【作者】 刘延鑫；

【导师】 王根林；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 本研究目的在于揭示与奶牛耐热性状相关候选基因的分子遗传机理,为标记辅助选择耐热性奶牛新品系提供基础资料。通过研究不同季节条件下奶牛部分生理指标、生化指标及产奶性状指标的变化规律；利用实时荧光定量PcR分析奶牛外周血淋巴细胞HSP70、ATP1A1、SOD3和GPX3基因mRNA表达水平与耐热指标的相关性,确定奶牛耐热性状的候选基因；选择牛遗传图谱上分别与耐热性状相关候选基因及影响产奶量和乳组成QTL连锁的7个微卫星标记,分析在奶牛样本群体的遗传参数,并运用最小二乘拟合一般线性模型与奶牛的耐热指标进行关联分析；采用PCR-LIS-SSCP和DNA测序技术检测奶牛耐热性状候选基因ATP1A1基因的SNPs,并研究了其多态性和奶牛耐热性状的关系。结果如下：1热应激对奶牛部分生理生化指标及产奶性状的影响为了研究不同季节条件下奶牛的部分生理指标、血液无机离子含量、红细胞膜Na+,K+一ATP酶活性、血液抗氧化指标和产奶性状的变化规律,选择3-5胎次的80头健康荷斯坦奶牛,根据夏季直肠温度和产奶量下降率两个指标分为耐热组(直肠温度在37.5-39.5℃之间,产奶量下降率<10%)和非耐热组(直肠温度>39.5℃,产奶量下降率>40%),分别于夏季8月份(牛舍日平均气温为32.5℃,THI为84.16)和同年秋季11月份(牛舍日平均气温为12.5℃,THI为56.80)条件下,测定直肠温度、耐热系数、呼吸频率、血液无机离子含量、血清SOD酶活、血清GSH-px酶活、血清MDA含量、红细胞膜Na+,K+-ATP酶活性及产奶性状等指标,结果表明：热应激对奶牛的部分生理、生化指标及产奶性状影响程度很大,导致直肠温度升高,呼吸频率增加,血清Na+和红细胞钾含量下降,红细胞膜Na+,K+-ATP酶活性下降,抗氧化能力受到损伤,产奶量下降和体细胞评分增高等,其中,直肠温度、耐热系数、红细胞钾含量、红细胞膜Na+,K+-ATP酶活、血清SOD酶活、血清GSH-px酶活、日产奶量在夏季耐热组与非耐热组间差异显著(P<0.05),表明其与奶牛耐热性关系密切,可以作为选育耐热性奶牛新品系和监测热应激程度的参考指标。2奶牛HSP70、ATP1A1、SOD3和GPX3基因mRNA表达水平与耐热性关系的研究为了研究奶牛HSP70、ATP1A1、SOD3和GPX3基因mRNA表达水平与耐热性的相关性,确定奶牛耐热性状的候选基因,为揭示奶牛耐热性状分子遗传机理提供参考。选取16头3胎次的高产健康荷斯坦奶牛,分为耐热组和非耐热组,分别于夏季8月份(牛舍日平均气温为32.5℃,THI为84.16)和同年秋季11月份(牛舍日平均气温为12.5℃,THI为56.80)条件下,采集奶牛尾静脉血液,并测定夏季直肠温度、产奶量下降率和红细胞钾含量作为耐热指标；应用RT-PCR和实时荧光定量RT-PCR技术分析不同温度条件和不同组别奶牛外周血淋巴细胞HSP70、ATP1A1、SOD3和GPX3基因mRNA表达水平的差异及其与奶牛耐热性状的关系。结果表明：与秋季相比,夏季奶牛HSP70、ATP1A1和GP.X3的]mRNA表达水平极显著上升(P<0.01), SOD3mRNA表达水平显著提高(P<0.05)；夏季高温期间,耐热组的ATP1A1和SO1D3基因的mRNA表达水平极显著高于非耐热组(P<0.01),GPX3 mRNA表达水平显著高于非耐热组(P<0.05),而HSP70mRNA表达水平在二组别间差异不显著(P>0.05)。相关分析表明奶牛外周血淋巴细胞四个基因mRNA表达水平与部分耐热指标呈显著相关(P<0.05)。因此,可以得出HSP70、ATP1A1、SOD3和GPX3参与了热应激过程中分子水平上的表达调控,可能对奶牛耐热性起作用,可作为影响奶牛耐热性状的候选基因进一步研究。