沼气池宏基因组文库中未培养微生物β-葡萄糖苷酶基因的克隆与鉴定  

Cloning and idientifcation of β-glucosidase gene of uncultured microorganisms from biogas digester

作　　者：唐咸来[1,2] 车志群[1,3,4] 李双喜[1,3,4] 蒋建林[1,3,4] 罗奉奉[1,3,4] 武波[1,3,4] 

机构地区：[1]广西大学生命科学与技术学院,南宁530005 [2]广西壮族自治区科学技术厅,南宁530005 [3]广西亚热带生物资源保护利用重点实验室,南宁530005 [4]微生物及植物遗传工程教育部重点实验室,南宁530005

出　　处：《广西植物》2008年第6期790-794,共5页Guihaia

基　　金：广西科技攻关项目(桂科攻033002-5)~~

摘　　要：以间接提取法提取了沼气池样品的微生物宏基因组DNA,用柯斯质粒载体pWEB:TNC构建了一个含三万个克隆的沼气池宏基因组文库,对文库中的克隆随机分析表明,该文库的外源片段平均长度为40 kb,文库的总容量为1 .2×106kb。对其中的一个在七叶苷平板上显色的阳性克隆pGXN100进行进一步亚克隆、测序和序列分析。结果表明,pGXN100上有一个全长为1 863bp的ORF,编码621个氨基酸组成的蛋白质。将该基因命名为Unglu100。与产气克雷伯菌属的一个β-葡萄糖苷酶基因AN292在核苷酸和氨基酸水平上分别有76%和85%的同源性,利用SMART软件进行预测表明,Unglu100可能是PTS中β-葡萄糖苷酶特异性的转运蛋白组件。The metagenomic DNA of uncultured microorganisms from biogas digester was extracted by indirect extraction, end-blunted and ligated with cosmid vector of pWEB： TNC. A metagenomic library containing 30 000 clones was constructed , the stochastic Analysis of the library indicated that： the average size of the foreign DNA fragments was 40 kb and the capacity of the DNA cloned in the library was 1.2× 10^5 kb. One of the positive clones pGXN100 that show black hydrolysis circles in Esculin plates was subcloned, sequenced and analysed. The results showed that the putative β-glucanase gene had a 1 863 bp ORF, encoding a protein with 620 amino acids sharing 76 % and 85% identity with another β-glucanase gene AN292 for DNA and protein levels respectively. The structure functional domain analysis of Unglu100 using SMART software showed it maybe one of beta-glucoside specific PTS system transport proteins.

关 键 词：沼气池 宏基因组文库 Β-葡萄糖苷酶 

分 类 号：Q943.2[生物学—植物学]