Molecular Characterization of the Coding Region and 5’ UTR of HSP70 Gene in Indian Riverine Buffalo Breeds

Background: HSP70 (Heat Shock Protein 70), plays a crucial role in nascent protein folding; the added challenges due to physiological factors demand stringent role-playing of such chaperones for tropical livestock such as water buffalo (Bubalus bubalis). Therefore to evaluate the variations at nucleotide level in HSP70 that could potentially unravel the molecular basis of thermal adaptation in the riverine buffalo breeds of India, the current study was targeted to sequence the CDS (Coding Sequence) and UTR (Untranslated Region) of the gene in a panel of 16 Indian riverine buffalo breeds. Methods: Blood samples were collected and genomic DNA was isolated followed by PCR standardized for the amplification of different fragments of the HSP70 gene using different sets of primer pairs covering the entire coding region and 5’UTR. Multiple amplicons generated to cover the entire gene were sequenced. Sequences were further analyzed manually for the identification of heterozygous animals to detect the polymorphic nucleotide sites and variation between breeds documented. Result: The HSP70 results suggest, the highly conserved nature of gene in buffalo. The only non-synonymous polymorphic site was found in the Toda buffalo breed (g.SNPC greater than T at position 14), resulting in amino acid change 5M greater than T. A total of 7 polymorphic sites were found in the 5’UTR flanking region. Additionally, two insertion/deletions (INDEL) of 30 and 1 nucleotide length were found in the 5’UTR.