Analog-sensitive蛋白激酶研究系统在植物病原真菌中的建立

Establishment of Analog-sensitive Protein Kinase Research System in Plant Pathogenic Fungi

常用的基因敲除技术无法应用于某些特殊激酶的研究,旨为在植物病原真菌中建立基于蛋白激酶人工改造的analog-sensitive蛋白激酶研究系统,为蛋白激酶的研究提供新的思路和方法。采用网站预测蛋白激酶的“守门员”残基,用载体回补法对其进行突变获得相应的类似物敏感型(analog-sensitive,as)突变体gpmk1-as与pmk1-as,并检测类似物敏感型蛋白激酶的功能及其对ATP类似物抑制剂1-NM-PP1的敏感性。结果显示,Gpmk1与Pmk1的守门员残基分别为Q103和Q104,此位点在多个代表性真菌中均十分保守;类似物敏感型突变体gpmk1-as的菌落生长速度与野生型PH-1一致,均快于Gpmk1敲除突变体,Pmk1-as能够产生与野生型Guy11一样成熟的黑化附着孢,而Pmk1敲除突变体无法产生;在5μmol/L的1-NM-PP1条件下,gpmk1-as的菌落生长速度下降,但PH-1正常生长,在10μmol/L的1-NM-PP条件下,pmk1-as无法形成附着孢,但Guy11可以形成成熟的黑化附着孢。结果表明,Gpmk1和Pmk1的类似物敏感型蛋白激酶均能行使正常的蛋白功能,却对ATP类似物抑制剂1-NM-PP1十分敏感。

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 5μmol/L 1-NM-PP1,the colony growth rate of Gpmk1-as decreased,but the growth of PH-1 was not affected.With 10μmol/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.