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  

Effects of RNAi Expression of TIP47 on Lipid Droplet Formation and the Expression of Related Genes in Goat (Capra hircus) Mammary Epithelial Cells

作　　者：张倡珲[1] 郝娟[1] 罗军[1] 石恒波[2] 史怀平[1] 许会芬[1] ZHANG Chang-Hui HAO Juan LUO Jun SHI Heng-Bo SHI Huai-Ping XU Hui-Fen(Shaanxi Provincial Key Laboratory of Agricultural Molecular Biology, College of Animal Science and Technology, Northwest A ＆ F University, Yangling 712100, China College of Life Science, Zhejiang Sci-tech University, Hangzhou 310018, China)

机构地区：[1]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,杨凌712100 [2]浙江理工大学生命科学学院,杭州310018

出　　处：《农业生物技术学报》2017年第2期250-257,共8页Journal of Agricultural Biotechnology

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)

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%(P<0.01)。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(P<0.05)和2.8(P<0.01)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。Tail-interacting protein 47 gene （TIP47）, known as mannose 6-phosphate receptors binding protein 1 （M6PRBP1）, is a member of the （perilipin/ADRP/TIP47, PAT） family, which plays a crucial regulatory role during lipid droplets （LD） biogenesis and secretion process. Milk fat, one of the important nutrients in goat （Capra hircus） milk, is especially rich in short and medium chain fatty acids, which can be absorbed by human （Homo sapiens） body easily. At the same time, the polyunsaturated fatty acids in goat milk have a good effect on anti-oxidation. However, the role of TIP47 on fatty acid metabolism in goat mammary gland is poorly understood. Thus, studying the function of the TIP47 on formation of the milk LD has a great significance to regulate the nutritional value of goat milk. RT- PCR was used to clone TIP47 CDS. The Block-iT short hairpin RNA （shRNA） interference system was used to design specific shRNAs according to the CDS of TIP47 mRNA and 293A cell line was used to package and amplify the adenovirus in this experiment. Then using qRT- PCR to detect the relative expression level of genes and oil red O was used to identify the changes of lipid droplets in intracellular. The full-length 1 314 bp CDS of TIP47 （GenBank No. HQ846827） was cloned onto pDsRedl-Clvector which could express the red fluoresence protein. Three pairs of pENTR/CMV-GFP/U6- 494/828/888 and pDsRedl-C1-TIP47 were co-transfected into 293A cells to filter out the most interference efficient shRNA. The results showed that shRNA-888 could markedly reduce the red fluorescence, so shRNA- 888 was considered to inhibit the expression of TIP47 significantly. Then shRNA-888 was packaged and amplified high-titer virus Ad-shRNA-888 into 293A cells. The titer of the Ad-shRNA-888 was 108 U/mL and the optimal multiplicity of infection （MOI） was 200 in goat mammary epithelial cells. Compared with the control, the expression of TIP47 and adipose triglyceride lipase （ATGL） mRNA were downregulated by 61% （P〈0.01） a

关 键 词：奶山羊 47 kD尾部相互作用蛋白基因(TIP47) RNA干扰 乳腺上皮细胞 

分 类 号：S826.94[农业科学—畜牧学]