| This study is aimed to investigate the molecular genetic mechanisms of candidate genes for heat tolerance traits in Chinese Holstein dairy cattle for accumulating basis to breed heat tolerance cows in China. In this paper, several physiological, biochemical and milk productivity parameters of dairy cows at different seasons were analyzed. The mRNA levels of HSP70, ATP1A1, SOD3 and GPX3 gene in lymphocytes of peripheral blood in dairy cows among various temperature groups and genotypes were studied by using real-time RT-PCR. and the correlation of these genes mRNA level with the heat tolerance traits was analysed. Genetic variation of seven microsatellite loci, which were closely linkaged with HSP70, ATP1A1, SOD. GPX3genes and QTL of major effect on milk yield and composition were analyzed in the dairy cows, relationships of the seven microsatellite loci with heat tolerance traits were also studied by least squares linear model. PCR Low Ionic Strength single-strand conformation polymorphism （PCR-LIS-SSCP） and DNA sequencing methods were used to analyze the polymorphisms within the coding region of bovine ATP1A1 gene, also, the association of the genetic polymorphisms of ATP1A1 gene with heat tolerance traits was studied, results as follows:1 Effect of heat stress on several physiological, biochemical and milk productivity parameters in dairy cowsThe study is aimed to compare several physiological indicators, the concentrations of inorganic ions in blood, Na+,K+-ATPase activity of erythrocyte membrane, anti-oxidative capability, and milk productivities in different seasons in dairy cows. Eighty Chinese Holstein cows were selected and divided into two groups according to rectal temperature （RT） and drop rate of milk yield （DRMY） in summer as compared with the milk yield in spring, the heat tolerance groups （RT in the range of 37.5℃to 39.5℃. DRMY<10%） and heat stress groups （RT> 39.5℃, DRMY> 40%） were classfied. These animals were subjected to a high temperature in August of summer （daily mean temperature 32.5℃temperature-humidity index （THI）=84.16） and a optimal temperature in November of autumn （daily mean temperature 12.5℃, THI=54.80）, the indicators of RT. heat tolerance coefficient （HTC）. respiratory rate （RR）. the activity of superoxide dismutase（SOD）, glutathione peroxidase （GSH-Px） and malondialdehyde （MDA） content in plasma. plasma Na+,K+, Cl-,red blood cell potassium. Na+,K+-ATPase activity of erythrocyte membrane were determined. Results showed that the RT. RR and MDA content of dairy cows in summer were significantly higher than those in autumn （P<0.05）, the activity of SOD. GSH-px in plasma and Na+,K+-ATPase activity of erythrocyte membrane, plasma Na+, red blood cell potassium, milk yields and were significantly lower than those in autumn （P<0.05）, in addition, RT, HTC, red blood cell potassium. Na+,K+-ATPase activity of erythrocyte membrane, and the activity of SOD, GSH-Px in plasma were significantly differ from heat tolerance groups and heat stress groups in summer （P<0.05）. Results indicated that RT, HTC. red blood cell potassium, Na+,K+-ATPase activity of erythrocyte membrane, and the activity of SOD, GSH-Px in plasma showed close relationships with heat tolerance traits, those indexes can be used as indicators of heat stress of dairy cows and for anti-heat stress traits in dairy cattle breeding.2 Relationships between the HSP70, ATP1A1, SOD3 and GPX3 gene expression and heat tolerance traits in dairy cowsThe present study was performed to investigate the relationships between the mRNA level of HSP70. ATP1A1. SOD3 and GPX3 gene in lymphocytes of peripheral blood and heat tolerance traits in dairy cows, so as to provide references for finding heat tolerance candidate gene and understanding the molecular genetic mechanism of heat tolerance in Chinese Holstein dairy cows. Sixteen healthy and high milk yields dairy cows, classified as the heat tolerance group （T） or heat stress group（S）, were sacrificed for blood sampling at a high temperature in August of summer （daily mean temperature 32.5℃temperature-humidity index （THI）=84.16） and a optimal temperature in November of autumn（daily mean temperature 12.5℃, THI=54.80）, and the heat tolerance traits such as heat tolerance coefficient （HTC）. drop rate of milk yield in summer and red blood cell potassium were measured. The mRNA expression levels of HSP70. ATP1A1, SOD3 and GPX3 genes in lymphocytes of peripheral blood among various temperature groups and groups were analyzed were investigated by RT-PCR and real-time RT-PCR. Results showed that the HSP70. ATP1A1 and GPX3 mRNA level at temperature 32.5℃was higher than that at optimal temperature 12.5℃in dairy cows （P<0.01）. the SOD3 mRNA level at temperature 32.5℃was higher than that at optimal temperature 12.5℃（P<0.05）. In summer, the expression level of ATP1A1 and SOD3 mRNA in heat tolerance goup was higher than that in heat stress group （P<0.01）, and the expression level of GPX3 mRNA was higher than that in heat stress group （P<0.05）, but there was no significant difference of HSP70 mRNA expression level in two groups （P>0.05）. Bivariate correlation analysis showed