Selection Of Peptides Which Interact With Proteins Of Barley Yellow Dwarf Virus By Yeast Two-Hybrid System

Barley yellow dwarf virus (BYDV) is reported to be the most important viral pathogen of cereals worldwide. In fact, BYDV is a group of at least five luteoviruses that cause similar symptoms in cereals but can be distinguished serologically and on the basis of vector specificity. BYDV-GPV, which was transmitted efficiently by the aphid species Schizaphis graminum and Rhopaloshiphum padi, causes severe problems in Chinese wheat crops but not recorded elsewhere. BYDV-GAV, the dominant strain in China, is transmitted by the vector of Macrosiphum avenae and Schizaphis graminum. As BYDV particles seem to be confined to the phloem, very low yields of virus can be obtained when attempt to extract them from infected plants, which increase the difficulty in BYDV molecular biology research.The development of the yeast two-hybrid system provided a genetic means to identify proteins that physically interact in vivo. The goal of the system is to isolate a protein or proteins that interact with a "bait" protein; this bait protein can be almost any protein from any organism. Over the past ten years, this system and its variants have been extremely useful for detecting and analyzing protein-protein interactions in many fields. This work is research for the interaction between "bait" protein and its partner.GPV-MP, GPV-CP and GAV-MP gene were amplified by PCR using specific primers designed on the basis of the sequence information obtained from GenBank, PCR product was cloned into a GAL4 DNA binding domain (BD)-expression vector pGBKT7 to construct recombinant plasmid pGBKT7-GAV-MP, pGBKT7-GPV-MP and pGBKT7-GPV-CP.The two types of hybrid plasmids, pGAD GH-RP (purchased from Clontech) and pGBKT7-GPV-MP, are co-transformed into the yeast strain CGI945 according to the manufacturer's instruction, and the transformants were selected on the correct solid synthetic dropout media. Use this assay, we obtain two short peptides, PHRGGLEWMGAIAGPV, TVSRRMRDCRQRIVCA respectively, after been sequenced and analyzed, co-transformed them with pGBKT7-GAV-MP and pGBKT7-GPV-CP, respectively. But we do not detect interaction between them. So the interaction of pGBKT7-GPV-MP and the two peptides maybe specific. The result will be pave the way for engineering peptide-mediate virus-resistance plant.