本文選題：豬　切入點：miRNA-　出處：《農(nóng)業(yè)生物技術(shù)學(xué)報》2017年09期 　論文類型：期刊論文

【摘要】：在生理病理過程中,micro RNAs(miRNAs)通過作用于相應(yīng)靶基因發(fā)揮著重要的作用。通過高通量測序結(jié)合熒光定量實驗驗證,本課題組前期初步確定miR-192靶向調(diào)控Discs大同源物(discs large homolog 5,DLG5)和活化白細胞黏附分子(activated leukocyte cell adhesion molecule,ALCAM)基因在斷奶仔豬抗大腸桿菌(Escherichia coli)感染過程中發(fā)揮了重要的作用。本研究采用雙熒光素酶報告系統(tǒng)和Western blot方法,驗證DLG5和ALCAM基因是否受到miR-192的特異性調(diào)控。構(gòu)建含有靶基因位點的熒光素酶報告基因重組載體,與PRL-TK和miRNA-192模擬物mimics、inhibitor或陰性對照共轉(zhuǎn)染293細胞,24 h后收集細胞檢測熒光素酶活性和靶基因蛋白表達變化。同時檢測3種大腸桿菌感染腸上皮細胞(intestinal epithelial cells,IPEC-J2)后靶基因的表達變化。本研究成功獲得熒光素酶報告基因重組載體,熒光結(jié)果顯示,miRNA-192 mimics顯著抑制2個報告重組載體的熒光素酶活性(P0.05),而miRNA-192inhibitor則顯著促進2個報告重組載體的熒光素酶活性(P0.05)。同時,miRNA-192模擬物處理組靶基因DLG5和ALCAM蛋白水平極顯著低于陰性對照組,再次驗證了上述結(jié)果。3種大腸桿菌感染后,2個靶基因的表達均顯著或極顯著上升。由結(jié)果可知,豬miR-192對DLG5和ALCAM基因具有靶向抑制作用,且DLG5和ALCAM基因的表達確實與大腸桿菌感染有關(guān)。本研究結(jié)果為miR-192及其靶基因在斷奶仔豬抵抗F18大腸桿菌感染過程中的功能和調(diào)控機制相關(guān)研究提供了一定的實驗基礎(chǔ)和理論依據(jù),進一步為豬抗大腸桿菌病有效遺傳標記的篩選提供了科學(xué)依據(jù)。
[Abstract]:Micro RNAsmiRNAsM plays an important role in physiological and pathological processes by acting on the corresponding target gene, which is verified by high throughput sequencing and fluorescence quantitative experiments. Our team initially identified that miR-192 targeting regulation of Discs large homolog 5 (DLG5) and activated leukocyte adhesion molecule leukocyte cell adhesion (ALCAM) gene play an important role in the resistance of weaned piglets to Escherichia coli infection. Double luciferase report system and Western blot method were used. To verify whether DLG5 and ALCAM genes were specifically regulated by miR-192, we constructed a recombinant vector of luciferase reporter gene containing target gene sites. The luciferase activity and target gene protein expression were detected in 293 cells cotransfected with PRL-TK and miRNA-192 mimicsl inhibitor or negative control for 24 h. The target genes were detected after three kinds of Escherichia coli infected intestinal epithelial cells were infected with intestinal epithelial cells IPEC-J2. The recombinant vector of luciferase reporter gene was successfully obtained in this study. The fluorescence results showed that miRNA-192 mimics significantly inhibited the luciferase activity of two reporter recombinant vectors, while miRNA-192inhibitor significantly promoted the luciferase activity of the two recombinant vectors. At the same time, the target gene DLG5 and ALCAM protein levels in the treatment group of miRNA-192 mimics were significantly increased. It was significantly lower than that in the negative control group. The results showed that the expression of two target genes was significantly or extremely significantly increased after infection of E.coli. The results showed that porcine miR-192 could inhibit the expression of DLG5 and ALCAM genes. The expression of DLG5 and ALCAM genes is indeed related to E. coli infection. The results of this study provide a certain experimental study on the function and regulation mechanism of miR-192 and its target genes in the process of weaning piglets resisting F18 E. coli infection. Basic and theoretical basis, It provides a scientific basis for screening effective genetic markers for resistance to Escherichia coli in pigs.
【作者單位】： 揚州大學(xué)動物科學(xué)與技術(shù)學(xué)院/江蘇省動物遺傳繁育與分子設(shè)計重點實驗室;揚州大學(xué)教育部農(nóng)業(yè)與農(nóng)產(chǎn)品安全國際合作聯(lián)合實驗室;
【基金】：國家自然科學(xué)基金(No.31372285和No.31572360) 江蘇省科技支撐計劃(No.BE2014357和No.BE2015329)
【分類號】：S828
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本文編號：1569602