水稻类受体激酶OsCR4 参与调控胚胎早期发育的分子机制

Plant embryogenesis includes two stages: the proembryo development and the embryo organ differentiation. Auxin has been well known to play important roles in embryo morphogenesis. Besides, differentiation of cell types also relies on cell-cell communication, which is mainly mediated by receptor-like kinases (RLKs). In the previous study supported by NSFC, we found that rice RLK OsCR4 is required for epidermal cell specification and interlock of pale and lemma. OsCR4 may also play key roles in early embryogenesis because OsCR4 RNAi plants displayed embryo defects at proembryo stage. In this project, we will continue to study the function of OsCR4 using oscr4 null allele mutant, which displays severe developmental phenotypes. To better understand the molecular mechanism of OsCR4, we purified OsCR4 protein complex in rice spikelets tissue using OsCR4-7Myc-6His tag, and identified auxin transport-, and auxin signaling related proteins by LC-MS-MS. These imply that OsCR4 may regulate cell differentiation and organ morphogenesis during early embryo development by integrating auxin signal transduction. We will analyze the effect of OsCR4 on the distribution and gradient of auxin, and on the activity of auxin transporter and AUX/IAA,through genetic, cell biology, and biochemistry methods. We will investigate the molecular mechanism by which OsCR4 regulates rice early embryogenesis through integrating auxin signaling.

植物的胚胎发育分为原胚发育和胚器官分化两个阶段，已知生长素在此过程中起重要调控作用，此外不同类型的细胞分化还依赖于细胞间的信息传递，类受体激酶介导这一过程。我们在前一个国家基金项目的研究中发现，水稻类受体激酶OsCR4不仅参与颖壳表皮细胞的分化和颖壳锁合结构的形成，而且还有可能在水稻早期胚胎发生中起重要作用。本项目中，我们将利用oscr4突变体继续进行研究，该突变体早期胚胎发育有严重缺陷。我们利用串联亲和层析在水稻小穗中纯化了OsCR4的蛋白复合体，从中鉴定出生长素运输及信号相关蛋白，暗示OsCR4可能与生长素的信号整合调控胚胎早期的细胞分化与器官建成。本项目将通过遗传、生化及细胞学分析OsCR4与生长素运输载体及信号组分AUX/IAA的关系，OsCR4对生长素梯度建成与分布的影响。对受体激酶OsCR4整合生长素信号途径调控早期胚胎发育的分子机制进行探索。

实验室前期对水稻类受体激酶OsCR4 RNAi 干涉突变体进行观察时发现其颖壳开裂，早期胚胎发育受到严重影响。本项目进一步利用OsCR4的T-DNA插入突变体进行细致研究。1）确定了oscr4 为单基因隐性突变体, 其杂合体无表型，纯合体三叶期生长受到严重抑制，不能结籽；2）对成熟胚进行切片观察，发现从杂合体中分离出来的oscr4 种子外表与野生型无差异，但胚根和胚芽都变小，细胞分化异常；3）对OsCR4组织特异性表达进行观察，发现其在胚芽，幼嫩根，及叶表皮细胞中表达；4）比较野生型和oscr4 中生长素的分布情况，发现DR5：GUS的染色在突变体中异常增强，且没有极性分布；5）对胞外域结构及胞内激酶活性进行研究，发现激酶活性和胞外的7个repeat结构域对于其生物学功能是必需的，而TNFR结构域对其生物学功能影响较小；6）比较了野生型和 oscr4 中地上部分表达的基因，发现WUS家族相关的基因表达升高；7）对叶片的观察表明oscr4 不仅表皮细胞分化出现问题，而且叶肉细胞的分化也出现了问题，表现为叶绿体的肿胀和缺乏类囊体。. 总之我们的研究结果表明受体激酶OsCR4对于水稻胚胎细胞层的分化、叶片叶绿体的发育及维管组织的建成至关重要，可能通过影响生长素的极性分布来调控的。