lncRNA介导的epiMAP位点特异DNA甲基化调控玉米苗期耐旱性的功能与机制研究

DNA甲基化在植物干旱等逆境胁迫适应性中具有重要作用，是基因组潜在的变异来源。前期研究中，通过全基因组关联分析筛选到玉米微管相关蛋白自然反义转录本（NATMAP65）上的DNA甲基化epiMAP位点与干旱胁迫下植株存活率和根系性状显著关联，但其与所在转录本间的调控关系及他们在植株耐旱性中的功能和作用机制尚不清楚。本研究基于CRISPR/dCas9开发特异位点DNA甲基化编辑系统，构建epiMAP位点DNA去甲基化、超甲基化和NATMAP65过表达、RNAi干扰载体，并在玉米中进行遗传转化。通过对转基因植株的耐旱性及生理生化指标鉴定，揭示epiMAP与NATMAP65间的调控关系及其在干旱胁迫应答中的生物学功能；针对NATMAP65进行RNA Pull-down等实验，发掘其结合蛋白，并结合转基因植株组蛋白修饰免疫共沉淀和转录组测序，解析lncRNA介导的DNA甲基化参与干旱胁迫应答机制。

DNA methylation, as a potential source of variation, plays an important role in plant adaption to stressed environment, such as drought. In our previous study, DNA methylation sites (epiMAP) on the first exon of microtubule-associated protein 65 (MAP65) natural antisense transcript was identified to be significantly associated with plant survival rate and root-related traits under drought stress by genome-wide association analysis (GWAS). In drought-associated recombination inbred lines, the expression level of epiMAP was significantly positively correlated with plant drought tolerance. Under drought stress, the trends of DNA methylation and gene expression changes on epiMAP were opposite. However, the regulatory relationship between them and their biological functions in plant drought tolerance remain largely unknown. In this study, a site-specific DNA methylation editing system will be developed based on CRISPR/dCas9 and applied in maize. Vectors for functional research, including candidate site DNA hypermethylation and hypomethylation, overexpression and RNAi interference of NATMAP65, will be constructed. Then the genetic transformation will be performed in maize. Different types of transgenic plants will be obtained and their phenotypic variations under drought stress will be observed. In addition, the relevant physiological and biochemical experiments will be carried out to find out the differences among different transgenic plants and wild type. The regulatory relationship between epiMAP DNA methylation and NATMAP65 will be clarified. Their biological functions in drought stress response, as well as the mechanism underlying, will be revealed by RNA pull down, transcriptome sequencing of transgenic plants and ChIP-qPCR. The results from this study will unveil the functional model of lncRNA-mediated DNA methylation involved in plant drought stress response.