MIR396对水稻小穗发育的调控机理

Stigma exsertion is regulated by hull closure which is determined by the development state of floral organs. Although the development of rice floral organs have been reported, the regulation mechanism mediated by multiple genes is poorly understood.It is important in theory and hybrid seed production to reveal the mechanism of hull closure. OsMIR396 gene was cloned previously in our group and overexpressed in rice. Several transgenic rice lines were selected. The phenotypical analysis indicated that overexpressed lines showed longer glume and opened-hull. Based on the bioinformatic prediction, we have screened one of the miR396 target gene OGP6. This project will focus on how the module consisting of miR396, OGP6 and its target gene to regulate the development of lemma and palea. The specific research include: (1) the expression pattern of OsMIR396 and OGP6 will be checked to confirm their spatial and temporal distribution, (2) phenotypical analysis of OsmiR396- and OGP6-overexpressed transgenic lines and ogp6 mutant line, (3) the screening of OGP intereaction proteins by Y2H and their biochemical characterization and screening of downstream regulated genes of OGP6, (4) the regulation of transgenes on glume developmented. The scientific objectives is to reveal the molecular mechanism of the glume development and open-hull mediated by mutiple genes networks. The feature of this project is that how a multi-gene molecular network control open-hull. Our preliminary findings have built the basic framework and this project is expected to reveal networks of genes control the developmental mechanisms of rice glume.

水稻花器官颖片的开闭控制柱头外露，而这一过程受其发育状态的直接影响。虽然水稻颖片发育相关基因已有报道，但多基因参与的调控机制知之甚少。揭示颖片开闭机制在理论上和杂交制种中具有重要意义。本项目将基于本实验室前期的miR396超表达水稻表现出护颖明显增长、颖壳开裂的稳定表型，重点研究该miR396与预测的可能靶基因OGP6及其调控基因所组成的分子模块对颖片发育的调控机制。研究内容包括这些基因超表达株系及其突变体的遗传学分析、转基因植物与突变体颖片发育的细胞学分析、OGP基因家族表达模式与生化特性，OGP互作蛋白筛选与鉴定、以及转基因植物颖片发育的生理调节。科学目标是揭示颖片发育基因调控网络的分子机制，特色是系统研究多基因协同控制开颖表型的分子网络。前期研究结果初步构筑了这些基因作用模式的基本框架，有望揭示多基因网络控制颖片发育机制。

为了阐明OsmiR396d过表达株系颖壳扣合异常、长护颖等表型产生的可能原因，我们在组织形态学以及分子生物学水平上进行了进一步的探索。首先我们对OsmiR396d过表达株系颖花进行了石蜡切片及扫描电镜的观察。我们发现在水稻颖花发育的早期，OsmiR396d过表达株系颖花就已呈现出异常花原基以及颖壳扣合异常的表型了。开颖，即开花，一般是由浆片吸水膨胀将内外稃片推开所导致的。我们推测OsmiR396d过表达株系颖花扣合异常的表型可能是由于异常的花内器官，或者异常的细胞分裂导致内外稃片不能正常扣合而引起的。通过扫描电镜观察，我们发现OsmiR396d过表达株系颖花长护颖的表面结构已经不同于野生型护颖的表面结构，而是更加接近野生型内外稃片的表面结构。.为了在分子水平上阐明OsmiR396d过表达株系颖花异常的可能机理，我们对OsmiR396d过表达株系及其对应野生型水稻幼穗组织进行了基因表达谱的芯片分析。在该芯片中，我们发现了一个感兴趣的基因，即OsJMJ706基因，该基因在OsmiR396d过表达株系中表达下调。OsJMJ706基因编码一个H3K9去甲基化酶，据报道该基因的突变体花器官发育异常，我们对该突变体的观察发现该突变体也呈现出颖壳扣合异常及长护颖的表型。OsCR4基因编码一个类受体激酶，据报道该基因反义表达后产生明显的颖壳扣合异常表型，但是在芯片中没有检测到该基因。OsJMJ706和OsCR4基因均在OsmiR396d过表达株系中表达下调。通过EMSA、ChIP及荧光素酶活性实验，我们发现GRF类转录因子能够结合OsJMJ706以及OsCR4基因启动子区含有GARE元件的DNA序列，并激活它们的转录。通过遗传互补实验，我们将OsJMJ706基因在OsmiR396d过表达水稻中过表达后，只能部分恢复OsmiR396d过表达水稻颖花发育异常的表型，不能恢复矮化表型，这就暗示OsJMJ706基因在OsmiR396d介导的调控水稻颖花发育过程中发挥着重要的作用。