Abstract: Anthocyanins are the main pigment of Pyrus pyrifolia, and the synthesis of anthocyanins is regulated by a series of transcription factors. PyWRKY26 is the main transcription factor involved in the anthocyanin synthesis. However, the structure of the protein is not clear. In this study, the coding sequence (CDS) full length of PyWRKY26 gene was cloned by polymerase chain reaction (PCR) from the pericarp of Starkrimson pear. PyWRKY26 was ligated with the prokaryotic expression vector pCOLD with His tag using homologous recombination technology, and the recombinant expression vector was transformed into Rosetta(DE3). Then, the recombinant protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The result showed that the CDS full length of PyWRKY26 gene was successfully amplified from the pericarp of Starkrimson pear and the recombinant expression vector of PyWRKY26-pCOLD was constructed. The PyWRKY26-pCOLD fusion protein was successfully expressed by 0.5 mmol/L IPTG for 16 h at 22 °C and purified by His-Ni column combined with SDS-PAGE. This study lays an important foundation for further studying the structure and function of PyWRKY26.
Keywords: Starkrimson pear; PyWRKY26 gene; gene cloning; prokaryotic expression; protein purification
合肥工业大学学报自科版