SDIR1互作蛋白ECA1在植物应对干旱胁迫过程中的功能分析

?.The ubiquitin/proteasome system has been demonstrated to play critical roles in drought stress response of plant. SDIR1 has been proved to be a membrane E3 ligase, functioning as a positive regulator in drought tolerance of plants. SDIR1 functions in both dicotyledon and monocotyledon. To deeply reveal SDIR1's function in plant plant drought stress response, yeast two-hybrid was applied to screen proteins interacting with SDIR1. ECA1, an Endoplasmic Reticulum (ER)-bound Ca2+ pump protein, was obtained.It is known that ECA1 can pump Ca2+ and Mn2+ into the ER to support plant growth under conditions of Ca2+ deficiency or Mn2+ toxicity. But the role of ECA1 in plant response to drought stress is unknown. The interaction between ECA1 and SDIR1 was further confirmed in vitro and in vivo through Pull-down, Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. ECA1 and SDIR1 can be co-localized in ER. Similar to SDIR1-overexpressing plants, eca1 mutant plants are tolerant to drought stress. The degradation of ECA1 through the 26S proteasome in the presence of SDIR1 and the stability of ECA1 protein in sdir1 mutant will be investigated. Furthermore, the phenotype of ECA1 overexpressing plants stressed by drought will be analyzed. Calcium concerntration and ABA-elicited [Ca2+]cyt oscillations in guard cells will be quantified in different type of plants. The study might provide some clues to figure out the role of SDIR1/ECA1 complex in calcium signaling and drought signaling of plant..This study will role out the new molecular mechanism of plant drought response.And as a guidance, it can be applied to develop the new strategy to improve drought tolerance of crop plants.

泛素介导的26S 蛋白酶体降解途径在植物干旱胁迫信号传导中起着重要的作用，已知拟南芥中含有环指蛋白结构域的E3 泛素连接酶SDIR1 是植物激素ABA调控途径中的正调控因子,在植物对干旱胁迫的反应中起重要作用。项目的前期工作表明：ECA1 是以SDIR1 为诱饵蛋白筛选得到的相互作用蛋白，其细胞生物学功能为ER 膜上的ATPase Ca2+泵，eca1突变体植物表现出明显的抗旱性。本项目将通过生物化学、分子细胞生物学和遗传学等方法分析ECA1 与SDIR1的相互关系，包括SDIR1和ECA1在植物体内和体外的相互作用，二者的亚细胞定位情况，分析SDIR1对ECA1的稳定性影响，以及SDIR1/ECA1 复合体参与的生理过程和在植物干旱胁迫信号途径中的调控机制。该研究不仅为阐明植物的干旱反应机理，还为农作物的改良及干旱育种提供理论依据。

泛素介导的26S 蛋白酶体降解途径在植物干旱胁迫信号传导中起着重要的作用，我们的前期工作表明拟南芥中含有环指蛋白结构域的E3 泛素连接酶SDIR1 是植物激素ABA调控途径中的正调控因子,在植物对干旱胁迫的反应中起重要作用。项目的前期工作表明：ECA1 是以SDIR1为诱饵蛋白筛选得到的相互作用蛋白，其细胞生物学功能为内质网膜上的ATPase Ca2+泵，本项目将通过生物化学、分子细胞生物学和遗传学等方法分析ECA1 与SDIR1的相互关系，包括SDIR1和ECA1在植物体内和体外的相互作用，二者的亚细胞定位情况，分析SDIR1对ECA1的稳定性影响，以及SDIR1/ECA1 复合体参与的生理过程和在植物干旱胁迫信号途径中的调控机制。我们发现SDIR1和ECA1在体内实验和体外实验可以发生互作；ECA1蛋白具有不稳定性，26S蛋白酶体抑制剂如MG132能抑制ECA1的降解过程；ECA1的体外泛素化依赖于SDIR1的E3活性，并且SDIR1能促进ECA1的降解；在sdir1-1突变体中ECA1蛋白的稳定性大大加强；SDIR1和ECA1互作参与了细胞质钙浓度的调控；相对于野生型植物而言，eca1突变体表现为盐敏感，表型类似SDIR1高表达植物；而ECA1高表达植物对盐不敏感，类似于sdir1-1突变体植物在盐处理条件下的表型。通过研究结果，我们提出了SDIR1-ECA1的工作模型：SDIR1是ABA信号转导通路中的正调控因子，SDIR1能促进ECA1的降解，ECA1作为SDIR1的底物，通过负调控ABA的正调控因子ABI5的RNA和蛋白水平，负调控了ABA响应基因的表达，进而负调控了植物对ABA的响应。该研究不仅为阐明植物的干旱反应机理，还为农作物的改良及干旱育种提供理论依据。

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