Cloning and bioinformatics analysis of iridoid oxidase gene in Gentiana rigescens / 中草药

ABSTRACT

Objective To obtain the key enzyme gene involving in the monoterpenoid biosynthesis pathway, an iridoid oxidase gene (GrIDO) and its promoter were cloned from the leaves of Gentiana rigescens, and its bioinformatics analysis were also performed. Methods The gene specific primers were designed according to the GrIDO gene of transcriptome in G. rigescens. The open reading frame (ORF) of GrIDO gene was cloned by RT-PCR method. The bioinformation of GrIDO gene was analyzed by online softwares. Meanwhile, the gene specific primers were designed according to the cloned GrIDO gene, and the promoter of GrIDO gene was amplified by PCR method. Its sequence analysis was also performed. Results The length of GrIDO gene ORF (GenBank accession number KP722034) was 1 557 bp, which encoded a protein with 518 amino acids. Its relative molecular weight was 58 920 with the theoretical isoelectric point of 8.40. GrIDO was the member of cytochrome P450 superfamily and may localize in chloroplast. GrIDO was a hydrophilic stable protein without signal peptide and composed of mainly α-helix (51.07%) and loops (42.69%). GrIDO protein had a high similarity with CrIDO of Catharanthus roseus (85.83%) and their genetic relationship was close. The cloned GrIDO promoter had a length of 720 bp (GenBank accession number KT428570), which possessed cis-regulatory elements TATA-box and CAAT-box, and had cis-acting elements involved in the abscisic acid and MeJA responsiveness, part of a light responsive element and MYB transcription factor binding site. Conclusion The expression of GrIDO gene were regulated by multifactors. The GrIDO gene and its promoter were cloned from G. rigescens. This study will lay foundations for the functional research of GrIDO gene in monoterpenoid biosynthesis pathway.