Cloning and bioinformatic analysis of FPPS gene from Paeonia lactiflora / 中草药

ABSTRACT

Objective: To clone the full-length cDNA sequence which encodes one of the key enzymes of terpene biosynthesis, farnesyl pyrophosphate synthase (FPPS) and analyze the bioinformation. Methods: Based on the transcriptome data of Paeonia lactiflora (Pl), a pair of specific PCR primers were designed. The objective PCR band of PlFPPS was successfully amplified using RT-PCR technique and then it was cloned to pMD18-T vector. After clone and plasmid PCR characterization, the recombinants were sequenced and then a series of bioinformatic analysis was carried out including sequence homology search, construction of molecular phylogenetic tree, domain search, and 3D structure prediction, etc. Results: Sequencing results showed that the full-length PlFPPS was 1 315 bp, which contained 60 bp 5'-UTR, 1 050 bp CDS, and 205 bp 3'-UTR. It encoded 349 amino acids and the accession number of GenBank was KP708571.Blastn and Blastp analysis revealed that PlFPPS (KP708571) and its encoding protein (AKJ26301) had high homology with FPPS genes and proteins from several plants at the nucleotide and amino acid levels. Phylogenetic tree analysis indicated that the genetic relationship of PlFPPS with FPPS from other plants was relatively far. It predicted that the molecular weight and isoelectric point of PlFPPS were 40200 and 5.33, which was a hydrophilic and stable protein, located in cytoplasm without transmembrane domain and signal peptide. It contained seven conservative domains like substrate binding pocket, substrate-Mg2+ binding site, catalytic site, aspartate-rich region 1, and aspartate-rich region 2.The proportion of α-helix in its 3D structure was high and the α-helix was linked by β-loop. The central cavity was composed of 10 core α-helixes. Conclusion: The full-length cDNA sequence of FPPS is cloned firstly from P. lactiflora and the bioinformatic analysis is then carried out.