Isolation and expression study of BBE encoding gene involved in sanguinarin biosynthesis pathway in greater celandine under drought stress

Different parts of the greater celandine herb contain important alkaloids such as chelodonin, chloritin, sanguinarine, and berberine. Sanguinarine is a four-member benzofenanthridine that is widely found in Papaveraceae, Fumariaceae and Rutaceae. This biologically active alkaloid, singuainarin, has a wide range of potentially useful pharmacological properties, such as anti-microbial, anti-inflammatory, and anti-tumor. The first step in biosynthesis pathway of sanguinarine alkaloid requires the transformation of S-reticulin to S-skolorine, which catalyzes by berberine beridge enzyme encoded by the BBE gene. In order to prepare plant materials for gene isolation, and the expression of BBE gene in four drought levels including severe, moderate, weak stress and non stress (40, 55, 70 and 100% field capacity, respectively) in three organs, root, stem and leaf, an factorial experiment was carried based on completely randomized design with three replications. RNA was extracted from root, stem and leaf to isolate the cDNA encoding BBE enzyme and study the gene expression. The cDNA encoding BBE enzyme with approximate length of 1500 bp was isolated from root and successfully integrated into PTG19-T plasmid and cloned at E. coli. The sequencing results showed that the isolated gene with a length of 1441 bp could translate to a peptide chain with 483 amino acids. The predicted BBE peptide chain had NX(S/T) motif as glycosylation sites, the flavin binding domain for the binding of flavinated histidine, SGGH motif, and the Glu sequence which plays an important catalytic role in the formation of berberine bridge in flavoprotein enzymes. Based on the mean comparison results, the highest and lowest expressions of this gene were obtained on root under weak drought stress (FC 70%) and on leaf and shoot under non-stress condition (100% FC), respectively. In general, it can be concluded that the weak drought stress can increase the BBE gene expression, and possibly this could result in higher production of sanguinarine in celandine.