中國農業科學 ?? 2015, Vol. 48 ?? Issue (12): 2354-2363.doi: 10.3864/j.issn.0578-1752.2015.12.008

? 植物保護 ? 上一篇    下一篇

利用酵母雙雜交系統篩選介體異沙葉蟬中與小麥矮縮病毒外殼蛋白互作的蛋白質

趙藝澤,劉艷,王錫鋒   

  1. 中國農業科學院植物保護研究所植物病蟲害生物學國家重點實驗室,北京 100193
  • 收稿日期:2014-12-12 出版日期:2015-06-16 發布日期:2015-06-16
  • 通訊作者: 王錫鋒,Tel:010-62815928;E-mail:[email protected] E-mail:[email protected]
  • 作者簡介:趙藝澤,E-mail:[email protected]
  • 基金資助:
    國家自然科學基金(31272017)、國家公益性行業(農業)科研專項(201303021)

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

ZHAO Yi-ze, LIU Yan, WANG Xi-feng   

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2014-12-12 Online:2015-06-16 Published:2015-06-16

摘要: 【目的】利用分離泛素酵母雙雜交膜系統(split-ubiquitin yeast membrane system),以小麥矮縮病毒Wheat dwarf virus,WDV)的外殼蛋白(CP)基因為誘餌對異沙葉蟬(Psammotettix alienus L.)cDNA文庫進行篩選,研究異沙葉蟬傳播WDV的分子機制。【方法】以筆者實驗室飼養的異沙葉蟬為材料,提取其總RNA后取100 ng進行純化,利用SMART法反轉錄合成ds cDNA,經過Sfi I酶切純化,連接到pPR3-N文庫載體上,構建得到以pPR3-N為載體的異沙葉蟬分離泛素酵母雙雜交膜系統cDNA文庫。同時,構建帶有Sfi I酶切位點的誘餌載體pDHB1-WDV CP,經功能檢測后用誘餌載體初步篩選pPR3-N空文庫,尋找適合篩庫的條件和確定His基因產物抑制劑3-氨基-1,2,4-三唑(3-AT)的使用濃度。然后用誘餌載體篩選異沙葉蟬cDNA文庫,對篩選結果進行分析,再通過共轉驗證和β-半乳糖苷酶檢測進一步驗證是否發生互作。利用Uniprot和KEGG在線網站,對篩到的蛋白進行gene ontology(GO)注釋和Pathway分析。【結果】初級文庫庫容量超過2.0×106 cfu,文庫實際擴增數量大于1.3×106 cfu,文庫重組率大于97%,擴增文庫插入片段平均長度大于1 000 bp,表明異沙葉蟬cDNA文庫的質量較高。酶切驗證顯示誘餌載體pDHB1-WDV-CP中CP的插入完整而準確。功能檢測表明融合蛋白能夠正確表達。3-AT濃度為5 mmol?L-1的篩選條件下,誘餌載體篩選異沙葉蟬cDNA文庫得到280個克隆,經測序和Blast比對分析最終得到12個可能與WDV的CP發生互作的異沙葉蟬蛋白質。將這12個蛋白質再次進行共轉驗證和β-半乳糖苷酶檢測,最終得到9個蛋白質與WDV CP互作。GO注釋顯示,9個蛋白參與的生物過程包括蛋白去磷酸化、碳水化合物代謝過程、先天性免疫應答、模式識別受體的信號通路、運輸、同向運輸和乙醇氧化等;分子功能包括金屬離子結合活性、蛋白磷酸酶活性、信號模式識別受體的活性、水解酶活性、磷酸離子載體活性和葉酸運輸活性等。參考KEGG數據庫,這些蛋白參與的代謝途徑有泛素介導的蛋白水解途徑、內吞作用、花生四烯酸代謝途徑、cAMP信號通路和模式識別受體的信號通路等。【結論】異沙葉蟬分離泛素酵母雙雜交膜系統cDNA文庫的成功構建與篩選,為研究異沙葉蟬與小麥矮縮病毒的互作機制研究奠定了基礎。

關鍵詞: 異沙葉蟬, 小麥矮縮病毒, SMART技術, cDNA文庫, 分離泛素酵母雙雜膜系統

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 1.3×106 cfu. 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

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