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

Abstract

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.

Keywords: Chinese Holstein cattle; Heat stress; Hsp90AA1 SNPs; Promoter activity; microRNA-mimic.

Fig. 1

Oscillograms (a) and alignment (b) of each genotype showing the mutations in the fragmentary amplification by P1and P2. The oscillograms indicated two mutations in the promoter and two mutations in the coding regions. Alignment showing substitutions of bases (the bases in the red color), and the locus of the bases in HSP90AA1 gene was marked with digits

Fig. 2

Association analysis between SNP g.− 87 G > C genotypes and promoter activity before heat shock (c) and after heat shock (d) in Hela cells, and assessment of relative mRNA expression of individual bearing genotypes GG and CC in non-stress and stress seasons (e). (a) and (b) are a plasmid and plasmid digestion. Luciferase activities were measured by using the Luciferase Assay System

Fig. 3

Correlation between SNP G > C g.− 87 and milk yield (a) and somatic cell count (b) between seasons. Data were analyzed by analysis of variance with a general linear model using comparative means in Statistix (version 8.1). P < 0.05 considered significant

Fig. 4

Association between SNPs A > G g.605, T > G g.1662, and G > Ag.2819 genotypes with milk yield (a) and somatic cell count (b) between seasons. Three SNPs were identified using sequencing technique by GenScript USA Inc. (Piscataway, NJ). Data were analyzed by analysis of variance with a general linear model using comparative means in Statistix (version 8.1). P < 0.05 considered significant

Fig. 5

Correlation between SNP A > G g.4172 genotypes and milk yield (a) and somatic cell count (b) between seasons. Data were analyzed by analysis of variance with a general linear model using comparative means in Statistix (version 8.1). P < 0.05 considered significant

Fig. 6

Polymorphism in g.4172 in 3′-UTR of Hsp90AA1 is predicted to be targeted by efu-miR-202 (a) and dme-miR-2279-5p (b). miRNAs with a seed sequence base pairing on g.4172 A/G were identified with microRNA database: miRBase website (http://www.mirbase.org/). The position of mutation; A and G bases were given in red color

Fig. 7

A 309 bp of HSP90AA1 with A allele or G allele in the 3′-UTR region inserted into pmirGLO miRNA target sites between restriction sites KpnI and Xhol (a). the plasmid was digested and visualized (b). miRNA mimics (20 nM) of miR-202, miR-2279, and a negative control (NC) were co-transfected with pmirGLO Dual-Luciferase vectors and an internal transfection control pRL-TK vector into Hella cells. After 48 h of transfection, luciferase activities (firefly luciferase activity was normalized to Renilla luciferase expression) in miR-202 or miR-2279-transfected cells were measured and compared to those in negative control-transfected cells. Transcripts with miR-202 seed sites (white) did not affect the luciferase reporter activity (c); however, transcripts with miR-2279 seed sites can specifically repress luciferase reporter activity. The luciferase expression registered with G mutant allele (white) was lower than those observed with control (black) and A wild-type allele (d). Representative data from triple independent experiments are shown with mean ± SE