Identification and expression profiling of a novel Kunitz trypsin inhibitor (KTI) gene from turmeric, Curcuma longa, by real-time quantitative PCR (RT-qPCR)

Kunitz trypsin inhibitor (KTI) is one of the widely studied protease inhibitors (PIs) and has been reported to take part in plant defense mechanism during pathogenesis. Numerous plant-origin recombinant KTIs have been described to exhibit anti-pathogenic properties and were used to fight pathogens in the field of pharmacology and agriculture. In this study, a novel Kunitz trypsin inhibitor gene, ClKTI, was isolated from a medicinal herb plant, turmeric, Curcuma longa. The full-length ClKTI gene is 754 bp long (Accession No. KF889322.1 in NCBI database) and it was obtained using 5′/3′ rapid amplification of cDNA ends (RACE) technique. ClKTI has an open reading frame of 639 bp length which encodes for 213 amino acids and contains the Kunitz-family motif, (V-X-D-X2-G-X2-L-X5-Y-X-I) and an altered reactive site motif, (G/E-I-S). Sequence similarity search using BLASTX showed that ClKTI shared the highest similarity to KTI from Theobroma cacao with 58% max identity while conserved domains search resulted in ClKTI having specific hits with Kunitz-family soybean trypsin inhibitor (STI). Phylogenetic studies suggested that ClKTI is related to T. cacao while protein homology modeling analysis indicated that it has 12 β-sheets with three disulfide bridges. Using real-time quantitative PCR, the ClKTI gene expression pattern in five different tissues (flower, basal stem, stem, rhizome and root) treated with methyl-jasmonate (MeJA) was studied where MeJA was suggested to regulate expression of PI genes in plants. The results indicated that the expression of ClKTI generally increased in the MeJA-treated tissues with the root tissues possessing the highest expression and stem tissues showed the highest expression fold-change.