利用酵母双杂交筛选与苹果褪绿叶斑病毒MP互作的寄主因子

Identification of Host Factors Interacting with the Movement Protein of Apple Chlorotic Leaf Spot Virus by Yeast Two-Hybrid System

为了找到与苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)运动蛋白(Movement protein,MP)互作的寄主因子,首先构建了ACLSV MP酵母双杂交诱饵载体,然后通过顺序转化的方法自前期已构建好的苏俄苹果(Malus sylvestris cv.R12740-7A)cDNA文库中筛选互作基因;在GenBank中对互作寄主基因进行BLAST分析,根据基因注释推测其在病毒与寄主互作过程中可能发挥的作用。结果表明,构建的酵母双杂交诱饵载体pGBKT7-MP无自激活性、无毒性。筛选到69个与ACLSV MP互作的苏俄苹果寄主因子,包括水解酶活性、病程相关蛋白、氧化还原酶活性、磷酸酶活性、催化活性、连接酶活性、苯丙氨酸解氨酶活性、过氧化物酶活性、DNA结合功能和未知蛋白,共10类不同功能的蛋白。通过生物信息学分析,推断蛋白磷酸酶、病程相关蛋白及3-磷酸甘油醛脱氢酶可能在互作过程中起重要作用。该研究为深入了解ACLSV致病特征及病原与寄主互作机制奠定基础。

In order to identify host factors which interact with the movement protein（MP）of Apple chlorotic leaf spot virus（ACLSV）,ACLSV MP was cloned into the bait vector pGBKT7 and used to screen a cDNA library of Malus sylvestris cv.R12740-7A,which had previously been constructed by yeast two-hybrid sequencing transformation.The protein functions of the identified host factors were determined according to their gene annotations in GenBank.The result showed that the bait plasmid pGBKT7-MP showed no virulence or self-activating effect on yeast strain Y2 H Gold.Sixty-nine interactor proteins were identified,which were divided into the following 10 classes according to their described functions：hydrolases;pathogenesis-related proteins;DNA binding proteins;phosphatases;ligases;proteins with catalytic activity;phenylalanine ammonia-lyases;peroxidases;NAD binding proteins;and proteins of unknown function.Bioinformatic analysis of gene homology suggested that phosphatases,pathogenesis-related proteins and glyceraldehyde-3-phosphate dehydrogenase A may play an important role in the interaction between virus and host.This study may provide a theoretical basis for the further study of viral pathogenesis and virus-host interaction mechanisms.