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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。
The specific primers were designed according to the gene fragments provided by Salvia miltiorrhiza transcriptome database and the reverse transcription polymerase chain reaction (RT-PCR) was used to clone 1-hydroxy-2-methyl-2- (E) The full-length cDNA sequence of the alkenyl-4-diphosphate synthase gene is designated SmHDS, GenBank Accession No. KJ746807. The sequence length was 2 529 bp with an open reading frame of 2 229 bp. It was deduced to encode 742 amino acids with a 170 bp 5’UTR and a 130 bp 3 ’UTR. Homology analysis of the obtained sequences using bioinformatics software showed that SmHDS was closely related to the HDS of Salvia miltiorrhiza. The result of prokaryotic expression analysis showed that SmHDS expressed the target protein in Escherichia coli with the same size as the predicted protein. At the same time, the four factors influencing protein expression were induction temperature, induction time, IPTG concentration and host cell density (A600) The optimal conditions for the expression of SmHDS protein were as follows: temperature 30 ℃, induction time 20 h, IPTG final concentration 0.2 mmol·L-1, and host strain A600 0.6. This provides a theoretical basis for further study of the role of 1-hydroxy-2-methyl-2- (E) -butenyl-4-diphosphate synthase in the pathway of tanshinones biosynthesis.