Cloning, Sequencing and Evaluation of the β-tubulin gene from Leishmania donovani as a Reference Gene for RT-qPCR
Leishmania is a group of trypanosomatid protozoan parasites that exist in two morphological forms: a promastigote form within their insect vector and an amastigote form in the mammalian host. To study gene expression in these two distinct lifecycle forms, real-time quantitative PCR (RT-qPCR) experiments were used to determine the copy number of control gene transcripts in the two lifecycle form populations. The goal of the current study was to clone the β-tubulin gene from Leishmania donovani and evaluate its role as a reference control for RT-qPCR gene expression studies. We cloned the β-tubulin gene from Leishmania donovani using primers designed based on the known sequences of the β-tubulin gene from the L. major database. Sequence analysis revealed a 1329bp ORF encoding a 443aa deduced protein with high homology to previously identified β-tubulin from other Leishmania species. The cloned L. donovani -tubulin gene, LdBTub, served as a reference control in RT-qPCR experiments with total RNA from L. donovani promastigotes and amasitigotes. Results showed that LdBTub is constitutively expressed by both parasite developmental forms at a constant level and is therefore a useful reference control for real time gene expression studies within these organisms.