柑橘类病毒CBCVd诱发寄主基因沉默拮抗CEVd的分子机制研究

柑橘裂皮类病毒（CEVd）引起的柑橘裂皮病严重危害我国柑橘产业。田间症状观察发现柑橘树皮裂纹类病毒（CBCVd）对CEVd存在明显的拮抗作用，但对其分子机理研究未见报道。项目组在前期工作中，成功构建了香橼-CBCVd/CEVd互作高效实验体系，通过类病毒单独/复合侵染香橼，寄主差异基因和小RNA分析，验证了拮抗作用与基因沉默相关。在此基础上，本项目拟用瞬时表达、免疫共沉淀、转基因、RNAi、基因编辑、VIGS等技术，构建杂合类病毒、验证vd-siRNA对类病毒RNA的切割，测定香橼DCL2/RDR1/AGO2降低/增强表达对类病毒积累的影响，分析AGO2结合的vd-siRNA和类病毒诱导的siRNA。从病原和寄主两个角度，最终阐明CBCVd对CEVd拮抗作用的分子机制，揭示柑橘基因沉默抗类病毒的功能。预期结果有望为预防柑橘裂皮病提供新思路、新方法，为开发具有类病毒抗性的种质资源奠定基础。

Citrus exocortis disease caused by CEVd is a serious threat to citrus industry in China. The field symptom observation showed that CBCVd had an obvious antagonistic effect on CEVd. However, the molecular mechanism was not reported. Here, we successfully constructed the efficient experimental system of citron-CBCVd/CEVd interaction. Through the analysis of viroid single/mix infection of citron on host differential response gene and vd-siRNA, it was verified that the antagonistic effect was related to host gene silencing. On this basis, the project plans to use viroid recombination, transient expression, immunocoprecipitation, transgenic, RNAi, gene editing, VIGS and other technologies to construct heterozygous viroid, to verify the cleavage of viroid RNA by vd-siRNA, to determine the effect of decreased / enhanced expression of citron DCL2 / RDR1 / AGO2 on the accumulation of viroid and to analyze the vd-siRNA combined with AGO2 and the siRNA induced by viroid. Finally, from the perspective of pathogen and host, molecular mechanism of antagonistic interaction will be elucidated, and mechanism of anti-viroid based on gene silencing of citrus will be revealed comprehensively and deeply. The results are expected to provide new ideas and methods for the prevention of citrus exocortis disease and lay a foundation for the development of germplasm resources with viroid resistance.