【作者】 张宇；

【导师】 刘勇；

【作者基本信息】 湖南大学 ， 化学， 2019， 博士

【摘要】 番茄褪绿病毒(Tomato chlorosis virus,ToCV)为长线形病毒科(Closteroviridae)毛形病毒属(Crinivirus)病毒,自2013年在我国山东爆发,现呈全国扩散趋势,严重威胁我国番茄等茄科重要经济作物的安全生产。加强该病毒的快速检测技术体系,病毒与寄主互作的分子机制等研究,可明确该病毒在我国的扩散分布,分析其对番茄等重要经济作物的潜在威胁,也可为该病毒病的科学防控提供技术手段和科学依据。利用原核表达的ToCV CP及HSP70蛋白作为抗原,制备获得CP蛋白和HSP70蛋白的多克隆抗体,稀释10,000倍,可特异性识别相应的抗原,为开发ToCV快速检测技术提供了技术资源。采用无缝克隆技术,构建了ToCV全长基因组cDNA侵染性克隆,利用农杆菌接种本氏烟BY2细胞和番茄,均接种不成功;农杆菌介导法接种本氏烟,接种成功率为15%。采用基因枪法,ToCV侵染性克隆质粒接种本氏烟和番茄,也均接种失败;利用PEG介导法接种本氏烟BY2细胞,ToCV侵染率可达60%。利用粉虱接种ToCV至番茄,采用相对定量技术测定接种病毒后番茄各部位的感病时间及病毒含量,结果表明10 dpi可在接种叶上检测到病毒,15 dpi接种叶以上的部位均可检测到病毒,接种20 dpi接种叶下部的叶片检测到病毒,且下部叶的含量最低,接种初期接种叶中病毒含量最低,随着病情发展,心叶的病毒含量最高。通过MDC染色、LC3蛋白含量,测定了ToCV感染番茄,对番茄自噬体途径的影响,结果表明,ToCV感染番茄,能够激活番茄的细胞自噬体途径。3-MA抑制番茄自噬体小泡的延伸,3-MA处理番茄叶片,可以促进番茄细胞自噬体途径ATG1上调表达,且促进ToCV的复制。而BFA促进ER和高尔基体解体,抑制自噬体途径启始;BFA处理番茄叶片,ATG1表达量和ToCV复制量均无差异。酵母双杂交结果表明,ToCV CP及CPm蛋白,均可与番茄自噬体途径ATG1蛋白发生互作。番茄ATG1蛋白参与自噬起始;因此,根据以上结果,推测ToCV激活番茄自噬体小泡形成启始阶段,促进不成熟自噬体小泡形成,以不成熟自噬体小泡双层膜作为复制位点,促进病毒复制。ToCV与番茄自噬体途径互作的详实分子机制,还需CO-IP和BiFC验证二者互作真实性,ATG1蛋白表达对ToCV复制的影响。更多还原

【Abstract】 Tomato chlorosis virus(ToCV),belonging to genus crinivirus of Closteroviridae,was severely epidemic threaten of important crops of Solanaceae in Shangdong province,China.Recently,ToCV was rapidly spread to most of Solanaceae crops planting region,and threated the planting safety of Solanaceae crops especially tomato.So the rapid detection method,molecular mechanisms of virus-host interaction was urgently manipulated to understand the distribution of ToCV,and its potential threaten to Solanaceae crops.The results would scientifically contribute to control ToCV in field.The polyantibodies of ToCV CP and HSP70 protein were prepared using prokaryotic expressed and purified ToCV CP and HSP70 protein as antigen.Western blotting and ELISA detection resulted showed diluted 10,000 × of polyantibodies of ToCV CP and HSP70 protein still recognize the ToCV.The ToCV full-genomic cDNA infectious clone was constructed using the seamless cloning.The ToCV infectious clone could not infect BY2 cell line of Nicotiana benthamiana,and tomato using Agrobacteria infiltration,N.benthamiana and tomato by particle bombardment.The ToCV infectious clone infected of 15% N.benthamiana plant using Agrobacteria infiltration.The infection ratio of the ToCV infectious clone was the highest upto 60% to BY2 cell line of N.benthamiana using PEG mediated method.The ToCV distribution in tomato plant was quantified by qRT-PCR.ToCV was inoculated to tomato plant by whitefly fed with ToCV infected tomato,and qRT-PCR results showed ToCV locate only in inoculated leafs in 10 dpi,and move to upper leafs in 15 dpi,move to lower leafs in 20 dpi.The highest concentration of ToCV was located in new shoots.The autophagy pathway possesses critical functions in plant viruses infecting host.The effect of ToCV influencing to tomato autophagy was tested by MDS staining and LC3 protein biosynthesis.The results showed ToCV could activate tomato autophagy,and further experiments testified inhibiting autophagosome vesicles mature by 3-MA,not inhibiting autophagosome biogenesis by BFA enhances ToCV replication.Yeast two-hybrid test verified ToCV CP and CPm could interact with autophagy ATG1 protein.Together,it could be deduced ToCV inhibits autophagosome vesicles mature,and uses double-membrane of unmatured autophagosome vesicles as replication cites to enhance ToCV replication.The unequivocal molecular mechanisms of ToCV interacting with host autophagy pathway w ould be uncovered by testifying the veracity of ToCV CP/CPm interacting with ATG1,which are key proteins involving in host autophagy pathway,and medulating ToCV replicaiton by ex pressing of ATG1.更多还原