温泉土壤宏基因组文库构建及普鲁兰酶筛选

目的]构建温泉土壤宏基因组文库并进行普鲁兰酶活性筛选,以期获得高活力、耐高温的新型普鲁兰酶基因.方法]采用间接法提取温泉土壤样品中宏基因组DNA,经Sephadex G200（含2％ PVPP）凝胶柱和电洗脱两步纯化后,扩增16S rRNA序列,通过序列比对和系统发育进化树分析温泉土壤微生物的多样性;然后以pCC1FOS为载体构建温泉土壤宏基因组文库,并对所获得的宏基因组文库进行活性筛选.结果]利用宏基因组技术提取温泉土壤样品中微生物的基因组DNA,构建了一个约含1 146个克隆的温泉土壤16S rRNA文库,经遗传进化分析,发现温泉土壤样品中的大部分微生物未被研究过,主要来源于厚壁菌门（Firmicutes）,其次为恐球菌—栖热菌门（Deinococcus-Thermus）和变形杆菌门（Proteobacteria）.用提取获得的宏基因组DNA成功构建了温泉土壤微生物宏基因组Fosmid文库,文库约含2.7万个克隆,平均插入片段长度为40.0 kb,文库外源DNA总容量为1.2×10^9bp;通过活性筛选共获得9个可表达普鲁兰酶活性的克隆.结论]宏基因组文库技术是获取极端环境微生物新酶的有效手段,可为进一步挖掘普鲁兰酶的应用潜力及研究其耐热机理奠定基础.

Construction of a metagenomic library from hot spring soil and cloning of pullulanase gene

Objective]A metagenomic library from hot spring soil was constructed and pullulanase activity was screened to find thermophilic pullulanase genes.Method]The metagenomic DNA from hot spring soil was extracted by indirect lysis method,and purified by Sephadex G200 column containing 2% acid-washed PVPP and by electroelution.16S rRNA sequences were amplified and phylogenetic trees were constructed to learn the hot spring soil microbial diversity.And then a metagenomic library was built by ligating the metagenomic DNA into pCC1FOS vector,and pullulanase activity was isolated by function-based screening strategy.Result]The metagenomic DNA was extracted from hot spring soil by employing metagenomic techniques.The hot spring soil 16S rRNA library composed of 1146 clones was constructed successfully.Through genetic evolution analysis,it was found that the majority of the prokaryotic microbes in this hot spring soil had not been studied,in which Firmicutes was the most abundant followed by Deinococcus-Thermus and Proteobacteria.On the basis of the soil metagenomic DNA,the hot spring soil metagenomic Fosmid DNA library was consisted of about 27000 clones with the average insert size of 40.0 kb and total capacity of 1.2×10^9 bp.And then nine clones exhibiting pullulanase activity were isolated via function-based screening.Conclusion] It is an effective method to find novel enzymes by construction of metagenomic library.The potential applications of pullulanase and thermophilic mechanisms of pullulanase could be further studied.