WRKY72调控水稻种子休眠的机制研究

As one of the most important phytohormones，abscisic acid (ABA) regulated the seed dormancy in plants. Our previous studies have shown that transcription factor WRKY72 was significantly induced by ABA and highly expressed in seed. Moreover, the seeds of WRKY72 over-expression transgenic rice emerged more sensitive to ABA stress compared with wild-type, that was seed dormancy became deepened and seed germination was retarded under ABA treatment, suggesting WRKY72 may be involved in ABA-mediated seed dormancy and germination processes. This project aims to clarify the biological function of WRKY72 in rice seed dormancy, and to explore the new molecular ABA-mediated seed dormancy signaling pathway in which WRKY72 participates. Analysis of tissue location and expression patterns response to various stresses indicated that WRKY72 may act as a positive regulator involved in ABA signal pathway. Through generating WRKY72 over-expression, wrky72-crispr mutant and function recovery transgenic lines, the sensitive of seed germination and seedling growth to ABA stress as well as physiological determination will be compared among the transgenic plants and wild-type, thus to interpret the biological function of WRKY72 in seed dormancy. On the other hand, comparative transcriptomic analysis of germinated seeds of WRKY72 over-expression lines under ABA and no ABA treatment will be performed, and the target genes involved in ABA signal pathway will be screened and detected whether direct regulated by WRKY72. Given the importance of abscisic acid signal network in rice seed dormancy and germination processes, the outcome of this project will not only expand the molecular mechanism of ABA-mediated seed dormancy, but also provide a theoretical basis for molecular breeding.

脱落酸(ABA)是调控水稻种子休眠的重要激素之一。前期研究结果显示转录因子WRKY72受ABA显著诱导，在种子里具有较高的表达量，其超表达材料的种子萌发与野生型相比对ABA的敏感性增加，表明WRKY72可能参与ABA调控的种子休眠过程。组织定位和各种逆境响应表达模式分析暗示WRKY72可能作为一个正调控因子参与ABA信号途径；通过发展 WRKY72超表达、wrky72-crispr突变体以及突变体功能回复材料，比较种子萌发及幼苗生长对ABA的敏感性及该过程中的生理测定在转基因植株与野生型间的差异，探究WRKY72在调控种子休眠过程中的生物学功能。另一方面，本项目还分别对WRKY72超表达株系受ABA处理和不受ABA处理的萌发后种子进行转录组学分析，筛选出受WRKY72直接调控的ABA信号通路的下游靶基因，发现WRKY72参与ABA调控种子休眠的新信号途径，为分子育种提供一定的理论基础。

种子萌发是影响谷物产量的重要农艺性状，受到各类植物激素和环境因素的精细调控。本研究首先分析了WRKY72在水稻的不同组织、种子不同发育阶段以及外施脱落酸（Abscisic acid，ABA）和（Gibberellic acid，GA）处理下的表达模式，结果表明WRKY72在水稻种子成熟并逐渐休眠的过程中具有较高的表达水平，并且其受ABA的诱导而受GA的抑制，表明WRKY72可能分别正调控ABA或负调控GA介导的种子休眠过程。WRKY72超表达材料的种子萌发延迟，而突变体材料种子的萌发与野生型的差异不大，可能存在着基因冗余现象。通过WRKY72超表达材料的萌发胚转录组学分析发现一系列差异表达基因参与调控GA的生物合成和种子萌发过程。通过荧光定量PCR，体内和体外DNA与蛋白互作实验发现WRKY72直接结合GA合成途径抑制因子富亮氨酸重复受体激酶LRK1的启动子并激活后者的转录。同时OxWRKY72萌发胚的GA含量下降，并且外施GA可恢复OxWRKY72的种子萌发和幼苗生长至野生型的水平。进一步研究发现WRKY72通过激活LRK1的表达进而抑制赤霉素合成关键酶OsKO2的表达，最终降低内源GA水平而抑制种子萌发，而外施OsKO2的产物贝壳杉烯酸同样可以恢复OxWRKY72的种子萌发和幼苗生长至野生型的水平。本项目首次揭示了GA合成途径的负调控通路“ WRKY72-LRK1-OsKO2”抑制水稻种子萌发的作用机制。