龙眼体胚发生过程中lmiRNA调控靶基因甲基化的作用机制研究

lmiRNAs (24 nt miRNA) mediated target genes expression by DNA methylation pathway plays an important regulatory role in plant development, which has become a new research hotspot in epigenetics, but its molecular mechanism is still unclear. In this project,using the system of longan somatic embryogenesis (SE), from the stand point of miRNA processing related proteins, the molecular mechanism of lmiRNAs mediated target genes expression by DNA methylation during SE in longan will be studied. Based on the previous work, we plan to isolate lmiRNA processing related-genes of DCL3, AGO4, and DRM2, to perform in situ hybridization analysis of these genes during longan somatic embryo development, to construct dcl3, ago4 and drm2 mutations in embryogenic cell line, to study the influence of different mutants on longan somatic embryo morphogenesis, to comparative analysis of DNA methylation, small RNA and gene expressions in different mutants lines by bisulfite-sequencing and Solexa sequencing for confirming the roles of DCL3, AGO4 and DRM2 in lmiRNA mediated DNA methylation pathway, to further verifie of lmiRNA mediated targets methylation in vitro. Finally, the genome-wide DNA methylation profiles will be provied, and the molecular mechanism of lmiRNAs mediated target genes expression by DNA methylation pathway during longan SE will be systematically clarified. And these results will further provide scientific evidences for regulating the in vivo and in vitro embryogenesis in longan.

lmiRNA（24nt miRNA）介导靶标甲基化在植物发育中的作用已成为目前表观遗传学研究的新热点，但其作用机制却未清楚。本研究利用龙眼体胚发生模式系统，从lmiRNA加工合成途径中相关蛋白质入手，研究龙眼体胚中lmiRNA介导靶标甲基化的作用机制。在前期工作基础上，进行lmiRNA加工合成途径中DCL3、AGO4、DRM2在龙眼体胚中的定位和定量；构建龙眼dcl3、ago4和drm2等突变胚性细胞系；研究不同突变体对龙眼体胚形态建成的影响；比较分析龙眼不同突变胚性细胞中的DNA甲基化水平及类型、sRNA种类、长度分布及表达和转录组中目的基因的转录水平，确认DCL3、AGO4和DRM2在lmiRNA介导靶标甲基化通路中的作用；筛选并体外验证lmiRNA介导的靶标甲基化。最终绘制龙眼体胚中全基因组甲基化图谱和lmiRNA介导靶标甲基化的通路框架图，为龙眼体胚发生的分子机制提供参考。

本研究利用龙眼体胚发生模式系统，研究龙眼体胚中lmiRNA介导靶标甲基化的作用机制。通过研究，我们确认了DCL3、AGO4、DRM2均定位于细胞核。定量表达分析表明，龙眼胚胎发生过程中DCL3基因呈下调表达趋势，AGO4基因在GE中转录水平显著上调，DRM2 基因表达呈先升后降趋势。dcl3、ago4、drm2突变胚性细胞系结果表明，抑制 DCL3和 AGO4基因表达在一定时间范围内加快了体胚发育进程。抑制DRM2，胚性愈伤到胚性紧实结构的时间延长，但到球形胚的过程却明显加快。龙眼体胚早期甲基化图谱结果表明，龙眼体胚发生早期是DNA甲基化减少的过程；DNA去甲基化可能通过调节不同途径（玉米素生物合成、脂肪酸生物合成、昼夜节律和线粒体自噬等）的基因促进龙眼体胚发生。龙眼体胚发生各阶段sRNA种类和数量分析表明，sRNA序列长度在18~30 nt之间，并以24 nt和21 nt的miRNA调控为主，体胚发生早期各阶段主要受24 nt miRNA转录调控。各阶段显著差异表达的miRNA及其靶基因定量表达表明，miR403-3p可能靶向调控甲基转移酶（Dlo_014588.1）；miR408-3p可能的靶向SERK4来调控GE形态建成，miR390a-5p可能靶向调控BR1和SERK1来调控GE形态建成；miR397a和miR397a-3p可能负调控LAC4-2来调控龙眼胚性发生，而在GE形成阶段miR397a则可能负调控LAC17-2、LAC17-5来调控龙眼体胚发生早期形态建成。我们的研究表明，龙眼体胚中lmiRNA介导靶基因甲基化可能通过两个途径，一是通过靶向DNA甲基转移酶从而改变DNA甲基化状态；二是lmiRNA可能影响体胚关键标记基因如SERK1、SERK4等基因从而参与龙眼体胚发生。