摘要
目的:以地衣芽孢杆菌高温α-淀粉酶基因(amyL)为报告基因,构建含不同启动子的枯草杆菌表达载体,转化枯草杆菌,并对重组菌的酶活进行分析,比较不同启动子对amyL基因在枯草杆菌中表达的影响。方法:以高温α-淀粉酶高产菌株B.licheniformis0204染色体DNA为模板,PCR扩增得到amyL并分别与PQ启动子和P43启动子进行连接构建表达载体pUB-PQ-amyL和pUB-P43-amyL,化学法转化枯草杆菌1A717,筛选得到重组转化子后对重组菌的表达产物进行SDS-PAGE和酶活检测。结果:重组菌摇瓶发酵105h后测定高温α-淀粉酶酶活,B.subtilis1A717(pUB-PQ-amyL)的最高酶活为280.1U/mL,B.subtilis1A717(pUB-P43-amyL)的最高酶活为190.5U/mL。结论:PQ启动子调控的高温α-淀粉酶最高表达水平是P43启动子调控的最高表达水平的1.47倍,说明PQ启动子能使amyL基因在枯草杆菌中更高效地表达。
Objective： Using amyL as a report gene,the Bacillus subtilis expression vectors were constructed with different promoters and transformed into B. subtilis,then the activity of the expressed production of the reombinant strains were studied to compare the regulation effects of different promoters on the amyL expression in B.subtilis.Method： The amyL gene was amplified by PCR from B.licheniformis 0204 chromosome DNA.The recombinant plasmids,pUB-PQ-amyL and pUB-P43-amyL were constructed using promoters PQ and P43,respectively,and transformed into B.subtilis 1A717 by chemical transformation.The recombinant strains were obtained through plate screening and the expressed production of the recombinant strains were analyzed by SDS-PAGE and enzymatic kinetic analysis.Result： By shake-flask fermentation,the specific activities of AmyL by transformants of pUB-PQ-amyL and pUB-P43-amyL were 280.1U/mL and 190.5U/mL respectively.Conclusion： The results show that synthetic promoter PQ mediates 1.47 folds production of AmyL comparison to that of the promoter P43 deriving from B.subtilis when using amyL as report gene.
出处
《生物技术》
CAS
CSCD
北大核心
2009年第5期11-13,共3页
Biotechnology
基金
国家高技术研究发展计划项目(No.2006AA020204)资助
