Polymorphisms In HSP70A1A And HSP90AA1 And Their Association With Heat Resistance And Performance In Chinese Holstein Cows

Ambient temperature and temperature-humidity interaction have negative effect on the productive and reproductive processes of dairy cows in tropical and sub-tropical regions.Whereas,thermal stress phenomenon has become a main issue in the area of climate change affecting animal performance.On the other hand,the lactating cows(Bos Taurus)are more susceptible to thermal stress because metabolic heat production associated with lactation processes predisposes cows to hyperthermia.Heat shock proteins are highly conserved family of chaperones proteins that are ubiquitously expressed in animal’s body during heat stress periods.Among heat shock protein genes,HSP70-1 and HSP90AA1 are the most abundant proteins in eukaryotic cells and are regarded as the most significant indicator of heat stress in lactating cows.Single nucleotide polymorphisms(SNPs)at certain HSP genes might associated with heat tolerance and thermos-regulation in Chinese Holstein cattle breeds.Thus,HSP genes family could be considering as candidate genes for thermal resistance during heat stress periods.However,few studies conducted have shown an association between SNPs as HSP70A1A and HSP90AA1 genes and heat resistance in Chinese Holstein cattle breed.therefore,this Study was designed to examine if the polymorphisms in an HSP70A1A and HSP90AA1 genes can affect the heat tolerance.We screened the SNP loci by sequencing the HSP70A1A and HSP90AA1 genes,attempted to find the association between SNPs,combined haplotypes and other two phenotype traits;milk yield and somatic cell count in Chinese Holstein.Additionally,the present study aims to detect the role of FOXO3a gene in the regulation of heat shock protein genes in bovine mammary epithelial cells in vitro and studies were carried out as follows:1-Functional analysis of Single Nucleotide Polymorphisms in HSP70A1Agene associated with heat tolerance in Chinese Holstein dairy cattle.One of the most significant issues in dairy production in recent times is falling milk yield and milk quality during periods of elevated humidity and temperature(stress season)in lactating cows.The first aim of this study was to identify novel SNPs in the HSP70A1A gene that may be associated with heat stress and thermoregulation.The second aim was to validate the relationship between the HSP70A1A gene genotypes and heat resistance for single nucleotide polymorphisms(SNPs)in the Chinses Holstein breed.A total of 146 multiparous cows were used in this study to investigate the relationship between HSP70-1 and heat tolerance.Using direct sequencing method,4 SNPs were identified in HSP70A1A,among this 4,2 SNPs in promoter region were novel and were first time reported in this study.Luciferase reporter assay method was conducted and showed that GG genotype in g.-133(mutant)obviously increased the promoter activity than did CC(wildtype).The DNAStar Protean Software was used to detected the hydrophilicity and secondary structure of the HSP70A1A protein.Hopp-Woods,Gamier and Homology-based modeling online methods were used to build a three-dimensional structure for HSP70A1A protein.Two SNPs in coding region were found to bring changes to amino acid sequence and affect milk yield and somatic cell count(SCC).Two SNPs were g.462 G/T and g.1877 G/C in the coding region that brought change to the amino acid sequence of the HSP70A1A protein 154 Gln→His and 626 Gly→Ala,respectively.Moreover,626 Gly→Ala was found to affect the secondary structure of the HSP70A1A protein.Heat resistance was assessed in lactating cows by measuring milk yield and somatic cell count(SCC)in different seasons or Temperature-Humidity Indexes(i.e.,non-stress season(March)and stress season(July)).This results suggested that the genetic polymorphisms in the HSP70A1A gene may influence the heat resistance,and it can be used as molecular marker in thermoregulation.Therefore,this study may prove beneficial of these markers in genetic selections for heat tolerance in Chinese Holstein.2-Genetic polymorphisms analysis of the bovine HSP90AA1 and their association with thermotolerance in Chinese Holstein cows.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 Chinses 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,3 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 wildtype GG(3.24±0.103).On the other hand,the result revealed that,one of the selected microRNA(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 wildtype allele G(1.008±0.0.129)in stress season.In summary,there was an association between genetic variations in the HSP90AA1 and thermo-resistance.This association could be used as a marker in genetic selection for heat tolerance in Chinese Holstein cattle breeds.3-Role of FOX03 in the regulation of mRNA expression of HSP70A1A and HSP90AA1 genes in bovine mammary epithelial cellsA transcription factor FOX03a is a member of forkhead box transcription factor family that is involved in many essential cellular processes.This study aims to detect the role of FOX03 a gene in the regulation of heat shock protein genes in bovine mammary epithelial cells in vitro.To identify the contribution of FOXO3a gene in regulatory HSP70A1A and HSP90AA1 genes,bovine mammary epithelial cell were cultured at 37C using DMEM/F-12 medium.In the first experiment,cells were classified into five groups control group,and four heat shocked groups.In the second experiment,we used chemical inhibitor LY294002(10 μmol/L)to inhibit PI3K/Akt pathway to seen any changes in FOX03a and HSP70A1A protein expression.Cells were classified into six groups;control group,two group exposure to heat stress only,cells group inhibited only,and two groups were heat treated and incubated with LY294002(10 μmol/L).The total mRNA and proteins were extracted after Oh,2hrs,4hrs and 8hrs of heat treated in the first experiment and after 4hrs,8hrs as well as from control group in the two experiments and from inhibiting group in the second experiment.The results of gene expression revealed that the level of expression of FOX03a was highly significant after 8hrs(3.806±0.243)of cells heat shocked treated compared to control group.Whereas,HSP70A1A and HSP90AA1 were seen increased gradually with increasing the time of recovery and significant of gene expression was observed after 8hrs of heat treated in both HSP70A1A(2.331 ± 0.189)and HSP90AA1(6.134±0.777)compared to their basal groups.Moreover,in first experiment,the western blot results showed significant protein concentrations after 8hrs of heat treated in FOX03a(1.545±0.162),HSP70A1A(1.617±0.039)and HSP90AA1(1.898±0.266)compared to their control groups.Additionally,in the second experiment the western blot revealed that the protein density in cells groups incubated with LY294002(10 μmol/L)was significantly reduced in FOX03a(0.494±0.011)and HSP70A1A(0.529±0.021)after 8hrs of heat treated in bovine mammary epithelial cell compared to the same heat stress treated only group after 8hrs are 2.793±0.225 and 1.766±0.069 folds,respectively.In summary,the transcription factor FOX03 a gene was observed response to heat stress,which in turn in overexpression of HSP70A1A and HSP90AA1 in bovine mammary cells.This result speculated that FOX03a gene could contribute to regulation expression of heat shock proteins...