干扰TIP47基因对奶山羊乳腺上皮细胞脂滴形成及相关基因表达的影响

发布时间：2018-04-09 08:46

本文选题：奶山羊　切入点：kD尾部相互作用蛋白基因(TIP)　出处：《农业生物技术学报》2017年02期

【摘要】：47 k D尾部相互作用蛋白(tail-interacting protein 47,TIP47)在脂滴形成与代谢过程中具有重要调控作用,研究其在乳脂滴形成过程中的作用对调控羊(Capra hircus)乳品质具有重要意义。本研究采用BLOCK-i T小发夹RNA(short hairpin RNA,sh RNA)腺病毒(Adenovirus)干扰系统,根据奶山羊TIP47基因序列,在线设计并合成干扰sh RNA。将有效sh RNA序列构建载体,并在293A细胞中包装腺病毒。利用腺病毒载体干扰乳腺上皮细胞TIP47基因表达后,检测胞内脂肪酸代谢基因及脂滴的变化。结果表明,筛选到有效sh RNA序列并获得高滴度TIP47基因的干扰腺病毒Ad-sh RNA-888。Ad-sh RNA-888感染山羊原代乳腺上皮细胞后发现,TIP47 m RNA表达量下调61%(P0.01)。干扰TIP47基因的表达后,甘油三酯水解酶基因(adipose triglyceride lipase,ATGL)下调了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)分别上调1.77(P0.05)和2.8(P0.01)倍,硬脂酰辅酶A去饱和酶基因(stearoyl-Co A desaturase,SCD)表达水平变化不显著。油红O结果显示干扰TIP47基因可以降低细胞脂滴(lipid droplets,LD)的形成。本研究对TIP47基因进行干扰,发现其在奶山羊乳腺细胞脂滴形成过程中有重要调控作用,为进一步研究TIP47基因对脂滴代谢的影响提供一定的理论依据。
[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.
【作者单位】：西北农林科技大学动物科技学院/陕西省农业分子生物学重点实验室;浙江理工大学生命科学学院;
【基金】：国家转基因生物新品种培育科技重大专项(No.2014ZX08009-051B) 国家公益性行业(农业)科研专项(No.201103038)
【分类号】：S827

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