番茄基于DNA-free基因编辑技术的2种类病毒抑制和脱毒的机理研究

番茄是世界上重要的蔬菜作物，我国鲜食番茄种植面积位居世界首位。类病毒（Viroid）是已知最小的专性寄生和检疫性病原体，可侵染番茄顶端分生组织和生殖器官，并通过营养器官和种子传播，对生产、育种和流通造成严重影响。常规方法对类病毒脱毒效果有限。病毒与类病毒生物特性相似，基因编辑已成功应用于病毒抑制，尚未应用于类病毒沉默。以弹状病毒为载体的基因编辑具有效率高和载体不遗传的特点，可应用于非转基因水平的类病毒沉默。项目以感染马铃薯纺锤体类病毒或/和啤酒花矮化类病毒的番茄“Alisa Craig”为试材，建立以弹状病毒为载体的CRISPR/LnsCas13a DNA-free类病毒编辑体系，以被编辑的带毒番茄茎尖为试材，构建高效茎尖超低温疗法脱毒体系，获得遗传稳定的脱毒种苗种子，同时初步揭示类病毒抑制和脱除的机制，为番茄无毒种子种苗可持续发展提供理论和实践基础。

Tomato（Solanum lycopersicum）is an important vegetable crop, the planting area of fresh tomato in China ranks first in the world. Viroid is the smallest known obligate parasite and quarantine pathogen. It can infect the apical meristem and reproductive organs of tomato, and spread through vegetative organs and seeds, causing serious impact on production, breeding and circulation. Conventional methods have limited effect on viroid elimination. The biological characteristics of virus and viroid are similar. Gene editing has been successfully used in virus suppression, but not in viroid silencing. Taking rhabdovirus as vector, the gene editing has the characteristics of high editing efficiency and non-heredity, which can be applied to DNA-free viroid silencing. In this research, CRISPR/Lnscas13a gene editing system with rhabdovirus as vector will be established in“Alisa Craig”tomato infected with Potato spindle tuber viroid or/and Hop stunt viroid. Taking shoot tips of edited tomato as materials, the present research will establish an efficient viroid-cryotherapy system, obtain genetically stable viroid-free plants and seeds, and preliminarily reveal the mechanism of viroid inhibition and elimination. This work provids theoretical and practical basis for the sustainable development of viroid-free tomato seeds and seedlings.