Molecular Characterization of HSP60 Gene in Poonchi Chicken

Background: Heat shock proteins are widely present in lower as well as higher forms of life ranging from bacteria and yeast to humans. HSP60 stabilizes the inner and outer membrane of the mitochondria and thereby preventing the apoptosis of cells. Aims: To amplify and sequence HSP60 gene in local Poonchi chicken and to study its genetic similarity and difference between different species of local chicken of Poonch. Place and Duration of Study: Division of Animal Genetics and Breeding, Sher-e-Kashmir University of Agricultural Sciences and Technology, R. S. Pura, Jammu, between August 2019 and February 2022. Methodology: 2 ml of blood was collected from wing vein of chicken. Total RNA was extracted from freshly collected chicken and cDNA was synthesized by reverse transcription from RNA. HSP60 gene was amplified from cDNA using specific primers designed by Primer 3 software. The amplified product was purified by GenElute Gel extraction kit and thereafter cloned and sequenced. A partial cDNA sequence of chicken HSP60 gene of 1002bp encoding 327 amino acids was obtained. The pairwise distance between sequences aligned with ClustalW method was estimated by MEGA X software. Results: PCR amplification revealed 1002-bp PCR product of HSP60 gene. The obtained sequence was of HSP60 gene as confirmed by BLAST. The percentage identity of chicken HSP60 cDNA sequence with other species and the percentage identity of deduced amino acid sequence with that of other species was taken from BLAST. Deduced amino acid sequence of 327 residues of chicken HSP60 gene was 98.47%, 96.94%, 96.64%, 99.30%, 98.17%, 99.00%, 96.33%, 98.47% and 99.08% homology to pigeon, ostrich, rabbit, guinea fowl, turkey, quail, guinea pig, geese and duck, respectively. The phylogenetic tree drawn by MEGA X software at nucleotide level showed the conserved nature of HSP60 gene. Maximum divergence from chicken partial cDNA was observed with guinea pig CDS with the value of 0.20695875 and minimum divergence was observed with guinea fowl with value of 0.02806869. Z test was conducted in order to test whether positive selection is operating on a gene. It was found selection is of purifying type. Conclusion: From the present study it can be concluded that chicken HSP60 is highly conserved among different species. The HSP60 gene might have evolved by purifying selection (dS > dN) and chicken CDS sequence is the closest to quail, ostrich, turkey, pigeon duck and the most divergent to guinea pig.