梭菌氢酶基因部分片段的同源克隆及敲除载体的构建  

Homologous Cloning of Clostridium Hydrogenase Gene and Construction of Gene Knockout Vector

作　　者：闫倩[1] 闫苗章[1] 王保莉[1] 曲东[2] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]西北农林科技大学资源环境学院,陕西杨凌712100

出　　处：《西北农业学报》2012年第7期61-66,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(40971158和41171204)

摘　　要：为从分子水平探索典型铁还原菌-梭菌的铁还原能力与其氢酶产氢之间的关系,以从水稻土中分离得到一株具有高铁还原能力和高产氢能力的梭菌为材料,通过同源克隆获得长度为761bp氢酶基因的部分序列。生物信息分析发现,该基因片段覆盖氢酶的活性中心,是氢酶的主要功能结构域。采用Overlap PCR的方法构建含有四环素抗性基因的氢酶基因敲除载体(pMD-19-HTH),以期进一步构建氢酶基因缺失的梭菌突变体,为分析氢酶产氢与铁还原的关系奠定基础。Dissimilatory iron-reducing bacteria grown in soil play an important role in bioremediation of organics and heavy metal pollution.The aim of this study is to explore the internal relationship between the iron-reducing ability and hydrogen-evolution of Clostridium,which is a typical iron-reducing bacteria.In this paper,a Clostridium strain isolated from paddy soil as the research object.Using homologous clone,a fragment（761 bp） of hydrogenase gene was obtained.Bioinformatics analysis showed that this gene fragment contained the active centers of hydrogenase,and was its major functional domain.Moreover,knockout vector（pMD-19-HTH） included tetracycline resistance gene was constructed by overlap PCR technique with the purpose of knockout hydrogenase function and lay the foundation for uncovering relationship between the iron-reducing ability and hydrogen-evolution.

关 键 词：氢酶 同源克隆 重叠PCR 敲除载体 

分 类 号：S154.3[农业科学—土壤学]