姜丹, 荣齐仙, 袁庆军, 张文婧, 张永清, 黄璐琦. 白花丹参HDS基因的全长克隆与原核表达分析J. 药学学报, 2014,49(11): 1614-1620.
引用本文: 姜丹, 荣齐仙, 袁庆军, 张文婧, 张永清, 黄璐琦. 白花丹参HDS基因的全长克隆与原核表达分析J. 药学学报, 2014,49(11): 1614-1620.
JIANG Dan, RONG Qi-xian, YUAN Qing-jun, ZHANG Wen-jing, ZHANG Yong-qing, HUANG Lu-qi. Cloning and prokaryotic expression analysis of HDS from Salvia miltiorrhiza bge.f.alba.J. Acta Pharmaceutica Sinica, 2014,49(11): 1614-1620.
Citation: JIANG Dan, RONG Qi-xian, YUAN Qing-jun, ZHANG Wen-jing, ZHANG Yong-qing, HUANG Lu-qi. Cloning and prokaryotic expression analysis of HDS from Salvia miltiorrhiza bge.f.alba.J. Acta Pharmaceutica Sinica, 2014,49(11): 1614-1620.

白花丹参HDS基因的全长克隆与原核表达分析

Cloning and prokaryotic expression analysis of HDS from Salvia miltiorrhiza bge.f.alba.

  • 摘要: 根据丹参转录组数据库提供的基因片段设计特异性引物, 采用逆转录聚合酶链式反应(RT-PCR)方法, 从白花丹参中克隆1-羟基-2-甲基-2-(E)-丁烯基-4-二磷酸合酶基因的全长cDNA序列, 命名为SmHDS, GenBank注册号KJ746807。序列长度为2 529 bp, 含有2 229 bp的开放阅读框, 推测编码742个氨基酸, 含有170 bp的5' UTR和130 bp的3' UTR。利用生物信息学软件对获得的序列进行同源性分析, 得出SmHDS与紫花丹参HDS的亲缘关系较近。原核表达分析结果表明SmHDS在大肠杆菌中表达出与预测蛋白大小相当的目标蛋白, 同时对影响蛋白表达的4个因素, 即诱导温度、诱导时间、IPTG浓度和诱导时宿主菌的密度(A600)进行了优化, 得出SmHDS蛋白表达的最佳条件为: 温度30 ℃、诱导时间20 h、IPTG终浓度0.2 mmol·L-1、宿主菌的密度(A600)值为0.6。这为进一步研究1-羟基-2-甲基-2-(E)-丁烯基-4-二磷酸合酶在丹参酮类化合物生物合成途径中的作用提供了理论依据。

     

    Abstract: According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, with the method of reverse transcription polymerase chain reaction(RT-PCR), this study cloned full-length cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene from Salvia miltiorrhiza bge.f.alba, this sequence is named as SmHDS and its GenBank registration number is KJ746807. SmHDS, 2 529 bp long, contains an ORF of 2 229 bp, encodes 742 amino acids, including 5' UTR 170 bp and 3' UTR 130 bp. Using bioinformatics software, having made a homology analysis of the obtained sequence, we can have a conclusion that SmHDS have a close genetic relationship with HDS of Salvia miltiorrhiza. Analysis result of prokaryotic expression revealed that in Escherichia coli, SmHDS expressed target proteins which in size are comparable with the protein predicted. Meanwhile, the 4 factors which can influence the protein expression were optimized, the 4 factors are inducing temperature, inducing time, IPTG concentrations and density of inducing host bacterium(A600). The optimal expression conditions of SmHDS were 30 ℃ until the A600 is 0.6, and add IPTG to a final concentration of 0.2 mmol·L-1, and the induction time of 20 h. It provides theoretical basis for the further study of the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase in the biosynthesis of tanshinone compounds.

     

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