Cloning and expression analysis of squalene synthase gene in Hedera helix / 中草药

ABSTRACT

Objective To obtain a full-length cDNA of squalene synthase gene from Hedera helix (HhSS) by cloning technique, and to carry out bioinformatics analysis and expression analysis. Methods Primers were designed based on H. helix transcriptome data, and HhSS was cloned by using RACE technologies. DNAMAN, PROTPARAM, TMHMM, PSORT, ScanProsite, SOPMA, and SWISS-MODEL were used for analysis of sequence and physical and chemical properties and domain of encoded protein. The relative expression of HhSS was detected by qRT-PCR. Results The cDNA sequence of HhSS (GenBank accession number KX056078) was 1 889 bp which obtained by RACE cloning. It contained an ORF from 248 bp to 1 477 bp and a 5'UTR with 247 bp and a 3'UTR with 412 bp. It encoded a 409-amino-acid protein with a molecular weight of 46 800and an isoelectric point (pI) of 5.68. The HhSS protein had the characteristic domain and transmembrane region of plant SS protein and closer relationship with Eleutherococcus senticosus, Panax ginseng et al. The qRT-PCR results indicated that it had positive correlation between relative expressing level of HhSS gene and contents of saponins in H. helix leaves. Conclusion The cloning and expression analysis results of HhSS provide a theoretical and technical basis for elucidating the role of HhSS in saponins biosynthetic pathway and metabolic regulation.