短命植物东方旱麦草Rubisco大亚基基因(rbcL)的克隆及表达分析

Cloning and Expression of rbcL Gene of Rubisco Large Subunit from Ephemeral Plant Eremopyrum orientale L.

利用PCR、DNA重组、原核与真核生物表达等技术从新疆短命植物东方旱麦草(Eremopyrum orientale)基因组中扩增出Rubisco大亚基基因rbcL(GenBank登录号为FJ346562),并构建了原核与真核表达载体pGEX4T-1-rbcL和pcDNA3-rbcL,随后分别在原核宿主BL21(DE3)和小鼠中进行了表达,并利用真核表达载体和纯化蛋白制备的抗体,对表达产物的特异性进行了Western印迹检测。结果表明:东方旱麦草的rbcL基因可读区包含1431 bp,与旱麦草rbcL基因的同源性达99.86%,推测编码476个氨基酸,分子量56 kD。该基因在BL21中以融合蛋白GST-RBCL形式表达并存在于包含体中,融合蛋白大小约为82 kD。RT-PCR检测到该基因在小鼠肝脏中的表达。利用真核表达载体与纯化融合蛋白进行联合免疫制备的RBCL抗体可与RBCL特异性地结合,这为短命植物东方旱麦草基因后续的免疫定位检测奠定了基础。

The rbcL （large subunit of ribulose-1,5-bisphosphosphate carboxylase/oxygenase） gene of ephemeral plant Eremopyrum orientale L. was amplified from the genomic DNA and the correct gene sequence was registered in GenBank （ Accession No. FJ346562 ）. The rbcL gene was cloned into prokaryotic expression vector pGEX4T-1 （ harbored with gene encoding GST and will form a fusion protein with foreign gene） and eukaryotic expression vector pcDNA3,and then expressed in the BL21 （DE3） and mouse,respectively. The expressed RBCL protein was detected by Western blot. The results showed that the ORF of rbcL included 1431 nucleotides and encoded 476 putative amino acids. It shares 99.86% homology with that of Eremopyrum triticeum. The fusion protein GST-RBCL with the size of 82 kD was expressed in inclusion body in BL21 （DE3） ;the rbcL gene was shown being transcribed in the mouse liver by RT-PCR analysis. Western blot results showed that the purified RBCL was able to specifically bind to its antibody. These preliminary results would provide evidence for further immunohistochemistry assay in E. orientale.