奶牛和水牛的MAP4K4和DGAT1基因对产奶性能和体细胞评分的影响与机理的研究

发布时间：2018-03-02 18:44

  本文选题：乳房炎　切入点：奶产量　出处：《华中农业大学》2017年博士论文　论文类型：学位论文

【摘要】：在我们实验室前期研究基础上,本研究选择了2个基因(MAP4K4和DGAT1)作为研究对象。第一部分对MAP4K4基因进行了SNP鉴定,并对500个荷斯坦奶牛样本进行了基因分型及性状关联分析;第二和三部分分别比较分析了MAP4K4和DGAT1基因启动子区序列在奶牛和水牛中的差异及其功能差异。主要结果如下:1.MAP4K4基因变异对牛奶中体细胞数等性状具有显著的影响。为了探讨MAP4K4基因单核苷酸多态性(SNP)与奶牛多个性状间的相关性,本研究测定和收集了500头荷斯坦奶牛的305天的产奶性状(奶产量、乳脂率、乳蛋白率和牛奶体细胞数等),利用一般线性模型对MAP4K4基因多态性与性状进行关联分析。分析结果发现位于第18外显子2个SNP(c.2061TG and c.2196TC)TT型奶牛的体细胞数评分(SCS)显著高于其它两种基因型奶牛,c.2061TG位点显著地影响奶牛乳蛋白率和奶牛产量。此外,还分析了MAP4K4基因其它位置的SNP,rs109082993 GA(内含子18)和rs210900613 TC(外显子21),分别与牛奶脂肪和牛奶产量显著相关。因此,MAP4K4可以作为奶牛乳房炎相关的重要候选基因,该基因上的SNP有可能作为分子辅助育种的分子标记,但需要在更大群体中验证。2.奶牛和水牛MAP4K4基因的不同启动子序列显著地影响MAP4K4的表达水平。本研究分析了奶牛和水牛MAP4K4基因5’-UTR区域中的核心启动子区及其它部分序列的差异,并分析了这些差异对基因表达和功能的影响。LPS刺激能引起牛乳腺上皮细胞中MAP4K4表达升高,并且这种上调表达呈现时间依赖效应(P0.05)。利用pGL3载体构建了不同长度的MAP4K4启动子序列(奶牛和水牛各5种),转染至293T、CHO以及牛乳腺上皮细胞中,利用免疫荧光报告系统分析了各启动子区域的活性。结果表明-1100至-778 bp区域对奶牛MAP4K4基因表达是必需的,而-608 to+176 bp区域对水牛MAP4K4基因表达是必需的,这两个区分别代表了奶牛和水牛MAP4K4基因启动子的核心区。奶牛该启动子区域活性高于水牛,奶牛MAP4K4基因相对于水牛的高水平表达可能是其体细胞数高、易患乳房炎的原因之一。3.DGAT1基因变异与牛奶乳脂率显著相关。DGAT1基因是脂肪组织中促进甘油三酯合成的重要基因,研究报道该基因K232A位点与牛奶乳脂率密切相关。在本研究的奶牛群中SNP位点(K232A)出现三个基因型;而在水牛群中只存在有利于乳脂率提高的基因型(赖氨酸K),水牛的牛奶脂肪含量较高。4.奶牛和水牛DGAT1基因核心启动子序列存在差异,但不影响其DGAT1基因表达水平。本研究分析了奶牛和水牛DGAT1启动子核心区并比较了其两个物种之间的活性。构建了含不同长度DGAT1启动子区序列的pGL-3载体(奶牛4个,水牛3个),转染至293T、CHO以及牛乳腺上皮细胞中,利用免疫荧光报告系统分析了各启动子区域的活性。结果表明-93 to-556bp区域对奶牛DGAT1基因表达是必需的、-84 to-590bp区域对水牛DGAT1基因表达是必需的,这两个区分别代表了奶牛和水牛DGAT1基因启动子的核心区,然而奶牛和水牛上这两个启动子区域的活性不存在显著差异,故推测奶牛和水牛的牛奶乳脂率的差异可能是基因编码区变异引起的。
[Abstract]:In our laboratory, based on the previous research, this study selected 2 genes (MAP4K4 and DGAT1) as the research object. The first part is the identification of SNP MAP4K4 gene, and 500 Holstein cows samples were analyzed and traits of gene; the second and third part compares the differences in cow and buffalo the promoter sequences and functions of promoter MAP4K4 and DGAT1 gene. The main results are as follows: 1.MAP4K4 gene mutation has a significant effect on somatic cell count in milk traits. In order to investigate MAP4K4 gene single nucleotide polymorphisms (SNP) and the correlation between dairy character, and this study was collected from 500 Holstein cows 305 days the milk production traits (milk yield, milk fat percentage, milk protein content and milk somatic cell count, etc.) of the MAP4K4 gene polymorphism and trait association analysis using general linear model analysis. It was found in the eighteenth exon 2 SNP (c.2061TG and c.2196TC) the number of somatic TT cows score (SCS) was significantly higher than that of other two genotypes of c.2061TG locus significantly affected cows, milk protein content and milk production of dairy cows. In addition, we analysis the MAP4K4 gene of SNP rs109082993 GA in other locations, including (18) and rs210900613 TC (exon 21), were significantly correlated with milk fat and milk yield. Therefore, MAP4K4 can be