三叶青是中国特有药用植物，可退热、抗炎、抑瘤，被誉为植物抗生素。三叶青多用无性繁殖，易染病毒，种性易退化。脱毒可恢复种性是学界共识。基于超低温保存的超低温疗法是一种新型高效脱毒技术。我们前期研究表明，超低温疗法可脱除三叶青病毒，也可促进其离体根膨大。膨大的脱毒离体根极易成活，可代替脱毒苗应用于生产，加快地下块根成熟，也可用于下游深加工，生产次生代谢物，是保护野生资源和缓解供需矛盾的有效途径。目前关于超低温疗法脱毒促进离体根膨大的研究尚未见报道。本课题将以怀玉山三叶青为研究对象，进行超低温疗法脱毒，再采用RNA-Seq技术对其膨大离体根进行转录组分析，筛选相关候选基因，克隆其全长cDNA序列，并通过遗传转化过量表达研究其功能和亚细胞定位，最后结合形态解剖、生理生化、DNA甲基化、氧化酶基因和激素相关基因表达等分析，诠释超低温疗法脱毒促进离体根膨大的机理，为三叶青种质保存及其栽培提供理论依据。

Tetrastigma hemsleyanum Diels et Gilg is a rare and endangered medicinal plant unique to China, which can resist fever, inflammation and tumor and is known as "plant antibiotics ". Tetrastigma hemsleyanum Diels et Gilg commonly uses asexual propagation, which is susceptible to viruses and prone to species degeneration. It is a consensus in academia that virus-free can recover variety. Cryotherapy based on cryopreservation is a new highly effective virus-free technique which has been developed in recent years and has two functions: germplasm conservation and virus free. Our previous studies have shown that cryotherapy can not only remove the viruses of Tetrastigma hemsleyanum Diels et Gilg, but also promote the thickening of its roots in vitro. Virus-free thickening roots in vitro are easy to transport, survive and germinate and can be directly used in production instead of virus-free plantlets, which can shorten the maturation time of underground tuber roots and can also be directly used in downstream deep processing to produce secondary metabolites. It is an effective way to protect their wild resources and solve the contradiction between supply and demand in the market. At present, there are no reports on the study of cryotherapy promoting the thickening of roots in vitro. This project will take Tetrastigma hemsleyanum Diels et Gilg from Huaiyushan Mountain as the research object. First, its virus-free by cryotherapy plantlets will be obtained. Then, RNA-Seq technique will be used to analyze the transcriptome of its thickening roots in vitro. Candidate genes related to thickening of roots in vitro will be screened out and their full-length cDNA sequences will be cloned. Their functions and subcellular localizations will be studied by over-expression of genetic transformation. Finally, combined with morphologic anatomy, physiological and biochemical indexes, DNA methylation, oxidant enzyme gene and hormone-related gene expression analysis, the mechanism of virus-free by cryotherapy promoting the thickening of roots in vitro will be preliminarily explained, which will provide a theoretical basis for the germplasm conservation and the container cultivation under alpine forests of Tetrastigma hemsleyanum Diels et Gilg.