从江香猪FGF10基因干扰载体的构建及相关基因表达分析  

Construction of Interference Vector of FGF10 Gene and Expression Analysis of Related Genes in Congjiang Xiang Pig(Sus scrofa)

作　　者：杨洋[1,2] 许厚强[1,2] 陈伟[1,2,3] 徐敏[1,2] YANG Yang;XU Hou-Qiang;CHEN Wei;XU Min(College of Animal Science,Guizhou University,Guiyang 550025,China;Key Laboratory of Animal Genetics,Breeding and Reproduction in the Platean Mountainous Region,Ministry of Education (Guizhou University)/Key Laboratory of Animal Genetics,Breeding and Reproduction,Guizhou Province,Guiyang 550025,China;College of Life Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]贵州大学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 [3]贵州大学生命科学学院,贵阳550025

出　　处：《农业生物技术学报》2018年第12期2066-2074,共9页Journal of Agricultural Biotechnology

基　　金：国家科技支撑计划(No.2015BAD03B02-3);黔科合重大专项(黔科合NY字[2013]6008号)

摘　　要：成纤维细胞生长因子10基因(fibroblast growth factor 10, FGF10)属于成纤维细胞生长因子家族成员之一,在脂肪组织的发育和代谢过程中具有重要的作用。为了进一步揭示FGF10基因在从江香猪(Sus scrofa)肌内脂肪中的调控作用,本研究通过在线软件设计了FGF10基因4对siRNA干扰序列和一对阴性对照序列,连入pGPU6/GFP/Neo载体后,筛选成功构建的干扰载体,并转染从江香猪肌内前体脂肪细胞,并采用qRT-PCR的方法筛选出最佳干扰载体,通过最佳干扰载体抑制FGF10基因的表达,采用qRT-PCR检测沉默FGF10对脂肪分化关键基因—过氧化物酶体增殖物激活受体γ基因(peroxisome proliferator activated receptorγ, PPARγ)、脂肪组织甘油三酯水解酶基因(adipose triglyceride lipase, ATGL)、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)、脂蛋白酯酶基因(lipoprotein lipase, LPL)、乙酰辅酶A羧化酶基因(acetyl-CoA carboxylase, ACC)以及激素敏感脂酶基因(hormone sensitive lipase, HSL)表达的影响。结果表明,本实验成功构建了从江香猪FGF10基因的干扰载体,并筛选出最佳干扰载体的干扰效率达74.30%;抑制FGF10基因表达后,相关脂肪分化关键基因ACC、ATGL、ADIPOQ、FABP4、FAS、LPL、PPARγ和HSL的表达量均显著降低,ACC与PPARγ的表达量显著低于空白对照组(P<0.05),ATGL、ADIPOQ、FABP4、FAS和LPL这5个基因的相对表达量均是极显著低于空白对照组(P<0.01),HSL基因的相对表达量与对照组无显著性差异(P>0.05)。本实验将构建的FGF10基因的干扰载体成功地转染了从江香猪肌内前体脂肪细胞,体外沉默FGF10基因可抑制猪肌内前体脂肪的分化,这可能与相关脂肪分化基因的低表达有关。本研究为进一步研究FGF10基因对脂肪沉积的调控机理奠定基础。The fibroblast growth factor 10 gene(FGF10) is a member of the fibroblast growth factor family,it plays an important role in the development and metabolism of white adipose tissue. In order to further revealthe role of FGF10 gene in the regulation of intramuscular fat in Congjiang Xiang pigs(Sus scrofa), in this experiment, four pairs of siRNA interference sequences and a pair of negative control sequences were designed by online software, and were connected to p GPU6/GFP/Neo vector. The successfully constructed interference vectors were transfected into Congjiang Xiang pig intramuscular preadipocytes and the best interference vector was screened by qRT-PCR. The expression of FGF10 gene was inhibited by the best interference vector. And then, the expressions of peroxisome proliferator activated receptorγ(PPARγ), adipose triglyceride lipase(ATGL), adiponectin(ADIPOQ), fatty acid binding protein 4(FABP4), fatty acid synthase(FAS), lipoprotein lipase(LPL), acetyl-CoA carboxylase(ACC) and Hormone-sensitive lipase(HSL) gene were detected by q RT-PCR.. The results showed that the interference vector of the FGF10 gene was constructed successfully, and the optimal interference vector was screened with the interference efficiency of 74.30%.After inhibiting the expression of FGF10 gene, the expression of ACC, ATGL, ADIPOQ, FABP4, FAS, LPL,PPARγ and HSL were decreased. The expression of ACC and PPARγ genes were significantly lower than the control group(P<0.05), and the expression of ATGL, ADIPOQ, FABP4, FAS and LPL genes were significantly lower than control group(P<0.01). There was no significant difference between the relative expression of HSL gene and the control group(P>0.05). In this experiment, the constructed interference vector of FGF10 gene was successfully transfected into the intramuscular preadipocyte cells of Congjiang Xiang pig, and the procine intramuscular preadipocytes differentiation can be inhibited by sliencing of FGF10 gene in vitro,which might be related to down-regulation of fat differentiation

关 键 词：从江香猪 FGF10基因 干扰载体 肌内前体脂肪细胞 基因表达 

分 类 号：S828[农业科学—畜牧学]