The Raphanus sativus L. antifungal protein 1 (Rs_AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the...The Raphanus sativus L. antifungal protein 1 (Rs_AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the prokaryotic expression vector pET_32b(+), respectively. Subsequent expression showed that the Rs_AFP1 was produced in E. coli as a 27 kD fusion protein only when the N_terminal signal peptide was removed. After treatment with thrombin to remove part of the N_terminal His.tag sequence, the bacterially expressed Rs_AFP1 was used for fungal growth inhibition assay which was conducted on Verticillium dahliae Kleb., a soil_born fungus causing the cotton wilt disease. Results showed that, in the liquid medium, the Rs_AFP1 fusion protein at a concentration of 0.3 g/L clearly inhibited the growth of V. dahliae and the germination of spores. Thus the bacterially expressed fusion protein had the antifungal activity against V. dahliae.展开更多
文摘The Raphanus sativus L. antifungal protein 1 (Rs_AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the prokaryotic expression vector pET_32b(+), respectively. Subsequent expression showed that the Rs_AFP1 was produced in E. coli as a 27 kD fusion protein only when the N_terminal signal peptide was removed. After treatment with thrombin to remove part of the N_terminal His.tag sequence, the bacterially expressed Rs_AFP1 was used for fungal growth inhibition assay which was conducted on Verticillium dahliae Kleb., a soil_born fungus causing the cotton wilt disease. Results showed that, in the liquid medium, the Rs_AFP1 fusion protein at a concentration of 0.3 g/L clearly inhibited the growth of V. dahliae and the germination of spores. Thus the bacterially expressed fusion protein had the antifungal activity against V. dahliae.
