茶树响应低温胁迫的microRNA发掘及其调控机制研究

Cold stress is an important limiting factor of tea plant cultivation for high yield and high quality in Chinese most tea-growing areas, which are particularly obvious with global climate deteriorating in recent years. The studies on molecular mechanism of tea plant cold-tolerance mainly focused on the screening and expression characteristics of functional genes but there were a few works in regulation of post-transcription. In this project, on the base of tea plant genome data, combined with bioinformatics, transcriptome deep sequencing, small RNA Solexa deep sequencing, miRNA microarray hybridization, gene expression profile and quantitative real-time PCR technique were utilized to screen and identify miRNAs responsive to low-temperature stress as well as study their functions through transgenic tobacco over expression with cold-resistant tea varieties as materials. Differential expression and interrelation of miRNAs and targeted genes were investigated under ABA, dehydration and low-temperature treatments whilst expression pattern differentia of miRNAs and targeted genes were compared in tea varieties, which was helpful for elucidating the function and expression characteristics of miRNAs and targeted genes related to cold-tolerance of tea plant and indicating the regulation mechanism responsive to low-temperature stress at post-transcriptome level. These researches could provide a new theory support for breeding cold-resistant tea varieties and screening stress-resistant genes as well as had an important significance for further realizing biological function of miRNAs related to stress-tolerance.

冻害是我国大部分茶区茶树优质高产栽培中重要限制因子，随着全球气候异常，近年表现尤为突出。茶树抗寒分子机制主要集中在功能基因发掘与表达特征方面的研究，而有关转录后调控研究甚少。本项目以耐寒茶树品种为材料，基于独有的茶树基因组数据，应用转录组深度测序、小RNA Solexa深度测序、miRNA芯片杂交、基因表达谱芯片和荧光定量PCR技术，并结合生物信息学手段，发掘与鉴定响应低温胁迫的茶树miRNAs，进行过表达分析，并采用降解组测序鉴定靶向基因。研究ABA、脱水和低温三种处理下miRNAs及靶向基因的表达差异与相互关系，比较品种间miRNA及靶向基因表达模式差异。这将有助于明确茶树中抗寒相关miRNAs及其靶基因功能与表达模式，在转录后水平上揭示出茶树响应低温胁迫的调控机制，为茶树抗寒品种选育、抗逆基因发掘提供新的理论支持，也对进一步认识植物抗逆miRNAs的功能具有重要意义。

茶树是一种多年生植物，主要分布于热带和亚热带地区。低温是限制茶树生长、产量和品质的主要因素。项目系统研究了茶树响应低温胁迫miRNA及其调控机理。通过高通量测序发现143个保守miRNA，属于25个家族，鉴定了131新miRNA。通过miRNA芯片，获得低温胁迫下茶树miRNA差异表达谱，其中80个miRNA显著差异表达。利用降解组测序，鉴定了149 miRNA 靶向的562目标基因，GO分析表明这些miRNA 可能在响应茶树低温胁迫中起着重要功能，这些miRNA主要通过动态调控AGO、转录因子、抗氧化酶，与渗透物质代谢的相关蛋白，抗性蛋白和植物激素的转录表达，从而形成比较复杂的miRNA介导的茶树抗寒基因调控网络。上述研究结果可为进一步了解茶树抗寒分子机理奠定基础。