甘蓝两种SRK短截蛋白的体外表达及其与THL1作用检测

In Vitro Expression of Two Truncated Form of SRK and Analysis on Its Interaction with THL1

为探明S–位点受体激酶(SRK)上与类硫氧还蛋白1(THL1)作用的氨基酸区域以及两者间作用方式,依据SRKE1上功能域分布构建了两种SRKE1短截体原核表达质粒pGEX-SRKE1A和pGEX-SRKE1B,分别在大肠杆菌BL21中获得了可溶性表达。通过体外孵育检测蛋白质相互作用的方法对SRKE1A、SRKE1B与THL1相互作用进行了检测,结果表明SRKE1A和SRKE1B均可与THL1结合,明确了THL1与SRK相互作用并不依赖于SRK激酶活性。两类SRK等位序列间比对结果表明Cys在两类SRK间的保守性存在明显差异,推测两类SRK材料亲和性的差异可能与Cys保守性不同有关。

To determine the amino acid region on SRK responsible for binding THL1 and their interaction manner,according to distribution of functional domains on SRKE1,two truncated form of SRKE1 prokaryotic expression plasmids pGEX-SRKE1A and pGEX-SRKE1B were constructed,and the recombinant protein were expressed in E.coli（BL21）.SRKE1A and SRKE1B were incubated with THL1 respectively,their interaction were analyzed in vitro.The result showed that SRKE1A and SRKE1B could interact with THL1,and their interaction did not depend on SRK kinase activity.Through alignment of SRKs from ClassⅠand ClassⅡ,the result showed that the conserved cysteine sites were different between the two Class SRKs,which suggests that self-incompatibility difference between two Class SRK probably has relation to the conserved Cys.