microRNA调控拟南芥光形态建成的分子机理研究

Seedling photomorphogenesis is a sophisticated developmental process that is controlled by the gene network comprising transcriptional and posttranscriptional regulation of gene expression. Studies of the gene regulation in photomorphogenic Arabidopsis are mainly focused on transcription factor-mediated gene activation or repression and the modifications or interaction of functional proteins, however, in which the regulation of microRNA (miRNA) has been less studied. Recent reports revealed that microRNA (miRNA) may participate in regulation of photomorphogenesis through modulating the transcriptome shift. Thus, the proposal is designed to explore the actions of miRNA in photomorphogenic Arabidopsis and the underlying regulatory basis. We will particular focus on three miRNAs (miR402, miR777 and miR858a) that are regulated by HY5, a key regulator of photomorphogenesis. Their expression patterns will be firstly characterized in roots, hypocotyls and cotyledons to reveal their possible roles in organ-specific photomorphogenic process. Then, transgenic plants over-expressing individual miRNA in hy5 mutant or knockout plants by CRISPR-Cas9 in wild type will be generated, and phenotypic examinations will clarify the miRNA regulation in photomorphogenic seedlings. Finally, by mRNA sequencing, transcriptomic comparisons will be used to identify differentially expressed genes in roots, hypocotyls, and cotyledons of wild type, hy5 mutant, and transgenic plants showing photomorphogenic changes. The related metabolic pathways will also be analyzed. Results from this study will help us to understand the regulation of organ-specific photomorphogenic process by miRNA, and further provide the theoretical basis for further understanding miRNA-mediated gene regulatory network in photomorphogenesis.

幼苗光形态建成是由转录和转录后水平共同调控的基因网络控制的一个复杂生长发育过程，其中基因转录调控和蛋白修饰及互作的研究较为广泛和深入，而关于miRNA调控作用的研究较少。近来研究表明microRNA（miRNA）可能是调控光形态建成的重要因子。本项目拟以作用于HY5下游的miRNAs (miR402、miR777和miR858a）为切入点，开展miRNA调控光形态建成的作用机理研究。通过分析它们在拟南芥幼苗根、下胚轴和子叶中的表达模式以理解其在器官特异性光形态发育过程中的调控作用；利用过量表达和CRISPR-Cas9基因敲除方法分析转基因幼苗光形态建成表型的变化；比较分析野生型、hy5突变体和转基因植物幼苗根、下胚轴及子叶中差异表达基因及miRNA靶基因的变化。这不仅有助于揭示miRNA调控幼苗器官特异性光形态发育的分子机制，也为深入理解光形态建成过程中miRNA基因调控网络奠定理论基础。

植物幼苗在光下从黄化状态到光形态建成的转变对于成活和后期的生长发育至关重要。大量研究表明幼苗光形态建成是由转录和转录后水平共同调控的基因网络控制的一个复杂生长发育过程，其中基因转录调控和蛋白修饰及互作的研究较为广泛和深入，而关于miRNA调控作用的研究较少。近来研究表明microRNA（miRNA）可能是调控光形态建成的重要因子。本项目拟以HY5调控的下游miRNAs (miR402、miR777、miR858和miR869）为切入点，开展miRNA调控光形态建成的作用机理研究。组织器官特异性表达分析表明这4个miRNA在拟南芥幼苗根、下胚轴和子叶中的模式各不相同，其中miR858和miR869为组成型表达。分别在光形态建成突变体hy5-215中过量表达这些miRNA，发现miR858和miR869能够回补hy5突变体表型，而miR402和miR777只能很小程度上回补hy5表型。利用STTM技术分别创制这4个miRNA的沉默突变体，在光照条件下能够模拟hy5长下胚轴的表型。这些结果表明miR858和miR869可能是调控光形态建成的重要因子。进一步对转基因过表达株系幼苗进行高通量测序分析，结果表明不同转基因株系中存在大量的差异表达基因，而在miR858和miR869转基因株系中的基因能在很大程度上回复至野生型水平，进一步说明这两个miRNA调控光形态建成的重要性。另外，在不同转基因株系中分析不同信号通路中的关键基因，表明光信号传导通路、生物钟和激素信号通路等多种途径共同调控幼苗光形态建成。这些结果为从基因转录后调控水平深入理解光形态建成的调控机制提供了理论依据，并为揭示光响应miRNA在植物生长发育过程中的功能奠定了基础。