Cell Stress and Chaperones

, Volume 23, Issue 4, pp 639–651 | Cite as

Genetic polymorphism in Hsp90AA1 gene is associated with the thermotolerance in Chinese Holstein cows

  • T. M. Badri
  • K. L. Chen
  • M. A. Alsiddig
  • Lian Li
  • Yafei Cai
  • G. L. WangEmail author
Original Paper


The heat shock protein 90 (Hsp90) is a copious and ubiquitous molecular chaperone which plays an essential role in many cellular biological processes. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the Hsp90AA1 gene and to determine their association with heat stress traits in Chinese Holstein cattle breed. Direct sequencing was used to identify new SNPs. Luciferase reporter assay methods were used to assess g.− 87G > C and g.4172A > G loci in the promoter activity and 3′-UTR, respectively. Quantitative real-time PCR was utilized to quantify the gene expression profile. Five SNPs were identified in 130 multiparous lactating cows: one SNP in the promoter, three SNPs in the coding region, and one in 3′-UTR were novel and reported for the first time in this study. As a result of promoter assay using dual luciferase assay system, the genotype CC showed the highest transcription activity region (13.67 ± 0.578) compared to the wild-type GG (3.24 ± 0.103). On the other hand, the result revealed that one of the selected microRNAs (dme-miR-2279-5p) was found to interact with the Hsp90AA1 3′-UTR sequence and to suppress the reporter activity markedly in the presence of the allele G (2.480 ± 0.136). The expression of Hsp90AA1 in cow bearing mutant allele C was higher (4.18 ± 0.928) than cows bearing wild-type allele G (1.008 ± 0.0.129) in stress season. In summary, there was an association between genetic variations in the Hsp90AA1 and thermoresistance. This association could be used as a marker in genetic selection for heat tolerance in Chinese Holstein cattle breeds.


Chinese Holstein cattle Hsp90AA1 SNPs Heat stress Promoter activity microRNA-mimic 



The authors would like to thank Dr. James Hadley from the UK for helping in language checking of this manuscript. We also want to express our thanks to Xigang and Tangquan dairy farms for allowing us to conduct this study on animals in their farms. The authors also would like to thank the National Science Foundation of China (grant no. 31372290) and the Science and Technology Sustainability Project of China (2012BAD12B00) for funding this project.

Supplementary material

12192_2017_873_MOESM1_ESM.docx (12 kb)
Table S1 (DOCX 12 kb)
12192_2017_873_MOESM2_ESM.doc (32 kb)
Table S2 (DOC 32 kb)
12192_2017_873_MOESM3_ESM.doc (33 kb)
Table S3 (DOC 33 kb)
12192_2017_873_MOESM4_ESM.doc (32 kb)
Table S4 (DOC 31 kb)
12192_2017_873_MOESM5_ESM.doc (30 kb)
Table S5 (DOC 29 kb)


