基于酵母双杂交系统筛选灰飞虱体内与大麦黄条点花叶病毒核衣壳蛋白互作的蛋白质

Screening of putative proteins in the small brown planthopper interacting with nucleocapsid protein of Barley yellow striate mosaic virus by yeast two-hybrid system

为了获取影响大麦黄条点花叶病毒(BYSMV)在灰飞虱体内增殖、积累和传播的相关介体因子,本研究利用分离泛素酵母双杂交膜系统,以BYSMV核衣壳蛋白(N)为诱饵对灰飞虱c DNA文库进行了筛选。将BYSMV N基因构建到诱饵载体p DHB1上进行表达检测和功能验证,结果表明重组载体p DHB1-N能在酵母内正常表达并行使功能。利用诱饵载体筛选p PR3-N空文库对文库筛选条件进行优化,确定3-氨基-1,2,4-三唑(3-AT)浓度为12 mmol·L-1的QDO平板为筛选文库培养基条件,去除可能存在的轻微筛库背景。在此筛选条件下以诱饵载体从灰飞虱c DNA文库中筛选得到57个阳性克隆,序列比对结果表明这些阳性克隆编码17种候选蛋白,包括表皮蛋白、泛素B、核糖体膜相关蛋白、细胞色素b5以及海藻糖转运蛋白等。经酵母双杂交共转验证和β-半乳糖苷酶检测进一步确认了这17个候选蛋白与BYSMV N发生互作。本研究成功从灰飞虱分离泛素酵母双杂交膜系统c DNA文库筛选到与BYSMV N互作的蛋白质,为进一步探索弹状病毒与介体昆虫的分子互作机制奠定了基础。

To reveal vector factors in Laodelphax striatellus Fallen(small brown planthopper,SBPH)that are related to proliferation,accumulation,and transmission of Barley yellow striate mosaic virus(BYSMV),the nucleocapsid protein of BYSMV was selected as bait to screen a c DNA library of SBPH using a split-ubiquitin yeast membrane system.Firstly,the bait plasmid was constructed by fusing the full-length BYSMV N gene into p DHB1.The result of the expression and the functional assay of the bait showed that the fusion bait protein was correctly expressed and functional in the yeast.In the pilot screen,the bait vector was co-transformed into NMY51 with empty library vector p PR3-N plated on TDO and QDO-SD medium with different 3-AT concentrations to remove the slight background.Then,the QDO-SD medium with 12 mmol·L-13-AT was selected for the library screening on which no growth appeared.As a result,57 positive colonies were obtained from the library screening and 17 proteins were identified by searching against NCBI database,such as cuticular protein,polyubiquitin-B,ribosome-associated membrane protein,cytochrome b5 and trehalose transporter.The interaction between BYSMV N and the 17 putative proteins were further confirmed by retransformation assay andβ-Galactosidase assay.In this study,vector proteins interacted with BYSMV N were successfully identified from the cDNA library of SBPH using the split-ubiquitin yeast membrane system,thus establishing a foundation for further exploration of the molecular interaction mechanism between rhabdovirus and the insect vector.