Bioinformatics analyses of rice calmodulin-lysine N-methyltransferases gene (OsCaM KMT) and characterization of its promoter from Oryza sativa L. sub sp. indica through transgenic approach

Calmodulin-lysine N-methyltransferases (CaM KMT) belong to a family of protein lysine methyltransferases that catalyzes methylation of non-histone proteins. Lysine present at 116 position in the protein sequence of calmodulin is apparently methylated by CaM KMT in most species, however not characterized in rice. In the present study rice calmodulin-lysine N-methyltransferase (OsCaM KMT) gene has been isolated from an indica rice variety ‘Swarna’. Derived amino acid sequences of CaM KMT gene from rice and other plants were used in constructing a phylogenetic tree to show the evolutionary relationship among those species. Motif analysis along with comparative protein modeling was done to find out structural relationships among rice, Arabidopsis, and human CaM KMT. Gene expression analysis revealed differential tissue specific expression of OsCaM KMT. The highest transcript expression was revealed in the root tip followed by flower and new leaf, while, the least expression was found in the old leaf. In silico analysis was performed considering the 2 kilobases (kb) upstream promoter region of OsCaM KMT gene to find out various transcription factor binding sites. Analyses of the putative cis-regulatory elements revealed their involvement in tissue specific and stress dependent responses. For further characterization, the promoter region (2 kb) of OsCaM KMT gene was isolated, cloned, and sequenced. Histochemical GUS detection of transgenic rice depicted strong OsCaM KMT promoter expression in midribs, stomata, and tip of leaf tissues, central cylinder as well as tip parts of the primary root and lateral roots, along with significant expression in pollen grains, ovary, and vascular bundles. OsCaM KMT gene expression was corroborated by the histological GUS detection in OsCaM KMT promoter::GUS fusion construct. Although several putative microRNAs (miRNAs) has been identified targeting OsCaM KMT coding DNA sequence (CDS) and UTR region, further studies are required to elucidate their tissue-specific regulation.