中文摘要：

【目的】利用分离泛素酵母双杂交膜系统（split-ubiquitin yeast membrane system）,以小麦矮缩病毒（Wheat dwarf virus,WDV）的外壳蛋白（CP）基因为诱饵对异沙叶蝉（Psammotettix alienus L.）c DNA文库进行筛选,研究异沙叶蝉传播WDV的分子机制。【方法】以笔者实验室饲养的异沙叶蝉为材料,提取其总RNA后取100 ng进行纯化,利用SMART法反转录合成ds c DNA,经过Sfi I酶切纯化,连接到p PR3-N文库载体上,构建得到以p PR3-N为载体的异沙叶蝉分离泛素酵母双杂交膜系统c DNA文库。同时,构建带有Sfi I酶切位点的诱饵载体p DHB1-WDV CP,经功能检测后用诱饵载体初步筛选p PR3-N空文库,寻找适合筛库的条件和确定His基因产物抑制剂3-氨基-1,2,4-三唑（3-AT）的使用浓度。然后用诱饵载体筛选异沙叶蝉c DNA文库,对筛选结果进行分析,再通过共转验证和β-半乳糖苷酶检测进一步验证是否发生互作。利用Uniprot和KEGG在线网站,对筛到的蛋白进行gene ontology（GO）注释和Pathway分析。【结果】初级文库库容量超过2.0×106 cfu,文库实际扩增数量大于1.3×106 cfu,文库重组率大于97%,扩增文库插入片段平均长度大于1 000 bp,表明异沙叶蝉c DNA文库的质量较高。酶切验证显示诱饵载体p DHB1-WDV-CP中CP的插入完整而准确。功能检测表明融合蛋白能够正确表达。在3-AT浓度为5 mmol？L-1的筛选条件下,诱饵载体筛选异沙叶蝉c DNA文库得到280个克隆,经测序和Blast比对分析最终得到12个可能与WDV的CP发生互作的异沙叶蝉蛋白质。将这12个蛋白质再次进行共转验证和β-半乳糖苷酶检测,最终得到9个蛋白质与WDV CP互作。GO注释显示,9个蛋白参与的生物过程包括蛋白去磷酸化、碳水化合物代谢过程、先天性免疫应答、模式识别受体的信号通路、运输、同向运输和乙醇氧化等;分子功能包括金属离子结合活性、蛋白磷酸酶活性、信号模式识别

英文摘要：

【Objective】To investigate the interaction between the leafhopper（Psammotettix alienus L.） and Wheat dwarf virus（WDV）, a c DNA library of leafhopper was constructed using a split-ubiquitin yeast membrane system. The protein interaction analysis was done by using WDV CP as bait protein to screen a c DNA library of P. alienus. 【Method】Total RNA of leafhopper was isolated from 2 g of insects. Poly A＋ RNA was enriched from 100 ng of total RNA and double-stranded c DNA was synthesized using SMART technology. After digestion with the Sfi I enzyme, the fragmented c DNA was ligated to prey vector p PR3-N, and then also digested with Sfi I enzyme to construct the split-ubiquitin yeast membrane system c DNA library. The full-length gene, WDV CP, amplified from wheat leaves infected by WDV was ligated into bait fusion vector, p DHB1. After functional assay, p DHB1-WDV CP vector was co-transformed into NMY51 with empty library vector in order to get an optional concentration of 3-AT. Then using the split-ubiquitin yeast membrane system, proteins interact with the bait p DHB1-WDV CP were screened from the c DNA library of P. alienus. Gene ontology（GO） and pathway information of proteins were analyzed from Uniprot and KEGG websites.【Result】Detection of the c DNA library showed that the unamplified library contained 2.0×106 independent clones, the titer of the amplified library was 1.3×106 cfu. The recombination rate was above 97%. The sizes of most inserts were above 1 kb in the c DNA library. The correct ligated fusion bait vector p DHB1-WDV CP was verified by restriction enzyme digestion analysis and sequencing. Functional assay showed that the fusion protein was functionally correctly expressed in the yeast and suited to this system. In library screen test, 280 clones were got from the c DNA library of P. alienus. Twelve proteins were selected for further research based on the functional analysis in terms of GO. Finally, 9 proteins confirmed by β-galactosidase assay were interacted with WDV CP. G