RNAi介导的转基因砧木诱导西瓜接穗抗CGMMV的机理

RNAi-mediated virus defense mechanism widely exists in plants in which a series of 20-25 nt siRNAs act as long-distance signal to induce systematic RNA silencing. Based on this mechanism, systematic RNA silencing should be graft transmissible and it is possible to prevent virus infection by grafting plant to transgenic rootstocks that can produce corresponding siRNA. Recent work in cherry confirmed this hypothesis. However, it is not clear if this grafting methods also works in other plants. Cucumber green mottle mosaic virus (CGMMV) is a destructive virus disease that mainly infected cucurbitaceous plants including water melon, cucumber and so on. Since 2006, the hazard of CGMMV became more serious. In this work, we will construct transgenic bottle gourd as rootstocks to graft water menlon. Changes of siRNAs and its association with the resistance in transgenic and non-transgenic plants and scions after CGMMV infection will be analyzed, which could further reveal the mechanism of rootstock-induced CGMMV resistance. Consequently, the proposal on exploring the mechanism of rootstock-induced CGMMV resistance will not only provide a typical case of application of transgenic rootstocks to control virus disease, but also further our understanding on siRNA-mediated plant virus resistance. The proposal will lay the foundation for developing and applying the new plant virus control strategy that using transgenic anti-virus rootstocks to prevent and cue virus disease.

在植物抵御病毒感染中普遍存在RNAi介导的防御机制，其特征是产生20-25 nt的siRNA，该沉默信号长距离运输可诱导系统性沉默。基于该原理构建转基因砧木，理论上可防御接穗部分相应病毒感染。此构想在樱桃PNRSV防治中已初步成功，能否用于其它病毒和作物有待研究。黄瓜绿斑驳花叶病毒（CGMMV）主要侵染葫芦科作物，常造成西瓜、黄瓜等作物的毁灭性损失，2006年传入我国后危害日重。本项目拟针对该病毒构建基于RNAi的转基因葫芦嫁接西瓜，检测转基因与非转基因植株中小RNA的差异，分析CGMMV感染前后砧木和接穗中小RNA的变化动态、相互关系及其与抗性的相关性，明确西瓜接穗诱导产生CGMMV抗性的机理。项目的完成，可为利用转基因砧木防治植物病毒病提供典型案例，也有助于深入理解小RNA介导的病毒抗性，为基于RNAi原理的植物病毒防控新技术——转基因砧木防治植物病毒病的开发应用提供科学依据。

黄瓜绿斑驳花叶病毒（CGMMV）是葫芦科作物上流行的主要病毒病之一，危害十分严重。已有研究表明植物可以将dsRNA切割成sRNA从而抵御病毒侵染。本研究针对CGMMV，利用该原理构建了15个RNA干扰载体；通过烟草瞬时表达系统初步评价了这些载体对CGMMV的抗性；获知不同的干扰片段与抗性之间的关系；利用农杆菌介导的遗传转化方法将其转化至葫芦和南瓜中，获得了具有CGMMV抗性的转基因植株并对其抗性进行了评价；对其抗病性以及病毒积累进行了研究，采用烟草嫁接系统对转基因嫁接植株中sRNA和植株抗病毒的关系进行了初步研究；利用嫁接技术将转基因南瓜组织嫁接入和非转基因植株中，并对其抗性及其病毒积累进行了初步评估。本研究为将转基因砧木介导接穗抵抗病毒的在生产中进行应用提供部分实践和理论依据。