利用酵母双杂交系统筛选介体异沙叶蝉中与小麦矮缩病毒外壳蛋白互作的蛋白质

doi:10.3864/j.issn.0578-1752.2015.12.008

摘要/Abstract

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.）cDNA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 cDNA，经过Sfi I酶切纯化，连接到pPR3-N文库载体上，构建得到以pPR3-N为载体的异沙叶蝉分离泛素酵母双杂交膜系统cDNA文库。同时，构建带有Sfi I酶切位点的诱饵载体pDHB1-WDV CP，经功能检测后用诱饵载体初步筛选pPR3-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cDNA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2.0×10⁶cfu，文库实际扩增数量大于1.3×10⁶cfu，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cDNA文库的质量较高。酶切验证显示诱饵载体pDHB1-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cDNA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、信号模式识别受体的活性、水解酶活性、磷酸离子载体活性和叶酸运输活性等。参考KEGG数据库，这些蛋白参与的代谢途径有泛素介导的蛋白水解途径、内吞作用、花生四烯酸代谢途径、cAMP信号通路和模式识别受体的信号通路等。【结论】异沙叶蝉分离泛素酵母双杂交膜系统cDNA文库的成功构建与筛选，为研究异沙叶蝉与小麦矮缩病毒的互作机制研究奠定了基础。

关键词: 异沙叶蝉, 小麦矮缩病毒, SMART技术, cDNA文库, 分离泛素酵母双杂膜系统

Abstract: 【Objective】To investigate the interaction between the leafhopper (Psammotettix alienus L.) and Wheat dwarf virus (WDV), a cDNA 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 cDNA 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 cDNA was synthesized using SMART technology. After digestion with the Sfi I enzyme, the fragmented cDNA was ligated to prey vector pPR3-N, and then also digested with Sfi I enzyme to construct the split-ubiquitin yeast membrane system cDNA library. The full-length gene, WDV CP, amplified from wheat leaves infected by WDV was ligated into bait fusion vector, pDHB1. After functional assay, pDHB1-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 pDHB1-WDV CP were screened from the cDNA library of P.alienus. Gene ontology (GO) and pathway information of proteins were analyzed from Uniprot and KEGG websites.【Result】Detection of the cDNA library showed that the unamplified library contained 2.0×10⁶ independent clones, the titer of the amplified library was 1.3×10⁶cfu. The recombination rate was above 97%. The sizes of most inserts were above 1 kb in the cDNA library. The correct ligated fusion bait vector pDHB1-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 cDNA 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. GO annotation analysis showed 9 putative proteins were involved in 10 biological processes including protein dephosphorylation, carbohydrate metabolic process, transport, etc. Molecular functions included metal ion binding, phosphate ion carrier activity, folic acid transporter activity, protein complex binding, etc. These proteins also were involved in ubiquitin mediated proteolysis, endocytosis, arachidonic acid metabolism, cAMP signaling pathway, PPAR signaling pathway.【Conclusion】A high-quality cDNA library was constructed and 9 proteins were interacted with WDV CP, which could be used for insect vector and WDV interaction analysis.

Key words: leafhopper (Psammotettix alienus L.), Wheat dwarf virus (WDV), SMART technology, cDNA library, split- ubiquitin yeast membrane system

赵艺泽，刘艳，王锡锋. 利用酵母双杂交系统筛选介体异沙叶蝉中与小麦矮缩病毒外壳蛋白互作的蛋白质[J]. 中国农业科学, 2015, 48(12): 2354-2363.

ZHAO Yi-ze, LIU Yan, WANG Xi-feng. Screening of Putative Proteins in Vector Psammotettix alienus L. that are Interacted with Coat Protein of Wheat dwarf virus by a Split-ubiquitin Yeast Membrane System[J]. Scientia Agricultura Sinica, 2015, 48(12): 2354-2363.

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