一个响应番茄黄化曲叶病毒侵染的番茄F-box基因的功能研究

As components of SCF (S-phase Kinase-associated Protein1 (SKP1)/CULLIN1(CUL1)/F-Box) ubiquitin-ligase complexes, F-box proteins confer substrate specificity within SCF complexes in protein degradation mediated by the ubiquitin-proteasome system, which control many processes in plants, including the modulation of plant immunity. But the function of F-box in tomato resistance to Tomato Yellow Leaf Curl Virus (TYLCV) had not been discussed. We identified a Slf1 gene which encoding an F-box protein in tomato (Solanum lycopersicon) was responded to the infection by TYLCV.RNA-seq and qRT-PCR revealed that the expression of Slf1 gene was up-regulated in TYLCV-resistant tomato inbred line, while the expression of Slf1 gene was down-regulated in TYLCV-susceptible tomato inbred line after the infection by TYLCV.In addition,the expression of Slf1 in resistant individuals from (the resiatant×the susceptible) F2 segregated population was significantly higher than that in the susceptible counterparts. Transient expression of Slf1 gene triggered the hypersusceptible response in Nicotiana benthamiana,suggesting a resistant role of Slf1 gene in tomato. In this study, we will clone this Slf1 gene through RT-PCR, and get insight into the role of Slf1 gene in the resistance of tomato to the TYLCV infection by virus-induced gene silencing of Slf1 gene and by over-expression of Slf1 gene in tomato, respectively. A possible interaction between Slf1 and TYLCV was suggested by the domain analysis of Slf1. For further understanding of the function of F-box gene in tomato- TYLCV interaction, we will explore the bimolecular fluorescence complementation system and yeast two-hybrid system to investigate the interaction of Slf1 protein with proteins encoded by TYLCV. Since TYLCV causes significantly reduction of production and quality of tomato, the identification of new host components with resistance to TYLCV will contribute to combat this viral disease and improvement of production and quality of tomato.

植物F-box蛋白在植物免疫防卫反应中具有底物识别特性，但在抗番茄黄化曲叶病毒（TYLCV）中的作用还未见报道。我们通过RNA-seq和qRT-PCR发现TYLCV侵染后，番茄一个F-box基因Slf1在抗病番茄中上调表达，在感病番茄中下调表达；抗感番茄杂交F2 分离群体接种TYLCV后，Slf1基因在抗病群体的表达量显著高于感病群体。Slf1基因瞬时表达可激发烟草的过敏性反应和防卫基因的表达。本研究拟通过病毒诱导的基因沉默、转基因过量表达明确Slf1基因在番茄抗TYLCV中的作用及其对茉莉酸、乙烯、水杨酸等激素介导抗病反应的影响。蛋白结构预测发现Slf1与TYLCV可能存在互作，本研究将利用双分子荧光互补和酵母双杂交明确Slf1蛋白与TYLCV编码蛋白是否存在互作及其具体作用的病毒蛋白,阐明F-box基因响应TYLCV侵染的作用模式，为防治番茄黄化曲叶病、提高番茄品质和产量提供理论依据。

本项目通过VIGS和过量表达番茄一个F-box基因Slf1后接种TYLCV，研究Slf1基因在抗TYLCV中的功能。VIGS沉默Slf1基因导致抗病番茄植株体内TYLCV的含量增加5.6倍，过量表达Slf1基因的转基因番茄株系体内TYLCV的含量比野生型番茄显著降低。但沉默Slf1基因没有导致番茄植株抗TYLCV能力的丧失，过量表达Slf1基因亦未产生对TYLCV免疫的转基因番茄株系，表明Slf1基因并不是预期的Ty-2抗病基因。酵母双杂交实验未发现Slf1蛋白直接与TYLCV编码蛋白互作。此外，沉默Slf1基因后，抗病相关基因PR1b和调控果实早熟的乙烯受体基因ERT4的表达水平分别显著下降至16.5%和84.9%，而脂氧合酶基因loxd和调控植物生长发育的生长素响应基因IAA19则分别上调表达3.6倍和5.1倍，表明Slf1基因参与了番茄对TYLCV的抗病反应，并参与了多个激素信号途径调节的代谢过程，具有科研和育种利用价值。