分子伴侣在黑芥子酶-特异蛋白互作系统中的作用机制研究

The mechanism of myrosinase and specifier proteins interacting to control and fine-tune glucosinolates breakdown appears to be essential for appropriate plant responses to various biotic as well as abiotic stresses. According to previous studies, cofactors are required for the interaction between myrosinase and specifier proteins. However, there have no such cofactors been reported until now. AKR2A was a newly found molecular chaperone in Arabidopsis.In our previous research, we found that TGG1 and TGG2 were interacted with AKR2A no matter in vitro or in vivo.The expression level and location of TGG1 and TGG2 were decreased statistically in akr2a mutants. Besides, based on iTRAQ analysis of akr2a-1, the expression level of specifier proteins ESP and ESM1 was also decreased. The H2Y analysis demonstrated that AKR2A also interacted with ESP and ESM1. Based on these results, we plan to reveal the action mechanism of AKR2A on myrosinase and specifier proteins interaction by three steps: ① studying the interaction mechanism among AKR2A, TGG1 and TGG2 by founding out how AKR2A interacted with TGG1 and TGG2 and affected their gene transcription, protein expression and location; ② investigating the interaction mechanism among AKR2A, ESP and ESM1; ③ studying the regulation mechanism of AKR2A on the interaction of myrosinase and specifier proteins by verifying the necessity of AKR2A and the interaction model of these three. The results will reveal more elements in glucosinolates metabolic pathway and push forward our understandings of glucosinolates metabolic regulatory network.

黑芥子酶-特异蛋白互作精细调控芥子油苷代谢的机制在植物应对各种生物和非生物胁迫的防卫反应中至关重要。研究表明特异蛋白与黑芥子酶的互作需要辅因子参与，但迄今为止尚无相关辅因子的报道。AKR2A是拟南芥中新发现的分子伴侣。我们研究发现AKR2A与黑芥子酶TGG1和TGG2在植物体内体外互作。akr2a突变体中黑芥子酶蛋白水平及定位均受抑制。经蛋白质组分析，akr2a突变体中特异蛋白ESP和ESM1水平显著下降而且AKR2A与二者在酵母中互作。基于此，本项目拟分三个步骤对AKR2A在黑芥子酶-特异蛋白互作中的作用机制进行研究：①解析AKR2A与黑芥子酶互作的分子机制；②解析AKR2A与特异蛋白互作的分子机制；③探索AKR2A在黑芥子酶-特异蛋白互作中的必要性及三者的互作模型。关于伴侣分子参与调控芥子油苷代谢的研究鲜有报道，本项目的研究将丰富芥子油苷代谢通路，推进芥子油苷代谢调控网络研究的进程。

芥子油苷是存在于十字花科植物中的一类重要的次生代谢物质，它主要以代谢产物的形式发挥生物学功能，黑芥子酶是其水解酶。特异蛋白是含有芥子油苷的植物体内存在的一类蛋白，自身无催化活性，与黑芥子酶一起精细控制其生成代谢产物的类型。已有的研究表明，特异蛋白与黑芥子酶无直接的互作关系，但却必须和黑芥子酶一起才能发挥功能，因此推测存在一种或多种辅因子连接黑芥子酶与特异蛋白的互作，但到目前为止尚无任何一个辅因子被发现的报道。我们发现分子伴侣AKR2A调控次生代谢物芥子油苷代谢酶TGG1和TGG2的合成和定位，从而影响了黑芥子酶的蛋白水平和功能。由于AKR2A同时调控特异蛋白ESM1，我们设计实验验证了AKR2A是否是TGG1、TGG2和特异蛋白互作的辅助因子，结果发现虽然三者可以被同时沉淀，说明了三者在植物体内是存在互作的，但体外AKR2A的存在却不能帮助黑芥子酶和特异蛋白发生互作，说明AKR2A可能并不能或者并不能独立作为黑芥子酶和特异蛋白互作的辅助因子。此外，本研究发现TGG1和TGG2能定位于叶绿体，而且AKR2A是二者定位于叶绿体的分子伴侣，这将使TGG1和TGG2成为拟南芥中首次被报道定位于质体的β-葡萄糖苷酶。进一步研究我们发现，tgg突变体中淀粉合成代谢通路显著变化，这个发现至关重要，TGG1和TGG2将很可能成为连接初生代谢和次生代谢的节点。本项目研究内容创新性明显，研究结果不仅丰富了伴侣蛋白调控的芥子油苷代谢通路研究，还首次发现了拟南芥中质体定位的β-葡萄糖苷酶，并初步发现了其在叶绿体中的功能，科学意义显著。