花生AhGPAT9与AhLPAAT4基因的原核表达及Western Blot分析

Prokaryotic Expression and Western Blot of AhGPAT9 and AhLPAAT4 of Peanut

酰基转移酶基因家族是甘油三酯合成的关键基因。本研究以前期克隆的AhGPAT9与AhLPAAT4基因部分序列设计引物,构建原核表达载体,转化进大肠杆菌BL21,经IPTG诱导表达,利用SDS-PAGE检测表明,重组蛋白在37℃,5h诱导条件下获得高效表达。重组蛋白经纯化和富集,对新西兰兔进行4次免疫,纯化获得的多克隆抗血清,通过间接ELISA检测,表明获得了效价比较高的多克隆抗体。通过对重组蛋白纯化后样品进行Western Blot分析,结果显示在纯化样品相应位置有明显信号,表明所制备的抗体具有很高灵敏度和特异性。并采用制备的抗体对AhGPAT9与AhLPAAT4蛋白在花生不同组织及种子不同发育时期的表达进行了Western Blot分析。为深入研究AhGPAT9与AhLPAAT4基因的功能提供了科学数据。

Acyltransferase family genes play important roles in triacylglycerol synthesis. The pro- karyotic expression vectors of AhGPAT9 and AhLPAAT4 genes were constructed, and the fusion proteins were super-expressed in E. coli BL21 under induction of IPTG. SDS-PAGE analysis showed that the fusion proteins were highly expressed after induction for 5h at 37℃. Then the fusion proteins were purified and used as antigens to inject rabbits for four times. The prepared polyclonal antiserumswere obtained and purified. The indirect ELISA analysis showed that the polyclonal antibodies have a high titer of 1：512000. The polyclonal antibodies were used to test the expression of fusion proteins in E. coli BL21 through Western Blot, and detected the specific bands. It suggested that the polyclonal an- tibodies have high specificity and sensitivity. Furthermore, the expressions of AhGPAT 9 and AhLPAAT4 proteins at different tissues and different seed developmental stages were determined by Western Blot. This study provided scientific data for the further study of AhGPAT9 and AhLPAAT4 enes.