Cloning and preliminary expression analysis on HDS gene in Dendrobium officinale / 中草药

ABSTRACT

Objective: To clone the 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS) gene from Dendrobium officinale, induce fusion protein in Escherichia coli, and explore the regular pattern about HDS gene in different tissues of D. officinale. Methods: RT-PCR and RACE technologies were used to clone the full-length cDNA of DoHDS. Using relevant softwares and online sites to analyze bioinformatics. Then the expression patterns of DoHDS were studied by real-time PCR. Constructing prokaryotic expression vector pET-28a (+)-DoHDS to induce the expression protein in E. coli BL21 (DE3). Results: The DoHDS gene was successfully obtained (GenBank accession number KJ161312), the full-length cDNA was 2666 bp and ORF was 2238 bp, coding the protein containing 745 amino acids. Relative real-time PCR analysis indicated that DoHDS showed the higher transcript abundance in the stems, 5 fold higher than protocorm-like bodies. The SDS-PAGE results showed that a relative molecular weight of 82700 recombinant protein was produced. Conclusion: The cDNA encoding HDS from D. officinale is cloned. The prokaryotic expression vector and different tissues expression patterns of DoHDS are constructed. It is helpful for the future research on the mechanism of terpenoid biosynthesis in medicinal plants D. officinale.