摘要
本研究旨在通过构建西农萨能奶山羊胰岛素诱导基因1(INSIG1)的重组腺病毒(Adenovirus,Ad)超表达载体,研究该基因超表达后对乳腺上皮细胞中脂质合成的影响,从而为该基因在山羊乳腺脂质合成调控中的功能研究奠定基础。根据GenBank(登录号:JQ665439)中山羊INSIG1基因序列设计引物,PCR扩增得到其CDS区序列。将目的基因连接到穿梭载体pAdTrack-CMV后获得pAdTrack-CMV-INSIG1质粒,将该质粒PmeⅠ线性化后转入大肠杆菌BJ5183感受态细胞进行同源重组,获得pAdEasy-INSIG1重组腺病毒超表达载体。该重组质粒经PacⅠ线性化后转染293A细胞进行病毒的包装及扩繁,利用LaSRT法测定其滴度。将获得的重组腺病毒AdINSIG1感染山羊原代乳腺上皮细胞,利用实时荧光定量PCR检测INSIG1及脂质合成相关基因的mRNA表达情况,利用GPO-Trinder酶学反应检测细胞中甘油三酯的含量。结果表明,成功构建了奶山羊INSIG1基因重组腺病毒超表达载体,获得了具有较高滴度(2×108 U·mL-1)的超表达腺病毒Ad-INSIG1;Ad-INSIG1感染山羊原代乳腺上皮细胞48h后,与Ad-GFP组相比,INSIG1基因的mRNA表达量上调约500倍,固醇调节元件结合蛋白1(SREBP1)和SREBP裂解活化蛋白(SCAP)的mRNA表达量无明显变化,参与脂肪酸从头合成(ACCα、FASN)及去饱和(SCD1)基因的mRNA表达量均显著下降(P<0.05);参与甘油三酯合成的3个关键基因中,GPAM及DGAT2的mRNA表达量显著下降(P<0.05),AGPAT6的表达无显著变化;同时,催化甘油三酯分解(ATGL)的基因mRNA表达量也显著下降(P<0.05);细胞内甘油三酯含量无显著变化。综上所述,在山羊原代乳腺上皮细胞中,INSIG1能够抑制脂质合成相关基因的表达,对山羊乳腺脂质合成具有调控作用。
The aim of this study was to construct an insulin-induced gene-1 （INSIG1） recombi- nant adenovirus overexpression vector of Xinong Saanen dairy goat, and determine its effect on lipid synthesis in goat mammary epithelial cells （GMEC）,to lay the foundation for its functional study in regulating lipid synthesis in goat mammary gland. The primers were designed according to the INSIG1 sequence in GenBank （accession number：JQ665439）,then its coding sequences （CDS） was cloned by PCR. The gene fragments were inserted into shuttle vector pAdTrack-CMV to obtain pAdTrack-CMV-INSIG1 vector. After being linearized by Pme I, pAdTrack-CMV-IN- SIG1 vector was transformed into Escherichia coli BJ5183 competent cells to obtain the recombi-nant adenovirus overexpression vector pAdEasy-INSIG1 by homologous recombination. Next, the pAdEasy-INSIG1 vector,linearized by Pac I ,was transfected into 293A cells for viral packaging and amplification. LaSRT was used for titer assay. Finally, after infecting the goat mammary gland epithelial cells（GMEC） with Ad-INSIG1 recombinant adenovirus,qRT-PCR was used to measure the mRNA expression of INSIG1 and genes related to lipid synthesis, and the GPO-Trinder en- zyme reaction was used to measure the cellular triacylglycerol （TAG） content. The result showed that INSIG1 overexpression recombinant adenovirus vector was successfully constructed,and the recombinant adenovirus Ad-INSIG1 with a high titer of 2× 10^8 U· mL-1 was obtained. Compared with Ad-GFP infected group,the mRNA expression of INSIG1 increased by about 500-fold after GMEC incubated with Ad-INSIG1 for 48 h. No obvious changes were observed on the mRNA ex- pression of sterol regulatory element binding protein 1 （SREBP1） and SREBP cleavage-activating protein （SCAP）, however,there was a significant decrease in the expression genes related to fatty acid de novo synthesis and desaturasion, acetyl-CoA carboxylas α （ACCα）, fatty acid synthase （FASN） and stearoyl-CoA desaturase 1 （SCD1） （P�
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2016年第9期1806-1816,共11页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金项目(31372281)
国家转基因新品种培育重大专项(2014ZX08009-051B)
