山羊瘤胃微生物宏基因组文库中一个新型纤维素酶基因-安徽农业大学.PDF

安徽农业大学学报, 2015, 42(2): 209-212 Journal of Anhui Agricultural University [DOI] 10.13610/ki.1672-352x012 网络出版时间：2015-3-2 15:11:00 [URL] /kcms/detail/34.1162.S1511.012.html 山羊瘤胃微生物宏基因组文库中一个新型纤维素酶 基因的克隆与表达 姜海琴，程建波，王力生，李吕木，范彩云* (安徽农业大学动物科技学院，合肥 230036) 摘 要：通过功能筛选方法，从安徽白山羊瘤胃微生物宏基因组文库中筛选得到了6 个纤维素酶的阳性克隆。 对其中的 1 个阳性克隆进一步进行亚克隆和序列分析，获得一个新型纤维素酶基因开放阅读框，通过BLAST 对基 因全序列进行分析比较，发现其与来自Bacteroides cellulosilyticus DSM 14838 的hypothetical protein 具有51%的序 列相似性和65%的同源性。并以pET28a(+)为载体、Escherichia coli BL21(DE3)为宿主菌，对新型酶基因进行高效 重组表达，并对该酶表达条件进行优化。 关键词：瘤胃微生物；宏基因组；纤维素酶 中图分类号：S852.6 文献标识码：A 文章编号：1672−352X (2015)02−0209−04 Cloning and expression of a new cellulase gene from the metagenomic library of the goat rumen JIANG Haiqin, CHENG Jianbo, WANG Lisheng, LI Lvmu, FAN Caiyun (School of Animal Science and Technology, Anhui Agricultural University, Hefei 230036) Abstract: Six positive clones with cellulase activities were isolated from a metagenomic library of the goat rumen using a functional screening method. One positive clone was subject to further subcloning and sequence analysis and a new cellulase gene open reading frame was obtained. After blast analysis, it was found that the cel- lulase gene showed 51% sequence similarity and 65% homology compared with the hypothetical protein of Bac- teroides cellulosilyticus DSM 14838. Using pET28a (+) as a carrier, Escherichia coli BL21 (DE3) as a host strain, the new gene was recombined and expressed efficiently and the expression conditions of the newly-cloned gene were optimized. Key words: rumen microbes; metagenomic; cellulase 纤维素是由 D-葡萄糖以 β-1,4-糖苷键连接而成 残基为单位进行切割生成纤维二糖，β-葡萄糖苷酶 的直链状大分子，是自然界存在的最丰富的可再生 切割纤维二糖生成葡萄糖[2] 。 资源的生物质。纤维素酶(cellulase)是能将纤维素水