苹果蔗糖转运蛋白MdSUT2对盐胁迫的应答途径和调控机理

Fruit tree production in saline-alkali soil has good economic benefits. However, salt stress seriously threatens the maintainable development of fruit tree industry. Fruit trees and other plant species have evolved elaborately responsive mechanisms to high. Osmotic adjustment plays a crucial role in the response of plant to salt responses, and soluble sugar is a kind of important osmotic adjustment substance. It is unclear yet, however, concerning the molecular mechanism and regulatory pathway by which soluble sugar responds to salt stress. Our preliminary investigations demonstrated that sucrose transporter MdSUT2 is involved in the high salinity-induced sugar accumulation. A serial of protein interaction was also identified. Based on these results, it is reasonable to draw a hypothesis concerning that MdSUT2 responds to salt stresses via a putatively regulatory pathway, i.e. salt stress→MdPAT-S/MdCBL-S→MdCIPK-S→MdSUT2→soluble sugar→ salt stress response. Based on this hypothesis, various techniques of molecular biology, genetics, biochemistry and physiology will be adopted in this study to further verify this pathway and elucidate the molecular mechanism underlying each step. First of all, several techniques, such as yeast two-hybrid, Pull-Down, Co-IP, BiFC and Western blotting and so on, will be conducted to verify if MdCIPK-S interacts with and phosphorylates MdSUT2 protein, and if MdCBL-S interacts with MdCIPK-S and MdPAT-S, respectively. Meanwhile, it will be verified if their interaction influences the function of MdCBL-S/MdCIPK-S complex in the regulation of MdSUT2. Finally, the whole regulatory pathway will be integrated, and molecular mechanism will be refined using a serial of transgenic apple plants such as MdCIPK-S and MdSUT2 transgenic lines.

在盐碱地发展果树生产具有良好的经济效益，但盐胁迫严重威胁果树产业的可持续发展。渗透调节在盐胁迫应答中具有重要地位，可溶性糖是重要的渗透调节物质，但其应答盐胁迫的分子机制和调控途径不清楚。根据前期研究，我们推测出苹果蔗糖转运蛋白MdSUT2应答盐胁迫的可能信号途径，即盐胁迫→MdPAT-S/MdCBL-S→MdCIPK-S→MdSUT2→可溶性糖→应答。在此基础上，拟采用分子生物学和遗传学及生理生化技术验证该调控途径，解析各步骤的分子机理。首先鉴定和解析MdCIPK-S与MdSUT2互作并对其磷酸化修饰，然后验证MdCBL-S与MdCIPK-S、MdPAT-S与MdCBL-S的互作，并鉴定它们如何影响MdCBL-S/MdCIPK-S复合体对MdSUT2的磷酸化修饰和转运功能；最后，通过鉴定MdCIPK-S和MdSUT2等转基因苹果材料的功能，进一步整合该调控途径，揭示其分子机理。

SUT是一类重要的蔗糖转运蛋白，其定位于液泡膜，参与植物的逆境应答以及品质形成。本课题组前期从苹果的全基因组序列中鉴定得到了一个蔗糖转移酶蛋白MdSUT2.2，通过对该基因转基因材料的表型分析发现，该基因能够响应盐胁迫以及干旱胁迫，进而促进液泡内的可溶性糖累积，提高果实品质以及胁迫抗性。以MdSUT2.2为诱饵，通过分离泛素酵母双杂交筛选，筛选得到了一个响应盐胁迫的蛋白激酶MdCIPK13为MdSUT2.2的互作蛋白，进一步研究发现，MdCIPK13能够磷酸化MdSUT2.2，从而稳定MdSUT2.2的蛋白表达。同样，通过筛选得到了MdCIPK22，一个响应干旱胁迫的蛋白激酶，同样磷酸化稳定了MdSUT2.2的蛋白表达。进一步向上筛选，通过转录组以及qRT-PCR数据，发现一个棕榈酰基转移酶MdPAT16，为苹果中PAT家族响应盐胁迫的关键蛋白，MdPAT16能够棕榈酰化MdCBL1，进而在蛋白水平上稳定MdCBL1的表达，同样MdCIPK13与MdSUT2.2为MdPAT16与MdCBL1的直接靶蛋白。即盐胁迫通过诱导MdPAT16的表达，以棕榈酰化的形式稳定下游MdCBL1-MdCIPK13互作体系的表达，进而磷酸化MdSUT2.2，促进液泡内可溶性糖含量累积，提高果实品质以及苹果的耐盐性。