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

唐咸来<sup>1; 2</sup>; 车志群<sup>1; 3; 4</sup>; 李双喜<sup>1; 3; 4</sup>; 蒋建林<sup>1; 3; 4</sup>; 罗奉奉<sup>1; 3; 4</sup>; 武 波<sup>1; 3; 4</sup>

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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
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
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

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1.2×10⁶ kb。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1 863 bp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。

关键词: 沼气池宏基因组文库 β-葡萄糖苷酶

DOI：

分类号:Q943.2

Fund project:

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

TANG Xian-Lai^1,2, CHE Zhi-Qun^1,3,4, LI Shuang-Xi^1,3,4, JIANG Jian-Lin^1,3,4, LUO Feng-Feng^1,3,4, WU Bo^1,3,4

1.College of Life Science and Biotechnology, Guangxi University, Nanning 530005, China;2.The Science and Technology Department of Guangxi, Nanning 530005, China;3.Guangxi Key Laboratory of Subtropical Bio-resource Conservation and Utilization, Nanning 530005, China;4.Key Laboratory of Education Ministry for Microbial and Plant Genetic Engineering, Nanning 530005, China

Abstract:

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 1.2×10⁶ 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.

Key words: biogas digester metagenome β-glucosidase