摘要
运动发酵单胞菌(Zymomonas mobilis)是目前发现的乙醇发酵能力最强的微生物之一,被认为是进行大规模乙醇生产的潜在菌种,但其可利用的碳源范围窄.对其进行分子生物学基础研究和基因工程改造的工作早在上世纪八、九十年代就开始了,但基于其独特的生物学特性,至今遗传操作手段仍不成熟.以大肠杆菌(E.coli)pBR328为出发质粒,分段插入带Z.mobilis乳酸脱氢酶基因ldh、分别作为左右同源臂、1.5 kb大小的DNA片段,构建了Z.mobilis定点整合型质粒pBR328-ldhR-ldhL(6 239 bp),此质粒两同源臂片段即ldhR和ldhL的中间有稀有酶切位点NotI和SfiI,以方便插入待整合片段.经NotI位点插入1 216 bp氯霉素抗性基因cml,将相应质粒pBR328-ldhR-cml-ldhL以环型和线型形式分别电击转化Z.mobilis ZM4菌株,都成功筛选到了ZM4 ldh::cml菌株,证明此定点整合质粒是方便可用的,为对Z.mobilis的定点遗传操作提供了很好的基础.
Zymomonas mobilis, one of the strains which have the highest ethanol yield and specific ethanol productivity, has been evaluated as a possible candidate for industrial ethanol production. However, its substrate range is limited. Early in 1980s-1990s people embarked on its molecular biology and gene engineering research. But genetically manipulating methods were still immature and inconvenient today, for its some special characteristics. In this paper, a specific integrating plasmid for Z. mobilis, pBR328-ldhR-ldhL with 6 239 bp size, was con- structed from E. coli pBR328 and two PCR fragments . These PCR fragments were about 1.5 kb and included partial ZM4 lactate dehydrogenase encoding ldh gene sequence used as homolo-gous arm ldhR and ldhL. There were two rare restriction sites NotI and SfiI between ldhR and IdhL for ligating into integrated fragments, pBR328-ldhR-cml-ldhL was constructed by inserting NotI-cml-NotI fragment into pBR328-ldhR-ldhL and electroporated into ZM4 competent cells by linear and circular molecules. ZM4 ldh：：cml strain could be successfully screened and identified in any case. This result indicated that pBR328-ldhR-ldhL was useful and convenient as a specific integrating vector.
出处
《南开大学学报（自然科学版）》
CAS
CSCD
北大核心
2009年第6期42-47,共6页
Acta Scientiarum Naturalium Universitatis Nankaiensis
基金
国家自然科学基金(30600369)
作者简介
邹少兰（1969-），女，湖北红安人，助理研究员。
