分子伴侣AKR2A调控芥子油苷代谢及其参与的先天免疫反应的机理

Plant innate immune response, which is an active defense respond against invasion of pathogens, plays an important role in plant defense system. It has been reported that PEN2 and PEN3 relied glucosinolate metabolites are required for Arabidopsis innate immune response. In our previous research, we found that PEN2 and PEN3 were interacted with AKR2A, which was a newly found molecular chaperone in Arabidopsis. Besides, just like pen2 and pen3, the akr2a mutants were defective in Flg22-induced Arabidopsis innate immune response and the related glucosinolate metabolites. The objectives of this project are: ① studying the interaction mechanism among AKR2A, PEN2 and PEN3 by founding out how AKR2A interacted with PEN2 and PEN3 and affected their gene transcription, protein expression and location; ② investigating the relationship among PEN2, PEN3 and AKR2A-regulated Arabidopsis innate immune response by application of Flg 22 or pst DC3000 and using of PEN2 or PEN3-overexpressing akr2a mutants; ③ studying the regulation mechanism of AKR2A on PEN2 and PEN3-related glucosinolate metabolisms by endogenous glucosinolates analysis and exogenous glucosinolates treatment experiments. Few studys have been reported about the regulation of molecular chaperone on glucosinolate metabolisms. This study is expected to reveal the regulating mechanisms of AKR2A to PEN2 and PEN3 and discover the function of this regulation in Arabidopsis innate immune response, which required glucosinolates metabolites.

植物通过起始先天免疫反应防御病原物，研究表明依赖于PEN2和PEN3的芥子油苷代谢对拟南芥先天免疫反应至关重要。AKR2A是拟南芥中最新发现的分子伴侣。我们研究发现AKR2A与PEN2和PEN3存在互作；而且akr2a突变体与芥子油苷代谢突变体pen2和pen3在Flg22处理后表型相似，均表现出先天免疫反应缺陷以及相关芥子油苷代谢受阻。项目拟以芥子油苷代谢和akr2a突变体等为材料，研究AKR2A对PEN2和PEN3调控的分子机理；通过对过量表达PEN2或PEN3的akr2a突变体表型分析，研究AKR2A如何通过调控PEN2和PEN3参与先天免疫反应；分离纯化不同组分的芥子油苷，内外源结合，研究AKR2A对PEN2和PEN3参与的芥子油苷代谢的调控。关于伴侣分子参与调控芥子油苷代谢的研究鲜有报道，本项目研究结果可以很好的丰富我们对芥子油苷代谢的调控网络及其在植物防卫反应中作用机理的认识。

芥子油苷是十字花科植物中的一种次生代谢产物，其代谢产物在植物应对生物和非生物胁迫的防卫反应中有重要作用。植物体中芥子油苷的代谢主要是由黑芥子酶完成。目前拟南芥中已鉴定出的黑芥子酶共有8个，包括TGG1-6 （THIOGLUCOSIDE GLUCOHYDROLASEs）和PEN2，PEN3（PENETRATION 2/3）。其中TGG1和TGG2只在地上部表达，是地上部主要的黑芥子酶，tgg1tgg2双突变体中黑芥子酶的活性只有野生型的0.5%，几乎完全检测不到。我们之前的研究发现伴侣蛋白AKR2A在酵母中与芥子油苷代谢酶PEN2和PEN3有相互作用，且参与依赖于芥子油苷代谢的拟南芥先天免疫反应。深入研究后我们发现AKR2A对拟南芥地上部最重要的两个芥子油苷代谢酶TGG1和TGG2的调控作用更为重要，因此我们及时把AKR2A对芥子油苷代谢酶调控的研究重点转为TGG1和TGG2，取得了较好的研究进展。主要结果有：1.利用酵母双杂交和免疫共沉淀技术证明了AKR2A与两个芥子油苷代谢酶的体内外互作关系；2.发现AKR2A相关突变体和转基因植株中两个芥子油苷代谢酶的蛋白水平受到显著影响；3.发现AKR2A影响两个芥子油苷代谢酶的蛋白定位；4.发现AKR2A对两个芥子油苷代谢酶的活性有显著影响；5.发现AKR2A突变体中芥子油苷代谢及依赖于芥子油苷代谢的拟南芥防卫反应和先天免疫反应均受到显著抑制。以上研究结果表明AKR2A通过与芥子油苷代谢酶的互作参与调控芥子油苷代谢，从而影响依赖于芥子油苷代谢的拟南芥防卫反应。项目研究结果丰富了芥子油苷代谢通路，推进了芥子油苷代谢调控网络研究的进程。