SMG1及其互作蛋白SIP1调控水稻籽粒大小的分子机理研究

Rice is one of the most important cereal crops worldwide. Rice grain size, which is specified by grain length, grain width and grain thickness, is one of the main factors of rice yield. Recent studies have isolated and characterized several factors for grain size in rice, but the genetic and molecular mechanisms of these factors are almost unknown. We have previously shown that the smg1 mutant exhibits small and light grain. SMG1 encodes OsMKK4, and overexpression of SMG1 produces large grains. To understand the molecular mechanisms of SMG1, we identified SMG1 interacting proteins (SIPs). The transgenic plants that down-regulated the expression of SIP1 by RNAi technology have small seeds than wild type, suggesting that SIP1 involved in the regulation of rice seed size. Genetic analyses indicate that SIP1 and SMG1 act in a common pathway to control grain size. Interactions between SIP1 and SMG1 were observed in living cells by using bimolecular fluorescence complementation. In this proposal, a combination of genetic, molecular biology, cell biology and biochemical approaches will be used to study expression pattern and subcellular localization of SIP1, reveal genetic interactions between SMG1 and SIP1, verify the physical interaction between SIP1 and SMG1 in vitro and in vivo, and screen the interaction proteins of SIP1. This study will elucidate the molecular mechanisms of SMG1 and SIP1 in grain size control, build up a genetic framework for SMG1-SIP1-mediated control of grain size, and also discover genes for engineering high yield crops.

水稻是重要的粮食作物。籽粒大小是重要的产量性状。近年来克隆了一些控制水稻籽粒大小的基因，但对其调控的分子机制尚不清楚。我们在以前的研究中发现了smg1小粒突变体，SMG1编码OsMKK4。过表达SMG1产生大的籽粒。为了揭示SMG1调控籽粒大小的功能，筛选得到了其相互作用的蛋白SIPs。其中SIP1的RNAi转基因植株籽粒变小，与smg1突变体表型相似。遗传分析表明SIP1与SMG1处于同一遗传途径。双分子荧光互补表明SIP1与SMG1在体内相互作用。本项目拟运用遗传学、分子生物学、细胞生物学和生物化学等方法，通过分析SIP1的表达模式和亚细胞定位、SIP1与SMG1的遗传关系以及SIP1与SMG1的相互作用、筛选鉴定SIP1互作蛋白及其在籽粒大小的功能研究，阐明SMG1和SIP1调控籽粒大小的分子机制，初步建立SMG1-SIP1粒型调控网络，该研究也将为水稻高产提供理论依据和基因资源。

水稻是世界上重要的粮食作物之一。水稻产量性状与粒型息息相关。目前已经克隆了一些水稻籽粒大小的调控基因，但对其分子机制知之甚少。我们实验室在先前的研究中分离了小粒基因SMG1，SMG1编码OsMKK4。MAPK信号途径包含MAPKKK、MAPKK和MAPK。为了寻找OsMKK4的上游激酶和下游蛋白，揭示SMG1调控水稻籽粒大小的功能，筛选得到了OsMKK4的互作蛋白OsMAPK6。酵母双杂交和双分子荧光互补等实验表明OsMAPK6可以与OsMKK4在体内体外相互作用。进一步研究表明OsMAPK6的显性负调控（DN-OsMAPK6）转基因植株种子变小，而过表达有持续激酶活性的OsMAPK6（CA-OsMAPK6）可以显著增加籽粒大小，证明OsMAPK6参与水稻籽粒大小的调控。磷酸化实验表明OsMKK4-DD可以在体外磷酸化OsMAPK6。遗传分析表明OsMAPK6与OsMKK4处于同一遗传途径。进一步通过酵母双杂交筛选到了与OsMAPK6相互作用的蛋白OsMKP1。OsMKP1编码一个丝裂原活化蛋白激酶磷酸酶，可以将磷酸化的MAPK去磷酸化。体外酵母双杂交和pull-down实验以及体内的双分子荧光互补和免疫共沉淀实验均证明OsMAPK6可以与OsMKP1相互作用。磷酸化实验表明OsMKP1能够将磷酸化的OsMAPK6去磷酸化，使其失去活性。同时，我们还发现并克隆了OsMKK4的上游基因OsMKKK10。生化分析表明OsMKKK10可以与OsMKK4在体内和体外相互作用，且OsMKKK10能够磷酸化OsMKK4。遗传分析显示OsMKKK10与OsMKK4和OsMAPK6作用于相同的遗传通路来调控水稻籽粒大小。总之，本研究建立了OsMKKK10-OsMKK4-OsMAPK6信号通路调控水稻籽粒大小的遗传网络，为水稻高产提供了理论依据和基因资源。