Detection of functional polymorphisms in the hsp70 gene and association with cold stress response in Inner-Mongolia Sanhe cattle

Abstract

The genetic mechanisms underlying the cattle resilience to severe cold temperatures are still unknown. In this study, we observed that four blood biochemical parameters were significantly altered, i.e., blood adrenocorticotropic hormone (ACTH), triiodothyronine (T3), thyroxine (T4), and potassium (K⁺) after expose to - 32 °C for 3 h. This was observed using 105 healthy Sanhe heifers with similar weight (398.17 ± 34.06 kg) and age (19.30 ± 4.91 months). A total of 20 single nucleotide polymorphisms (SNPs) were identified in 5'-flanking region of the hsp70 gene in Sanhe cattle, while only 10 SNPs were segregating when comparing genetic variations between Sanhe cattle and 285 Chinese Holstein samples. Statistically significant associations between the genomic markers SNP-42^-, SNP-105⁺, SNP-181⁺, and SNP-205⁺ with blood T3 and between SNP-105⁺ and blood T4 were observed by applying the general linear model procedure and Bonferroni t test. Furthermore, we demonstrated that the T alleles of SNP-42^- and SNP-205⁺ in the GC box and Kozak sequence of the hsp70 gene, respectively, significantly decreased the green fluorescent proteins activity in vitro GFP reporter assays. These findings suggest that these two SNPs are causative polymorphisms involved in the regulation of hsp70 promoter activity and might contribute to the observed association between the hsp70 gene and T3 and T4 levels in Sanhe cattle. Thus, hsp70 gene is a promising candidate gene to be validated in independent cattle populations and functional studies related to cold stress resilience in cattle.

Keywords: Climate resilience; Functional polymorphisms; Inner-Mongolia Sanhe cattle; Severe cold stress; hsp70 gene.

Fig. 1

Sanhe breed animals during severe cold stress challenge

Fig. 2

Nucleotide sequences of 5′-flanking region of the hsp70 gene in Sanhe cattle. Gray shadings indicate deletions in Hereford sequence (AC_000180.1). Underlined sequences represent elements (HSE heat-shock element, C/E CCAAT enhancer). Boxed sequences indicate the transcription start site. Capital and bold letters indicate the variations within Sanhe cattle. Italics indicate nucleotides (ATG) that code for the first amino acid

Fig. 3

GFP reporter expression analysis of SNP-42- and SNP-205+ polymorphisms. a Illustration of three plasmids constructed for promoter activity analysis of the hsp70 gene. b Statistical analysis of flow cytometry data for the three plasmids. Comparisons of p42C-205C, p42T-205C, and p42C-205T showed lower fluorescence intensity of GFP, by 21.38% and 13.90%, respectively. Data is given as means ± SD (*P < 0.05; n = 5/group). c Representative fluorescence images for GFP by microscope