烟草翻译控制肿瘤蛋白NtTCTP在马铃薯Y病毒侵染过程中的功能研究

Potato virus Y is one of most important virus which causes serious losses in yield and qualityin for many economical crops, such as tobacco. To completely control PVY disease, breeding resistant tobacco varieties is the key point on the basis of anti-PVY genes identifications. Our previous research showed that tobacco Translationally Controlled Tumor Protein (NtTCTP) exhibited high resistance against PVY although the molecular mechanism was unrevealed. On the basis of tobacco yeast cDNA library construction, we will utilize yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP) and pull-down technique to screen and verify the proteins which could interact with NtTCTP. The transgenic tobacco plants of the interacting proteins, including over-expression and knock out by CRISPR/Cas9, will be created for clarify the anti-virus performance by measuring replication, symptom development and other physical factors. Theses results will be good evidences to illustrate the molecular mechanism of PVY resistance mediated by NtTCTP

马铃薯Y病毒（potato virus Y，PVY）在生产上严重危害烟草等经济作物的产量和品质，多年来造成了重大的经济损失。克隆和鉴定植物中抗PVY基因并以此为基础创制抗性材料是从根本上解决此类病毒病害的关键。申请人在前期研究中发现烟草翻译控制肿瘤蛋白基因NtTCTP对PVY的侵染有明显的抗性，但其机制尚不清楚。本项目针对NtTCTP介导的PVY抗性机制这一科学问题，构建烟草cDNA文库，利用酵母双杂交技术筛选烟草中与NtTCTP互作的蛋白，并通过pull-down、BiFC和Co-IP等方法对互作结果进行验证。进一步构建互作蛋白基因的过表达和基因编辑载体并转化烟草，研究互作蛋白的基因在烟草中过量表达和敲除条件下对PVY侵染的影响，进而阐述NtTCTP介导的PVY抗性分子机制。

抗性基因资源的利用是现阶段解决植物病毒病害的主要方法。前期研究发现，烟草NtTCTP基因与PVY的抗性有直接关系，是可以利用的抗性基因资源。本项目针对NtTCTP如何介导的PVY抗性这一科学问题，构建酵母cDNA文库，利用酵母双杂交技术筛选到4个与NtTCTP互作的蛋白，通过IP、BiFC的方法明确了NtFBA1与NtFBA6与NtTCTP的互作关系。利用不同的缺失突变体，确定FBA6前160aa是与TCTP的互作核心区段。以过表达以及RNAi转基因植物材料，明确了FBA6的表达和缺失不影响TCTP的表达，但是NtFBA过表达可以促进病毒积累，而沉默NtFBA6会影响PVY的积累并推迟发病时间。本项目研究为进一步发掘抗性基因资源从而实现抗病育种奠定基础。