秦川肉牛脂肪细胞PLIN 2基因干扰siRNA筛选及过表达载体效率检测  

The Selection of siRNA Efficiency and Detection of Overexpression Vector for PLIN 2 Gene of Adipocytes in Qinchuan Beef Cattle

作　　者：李佩韦 张薇依 成功[1,3] 昝林森[1,3] LI Peiwei;ZHANG Weiyi;CHENG Gong;ZAN Linsen(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China;Shaanxi Institute of Zoology,Xi’an 710032,China;National Beef Cattle Improvement Center,Yangling 712100,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]陕西省动物研究所,西安710032 [3]国家肉牛改良中心,杨凌712100

出　　处：《中国畜牧兽医》2021年第8期2727-2735,共9页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31972994);国家重点研发计划(2018YFD0501700);国家肉牛牦牛产业技术体系建设(CARS-37);陕西省农业科技创新转化项目(NYKJ-2018-YL09);宁夏回族自治区重点研发计划(2019BEF02004)。

摘　　要：试验旨在获得秦川牛肉用新品系(以下简称“秦川肉牛”)具有高干扰效率的脂肪细胞分化相关蛋白2(PLIN2)基因干扰siRNA及高过表达效率的基因超表达载体,为后续研究PLIN 2基因在秦川肉牛脂肪细胞分化过程中的功能提供试验依据。通过采用siRNA介导的靶基因沉默技术合成靶向牛PLIN2 mRNA序列的si-PLIN2及构建真核pcDNA3.1(+)-PLIN2过表达载体,利用FuGENE■ 6低毒性转染试剂配制转染复合物,转染秦川肉牛脂肪细胞并进行诱导分化。采用实时荧光定量PCR检测技术检测各试验组与对照组中PLIN 2基因的相对表达量,并计算目的基因干扰及过表达效率;采用Western blotting方法检测蛋白水平的表达情况;采用BODIPY脂滴染色的方法观察表型。结果表明,转染秦川肉牛前体脂肪细胞后干扰组3对si-PLIN2(si-PLIN2_01、si-PLIN2_02和si-PLIN2_03)与对照组(si-PLIN2-NC)相比,PLIN 2基因表达量下降(P<0.01),干扰效率分别为71%、54%和50%;蛋白水平检测发现,si-PLIN2_01、si-PLIN2_03组蛋白水平表达与对照组(si-PLIN2-NC)相比出现下降趋势;脂滴染色后发现,干扰组与对照组相比,脂滴数目明显减少。过表达组pcDNA3.1(+)-PLIN2与对照组相比,PLIN2基因表达量显著上调,达35倍,脂滴数目明显增多。综上所述,siRNA介导的PLIN 2基因沉默或pcDNA3.1(+)介导的体外表达载体均可以成功实现对PLIN 2基因在秦川肉牛脂肪细胞分化过程中的干扰或超表达,并影响脂肪细胞分化过程中脂滴的形成数量。本研究为进一步探究PLIN2在秦川肉牛脂肪细胞分化过程中的功能奠定基础。In order to obtain the high interference efficiency siRNA of adipocyte differentiation associated protein 2(PLIN2)gene and the high overexpression efficiency gene overexpression vector of Qinchuan new beef line(hereinafter referred to as“Qinchuan beef cattle”),and provide experimental basis for the follow-up study of PLIN 2 gene function in the process of adipocyte differentiation of Qinchuan beef cattle,in this study,si-PLIN2 targeting bovine PLIN2 mRNA sequence was synthesized by siRNA mediated target gene silencing and eukaryotic pcDNA3.1(+)-PLIN2 overexpression vector was constructed.FuGENE■6 low toxic transfection reagent was used to prepare transfection complex,transfect Qinchuan beef cattle adipocytes and induce differentiation.Real-time quantitative PCR was used to detect the relative expression of PLIN 2 gene in each experimental group and control group,and the interference and overexpression efficiency of the target gene were calculated.Western blotting was used to detect the protein expression and the phenotype was observed by BODIPY staining.The results showed that after transfection of preadipocytes in Qinchuan beef cattle,three groups of si-PLIN2(si-PLIN2_01,si-PLIN2_02 and si-PLIN2_03)compared with control group(si-PLIN2-NC),the expression of PLIN 2 gene decreased(P<0.01),and the interference efficiency was 71%,54%and 50%,respectively.The protein level of the si-PLIN2_01 and si-PLIN2_03 groups showed a downward trend compared with control group.Compared with control group,the number of lipid droplets in the interference group decreased significantly.Compared with control group,the expression of PLIN 2 gene in overexpression group pcDNA3.1(+)-PLIN2 was significantly increased by 35 times(P<0.01),and the number of lipid droplets was significantly increased.In conclusion,the silencing of PLIN 2 gene mediated by siRNA or the expression vector mediated by pcDNA3.1(+)in vitro could successfully interfere with or overexpress PLIN 2 gene in the process of adipocyte differentiation in Qinchuan beef ca

关 键 词：秦川肉牛 PLIN2 基因干扰 载体构建 

分 类 号：S813.3[农业科学—畜牧学]