  1. Basiricò L, Morera P, Primi V, Lacetera N, Nardone A, Bernabucci U (2011) Cellular thermotolerance is associated with heat shock protein 70.1 genetic polymorphisms in Holstein lactating cows. Cell stress and Chaperones 16(4):441–448. CrossRefPubMedGoogle Scholar
  2. Bedulina D, Evgen'ev M, Timofeyev M, Protopopova M, Garbuz D, Pavlichenko V, Luckenbach T, Shatilina Z, Axenov-Gribanov D, Gurkov A (2013) Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal. Mol Ecol 22(5):1416–1430. CrossRefPubMedGoogle Scholar
  3. Berry I, Shanklin M, Johnson H (1964) Dairy shelter design based on milk production decline as affected by temperature and humidity. Transactions of the ASAE 7: 329–331. doi:
  4. Buffington D, Collazo-Arocho A, Canton G, Pitt D, Thatcher W, Collier R (1981) Black globe-humidity index (BGHI) as comfort equation for dairy cows. Transactions of the ASAE 24(3):711–714. CrossRefGoogle Scholar
  5. Charoensook R, Gatphayak K, Sharifi AR, Chaisongkram C, Brenig B, Knorr C (2012) Polymorphisms in the bovine HSP90AB1 gene are associated with heat tolerance in Thai indigenous cattle. Trop Anim Health Prod 44(4):921–928. CrossRefPubMedGoogle Scholar
  6. Chen B, Zhong D, Monteiro A (2006) Comparative genomics and evolution of the HSP90 family of genes across all kingdoms of organisms. BMC Genomics 7: 156. doi:
  7. Collier R, Collier J, Rhoads R, Baumgard L (2008) Invited review: genes involved in the bovine heat stress response. J Dairy Sci 91(2):445–454. CrossRefPubMedGoogle Scholar
  8. Collier RJ, Stiening C, Pollard B, VanBaale M, Baumgard L, Gentry P, Coussens P (2006) Use of gene expression microarrays for evaluating environmental stress tolerance at the cellular level in cattle. J Anim Sci 84: E1-E13. doi:
  9. Craven R, Egerton M, Stirling, C (1996) A novel Hsp70 of the yeast ER lumen is required for the efficient translocation of a number of protein precursors. EMBO J 15: 2640.Google Scholar
  10. Deb R, Sajjanar B, Singh U, Kumar S, Brahmane M, Singh R, Sengar G, Sharma A (2013) Promoter variants at AP2 box region of Hsp70.1 affect thermal stress response and milk production traits in Frieswal cross bred cattle. Gene 532(2):230–235. CrossRefPubMedGoogle Scholar
  11. Erhardt E, Weimann C (2007) Use of molecular markers for evaluation of genetic diversity and in animal production. Arch Latinoam Prod Anim 15Google Scholar
  12. Hansen P (2004) Physiological and cellular adaptations of zebu cattle to thermal stress. Anim Reprod Sci 82:349–360. CrossRefPubMedGoogle Scholar
  13. Hu H, Zhang Y, Zheng N, Cheng J, Wang J (2016) The effect of heat stress on gene expression and synthesis of heat-shock and milk proteins in bovine mammary epithelial cells. Anim Sci J 87(1):84–91. CrossRefPubMedGoogle Scholar
  14. Jin Y, Chen Z, Liu X, Zhou X (2013) Evaluating the microRNA targeting sites by luciferase reporter gene assay. MicroRNA Protocols.
  15. Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42(D1):D68–D73. CrossRefPubMedGoogle Scholar
  16. Kumar R, Gupta I, Verma A, Verma N, Vineeth M (2015) Genetic polymorphisms within exon 3 of heat shock protein 90AA1 gene and its association with heat tolerance traits in Sahiwal cows. Veterinary world 8(7):932–936. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kumar R, Gupta ID, Verma A, Singh SV, Verma N, Vineeth M, Magotra A, Das R (2016) Novel SNP identification in exon 3 of HSP90AA1 gene and their association with heat tolerance traits in Karan fries (Bos taurus × Bos indicus) cows under tropical climatic condition. Trop Anim Health Prod 48(4):735–740. CrossRefPubMedGoogle Scholar
  18. Lacetera N, Bernabucci U, Scalia D, Basiricò L, Morera P, Nardone A (2006) Heat stress elicits different responses in peripheral blood mononuclear cells from Brown Swiss and Holstein cows. J Dairy Sci 89(12):4606–4612. CrossRefPubMedGoogle Scholar
