拟南芥XPO1A介导DCAF2蛋白核质转运响应干旱胁迫的机理

Nucleocytoplasmic trafficking of proteins mediated by nuclear transport receptor (NTR) is one of the important signal transduction cascades in Eukaryotes, which helping to adapt to the exogenous environmental factors, biotic and abiotic stresses. In previous study, we identified a hypersensitive mutant, xpo1a-4, to drought stress, XPO1A has been reported to be a nuclear export receptor,while the molecular mechanism of XPO1A involved in drought stress remains unknown. We have screened a interactive protein DCAF2 with XPO1A protein using co-immunoprecipitation/mass- spectrography (IP/MS) and yeast two hybrid methods. In this project, we will focus on: 1) Confirmation of the interaction between XPO1A and DCAF2 and identification of the interactive protein sites; 2) the effection of the interaction between XPO1A and DCAF2 on the nucleocytoplasmic distribution of DACF2 protein; 3) analysis of the DCAF2 gene responding to the abiotic stress and its relation to XPO1A gene; 4) the mechanism of the interaction of XPO1A and DCAF2 responses to drought stress. The emphasis of the project is to illustrate the signal cascade XPO1A-DCAF2-ABI5, which will help not only to understand the molecular mechanism of XPO1A responses to drought stress in Arabidopsis, but also to reveal the roles of nucleocytoplasmic trafficking in regulating ABA signaling.

核质转运受体（NTR）介导的蛋白质核质转运是真核生物信号调节的重要机制之一，并以此响应外界生物或非生物胁迫作用。利用正向遗传学技术，我们前期鉴定了一个干旱敏感突变体xpo1a-4, XPO1A是细胞核输出蛋白受体，但其参与干旱胁迫的作用机理尚不清楚。通过蛋白质免疫共沉淀-质谱分析和酵母双杂交实验，筛选到一个XPO1A互作底物蛋白DCAF2。本项目将在此基础上继续探讨：1）XPO1A与DCAF2互作验证及互作位点分析；2）XPO1A与DCAF2互作对DCAF2核质分布的影响；3）DCAF2响应逆境胁迫分析及其与XPO1A的关系；4）XPO1A与DCAF2互作响应干旱胁迫的机理。重点是解析XPO1A-DCAF2-ABI5的信号级联过程，探明此过程不仅可以阐明XPO1A响应干旱胁迫的作用机制，而且对揭示核质转运在ABA信号转导中的调节功能有重要的启示作用。

蛋白质核质转运是真核生物信号调节的重要机制之一，以此响应植物生长发育和外界环境胁迫。研究以蛋白质核转运受体XPO1A为对象，通过免疫共沉淀的方法筛选XPO1A互作蛋白，并阐明互作蛋白核质转运参与的生物学过程。本研究鉴定到2个XPO1A互作底物蛋白，分别是XIW1(XPO1-Interacting WD40 protein 1) 和HDA6(Histone deacetylase 6)。主要研究结果及其科学意义如下：.(1)XPO1A-XIW1-ABI5信号级联解析及其对ABA信号的响应。研究表明拟南芥XIW1是一个核质分布蛋白，XIW1的出核转运依赖于蛋白质核转运受体XPO1A和XPO1B。正常生理条件下XIW1主要分布在细胞质，外源ABA和逆境处理可促进XIW1在细胞核内积累。XIW1在细胞核内与ABI5直接互作，起到保护ABI5不被蛋白酶体降解的作用，从而提高ABI5蛋白水平，促进ABA响应和干旱胁迫耐受性。此研究解析了XPO1-XIW1-ABI5信号级联过程，阐明了一种通过蛋白核质转运调节ABA信号途径的新机制；同时，过量表达XIW1或者促使XIW1在细胞核内表达，均可提高植物的抗旱性，有一定的应用前景。.(2)XPO1A与HDA6互作影响基因组甲基化水平。研究发现xpo1a突变体中转基因35S启动子区域呈现高甲基化状态，而其组蛋白乙酰化水平显著降低。通过生化分析，发现XPO1A能够与HDA6直接互作并介导HDA6的出核转运，从而使细胞核内的HDA6维持在一定的水平。由于HDA6招募DNA甲基转移酶促进基因沉默，因此XPO1A的突变导致HDA6的出核受到抑制，细胞核内的HDA6水平显著增高，造成靶基因的沉默增强。研究揭示了表观遗传调控的一种全新作用机制，即通过改变表观遗传因子的核质分布影响下游基因的表达。