油菜素内酯调控水稻籽粒大小的分子机制

Phytohormone brassinosteroid (BR) has significant function in regulation of rice grain size. So far, most of the identified rice BR mutants or transgenic plants present grain size alterations. BR was generally thought to positively regulate grain size. However, except for those known genes involved in primary BR signaling pathway, we have also identified a number of BR deficient mutants which have enlarged seeds, and the enhanced BR signaling mutants which have decreased grain size. These data suggest that there should exist one or several branched pathways downstream of BR primary signaling pathway which are specialized in regulation of rice grain size. In this porposed project, we will combine grain size selection and BR sensitivity test to carry out an extensive screening using different systems. First, we will identify the grain size mutants directly from our rice mutant library, then test their BR sensitivities for a secondary screening. Second, we will make the suppressor screening for the dlt mutant, corresponding gene of which is the most downstream component of BR signaling pathway. Third, we will perform a yeast two-hybrid screening for identification of substrates of GSK2, a GSK3-like kinase that is also a downstream component of BR primary signaling pathway (DLT is one of GSK2 target). Last, we will characterize the involvement of those identified grain size genes in BR responses through examing the BR sensitivity of their transgenic plants. These various screens will lead to discovery of the potential genes specifically involved in both grain size regulation and downstream BR signal transduction. Further studies on these genes function as well as their regulatory relationship among each other will bring deep insight into the mechanism of BR regulation on rice grain size, thus lay a solid foundation for rice yield improvement.

油菜素内酯（BR）在调控水稻籽粒大小过程中具有重要功能，绝大部分已鉴定的水稻BR突变体或转基因株系都表现有籽粒大小的变化。一般认为BR对籽粒大小具有促进功能，然而我们前期工作发现，除了BR主信号通路上的基因之外，许多导致BR缺陷的突变体或转基因株系其籽粒显著增大，反之，对BR超敏感的突变体籽粒却明显减小，说明BR对水稻籽粒大小的调控在BR信号通路下游具有特异支路。本项目拟结合籽粒大小表型与BR敏感性测试对突变体与水稻基因进行多种筛选，包括：1）突变体库直接筛选；2）BR信号通路下游基因DLT突变体抑制子筛选；3）BR主信号通路末端组分GSK2激酶底物的酵母双杂交筛选；4）已知籽粒大小QTL基因转基因株系的BR敏感性筛选，以挖掘既调控籽粒大小又参与BR响应的下游特异基因。对这些基因相互调控及相互作用分子模式进行全面研究，为深入诠释BR调控水稻籽粒大小的分子机制和水稻育种改良奠定基础。

油菜素内酯（Brassinosteroid, BR）是植物内源合成的一类重要的生长促进型甾醇类激素，参与到植物生长发育的各个方面，包括细胞伸长、维管组织分化、开花、衰老、抗逆、抗病等。其信号传导途径近年来在双子叶模式植物拟南芥中取得了快速进展，并已有了比较完整的认识，然而在单子叶模式植物水稻中，其相关的研究还很零散。目前相关植物突变体和转基因工作均已显示出BR在提高植物产量中的巨大应用潜力，因此在重要的粮食作物水稻中开展有关BR信号传导及下游功能机制的研究具有重要的理论和应用意义。通过对本研究组构建的水稻突变体库进行筛选，获得一个半矮化少分蘖突变体（dwarf and low-tillering, dlt），表现出典型的BR温和突变体的多向性表型，包括矮化深绿、叶片短而宽厚、叶夹角变小、株型紧凑、第二茎节特异性缩短等。对突变基因进行图位克隆发现DLT编码植物特异的GRAS蛋白家族中的一员，突变体在此基因内部含有62bp缺失。将野生型DLT基因导入突变体中能完全互补突变体的表型，对野生型DLT进行RNA干涉得到和突变体类似的表型，而过表达DLT则引起和突变体完全相反的表型，这些转基因实验充分证明DLT控制着突变体的诸多表型。叶夹角和胚芽鞘对活性BR（Brassinolide, BL）的敏感性实验证明，dlt是一个BR不敏感的突变体。定量RT-PCR分析发现BR合成基因及DLT自身的转录水平在突变体中都明显升高，而且外源施加BL对这些基因表达的抑制程度在突变体中都明显减弱，同时突变体中多个BR响应基因的表达水平都有所变化。进一步对DLT启动子的分析发现了三个BR反应元件，并在体外用凝胶阻滞实验证明了OsBZR1能够结合此元件，说明DLT可能在转录水平上受OsBZR1直接调控，从而介导BR的信号传导过程。