东乡野生稻OsEBS基因增加穗粒数以及提高生物量的机理

Genetic improvement is the most important impetus to crop yield and quality. Molecular Design Breeding (MDB)，which mainly counts on the understanding of key genes functions, has become an effective way for genetic improvement right now, so the studies on the molecular basis of yield and important agronomic traits is vital in rice.OsEBS, a gene involved in enhancing plant biomass and spikelet number, has been identified and characterized from Dongxiang wild rice (O.rufipogon Griff) in our study previously. An alignment of amino acid sequence of OsEBS indicated it is homology to Bip (luminal binding protein), which is a type of Hsp70 located in the endoplasmic reticulum. Compared with the three conserved domains in HSP70, OsEBS contained only an ATPase domain with lacking the peptide-binding domain and the C-terminus. Subcellular localization result showed that it was localized to the cytoplasm rather than the endoplasmic reticulum. In addition, there was no HSEs cis-element in the promoter of OsEBS, and which expression wasn't induced under heat shock. So, we can get the conclusion that OsEBS with a conserved ATPase domain is a variant HSP70, and has obtained a new function different from molecular chaperone. In this project, more detail of OsEBS function will be identified. By yeast two-hybrid system, the interacting genes downstream OsEBS will be detected, and the molecular mechanism of OsEBS on controlling grain number per spike and biomass will be revealed; In addition, by analyzing genomic structure variation, we will clarify the evolutionary relationship of OsEBS gene between monocots and dicots, wild rice and cultivated rice. This study will provide an important element for the Molecular Design Breeding, moreover, supply gene resources and theoretical basis to the super rice breeding in China.

遗传改良是提升作物产量和品质的最重要推动力，如今分子设计育种逐渐成为遗传改良的一种有效途径，其核心是对关键基因功能的认识，因此水稻产量及重要农艺性状基因的分子基础研究至关重要。我们从东乡野生稻(O.rufipogon Griff)中克隆了一个增加穗粒数和提高生物量的基因OsEBS。该基因与HSP70-Bip蛋白同源，但只含有N端的ATPase结构域，定位在细胞膜和细胞质中，是一个由HSP70变异而来的新基因。转基因研究表明，OsEBS在单双子叶植物中具有保守的生物学功能。本研究将进一步分析OsEBS的功能，利用酵母双杂等方法找出其下游互作基因，揭示OsEBS控制穗粒数和生物量的分子机制；通过基因组结构分析，剖析OsEBS在我国水稻品种中的遗传变异，阐明OsEBS在单双子叶植物间、野栽间及品种间的进化关系。为我国新兴起的品种分子设计提供重要的元件，也为我国超级稻育种提供基因资源和理论依据。

资源匮乏是造成当前超高产水稻育种徘徊不前的根本原因之一。普通野生稻（Oryza rufipogon Griff.）是亚洲栽培稻(O.sativa L.)的祖先种，从野生稻中发掘栽培稻中已丢失或削弱了的优异基因，对我国水稻育种研究具有特殊的意义。本研究从江西东乡普通野生稻中克隆到一个提高生物量及产量的基因OsEBS，并开展了相关特征研究和进化分析，获得以下主要研究结果：. 1.细胞学分析表明, OsEBS生物量的提高是细胞数目增加的结果。氨基酸序列比对发现, OsEBS与定位于内质网的HSP70-Bip蛋白同源。与具有三个保守结构域的HSP70相比，OsEBS蛋白缺失C端的底物结合域和末端定位信号, 只含有N端的ATPase结构域，定位在细胞质中。进一步分析发现, OsEBS基因上游调控区内不含HSP70所具有的的热激活元件HSE, OsEBS基因的表达也不能被热激诱导。因此推测OsEBS是一个由HSP70变异而来的新基因，产生了不同于典型分子伴侣的新功能。. 2.通过分析HSP70蛋白家族的进化关系以及相应的蛋白质结构域发现，OsEBS在丢失了底物结合域和C末端后，氨基酸序列变异加速；进一步系统进化分析发现类似于OsEBS的结构域丢失事件是禾本科植物特有的。推测该结构域丢失事件发生在单子叶植物和双子叶植物分化后，并且保守存在于多种禾本科植物中。. 3.利用酵母双杂交系统筛选到一个生长素响应因子ABP1与OsEBS互作。已有实验证明ABP1在细胞生长调控中甚至是细胞对生长素的敏感性上起作用。本研究中，生长素含量检测发现，渗入系BIL112的生长素含量极显著的高于对照桂朝二号。由此推测OsEBS极有可能通过与ABP1的互作，影响生长素响应基因的表达，调控细胞增长，最终提高生物量和穗粒数。.