作 者：
杨昌恒;李琪;黄维;林亚秋;王永;向华;朱江江
单 位：
青藏高原动物遗传资源保护与利用教育部/四川省重点实验室;西南民族大学青藏高原研究院
关键词：
山羊;DGAT1;过表达;肌内前体脂肪细胞;甘油三酯
摘 要：
旨在克隆山羊DGAT1基因序列,明确DGAT1基因在山羊不同组织中的表达模式,并进一步揭示过表达DGAT1基因对山羊肌内前体脂肪细胞脂质代谢的影响。本试验以10月龄健康简州大耳公羊(n=7)为试验动物。采用RT-PCR法克隆山羊DGAT1基因序列,并对序列进行生物信息学分析;利用实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)检测DGAT1在山羊不同组织中的相对表达水平;采用双酶切法构建pcDNA3.1-DGAT1真核表达载体并转染至山羊肌内前体脂肪细胞;使用RT-qPCR检测DGAT1过表达效率及脂质代谢相关基因的表达情况;通过油红O染色法观察过表达DGAT1对脂滴形成的影响,利用GPO-Trinder酶学反应检测甘油三酯含量。结果显示,获得山羊DGAT1基因序列全长1 651 bp(GenBank登录号:MT221183),包含5′UTR 125 bp, CDS 1 470 bp, 3′UTR 56 bp,编码489个氨基酸残基;山羊DGAT1基因在小肠中的表达量最高,在脾中表达量最低;RT-qPCR检测结果显示,DGAT1在细胞中过表达极显著(P<0.01),GPAM基因的相对表达水平显著上调(P<0.05),ADRP和ACOX1基因极显著上调(P<0.01),而AGPAT6基因相对表达水平显著下调(P<0.05),MLYCD和HSL基因极显著下调(P<0.01);油红O染色结果显示,过表达DGAT1后脂滴聚积相较于对照组极显著增多(P<0.01),甘油三酯测定结果显示,过表达DGAT1基因可极显著增加山羊肌内脂肪细胞甘油三酯含量(P<0.01)。本研究成功获得山羊DGAT1基因CDS区序列并构建了pcDNA3.1-DGAT1真核表达载体,过表达DGAT1可显著促进山羊肌内前体脂肪细胞脂质沉积,并显著影响脂质代谢相关基因的表达,这些结果为进一步阐明DGAT1对调控山羊肌内脂肪代谢的作用机制提供了重要数据。
译 名：
Cloning of Goat DGAT1 Gene and Its Regulation on Lipid Deposition in Preadipocytes
作 者：
YANG Changheng;LI Qi;HUANG Wei;LIN Yaqiu;WANG Yong;XIANG Hua;ZHU Jiangjiang;Institute of Qinghai-Tibetan Plateau, Southwest Minzu University;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education/Sichuan Province;
关键词：
goat;;DGAT1;;overexpression;;intramuscular preadipocytes;;triglycerides
摘 要：
The aim of the study was to clone the sequence of DGAT1 gene, clarify its expression pattern in different tissues of goat, and further reveal the role of DGAT1 gene in regulating lipid metabolism in goat intramuscular preadipocytes. In this study, seven healthy 10-month-old Jianzhou male goats were used as experimental animals. The sequence of DGAT1 gene was cloned by RT-PCR and analyzed using bioinformatics. The relative expression level of DGAT1 in different tissues of goat were determined by real-time quantitative PCR(RT-qPCR). The eukaryotic expression vector pcDNA3.1-DGAT1 was constructed by double enzyme digestion and transfected into goat intramuscular preadipocytes. RT-qPCR was used for detecting the expression of DGAT1 and lipid metabolism-related genes. The effect of overexpression of DGAT1 on lipid droplet formation was observed by Oil red O staining method, and the GPO-Trinder enzyme reaction was used to measure triglyceride content. A length of 1 651 bp of DGAT1 gene was cloned(GenBank accession number: MT221183), including 125 bp 5′UTR, 1 470 bp CDS, and 56 bp 3′UTR, encoding 489 amino acid residues. The goat DGAT1 gene had the highest expression in small intestine and the lowest in spleen. RT-qPCR results showed that DGAT1 was extremely significantly overexpressed in cells(P<0.01) by pcDNA3.1-DGAT1 treatment, as a results, the expression of GPAM(P<0.05), ADRP(P<0.01) and ACOX1(P<0.01) were significantly up-regulated, while the expression of AGPAT6(P<0.05), MLYCD(P<0.01) and HSL(P<0.01) were significantly down-regulated. Oil red O staining results showed that the lipid droplet accumulation after overexpression of DGAT1 was significantly(P<0.01) increased compared with that in the control group, as well as the results of triglyceride determination(P<0.01) in intramuscular adipocytes in goats. This study successfully obtained the goat DGAT1 gene CDS region sequence and constructed the pcDNA3.1-DGAT1 eukaryotic expression vector, overexpression of DGAT1 could significantly promote lipid deposition of intramuscular preadipocytes in goats and significantly affect the expression of lipid metabolism-related genes. These results provide important data for further clarifying the mechanism of DGAT1 in regulating intramuscular lipid metabolism in goats.
