猪腺苷酸活化蛋白激酶α基因(AMPKα)特异性siRNA载体的构建及干扰效果评价  

Construction and Evaluation of Porcine Adenosine Monophosphate Activated Protein Kinase α Gene(AMPKα) siRNA Expression Vectors

作　　者：黄晶[1] 任阳[1] 余秀锋[1] 周锡红[1] 汪以真[1] 

机构地区：[1]浙江大学饲料科学研究所,农业部动物营养与饲料重点开放实验室,浙江省饲料及动物营养重点实验室,杭州310029

出　　处：《农业生物技术学报》2013年第9期1119-1124,共6页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(NO.2012CB124705)

摘　　要：腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK),在真核细胞生物中广泛存在,是调控机体能量代谢的关键酶。本研究旨在构建并筛选猪(Sus scrofa)AMPKα基因的有效siRNA干扰载体,并探讨AMPKα基因对脂肪分解关键酶基因表达的影响。根据猪AMPKα基因(GenBank登录号:NM-001167633)全长cDNA序列,设计合成其特异性发夹siRNA干扰片段,将其克隆插入pSilencer 4.1-CMV neo Vector干扰载体中,构建猪AMPKα基因的siRNA真核表达载体RA1和RA2,并通过PCR、双酶切和测序对其进行鉴定。构建成功后转染猪肌内脂肪前体细胞并检测其干扰效果及对脂肪分解关键酶——激素敏感脂肪酶(hormone sensitive lipase,HSL)、脂肪甘油三酯脂肪酶(adipose triglyceride lipase,ATGL)和肉毒碱棕榈酰转移酶(carnitine palmitoyl transferase,CPT1)基因表达的影响。结果表明,所构建的猪AMPKα基因的特异性siRNA表达载体RA1和RA2均可降低猪AMPKαmRNA表达(P<0.05),而且RA2干扰效果优于RA1。用RA2干扰AMPKα后,猪肌内脂肪前体细胞中AMPKα及脂肪分解关键酶HSL、ATGL和CPT1基因表达量显著下降(P<0.05)。提示,抑制AMPKα基因表达后,猪肌内脂肪前体细胞中脂肪分解和脂肪酸氧化速率降低。本研究结果为深入探讨AMPKα对猪脂肪代谢的影响机制及通过营养调控基因表达进而调控猪体脂沉积提供基础资料。The adenosine monophosphate activated protein kinase（AMPK）is a key regulator of cata- bolic and anabolic processes in energy metabolism. This study was conducted to construct and select the effective siRNA interference vector for porcine AMPKot gene. Two pairs of porcine AMPK a-specif- ic double-strand siRNA（dsRNA） primers were designed and inserted into pSilencer 4.1-CMV neo Vector after annealing. The vectors were then transfected into porcine intramuscular preadipocytes by lipofection 2000, AMPKa and lipolitic enzyme genes HSL（hormone sensitive lipase）, ATGL（adipose triglyceride lipase） and CPTl（carnitine palmitoyl transferase） mRNA expressions in these cells were detected by Real-time PCR. The results showed that the AMPlx^-specific siRNA vectors RA1 and RA2 were successfully constructed and transfected into porcine intramuscular preadipocytes. The efficiency of RA2 was significantly higher than that of RA 1. The mRNA expressions of AMPKoL and the lipolitic enzyme genes HSL, ATGL and CPTI decreased significantly after treatment of porcine intramuscular preadipocytes by vector RA2（P^0.05）. It indicated that decomposition of fat and fatty acid oxidation rate decreased in porcine intramuscular preadipocytes when AMPKa mRNA expressions was inhibited. These data will provide insight in the function of AMPK in fat de- position and manipulating gene expression of AMPK in regulating fat metabolism and meat quality in pigs.

关 键 词：腺苷酸活化蛋白激酶 载体构建 猪 干扰效果 

分 类 号：Q591[生物学—生物化学]