拟南芥DIF（DRIP1-Interacting Factor）在胁迫信号应答中的功能分析

As a single-subunit E3 ligase, DRIP1 (DREB2A-Interacting Protein 1) regulates negatively plant response to water stress through DREB2A, which is a key transcription factor in regulating water stress signaling in Arabidopsis. There are still questions of how DRIP1 senses the signaling and how DIRP1 activity is regulated. In this assay, RNA microarray and yeast two-hybrid system were used for gene screening. One gene was used for further analysis because of both its up-regulated expression in early drought stress and the interaction between its encoded protein and DRIP1, and was designated as DIF (DRIP1-Interacting Factor). Real-time PCR assay displayed that DIF was up-regulated by ABA, NaCl and cold besides drought, which implies that, on one side, DIF may be involved in water stress response by transcriptional DREB2A by its regulation of DRIP1; one the other hand, DIF may also be involved in other stress response besides water stress. In this study, biochemics, genetics and cell biology were applied to analyze the regulation of DIF on DRIP1 E3 activity, explain the mechanism of how DIF is involved in water stress response by ubiquitination and display which additional stress signals DIF responses to. In the course of exploring, new signals should be discovered in the stress signal pathway and a knowledge of stress sense and response in plant should be deepened.

拟南芥DREB2A互作蛋白DRIP1属于RING类单亚基泛素化E3连接酶，它通过泛素化DREB2A这个水分胁迫应答途径中的关键转录因子，负调控植物对水分胁迫的响应。但它是如何感知信号，其E3连接酶活性的调控机理尚不清楚。本研究通过基因芯片数据和酵母双杂交实验筛选到了一个干旱前期表达上调的基因，它编码的蛋白与DRIP1在酵母中有较强的互作，命名为DIF(DRIP1-Interacting Factor)。qPCR实验表明，除干旱外，DIF在ABA、NaCl和冷处理表达量明显上调。本研究通过生物化学、遗传学、细胞生物学方法研究DIF对DRIP1活性的调控，揭示DIF参与的DRIP1 E3泛素化反应调控的水分胁迫应答机制和其对其它胁迫信号的应答过程。本研究将有可能发现逆境信号传递过程中新的信号因子，加深人们对植物逆境信号感知和传递的认识。

DREB类蛋白是植物响应水分胁迫的应答过程中重要的转录因子，调控靶基因的表达使植物对不利的外界环境作出适应。有研究表明RING类E3连接酶DRIP1和DRIP2可以通过泛素化DREB2A使其被26S蛋白酶体降解，维持DREB2A在细胞中稳定的蛋白丰度；并推测干旱、热胁迫条件下DRIP1和DRIP2的E3泛素连接酶活性被抑制，DREB2A在细胞内大量积累从而启动下游含DRE/CRT元件的胁迫应答基因表达。研究胁迫条件下DRIP1和DRIP2的E3泛素化连接酶活性是如何调控的，是否存在其它的调控因子，以及该调控因子在胁迫应答过程中起到什么样的作用有待研究。.我们通过酵母双杂交方法筛选到一个能特异与DRIP1互作的蛋白，我们将其命名为DIF1（DRIP1-Interacting Factors 1），其同源基因表达蛋白命名为DIF2。我们发现DIF1/2除受到干旱胁迫诱导上调表达以外，还受到ABA、NaCl及AlCl3诱导；DIF1pro::GUS和DIF2pro::GUS转基因植株组织染色实验也证明DIF1/2受上述因素诱导表达。并且DIF1和DIF2在拟南芥的幼胚、莲座叶、花蕾以及角果中均有表达。DIF1-GFP和DIF2-GFP原生质体亚细胞定位实验中GFP荧光在细胞核及细胞质中均能够观察到，表明DIF1和DIF2蛋白可能在细胞内遍在分布；而在双分子荧光互补实验中，DIF1和DIF2与DRIP1的互作信号仅在烟草细胞的细胞核中观察到，表明DIF1和DIF2与DRIP1在细胞核中互作，这一结果与DRIP1蛋白为核蛋白结果一致。.对dif1-1, dif2-2和 dif1-1 dif2-2三种突变体ABA敏感性的实验分析发现，dif1-1与野生型植株无显著差异，dif2-2和dif1-1 dif2-2双突变体较野生型在萌发阶段表现出不同程度对外源ABA处理的不敏感；同时在高浓度葡萄糖培养基上，三种突变体均表现出对葡萄糖信号不敏感；相反，DIF1和DIF2过表达植株则对外源ABA和葡萄糖更敏感。土壤干旱实验也发现DIF1和DIF2过表达材料对干旱胁迫有更好的耐受性，而相应的突变体则较野生型材料对干旱胁迫更加敏感。以上结果暗示着DIF1和DIF2可能正调控植物水分胁迫的应答，但是其分子作用机制还有待进一步的研究。