3奶牛微卫星标记与耐热性状的相关分析为了探讨微卫星DNA作为奶牛耐热性状遗传标记的可行性。根据牛基因组遗传图谱,选择奶牛不同染色体上分别与HSP70、ATP1A1、SOD、GPX3基因和影响产奶量和乳组成的QTL连锁的微卫星座位BMS468.BM1258、BM1815、BM723、SOD1. BMS2258和D14S31,对160头3-5胎次中国荷斯坦奶牛进行多态性检测,采用最小二乘拟合一般线性模型分析微卫星座位与耐热性状部分指标之间的关联效应。结果表明：所选7个微卫星座位在该奶牛群体中均表现出丰富的遗传多态性,多态信息含量、遗传杂合度和有效等位基因数的平均值分别为0.64089、0.6866和3.2789。微卫星座位BMS468与耐热系数、产奶量下降率和红细胞钾含量显著相关的最有利基因型为134bp/128bp(P<0.05),与红细胞膜Na+,K+-ATP酶活显著相关的最有利基因型为140bp/134bp(P<0.05)；微卫星座位BM1258与产奶量下降率显著相关,最有利基因型为101bp/99bp(P<0.05)；微卫星座位BM1815与耐热系数、红细胞钾含量和红细胞膜Na+,K+-ATP酶活显著相关,最有利基因型为186 bp /148 bp(P<0.05)；微卫星座位BM723与产奶量下降率、红细胞膜Na+,K+-ATP酶活和红细胞钾含量显著相关,最有利基因型为161 bp/111 bp(P<0.05)；微卫星座位BMS2258与产奶量下降率和血清GSH-px酶活显著相关,最有利基因型为182 bp/164 bp(P<0.05)；微卫星座位SOD1与耐热系数和产奶量下降率显著相关,最有利基因型为148 bp/148 bp(P<0.05),与血清SOD酶活显著相关的最有利基因型为148 bp/146 bp(P<0.05);微卫星座位D14S31与产奶量下降率显著相关,最有利基因型为176 bp/152 bp(P<0.05).本研究发现了对奶牛耐热性状有显著效应、多态性较高的微卫星基因座,为开展奶牛耐热性的标记辅助选择提供了有价值的遗传标记。4 ATP1A1基因多态性及其与奶牛耐热性状的关联分析为了研究ATP1A1基因多态性及其与奶牛耐热性状的相关性,寻找影响奶牛耐热性状的QTL,为分子标记辅助选择奶牛耐热性新品系提供相应依据。以160头3-5胎次的健康高产中国荷斯坦奶牛为实验材料,采取PCR-LIS-SSCP和DNA测序等方法进行多态位点检测及基因型分析,研究单个多态位点基因型及不同多态位点的合并基因型与奶牛耐热性状的关系。结果表明,与GenBank ATP1A1基因(NC 007301.3)序列比较,ATP1A1基因外显子5、14和17各发现一个沉默SNP突变,分别为-4357bp处A/G、-14103bp处G/A和-15739 bp处C/A；内含子5发现1个SNP,为-4397bp处T/G；内含子14发现3个SNP,分别为-14242bp处C/T、-14829处T/-和-14997处G/A,统计分析表明4个多态位点的不同基因型个体间的耐热系数、产奶量下降率、红细胞钾含量和红细胞膜Na+，K+-ATP酶活性存在显著差异；在P5和P17位点,AA基因型为各自位点上的耐热性状最有利基因型,在P14和P15位点,AC基因型为各自位点上的耐热性状最有利基因型。合并基因型与耐热性状的关联分析表明,最佳合并基因型AAACACAA是4个多态位点上单个最优基因型的组合,但高于最好的单个基因型效应。本研究表明ATP1A1基因4个多态位点附近可能存在影响奶牛耐热性状的QTL,ATP1A1基因可能是影响奶牛耐热性状的重要基因之一更多还原

【Abstract】 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.更多还原