發(fā)布時間：2018-03-03 14:02

  本文選題：DNA甲基化　切入點(diǎn)：溫度脅迫　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：溫度是影響植物生長發(fā)育和自然分布的主要限制因子之一。在長期的進(jìn)化過程中植物形成了響應(yīng)溫度脅迫的抗性機(jī)制。有研究表明表觀遺傳可以參與到植物響應(yīng)溫度脅迫的抗性機(jī)制中,基因組的表達(dá)調(diào)控受染色質(zhì)結(jié)構(gòu)改變的影響,而組蛋白的翻譯后修飾和DNA甲基化調(diào)控是控制染色質(zhì)結(jié)構(gòu)變化的主要因素。在高等生物體中,DNA甲基化修飾是調(diào)控基因表達(dá)的一個重要的表觀遺傳學(xué)。而RNA介導(dǎo)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(dá)調(diào)控的重要機(jī)制。本研究以擬南芥為材料,利用正向遺傳學(xué)與反向遺傳學(xué)策略,分離鑒定了兩個與溫度脅迫相關(guān)的基因,主要結(jié)果如下:(1)通過對溫度脅迫芯片數(shù)據(jù)、擬南芥表觀遺傳學(xué)圖譜和si RNA數(shù)據(jù)庫的分析篩選到可能受高溫脅迫誘導(dǎo)和表觀遺傳調(diào)控的基因AT2G21150,該基因編碼一個植物光周期調(diào)控相關(guān)蛋白XCT。(2)利用DNA甲基化測序(BSP)和q RT-PCR技術(shù)分析該基因啟動子區(qū)域的甲基化水平和基因表達(dá)量,結(jié)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(fā)現(xiàn),在種子萌發(fā)期和幼苗期突變體植株表現(xiàn)出對高溫脅迫的敏感表型。(4)對該基因的時空表達(dá)模式進(jìn)行檢測,發(fā)現(xiàn)該基因在種子中的表達(dá)水平最高。該基因的表達(dá)還明顯受高溫誘導(dǎo),在高溫脅迫中,基因啟動子區(qū)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(diào),推測可能是si RNA介導(dǎo)的Rd DM途徑通過降低甲基化水平促進(jìn)了基因的表達(dá)。(5)體外瞬轉(zhuǎn)煙草實(shí)驗(yàn)進(jìn)一步驗(yàn)證了RNA介導(dǎo)的DNA甲基化途徑參與了高溫響應(yīng)過程。(6)對EMS誘變的突變體庫進(jìn)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(qū)別不大,但長日照條件下表現(xiàn)出植株矮小、花青素積累的表型。(7)在種子萌發(fā)期對h46進(jìn)行高溫處理,突變體表現(xiàn)出熱敏感的表型,而在幼苗期和成苗期卻表現(xiàn)出抗熱表型。(8)對該突變體進(jìn)行圖位克隆,發(fā)現(xiàn)突變位點(diǎn)位于一號染色體上,下一步驗(yàn)證該突變基因,進(jìn)行互補(bǔ)驗(yàn)證。該突變體具有較強(qiáng)的非生物脅迫抗性,為進(jìn)一步研究植物與高溫脅迫的關(guān)系提供了依據(jù)。
[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(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q945.78;Q943.2

【參考文獻(xiàn)】

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1 李娜;張e，

本文編號：1561329