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

Abstract

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

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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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

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