used as a candidate gene involved in bovine mastitis, the gene SNP may be molecular markers as molecular assisted breeding, but the need to verify the different promoter.2. and MAP4K4 gene in cow buffalo the sequence in larger groups significantly affect the expression level of MAP4K4. This study analyzed the differences between the core and the cow buffalo MAP4K4 gene 5 '-UTR region in the promoter region and the other part of the sequence, and analyzes the Difference of gene expression and function of.LPS stimulation can lead to increased expression of MAP4K4 in bovine mammary epithelial cells, and the expression showed a time dependent effect (P0.05) was constructed. The different length of MAP4K4 promoter sequence by pGL3 vector (cow and buffalo 5), was transfected into 293T, CHO and bovine mammary epithelial cells in the immunofluorescence analysis reporting system of each promoter activity. The results show that the -1100 to -778 BP region on the expression of bovine MAP4K4 gene is required, and the -608 to+176 BP region on the expression of buffalo MAP4K4 gene is required, these two areas represent the core area of the cow and the buffalo MAP4K4 gene promoter. The cows on the promoter region of bovine MAP4K4 gene was higher than that of buffalo, relative to the high level expression of buffalo may be the number of somatic cells is high, easy to cause one of the.3.DGAT1 gene variation and bovine mastitis Milk fat percentage was significantly related to.DGAT1 gene is an important gene of triglyceride synthesis in adipose tissue, the reported gene K232A and milk fat percentage are closely related. In this study the herd SNP loci (K232A) of three genotypes; and in the buffalo group only in favor of genotype increased milk fat rate the (lysine K), milk fat content high.4. cows and buffalo DGAT1 gene core promoter sequences of buffalo are different, but does not affect the expression level of DGAT1 gene. This study analyzed the cow and the buffalo DGAT1 core promoter region were compared between the two species of the constructed pGL-3 vector containing activity. The different length of DGAT1 promoter sequence (cow 4, buffalo 3), was transfected into 293T, CHO and bovine mammary epithelial cells by immunofluorescence, reporting system analysis of the promoter region. The results show that the activity of -93 t O-556bp region on the expression of bovine DGAT1 gene is required, the -84 to-590bp region on the expression of buffalo DGAT1 gene is required, these two areas represent the core area of the cow and the buffalo DGAT1 gene promoter, but the cow and the two buffalo promoter activity does not exist significant differences, suggesting that differences in dairy cows buffalo and the milk fat percentage may be caused by the variation of gene encoding region.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S823
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本文编号：1557721