基因串联策略强化枯草芽孢杆菌终止子的功能及其在基因表达中的应用  

Gene tandem strategy strengthens the function of terminators and its application in gene expression in Bacillus subtilis

作　　者：林巧 周哲敏[1,2] 崔文璟[1,2] Qiao Lin;Zhemin Zhou;Wenjing Cui(School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu Province,China;Key Laboratory of Industrial Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu Province,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《微生物学报》2021年第8期2517-2529,共13页Acta Microbiologica Sinica

基　　金：国家自然科学基金(21878125);国家重点研发计划(2017YFE0129600)。

摘　　要：在底盘微生物中使用高性能终止子能够显著增强基因终止效率,维持新合成mRNA的稳定性,提升外源基因表达性能。而目前缺乏专一用于枯草芽孢杆菌的终止子元件,限制了复杂功能基因电路的设计。【目的】在枯草芽孢杆菌中挖掘新的高性能终止子,并进一步重新设计,丰富适用于这一底盘的高性能人工终止子。【方法】将枯草芽孢杆菌终止子和枯草芽孢杆菌噬菌体终止子分别构建至终止子检测质粒中,测定各终止子的终止效率(terminator efficiency,TE)。将不同强度单终止子按强弱、强强、弱弱组合构建串联终止子,测定不同组合体的终止性能。利用高性能串联终止子进行天冬氨酸氨基裂解酶(Asp A)和β-葡萄糖苷酸酶(Gus A)的表达功能验证,检测高性能终止子对基因表达水平的作用。【结果】经过检测,TB5终止子终止效率最强,TE为98%。含有TB5终止子的表达体系中GFP水平比对照上调2.2倍,RFP的表达水平下调27倍。双串联组合体终止子中,TH1.5b-TB5(TE=97%)和TB5-TB5(TE=98%)串联终止子可将RFP的表达水平下调30倍。而三串联终止子中,TB2-TB5-TB5组合体与相应的双组合体相比,GFP的表达水平虽无进一步提升,但RFP的表达仅有参照的1/300。最后,通过AspA和GusA的重组表达确证了串联终止子TH1.5b-TB5和TB10-TB5对基因表达水平的提升作用。【结论】高终止效率的终止子可以显著提升上游基因的表达水平,通过一定规律的基因串联能够进一步提升终止子的工作效率。这种人工再设计的终止子可方便、快捷地构建至细菌基因表达系统,实现基因表达的高效调控。Using high-property terminators enable significant enhancement of transcription termination,m RNA stability,heterologous gene expression in classic microorganisms.However,design of complex gene circuits was limited by the lack of terminators specifically adapted to Bacillus subtilis.[Objective]Exploiting new high-performance terminators from Bacillus subtilis and further redesigning to enrich the artificial terminators suitable for this chassis.[Methods]The terminators from Bacillus subtilis and Bacillus subtilis phage were respectively constructed into the terminator measurement plasmid to determine the termination efficiency(TE).Multiple tandem terminators were constructed with combinatorial patterns of strong-weak,strong-strong,and weak-weak and subsequently TEs of those tandem terminators were individually determined.The tandem terminators with high TE were harnessed to verify the function of heterologous expression of L-aspartate amino lyase(AspA)and β-glucuronidase(GusA)in B.subtilis.[Results]The TE of terminator TB5 is 98%,which is strongest among the natural terminators in B.subtilis.Meanwhile,expression level of GFP harboring TB5 was up-regulated by 2.2 folds,and the expression level of RFP was down-regulated by 27 folds.For the dual-tandem terminators,the expression levels of RFP regulated by TH1.5b-TB5(TE=97%)and TB5-TB5(TE=98%)tandem terminators were decreased by 30 folds.For the triple-tandem terminators,TB2-TB5-TB5 combination no longer increase the expression level of GFP compared with the corresponding dual combination.Nevertheless,the expression of RFP is only 1/300 of the control.Finally,heterologous expression of AspA and GusA confirmed that two tandem terminators,TH1.5b-TB5 and TB10-TB5,significantly improved the gene expression in B.subtilis.[Conclusion]Strong synthetic terminators are able to reinforce the heterologous gene expression in surrogate host.Importantly,the efficiency of termination is further improved by rational combination of natural terminators in tandem manner.These artifici

关 键 词：枯草芽孢杆菌 转录终止子 串联终止子 基因表达 

分 类 号：Q78[生物学—分子生物学]