极端嗜热厌氧纤维素分解菌基因文库的构建及其内切葡聚糖酶基因片段的克隆

Construction Genomic Library of Extremely Thermophilic Anaerobic Bacterium BANG 2 and Cloning of Endoglucanase Gene Fragments

以从云南邦拿掌温泉中分离、纯化的高温厌氧纤维素分解菌邦2菌(Cadicellulosiruptor)为材料,制备其总DNA,经限制性核酸内切酶EcoRⅠ部分酶切后,在T4DNA连接酶的作用下与经EcoRⅠ完全酶切、去磷酸化的质粒载体pUC18连接,然后转化E.coliJM109,建立了邦2的基因文库,经筛选鉴定得到6.3×103个重组子;重组子经刚果红平板验证:约有23.5%菌落呈现透明圈;重组子经EcoRⅠ酶切验证显示:重组质粒均含有外源DNA插入片段.结果表明已克隆到邦2菌纤维素酶系中的内切葡聚糖酶基因(ED基因)片段.

An extremely thermophilic anaerobic bacterium was isolated from Bangnazhang hotspring in Yunnan province, named Bang2. The strain Bang2 was initially identified as Cadicellulo siruptor.Genomic DNA of Bang2 was extracted and was partially digested by restriction enzyme EcoRⅠ,and ligated with plasmid pUC18 by T_(4)DNA ligase. After transformation E.coli JM109,the genomic library was constructed and 6.3×10^(3)recombinants was obtained.The ratio of the recombinants which formed transparent zones on the Congo Red plates was 23.5%. The results after digested by restriction enzyme EcoRⅠrevealed that the recombinants which had been picked out contained foreign DNA fragments. The results showed that the endoglucanase gene has been cloned.