Cloning, bioinformatic analysis and expression analysis of RgGGPPS2 gene from Rehmannia glutinosa / 中草药

ABSTRACT

Objective: In order to investigate the functional genes involved in terpenoids biosynthesis pathway of Rehmannia glutinosa, a new geranylgeranyl pyrophosphate synthase gene (RgGGPPS2) was isolated from R. glutinosa. Meanwhile, the bioinformatic analysis, prokaryotic expression, purification, and expression patterns of RgGGPPS2 gene were carried out. Methods: Based on the transcriptome data of R. glutinosa, specific primers of RgGGPPS2 gene were designed, and an open reading frame (ORF) of RgGGPPS2 gene was isolated from R. glutinosa. By constructing the prokaryotic expression vector pET32a-RgGGPPS2, the recombinant RgGGPPS2 protein was expressed in Escherichia coli BL21 (DE3) cells under IPTG induction. The expression pattern of RgGGPPS2 in different tissues was detected by real-time PCR. Results: RgGGPPS2 had an ORF of 867 bp, which encoded a protein of 289 amino acid residues. Bioinformatic analysis indicated that RgGGPPS2 protein contains the two conserved motifs FARM (first aspartate-rich motif, DDxxxxD) and SARM (second aspartate-rich motif, DDxxD). Phylogenetic analysis revealed that RgGGPPS2 protein showed the highest homology with GGPPS protein from Sesamum indicum, Catharanthus roseus and other dicots. Through the construction of prokaryotic expression vector pET-32a-RgGGPPS2, the recombinant RgGGPPS2 protein was successfully expressed in E. coli BL21 (DE3) cells and the recombinant RgGGPPS2 protein was purified by Ni2+ affinity chromatography. Real-time PCR analysis indicated that RgGGPPS2 was expressed in high transcript level in roots, lower level in leaves and the lowest level in stems. Conclusion: The RgGGPPS2 gene was isolated from R. glutinosa and the recombinant RgGGPPS2 protein was obtained. The results of this study provide a foundation for functional characterization of RgGGPPS2 gene involved in terpenoids biosynthesis pathway of R. glutinosa.