本文選題：miRNA　切入點：活體再生　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：miRNA是一種廣泛存在于真核生物和病毒中的非編碼小RNA,長度一般在19～24nt之間,通過切割靶基因mRNA或抑制靶基因mRNA翻譯導(dǎo)致靶基因mRNA沉默的方式來負向調(diào)控靶基因的表達。miRNA在植物體中發(fā)揮了重要的作用,主要包括參與了新陳代謝、信號傳導(dǎo)、應(yīng)激反應(yīng)、能量代謝、物質(zhì)代謝等過程中。然而有關(guān)于植物活體再生的miRNA研究較少,這方面還有待于進一步的研究。本研究選取了番茄作為研究對象,以番茄活體植株傷口上的再生體系作為研究材料來確認參與其中的miRNA。使用Trizol法提取總RNA,構(gòu)建了活體再生過程的小RNA文庫,通過對高通量測序后得到的結(jié)果進行差異性分析和靶基因預(yù)測發(fā)現(xiàn)了相關(guān)miRNA,之后選取了 RT-PCR和熒光定量PCR的分子生物學(xué)方法對生物信息學(xué)分析得到的結(jié)果進行了進一步的驗證,主要結(jié)果如下:預(yù)測到92個已知的miRNA和91個新的miRNA,其中已知的miRNA中包括分屬于18個miRNA家族的50個保守miRNA和分屬于26個miRNA家族的42個不保守的miRNA;找到了 Sly-miR482e-3p,Sly-miR171a,Sly-miR164a-5p,Sly-miR156a,novel-56,novel-128這6個與活體再生有關(guān)的miRNA;通過RT-PCR中成功識別到這6個miRNA;通過熒光定量PCR中得到了相對表達量,分析后發(fā)現(xiàn)這6個kmiRNA在活體再生的過程中表達量相對有不同程度的降低。本研究實驗過程中使用了莖環(huán)法對miRNA進行處理,成功解決了 miRNA長度太短無法直接擴增的問題。建立了番茄miRNA的莖環(huán)RT-PCR檢測體系,為研究參與番茄其他生長發(fā)育過程中的miRNA研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

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本文編號：1586875