红树林土壤微生物宏基因组文库的构建及两种新颖的β-葡萄糖苷酶基因的鉴定

摘要

宏基因组技术是挖掘微生物新酶的重要途径。通过构建红树林土壤微生物Fosmid文库，共获得了约100000个阳性克隆，该文库外源插入片段平均长度约为30 kb，文库容量达3 Gb。通过对文库中10000个克隆进行功能筛选，获得了17个具有β-葡萄糖苷酶活性的克隆。对其中2个表达β-葡萄糖苷酶的克隆进行亚克隆，获得2个新颖的β-葡萄糖苷酶的基因，分别命名MhGH3和bgl66。生物信息学分析表明，MhGH3基因由1992个碱基对组成，bgl66基因由2025个碱基对组成。在氨基酸水平上， MhGH3和bgl66编码的蛋白质与GenBank数据库中已知β-葡萄糖苷酶的一致性分别为64%和55%。%Metagenomics provides a means of exploring new enzymes in microorganisms. A mangrove soil metagenome library with 100 000 clones was constructed. The average length of inserted DNA fragment is about 30 kb, and the total capacity of inserted DNA reached about 3 Gb. Seventeen clones withβ-glucosidase activity were detected by screening 10 000 fosmid clones. The subcloning and sequencing revealed two novelβ-glucosidase genes(MhGH3 and bgl66). Bioinformatics analysis indicated that MhGH3 and bgl66 composed of 1 992 bp and 2 025 bp in length, respectively. The deduced amino acid sequence of MhGH3 and bgl66 showed the highest sequence identity of 64%and 55%with the knownβ-glucosidase in GenBank datebase, respectively.