that HSP70, ATP1A1, SODS and GPX3 gene mRNA expression in lymphocytes of peripheral blood were correlated with several indicators of heat tolerance traits in dairy cows （P<0.05）. It suggested that HSP70, ATP1A1, SOD3 and GPX3 gene might play a key role in anti-heat stress and might be candidate genes for heat tolerance traits in dairy cows.3 Correlation analysis between microsatellite loci and heat tolerance traits in Chinese Holstein dairy cowsThe aim of the present work was to research whether the microsatellite DNA could improve heat tolerance traits of dairy cows as the genetic marker. According to genetic map of cattle,7 dairy cattle microsatellite loci （BMS468, BM1258, BM1815, SOD1, BM723, BMS2258 and D14S31） closely linkaged with HSP70, ATP1A1, SOD, GPX3 gene and QTL of major effect on milk yield and composition in different chromosome were detected in 160 Chinese Holstein dairy cows of 3-5 parity. The genetic analysis and correlation analysis between microsatellite markers and heat tolerance traits were calculated. The genetic analysis showed that 7 microsatellite loci had a rich diversity in Chinese Holstein dairy cows. The mean values of the PIC, He, and Ne were 0.64089,0.6866 and 3.2789, respectively. Least square analysis evidenced that BMS468 had a significant effect on the heat tolerance coefficient （HTC）, drop rate of milk yield in summer and red blood cell potassium （P<0.05）, and 134 bp/128 bp were the favorable genotypes for respectively, BMS468 also had a significant effect on the Na+,K+-ATPase activity of erythrocyte membrane（P<0.05）, and the 140 bp/134 bp were the favorable genotypes. BM1258 had a significant effect on drop rate of milk yield in summer （P<0.05）, and the genotype 101 bp /100 bp was the most favorable genotype. BM1815 had a significant effect on the HTC, red blood cell potassium and Na+,K+-ATPase activity of erythrocyte membrane （P<0.05）. and the genotypes 186 bp/148 bp were the favorable genotypes, respectively （P<0.05）. BM723 had a significant effect on the drop rate of milk yield in summer, Na+,K+-ATPase activity of erythrocyte membrane and red blood cell potassium（P<0.05）, and the genotypes 161 bp/111 bp was the most favorable genotypes, respectively. BMS2258 had a significant effect on drop rate of milk yield in summer and GSH-px activity in plasma （P<0.05）, and the genotype 182 bp/164 bp was the most favorable genotype, respectively. SOD1 had a significant effect on the HTC and drop rate of milk yield in summer and SOD activity in plasma （P<0.05）, and the genotypes 148 bp/148 bp was the most favorable genotypes, respectively. SOD1 also had a significant effect on the SOD activity in plasma （P<0.05）, and the 148 bp 1146 bp were the favorable genotypes. D14S31 had a significant effect on drop rate of milk yield in summer （P<0.05）, and the genotype 176 bp/152 bp was the most favorable genotype. It indicated that the 7 microsatellite loci with a rich diversity and have a significant effect on heat tolerance traits in dairy cows, and it can provided valuable genetic markers for marker-assisted selection of dairy cattle.4 Genetic polymorphisms of bovine A TP1A1 gene and its association with heat tolerance traits in dairy cowsThe bovine ATP1A1 gene polymorphisms were detected, and its association with heat tolerance traits were analyzed in order to find the quantitative trait locus （QTL） which providing a basis for molecular marker-assisted selection in dairy cattle breeding. PCR Low Ionic Strength single-strand conformation polymorphism （PCR-LIS-SSCP） and DNA sequencing methods were used to analyze the polymorphisms within the coding region of bovine ATP1A1 gene. According to NC007301.3, seven novel SNPs were identified, the A/G SNP at -4357bp was in exon 5; the G/A SNPs at -14103bp was in exon 14 and C/A SNP at -15739bp was in exon 17 of ATP1A1 gene, but all of them caused no amino acids exchange, the T/G SNP at -4397bp was in intron 5, the C/T at -14242 bp, the T/-SNP at-14829 and the G/A SNP at -14997bp were in intron 14 of ATP1A1 gene, respectively. The association of the ATP1A1 gene polymorphisms with heat tolerance traits was studied, we found that the individuals with genotype AA at P5 and P17 loci showed significantly higher heat resistance than those of other genotype （P<0.05）, and the most favorable genotype for heat tolerance traits was AC at P14 and P15 loci. Further, the association of the combined genotypes （haplotypes） of the four polymorphism loci of ATP1A1 gene with heat tolerance traits shows that most favorable haplotype was AAACACAA, the combination favorable genotypes of four polymorphism loci, but the genetic effect of AAACACAA was not the simple addition of different genotypic effects. It showed that there may exist QTL of heat tolerance traits near the four polymorphism loci of ATP1A1 gene, and ATP1A1 gene might be one of the candidate genes having an important influence on the heat tolerance traits in dairy cows, our findings also implied that the ATP1A1 gene polymorphism here could be potential genetic markers for anti-heat stress trait in dairy cattle breeding. |
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