[Cloning and characterization of geranylgeranyl diphosphate synthase gene of Salvia miltiorrhia].
Từ khóa
trừu tượng
OBJECTIVE
To obtain geranylgeranyl diphosphate synthase gene of Salvia miltiorrhiza, and conduct bioinformatic and transcript expression analysis of the cloned SmGGPS1 gene.
METHODS
The degenerate primers were designed based on the conservative regions of GGPS protein sequences from public databases. The target gene was obtained from root of S. miltiorrhiza by use of homologous cDNA amplification and RACE technologies. The sequence alignment was performed using BLAST. The open reading frame was identified by use of the ORF Finder. The protein domains were defined by use of Prosite software and the signal peptide sequence was predicted by Target P1.1. MEGA4.0 was used to conduct multiple amino acid sequence alignment and construct the phylogenetic tree. Roots and leaves at the seedlings stage and roots, stems, leaves, buds and flowers in the flowering stage were sampled for transcript analysis. Semi-quantitative RT-PCR was used to detect the gene expression level. The complete gene of GGPS was obtained from S. miltiorrhiza genomic DNA by PCR using the cDNA-derived specific primer. The gene structure of GGPS was analyzed by comparison of the genomic DNA and its cDNA.
RESULTS
The obtained 1 298 bp SmGGPS1 cDNA sequence contains an 1095 bp ORF, encoding 364 amino acids. It is predicted that it has a plastid targeting signal peptide of approximately 52 amino acid at the N-terminal end. It is to believe that this is the polyprenyl synthetase signature, and nucleic acid sequence comparison revealed that SmGGPS1 ORF has more than 60% identity to the reported GGPS. RT-PCR semi-quantitative analysis showed that the gene expresses in the all tested tissues, and with much higher level of expression in the leaves in the flowering stage. SmGGPS1 has a 397 bp intron.
CONCLUSIONS
For the first time the cloning of geranylgeranyl diphosphate synthase gene from S. miltiorrhiza was reported, and it provides a good basis for further functional study of SmGGPS1.
