGEM-3zf(+)载体上,电转化至大肠杆菌E.coliDH5α中构建基因文库,利用七叶苷平板筛选到120个能产黑色水解圈的阳性克隆。将其中一个含3.5kb左右大小的外源片段的阳性克隆子进行测序,结果发现其中含有一个1347bp大小的完整ORF框,进一步分析表明该片段编码448个氨基酸组成的蛋白质,氨基酸序列与来自Clostridiumcellulovorans 743B(登录号为EES30417.1)的β-葡萄糖苷酶具有48%的同源性,其预测的等电点和分子量分别为4.94和53978.6Da,可能为一个新的β-葡萄糖苷酶基因。An anaerobic bacteria strains WGCT02 for product β-glumsidase was isolated from biogas digester and was identified as a spedes of Clostridium sp. （named Clostridium sp. WGCT02）based on its 16S rDNA sequence. After digestion of the chommasomal DNA with BamHI, the 2-- 10 kb DNA fragment was gel-purified and ligated to pGEM-3zf（ ＋ ）vector to prepare the genornic library. Approximately 120 clones were screened by media with Esculin, and one of the positive clone was analyzed by sequencing. The results showed that an ORF of 1347 bp was included, encoding a protein of 448 amino acids. This β-glumsidase gene was analyzed by NCBI gene data base in amino acid level, it indicated that had 48 % identity with the sequence of the beta-galactosidase from Clostridium cellulovorans 743B （EES30417.1）. The predicted pI and Mw were 4.94 and 53978.6 Da respectively. It might be a new β-glucosidase gene.

关 键 词：WGC702 筛选 基因文库 Β-葡萄糖苷酶基因 克隆 

分 类 号：Q78[生物学—分子生物学]