枯草芽孢杆菌核黄素操纵子与ribC基因的修饰与遗传效应

The genetic modification effect of rib operon and ribC gene in Bacillus subtilis

摘要:

【目的】研究核黄素操纵子(rib)组成型高表达，以及ribC基因低水平表达对枯草芽孢杆菌过量合成核黄素的影响。【方法】在染色体原位修饰启动子，用mRNA稳定子替换mRNA前导区，使rib操纵子组成型高表达；修饰ribC基因的启动子，降低ribC基因的表达水平。采用qRT-PCR方法，表征基因的相对表达水平；通过摇瓶发酵，测定重组菌的生物量和核黄素产量，表征相关基因修饰所表现的遗传效应。【结果】用gsiB mRNA稳定子替换核黄素操纵子的mRNA前导区，使其相对表达水平提高了约1 500倍。ribC基因启动子?35区的首个碱基由“T”突变为“C”，使ribC基因的表达水平下降了97%以上。得到的重组菌株LX34在补加蔗糖20 g/L的LB培养基上摇瓶发酵36 h，可积累核黄素2.1 g/L，同时生物量没有明显下降。【结论】使用gsiB mRNA稳定子，能够有效地提高目标基因或操纵子的表达水平；启动子?35区首个碱基的点突变，能够有效降低ribC基因的表达水平；rib操纵子过量表达和ribC基因低水平表达，使细胞能够过量合成并积累核黄素。

Abstract:

[Objective] This research focus on the influence of rib operon constitutively over-expression and ribC gene low levels expression on the synthesis and accumulation of riboflavin in Bacillus subtilis. [Methods] For the constitutively over-expression of rib operon, its promoter was modified in situ, and the mRNA leader region was replaced by mRNA stabilizer. Using point mutations of its promoter ?35 region, ribC gene transcription level was reduced. The transcription levels of the target genes were analysed by qRT-PCR method. The genetic effects of the modified genes were assessed by measuring the biomass and riboflavin production of the recombinant in shaking flask fermentation. [Results] The relative transcription levels of gsiB stabilizer-modified rib operon have increased about 1 500 times. The first base mutations of ribC promoter ?35 region can lead to its expression levels decreased by more than 97%. With the LB medium supplemented with sucrose 20 g/L, fermentation was completed in 36 h and the resulting recombinant strains LX34 can accumulate riboflavin 2.1 g/L, meanwhile no significant decline in the biomass. [Conclusion] The gsiB mRNA stabilizer can effectively improve transcription levels of the target gene or operon. A point mutation in the first base of promoter ?35 region can effectively reduce the transcription of ribC gene. The over-expression of rib operon and the expression reduced significantly of ribC gene result in the accumulation of riboflavin.