Cloning and Functional Characterization of Full-length cDNA Encoding Squalene Synthase from Fritillaria thunbergii Miq / 中国生物化学与分子生物学报

ABSTRACT

Squalene is a key metabolic intermediate for sterols and various other triterpenoids. Its biosynthesis is catalyzed by squalene synthase (SQS), which converts two molecules of farnesyl pyrophosphate to squalene. The biosynthetic pathway of Fritillaria thunbergii Miq isosteroid alkaloids is similar to that of triterpenoids. In this study, a full-length cDNA of squalene synthase from Fritillaria thunbergii Mig (FtSQS) was cloned using rapid amplification from cDNA ends (RACE) technology. GenBank accession number was KF551097. 2. Bioinformatics methods were used to characterize the FtSQS in detail, including the detection of conserved regions, sequence homology analysis, secondary and tertiary structure prediction, and phylogenetic tree analysis. The results showed that its open reading frame (ORF) was 1 230 bp and encoded 409 amino acids. Protein-Blast alignment found that amino acid homology with SQS of Indian pine, Truncate alfalfa, Purple shirt, Potato, Bupleurum, Golden iron lock and Arabidopsis reached 73. 84%, 73. 23%, 72. 24%, 70. 66%, 70. 66%, 69. 44%, 68. 14%. Promoter analysis indicated that the 5' upstream region of FtSQS possessed various potential elements associated with physiological and environmental factors. To obtain a soluble recombinant protein, 24 hydrophobic amino acids were deleted from the carboxyl terminus, and the C-terminal truncated mutant FtSQS (FtSQSATM) was expressed in E. coli BL21 (DE3). SDS-PAGE analysis suggested that approximately 66 kD recombinant protein was checked. The in vitro enzymatic reaction proved that FtSQS could catalyze farnesyl pyrophosphate to generate squalene. Expression level of FtSQS mRNA in leaves was the highest, followed by stem and root, but in bulb was much lower than that in other tissues. It suggests that leaves are active organ for biosynthesis of peimine. The identification and function of FtSQS provides an important basis for the study of secondary metabolites of Fritillaria thunbergii Miq.