不同核糖开关对下游靶基因调控效能的对比研究

Different riboswitches on regulatory efficiency of downstream target genes:a comparative study

目的比对分属于激活和抑制型中的不同核糖开关的调控功效,为实现基因电路的精准调控奠定基础。方法构建不同核糖开关(add A与M6,TPP与btu B)调控的绿色荧光蛋白amcyan表达载体,通过荧光表达量、RTq PCR分析在不同配体物浓度调控下的amcyan表达,与未含核糖开关载体的表达量进行比较,进行动态调控效能分析。结果在add A与M6激活型核糖开关的调控下,随配体物浓度的增加,绿色荧光表达量增加,且相比M6核糖开关,add A核糖开关拥有更大的动态调控效能;相反,在TPP与btu B抑制型核糖开关的调控下,随配体物浓度的增加,绿色荧光表达量减少,但btu B核糖开关的动态调控效能略大于TPP核糖开关。结论相同作用机制的不同核糖开关调控功效存在差异,拥有动态调控效能优势的激活型开关add A与抑制型开关btu B更适合在大肠杆菌中用于代谢精确调控与靶基因精准表达。

Objective To compare the regulation effects of different activated and inhibitory riboswitches,and to facilitate the precise regulation of gene circuits. Methods A green fluorescent protein amcyan expression vector regulated by different riboswitches（ add A,M6,TPP and btu B） was constructed,and the expression level of amcyan under different ligand concentrations was analyzed by RT-q PCR and relative fluorescence intensity,and then compared with the expression level of a vector without any riboswitch. The dynamic control performance was analyzed. Results Under the control of add A and M6 activated riboswitches,the expression of green fluorescent protein increased with ligand concentrations,and add A riboswitch had more dynamic regulatory effect than M6 riboswitch. However,under the control of TPP and btu B inhibitory riboswitches,the expression of green fluorescence decreased with the increase in ligand concentrations,and the dynamic regulation of btu B riboswitch was slightly greater than that of TPP riboswitch. Conclusion The regulation efficacy of different riboswitches which have the same mechanism varies. Activated riboswitch add A and inhibitory riboswitch btu B with dynamic regulation and control advantages are more suitable for precise metabolism regulation and target gene expression in Escherichia coli.