水稻胚乳特异表达蛋白U496A在籽粒灌浆过程中的作用研究

OsU496A is an integral membrane protein with unknown function in Oryza sativa. OsU496A comprises an extracellular domain, a single transmembrane helix, and a cytoplasmic tail, which very similarities to integrins from animals by protein structure prediction. And there are also a few of conserved sequences between OsU496A and integrins. As a substantial middle member of the extracellular matrix-plasma membrane-cytoskeleton continuum, integrins play critical roles in a variety of biological processes, including cell proliferation, adhesion and apoptosis. The cell wall, plasma membrane (especially integral membrane proteins as a linker) and the cytoskeleton of endosperm cells have been proved respectively to be involved in the endosperm cell proliferation, development and apoptosis during grain filling of rice. But how the three of them work together in endosperm cells and regulate the endosperm development remains unknown. Our preliminary results showed that the gene encoding OsU496A was specifically expressed in the endosperm and has an important role in the grain filling of rice. In the present study, OsU496A overexpression and RNA interference (RNAi) knockdown transgenic rice lines will be created for the study on the phenotype and the physiological and biochemical indexes related to grain filling of transgenic rice lines. The interaction protein network of OsU496A will also be constructed for further study on the possible mechanism of cell wall-plasma membrane-cytoskeleton continuum mediated by OsU496A in the endosperm cell proliferation, development, apoptosis and grain filling. This study will provide a basis for elucidating further the physiological mechanism of endosperm development and grain filling of rice and be likely to help improve grain yield and quality of rice.

水稻U496A是一个功能未知的膜整合蛋白。结构预测表明OsU496A具有类似动物整合素的蛋白结构和较保守的同源序列。作为质膜上介导胞外基质-质膜-细胞骨架连续体的中间成员，整合素在动物细胞的增殖、粘附和凋亡中起着重要的作用。在水稻籽粒灌浆过程中，胚乳细胞的增殖、发育、凋亡过程与细胞壁、质膜（尤其是起联接作用的膜蛋白）和细胞骨架关系密切，但不清楚这三者是如何协同调控胚乳发育的。前期研究结果表明，OsU496A特异表达在稻粒胚乳部位，参与了水稻籽粒充实过程的调控。本项目拟以OsU496A干扰表达和过表达转基因水稻株系为试验材料，比较研究各转基因水稻株系间与籽粒灌浆相关的表型和生理生化变化，并构建OsU496A相互作用蛋白网络，以期阐明水稻U496A蛋白在籽粒灌浆过程中的作用，尤其是其介导的细胞壁-质膜-细胞骨架连续体在稻粒胚乳发育和籽粒充实过程中的作用，为进一步提高水稻的产量和品质奠定基础。

水稻籽粒灌浆过程是稻米产量形成和品质构建的关键阶段。此过程受包括生长素和细胞分裂素在内的多种植物激素的调控，其生理和分子机理的探究一直是研究的重点和难点。水稻籽粒灌浆过程实质是同化物（主要是糖类物质）向籽粒胚乳输送和累积的过程。作为一个在水稻胚乳中特异性表达的未知功能蛋白，OsU496A很可能参与了水稻籽粒灌浆过程的调控。以OsU496A干扰表达和过表达转基因水稻株系为材料的研究结果表明，OsU496A-RNAi水稻颖果变小，籽粒长度显著变短，粒重显著降低；而OsU496A-OX籽粒则表现为颖果增大，粒长变长，粒重显著增加。相应地，不同OsU496A转基因水稻籽粒中可溶性糖和淀粉含量，蔗糖－淀粉转化关键酶的表达水平和活性也出现了差异。进一步研究表明，OsU496A参与了水稻籽粒内IAA的合成与运输过程以及细胞分裂素的氧化分解过程，导致籽粒内IAA和CTK含量增加，进而参与了IAA和CTK介导的籽粒灌浆过程。并且，外源IAA和6-BA处理可促进野生型水稻籽粒灌浆过程中OsU496A的表达。与野生型相比，外源激素处理可显著增加OsU496A-RNAi籽粒重量，而对OsU496A-OX籽粒增重不明显。水稻籽粒灌浆动态分析表明，外源激素处理显著提高了OsU496A-RNAi和野生型水稻最大灌浆速率Gmax，同时显著缩短了OsU496A-RNAi籽粒活跃灌浆期D和达到最大灌浆速率的时间Tmax，从而显著地提高其灌浆效率。进一步分析表明，OsU496A可通过参与生长素和细胞分裂素对蔗糖－淀粉转化关键酶（特别是SuS和SSS）的表达和活性的调控，影响蔗糖的转化与淀粉合成，进而调控水稻籽粒灌浆进程。此外，酵母双杂交实验结果表明，OsU496A与E3泛素连接酶ARI2间存在相互作用。推测OsU496A可能是通过与E3泛素连接酶互作，促进IAA转录抑制因子的降解，从而激活生长素转录因子，引起下游生长素调控基因的表达，从而正向调控水稻籽粒灌浆进程；另一方面，OsU496A可通过抑制细胞分裂素氧化酶基因的表达，从而增加了籽粒内细胞分裂素含量，进而正向调控细胞分裂素介导的水稻籽粒灌浆过程。本研究初步探明了OsU496A在生长素和细胞分裂素调控的水稻籽粒灌浆过程中的作用，可为进一步解析OsU496A的功能，揭示水稻籽粒灌浆的生理和分子机制以及提高稻米的产量和品质奠定基础。