酵母双杂交筛选OIPK相互作用蛋白

Analysis of Proteins Interacted with OIPK by Yeast Two-hybrid Method

前期研究发现烟草中一个壳寡糖诱导的Ser/Thr蛋白激酶(Oligochitosan induced protein kinase,OIPK)在壳寡糖诱导的烟草抗性反应中起着重要作用.目前在烟草中发现OIPK基因的两个选择性剪接体:OIPKL和OIPKS,其中OIPKL是3个选择性剪接体中序列最长的.为进一步研究OIPK在壳寡糖诱导植物抗性中起的作用,本研究采用酵母双杂交方法,以pGBKT7-OIPKL为诱饵质粒,筛选构建在pACT2载体上的烟草cDNA文库,寻找OIPKL的相互作用蛋白.结果表明OIPKL蛋白在酵母中可以和烟草的S-腺苷甲硫氨酸合成酶、泛素激活酶E1、丙酮酸激酶、过氧化物酶相互作用.实验结果为研究壳寡糖诱导抗性机理提供了一定依据.

In our previous study,an oligochitosan-induced protein kinase（OIPK）was identified to be involved in oligochitosan-induced defense reactions in tobacco.Two alternatively spliced variants of OIPK gene,referred to as OIPKL and OIPKS,were found in tobacco and OIPKL was the longest one.To further investigate the function of OIPK in the signal transduction pathway of oligochitosan induced plant resistance,yeast two-hybrid screening was performed to find out its interaction partners.OIPKL cDNA was inserted downstream of the DNA-binding domain of the bait vector pGBKT7 and the recombinant pGBKT7-OIPKL plasmid was used as a bait plasmid to screen a tobacco By-2 cell suspension library constructed in pACT2 vector.Four proteins were identified to be interacted with OIPKL protein in the yeast two-hybrid system,including S-adenosyl-L-methionine synthetase,ubiquitin-activating enzyme E1,pyruvate kinase and peroxidase.The results provide evidence for the mechanism of oligochitosan induced plant resistance.Fig 3,Ref 26