十字花科黑腐病菌中十个小RNA的功能鉴定

发布时间：2018-03-02 07:03

本文关键词： 十字花科黑腐病菌小RNA 功能鉴定过量表达缺失　出处：《广西大学》2015年硕士论文　论文类型：学位论文

【摘要】：细菌小RNA,也叫非编码RNA,是一类大小处于50～500 nt之间的RNA分子。细菌小RNA可以独立地行使一些特定的功能,但是不具备编码蛋白的能力,与核糖体RNA和转运RNA有明显的差异。小RNA广泛存在于各种生物中,对细胞的生理过程起着重要的调节作用。研究表明,它们作为转录后调控因子在细菌的糖代谢、碳源代谢、群体感应、应激反应、细胞运动和致病性等方面发挥着重要的调控作用。目前,小RNA的鉴定工作以及对其功能进行研究是当前微生物学研究领域的前沿和热点。目前,有效鉴定小RNA方法已经成功建立,已经成功鉴定了近1000个细菌小RNA,但是它们的生物学功能几乎没有被确定。小RNA在植物病原细菌致病过程中的作用和重要性仍然需要更深入的研究。十字花科黑腐病菌(Xanthomonas campestris pathovar campestris,下文简称为Xcc)是引起十字花科植物黑腐病的病原菌,它是研究病原菌与植物相互作用领域的一个极其重要的模式菌株。近来,本课题组采用RNA深度测序(RNA-seq)方法,对Xcc 8004菌株在MMX基本培养基中生长至对数期时表达的小RNA进行了鉴定,结果发现了500多个候选。但是这些候选小RNA的生物学功能仍不清楚。本研究选取了十个候选小RNA作为研究对象,首先采用三引物RT-PCR法确证它们的存在,然后分别构建这十个候选小RNA的缺失突变体和过量表达株,然后对构建成功的缺失突变体和过量表达株的相应表型均进行检测和分析讨论从而得知它们具有怎么的生物学功能。三引物RT-PCR结果分析得知这十个候选小RNA的确是真实存在的。然后,通过链特异性RT-PCR的方法,我们可以进一步得知这十个小RNA的主要转录方向。目前,本研究已成功构建了这10个目的小RNA的9个缺失突变体和10个过量表达菌株,然后,对成功构建的所有缺失突变体和过量表达株均进行了一系列的表型检测,分别为：(1)在丰富培养基和基本培养基内的生长情况、(2)胞外多糖产量和胞外酶活性、(3)游动性、(4)生物膜形成、(5)在非寄主植物上引起的高敏反应(HR反应)、(6)在寄主植物上的致病能力等表型进行了检测,然后把检测的所有表型与野生型菌株和带有空的过量表达载体pBBad18K的野生型菌株的表型作出了相应的比较和分析。结果发现：(1)小RNA sRXcc-50的缺失突变体DM50在丰富培养基中的生长稳定期的菌体密度小于野生型菌株；(2)小RNA sRXcc-49、sRXcc-51、sRXcc-52、sRXcc-55的缺失突变体DM49、DM51、 DM52、DM55在基本培养基中的对数期和稳定期的菌体密度明显小于野生型菌株；(3)小RNA sRXcc-53的过量表达株OE53在基本培养基中的对数期和稳定期的菌体密度明显低于对照组WT/pBBad18K;(4)小RNA sRXcc-55的过量表达株OE55在基本培养基中的对数期和稳定期的菌体密度明显高于对照组WT/pBBad18K;(5)小RNA sRXcc-49的缺失突变体DM49在辣椒叶片上出现HR反应的时间比野生型Xcc 8004早；(6)小RNA sRXcc-52、sRXCC-55、sRXCC-56的过量表达株OE 52、OE 55、ROE56在辣椒叶片上出现HR反应的时间比对照组WT/pBBad18K早；(7)其它的缺失突变体和过量表达株的所有表型检测结果几乎与对照菌株的完全一样。检测结果表明：(1)小RNA sRXCC-49、sRXCC-50、sRXCC-51、sRXCC-52、 sRXCC-53、sRXCC-55与Xcc的生长有关；(2)小RNA sRXCC-49、sRXCC-52、 sRXCC-55、sRXCC-56在Xcc8004的过敏反应中可能起着重要的调控作用。综上所述,本工作验证了十个Xcc候选小RNA的存在,确定了它们的转录方向,并对它们的生物学功能进行了鉴定。结果发现了六个小RNA (sRXCC-49、sRXCC-50、sRXCC-51、sRXCC-52、sRXCC-53、sRXCC-55)与生长有关,四个(sRXCC-49、sRXCC-52、sRXCC-55、sRXCC-56)与HR反应有关。这些结果增加了人们在小RNA功能研究领域的知识,为深入探讨小RNA在Xcc中的具有怎样的功能以及如何参与机理调控提供了重要的素材。
[Abstract]:Bacterial small RNA, also called non RNA encoding, is a kind of size in the RNA molecule 50~500 NT. Bacterial small RNA can independently exercise some specific function, but does not have the ability of encoding protein, there are obvious differences between the ribosomal RNA and transfer RNA. RNA exists widely in all kinds of biological, physiological the process of cells play an important role. The research results show that as the post transcriptional regulation factor in the sugar metabolism of bacteria, carbon metabolism, quorum sensing, stress response, play an important role in cell motility and pathogenicity. At present, identification of small RNA and the function of the frontier and hot point in the research field of microbiology. At present, the effective identification of small RNA method has been successfully established, has successfully identified nearly 1000 small bacteria RNA, but their biological functions have hardly been determined. Small RNA in plants The role and importance of complex pathogenic bacteria in the pathogenic process still need further research. XpsE (Xanthomonas campestris pathovar campestris, hereinafter referred to as Xcc) is the pathogen of crucifers black rot, it is a very important research field of pattern strains of pathogenic bacteria and plant interactions. Recently, our research group used RNA deep sequencing (RNA-seq) method of Xcc 8004 strain in MMX medium and grown to logarithmic phase when the expression of RNA was identified and found