牛瘤胃未培养细菌中一个β-葡萄糖苷酶基因umbgl3A的克隆及鉴定  

Cloning and identification a β-glucosidase gene umbgl3A of uncultured bacteria from bovine rumen

作　　者：赵广存[1] 段承杰[1] 庞浩[2] 封毅[2] 许跃强[2] 莫新春[2] 唐纪良[1] 冯家勋[1] 

机构地区：[1]广西大学广西亚热带生物资源保护利用重点实验室 [2]广西大学生命科学与技术学院,广西南宁530005

出　　处：《西南农业学报》2005年第4期472-476,共5页Southwest China Journal of Agricultural Sciences

基　　金：国家863计划(2004AA214140);国际科技合作重点项目计划(2002AA217121)

摘　　要：通过构建牛瘤胃未培养细菌的宏基因组文库,共获得12000个克隆,文库外源DNA总容量为4.2×108bp,筛选得到九个表达β-葡萄糖苷酶活性的克隆,并对其中的一个表达β-葡萄糖苷酶活性的克隆pGXN1009进行亚克隆,将β-葡萄糖苷酶基因定位在4.3kb的片段上。测序结果表明在4.3kb的片段上有一个全长为1956bp的ORF,编码一个可能的β-葡萄糖苷酶基因um-bgl3A,其编码产物与一个来源于小鼠大肠未培养细菌的β-葡萄糖苷酶(GenBankAcessionNO:AAX16378.1)一致性为63%、相似性为79%;与哈氏噬纤维菌(Cytophagahutchinsonii)的β-葡萄糖苷酶(GenBankAcessionNO:ZP-00308419)的一致性为50%、相似性为67%。利用SMART软件对Umbgl3A结构组件预测表明,Umbgl3A包含一个家族3糖基水解酶(glycosylhydrolase)功能域和一个家族3C糖基水解酶功能域。遗传进化分析表明umbgl3A基因可能是来自牛瘤胃微生物噬纤维菌属未培养的一个种。A metagenomic library of uncultured bacteria from bovine tureen fluid content was constructed, which contained 12 000 clones and the capacity of inserted DNA,of which was 4.2 × 10^8 bp. Nine clones expressing β-glucosidase activity were isolated and a clone designated pGXN1009 was subcloned and a putative β-gluoasidase gene was localised to a 4.3 kb fragment. Sequencing of this 4.3 kb DNA fragment revealed that there is a β-gluoasidase gene umbgl3A with an ORE of 1956 bp, encoding product shared 63 % identity and 79 % similarity with a beta-glucosidase（GenBank NO： AAX16378.1 ） of uncultured murine large bowel bacterium BAC 31B, and shared 50 % identity and 67 %similarity with a beta-gluoasidase-related glyoasidases （GenBank NO：ZP-00308419.1 ） of Cytophaga hutchinsonii, Umbegl3A possessed a domain of glycosyl hydrolase family 3 and a domain of glycosyl hydrolase family 3C. Phylogentic analysis of Umbgl3A indicated umbgl3A may come from a species of Cytophaga.

关 键 词：牛瘤胃 未培养细菌 宏基因组 β-葡萄耱苷酶 

分 类 号：Q781[生物学—分子生物学]