A technology to capture DNA binding proteins based on biolistic transformation

Interactions between DNA and proteins are critical to almost all cellular functions, and the identification of proteins that bind to a DNA sequence of interest (gene-centered approach) is a widely studied area. However, a limited number of gene-centered methods have been developed. In the present study, we built a protein capture technology based on biolistic transformation (PCaT) to capture proteins bound by a target DNA. A promoter sequence or a DNA fragment was labeled with biotin and introduced into the host plants using biolistic transformation. The DNA and its bound proteins were cross-linked using formaldehyde in the transformed plants, and the labeled DNAs were captured using streptavidin magnetic beads, which enabled harvesting of the DNA-bound proteins. After mass spectrum analysis, the proteins associated with the target DNA were identified. Using this method, we identified the upstream regulators of a WRKY gene from Populus davidiana×P. bolleana. The reliability of the identification of the upstream regulators of WRKY was confirmed using chromatin immunoprecipitation and electrophoretic mobility shift assays. Taken together, the results showed PCaT provides a novel DNA and protein interactome discovery strategy. In addition, based on PCaT, a quick procedure to determine the upstream regulators of a gene of interest was proposed.