【作者】 李拓；

【导师】 王金玉； 储明星；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 本研究选取了河北省四个牛场共计210头中国荷斯坦母牛作为研究对象,应用分子生物学实验技术研究与奶牛乳房炎相关的AHCY (S-adenosylhomocysteine hydrolase)基因、TLR10基因、PRKDC (DNA-dependent protein kinase)基因和SLC11A1 (solute carrier family 11 member 1)基因的单核苷酸多态性(single nucleotide polymorphisms, SNPs) ,并采用最小二乘拟合一般线性模型分析候选基因与荷斯坦母牛乳中体细胞评分(somatic cell score, SCS)之间的关系及影响因素。研究结果表明:(1)采用PCR产物直接进行非变性聚丙烯酰胺凝胶电泳的方法对本研究奶牛群体AHCY基因和TLR10基因编码区进行单核苷酸多态性检测,结果表明检测的两个基因外显子都不存在多态性现象。(2)利用PCR-SSCP结合测序技术对所研究奶牛群体PRKDC基因进行多态性筛查,结果发现G34126A、C77353T、T77384G、C77406T和77408 bp处C缺失五个多态位点,其中G34126A突变导致Ser1071Asn替换。χ2检验显示本研究奶牛群体在这五个位点均处于Hardy-Weinberg平衡。G34126A多态位点经SSCP检测发现3种基因型AA(0.186)、AB(0.433)和BB(0.381);该多态性与SCS的关联分析表明,AA基因型SCS最小二乘均值显著低于AB和BB基因型所对应的值(P<0.05),AB和BB基因型SCS最小二乘均值差异不显著(P>0.05),AA基因型为乳房炎抗性的有利基因型。C77353T、T77384G和C77406T同时77408 bp处C缺失多态位点经SSCP检测发现5种基因型CC(0.786)、CD(0.129)、DD(0.009)、CE(0.067)和DE(0.009);关联分析显示,CC、CD、DD、CE和DE基因型SCS最小二乘均值差异均不显著(P>0.05)。(3)对SLC11A1基因的编码区进行多态性检测,发现其编码区在C723T、C1144G、A1749G、T1782G、1803-4bp处GT缺失5个突变位点,其中C723T突变造成Ala217Val替换,C1144G突变造成Pro356Ala替换。χ2检验显示本研究奶牛群体在这五个位点均处于Hardy-Weinberg平衡。C723T突变位点经Bpu10I酶切产生2种基因型AA(0.791)和AB(0.209); C1144G突变位点经PstI酶切产生3种基因型CC(0.628)、CD(0.343)和DD(0.019);关联分析表明,AA型SCS均值显著低于AB型的(P<0.05)。A1749G、T1782G、1803-4bp处GT缺失3个突变位点经SSCP检测发现3种基因型EE(0.686)、EF(0.300)和FF(0.014),该多态性与SCS的关联分析表明,EE、EF和FF基因型的SCS均值差异不显著(P>0.05)。对多态位点进行单倍型分析,发现在荷斯坦母牛210个个体中共检测到7种单倍型,其中单倍型ACG频率超过了70%。(4)方差分析结果表明公牛、牛场、胎次对牛乳中体细胞评分都有显著影响(P<0.05)。更多还原

【Abstract】 210 Chinese Holstein cow blood samples from 4 dairy farms of Hebei Province were applied to analyze the single nucleotide polymorphisms of AHCY, TLR10, PRKDC and SLC11A1 gene. The correlation between the different genotypes and somatic cell scores (SCS) was analyzed using least-squares analysis, the results as follows:(1) Non-denatured PAGE(Polyacrylamide Gle Electorphoresis) method was used to detect the mutation of AHCY and TLR10 genes, no polymorphism was found in both two genes in these population.(2) PCR-SSCP and DNA sequencing technologies were used to detect the polymorphism of PRKDC gene in 210 Holstein cows, five polymorphic sites were found, G34126A, C77353T, T77384G, C77406T and C deletion at 77408 bp were found, and G34126A mutation caused an amino acid change Ser1071Asn. Chi-square test showed that Holstein cows were in Hardy-Weinberg equilibrium in all sites. AA(0.186), AB(0.433) and BB(0.381) genotypes were detected at G34126A site in Holstein cows using PCR-SSCP. CC(0.786), CD(0.129), DD(0.009), CE(0.067) and DE(0.009) were found at C77353T, T77384G, C77406T and C deletion of 77408 bp sites in Holstein cows by PCR-SSCP. The relationship between polymorphisms of PRKDC gene and SCS (somatic cell score) in Holstein cows was analyzed by least squares linear model. Least squares mean of SCS for AA was significantly lower than that for AB or BB (both P<0.05), and the difference of the least squares means for SCS between AB and BB was not significant (P>0.05). AA genotype is predominant genotype to mastitis. The difference of the least squares means for SCS between CC, CD, DD, CE and DE was not significant (P>0.05). (3) Polymorphisms of SLC11A1 gene in 210 Holstein cows were detected by PCR-SSCP and PCR-RFLP, five polymorphic sites of SLC11A1 gene at C723T, C1144G, A1749G, T1782G, and GT deletion of 1803-4bp were found in Holstein cows and C723T, C1144G mutations resulted in amino acid change: Ala217Val, Pro356Ala, respectively. Chi-square test showed that Holstein cows were in Hardy-Weinberg equilibrium in all sites. AA(0.791), AB(0.209) genotypes were detected at C723T site using PCR-RFLP with Bpu10 I, CC(0.628), CD(0.343) and DD(0.019) were detected at C1144G site with Pst I. LSM of SCS for AA was significantly lower than that for AB (P<0.05), AA genotype is the predominant genotype to mastitis. EE(0.686), EF(0.300) and FF(0.014) were found at A1749G, T1782G and GT deletion of 1803-4bp polymorphic sites in Holstein cows by PCR-SSCP. LSM of SCS among all genotypes was nonsignificant (P>0.05). In 210 Holstein cows, 7 haplotypes were reconstructed. The frequency of ACG was more than 70%.(4) Least square analysis showed that bull, cattle farm, parity had significant effect on SCS(P<0.05).更多还原