本文選題：奶山羊　切入點：miRNA篩選　出處：《西北農林科技大學》2017年博士論文　論文類型：學位論文

【摘要】：羊奶中蛋白質、維生素、鈣、礦物質、總脂肪含量高,羊奶所含的有益脂肪酸含量(如短、中鏈脂肪酸,不飽和脂肪酸和共軛酸等)均顯著高于牛奶,羊奶中的乳脂代謝非常復雜,它受多種因素調控。Mi RNA(micro RNA)可通過調控其靶基因的表達影響脂肪酸代謝過程。本研究對奶山羊不同泌乳時期(泌乳前期、泌乳盛期、泌乳后期和干奶期)乳腺組織進行mi RNA篩選后,對催乳素處理的乳腺上皮細胞做進一步篩選,發(fā)現(xiàn)6個表達差異的mi RNA。在此基礎上,對這些篩選出來的mi RNA在功能、調控關系以及分子機制方面進行深入研究。得到如下主要研究結果:1.奶山羊乳腺組織mi RNA表達譜分析及乳脂代謝關鍵mi RNA篩選研究以奶山羊泌乳前期、泌乳盛期、泌乳后期和干奶期的乳腺組織進行mi RNA表達量檢測,基于mi RBase數(shù)據(jù)庫提供的793條牛的mi RNA和267條羊的mi RNA進行第一輪的篩選,以p值小于0.05且差異在4倍以上為標準,篩選得到156個表達差異的mi RNA。隨后使用與發(fā)動泌乳密切相關的因子——催乳素(2.5μg/ml)處理乳腺上皮細胞,進行第二輪篩選。發(fā)現(xiàn)mi R-30e-5p,mi R-15a,mi R-181b,mi R-148a,mi R-17-5p和mi R-135b這6個mi RNA在催乳素處理的乳腺上皮細胞中表達差異顯著。2.Mi R-181b通過IRS2(Insulin receptor substrate 2)抑制TAG合成并調控Hippo信號通路基因在乳腺上皮細胞過表達mi R-181b可以抑制乳脂代謝,而抑制mi R-181b可以促進乳脂代謝。熒光素酶報告試驗和Western Blot試驗證明了mi R-181b的靶基因為IRS2。此外,在乳腺上皮細胞中,mi R-181b可以調控Hippo信號通路中的多個作用元件,如LATS1和YAP1。簡而言之,研究發(fā)現(xiàn)mi R-181b通過調控Hippo通路上的多個元件及靶向IRS2抑制脂代謝。3.Mi R-30e-5p和mi R-15a通過靶向LRP6和YAP1基因協(xié)同調控乳脂代謝QRT-PCR和Western Blot試驗證明了mi R-30e-5p和mi R-15a分別通過靶向Yes相關蛋白(YAP1)和LDL受體相關蛋白6(LRP6)來促進脂質分化。其中,mi R-30e-5p可以調控β-catenin基因,β-catenin進而介導YAP1的表達。在乳腺上皮細胞脂代謝中YAP1基因的m RNA和蛋白水平表達受mi R-30e-5p調控。此外,在乳腺上皮細胞中過表達mi R-30e-5p和mi R-15a可以促進脂代謝,與之相反抑制mi R-30e-5p和mi R-15a可以減少脂代謝。此外,mi R-30e-5p通過調控mi R-15a表達水平來協(xié)同調控YAP1在乳腺上皮細胞中的乳脂代謝功能。4.PRL通過甲基化修飾抑制mi R-135b功能QRT-PCR和Western Blot試驗證明LATS2作為mi R-135b的靶基因來行使乳脂代謝功能。PRL處理乳腺上皮細胞時,對下調mi R-135b的表達有一定的時間和濃度梯度效果。此外,mi R-135b的5'端發(fā)現(xiàn)一個Cp G島,通過調控Cp G島的甲基化來抑制mi R-135b表達。進一步對PRL處理的乳腺上皮細胞中mi R-135b的功能和作用途徑進行研究發(fā)現(xiàn),DNMT I(DNA甲基化轉移酶I)表達水平顯著提高,而mi R-135b表達水平下調。此外,當mi R-135b的表達受到抑制時,LATS2表達水平上調。這就導致乳腺上皮細胞中乳脂代謝水平提高,從而行使維持泌乳等功能。5.Mi R-148a和mi R-17-5p通過PPARGC1A和PPARA基因協(xié)同調控乳腺上皮細胞乳脂代謝表達譜結果顯示mi R-148a、mi R-17-5p、PPARGC1A和PPARA在泌乳盛期和干奶期中表達差異顯著。熒光素酶和Western Blot試驗發(fā)現(xiàn),PPARA(脂肪酸調控重要因子)和PPARGC1A(調控脂代謝基因)分別是mi R-148a和mi R-17-5p的靶基因,而mi R-148a可以調控PPARA,mi R-17-5p可以調控PPARGC1A。過表達mi R-148a和mi R-17-5p促進甘油三酯(TAG,Triglyceride)的合成,而抑制mi R-148a和mi R-17-5p降低TAG的產生。重要的發(fā)現(xiàn)是mi R-148a和mi R-17-5p的協(xié)同作用促進了TAG的合成。即在乳腺上皮細胞中,mi R-148a與mi R-17-5p分別調控PPARGC1A和PPARA來協(xié)同完成TAG的合成,。本研究篩選的6個mi RNA及其靶基因對乳脂代謝的影響,為深入闡明奶山羊乳脂代謝調控機制提供了理論和試驗依據(jù),為解析羊奶脂肪酸成分形成機理奠定了基礎。
[Abstract]:Goats'milk protein, vitamins, minerals, calcium, total fat content is high, beneficial fatty acids goats' milk contains (such as short chain fatty acid, unsaturated fatty acid and conjugated acid) were significantly higher than that of milk, milk fat metabolism in goats'milk is very complex, it is regulated by a variety of factors (.Mi RNA micro RNA) can regulate the expression of their target genes that affect the metabolism of fatty acids. The study of dairy goats in different lactation periods (early lactation, lactation period, late lactation and dry period) of MI RNA breast tissue after screening, further screening of prolactin treatment of mammary epithelial cells, found 6 differentially expressed mi RNA. on the basis of these, the selected mi RNA in function, regulation relationship and molecular mechanism were studied. The main results are as follows: the expression spectrum analysis and fat metabolism key mi RNA 1. mi RNA of dairy goat mammary tissue Study on screening of dairy goat in early lactation, lactation stage, MI RNA expression detection in late lactation and dry period of breast tissue, screened in the first round of the MI RNA 793 mi RNA mi RBase cattle and 267 sheep database based on the p value of less than 0.05 and the difference in more than 4 times as a standard, screened 156 differentially expressed mi RNA. and then use the launch of lactation related factors: prolactin (2.5 g/ml) treatment of mammary epithelial cells, the second round of screening. Mi R-30e-5p, MI R-15a, MI R-181b, MI R-148a, MI R-17-5p and MI R-135b expression of the 6 mi RNA in prolactin treatment of mammary epithelial cells