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2025年4月5日 星期六
农业生物技术学报  2019, Vol. 27 Issue (3): 471-480    DOI: 10.3969/j.issn.1674-7968.2019.03.011
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
FGF10基因真核表达载体的构建及其在从江香猪不同组织中的表达
徐敏1, 许厚强1,*, 杨洋1, 陈伟1,2
1 贵州大学 动物科学学院/高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室,贵阳 550025;
2 贵州大学 生命科学学院,贵阳 550025
Construction of Eukaryotic Expression Vector of FGF10 Gene and Its Expression in Different Tissues of Congjiang Xiang Pig (Sus scrofa)
XU Min1, XU Hou-Qiang1,*, YANG Yang1, CHEN Wei1,2
1 College of Animal Science, Guizhou University/Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education/Guizhou Key Laboratory of Animal Genetics, Breeding and Reproduction, Guizhou 550025, China;
2 College of Life Science, Guizhou University, Guiyang 550025, China
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摘要 成纤维细胞生长因子10基因(fibroblast growth factor 10, FGF10)属于6个FGFs亚家族中的 FGF7亚家族,是特异地表达于白色脂肪组织的唯一FGF,在脂肪组织的发育和代谢过程中具有重要的作用。本实验旨在研究从江香猪(Sus scrofa)肌内前体脂肪细胞中FGF10对脂肪代谢的调控功能,实验采用qRT-PCR的方法检测了从江香猪不同组织中的表达情况,并根据克隆得到的FGF10的CDS区,构建了真核过表达载体pEGFP-C1-FGF10;分离培养从江香猪肌内前体脂肪细胞运用胶原酶消化法分离培养从江香猪肌内前体脂肪细胞,并进行了诱导培养,采用油红O染色法进行鉴定;运用qRT-PCR技术检测FGF10基因在不同诱导阶段的从江香猪肌内前体脂肪细胞中的表达;利用脂质体法将重组质粒pEGFP-C1-FGF10瞬时转染从江香猪肌内前体脂肪细胞,并检测过表达FGF10基因后脂肪相关代谢沉积的氧化物酶体增殖物激活受体γ基因(peroxisome proliferator activated receptor γ, PPARγ)、脂联素基因(adiponectin, ADIPOQ)、脂肪酸结合蛋白4基因(fatty acid binding protein 4, FABP4)、脂肪酸合成酶基因(fatty acid synthase, FAS)、脂肪组织甘油三酯水解酶(adipose triglyceride lipase, ATGL)、激素敏感脂酶基因(hormone sensitive lipase, HSL)、脂蛋白酯酶基因(lipoprotein lipase, LPL)以及乙酰辅酶A羧化酶基因(acetyl-CoA carboxylase, ACC)的表达水平。FGF10基因表达量最低是在背最长肌中,表达量最高是在胃中(P<0.05),其次是在肾脏和脂肪中;在诱导的早期阶段,FGF10基因表达量逐渐增加,在诱导分化48 h的表达量最高;经双酶切和测序鉴定,成功构建了pEGFP-C1-FGF10表达载体,且在从江香猪肌内前体脂肪细胞中成功表达;与空白对照相比,ACCFABP4FASLPLPPARγ的表达量均增加,ADIPOQATGLHSL的表达量均减少。FGF10对脂肪的沉积具有一定的调控作用,这种作用可能是通过对相关脂肪代谢基因的调控实现的。本研究为揭示FGF10对脂肪的调控作用提供了基础数据。
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关键词 从江香猪FGF10基因真核表达载体肌内前体脂肪细胞诱导分化脂肪沉积    
Abstract:The fibroblast growth factor 10 gene (FGF10) is a member of the fibroblast growth factor family, belonging to the FGF7 subfamily of six subfamilies of FGFs, it is the only FGF specifically expressed in white adipose tissue, and plays an important role in the development and metabolism of adipose tissue. The purpose of this study was to investigate the regulation of fat metabolism of FGF10 gene in the intramuscular preadipocytes of Congjiang Xiang pig (Sus scrofa), and the function of FGF10 in regulating fat deposition was revealed. The qRT-PCR was used to detect the relative expression level of FGF10 gene in different tissues of Congjiang Xiang pig. According to the CDS region of the cloned FGF10 gene, the expression vector pEGFP-C1-FGF10 was constructed. The intramuscular preadipocyte cells had been digested by type collagenase. The oil red O staining method was used identification after induction culture. qRT-PCR was used to detect the expression of FGF10 gene in precursors adipocytes in the intramuscular preadipocytes of Congjiang Xiang pig at different induction stages. The recombinant plasmid pEGFP-C1-FGF10 was transfected by liposome method in the intramuscular preadipocytes of Congjiang Xiang pig, and then detected the expression levels of peroxisome proliferator activated receptor γ gene (PPARγ), adiponectin gene (ADIPOQ), fatty acid binding protein 4 gene (FABP4), fatty acid synthase gene (FAS), adipose triglyceride lipase gene (ATGL), lipoprotein lipase gene (LPL), hormone-sensitive lipase gene (HSL)and acetyl-CoA carboxylase gene (ACC) after overexpression of FGF10. The results showed that the extremely higher expression levels of FGF10 gene in the stomach and significantly higher than other tissues (P<0.05), followed by high expression in the kidney and fat, which were the lowest in the dorsal longest muscle. At the early stage of induction, the expression level of FGF10 gene gradually increased, and the expression level of FGF10 gene was the highest at the induction and differentiation of 48 h. The pEGFP-C1-FGF10 expression vector was successfully constructed by double enzyme digestion and sequencing, and was successfully expressed in the intramuscular preadipocytes of Congjiang Xiang pig. Compared with blank controls, the expression levels of ACC, FABP4, FAS, LPL, and PPARγ gene had all increased, while the expression levels of ADIPOQ, ATGL, and HSL gene had all decreased. The results of this study showed that FGF10 gene had certain regulation effect on the fat deposition, this study provides basic data for revealing FGF10 gene's regulation of fat metabolism.
Key wordsCongjiang Xiang pig    Fibroblast growth factor 10 gene (FGF10)    Eukaryotic expression vector    Intramuscular preadipocyte cell    Induction differentiation    Fatty deposits
收稿日期: 2018-09-12     
ZTFLH:  S828  
基金资助:国家科技支撑计划(No. 2015BAD03B02-3)和黔科合重大专项(黔科合NY字[2013]6008号)
通讯作者: *gzdxxhq@163.com   
引用本文:   
徐敏, 许厚强, 杨洋, 陈伟. FGF10基因真核表达载体的构建及其在从江香猪不同组织中的表达[J]. 农业生物技术学报, 2019, 27(3): 471-480.
XU Min, XU Hou-Qiang, YANG Yang, CHEN Wei. Construction of Eukaryotic Expression Vector of FGF10 Gene and Its Expression in Different Tissues of Congjiang Xiang Pig (Sus scrofa). 农业生物技术学报, 2019, 27(3): 471-480.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.03.011     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I3/471
 
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