Molecular Cloning and Characterization of a Cyclotide Gene Family in Viola modesta Fenzl

Cyclotides are small disulfide-rich proteins that have the unusual feature of a cyclic backbone. Cyclotides have a range of interesting biological activities and are found in a variety of tropical plants from the Rubiaceae, Violaceae, Cucurbitaceae and Fabaceae families. We have cloned and characterized cyclotides in Viola modesta, a Viola species native to western Asia, which was collected from the Kurdistan Province of Iran. Fifteen cyclotide sequences were obtained using homology based PCR strategy. Sequence analysis showed that 14 of them had continued open reading frames and showed high level of similarity to cyclotide genes from other species of the Violaceae. After analyzing the full endoplasmic reticulum signals of V. modesta cyclotides, two conserved sequences, AAFALPA and ATAFALP, were detected. Analysis of isolated cyclotide sequences showed that they all belonged to bracelet family and were separated into two subclasses. Phylogenetic analysis of cyclotide genes from V. modesta and other Viola species revealed that most V. modesta genes showed close relationship with their homologs from the Violaceae, while the V. modesta genes formed two separate clades. Transcription analysis by semi-quantitative RT-PCR revealed that Vmcyc1 and Vmcyc7 were differentially expressed in all tested tissues including roots, stems, leaves, flowers, seeds, peduncles, and capsules with the highest transcript level in the capsules.