番茄活体再生相关miRNA的鉴定

发布时间：2018-03-09 04:02

  本文选题：miRNA　切入点：活体再生　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：miRNA是一种广泛存在于真核生物和病毒中的非编码小RNA,长度一般在19～24nt之间,通过切割靶基因mRNA或抑制靶基因mRNA翻译导致靶基因mRNA沉默的方式来负向调控靶基因的表达。miRNA在植物体中发挥了重要的作用,主要包括参与了新陈代谢、信号传导、应激反应、能量代谢、物质代谢等过程中。然而有关于植物活体再生的miRNA研究较少,这方面还有待于进一步的研究。本研究选取了番茄作为研究对象,以番茄活体植株伤口上的再生体系作为研究材料来确认参与其中的miRNA。使用Trizol法提取总RNA,构建了活体再生过程的小RNA文库,通过对高通量测序后得到的结果进行差异性分析和靶基因预测发现了相关miRNA,之后选取了 RT-PCR和荧光定量PCR的分子生物学方法对生物信息学分析得到的结果进行了进一步的验证,主要结果如下:预测到92个已知的miRNA和91个新的miRNA,其中已知的miRNA中包括分属于18个miRNA家族的50个保守miRNA和分属于26个miRNA家族的42个不保守的miRNA;找到了 Sly-miR482e-3p,Sly-miR171a,Sly-miR164a-5p,Sly-miR156a,novel-56,novel-128这6个与活体再生有关的miRNA;通过RT-PCR中成功识别到这6个miRNA;通过荧光定量PCR中得到了相对表达量,分析后发现这6个kmiRNA在活体再生的过程中表达量相对有不同程度的降低。本研究实验过程中使用了茎环法对miRNA进行处理,成功解决了 miRNA长度太短无法直接扩增的问题。建立了番茄miRNA的茎环RT-PCR检测体系,为研究参与番茄其他生长发育过程中的miRNA研究奠定了基础。
[Abstract]:MiRNA is a non-coding small RNAs widely found in eukaryotes and viruses. By cutting the target gene mRNA or inhibiting the target gene mRNA translation resulting in the target gene mRNA silencing, the expression of the target gene plays an important role in plants, including metabolism, signal transduction, stress response. Energy metabolism, substance metabolism and so on. However, there are few miRNA studies on plant regeneration in vivo, which need to be further studied. In this study, tomato was selected as the research object. The regeneration system of tomato plants in vivo was used as the research material to identify the participating miRNAs. The total RNAs were extracted by Trizol method, and the small RNA library was constructed for the process of in vivo regeneration. The related miRNAs were found through the difference analysis of the results of high-throughput sequencing and the prediction of target genes. Then the molecular biological methods of RT-PCR and fluorescent quantitative PCR were selected to further verify the results obtained by bioinformatics analysis. The main results are as follows: 92 known miRNA and 91 new miRNAs were predicted, of which the known miRNA included 50 conserved miRNA belonging to 18 miRNA families and 42 unconserved miRNAs belonging to 26 miRNA families; Sly-miR482e-3pSly-miR171aSly-miR164a-5pSly-miR156a Novel-128were found. MiRNAs related to in vivo regeneration; these six miRNAs were successfully identified by RT-PCR; the relative expression levels were obtained by fluorescence quantitative PCR. It was found that the expression of the six kmiRNA decreased in different degrees during the process of in vivo regeneration. In this study, miRNA was treated with stem ring method. The problem that the length of miRNA is too short to be directly amplified was solved successfully, and the detection system of RT-PCR in stem ring of tomato miRNA was established, which laid a foundation for the study of miRNA involved in other growth and development of tomato.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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