Cloning, molecular properties and differential expression analysis of HMGS gene from Sanghuangporus baumii / 中草药

ABSTRACT

Objective: To clone and characterize a 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) gene which involved in the triterpenoid biosynthesis pathway in Sanghuangporus baumii. Methods: The HMGS gene cDNA full-length sequence was cloned by RACE technology. Characteristics including the physicochemical properties and conserved domain of the deduced HMGS protein were determined by a series of bioinformatics tools. The entire protein-coding cDNA of HMGS was cloned into the expression vector pET-32a (+). Then the recombinant plasmid was transformed into E. coli BL21 (DE3) cells. With IPTG induction, SDS-PAGE was used to investigate the situation of expression. Additionally, qRT-PCR technology was performed to measure the transcript levels of HMGS gene in the triterpenoid pathway during different developmental stages of S. baumii. Results: The full-length nucleotide sequence of HMGS was 1 930 bp, containing a complete open reading frame of 1 458 bp which encoded a polypeptide of 485 amino acids. Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52 750, and theoretical isoelectric point was 5.60. This protein was a hydrophilic protein, without transmembrane and signal peptide sequence. The constructed phylogenetic tree showed that HMGS from S. baumii had the highest similarity with HMGS from Fomitiporia mediterranea. The prokaryotic expression vector pET-32a-HMGS was sucessfully obtained. SDS-PAGE results showed that a significant protein band was in consistent with molecular weight of the predicted protein. Moreover, the results showed that the transcript levels of HMGS gene were in dynamic change. The transcript levels in the mycelium stage were higher than that in the fruiting body stage. For instance, the highest transcript level of HMGS was at 14 d and was 2.33-fold higher than the 5 d. Conclusion: Molecular characterization of HMGS will be useful for further functional elucidation of the gene involving in triterpenoid biosynthesis pathway in S. baumii.