灰飞虱与水稻条纹病毒互作过程中pc2受体蛋白GN-Rp的功能研究

Rice stripe virus (RSV) has been reported to cause severe rice stripe disease in rice fields in China, and RSV is transmitted by small brown planthopper (Laodelphax striatellus Fallén, SBPH). At present, the transmission mechanisms of RSV by SBPH remain unclear. Our preliminary study showed that RSV-pc2 and its receptor protein GN-Rp had important contributions to RSV infecting and escaping from midgut barrier, but the mechanism is unknown. The project was planned to take transmission determinants pc2 as the breakthrough, the pc2's receptor protein GN-Rp was screened, GN-Rp gene was cloned, and its antibody was prepared, then the interaction between two proteins was verified using various methods, analyzing the relations between the three-dimensional structure of GN-Rp and its function for RSV transmission in the protein level. Afterward, RNAi, antibodies blocking, virus-feeding experiments and tissue localization were performed to reveal the functions of pc2 and GN-Rp in RSV transmission (acquisition) by SBPH in a biological level. Meanwhile, the GN-Rp gene as bio-marker for RSV transmission in SBPH was also developed, and rice cultivar that SBPH had difficulties in acquiring RSV was prepared. At the same time revealing the molecular mechanisms that RSV escaped from midgut barrier, the project could provide techniques and means for sustainable control of rice stripe disease.

重要致灾病害水稻条纹叶枯病是由灰飞虱传播水稻条纹病毒（RSV）引起的。目前，灰飞虱传毒的分子机制还不清楚。前期发现，RSV的pc2蛋白及其受体蛋白GN-Rp对RSV侵染和跨越介体中肠屏障具有重要贡献，但机制不详。本项目拟从传毒决定因子pc2入手，筛选pc2受体蛋白GN-Rp，克隆GN-Rp基因并制备抗体，多种方法验证pc2与GN-Rp之间的互作，从蛋白质水平解析GN-Rp三维结构与传毒功能之间的关系；通过RNA干扰、抗体封闭技术、饲毒传毒实验、组织细胞定位等研究，从生物学水平明确pc2及GN-Rp在灰飞虱的传毒（获毒）过程中的作用。同时将GN-Rp基因开发为鉴别灰飞虱传毒能力的生物标记，并制备灰飞虱不可获毒的水稻材料。本项目在揭示RSV跨越介体中肠屏障的分子机理的同时，为水稻条纹叶枯病的绿色可持续防控提供了技术手段。

水稻条纹叶枯病是由灰飞虱传播水稻条纹病毒（RSV）引起的，研究灰飞虱传毒机制是防治该病害的关键。前期研究显示，RSV编码的糖蛋白pc2在水稻中可以被切割成2个蛋白GN和GC，推测其为RSV参与灰飞虱传播的决定因子。为此，本项目围绕pc2是传毒因子及其受体蛋白的鉴定展开研究。.发现GN和GC均可与RSV核衣壳蛋白NCP互作，免疫荧光定位发现GN可附着于灰飞虱中肠，且与NCP在中肠上皮细胞共定位。饲喂灰飞虱过量纯化的GN蛋白先期封闭中肠潜在受体后饲毒RSV病叶，则病毒侵染昆虫中肠上皮细胞受阻，灰飞虱获毒率显著下降。同时发现NCP、GN、GC和Rab5可共定位于中肠上皮细胞的囊泡（即早期内涵体）中，这些结果证明了RSV编码的pc2蛋白为参与灰飞虱传播的决定因子。.以GN为诱饵，筛选灰飞虱cDNA文库，获得一个受体蛋白，命名为GN-Rp，完成基因克隆和抗体制备。利用杆状病毒表达系统表达GN-Rp，并利用免疫组化分析了其亚细胞定位，发现GN-Rp蛋白主要定位于细胞膜，证实其为膜蛋白。.通过RNAi干扰昆虫体内GN-Rp表达，然后进行饲毒实验，结果显示，灰飞虱获毒能力显著下降，这说明GN与GN-Rp的互作参与了病毒侵染灰飞虱中肠屏障，对于灰飞虱获毒（RSV）至关重要。干扰GN-Rp的表达进行传毒实验，结果显示，灰飞虱传毒能力未受影响，说明GN-Rp并没有参与RSV与灰飞虱唾液腺屏障的互作。.利用产生pc2-siRNA的转基因水稻饲喂带毒灰飞虱沉默GN基因，结果RSV的积累量也显著降低，同时传毒能力也下降了，说明pc2对于RSV在灰飞虱体内复制和积累也是有帮助的。.制备GN过表达转基因水稻，以其饲喂无毒灰飞虱，在让灰飞虱取食RSV病叶，结果显示，与对照组相比，取食GN转基因水稻的灰飞虱获毒率显著降低，虫体内RSV的积累量也减少了。结果说明饲喂过量表达GN的水稻可以抑制灰飞虱获取RSV。.本项目鉴定了pc2为RSV编码的传毒因子，为阻断灰飞虱传毒新策略的开发提供了理论基础。发表SCI论文3篇，培养硕士生2名。项目期间，项目负责人获国家科技进步奖二等奖1项，省部级科技进步一等奖1项，达到了预期目标。