significantly.2.Mi R-181b by IRS2 (Insulin receptor substrate 2) inhibit TAG synthesis and regulation of Hippo signaling pathway genes in mammary epithelial cells overexpressing mi R-181b can inhibit the fat metabolism, and inhibition of MI R-181b In order to promote fat metabolism. The luciferase reporter assay and Western Blot tests showed that the target genes of MI R-181b because of IRS2. addition in mammary epithelial cells, MI R-181b can be a role of regulatory element in the Hippo signaling pathway, such as LATS1 and YAP1.. In short, studies have found that MI R-181b by a plurality of elements on the regulation of the Hippo pathway and target to IRS2.3.Mi and MI R-30e-5p inhibited the lipid metabolism of R-15a gene to LRP6 and YAP1 by QRT-PCR and fat metabolism target synergistic regulation of Western Blot MI and MI R-30e-5p test proved that R-15a were targeted by Yes associated protein (YAP1) and LDL receptor related protein 6 (LRP6) to promote differentiation. Among them, MI R-30e-5p can regulate beta -catenin gene, expression of beta -catenin and YAP1 mediated by Mi R-30e-5p. The regulation of the expression of YAP1 gene in mammary epithelial cells of lipid metabolism in M RNA and protein level. In addition, in breast epithelial cells. Overexpression of MI in R-30e-5p cell and MI R-15a can promote lipid metabolism, and inhibition of MI R-30e-5p and MI in R-15a can reduce lipid metabolism. In addition, the MI expression level of R-30e-5p by regulating mi R-15a collaborative fat metabolism function of.4.PRL regulation of YAP1 in mammary epithelial cells through inhibition of MI methylation modification of R-135b function of QRT-PCR and Western the experimental results show that the LATS2 Blot as the target gene of MI R-135b to exercise fat metabolism function of.PRL treatment of mammary epithelial cells, there is a certain amount of time and concentration gradient effect on the expression of MI R-135b mi R-135b 5'. In addition, the end found a Cp G Island, G island methylation through the regulation of Cp to inhibit the expression of R-135b Mi study found that the further function and effect on the treatment of PRL in mammary epithelial cells mi R-135b pathway, DNMT I (DNA methyltransferase I) expression level increased significantly, while the MI R-135b table of water Flat down. In addition, the expression of R-135b when MI is inhibited, LATS2 expression level increased. This leads to fat metabolism in mammary epithelial cells increased, thereby maintaining lactation function.5.Mi R-148a exercise and MI R-17-5p by PPARGC1A and PPARA gene co regulation of mammary epithelial cells in milk fat metabolism expression showed that MI R-148a, MI R-17-5p PPARGC1A, and PPARA at peak lactation and dry milk period significantly. The expression of luciferase and Western found Blot test, PPARA (an important factor regulating fatty acid (PPARGC1A) and the regulation of lipid metabolism genes) are target gene mi R-148a and MI R-17-5p, and MI R-148a PPARA mi R-17-5p can control, can control the PPARGC1A.. The expression of MI R-148a and MI R-17-5p (TAG, Triglyceride) promotes triglyceride synthesis and inhibition of MI R-148a and MI R-17-5p decreased TAG. The important finding is the MI R-148a and MI R-17- The synergistic effect of 5P and promote the synthesis of TAG. Namely in mammary epithelial cells, MI R-148a and MI R-17-5p and PPARA PPARGC1A respectively control to execute TAG synthesis. The research of 6 mi RNA and its target gene screening on fat metabolism, and provides a theoretical and experimental basis for the elucidation of dairy goat milk the metabolic regulation mechanism, laid the foundation for the mechanism of acid composition analysis goats'milk fat formation.

【學位授予單位】：西北農林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S823

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