与瓜类褪绿黄化病毒P22蛋白互作的寄主因子的筛选

Screening of the Host Factors Interacting with P22 Protein of Cucurbit chlorotic yellows virus

为阐明瓜类褪绿黄化病毒(CCYV)的侵染机制,筛选与P22互作的寄主因子。采用免疫沉淀技术结合质谱分析共获得128个寄主蛋白。KEGG分析发现,所获得的寄主因子主要参与到寄主体内的碳水化合物代谢、能量代谢以及信号转移等代谢途径中。将所获得的128个基因进行GO分析,发现筛选到的与P22蛋白相互作用的寄主蛋白分为39个功能组,分别属于生物过程、分子功能和细胞组分三大类别。在生物过程类别中光合作用、蛋白质折叠、葡萄糖代谢过程及碳代谢过程为主要功能组。在分子功能类别中,ATP结合和金属离子结合为主要功能组。在细胞组分类别中生物膜、细胞质和光系统Ⅱ氧化复合体为主要功能组。结合质谱分析结果,根据富集肽段数目和蛋白质功能筛选出8个候选寄主蛋白。KEGG数据库分析表明,8个候选寄主蛋白可能参与或涉及的代谢通路大类可以归为碳水化合物代谢、能量代谢以及信号转移等3个代谢途径。利用酵母双杂交验证8个寄主因子与P22互作。结果表明,甘油醛-3-磷酸脱氢酶A(GAPDH-A)能够与P22蛋白在酵母中互作,推测P22可能影响寄主抗性并参与寄主黄化症状的产生。筛选鉴定了与P22互作的寄主因子,为阐明P22蛋白在CCYV侵染过程中的作用机制奠定了基础。

In order to clarify the infection mechanism of Cucurbit chlorotic yellow virus(CCYV),we screened host factors interacting with P22.We used immunoprecipitation and mass spectrometry to screen 128 host factors in Nicotiana benthamiana interacting with P22.KEGG analysis showed that these genes were mainly involved in the pathways of carbohydrate metabolism,energy metabolism and signal transduction.The obtained 128 host factors could be divided into 39 functional groups by GO analysis,which belonged to three categories of biological process,molecular function and cell component.In the category of biological processes,photosynthesis,protein folding,glucose metabolic process and one-carbon metabolic process were the main functional groups.In the category of molecular function,ATP binding and metal ion binding were the main functional groups.And in the category of cell components integral component of membrane,cytoplasm and photosystemⅡoxygen evolving complex were the main functional groups.Combined with the results of mass spectrometry,8 candidate host proteins were selected according to the number of enriched peptide segments and protein function.KEGG database analysis showed that the metabolic pathways involved in the 8 candidate host proteins could be classified into three metabolic pathways:carbohydrate metabolism,energy metabolism and signal transfer.Yeast two-hybrid assay was used to verify the interaction between 8 host proteins and P22.The results showed that glyceraldehyde 3-phosphate dehydrogenase-A(GAPDH-A)interacted with P22 protein.P22 may affect the resistance of host and cause chlorosis and yellowing symptoms.Taken together we identified the host factors that interact with P22 and this study would lay the foundation for uncovering the mechanism of P22 protein in the CCYV infection.