葡萄浆果内坏死病毒侵染的‘贝达'葡萄cDNA文库构建及其利用

 葡萄浆果内坏死病毒(grapevine berry inner necrosis virus, GINV)侵染葡萄可引起葡萄叶片表现明显的褪绿斑驳症状,其致病的分子机制,特别是病毒与寄主互作蛋白的研究尚未见报道。本研究构建了GINV侵染的‘贝达'葡萄叶片的cDNA文库,并以GINV 外壳蛋白(coat protein,CP)为诱饵筛选文库中与其互作的寄主因子。本研究构建的cDNA文库库容量达1.6×10⁷,插入片段平均大小在1.0 kb以上,质量符合cDNA文库筛库要求。筛选到了55个候选寄主蛋白与GINV CP在酵母中互作。经测序分析及回转验证,结果确定了17个寄主互作蛋白,其涉及叶绿体相关类蛋白、泛素化相关蛋白、防御相关蛋白等。本研究可为深入研究GINV致病性及其与寄主互作机制提供依据。

Construction and screening of a yeast two-hybrid cDNA library from Beta grapevine infected by grapevine berry inner necrosis virus

Grapevine berry inner necrosis virus (GINV) can cause obvious chlorotic mottling symptoms on grape leaves. Currently, the molecular mechanism of its pathogenesis, especially the interaction between GINV and host protein has not been reported. In this study, the cDNA library of GINV-infected Beta grapevine leaves was constructed, and GINV coat protein (CP) was used as the bait to screen the host factors in the library. The cDNA library constructed in this study contains 1.6×10⁷ clones with average size of inserted fragments being more than 1.0 kb, and the quality meets the requirements of cDNA library screening. A total of 55 candidate genes interacted with GINV CP in yeast. Sequencing and verification of positive clones showed that these genes encoded a total of 17 host proteins, including chloroplast photosynthesis-related proteins, ubiquitination-related proteins and defense-related proteins, and so on. This study provides a basis for further research on GINV pathogenicity and mechanism of interaction