人参新的mlncRNA基因HTAR在高温胁迫响应中的调控作用研究

Recently, non-coding RNA has been a research hotspot in life science. They regulate gene expression through epigenetic, transcriptional or posttranscriptional regulation. In animals, long non-coding RNAs have been reported to function in many aspects, such as growth, development and disease response and so on. However, it has just begun in plants. As an important medicinal plant, Panax gingseng is much less studied yet. We identified a novel high temperature associated mlncRNA (HTAR) gene using transcriptome analysis. The gene HTAR transcribes bidirectionally. The pair of transcripts overlaps partially to form double strand RNAs, which generates a large number of endogenous siRNAs. Some of the siRNAs are 21nt phased. In this work, we will make an intensive research on the regulatory role of HTAR invovled in high temperature response. The main work is included as follows: cloning and functional analysis of HTAR promoters bidirectionally, target genes selection using degradome analysis and cleavage site confirmation, differential expression profiles of HTAR and target genes with and without high temperature treatment by RNA-seq, cloning and functional analysis of target genes, functional analysis of HTAR bidirectional transcripts through transgenic ginseng. The results will provide basic theoretical framework for the molecular breeding of elite heat-tolerant ginseng varieties, and also facilitate the basic gene function and mechanism research of other tranditional important Chinese herbs.

非编码RNA在生命体内的功能及调控机制是目前生命科学研究的前沿领域之一。与动物相比，植物mlncRNA的研究才刚刚起步。人参是名贵中药材，但人们对其适应非生物逆境的调控机理还知之甚少。基于转录组学分析，本课题组从人参鉴定得到了一个新的对高温胁迫响应的mlncRNA基因HTAR。该基因双向转录，部分互补形成双链RNA，进而产生大量的内源性siRNA，部分按21nt相位排列。在此基础上，本项目拟深入解析HTAR参与高温胁迫响应的分子机制，主要包括以下步骤：HTAR双向转录启动子的克隆及功能研究，降解组测序筛选靶基因并验证其剪切位点，RNA-seq分析高温胁迫处理后HTAR及其靶基因的表达差异，靶基因的克隆及功能验证，转基因人参验证HTAR正反向转录本的功能等。本项目将为利用植物基因工程技术培育人参耐高温新品种，进而扩大其产业种植区域奠定了重要的理论依据。

越来越多的研究表明，非编码RNA在植物生长发育过程中发挥着重要的作用。与其他作物相比，人参的基础研究还很薄弱。本项目完成了HTAR正反向转录本序列及启动子的克隆、HTAR-siRNA靶基因的验证及克隆、转录组测序分析人参幼苗高温胁迫处理后HTAR正反向转录本及其靶基因的表达差异，建立了人参再生体系，并对根癌农杆菌介导的人参遗传转化条件进行了优化。此外，还建立了人参不定根的诱导和增殖培养体系。本项目的实施初步揭示了HTAR参与人参高温胁迫响应的调控机制，为进一步深入解析人参生长发育调控的分子机理奠定了重要的基础。