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Molecular Characterization of the Coding Region and 5’ UTR of HSP70 Gene in Indian Riverine Buffalo Breeds
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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>T at position 14), resulting in amino acid change 5M>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.
The distribution of domestic water buffaloes (Bubalus bubalis) is limited to the tropical region of Southeast Asia. Hence, they are well adapted to different agro-climatic conditions of India and India possesses 57% of the world’s buffalo population. India is home to 17 different registered breeds very well adapted to their respective agro-ecological production system. Despite the black coat colour and poorly developed sweat glands, buffalo has very well acclimatized itself to the hot and humid climate, emerging as a major source of income for the resource-poor farmers. Understanding the genetics of heat stress has led to the identification of various genes responsible for climatic adaptation, among which the heat shock protein (HSP) family members play a major role. One of the members, Heat Shock Protein 70 (HSP70) is a highly evolutionary conserved protein, expressed in both stress and non-stressed conditions (Rosenzweig et al., 2019; Jolly and Morimoto 2000; Morimoto 1998). Since HSP70 also acts as a molecular chaperone and assists in the folding of other proteins, under stressful conditions, the highest levels of HSP70 among the major HSPs were reported (Singh et al., 2019). It plays a major role in the folding of nascent peptide chains on ribosomes thus preventing the aberrant folding by protecting the hydrophobic surface, which would normally be exposed to solvent (Gaviol et al., 2008). The HSP70 gene of buffalo is encoded by a single coding-exon with a transcript length of 1.93 Kb and has not been extensively explored, therefore this study was designed to characterize the promoter and coding region of different Indian buffalo breeds by sequencing and document the polymorphism.
MATERIALS AND METHODS
The various amplicons were electrophoresed on 1.5% agarose gel and single-band PCR products of 1.2 kbp were used for dideoxy sequencing after exonuclease and alkaline phosphatase treatment. Sequences were further analyzed by using different software. The chromatograms were screened manually for the identification of heterozygous animals to detect the polymorphic nucleotide sites using Chromas Lite 2.0 software (http://chromas-lite.software. informer.com/2.0). The amplicons were edited and trimmed using the Editseq software and aligned using MegAlign, both of which are part of Lasergene 12 package of DNASTAR (https://www.dnastar.com/software/lasergene/).
RESULTS AND DISCUSSION
In this study, 21 variations were identified when Bubalus bubalis compared with Bos taurus, amongst 11 were transversions and the rest were translation type of variations. Whereas only 18 nucleotide variations were reported by Sodhi and coworkers (2013) when comparing the HSP70 of Bubalus bubalis with Bos taurus, since the study analyzed the panel with a lesser number of breeds than the present study. Similarly, higher numbers of nucleotide transversions (11 out of 22 variations) were found, when compared Bubalus bubalis with Bos indicus. Higher numbers of variable sites were identified in Bubalus bubalis vs Bos taurus compared to Bubalus bubalis vs. Bos indicus, which shows similarity with the previous reports (Sodhi et al., 2013).
Important findings were extracted related to the 5’ untranslated region (5’ UTR) in the buffalo HSP70 gene. As 5’ UTR is known for regulating gene expression, therefore, variation in this region plays a significant role by governing the rate of transcription. Within 16 different breeds of buffaloes, a total of 5 polymorphic sites were found in the 5’ flanking region (Table 1). As compared to cattle, nucleotide sequences in the 5’ UTR of Bubalus bubalis showed significant variation concerning nucleotide changes, but 5’ UTR of buffalo was 32 nucleotides longer (204 nucleotides) compared to Bos taurus (172 nucleotides, accession number- NM_174550). Two insertions/deletions (INDEL) of 30 and 1 nucleotide at positions -105 to -75 and -17, respectively were the reason for the longer length of 5’UTR in the Bubalus bubalis (Fig 3). Three transversions and two transitions along with the INDELs were also identified in this comparative analysis between the two species. These findings are similar to those reported by Sodhi et al. (2013).
Further, comparative sequence analysis for bubaline HSP70 gene coding region with other twelve different species revealed maximum homology of 98.8% with taurine cattle indicating the overall high similarity of the gene among the mammalian species. Phylogenetic analysis of bubaline HSP70 gene with different species showing the closeness of bubaline with the Bos indicus and Bos taurus (Fig 4). All ruminant species are grouped in a single major clade, while two other livestock species horse (Equus cabalus) and pig (Sus scrofa) being placed distantly.
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