【作者】 林嘉鹏；

【导师】 刘明军； 罗淑萍；

【作者基本信息】 新疆农业大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 Toll-样受体(Toll like receptors,TLRs)可以通过识别病原微生物启动天然免疫并激活适应性免疫。本研究以中国荷斯坦奶牛和新疆褐牛为研究对象,对TLR4基因外显子3进行了序列分析,并对发现的多态性位点进行PCR-RFLP分析,共发现TLR4 E3+1656、TLR4E3+2021和TLR4 E3+2414三个多态位点,其中TLR4 E3+1656位点发生T/C的碱基突变,但没有引起氨基酸变化;TLR4 E3+2021位点C/T的碱基突变,导致编码氨基酸由苏氨酸变为异亮氨酸;TLR4 E+2414位点发生G/A突变,引起氨基酸由精氨酸变为谷氨酸。但由于TLR4 E+2414位点只发现2个AB基因型个体,其余均为BB基因型个体,不具有统计意义,因此只对TLR4 E3+1656和TLR4E3+2021位点χ2检验表明:两个品种这两个位点的突变均处于Hardy-Weinberg平衡状态(P>0.05)。运用最小二乘法分析表明:TLR4 E3+1656位点的各基因型个体间的平均SCS差异不显著(P>0.05);而TLR4 E3+2021位点中AA与AB基因型SCS水平差异显著(P<0.05),AA与BB基因型SCS水平差异极显著(P<0.01),但AB与BB基因型SCS水平差异不显著(P>0.05),说明TLR4 E3+2021位点的AA基因型可能与抗乳房炎的抗性有关,A等位基因是乳房炎抗性的有利基因。为进一步研究TLR4 E3+2021位点不同基因型对奶牛SCS的影响,采用Quantitative Real-Time PCR技术检测了新疆褐牛不同基因型个体中TLRs信号通路下游的部分基因mRNA的表达水平,结果显示:NF-κB1、NF-κB2和IL-1β基因mRNA表达水平与TLR4 E3+2021位点AB和BB两种基因型差异显著(P<0.05),TNF、TLR4、IL-10、IL-8、MD-2和Myd88基因mRNA表达水平与TLR4 E3+2021位点AB和BB两种基因型差异不显著(P>0.05)。这些结果提示我们TLR4 E3+2021位点碱基的改变可能引起了核内转录因子NF-κB表达量的变化,从而进一步导致IL-1β的mRNA表达量改变。更多还原

【Abstract】 Toll like receptors (TLRs) recognize pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, Chinese holstei and XinJiang Brown Battle were used as experimental example. The genetic polymorphisms of TLR4 gene exonⅢwere detected by sequencing in the first and by RFLP in the second. The results showed that T/C polymorphic sites did not cause the AA variation in TLR4 E3+1656; C/T polymorphic sites mutation exists in TLR4 E3+2021, which induced that changed Thr into Ile. Statistical results ofχ2 test indicated that two polymorphism sites in the two populations fitted with Hardy–Weinberg equilibrium(P>0.05). The associations were observed between genotypes and SCS by the least square analysis. The results showed that SCS was not significantly affected by different genotypes in TLR4 E3+1656 (P>0.05);both of a significant different effect between genotype AA and AB was found (P<0.05) ,the difference was significant between genotype AA and BB was found (P<0.01) and no significant difference were detected between genotype AB and BB(P>0.05)in TLR4 E3+2021 site. The AA genotype in TLR4 E3+2021 site would be the best genotype to resist mastitis. In short, the allele A might play an important role in mastitis resistance in bovine. To research the effect of different genotypes of TLR4 E3+2021 site on SCS, part of the mRNA of the gene expression where in TLRs signaling pathway downstream were detected by Quantitative Real-Time PCR. The level of NF-κB1、NF-κB2 and IL-1βtranscripts was significantly correlated with genotype of AA and BB in TLR4 E3+2021 (P<0.05),but the level of TNF、TLR4、IL-10、IL-8、MD-2 and Myd88 transcripts was no significantly correlated with genotype of AA and BB in TLR4 E3+2021(P>0.05). These give the implication: The changes of TLR4 E +2021 base cause the change of NF-κB expression, which would lead to the change of IL-1βexpression of mRNA.更多还原