  19. Lee W, Wen H, Chang C, Chen M, Lin MT (2006) Heat shock protein 72 overexpression protects against hyperthermia, circulatory shock, and cerebral ischemia during heatstroke. J Appl Physiol 100(6):2073–2082. CrossRefPubMedGoogle Scholar
  20. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25(4):402–408. CrossRefPubMedGoogle Scholar
  21. Marcos-Carcavilla A, Mutikainen M, González C, Calvo JH, Kantanen J, Sanz A, Marzanov NS, Pérez-Guzmán MD, Serrano M (2010) A SNP in the HSP90AA1 gene 5′ flanking region is associated with the adaptation to differential thermal conditions in the ovine species. Cell Stress and Chaperones 15(1):67–81. CrossRefPubMedGoogle Scholar
  22. Michael CA (2008) Simple calculator to determine whether observed genotype frequencies are consistent with Hardy–Weinberg equilibrium. http://www. Tufts. Edu/~mcourt01/documents. Court lab-HW calculator. XlsGoogle Scholar
  23. Morimoto RI (1998) Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 12(24):3788–3896. CrossRefPubMedGoogle Scholar
  24. National Research Council (NRC) (1971) A guide to environmental research on animals. Natl. Acad. Sci, Washington, DCGoogle Scholar
  25. Page TJ, Sikder D, Yang L, Pluta L, Wolfinger RD, Kodadek T, Thomas RS (2006) Genome-wide analysis of human HSF1 signaling reveals a transcriptional program linked to cellular adaptation and survival. Mol BioSyst 2(12):627–639. CrossRefPubMedGoogle Scholar
  26. Pirkkala L, Nykänen P, Sistonen L (2001) Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J 15: 1118–1131. doi:
  27. Sailo L, Gupta I, Verma A, Singh A, Vishwas M, Chaudhari RD, Upadhyay R, Goswami J (2015) Single nucleotide polymorphism in HSP90AB1 gene and its association with thermo-tolerance in Jersey crossbred cows. Animal science 9.Google Scholar
  28. Salces-Ortiz J, González C, Moreno-Sánchez N, Calvo JH, Pérez-Guzmán MD, Serrano MM (2013) Ovine HSP90AA1 expression rate is affected by several SNPs at the promoter under both basal and heat stress conditions. PLoS One 8(6):e66641. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Shergojry S, Ganayi B, Ramesha K, Pirzada A (2012) Single strand conformation polymorphism (SSCP) of HSP90AA1 gene in Deoni cattle. Animal Science Reporter 6:65–69Google Scholar
  30. Trott JF, Freking BA, Hovey RC (2014) Variation in the coding and 3′ untranslated regions of the porcine prolactin receptor short form modifies protein expression and function. Anim Genet 45: 74–86., 1.
  31. Wan Y, Ma C, Wei P, Fang Q, Guo X, Zhou B, Jiang R (2017) Dynamic expression of HSP90B1 mRNA in the hypothalamus of two Chinese chicken breeds under heat stress and association analysis with a SNP in Huainan chickens. Czech Journal of Animal Science 62: 82–87.
  32. Yang C, Wang L, Liu C, Zhou Z, Zhao X, Song L (2015) The polymorphisms in the promoter of HSP90 gene and their association with heat tolerance of bay scallop. Cell Stress and Chaperones 20(4):297–308. CrossRefPubMedGoogle Scholar
  33. Yang F, Li L, Liu H, Cai Y, Wang G (2012) Polymorphism in the exon 4 of β-lactoglobulin variant B precursor gene and its association with milk traits and protein structure in Chinese Holstein. Mol Biol Rep 39(4):3957–3964. CrossRefPubMedGoogle Scholar
  34. Yeh F, Yang R, Boyle T (1999) POPGENE 32-version 1.31. Population genetics software. University of Alberta, Edmonton, AB Canada T6G 2H1.Google Scholar

Copyright information

© Cell Stress Society International 2018

Authors and Affiliations

  • T. M. Badri
    • 1
    • 2
  • K. L. Chen
    • 1
  • M. A. Alsiddig
    • 1
  • Lian Li
    • 1
  • Yafei Cai
    • 1
  • G. L. Wang
    • 1
    Email author
  1. 1.Department of Animal Genetic, Breeding and Reproduction, College of Animal Science and TechnologyNanjing Agricultural UniversityNanjingChina
  2. 2.Department of Animal Genetic and Breeding, College of Animal ProductionUniversity of BahriKhartoum NorthSudan

Personalised recommendations