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。

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.