Molecular Cloning and Expression of Squalene Epoxidase from a Medicinal Plant, Bupleurum chinense / 中草药·英文版

ABSTRACT

Objective: In plant, squalene epoxidase (SE) catalyzes the first oxygenation step in the biosynthetic pathway of triterpenoid and phytosterol, representing one of the rate-limiting enzymes in this pathway. Bupleurum chinense is an important medicinal herb with its major active constituents such as triterpenoid saponins and saikosaponins. In order to obtain the series of enzymatic genes involved in saikosaponin biosynthesis, a cDNA of SE, designated BcSE1, was cloned from B. chinense. Methods: The BcSE1 gene was cloned by homology-based PCR and 5'/3' RACE methods from the adventitious roots of B. chinense. The physical and chemical parameters of BcSE1 protein were predicted by protparam. In order to discover hints in amino acid sequences on the dominant functions in the biosynthesis of saponin or phytosterol, sequences of SE from other plants were downloaded from NCBI for sequences alignment and phylogenetic analysis. BcSE1 was cloned into a yeast mutant KLN1 (MATa, erg1URA3, leu2, ura3, and trp1) to verify the enzyme activity of BcSE1. Additionally, the tissue-specific expression and methyl jasmonate (MeJA) inducibility of BcSE1 were investigated using quantitative real-time PCR. Results: The predicted protein of BcSE1 is highly similar to SEs from other plants sharing amino acid sequence identities of up to 88%. The BcSE1 can functionally complement with yeast SE gene (ERG1) when expressed in the KLN1 mutant (MATa, erg1URA3, leu2, ura3, and trp1). Using as controls with β-amyrin synthase (β-AS) which is presumed to catalyze the first committed step in saikosaponin biosynthesis and a cycloartenol synthase (CAS) relating to the phytosterol biosynthesis, the transcript of BcSE1 was significantly elevated by MeJA in adventitious roots of B. chinense and the transcript of BcSE1 was most abundant in the fruits and flowers of plants, followed by that in the leaves and roots, and least in stems. Conclusion: It is the first time to illustrate the molecular information of SE in B. chinense and to clone the full-length SE gene in plants of genus Bupleurum L.