水稻闭颖相关基因OsHRT2的功能研究

Rice floret opening is initiated by the swelling of a pair of lodicules which lying at the base of ovary. Swelled lodicules lever away the lemma and palea, at the same time, the filaments quickly elongate which allows anthers to emerge for pollination. Previous studies focused on the histomorphology and physiology changes of lodicule cells during anthesis. However, the molecular mechanism of rice anthesis remains obscure, as lacking of proper genetic materials. Most of the cleistogamous mutants have been identified are lodiculeless and unsuitable for further genetic study. Here, we identified a novel cleistogamous mutant hrt2 in rice. hrt2 mutant has normal lodicules and other floret organs and has no problem to propagate. Our preliminary data showed that OsHRT2 highly expressed in panicles, especially in lodicules. The expression level of OsHRT2 can be induced by high temperature and exogenous MeJA. In this study, we are going to characterize the biological function of OsHRT2 in rice anthesis with genetics, biochemistry and molecular biological approaches. We hope our work will lead new insights into the molecular mechanisms of floret opening in rice and help breeders resolve the problems in agricultural production, such as asynchronous floret opening in hybrid breeding and gene flow in transgenic rice growth.

水稻颖花开放是由分布于子房基部的一对浆片吸水膨胀启动的，浆片膨胀撑开内稃和外稃，同时花丝伸长花药外露开裂散粉最终完成授粉。通过对水稻颖花开放前后浆片的组织形态，细胞结构及水势调节相关物质的变化等方面的研究使人们对开颖过程中浆片细胞的生理生化过程及行为模式有了初步的认识，但浆片膨胀介导的水稻开颖的分子机制的研究却依然匮乏，主要原因之一是缺乏合适的闭颖突变体。目前已报道的的闭颖突变体均有比较严重的花器官发育缺陷，难以适合后期水稻开颖的分子机制研究的开展。本项目前期鉴定的闭颖突变体hrt2浆片发育正常，能结实并获得子代，该突变基因编码一个高温响应的转录因子，能在小穗及浆片中特异性高表达。本项目将以OsHRT2为切入点，深入研究水稻开颖的信号通路及其调控机制。为解决水稻杂交制种中花时不遇及防范转基因水稻基因漂流等重要农业生产问题提供理论基础和基因资源。

水稻颖花开放是一个复杂的过程。水稻颖壳的张开是由浆片膨胀驱动的，浆片膨胀后，推开内稃和外稃之间的勾合，颖壳打开。本研究以闭颖自花授粉的水稻突变体hrt2（heat responsive transcription factor 2）为材料研究水稻开颖机制。组织切片结果表明hrt2颖花浆片相比野生型华阳瘦薄，开颖时华阳的浆片膨胀后外缘嵌入內、外稃之间的缝隙撑开它们之间的钩合，颖花开放；而hrt2由于浆片瘦小膨胀后外缘只有微小部分延伸入缝隙而不足以撑开钩合，导致闭颖。实时荧光定量PCR表明相比野生型，hrt2开花前与开花时颖花中OsJAZs基因的表达减弱，而在OsHRT2过表达植株中则显著上调，表明OsHRT2可能通过茉莉酸途径调控水稻开花,OsHRT2正调控OsJAZs的表达。双分子荧光报告试验表明OsHRT2能提高OsJAZ11启动子驱动的荧光素酶的转录，表明OsJAZs基因可能就是OsHRT2的调控目标。同样利用双荧光素酶报告实验，OsHRT2还能在水稻原生质体中促进扩张蛋白，细胞纤维素酶等基因启动子的转录活性，这些基因的表达利于细胞壁的松弛，有助于浆片细胞的增大和膨胀。通过对OsHRT2进行过量表达，发现OsHRT2的过量表达植株表现出叶片下垂的BR增强表型，检测BR合成相关基因的表达显示过表达植株中部分BR合成基因上调。通过双分子荧光互补试验，在烟草中表明OsHRT2与BR信号传递相关基因OsGSK2、OsDLT存在相互作用。因此OsHRT2可能通过与OsGSK2、OsDLT相互作用参与BR途径，调控浆片细胞，器官的大小。综上所述，课题组总结了OsHRT2的功能模式。OsHRT2在水稻幼穗发育早期，通过参与调控BR的积累，促进浆片组织的增大；在临近开花时，通过增强茉莉酸途径促进浆片细胞的代谢，渗透调节物质的积累增大浆片细胞的渗透势，促进浆片从小穗轴吸水；同时，OsHRT2促进扩张蛋白，纤维素酶等基因的转录，促进浆片细胞壁松弛；两条路径协同作用促进水稻开颖。