Analog-sensitive 蛋白激酶研究系统在植物病原真菌 中的建立.

Common gene knockout techniques cannot be applied to the study of some specific kinases. In order to provide new ideas and methods for the study of protein kinases, an analog-sensitive protein kinase system based on artificial modification of protein kinases was established in plant pathogenic fungi. The“ gatekeeper” residues of protein kinase were predicted via web technology, and the analog-sensitive (as) mutants gpmk1-as and pmk1-as were obtained by vector complement method. The function of analog-sensitive protein kinase and its sensitivity to ATP analogue inhibitor 1-NM-PP1 were detected. The results showed that the“ gatekeeper” residues of Gpmk1 and Pmk1 were Q103 and Q104, respectively, which were very conserved in multiple representative fungi. The colony growth rate of the analog-sensitive mutant gpmk1-as was consistent with that of the wild type PH-1, both faster than that of the Gpmk1 knockout mutant. pmk1-as produced appressorium being same with that of the wild type Guy11, but the Pmk1 knockout mutant could not do so. With 5 μmol/L 1-NM-PP1, the colony growth rate of Gpmk1-as decreased, but the growth of PH-1 was not affected. With 10 μmol/L 1-NM-PP, pmk1-as could not form appressorium, but Guy11 formed mature black appressorium. The results showed that both Gpmk1 and Pmk1 analog-sensitive protein kinases could perform normal protein functions, but were very sensitive to ATP analogue inhibitor 1-NM-PP1. [ABSTRACT FROM AUTHOR]