more than 500 candidate. But the biological function of these candidate RNA remains unclear. This study selected ten candidate small RNA as the research object, firstly using three primer RT-PCR method confirmed their existence, then construct the deletion mutants of the ten candidate RNA and overexpression strains, and to build a successful. The corresponding phenotype loss mutants and overexpressing strains were detected and analyzed so as to know how they have biological function. Three primer RT-PCR results of the ten candidate RNA is indeed real. Then, through the method of chain specific RT-PCR, we can further learn the main transcription direction of the ten small RNA at present, this study has successfully constructed 9 deletion mutants of the 10 to 10 small RNA and overexpression strains, then, were all constructed deletion mutants and overexpression strains were phenotypic assays, a series of: (1) in rich medium and medium growth the situation of the media, (2) extracellular polysaccharide production and intracellular enzyme activity (3), motility, biofilm formation (4), (5) Gao Min reaction due to non host plants on (HR reaction), (6) in the host plant pathogenic ability The phenotype was detected, then the phenotype of wild type strains and wild type strains of all phenotypic detection and with empty excess expression vector of pBBad18K has made the comparison and analysis of the corresponding. Results showed that: (1) RNA sRXcc-50 deletion mutant DM50 in rich growth stable cell density in the medium is smaller than the wild strain; (2) RNA sRXcc-49, sRXcc-51, sRXcc-52, sRXcc-55 deletion mutant of DM49, DM51, DM52, DM55 in basic medium in the log phase and stable phase of cell density was significantly less than that of wild type strain; (3) overexpression of sRXcc-53 small RNA strains of OE53 in basic medium in logarithmic phase and the stable cell density was significantly lower than the control group (WT/pBBad18K; 4) overexpression of small RNA sRXcc-55 strains of OE55 in basic medium in the log phase and stable phase of cell density was significantly higher than the control group (WT/pBBad18K; 5) DM49 small RNA sRXcc-49 deletion mutant HR reaction in pepper leaves on time earlier than the wild type Xcc 8004; (6) RNA sRXcc-52, sRXCC-55, sRXCC-56 overexpression strains OE 52, OE 55, ROE56 HR reaction in pepper leaves on time than the control group WT/pBBad18K; (7) all phenotype the detection results of deletion mutants and overexpression strains and other strains were almost the same. The detection results show that: (1) RNA sRXCC-49, sRXCC-50, sRXCC-51, sRXCC-52, sRXCC-53, sRXCC-55 and Xcc growth; (2) RNA sRXCC-49, sRXCC-52, sRXCC-55, sRXCC-56 may play an important role in regulation the allergic reaction in the Xcc8004. In summary, this work verified the ten Xcc candidate RNA, to determine the direction of transcription of them, and their biological functions were identified. Results showed that six small RNA (sRXCC-49, s RXCC-50, sRXCC-51, sRXCC-52, sRXCC-53, sRXCC-55) and growth, four (sRXCC-49, sRXCC-52, sRXCC-55, sRXCC-56) associated with the HR reaction. These results increase people's knowledge in the field of function of small RNA, for the in-depth study of RNA function in Xcc is how and how to participate in the regulation mechanism provides important material.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.4

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