水稻白叶枯病抗性基因Xa21和稻瘟病抗性基因Pi-d2激酶蛋白质在毕赤酵母中的表达及条件优化

Expression optimization of rice bacterial blight resistance gene Xa21 and blast resistance gene Pi-d2 protein kinases in Pichia Pastoris

【目的】白叶枯病和稻瘟病是最主要的水稻病害,Xa21是水稻白叶枯病抗性基因,Pi-d2是稻瘟病抗性基因,二者都编码类受体激酶蛋白质。本研究旨在毕赤酵母系统中表达XA21和PI-D2激酶蛋白质。【方法】用Xa21和Pi-d2的激酶区PCR产物,构建了pPICZαA-Xa21K、pPICZαA-Pi-d2K重组质粒,酶切及测序验证后,将重组质粒线性化,转化到毕赤酵母菌株中,系统地比较了不同酵母菌株(KM71、GS115、X33),不同甲醇浓度(1%、2%、3%),不同pH(pH5、pH6、pH7、pH8)值,不同诱导时间(24h、48h、72h)条件下激酶蛋白质的表达情况。【结果】XA21和PI-D2激酶蛋白质可以在毕赤酵母中表达,但表达的蛋白质不能分泌到培养基上清中,而只能在菌体中检测到,对表达条件的系统比较发现,毕赤酵母菌株KM71和X33、2%的甲醇诱导浓度、pH5和48h以上的诱导时间有利于激酶蛋白质的表达,最后我们在酵母裂解物上清中获得了纯化的考染可见的激酶蛋白质。【结论】在毕赤酵母中表达了XA21和PI-D2激酶蛋白质,为下一步生化特性研究奠定了基础。

[Objective]Bacterial blight and blast are the most severe rice diseases.Xa21 confers resistance to bacterial blight,while Pi-d2 confers resistance to rice blast.Both Xa21 and Pi-d2 encode receptor-like kinase proteins.The aim of this study was to express kinase domain of XA21 and PI-D2 proteins in Pichia pastoris yeast system.[Methods]We amplified the coding regions for the kinase domains of Xa21 and Pi-d2 and constructed recombinant plasmids pPICZαAXa21K and pPICZαA-Pi-d2K,respectively.Restriction enzyme digestion and sequence verified plasmids were linearized and transformed into yeast strains.We compared the expression of recombinant proteins in three Pichia pastoris strains （KM71,GS115 and X33）,with various methanol concentration （1%,2% and 3%）,pH （pH5,pH6,pH7 and pH8） and induction time （24 h,48 h and 72 h）.[Results]The recombinant kinase domain of XA21 and PI-D2 proteins were expressed in yeast,however,they were detected only in pellet of yeast cells but not in the supernatant of medium.This indicated that the recombinant proteins were not secretive.Comparison results revealed that Pichia pastoris strains （KM71 and X33）,2% methanol,pH5 and 48 h or longer induction time were optimal for the expression of the two rice kinase domain proteins.Finally,we purified soluble recombinant proteins and detected them by SDS-PAGE.[Conclusion]We obtained purified domain of XA21 and PI-D2 proteins from Pichia pastoris expression strain,which will facilitate the investigations of their biochemical properties.