慢病毒介导shRNA干扰FoxO1促进猪成肌细胞中MyHCⅠ的表达  

Lentivirus Mediated shRNA Interference Silencing FoxO1 Gene Promoted the Expression of MyHCⅠin Pig Myoblasts

作　　者：史新娥[1] 郑雪莉[1] 刘月光[1] 袁媛[1] 张辉[1] 杨公社[1] 

机构地区：[1]西北农林科技大学动物科技学院动物脂肪沉积与肌肉发育实验室,陕西杨凌712100

出　　处：《中国生物化学与分子生物学报》2011年第6期582-588,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：转基因生物新品种培育重大专项(No.2009ZX08009-157B);西北农林科技大学"创新团队建设计划"资助~~

摘　　要：叉头框转录因子O亚族1(forkhead box transcription factor O1,FoxO1)是调控骨骼肌生长、代谢及细胞分化过程的重要转录因子,在肌纤维类型转化过程中发挥重要作用.为深入研究其对肌纤维类型的影响,构建FoxO1短发夹RNA(short hairpin RNA,shRNA)干扰慢病毒载体并感染猪成肌细胞,用real-time PCR和Western印迹法检测FoxO1和肌球蛋白重链Ⅰ(myosin heavy chainⅠ,MyHCⅠ)mRNA和蛋白的表达情况.测序结果证实,Lenti H1 RNAi FoxO1质粒构建正确,获得Lenti H1 RNAi FoxO1病毒颗粒滴度为8×107U/mL.病毒感染猪成肌细胞后,与对照组相比,siFoxO1a组FoxO1在转录水平上降低了58%,蛋白水平降低了77%,而MyHCⅠmRNA表达量提高了2.58倍.结果表明,本研究成功构建了猪FoxO1基因shRNA干扰慢病毒载体,且沉默FoxO1促进猪成肌细胞中MyHCⅠmRNA的表达.Forkhead box transcription factor O-1（FoxO1） is an important transcription factor which regulates growth,metabolism and cell differentiation of skeletal muscle.FoxO1 plays a key role in the transformation of muscle fibers.To study its effect on muscle fibers type,the lentivirus mediated short hairpin RNA（shRNA） targeted to FoxO1 gene was constructed.After the pig myoblast was infected with the constructed lentivirus,the expression of FoxO1 and myosin heavy chainⅠ（MyHCⅠ）were evaluated by real-time PCR and Western blotting.The sequencing results suggested that the vector of lenti H1 RNAi FoxO1 was successfully constructed,and the functional titer of concentrated virus was 8×107 U/mL.Compared with control group,the FoxO1mRNA and protein levels in siFoxO1 group were reduced by 58% and 77%,respectively.The expression of MyHCⅠin mRNA level was up-regulated by 2.58 folds.These results indicated the lenti H1 RNAi FoxO1 vector was constructed successfully,and FoxO1 gene silence promoted the expression of MyHCⅠ.

关 键 词：叉头框转录因子O亚族1(FoxO1) RNA干扰 慢病毒 成肌细胞 猪 

分 类 号：S811.3[农业科学—畜牧学] Q956[农业科学—畜牧兽医]