拟南芥高温胁迫响应基因的鉴定和功能研究

发布时间：2018-03-03 14:02

本文选题：DNA甲基化　切入点：温度胁迫　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：温度是影响植物生长发育和自然分布的主要限制因子之一。在长期的进化过程中植物形成了响应温度胁迫的抗性机制。有研究表明表观遗传可以参与到植物响应温度胁迫的抗性机制中,基因组的表达调控受染色质结构改变的影响,而组蛋白的翻译后修饰和DNA甲基化调控是控制染色质结构变化的主要因素。在高等生物体中,DNA甲基化修饰是调控基因表达的一个重要的表观遗传学。而RNA介导的DNA甲基化(RNA-directed DNA methylation,Rd DM)途径是植物在非生物胁迫下基因表达调控的重要机制。本研究以拟南芥为材料,利用正向遗传学与反向遗传学策略,分离鉴定了两个与温度胁迫相关的基因,主要结果如下:(1)通过对温度胁迫芯片数据、拟南芥表观遗传学图谱和si RNA数据库的分析筛选到可能受高温胁迫诱导和表观遗传调控的基因AT2G21150,该基因编码一个植物光周期调控相关蛋白XCT。(2)利用DNA甲基化测序(BSP)和q RT-PCR技术分析该基因启动子区域的甲基化水平和基因表达量,结果表明两者均受到高温胁迫的影响。(3)利用AT2G21150的T-DNA插入突变体(xct-2、xct-3),研究发现,在种子萌发期和幼苗期突变体植株表现出对高温胁迫的敏感表型。(4)对该基因的时空表达模式进行检测,发现该基因在种子中的表达水平最高。该基因的表达还明显受高温诱导,在高温胁迫中,基因启动子区域的甲基化水平明显下降而且24-nt si RNA的积累量明显下调,推测可能是si RNA介导的Rd DM途径通过降低甲基化水平促进了基因的表达。(5)体外瞬转烟草实验进一步验证了RNA介导的DNA甲基化途径参与了高温响应过程。(6)对EMS诱变的突变体库进行筛选,鉴定了一个成苗抗热突变体,命名为h46。该突变体短日照条件下与野生型区别不大,但长日照条件下表现出植株矮小、花青素积累的表型。(7)在种子萌发期对h46进行高温处理,突变体表现出热敏感的表型,而在幼苗期和成苗期却表现出抗热表型。(8)对该突变体进行图位克隆,发现突变位点位于一号染色体上,下一步验证该突变基因,进行互补验证。该突变体具有较强的非生物胁迫抗性,为进一步研究植物与高温胁迫的关系提供了依据。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945.78;Q943.2

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