鼠伤寒沙门菌Hfq依赖型sRNA GcvB对Ⅲ型分泌系统相关基因调控作用的研究

Study on Hfq Dependent sRNA GcvB of Salmonella Typhimurium on the Regulation of Type Ⅲ Secretory System Related Genes

试验旨在研究鼠伤寒沙门菌伴侣蛋白Hfq依赖型sRNA GcvB对spaP和invC基因mRNA所具有的调控作用。利用λ-Red同源重组技术构建spaP和invC基因LacZ融合菌株,并利用P_(22)噬菌体转导技术分别构建GcvB和Hfq基因单缺失和双缺失菌株及GcvB功能区域缺失株,通过β-半乳糖苷酶活性试验测定在伴侣蛋白Hfq、sRNA GcvB及Hfq&GcvB缺失株中spaP和invC基因β-半乳糖核苷酶活性;实时荧光定量PCR测定spaP和invC基因mRNA表达量。结果显示,利用P_(22)噬菌体转导技术成功构建出ΔGcvB(696 bp)、ΔHfq(2369 bp)、ΔGcvBΔHfq(2369/696 bp)、GcvBΔR1(757 bp)、GcvBΔR2(951 bp)、GcvBΔR3(986 bp)缺失株;β-半乳糖苷酶活性检测结果显示,与野生型菌株相比,spaP基因β-半乳糖核苷酶活性在缺失株GcvBΔR2、GcvBΔR3中极显著下调(P<0.01),在缺失株ΔGcvB、ΔHfq、ΔGcvBΔHfq、GcvBΔR1中极显著上调(P<0.01);而invC基因β-半乳糖核苷酶活性在缺失株GcvBΔR2、GcvBΔR3中极显著下调(P<0.01),在缺失株GcvBΔR1中显著下调(P<0.05),在缺失株ΔGcvB、ΔHfq、ΔGcvBΔHfq中极显著上调(P<0.01)。实时荧光定量PCR结果显示,与野生型菌株相比,spaP基因在ΔGcvB、ΔHfq、ΔGcvBΔHfq和GcvBΔR1缺失菌株中转录水平极显著上调(P<0.01),而GcvBΔR2缺失菌株转录水平极显著下调(P<0.01),GcvBΔR3缺失菌株转录水平显著下调(P<0.05),invC基因ΔGcvB、ΔHfq、ΔGcvBΔHfq缺失菌株转录水平极显著上调(P<0.01),GcvBΔR1、GcvBΔR2缺失菌株均极显著下调(P<0.01),GcvBΔR3缺失菌株均显著下调(P<0.05)。通过以上试验结果得出,sRNA GcvB及伴侣蛋白Hfq对invC和spaP基因存在一定的调控作用。本试验结果为sRNA GcvB及伴侣蛋白Hfq调控提供了理论依据,丰富了GcvB的靶基因数目。

The purpose of this experiment was to study the regulatory effect of Salmonella Typhimurium chaperone protein Hfq-dependent sRNA GcvB on spaP and invC gene mRNA.λ-Red homologous recombination technology was used to construct spaP and invC genes LacZ fusion strains,P_(22) phage transduction technology was used to construct GcvB and Hfq genes single deletion and double deletion strains and GcvB functional region deletion strains.β-galactosinosidase activity experiment was performed to determine the protein expression activity of spaP and invC genes in chaperone proteins Hfq,sRNA GcvB and Hfq&GcvB deletion strains.Real-time PCR was used to determine the mRNA expression of spaP and invC genes.The results showed that P_(22) phage transduction technology successfully constructedΔGcvB(696 bp),ΔHfq(2369 bp),ΔGcvBΔHfq(2369/696 bp),GcvBΔR1(757 bp),GcvBΔR2(951 bp),GcvBΔR3(986 bp)deletion strains.β-galactosinosidase activity test results showed that compared with wild-type strains,theβ-galactosinosidase activity of spaP gene was extremely significantly down-regulated in the deleted strains GcvBΔR2 and GcvBΔR3(P<0.01),and theβ-galactosinosidase activity of the invC gene was extremely significantly up-regulated in the deleted strainsΔGcvB,ΔHfq,ΔGcvBΔHfq and GcvBΔR1(P<0.01).The invC gene was extremely significantly down-regulated in the deletion strain GcvBΔR2 and GcvBΔR3 protein activity(P<0.01),and the GcvBΔR1 protein activity was significantly down-regulated in the deletion strain(P<0.05).The protein activity in the deletion strainsΔGcvB,ΔHfq andΔGcvBΔHfq was extremely significantly up-regulated(P<0.01).Real-time PCR results showed that compared with the wild-type strain,the transcription level of the spaP gene inΔHfq,ΔGcvB,ΔGcvBΔHfq and GcvBΔR1 deletion strains was extremely significantly up-regulated(P<0.01),while the transcription level of GcvBΔR2 deletion strain was extremely significantly down-regulated(P<0.01).The transcription level of GcvBΔR3 deletion strains was significantly down-regulated(P<0.05),the transcription level of invC geneΔHfq,ΔGcvBΔHfq andΔGcvB deletion strains was extremely significant up-regulated(P<0.01),GcvBΔR1 and GcvBΔR2 deletion strains were all extremely significantly downward-regulated(P<0.01),GcvBΔR3 deletion strains were all significant down-regulated(P<0.05).According to the above experimental results,sRNA GcvB and chaperonin Hfq had a certain regulatory effect on invC and spaP genes.This experiment provided a theoretical basis for the regulation of sRNA GcvB and chaperonin Hfq,and enriched the number of target genes of GcvB.