一个用于黄单胞菌核开关筛选鉴定的GUS报告载体的构建

Construction of a GUS Reporter Vector for Riboswitch Screening and Identification in Xanthomonas

核开关(riboswitch)是存在于信使RNA(messenger RNA,mRNA)分子的5′非翻译区(5′-untranslated region,5′UTR)的一段具有调控功能的RNA片段,它通过与特定的代谢物分子直接结合来调控所在mRNA自身的表达。核开关广泛存在于细菌中,在调控细菌的基础代谢以及致病等生理过程中发挥非常重要的作用。尽管目前在大肠杆菌(Escherichia coli)等模式细菌中已经鉴定了许多核开关,然而,至今在重要的植物病原细菌黄单胞菌属(Xanthomonas)细菌中只鉴定了1个核开关。为了实现在全基因组水平上对黄单胞菌的核开关进行高通量筛选鉴定,本研究设计和构建了1个β-葡萄糖苷酸酶(β-glucuronidase,GUS)报告载体pL3-SD^(+)gusA。该载体既适用于在黄单胞菌属细菌中对候选的核开关进行验证,也可以通过构建基因组DNA表达文库而实现对黄单胞菌核开关的高通量筛选鉴定。

Riboswitch is a regulatory RNA element located in the 5′-untranslated region(5′UTR)of a messenger RNA(mRNA),it regulates the expression of the mRNA by directly binding to a specific metabolite molecule.Riboswitches are widely distributed in bac-teria and have been shown to play an important role in various physiological processes including the primary metabolism and pathogene-sis.Although a large number of riboswitches have been identified in model bacteria such as Escherichia coli,only one riboswitch was found in the important plant pathogenic bacterial genus Xanthomonas,majorly due to lacking a simple and highly efficient method for ri-boswitch screening and identification in Xanthomonas.For a high throughput and genome-wide riboswitch screening and identification in Xanthomonas,in this work,a GUS reporter vector,pL3-SD^(+)gusA,was designed and constructed.This vector is suitable for the validation of riboswitch candidates in Xanthomonas,it also can achieve a high throughput riboswitch screening and identification in Xanthomonas through construction of a genomic DNA expression library.