过表达与干扰LXRβ基因对奶山羊乳腺上皮细胞脂质代谢的影响

发布时间：2018-05-26 08:08

本文选题：奶山羊 + LXRβ　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：肝脏X受体β(Liver X receptorβ,LXRβ)是核受体LXRs基因的亚型之一,是参与调控机体脂质代谢的关键转录因子,它通过与视黄醛X受体共价结合,组成异二聚物后靶定至基因的调控区域,如固醇调节元件结合蛋白-1和脂肪酸合酶的启动子上,影响其转录,进而影响到机体脂质的代谢。本研究通过克隆得到奶山羊LXRβ基因的编码区,将其重组至腺病毒载体中,进一步转染重组质粒进入293A细胞,获取高滴度的腺病毒,同时在线设计并合成siLXRβ,在奶山羊乳腺上皮细胞中分别超表达和干扰LXRβ基因后,通过RT-qPCR和Western Blot等方法检测细胞内脂代谢基因表达量及胞内脂质含量的变化,为进一步明确该基因在乳脂代谢调控中的功能奠定理论基础。以下为本研究获得的主要研究结果:(1)通过提取西农萨能奶山羊乳腺组织总RNA,根据GenBank中收录的羊(XM_018062857)的LXRβ基因序列设计引物,利用RT-PCR技术克隆得到山羊LXRβ基因CDS序列,其长度为1 368 bp,编码455个氨基酸。山羊LXRβ基因CDS区序列与牛(Bos taurus)、绵羊(Ovisaries)、人(Homo sapiens)和小鼠(Mus musculus)的相似性分别为98%、98%、87%和85%,氨基酸序列相似性分别为99%、99%、90%和91%。(2)成功获得含有目的基因的重组质粒pAdEasy-LXRβ,转染293A细胞后得到高滴度腺病毒,滴度为109 U/mL。通过在山羊乳腺上皮细胞中添加不同体积的病毒液,确定其最佳感染复数(MOI)为200。病毒处理细胞48h后,RT-qPCR结果显示LXRβ基因表达量上升100倍左右,被T0901317激活后,SREBP-1、FASN、SCD、ACSL1、ACSL3、ACCα及ELVOL6基因均显著上调(P0.05),FADS1基因的表达水平无明显变化;Western blot结果表明FASN蛋白表达量显著增加;油红O和甘油三脂检测结果说明LXRβ基因可以显著上调细胞内的脂滴数量和胞内甘油三酯的含量;脂肪酸组分分析表明细胞中C18:1的含量显著上升(P0.05)。(3)根据LXRβ基因CDS序列,设计特异针对其的siRNA并转染山羊乳腺上皮细胞,RT-qPCR结果表明LXRβ基因表达量显著下调了88%,干扰LXRβ表达后,不添加激动剂时DGAT2基因下调30%,SREBP1基因上调50%,其他脂代谢基因变化不显著,添加激动剂之后相对于对照组SREBP1和FSAN基因分别下调15%和35%,ASCL1基因表达量上调30%左右,ACSL3、FADS1、ELOVL6以及SCD基因表达量变化不显著;甘油三脂检测结果表明当LXRβ表达量下降时,胞内甘油三脂含量随之减少。综上所述,本试验成功克隆得到了奶山羊LXRβ基因的CDS区,构建了超表达载体,通过感染山羊乳腺上皮细胞超表达LXRβ基因并添加激动剂可导致众多脂代谢基因的mRNA表达水平显著上调,胞内脂滴含量增加,干扰该基因的表达对脂代谢基因的mRNA表达以及胞内甘油三酯合成有一定的抑制作用。为明确LXRβ基因在奶山羊乳腺细胞中乳脂代谢中的功能提供理论依据。
[Abstract]:Liver X receptor 尾 -Liver X receptor 尾 -LXR 尾) is one of the subtypes of nuclear receptor LXRs gene and a key transcription factor involved in regulating lipid metabolism. For example, the promoter of steroid regulatory element binding protein 1 and fatty acid synthase affect its transcription, and then affect the metabolism of body lipid. In this study, the coding region of LXR 尾 gene of dairy goat was cloned, and the recombinant plasmid was transfected into 293A cells to obtain high titer adenovirus. At the same time, siLXR 尾 was designed and synthesized online. After overexpression and interference of LXR 尾 gene in dairy goat mammary epithelial cells, the changes of lipid metabolism gene expression and intracellular lipid content were detected by RT-qPCR and Western Blot. In order to further clarify the role of the gene in regulation of milk fat metabolism lay a theoretical foundation. The following is the main result of this study: (1) by extracting the total RNAs from the breast tissue of Sinon Sanen dairy goat, we designed primers according to the LXR 尾 gene sequence of XM018062857 (included in GenBank) and cloned the CDS sequence of LXR 尾 gene from goat by RT-PCR technique. Its length is 1 368 BP, encoding 455 amino acids. The similarity of CDS region of goat LXR 尾 gene with Bos taurus, Ovisariesus, Homo sapiensus and mouse Mus musculus was 98% and 85%, respectively. The amino acid sequence similarity was 990.9999% and 91.1%, respectively. The recombinant plasmid pAdEasy-LXR 尾 containing the target gene was successfully obtained and transfected into 293A cells. Get a high titer adenovirus, The titer was 109 UmL. By adding different volumes of virus to goat mammary epithelial cells, it was determined that the optimal number of infected moi was 200. After 48 hours of virus treatment, RT-qPCR showed that the expression of LXR 尾 gene increased about 100fold, and the expression level of FASN protein increased significantly after activation by T0901317. The expression level of ACSL1ACSL3ACSL3ACC 伪 and ELVOL6 were significantly up-regulated by T0901317. The results of Western blot showed that FASN protein expression was significantly increased. The results of oil red O and triglyceride detection showed that LXR 尾 gene could significantly up-regulate the number of lipid droplets and the content of intracellular triglycerides in cells, and fatty acid component analysis showed that the content of C18: 1 increased significantly (P0.05. 3) according to the CDS sequence of LXR 尾 gene. The results of RT-PCR showed that the expression of LXR 尾 gene was down-regulated by 888.After interfering with the expression of LXR 尾, the DGAT2 gene was down-regulated by 30% SREBP1 gene and no significant change of other lipid metabolism genes was observed without the addition of agonist. Compared with the control group, the expression of SREBP1 and FSAN genes were down-regulated by 15% and 35%, respectively, and the expression of ACSL3, FADS1, ELOVL6 and SCD were up regulated by about 30%, and the results of triglyceride test showed that the expression of LXR 尾 was decreased. The intracellular triglyceride content decreased. In conclusion, the CDS region of LXR 尾 gene of dairy goat was cloned successfully, and the superexpression vector was constructed. The overexpression of LXR 尾 gene in goat mammary epithelial cells and the addition of agonist could significantly upregulate the expression of mRNA and increase the content of lipid droplets in many lipid metabolism genes. Interfering with the expression of this gene can inhibit the mRNA expression and intracellular triglyceride synthesis of lipid metabolism gene. To provide theoretical basis for the function of LXR 尾 gene in milk fat metabolism of milk goat breast cells.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

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