本文選題：乳房炎　切入點：奶產(chǎn)量　出處：《華中農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

【摘要】：在我們實驗室前期研究基礎上,本研究選擇了2個基因(MAP4K4和DGAT1)作為研究對象。第一部分對MAP4K4基因進行了SNP鑒定,并對500個荷斯坦奶牛樣本進行了基因分型及性狀關聯(lián)分析;第二和三部分分別比較分析了MAP4K4和DGAT1基因啟動子區(qū)序列在奶牛和水牛中的差異及其功能差異。主要結果如下:1.MAP4K4基因變異對牛奶中體細胞數(shù)等性狀具有顯著的影響。為了探討MAP4K4基因單核苷酸多態(tài)性(SNP)與奶牛多個性狀間的相關性,本研究測定和收集了500頭荷斯坦奶牛的305天的產(chǎn)奶性狀(奶產(chǎn)量、乳脂率、乳蛋白率和牛奶體細胞數(shù)等),利用一般線性模型對MAP4K4基因多態(tài)性與性狀進行關聯(lián)分析。分析結果發(fā)現(xiàn)位于第18外顯子2個SNP(c.2061TG and c.2196TC)TT型奶牛的體細胞數(shù)評分(SCS)顯著高于其它兩種基因型奶牛,c.2061TG位點顯著地影響奶牛乳蛋白率和奶牛產(chǎn)量。此外,還分析了MAP4K4基因其它位置的SNP,rs109082993 GA(內含子18)和rs210900613 TC(外顯子21),分別與牛奶脂肪和牛奶產(chǎn)量顯著相關。因此,MAP4K4可以作為奶牛乳房炎相關的重要候選基因,該基因上的SNP有可能作為分子輔助育種的分子標記,但需要在更大群體中驗證。2.奶牛和水牛MAP4K4基因的不同啟動子序列顯著地影響MAP4K4的表達水平。本研究分析了奶牛和水牛MAP4K4基因5’-UTR區(qū)域中的核心啟動子區(qū)及其它部分序列的差異,并分析了這些差異對基因表達和功能的影響。LPS刺激能引起牛乳腺上皮細胞中MAP4K4表達升高,并且這種上調表達呈現(xiàn)時間依賴效應(P0.05)。利用pGL3載體構建了不同長度的MAP4K4啟動子序列(奶牛和水牛各5種),轉染至293T、CHO以及牛乳腺上皮細胞中,利用免疫熒光報告系統(tǒng)分析了各啟動子區(qū)域的活性。結果表明-1100至-778 bp區(qū)域對奶牛MAP4K4基因表達是必需的,而-608 to+176 bp區(qū)域對水牛MAP4K4基因表達是必需的,這兩個區(qū)分別代表了奶牛和水牛MAP4K4基因啟動子的核心區(qū)。奶牛該啟動子區(qū)域活性高于水牛,奶牛MAP4K4基因相對于水牛的高水平表達可能是其體細胞數(shù)高、易患乳房炎的原因之一。3.DGAT1基因變異與牛奶乳脂率顯著相關。DGAT1基因是脂肪組織中促進甘油三酯合成的重要基因,研究報道該基因K232A位點與牛奶乳脂率密切相關。在本研究的奶牛群中SNP位點(K232A)出現(xiàn)三個基因型;而在水牛群中只存在有利于乳脂率提高的基因型(賴氨酸K),水牛的牛奶脂肪含量較高。4.奶牛和水牛DGAT1基因核心啟動子序列存在差異,但不影響其DGAT1基因表達水平。本研究分析了奶牛和水牛DGAT1啟動子核心區(qū)并比較了其兩個物種之間的活性。構建了含不同長度DGAT1啟動子區(qū)序列的pGL-3載體(奶牛4個,水牛3個),轉染至293T、CHO以及牛乳腺上皮細胞中,利用免疫熒光報告系統(tǒng)分析了各啟動子區(qū)域的活性。結果表明-93 to-556bp區(qū)域對奶牛DGAT1基因表達是必需的、-84 to-590bp區(qū)域對水牛DGAT1基因表達是必需的,這兩個區(qū)分別代表了奶牛和水牛DGAT1基因啟動子的核心區(qū),然而奶牛和水牛上這兩個啟動子區(qū)域的活性不存在顯著差異,故推測奶牛和水牛的牛奶乳脂率的差異可能是基因編碼區(qū)變異引起的。
[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.

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S823
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本文編號：1557721