禾谷缢管蚜RpGST1基因的克隆、序列分析及原核表达

Cloning,sequence analysis and prokaryotic expression of glutathione S-transferase gene from Rhopalosiphum padi( Hemiptera: Aphididae)

为揭示禾谷缢管蚜耐药性的分子机理,采用RT-PCR方法克隆了禾谷缢管蚜谷胱甘肽-S-转移酶(glutathione S-transferases,GSTs)的c DNA序列,命名为Rp GST1(Gen Bank登录号KP192850),并构建原核表达载体p ET32-Rp GST1,对Rp GST1基因进行原核表达、SDS-PAGE和Western blotting检测。结果显示,禾谷缢管蚜Rp GST1基因的编码区长651 bp,编码216个氨基酸,分子量约为24.06 k D,理论等电点为6.20;Rp GST1基因在大肠杆菌中成功表达出一个分子量约为45 k D的融合蛋白,与预测的融合蛋白分子量大小一致。通过克隆Rp GST1基因的c DNA序列并进行序列比对分析,表明构建了GST基因的原核表达载体并成功表达。

In order to clarify the resistance mechanism of Rhopalosiphum padi to pesticides at molecular level,the c DNA sequences of GST genes of R. padi were cloned using RT-PCR and designated as Rp GST1( Gen Bank No. KP192850). The prokaryotic expression of Rp GST1 gene was done after construction of its prokaryotic expression vector p ET32-Rp GST1, SDS-PAGE and Western blotting analysis. The results showed that Rp GST1 gene included an open reading frame of 651 bp encoding 216 amino acids with the predicted molecular mass of 24. 06 k D,and the theoretical isoelectric point was6. 20. The molecular weight of the recombinant Rp GST1 is 45 k D,which is consistent with the predicted result. The c DNA sequences of GST gene from R. padi were successfully cloned and comparatively analyzed. The fusion protein was expressed in vitro by p ET system.