Molecular Cloning, Sequencing and In-silico Analysis of N, P, C domains and full length Calreticulin-3 gene of Brassica juncea for understanding its role in downstream regulation
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Date
2019-02
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Rapeseed and mustard (Brassica juncea) holds immense agricultural and economic importance. The productivity of rapeseed and mustard is severely affected by Alternaria blight disease which is caused by the fungal pathogen, Alternaria brassicae. So far there is no source of resistance against this disease. However, biotechnological approaches could be used to develop disease resistance provided that the key candidate genes/proteins involved in imparting defence towards A. Brassicae pathogen are identified. In the recent past, the role of calreticulin has been demonstrated in during defence responses of plants towards various pathogens. Therfore it could prove an attractive candidate for improving defense response of Brassica plants towards Alternaria blight disease. Hence, the present studies were conducted to understand the similar role of Brassica juncea calreticulin-3 with the objective of isolating and cloning of N, P and C domains and full length Calreticulin-3 gene of Brassica juncea. Total RNA was isolated from 30-day old leaves of B. juncea var. PAB9511 followed by synthesis of cDNA and its confirmation by amplification of internal control Actin. The primers were designed from the mRNA sequence of Calreticulin-3 of Brassica rapa using Primer3 and BLAST tools. The PCR conditions were optimised for obtaining 1300 bp, 645 bp, 288 bp and 339 bp amplicons of full length calreticulin-3, N, P and C domains respectively. The N, P, C domains and full length calreticulin-3 gene were cloned into TA cloning vector, pGEMT-Easy vector and confirmed by colony PCR and restriction digestion using enzymes Xba I and EcoR I . The full length calreticulin 3 gene along with N, P and C domains were sequenced. The restricted fragments of each insert were then successfully cloned into mammalian expression vector, pCDNA3.1 and again confirmed by colony PCR and restriction digestion. The BLASTn results revealed that the full length calreticulin-3 of Brassica juncea showed 98% sequence identity with the mRNA sequence of calreticulin-3 of Brassica rapa subspecies pekinensis. Also, the phylogenetic tree revealed more than 90% sequence similarity between calreticulin-3 of Brassica juncea to the calreticulin-3 of Brassica rapa, Brassica napus and Brassica oleracea. The SmartBLAST analysis revealed 86% homology between calreticulin-3 protein of Brassica juncea and Arabidopsis thaliana. The domain analysis assured the presence of calreticulin domain in this sequence and motif analysis revealed the presence of many motifs having significant involvement in responding to various biotic and abiotic stresses. The in-silico prediction of phosphorylation sites revealed the presence of 17 serine residues, 5 tyrosine residues and 9 threonine residues that could be involved in phosphorylation/ dephosphorylation indicating their involvement in signaling transduction.The prediction of interacting partners of calreticulin-3 of Brassica rapa showed that it could interact with 70 KDa heat shock protein, Brassinosteroid insensitive-1 and dolichyl-diphospho-oligosaccharide-protein glycotransferase. The role of these proteins in plant response to various stresses has been demonstrated by recent studies. These could also be hypothesized to be the interacting partners for calreticulin-3 of Brassica juncea. The experimental studies of effect of over expression of calreticulin-3 in Brassica plants could be tested for its role in conferring resistance against Alternaria blight and other different pathogenic infections or environmental stresses.
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