人参中核黄素激酶基因的克隆与原核表达

Cloning and prokaryotic expression of riboflavin kinase gene in Panax ginseng

目的从人参中克隆出核黄素激酶基因,进行相关的生物信息学分析,构建原核表达载体并在大肠杆菌诱导表达。方法根据人参转录组数据库筛选出序列comp61599_c0_seq12,经相关软件对其进行序列分析;该序列3’末端缺失,利用3’RACE技术获得3’端序列;通过PCR技术获得人参核黄素激酶基因的全长c DNA序列,并构建原核表达载体p ET-32a-Pg RFK,通过热激法转化到大肠杆菌BL-21中进行诱导表达。结果从人参中成功克隆出1条长度为1 200 bp的核黄素激酶基因,其编码399个氨基酸,同时成功构建该基因的原核表达载体,SDS-PAGE电泳结果显示该蛋白大小与预测蛋白相对分子质量一致。结论成功克隆了人参核黄素激酶基因,并在大肠杆菌中成功表达。

Objective To clone the riboflavin kinase gene of Panax ginseng and perform bioinformatics analysis to construct the prokaryotic expression vector and induce its expression in Escherichia coli. Methods The sequences of comp61599_c0_seq12 were screened from transcriptome database in P. ginseng, and software and online resources were used for its bioinformatics analysis. The 3＇ end of sequence was lost and the full-length of 3＇ end sequence was obtained by 3＇ RACE technology. The complete sequences of c DNA of riboflavin kinase in ginseng was obtained by PCR amplification, and the prokaryotic expression vector of p ET-32 a-Pg RFK was constructed and transformed into E. coliBL-21 for inducing expression. Results A riboflavin kinase gene with length of 1 200 bp was successfully cloned from P. ginseng which encoded 399 amino acid and its prokaryotic expression vector was successfully constructed. The molecular weight of SDS-PAGE electrophoresis results was consistent with prediction. Conclusion The Pg RFK gene was successfully cloned and expressed in E. coli.