Yeast Two-hybrid System Of Barley Yellow Dwarf Virus (of Bydv) And Wheat Interaction

Yeast two-hybrid system is a very effective method in studying protein-protein interaction. This system can analyze the protein's interaction of physiological situation in eukaryotic cells rapidly and sensitively, and target protein's coding gene can be acquired quickly, with the characteristics of conveniece and high sensitivity, which had been used in many aspects of plant virology. In the research, the system was used to study the interaction between Barley Yellow Dwarf Virus (BYDV) and wheat, and exploration had been made for the movement mode of virus' building .Synthesizing the primers of CP and MP gene's upstream and downstream according to the known BYDV-GPV strain's coat protein and movement protein gene sequence. Target fragments were aquired by means of PCR amplification, which were cleaved with restrict endonuclase Sal I I Pst I, ligation, transformation, recombinant plasmids' restrict identification and gene sequencing, Yeast expression vectors pGBKT7-GPV-CP and pGBKT7-GPV-MP were constructed, for expressing the bait fusion protein in yeast two-hybrid analysis.Yeast strains AH109 Y187 and vector pGADT7-Rec were tested using SD selective medium and yeast cotransformation positive compared experiments, The result indicated the strains and vector both corresponded with the requests of two-hybrid analysis. Then pGBKT7-GPV-CP or pGBKT7-GPV-MP, wheat cDNA library and pGADT7-Rec were transferred into yeast competent cells, which were spreaded on the SD selective medium, by this way, galactosidase's color-producing testing was made. In the end, there was no interaction tested in the result, which can indicate that there might be no host factor which can interact with BYDV in wheat cDNA library. The result of screening by means of yeast mating coincided with the cotransformation, which proved the conclusion above.