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TLR2基因的SNPs及其与奶牛乳腺炎相关性研究
Study on the Relationships between SNPs of TLR2 Gene and Mastitis of Dairy Cow
【作者】 张翠霞；
【导师】 赖松家；
【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2008， 硕士
【摘要】 奶牛乳腺炎是奶业经济损失最严重的疾病,遗传因素是影响奶牛乳腺炎发生的因素之一。TOLL样受体家族(Toll-like receptor,TLR)能在病原体入侵机体早期启动天然免疫,在抗感染中起着重要作用。本研究采用PCR-RELP、CRS-RFLP和基因测序技术研究了297头中国荷斯坦牛奶牛TLR2基因SNPs及其与奶牛乳腺炎相关性。结果如下:扩增TLR2基因的编码区部分序列,测序结果表明:在10098、10111和11541三个位点发现多态性。10098位点碱基突变由T/G,导致编码蛋白质的天冬氨酸突变为谷氨酸,PCR-RELP结果发现AA(295bp)、AB(295bp/186 bp/109 bp)和BB(186 bp/109 bp)3种基因型,A等位基因占优势。10111位点存在A/G突变,导致编码蛋白质的甘氨酸突变为丝氨酸,CRS-RFLP分析发现CC(90bp/20bp/20bp)和CD(110bp/90bp/20bp/20bp)2种基因型,C等位基因占优势。11541位点存在T/C突变,没引起编码蛋白变异,CRS-RFLP分析发现EE(90bp/21bp)、EF(101bp/90bp/21bp)和FF(101bp)3种基因型,F等位基因占优势。群体遗传学分析表明:中国荷斯坦奶牛在TLR2基因三个多态位点均达到Hardy-Weinberg平衡状态,研究群体在10098多态位点达到中度多态,在10111和11541多态位点达到低度多态。利用SAS的一般线性模型,对TLR2基因的3个多态位点的基因型与体细胞评分(SCS)进行相关分析。结果表明10098位点不同基因型对SCS差异显著(P＜0.05),AA、AB基因型个体的SCS值显著高于BB型个体(P＜0.05),AA基因型个体与AB型个体差异不显著(P＞0.05),而TLR2基因10111位点和11541位点不同基因型与研究群体乳腺炎SCS指标差异不显著(P＞0.05)。三个位点联合分析发现5个等位基因,共组成7种基因型。H1H4和H1H1基因型个体的SCS值都显著高于HIH2和H2H2基因型个体(P＜0.05),说明HIH2和H2H2基因型个体具有更好的抗乳腺炎潜力。
【Abstract】 Mastitis is the most severe disease resulted in great economic loss.Genetic factor is one of several factors affecting the occurrence of cow mastiffs.Toll-like receptor (TLR),which plays an important role on anti-infection,can priming innate immune on the early stage of pathogen invade.In this study,we investigated the nucleotide sequence variations of TLR2 gene among 297 Chinese Holstein using PCR-RFLP, CRS-RFLP methods and gene sequencing.The results were as follows:Parital sequence of encode region of TLR2 gene was amplified,sequencing result indicated that 3 SNPs were detected at 10098bp(T/G),10011 lbp(A/G) and 11541bp (T/C) respecively.10098bp(T/G) mutation lead to amino acid replacement of Asp→Glu.3 genetypes named AA(295bp)、AB(295bp/186 bp/109 bp) and BB(186 bp/109 bp) were detected by PCR-RFLP analysis.Allele A was the dominant allele. 10111bp(A/G) mutation lead to amino acid replacement of Gly→Ser.2 genetypes named CC(90bp/20bp/20bp) and CD(110bp/90bp/20bp/20bp) were detected by CRS-RFLP analysis.11541bp(T/C) mutation lead to no amino acid replacement.3 genetypes named EE(90bp/21bp)、EF(101bp/90bp/21bp) and FF(101bp) were detected by CRS-RFLP analysis.Allele F was the dominant allele.Population genetics analysis showed that all the three variations remained Hardy-Weinberg balance situation.Among these,site 10098 reached to moderate polymorphism,while sites 10111 and 11541 were lower polymorphism.The association between the three polymorphism sites residing in second extron of TLR2 gene and body somatic cell score(SCS) were analyzed GLM model.The genotypes of site 10098 had significant effect on SCS trait among 297 samples (P＜0.05).The SCS values for genotypes AA and AB were significantly higher than genotype BB(P＜0.05),but there was no difference between genotypes AA and AB(P＞0.05).The genotypes for sites 10111 and 11541 were no significant association with SCS trait(P＞0.05).Combined with the three sites,five alleles were detected, which further determined seven genotypes.The SCS values for H1H4 and H1H1 genotypes were significantly higher than HIH2 and H2H2 genotypes(p＜0.05).The results segguested that the individuals being along with HIH2 and H2H2 genotypes could be used as mastitis resistance markers adopted in breeding program.