布鲁氏菌sRNA AbcR1和靶基因的相互作用研究

发布时间：2018-04-15 04:28

本文选题：布鲁氏菌 + sRNA　；参考：《华中农业大学》2015年硕士论文

【摘要】：布鲁氏菌是一种革兰氏阴性兼性胞内寄生菌,能够引起人类和家畜的布鲁氏菌病,通过皮肤黏膜、呼吸道和消化道传播。羊、牛、猪等宿主被感染后,会出现流产和不孕不育等症状,而人则会出现关节炎、睾丸炎、波浪热、多汗等特征。布鲁氏菌感染机体后,能在机体内建立免疫逃避机制。最近研究表明,细菌中存在一类非编码的小RNA(small non-coding RNA,s RNA),具有潜在的调控细菌代谢以及毒力相关基因作用。本课题组已有的RNA-seq分析预测表明,羊种布鲁氏菌的s RNA Abc R 1极可能是具有调控功能的s RNA。布鲁氏菌的s RNA靶定其m RNAs然后发挥转录后调控作用。通过查阅文献同源性比对以及生物信息学预选定测Abc R 1的三个潜在靶基因BMEII0923,BMEII0372,BMEI1250。它们与布鲁氏菌Mer R蛋白家族转录后调控、短链脱氢酶的活性、周质蛋白有关。通过RACE实验检测靶基因的TSS位点,测序验证后克隆相关靶基因,然后利用具有绿色荧光GFP报告系统的大肠杆菌质粒检测系统,将s RNA构建到具有氨苄青霉素抗性的质粒载体p ZK001上,将备选targets构建到具有氯霉素抗性的质粒载体p XG-10SF上,p XG-10SF上具有superfolder GFP基因,可表达target::GFP融合蛋白。通过共转化两种质粒得到菌落,检测GFP在特定激发光下的荧光强度;超声波破碎菌体后,Western blot检测GFP蛋白的表达量;q RT-PCR检测靶基因的转录水平,由此推断GFP蛋白的转录和表达情况,从而分析s RNA对于target的调控作用。相关结论如下:1.Target RNA2作为专门的细菌s RNA靶位点预测工具,能够用于布鲁氏菌靶位点的分析,例如s RNA Abc R1靶位点的相互作用预测,但预测结果需要更进一步的实验验证。2.羊种布鲁氏菌Brucella menlitensis的s RNA Abc R1经验证是具有调控功能的非编码小RNA,能够下调靶基因BMEII0923的表达,也能够上调靶基BMEII0372的表达,但对于BMEI1250的调控作用不明显。3具有GFP的质粒报告系统能够直观快速的反映羊种布鲁氏菌中目的s RNA和潜在靶基因的互作,该报告系统能够用于布鲁氏菌s RNA靶位点的筛选。实验结果表明,此大肠杆菌质粒报告系统可以用于验证布鲁氏菌s RNA和targets的互作,相比传统的western blot验证和q RT-PCR,具有快速直观的特点。此方法可以便捷的验证布鲁氏菌毒力代谢调控相关的s RNA和对应靶位点的互作,为揭示布鲁氏菌s RNA调控毒力以及代谢的机理提供新的思路。
[Abstract]:Brucellosis is a gram-negative facultative intracellular parasite that causes brucellosis in humans and livestock and is transmitted through skin, respiratory and digestive tract.Sheep, cattle, pigs and other hosts are infected with abortion and infertility symptoms, while people will develop arthritis, orchitis, wave fever, sweating and other characteristics.After brucella infection, the immune escape mechanism can be established in the organism.Recent studies have shown that there is a class of non-coding small RNA(small non-coding RNAs RNAs in bacteria, which have the potential to regulate bacterial metabolism and virulence related genes.The RNA-seq analysis and prediction of Brucella species showed that s RNA Abc R 1 of Brucella was probably a regulatory function of s RNA Abc R 1.S RNA of Brucella targeted its m RNAs and then played a post-transcriptional regulatory role.Three potential target genes, BMEII0923, BMEII0372 and BMEI1250, were selected by literature homology comparison and bioinformatics to detect Abc R1.They are related to the posttranscriptional regulation of Mer R protein family, the activity of short chain dehydrogenase and the pericytoplasmic protein of Brucella.The TSS site of the target gene was detected by RACE experiment, the relevant target gene was cloned by sequencing, and then the plasmid detection system of E. coli was used to detect the target gene by using the green fluorescent GFP report system.S RNA was constructed into ampicillin resistant plasmid vector p ZK001, and alternative targets was constructed into chloramphenicol resistant plasmid vector p XG-10SF with superfolder GFP gene on p XG-10SF, which could express target::GFP fusion protein.The colony was obtained by co-transformation of two plasmids to detect the fluorescence intensity of GFP under specific excitation light, and the expression of GFP protein was detected by Western blot after supersonic cell fragmentation, and the transcription level of target gene was detected by Q RT-PCR.The transcription and expression of GFP protein were deduced, and the regulation of s RNA on target was analyzed.The relevant conclusions are as follows: 1. Target RNA2, as a special tool for predicting the target sites of bacteria s RNA, can be used for the analysis of target sites of Brucella, such as the interaction prediction of target sites of s RNA Abc R1, but the prediction results need further experimental verification.S RNA Abc R1 of Brucella menlitensis in sheep has been proved to be a non-coding small RNAs with regulatory function, which can down-regulate the expression of target gene BMEII0923 and up-regulate the expression of target BMEII0372.However, the plasmid reporting system with GFP can reflect the interaction of target s RNA and potential target gene in Brucella species directly and quickly. The system can be used to screen target sites of Brucella spp. S RNA.The results showed that the plasmid reporting system could be used to verify the interaction between RNA and targets of Brucella. Compared with the traditional western blot and Q RT-PCR, the system had the characteristics of rapid and intuitive.This method can easily verify the interaction of s RNA and corresponding target sites in the regulation of brucella virulence metabolism, and provide a new idea for revealing the virulence and metabolic mechanism of brucella s RNA.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.614

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