利用酵母双杂交筛选与苹果褪绿叶斑病毒CP互作的寄主因子

doi:10.3864/j.issn.0578-1752.2014.24.005

摘要/Abstract

摘要：

【目的】利用酵母双杂交系统筛选与苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus, ACLSV）外壳蛋白（coat protein, CP）互作的寄主因子，利用生物信息学方法分析其在病毒与寄主互作过程中可能发挥的作用。【方法】首先构建ACLSV CP的酵母双杂交诱饵载体pGBKT7-CP，转化至酵母菌株Y2H gold中，测定转化菌在SD/-Trp、SD/-Trp/-His和SD/-Trp/-Ade-X-α-Gal平板上的生长情况，从而判断其在酵母双杂交过程中对报告基因的自激活活性。将pGBKT7-CP及pGBKT7分别转化至酵母菌株Y2H gold，培养不同时间后，用紫外分光光度计测定OD₆₀₀值，分析pGBKT7-CP对酵母菌生长的影响；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文库中钓取与ACLSV CP互作的因子，最终在SD/-Ade/-His/-Leu/-Trp/X-α-Gal平板上筛选生长良好的蓝色阳性菌落，测定序列，并在GenBank中对其进行BLAST分析，根据基因注释推断其在病毒与寄主互作过程中可能发挥的作用。【结果】成功构建了酵母双杂交诱饵载体pGBKT7-CP，转化至酵母菌株Y2H gold后，转化菌只能在SD/-Trp平板上正常生长，不能在SD/-Trp/-His和SD/-Trp/-Ade-X-α-Gal平板上生长，表明重组质粒自身不能激活报告基因His及Ade的表达，没有自激活活性。酵母双杂诱饵载体pGBKT7-CP与对照空质粒转化菌生长情况相似，证明诱饵载体对酵母菌没有毒性；筛选到了69个互作寄主基因，包括转录因子活性、碳固定活性、细胞内铁离子平衡、防御反应、水解酶活性、氧化酶活性、氧化还原酶活性、光系统II组件、绑定蛋白等10类蛋白，经生物信息学分析，其中8个寄主基因（Malus3-1、Malus4-2、Malus4q1、Malus31-3、Malus49q2、Malus15-1、Malus6-2、Malus91-4）可能在病毒与寄主互作过程中起重要作用。【结论】分析筛选到的重要寄主蛋白功能，推测ACLSV CP与BZIP类、MYB类转录因子、病程相关蛋白（PR-5、PR-8、PR-10）互作，可能调控了寄主对病毒的抗性作用；与光系统II（PSII）的稳定性/装配因子蛋白、铁结合蛋白互作，影响植物光系统的稳定性及叶绿素的形成，从而降低光合作用。这将为揭示ACLSV导致寄主产生褪绿叶斑及树体衰退的原因提供理论依据。

关键词: 苹果褪绿叶斑病毒, 苏俄苹果, 酵母双杂交, 互作基因

Abstract:

【Objective】 The objective of this study is to screen the host factors interacting with coat protein (CP) of Apple chlorotic leaf spot virusthe method of bioinformatics. 【Method】 Firstly, the bait vector of pGBKT7-CP was constructed and transformed into Y2H gold. The growth of the transformed yeast was assayed on the culture plate of SD/-Trp, SD/-Trp/-His and SD/-Trp/-Ade-X-α-Gal to make clear the self-activating effect of pGBKT7-CP. The plasmids of pGBKT7-CPand pGBKT7 were transformed into Y2H gold, the OD₆₀₀ of which was tested by ultraviolet spectrophotometer at different stages. The effect of pGBKT7-CP on the yeast was analyzed. Secondly, the interactive proteins of CP were screened from cDNA library of Malus sylvestristhe interaction between host and virus was deduced based on gene annotation. 【Result】 The bait vector of pGBKT7-CP was constructed. The transformed yeast Y2H gold was able to grow on the plate of SD/-Trp, but not able to grow on the plate of SD/-Trp/-His and SD/-Trp/-Ade-X-α-Gal, which implied that the recombined plasmid had no ability to activate the reporter gene of His and Ade. The growth of pGBKT7-CPand the control was similar, which predicated that the recombined plasmid had no virulence to the yeast. Sixty-nine interactive factors were obtained, which were divided into 10 types of proteins based on their functions, including transcription factor activity, carbon fixation activity, cellular iron ion homeostasis, defense response, hydrolase activity, oxidase activity, oxidoreductase activity, photosystem II assembly and protein binding activity. Eight host genes (Malus 【Conclusion】According to the results, the function of the important proteins was analyzed. It was presumed that ACLSV CP interacting with BZIP, MYB transcription factors, pathogenesis-related protein (PR-5, PR-8, PR-10), which may regulate the host resistance to virus. CP interacting with photosystem II stability/assembly factor protein and iron-binding protein may affect the stability of photosystem and the formation of chlorophyll, thereby reducing photosynthesis. This will provide a theoretical basis for revealing the mechanism of chlorotic green leaf spot and tree decline caused by ACLSV.3-1, Malus 4-2, Malus 4q1, Malus 31-3, Malus 49q2, Malus 15-1, Malus 6-2, and Malus 91-4) may play a great role in the interaction between host and virus. cv. R12740-7A constructed early by yeast two-hybrid sequencing transformation. At last, the positive clones were selected from the culture plate of SD/-Ade/-His/-Leu/-Trp/X-α-Gal and sequenced. BLAST analysis was made for the host factors in the GenBank and the function in (ACLSV) by yeast two-hybrid system and analyze their function in the interaction between host and virus by

Key words: Apple chlorotic leaf spot virus, Malus sylvestris cv. R12740-7A, yeast two-hybrid system, interaction gene

何乙坤，钟敏，胡同乐，王树桐，段豪，丁丽，王亚南，曹克强. 利用酵母双杂交筛选与苹果褪绿叶斑病毒CP互作的寄主因子[J]. 中国农业科学, 2014, 47(24): 4821-4829.

HE Yi-kun, ZHONG Min, HU Tong-le, WANG Shu-tong, DUAN Hao, DING Li, WANG Ya-nan, CAO Ke-qiang. Screening of the Host Factors Interacting with CP of Apple chlorotic leaf spot virus by Yeast Two-Hybrid System[J]. Scientia Agricultura Sinica, 2014, 47(24): 4821-4829.

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