OsPIN3b调控根向重力性及淀粉粒沉积的分子机制研究

As sessile organisms, plants have to be able to quickly adapt to environment changes. Part of this adaptability entails roots grow toward the vector of gravity to take up nutrient and water. Although the genes involved in root gravitropic responses in Arabidopsis have been well studied, how the response is regulated in rice is still largely unknown. We cloned and characterized a auxin efflux carrier gene OsPIN3b, OsPIN3b over expression leads to loss of gravitropism and abnormality of starch granules sediment in rice. The OsPIN3b-related mutant rice obtained previous was used as a material to analyze the effect of OsPIN3b on the distribution of auxin in roots. The transcription factors and proteins interacting with OsPIN3b were searched by immunoprecipitation and yeast one/two-hybridization to determine the signaling pathway of OsPIN3b involved in regulating root gravitropism, revealing the molecular mechanism of OsPIN3b regulating root gravitropism. The starch content, the number and morphology of starch granules in the mutants were analyzed and determined. The relationship among the interaction of OsPIN3b and OsACTIN, the level of actin polymerization and the sedimentation of starch granules in the mutant roots were examined to explore the molecule mechanism of OsPIN3b regulating starch granules sedimentation.

作为固着生物，植物必须快速适应环境的改变，这种适应能力包括根向重力生长以吸收营养和水分。虽然拟南芥中涉及根向重力响应的基因已经有很多报道，但是水稻根中有关向重力响应是如何被调控的报道还比较少。我们克隆了一个生长素输出载体蛋白基因OsPIN3b，过表达OsPIN3b导致水稻根向重力性丧失以及淀粉粒沉积异常。本研究以前期获得的OsPIN3b相关突变体水稻为材料，分析OsPIN3b对根中生长素分布的影响；通过免疫沉淀、酵母单/双杂交等技术寻找与OsPIN3b相互作用的转录因子及蛋白，以确定OsPIN3b参与调控根向重力响应的信号通路，揭示OsPIN3b调控根向重力响应的分子机制。分析测定突变体中淀粉含量、淀粉粒数量及形态，检测OsPIN3b-OsACTIN之间的互作以及肌动蛋白聚合程度与突变体根中淀粉粒沉积的关系，初步探索OsPIN3b调控淀粉粒沉积的分子机制。

根向重力生长以吸收水分及营养对植物的生长发育至关重要，本项目前期发现过表达OsPIN3b导致水稻根向重力性丧失，根尖小柱细胞中的淀粉粒沉积异常。本项目主要进行并完成了三方面的研究：1）获得的OsPIN3b-GFP水稻，并确定OsPIN3b在水稻根尖细胞呈极性定位。酵母系统的分析显示OsPIN3b具有生长素输出功能。重力响应过程中，过表达OsPIN3b根尖的IAA浓度及IAA流速均异于野生型，生长素运输抑制剂NPA处理使得野生型水稻根向重力性丧失，类似于过表达OsPIN3b的表型。2）酵母双杂交、CoIP及双荧光酶互补实验鉴定出LRR型类受体激酶OsPK(X)能够与OsPIN3b相互作用，体外磷酸化实验表明其可以磷酸化OsPIN3b。酵母双杂交筛选到的小GTP酶OsRab(X)不能与OsPIN3b直接相互作用，但能与OsPK(X)直接互作。OsPK(X)可能作为中间体与OsPIN3b和OsRab(X)形成复合体，该复合体可能在OsPIN3b的定位中发挥功能。GUS染色结果显示，响应生长素的关键作用元件位于OsPIN3b基因ATG上游638 bp以内。酵母单杂交以及AP2类转录因子OsAP(X)相关突变体中OsPIN3b的表达量分析表明OsAP(X)调控OsPIN3b的转录。3）OsPIN3b与微丝蛋白OsActin7或OsActin7-1不存在相互作用，肌动蛋白聚合抑制剂Lat B不能完全恢复过表达OsPIN3b水稻根的向重力性。过表达OsPIN3b水稻根中淀粉粒的异常沉积可能是由于生长素不对称分布存在缺陷引起的。除此以外，本项目还确定了OsPIN3b在种根长度和侧根长度方面的功能。研究结果为水稻根向重力响应以及PIN蛋白极性定位和磷酸化修饰的分子机制提供了理论依据，也可为分子设计育种提供候选基因。