Prokaryotic expression of Arabidopsis AtMYB50 and AtMYB61 fusion proteins

Total RNA was extracted from Arabidopsis seedlings,and cDNA fragments of AtMYB50 and AtMYB61genes were amplified by RT-PCR.cDNA fragments were subsequently cloned into pEASY-Blunt vectors,and then were transformed into Trans-T1phage resistant chemically competent cells.The analysis of bacterial colony PCR and cDNA sequencing were performed to confirm that cDNA of the Arabidopsis thaliana AtMYB50and AtMYB61genes was successfully cloned.Their cDNA fragments with sticky ends were isolated by using restriction enzyme digested.The cDNA fragments were cloned into prokaryotic expression vectors pET-32a+and pGEX-4T-1.In addition,the recombinant plasmids were transformed into BL21(DE3)plysS chemically competent cells.The expression of MYB61and MYB50was induced to generate their fusion proteins by IPTG.The protein expression conditions such as the inducing time and temperature and the IPTG concentration were optimized,and the water solubility of the protein was tested.