毛白杨4CL-like基因的克隆与原核表达分析

Cloning and prokaryotic expression of Populus tomentosa Carr. 4CL-like gene

为得到毛白杨4CL-like基因并对其原核表达特性进行分析,利用毛白杨叶片的总RNA反转录得到的总cDNA为模板,利用PCR技术克隆得到毛白杨4CL-like基因(登录号:XP_002315339.1)。利用MEGA软件对4CL-like基因及其他9个来自不同物种的4CL基因进行进化树分析。最后通过构建pET30-4CL-like原核表达载体对该基因进行原核表达分析。结果显示,克隆得到的4CL-like基因CDS区全长为1 758 bp,编码585个氨基酸。系统进化分析表明,该基因与葡萄中的4CL-like5有很高的同源性。另外,SDS-PAGE分析表明,4CL-like基因在大肠杆菌BL21中成功表达,所表达融合蛋白的分子量约为60 ku。

In order to clone and analyze the prokaryotic expression of 4CL-like gene from Populus tomentosa Carr., total RNA were isolated, and 4CL-like gene were cloned by PCR. Phylogenetic analysis of the 4CL-like gene compared with other nine 4CL genes form different species with MEGA. Then it was over-expressed in prokaryotic systems. The expression construct pET30-4CL-like was preformed. Based on the results, the bioinformational analysis showed that the CDS region of the nucleotide sequence was 1 758 bp, which could code 585 predicted aminoacids. Phylogenetic analysis showed that this gene was most similar to 4CL-like5 in Vitis vinifera. The SDS-PAGE analysis showed that the 4CL-like .gene was transformed into E. coli BL21 and expressed successfully, fusion protein with molecular weight of 60 ku was successfully expressed in transformant.