Quantitative Real-Time PCR Analysis of Colchicine Pathway Genes

Modern synthetic drugs consistently show side effects, which underlines the need for the development and manufacturing of plant-based drugs [47]. Colchicine is a naturally cultivated, plant-based alkaloid extracted from the dried tubers of Gloriosa superba L., and the bulb-like corms of Colchicum autumnale L. It is commonly known for its use in treating gout, but more notably recognized for its antimitotic functions, making it a potential anticancer drug. Contrary to its vast usage in medicine, colchicine’s exact mechanism of action is still being explored. Previous transcriptome studies of G. superba and C. autumnale provide a putative pathway mechanism and candidate genes to help in the elucidation of the colchicine biosynthetic pathway [23]. Evaluation of candidate genes using quantitative real-time polymerase chain reaction (qRT-PCR) analysis in G. superba can validate gene expression and provide further knowledge of the biosynthetic mechanism of colchicine. Previous studies indicate that the highest level of colchicine production occurs in the rhizome of G. superba [38]. Comparative analysis of gene expression levels in different tissues of G. superba can validate differential gene expression. Normalization is a key step in effectively analyzing gene expression in qRT-PCR. Twelve candidate reference genes were selected and analyzed to determine the most stable gene to use for normalization against the colchicine pathways genes. One stable reference gene, UBC22a, was selected to normalize gene expression levels of two candidate N-methyltransferase genes and ten candidate O-methyltransferase genes in the leaf, root, and rhizome of G. superba. Tested candidate colchicine genes, NMT-46812, OMT-46280, OMT-36240, OMT-13320, OMT-2006, OMT-16875, and OMT-10195 were highly expressed in the rhizome of G. superba, while NMT-31585, OMT-36043, OMT-31794, OMT-6585, and OMT-238 genes were not, thus the highly expressed genes within the rhizome are good candidates for involvement in colchicine biosynthesis. The confirmation of candidate colchicine pathway gene expression is just the first step towards the emergence of advanced, large-scale production of colchicine, and the discovery of novel derivatives. A complete elucidation and validation of the colchicine biosynthetic pathway can open up the opportunity of colchicine pathway reconstitution, pathway engineering, and many other biotechnological approaches to increase colchicine production.