紫黄质脱环氧化酶在甘蔗花叶病毒侵染玉米过程中的作用研究

The development of viral diseases is the consequences of interactions between viruses and their host plants. It is essential to identify host factors interacting with viral proteins and illuminate the roles they play during virus infection. Our preliminary results showed that the helper component-proteinase (HC-Pro) encoded by Sugarcane mosaic virus (SCMV), the major causal agent of maize dwarf mosaic disease, specifically interacted with violaxanthin de-epoxidase (VDE) in vitro and in vivo. In addition, the key amino acids of VDE responsible for the interaction have been determined. Since HC-Pro is a multifunctional protein involved in a variety of activities (including the suppression of gene silencing) during viral infection of host plant, it is of importance to investigate the role of the interactor VDE plays during the infection of maize by SCMV. The subcellular localization of HC-Pro and VDE after SCMV infection will be probed by using transformation of maize protoplast; and a series of experiments will be performed to determine whether VDE could influence any activities (including the gene silencing suppressor activity) of HC-Pro and to reveal the underlying action mechanism. As a result, these efforts should help to reveal the molecular mechanism of dwarf mosaic disease development and contribute to control viral diseases by approach of gene regulation.

病毒病的发生是病毒与寄主植物相互作用的结果。鉴定与病毒互作的寄主因子并解析其在病毒侵染过程中的作用具有重要的科学意义。我们的前期研究结果表明，玉米矮花叶病主要毒原甘蔗花叶病毒（SCMV）的蚜传辅助组分-蛋白酶（HC-Pro）蛋白可与玉米紫黄质脱环氧化酶（violaxanthin de-epoxidase, VDE）在体外和植物细胞中发生特异性互作，已经明确了VDE与HC-Pro互作的关键氨基酸，初步证明VDE可以抑制SCMV的积累。VDE 是叶黄素循环中的关键酶类，可以响应强光、热、冷、旱、涝等非生物胁迫，从而增强植物的耐性；我们将通过玉米原生质体转化等多种方法研究SCMV侵染玉米后HC-Pro和VDE的亚细胞定位情况，以及VDE可以影响HC-Pro的何种活性；阐明VDE在SCMV侵染过程中的作用及其分子机制，揭示玉米矮花叶病发生的分子机制，为采用基因调控措施控制该病害提供理论依据。

该项研究证实了甘蔗花叶病毒（SCMV） HC-Pro与玉米紫黄质脱环氧化酶（Zea mays violaxanthin de-epoxidase, ZmVDE）在体内发生互作。ZmVDE是核基因编码的叶绿体蛋白。 ZmVDE的前体（ZmVDEp）成熟蛋白（ZmVDEm）可与HC-Pro互作。用Y2H和Co-IP对ZmVDE与SCMV HC-Pro的互作特异性进行了研究，结果表明二者的互作具有病毒和寄主特异性。此外，ZmVDEm中第292位的谷氨酰胺是互作所必需的关键氨基酸，HC-Pro中的101—460位氨基酸区域是互作的关键结构域。接种SCMV 10天后，ZmVDE的转录水平和蛋白水平都上调表达，瞬时沉默ZmVDE基因，SCMV的积累量显著增加。在SCMV侵染的玉米细胞中，ZmVDE的亚细胞定位发生了改变，主要分布于聚集体上。将ZmVDE与SCMV HC-Pro及GFP在本生烟叶片中瞬时共表达后的荧光强度、免疫印迹和Northern blot检测结果均表明ZmVDE能够减弱SCMV HC-Pro的RNA沉默抑制活性，且该特性依赖于二者之间的相互作用。该研究发现了ZmVDE参与病毒侵染过程中的次级防卫反应，说明ZmVDE除响应非生物胁迫外还能响应生物胁迫。研究还发现ZmVDE也能够与玉米褪绿斑驳病毒（MCMV）的致病相关蛋白发生互作并显著地影响病毒的侵染。综上所述，ZmVDE是一种多功能的蛋白，可能对于多种病毒侵染均发挥重要作用，影响侵染的结果及所致病害的严重程度。对于其各种功能及发挥作用的分子机制值得进一步加以研究。