本文選題：DNA甲基化　切入點：溫度脅迫　出處：《山東農業大學》2017年碩士論文　論文類型：學位論文

【摘要】：溫度是影響植物生長發育和自然分布的主要限制因子之一。在長期的進化過程中植物形成了響應溫度脅迫的抗性機制。有研究表明表觀遺傳可以參與到植物響應溫度脅迫的抗性機制中,基因組的表達調控受染色質結構改變的影響,而組蛋白的翻譯后修飾和DNA甲基化調控是控制染色質結構變化的主要因素。在高等生物體中,DNA甲基化修飾是調控基因表達的一個重要的表觀遺傳學。而RNA介導的DNA甲基化(RNA-directed DNA methylation,Rd DM)途徑是植物在非生物脅迫下基因表達調控的重要機制。本研究以擬南芥為材料,利用正向遺傳學與反向遺傳學策略,分離鑒定了兩個與溫度脅迫相關的基因,主要結果如下:(1)通過對溫度脅迫芯片數據、擬南芥表觀遺傳學圖譜和si RNA數據庫的分析篩選到可能受高溫脅迫誘導和表觀遺傳調控的基因AT2G21150,該基因編碼一個植物光周期調控相關蛋白XCT。(2)利用DNA甲基化測序(BSP)和q RT-PCR技術分析該基因啟動子區域的甲基化水平和基因表達量,結果表明兩者均受到高溫脅迫的影響。(3)利用AT2G21150的T-DNA插入突變體(xct-2、xct-3),研究發現,在種子萌發期和幼苗期突變體植株表現出對高溫脅迫的敏感表型。(4)對該基因的時空表達模式進行檢測,發現該基因在種子中的表達水平最高。該基因的表達還明顯受高溫誘導,在高溫脅迫中,基因啟動子區域的甲基化水平明顯下降而且24-nt si RNA的積累量明顯下調,推測可能是si RNA介導的Rd DM途徑通過降低甲基化水平促進了基因的表達。(5)體外瞬轉煙草實驗進一步驗證了RNA介導的DNA甲基化途徑參與了高溫響應過程。(6)對EMS誘變的突變體庫進行篩選,鑒定了一個成苗抗熱突變體,命名為h46。該突變體短日照條件下與野生型區別不大,但長日照條件下表現出植株矮小、花青素積累的表型。(7)在種子萌發期對h46進行高溫處理,突變體表現出熱敏感的表型,而在幼苗期和成苗期卻表現出抗熱表型。(8)對該突變體進行圖位克隆,發現突變位點位于一號染色體上,下一步驗證該突變基因,進行互補驗證。該突變體具有較強的非生物脅迫抗性,為進一步研究植物與高溫脅迫的關系提供了依據。
[Abstract]:Temperature is one of the main limiting factors of plant growth, development and natural distribution. During the long evolutionary process, plants have developed resistance mechanisms in response to temperature stress. Some studies have shown that epigenetics can participate in plant response. In the mechanism of resistance to temperature stress, The regulation of genome expression is affected by chromatin structural changes. The posttranslational modification of histone and the regulation of DNA methylation are the main factors controlling chromatin structure changes. In higher organisms, methylation of histone is an important epigenetic regulation of gene expression. RNA-directed DNA methylation (Rd) pathway of DNA methylation is an important mechanism of gene expression regulation in plants under abiotic stress. Using the strategy of forward genetics and reverse genetics, two genes related to temperature stress were isolated and identified. The main results were as follows: 1. Analysis of epigenetics map and si RNA database of Arabidopsis thaliana showed that AT2G21150, a plant photoperiod regulatory protein encoded by AT2G21150, which could be induced by high temperature stress and regulated by epigenetic regulation, was sequenced by DNA methylation. And Q RT-PCR techniques were used to analyze the methylation level and gene expression in the promoter region of the gene. The results showed that both of them were affected by high temperature stress. During seed germination and seedling stage, the mutant plants showed a sensitive phenotype to high temperature stress. The spatiotemporal expression pattern of the gene was detected. It was found that the expression level of the gene was the highest in the seeds, and the expression of the gene was also significantly induced by high temperature. Under high temperature stress, the methylation level of gene promoter region decreased and the accumulation of 24-nt si RNA decreased significantly. We speculated that the Rd DM pathway mediated by si RNA promoted gene expression by reducing methylation level. The transient tobacco experiment in vitro further demonstrated that RNA mediated DNA methylation pathway was involved in the high temperature response process. 6) EMS mutagenesis was induced by EMS. For screening, A heat-resistant mutant named H46 was identified, which was not different from wild type under the condition of short sunlight, but it showed that the plant was short and the phenotype of anthocyanin accumulation was high temperature treatment at the germination stage of seeds. The mutant showed thermo-sensitive phenotype, while it showed heat-resistant phenotype at seedling stage and seedling stage. It was found that the mutant was located on chromosome 1. The next step was to identify the mutant gene. The mutants have strong abiotic stress resistance, which provides a basis for further study on the relationship between plant and high temperature stress.
【學位授予單位】：山東農業大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q945.78;Q943.2

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1 李娜;張e，

本文編號：1561329