Quantitative Real-Time PCR Analysis of Colchicine Pathway and Rhizome Developmental Genes
Colchicine is an alkaloid with a long history of use as a treatment for several conditions, such as gout and Familial Mediterranean Fever, but has shown potential as an anti-cancer drug due to its anti-mitotic properties. As demand for colchicine increases, traditional production methods lag behind as naturally-sourced colchicine is highly susceptible to environmental conditions, low yielding, and labor intensive. Synthetic colchicine has been used to meet growing demand but causes negative side effects due to toxicity. Therefore, developing a platform for natural colchicine production is necessary. The Colchicaceae family member, Gloriosa superba L., is one of the main commercial sources of colchicine, however G. superba’s colchicine biosynthesis pathway is not fully understood. Biorhizomes, grown from rootstocks of G. superba, were developed for the purpose of biosynthesizing important pharmaceutical compounds, such as colchicine. Transcriptomic and genomic studies of G. superba sequences have identified putative genes potentially involved in the colchicine biosynthesis pathway and rhizome development, respectively. Using quantitative real-time polymerase chain reaction (qRT-PCR), a comparative analysis of gene expression in G. superba may reveal differential gene expression of selected candidate genes. Reference genes were selected and analyzed for normalization of qRT-PCR data. The most stable reference gene, UBC22a, was selected and used to normalize gene expression data from three candidate colchicine pathway genes (Step 2 Cytochrome P450, Step 4 Cytochrome P450, and N-methyltransferase) and two candidate rhizome developmental genes (REVOLUTA and sucrose synthase). The genes CYP-30999, CYP-5091, NAT-70209, SuSy-69933 and REV-15982 were expressed in leaf and root tissue, while CYP75A109, CYP75A110, NAT-30717, SuSy-2693 and REV-11056 were expressed in rhizome tissue. Genes with high expression in rhizome tissue are likely candidates for involvement in colchicine biosynthesis or rhizome development. This study has confirmed candidate genes CYP75A109, CYP75A110, NAT-30717, SuSy-2693, and REV-11056 as potentially involved in the colchicine biosynthesis pathway and rhizome development, respectively. Further validation of these candidate genes using functional analysis studies may provide additional insights into their involvement in their respective pathways and associated enzymatic products. After constructing the colchicine pathway, synthetic biotechnologies can be developed for large-scale production of colchicine and its derivatives. Furthermore, an understanding of genes involved in G. superba rhizome development allows for genetic modification to overexpress these genes in biorhizomes, leading to larger biomass, increased production of colchicine, and ultimately a platform for continuous natural colchicine production.