Novel polymorphisms in UTR and coding region of inducible heat shock protein 70.1 gene in tropically adapted Indian zebu cattle (Bos indicus) and riverine buffalo (Bubalus bubalis)

Abstract

Due to evolutionary divergence, cattle (taurine, and indicine) and buffalo are speculated to have different responses to heat stress condition. Variation in candidate genes associated with a heat-shock response may provide an insight into the dissimilarity and suggest targets for intervention. The present work was undertaken to characterize one of the inducible heat shock protein genes promoter and coding regions in diverse breeds of Indian zebu cattle and buffaloes. The genomic DNA from a panel of 117 unrelated animals representing 14 diversified native cattle breeds and 6 buffalo breeds were utilized to determine the complete sequence and gene diversity of HSP70.1 gene. The coding region of HSP70.1 gene in Indian zebu cattle, Bos taurus and buffalo was similar in length (1,926 bp) encoding a HSP70 protein of 641 amino acids with a calculated molecular weight (Mw) of 70.26 kDa. However buffalo had a longer 5' and 3' untranslated region (UTR) of 204 and 293 nucleotides respectively, in comparison to Indian zebu cattle and Bos taurus wherein length of 5' and 3'-UTR was 172 and 286 nucleotides, respectively. The increased length of buffalo HSP70.1 gene compared to indicine and taurine gene was due to two insertions each in 5' and 3'-UTR. Comparative sequence analysis of cattle (taurine and indicine) and buffalo HSP70.1 gene revealed a total of 54 gene variations (50 SNPs and 4 INDELs) among the three species in the HSP70.1 gene. The minor allele frequencies of these nucleotide variations varied from 0.03 to 0.5 with an average of 0.26. Among the 14 B. indicus cattle breeds studied, a total of 19 polymorphic sites were identified: 4 in the 5'-UTR and 15 in the coding region (of these 2 were non-synonymous). Analysis among buffalo breeds revealed 15 SNPs throughout the gene: 6 at the 5' flanking region and 9 in the coding region. In bubaline 5'-UTR, 2 additional putative transcription factor binding sites (Elk-1 and C-Re1) were identified, other than three common sites (CP2, HSE and Pax-4) observed across all the analyzed animals. No polymorphism was found within the 3'-UTR of Indian cattle or buffalo as it was found to be monomorphic. The promoter sequences generated in 117 individuals showed a rich array of sequence elements known to be involved in transcription regulation. A total of 11 nucleotide changes were observed in the promoter sequence across the analyzed species, 3 of these changes were located within the potential transcription factor binding domains. We also identified 4 microsatellite markers within the buffalo HSP70.1 gene and 3 microsatellites within bovine HSP70.1. The present study identified several distinct changes across indicine, taurine and bubaline HSP70.1 genes that could further be evaluated as molecular markers for thermotolerance.

Keywords: A; AA; ADP; AMC; AP-2; ATP; ATP1A1; ATPase Na(+)/K(+) transporting alpha 1 polypeptide; Activator protein-2; Adenine; Adenosine diphosphate; Adenosine triphosphate; Ala; Alanine; Amino acid; Amino-terminus/terminal; Amritmahal cattle; Arginine; Asp; Aspartic acid; B. bubalis; B. indicus; B. taurus; Base pair; Bos indicus; Bos taurus; Bubalus bubalis; Buffalo; C; CDS; Coding sequence; Complementary deoxyribonucleic acid; Cytosine; D; DNA; DNase; DTT; Degree celsius; Deoxyribonuclease; Deoxyribonucleic acid; Deoxyribonucleotide triphosphate; Dithiothreitol; E; E twenty-six (ETS)-like transcription factor 1; EDTA; EEVD; Elk-1; Ethylene diamine tetra acetic acid; F; Fin; Forward; Forward internal; G; GAC; GC content; GIC; GTP; Gaolao cattle; Gap4; Genome Assembly Program; Gir cattle; Glu; Glutamic acid; Glutamic acids, glutamic acids, valine and aspartic acids; Gly; Glycine; Guanine; Guanine–cytosine content; Guanosine-5′-triphosphate; H; HAC; HSE; HSF2; HSP; HSP70.1; HSP70.2; Hariana cattle; Heat shock factor 2; Heat shock protein; Heat shock protein 70.1; Heat shock protein 70.2; Histidine; INDEL; Insertion or the deletion; K; KJC; KYC; Kangayam cattle; Kankrej cattle; Kilo Dalton; L; LINEs; Liter; Long interspersed nucleotide repetitive elements; Lysine; M; MATCH; MEGA; MGC; MS; Magnesium chloride; Malnad Gidda cattle; Messenger ribonucleic acid; Met; Methionine; MgCl(2); Microliters; Micromolar; Microsatellite; Milimolar; Millimoles; Minutes; Molar; Molecular characterization; Molecular evolutionary genetic analysis; Molecular weight; Musculoaponeurotic fibrosarcoma oncogene homolog; Mw; N-terminal; NCBI; NF-Y; NIC; NJ; Nanogram; National center for biotechnology information; Native cattle; Neighbor-joining; Nimari cattle; Nuclear transcription factor Y; ONC; ORF; Ongole cattle; Open reading frame; PCR; Paired box gene 4; Pax-4; Pfam database; Phragment assembly program; Phrap; Picomol; Pmol; Polymorphism; PrF; PrR; Promoter forward; Promoter reverse; Protein families database; R; RAC; RFX; RNA; RNAse; RSC; RT; Rathi cattle; Red Sindhi cattle; Regulatory factor X; Reverse; Reverse transcriptase; Ribonuclease; Ribonucleic acid; SAC; SBD; SINEs; SMART; SNP; Sahiwal cattle; Searching transcription factor binding sites; Seconds; Short interspersed nucleotide repetitive elements; Simple modular architecture research tool; Single-nucleotide polymorphism; Substrate binding domain; T; TESS; TFBS; TFSearch; THC; TRANSFAC; Taq; Tharparkar cattle; Thermus aquaticus; Thr; Threonine; Thymine; Transcription element search system; Transcription factor binding sites; Transcription factor database; U; UMC; UTR; Umblachery cattle; Unit; Untranslated region; bp; cDNA; dNTP; heat shock element; kDa; mM; mRNA; matrix search for transcription factor binding sites; min; mmol; ng; polymerase chain reaction; sec; v-Maf; °C; μM; μl.