本文選題：奶山羊　切入點：kD尾部相互作用蛋白基因(TIP)　出處：《農(nóng)業(yè)生物技術(shù)學(xué)報》2017年02期

【摘要】：47 k D尾部相互作用蛋白(tail-interacting protein 47,TIP47)在脂滴形成與代謝過程中具有重要調(diào)控作用,研究其在乳脂滴形成過程中的作用對調(diào)控羊(Capra hircus)乳品質(zhì)具有重要意義。本研究采用BLOCK-i T小發(fā)夾RNA(short hairpin RNA,sh RNA)腺病毒(Adenovirus)干擾系統(tǒng),根據(jù)奶山羊TIP47基因序列,在線設(shè)計并合成干擾sh RNA。將有效sh RNA序列構(gòu)建載體,并在293A細(xì)胞中包裝腺病毒。利用腺病毒載體干擾乳腺上皮細(xì)胞TIP47基因表達(dá)后,檢測胞內(nèi)脂肪酸代謝基因及脂滴的變化。結(jié)果表明,篩選到有效sh RNA序列并獲得高滴度TIP47基因的干擾腺病毒Ad-sh RNA-888。Ad-sh RNA-888感染山羊原代乳腺上皮細(xì)胞后發(fā)現(xiàn),TIP47 m RNA表達(dá)量下調(diào)61%(P0.01)。干擾TIP47基因的表達(dá)后,甘油三酯水解酶基因(adipose triglyceride lipase,ATGL)下調(diào)了15%,而脂肪酸合酶基因(fatty acid synthase,FASN)和激素敏感脂酶基因(hormone-sensitive lipase,HSL)分別上調(diào)1.77(P0.05)和2.8(P0.01)倍,硬脂酰輔酶A去飽和酶基因(stearoyl-Co A desaturase,SCD)表達(dá)水平變化不顯著。油紅O結(jié)果顯示干擾TIP47基因可以降低細(xì)胞脂滴(lipid droplets,LD)的形成。本研究對TIP47基因進(jìn)行干擾,發(fā)現(xiàn)其在奶山羊乳腺細(xì)胞脂滴形成過程中有重要調(diào)控作用,為進(jìn)一步研究TIP47基因?qū)χ未x的影響提供一定的理論依據(jù)。
[Abstract]:47 KD tail interacting protein 47) plays an important role in lipid droplet formation and metabolism, and it is important to study its role in the regulation of milk quality of sheep capra hircus.In this study, BLOCK-i T small hairpin RNA(short hairpin RNAs) adenovirus Adenovirus-based interference system was used to design and synthesize interference shRNAs online according to the TIP47 gene sequence of dairy goats.The effective sh RNA sequence was constructed and the adenovirus was packaged in 293A cells.After interfering the expression of TIP47 gene with adenovirus vector, the changes of fatty acid metabolism genes and lipid droplets in breast epithelial cells were detected.The results showed that after screening the effective sh RNA sequence and obtaining the interfering adenovirus Ad-sh RNA-888.Ad-sh RNA-888 with high titer of TIP47 gene, it was found that the expression of TIP47m RNA in goat primary mammary epithelial cells decreased by 61% and P0.01a.After interfering with the expression of TIP47 gene, triglyceride hydrolase gene (TGA) was down-regulated by 15 times, while fatty acid synthase gene fatty acid synthase (FASN) and hormone sensitive lipase gene (HSLs) were up-regulated by 1.77g / P0.05) and 2.8P0.01), respectively.The expression level of stearoyl-Co A desaturase gene SCD was not significantly changed.The results of oil red O showed that interfering with TIP47 gene could reduce the formation of lipid dropletsus.This study interferes with the TIP47 gene and finds that it plays an important role in the lipid droplet formation of mammary gland cells of dairy goats, which provides a theoretical basis for further study of the effect of TIP47 gene on lipid drop metabolism.
【作者單位】： 西北農(nóng)林科技大學(xué)動物科技學(xué)院/陜西省農(nóng)業(yè)分子生物學(xué)重點實驗室;浙江理工大學(xué)生命科學(xué)學(xué)院;
【基金】：國家轉(zhuǎn)基因生物新品種培育科技重大專項(No.2014ZX08009-051B) 國家公益性行業(yè)(農(nóng)業(yè))科研專項(No.201103038)
【分類號】：S827

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