拟南芥根系发育相关长链非编码RNA的系统挖掘及调控机制研究

LncRNAs (long non-coding RNAs), longer than 200 nt, are widely distributed among diverse eukaryotic species. In animals, lncRNAs could regulate the expression of specific genes transcriptionally or post-transcriptionally. The functions of these lncRNAs are implicated in many important biological processes, such as cell differentiation and immune response. However, in contrast to the research progresses in animals, we currently know little about the biological functions of the plant lncRNAs and the underlying regulatory mechanisms. Based on the published 6,480 lncRNAs and RNA-seq data in Arabidopsis (Arabidopsis thaliana), the research project proposed here starts from the identification of the highly expressed lncRNAs in specific tissues (epidermis, cortex, endodermis, quiescent center and stele) of the root tip. By utilizing T-DNA insertion mutants and by employing experimental approaches such as transgenic studies and LCM (laser capture microdissection), the involvement of specific lncRNAs in root development will be deeply investigated. For the studies on the molecular mechanisms underlying lncRNA-involved regulation of root development, two pieces of research contents are proposed: (i) the regulation of the expression of the lncRNA themselves; (ii) try to decipher the regulatory pathway "lncRNA (target mimic)--miRNA--target" implicated in root development. The accomplishment of this research project will uncover the biological role of specific lncRNAs in plant root development, and could serve as a basis for further studies on the regulatory mechanisms of plant lncRNAs.

真核生物中存在大量长度大于200 nt的非编码RNAs，称为长链非编码RNAs（long non-coding RNAs，lncRNAs）。在动物中，lncRNAs通过染色质修饰等途径实现对特定基因的转录或转录后调控，功能则涉及细胞分化、免疫等诸多重要的生物学过程。然而，我们迄今对植物lncRNAs的调控机制及生物学功能知之甚少。本项目充分利用已报道的拟南芥lncRNAs及RNA测序数据，搜寻在根尖特定组织高表达的lncRNAs。基于T-DNA插入突变体及相关转基因实验，探索特定lncRNAs与根系发育的关系。对lncRNAs参与根系发育调控机制的探讨，则从“lncRNAs自身表达调控”和“lncRNAs作为模拟靶标调控microRNAs及microRNAs下游靶基因”这两方面展开。本研究旨在揭示lncRNAs参与植物根系发育调控的生物学功能，并为lncRNAs调控机制的深入研究打下基础。

围绕长链非编码RNA对拟南芥根系发育的生物学意义及具体分子调控机制的研究，项目基本按照原定的三个科学问题开展了较为系统、深入的探索性研究工作。主要包括：1、根尖组织特异高表达lncRNAs的筛选及生物学功能研究。最终在拟南芥根尖表皮组织中发现141条特异高表达的lncRNAs，在皮层组织中发现369条特异高表达的lncRNAs，在内皮层组织中发现243条特异高表达的lncRNAs。我们正通过T-DNA插入突变体的表型鉴定、转基因回复、超表达等实验研究个别lncRNAs对拟南芥根系发育的生物学功能。2、LncRNAs相关调控机制及生物学功能的揭示。发现了一类依赖于RDR2和DCL3活性，从lncRNA前体加工而来的sRNAs。不同于以往的报道，这些lncRNA前体由RNA Pol II转录。此外，我们对模式植物拟南芥、水稻在重金属（铜、镉、砷）胁迫条件下的非编码RNA的响应及相关分子调控机制展开了研究。3、miRNA等sRNAs的系统挖掘以及新调控模式的研究。开发了一种用于系统挖掘植物miRNA的生物信息学软件PmiRDiscVali；基于miRNA前体加工过程中产生的降解信号，该软件能够对预测miRNAs做出可靠性评判。提出了研究植物细胞核中miRNA/sRNA—target互作的研究方法，为后续sRNA—lncRNA互作调控研究打下基础。