调控水稻穗发芽转录因子OsHRT3的功能分析

Seed germination in the ear or panicle, usually under high humidity conditions before harvest, is termed as pre-harvest sprouting (PHS) or vivipary. PHS of cereal grains not only causes reduction of grain yield but also grain quality, thus resulting in significant economic loss. The molecular mechanisms of PHS in rice are poorly understood because only few PHS mutants were identified. Previously, we isolated a high temperature responsive transcript factor, OsHRT3. The impairment of this gene leads to PHS in rice. OsHRT3 is a seed specific transcript factor which can be greatly induced by high temperature and ABA but slightly reduced by high humidity and GA. Over-expression of OsHRT3 results in GA deficient phenotypes such as dwarf, late flowering and less tillerings. The deficient phenotypes can be rescued by application of GA. Furthermore, OsHRT3 can suppress the reporter genes’ expression driven by the promoter of key enzymes involved in GA biosynthesis, such as GA20ox and GA3ox. The ABA sensitivity of hrt3 mutant is much weaker than WT. These results indicated that OsHRT3 might be an important regulator of rice seed dormancy through mediating cross-talk between ABA and GA pathways. In this study, we are going to characterize the biological function of OsHRT3 in seed dormancy with genetics, biochemistry and molecular biological approaches. We will screen natural variation in the genes involved in the OsHRT3 signaling pathway, and try to find favorable allelic variation which can be used for resistant pre-harvest sprouting breeding. We hope our work will provide new genetic resources for molecular breeding of rice quality.

水稻种子在成熟后期因高温高湿天气在穗上发芽，会严重影响稻米的产量和品质，造成极大的经济损失。我们实验前期获得一个灌浆期高温响应的转录因子OsHRT3，其突变体材料hrt3更易穗发芽，该基因超表达的植株表现出矮化、晚花等GA缺陷的表型，上述表型可被外源施加的GA回复。烟草瞬时表达结果显示，该基因可结合GA合成途径中的关键酶基因GA20ox和GA3ox启动子并抑制其表达。表明该基因可能通过负调控GA的合成促进种子休眠。该基因只在种子中表达，且表达受高温和ABA的强烈诱导，但受高湿和GA的抑制。本项目拟通过遗传、生化和分子生物学等方法对OsHRT3的生物学功能开展研究，构建ABA与GA参与的水稻休眠的调控网络，解析穗芽发生过程中遗传与环境的互作关系。对参与OsHRT3信号通路的各个基因进行自然变异的筛选，挖掘可用于抗穗发芽育种的有利等位变异，为水稻品质分子设计育种提供新的基因资源。

水稻种子在成熟后期常因高温高湿天气造成穗上发芽。穗发芽会严重影响稻米的产量和品质，造成极大的经济损失，但其分子机制不清。前期我们获得一个灌浆期特异表达的高温响应转录因子OsHRT3，其突变体材料hrt3更易穗发芽。该基因超表达的植株表现出矮化、晚花等GA缺陷的表型，上述表型可被外源施加的GA回复。通过检测过表达植株和野生型体内GA含量，我们发现 GA含量在过表达植株中明显减少，表明过表达OsHRT3基因确实能抑制GA的合成。qRT-PCR和双荧光素酶报告实验表明OsHRT3能够抑制GA合成关键基因GA20ox2的表达。进一步我们通过酵母单杂交和双荧光报告实验确定OsHRT3能够结合GA20ox2基因启动子位置在-992至-879bp。EMSA和ChIP实验也证实OsHRT3直接调控GA20ox2的表达。我们通过IP-MS筛选与OsHRT3相互作用的蛋白，筛选到一类TOPLESS相关转录共抑制因子蛋白（TPR1-TPR3），进一步通过BiFC和Co-IP证实TPR3可以与OsHRT3相互作用。因此这些结果表明OsHRT3通过与TOPLESS相关蛋白相互作用，形成转录抑制复合体直接抑制GA合成关键基因GA20ox2的表达从而调控种子休眠。通过本项目的研究将有助于我们了解水稻调控穗发芽的分子机理以及调控网络，并为今后培育穗发芽的优质水稻提供新种质。