拟南芥受十字花科黑腐病菌诱导或抑制的miRNA的鉴定

发布时间：2018-01-25 00:48

  本文关键词： 拟南芥 十字花科黑腐病菌 RNA高通量测序 miRNA差异表达　出处：《广西大学》2017年硕士论文　论文类型：学位论文

【摘要】：miRNA(microRNA)是存在于真核生物细胞中长度～22nt(nucleotide)的内源非编码RNA。miRNA参与生长发育、组织分化、肿瘤发生、细胞凋亡等一系列重要的生命过程。此外,miRNA在植物对抗病原菌入侵的防卫反应中起重要作用。拟南芥是研究寄主植物与病原菌互作分子机理的模式植物。目前人们已经在拟南芥中发现了多个参与对抗丁香假单胞菌(Pseudomonas syringaepv.Syringae,Pst)入侵的miRNA。十字花科黑腐病菌(Xanthomonas campestrispv.Campestris,c)是引起十字花科植物黑腐病的病原菌,同时也可以侵染拟南芥。但是,参与对抗Xcc侵染的拟南芥miRNA至今未见报道。研究表明,在对抗病原菌入侵的防卫反应中起重要作用的miRNA的表达往往会受到病原菌的诱导或者抑制。本研究的目的是鉴定拟南芥中受Xcc诱导或者抑制的miRNA,为鉴定在对抗Xcc侵染过程中起重要作用的miRNA打下基础。为此,我们首先采用高通量RNA测序(RNA-seq)技术,测定了拟南芥在没有感染以及在感染Xcc后的不同时期的miRNA表达谱,并通过差异表达分析确定受Xcc诱导或抑制的候选的miRNA;然后采用定量Northern杂交方法对这些候选miRNA进行验证。在进行高通量RNA测序前,我们首先建立了一种适合于测定拟南芥与病原菌互作过程中寄主和病原菌转录组变化的接种方法——真空抽滤法。随后,我们测定了哥伦比亚0生态型(Col-O)拟南芥在没有感染以及在感染Xcc和hrcV基因突变体(△hrcV)后2、6、12和24小时的miRNA表达谱;通过表达差异分析,确定了 10个受Xcc诱导的候选miRNA。定量Northern杂交验证结果显示,miR166和miR393在接种Xcc后2小时时的达量显著升高,而miR160和miR156在接种后24小时时的表达量显著升高,说明这些miRNA的表达受Xcc诱导。此外,通过Northern杂交,我们还发现了 3个新的拟南芥miRNA。本研究采用RNA-Seq技术对拟南芥中受Xcc诱导或抑制的miRNA进行了系统鉴定,发现miR156、miR160、miR166和miR393受Xcc诱导。这是首次在拟南芥中发现受Xcc诱导或抑制的miRNA,为进一步研究miRNA在拟南芥—Xcc互作过程的作用的打下了基础。
[Abstract]:MiRNA-microRNAs are endogenous non-coding RNA.miRNA that exist in eukaryotic cells with a length of 22 NT nucleotide. they are involved in the growth and development of Eukaryotic cells. Tissue differentiation, tumorigenesis, apoptosis and other important life processes. MiRNA plays an important role in plant defense against pathogen invasion. Arabidopsis thaliana is a model plant to study the molecular mechanism of host plant and pathogen interaction. Several ginseng have been found in Arabidopsis thaliana. Against Pseudomonas clove. Pseudomonas syringaepv.Syringae. Xanthomonas campestrispv.Campestris. C) is the pathogen causing black rot of cruciferous plants and can also infect Arabidopsis thaliana. However, the miRNA involved in the resistance to Xcc infection has not been reported. The expression of miRNA, which plays an important role in defense response to pathogen invasion, is often induced or inhibited by pathogens. The purpose of this study was to identify the Xcc induced or inhibited mi in Arabidopsis thaliana. RNA. In order to lay a foundation for the identification of miRNA which plays an important role in the process of combating Xcc infection, we first adopted the technique of high-throughput RNA sequencing RNA-seq. The miRNA expression profiles of Arabidopsis thaliana at different stages of infection and after infection with Xcc were determined, and the candidate miRNAs induced or inhibited by Xcc were identified by differential expression analysis. Then the candidate miRNA was verified by quantitative Northern hybridization before high-throughput RNA sequencing. We first established a inoculation method, vacuum filtration method, which was suitable for the determination of transcriptional changes in host and pathogen during the interaction between Arabidopsis thaliana and pathogenic bacteria. We measured the level of Xcc and hrcV gene mutants (hrcV6) in Arabidopsis thaliana without infection and after infection with Xcc and hrcV gene mutants (HRCV). MiRNA expression profiles were observed at 12 and 24 hours. Ten candidate miRNAs induced by Xcc were identified by differential expression analysis. The results of quantitative Northern hybridization showed that 10 candidate miRNAs were induced by Xcc. The levels of miR166 and miR393 increased significantly at 2 hours after Xcc inoculation, while the expression of miR160 and miR156 increased significantly at 24 hours after inoculation. These results indicated that the expression of miRNA was induced by Xcc. In addition, the expression of these miRNA was induced by Northern hybridization. We also found three new Arabidopsis miRNAs. MiRNA induced or inhibited by Xcc in Arabidopsis thaliana was systematically identified by RNA-Seq technique. It was found that miR156, miR160, miR166 and miR393 were induced by Xcc. This is the first time that miRNA induced or inhibited by Xcc was found in Arabidopsis thaliana. The results provide a basis for further study on the role of miRNA in Arabidopsis-Xcc interaction.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.4

【参考文献】

相关期刊论文 前1条

1 Gang Wu;;Plant MicroRNAs and Development[J];Journal of Genetics and Genomics;2013年05期

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