玉米干旱响应的自然反义转录本鉴定及其启动子克隆与功能分析

Drought stress affects the water relations of plants at cellular, tissue and organ levels, causing damage and adaptation reactions. Plants response and adapt to drought stress by the induction of various morphological, biochemical, physiological and epigenetic responses including DNA methylation, histone modification, chromatin remodeling and non-coding RNA, regulating gene expressions in the transcriptional and post transcriptional levels. In privous study, a recombinant inbred line (RIL) population and core association population with highly dense SSR and SNP maps were created for drought tolerance study. To identify drought response natural antisense transcripts in maize, strand specific RNA-seq will be performed on the biparental inbreds and two recombinant inbred lines from RIL with drought-tolerant and drought-sensitive characters. The mechanism of NAT expression in response to drought tolerance and regulations between NAT and corresponding sense transcript will be revealed. Potential and key NAT screened by association analysis in linkage and core association mapping populations will be selected to verify their gene expression and DNA methylation variation in different development stages and tissues of drought-tolerant and drought-sensitive inbreds under two water regimes. Then, biological function for promotors of key NAT will be validated through genetic transformation. Furthermore, the correlation and causations of NAT expression and drought inducible promotor with different DNA methylation level will be discussed. The results of project will provide new clue to elucidate molecular mechanism response to drought stress, and lay a foundation for molecular breeding on maize.

干旱胁迫下玉米会通过信号传递，产生DNA甲基化、组蛋白共价修饰、染色体重塑和非编码RNA等表观遗传变异，从转录和转录后水平调控基因表达，调节对水分的吸收、利用，以适应胁迫环境。本研究对耐旱RIL群体两亲本以及筛选的强耐旱、干旱敏感重组自交系进行链特异转录组测序，发掘干旱胁迫应答的自然反义转录本（Natural antisense transcript，NAT）及其在不同耐旱性亲代与子代间表达差异，揭示玉米干旱胁迫应答NAT表达的遗传模式及其与正义转录本表达的调控关系；通过分析NAT基因区域遗传变异在连锁与核心关联群体中与玉米耐旱性状的相关性，筛选干旱应答关键NAT，针对这些基因进行启动子克隆与功能分析，探索NAT和正义转录本启动子功能与其DNA甲基化、时空表达的相互关系，揭示干旱诱导的NAT表达及调控机制，为阐明玉米耐旱形成的分子遗传机理提供新的线索，同时为玉米抗逆分子育种提供理论指导。

玉米在干旱胁迫下通过信号传递，产生DNA甲基化、组蛋白共价修饰、染色体重塑和非编码RNA等表观遗传变异，从转录和转录后水平调控基因表达，调节对水分的吸收、利用，以适应胁迫环境。本研究对耐旱RIL群体两亲本以及筛选的强耐旱、干旱敏感重组自交系进行链特异转录组测序，共鉴定出1769对正义/反义转录本（Natural antisense transcript，NAT），分析了不同玉米自交系间、亲代与子代间自然反义转录本表达异同及遗传关系；对干旱应答关键基因ZmLOX1、ZmWRKY30正义/自然反义转录本进行表达特征和启动子DNA甲基化水平分析，揭示了干旱胁迫应答的自然反义转录本表达与其对应启动子区域DNA甲基化水平间的相互关系。此外，对ZmWRKY30自然反义转录本对应基因启动子及其正义/自然反义转录本cDNA全长克隆与功能分析，揭示ZmWRKY30反义转录本对干旱胁迫的答应机制及其与正义转录本间的调控关系，为阐明玉米耐旱形成的分子遗传机理提供新的线索，同时为玉米抗逆分子育种提供理论指导。