苹果茎沟病毒（Apple stem grooving virus, ASGV）CP基因介导的RNAi 转基因对ASGV侵染和脱毒的影响研究

Apple stem grooving virus (ASGV) is one of the most important latent viruses influencing on apple. Production of virus-free plants is an effective approach to control virus diseases. But ASGV is very difficult to be eradicated by conventional methods, and it often happens in the field that virus-free plants may be secondly infected by the same viruse, thus causing a great threat to the development of virus-free plant production. Plant antiviral genetic engineering has provide an efficient and stable way to control viruses, and many researches have been reported on genetic transformation by viral coat protein-RNA silencing. Through construction of hpRNA expression vector targeting CP gene of ASGV and its transformation in apple plants by agrobacterium-mediated method, the aim of this study is to study on effect of RNAi transformation of ASGV CP gene on its expression, infection and virus eradication in apple; Transgenic virus-free apple plants will be inoculated with ASGV by grafting and infectious clone, and the interference efficiency will be detected. In situ hybridization and Immunohistochemistry will be used for virus localization. Virus eradication by shoot tip culture and cryotherapy will be conducted and its mechanisms will be studied; For ASGV-infected apple leaves, experiment of virus localization will be conducted during different stages of the leaf regeneration process, and virus eradication by leaf regeneration will be studied as well as its mechanisms. This research will have an important theoretical significance both on plant antiviral genetic engineering and production of virus-free plants and will provide a new strategy or approach for the long-term and efficient prevention of plant virus diseases, especially fruit virus diseases.

苹果茎沟病毒是影响苹果生产的重要潜隐性病毒。培育无毒苗是防治病毒病的常用途径。但ASGV用常规方法很难脱除，且脱毒苗在田间容易受到病毒二次感染。植物抗病毒基因工程为植物病毒病有效稳定的防治提供了新途径。由病毒CP基因介导的RNAi转基因获得抗病毒材料，已在多种作物上进行了研究报道。本项目拟构建ASGV病毒CP基因的hpRNA干扰载体，农杆菌介导法转化苹果，研究RNAi对ASGV病毒的表达、侵染和脱毒的影响；通过嫁接和侵染性克隆侵染法接种转基因无毒苗，检测干扰效果和病毒含量，用原位杂交法和免疫组织化学染色法进行病毒定位，用茎尖培养和超低温疗法脱毒，揭示脱毒机理；对带ASGV病毒的苹果叶片，在转化后不同时期对叶片进行病毒定位和叶片再生脱毒，揭示其脱毒机理。该研究不论对植物脱毒还是植物抗病毒基因工程均有重要的理论指导意义，将为植物病毒病，尤其是果树病毒病长期有效的防治提供新的策略和途径。

苹果茎沟病毒是影响苹果生产的重要潜隐性病毒，用常规方法很难脱除，且脱毒苗在田间容易受到病毒二次感染。由病毒CP基因介导的RNAi转基因获得抗病毒材料，已在多种作物上进行了研究报道。本项目构建了2个ASGV病毒CP片段hpRNA干扰载体，并成功转化烟草和苹果，获得了RNAi转基因植株；构建了ASGV病毒的侵染性克隆，证明了其具有侵染活性，为ASGV病毒接种提供了新的技术途径；采用摩擦接种、试管苗嫁接和农杆菌介导侵染性克隆接种等方法对转基因植株进行传毒，用野生型植株作为对照，通过病毒检测鉴定RNAi转基因的干扰效果，结果表明RNAi转基因能够提高本氏烟和苹果对ASGV病毒侵染的抗性，即存在干扰效果。免疫组织化学染色法病毒定位和荧光定量结果表明RNAi转基因能够抑制ASGV病毒在苹果茎尖和叶片中的侵染分布，降低病毒含量。该研究为RNAi转基因和脱毒两者的结合提供了前期理论依据，为难以脱除的病毒种类提供了一种新的防治策略和方法；另外还尝试用外源褪黑度处理提高了ASGV的茎尖培养脱毒效率（达95%），为生产无毒苗提供一种新的可能途径；分析了河南省不同地方苹果上主要病毒种类为ASGV、ASPV、ACLSV和ApNMV，发现了citrus leaf blotch virus (CLBV)病毒的两个株系，得到了全基因组序列，这些研究为苹果上病毒种类和分子变异研究提供了理论依据。