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.