水稻应答高温干旱复合胁迫的miRNA分子调控机制研究

High temperature and drought are especially considered as key stress factors with high potential impact on crop yield in numerous abiotic stresses. High temperature is often accompanied with drought which produces the combined stress to crops.The combined stress will usually appear synergistic effect or antagonistic effect compared with the single stress. However,the molecular mechanism of rice response to the combined stress of high temperature and drought has not been understood.In this project,on the base of rice genome data,combined with bioinformatics,small RNA Solexa deep sequencing,miRNA micro-array hybridization and quantitative real-time PCR technique were utilized to screen and identify miRNA responsive to combined stress of high temperature and drought as well as study their functions through transgenic rice over expression with rice varieties of heat-tolerant and drought-tolerant..Differential expression and interrelation of miRNA and targeted genes were analyzed under high temperature stress,drought stress and combined stress of high temperature and drought whilst expression pattern differential of miRNA and targeted genes were compared in rice varieties.The above studies will be helpful for elucidating the function and expression characteristics of miRNA and targeted gene related to the combined stress of high temperature and drought of rice and indicating the molecular regulation mechanism responsive to the combined stress of high temperature and drought at post-transcriptome level.At the same time,these researches could provide a new theory support for breeding combined resistant of heat and drought rice varieties and screening stress-resistant genes.

在众多影响粮食产量的因素中，高温和干旱被认为是主要的环境胁迫因子。高温往往伴随着干旱对作物产生复合胁迫，与单一胁迫相比复合胁迫通常会出现协同效应或拮抗效应，而水稻对高温干旱复合胁迫响应的分子机理尚未明确。本项目以耐热耐旱水稻品种为材料，基于水稻基因组数据，借助小RNA Solexa深度测序、miRNA芯片杂交和荧光定量PCR技术，结合生物信息学手段，分离鉴定水稻响应高温干旱复合胁迫的miRNA，进行过表达分析，并采用降解组测序鉴定靶向基因。比较水稻在单一高温、单一干旱、复合高温干旱三种胁迫处理下miRNA及靶向基因的表达差异与相互关系，及其在不同品种间的差异。通过这些研究，有助于明确水稻响应高温干旱复合胁迫相关miRNA 及其靶基因功能与表达模式，进而在转录后水平上揭示出水稻响应复合胁迫的分子机制，同时为水稻耐热抗旱复合抗性育种、抗逆基因发掘提供新的理论支持。

本项目通过小RNA测序和降解组测序，结合生物信息学手段，分离鉴定了水稻响应高温干旱复合胁迫的miRNAs及其靶基因，从而在转录后水平上初步揭示出水稻响应复合胁迫的分子机制。首先，成功构建水稻small RNA文库，通过高通量测序，获得了一批保守的和新的miRNAs以及48条水稻响应高温干旱复合胁迫的miRNAs，并利用生物信息学对水稻响应高温干旱复合胁迫的miRNAs进行靶基因预测及功能富集分析。其次，成功构建了高温干温复合胁迫下水稻降解组测序文库，并进行深度测序，通过切割位点的分析对差异miRNAs的靶基因进一步鉴定，并利用荧光定量PCR技术，比较分析单一逆境与复合逆境下miRNAs、靶基因表达差异与相互关系。再次，成功构建了osa-miR162a的过表达载体，利用水稻的遗传转化体系，进行转基因实验，获得了osa-miR162a的过表达转基因植株，进一步对T1代转基因水稻和野生型水稻苗期生理指标测定，结果表明超表达 osa-miR162a 转基因水稻植株在一定的程度上提高了对逆境胁迫的耐受能力。通过对水稻响应高温干旱复合胁迫的miRNAs及其靶基因功能的分析，初步绘制了水稻响应高温干旱复合胁迫可能的miRNAs转录后调控的分子调控网络图，为水稻耐热抗旱复合抗性育种、抗逆基因发掘提供新的理论支持。项目资助发表论文1篇，待发表2篇。培养硕士生2名，其中1名已经取得硕士学位，1名在读。