腺病毒介导shRNA干扰绵羊MSTN基因效果及对生肌调节因子和干扰素反应基因表达的影响

Adenovirus-mediated shRNA Interference of MSTN Gene and Its Effect on Expression of Myogenic Regulatory Factor and Interferon Response Gene in Sheep

旨在进一步揭示MSTN在绵羊成肌细胞中的调控机制,制备可有效失活MSTN基因的工具,为通过RNA干扰技术提高绵羊产肉量提供方法和理论依据。本研究以绵羊成肌细胞为试验材料,构建特异靶向绵羊MSTN基因的shRNA干扰质粒载体,将干扰效果好的质粒进一步包装为重组腺病毒,转染细胞后采用qRT-PCR和Western blot检测MSTN基因以及生肌调节因子和干扰素反应基因的表达。结果表明,质粒ShR218和ShR511干扰MSTN基因效率分别达到35%和48%,双元干扰质粒ShR3+4干扰效率最高达到85%。成功包装shRNA重组腺病毒载体Sh511和Sh3+4,病毒滴度达到1×10~8 pfu·mL^(-1),对成肌细胞的感染效率达到90%以上。Sh511和Sh3+4对MSTN基因mRNA的表达抑制分别达到53%和76%,对蛋白表达抑制分别达到55%和64%。MSTN基因沉默后,伴随着Myf5、MyoD、MyoG、Myf6基因mRNA水平的极显著性下调(P<0.01),但只引起MyoG蛋白水平极显著升高(P<0.01),未引起Myf5、MyoD、Myf6蛋白水平的显著变化;腺病毒感染成肌细胞未引起OAS1基因mRNA水平的显著变化,但引起IFNGR1基因mRNA水平的极显著升高(P<0.01),对二者蛋白水平均无显著影响。本研究成功构建靶向MSTN基因的shRNA腺病毒载体,能有效抑制成肌细胞MSTN的mRNA和蛋白表达,并影响生肌调节因子Myf5、MyoD、MyoG、Myf6基因和干扰素受体基因IFNGR1表达。

The aim of this study was to further reveal the regulation mechanism of MSTN in sheep myoblasts, prepare the tool silencing MSTN, and provide the methods and theoretical basis for increasing the yield of muscle mass by using RNA interference technology. In this study, sheep myoblasts were used as experimental materials. The short hairpin RNA （shRNA） expression plasmid vector specific targeting to MSTN gene were constructed, then the plasmids with better interference effect were packaged for recombinant adenovirus, the expression of MSTN, myogenic regulatory factor and interferon response genes in myoblasts after infected by recombinant adenovirus were detected by qRT-PCR and Western blot. The result showed that the interference efficiency of plasmid ShR218, ShR511 were 3 5% and 48%, respectively, the interference efficiency of ShR32c4 was 85%. Recombinant adenovirus vector Sh511 and Sh3 ＋4 were successfully packaged, the virus titer reached 1 × 10^8 pfu· mL^-1 , the infection rate to myoblasts reached more than 90%. The mRNA expression of MSTN gene had been decreased by 53% and 76%, and the protein level had been decreased by 55% and 64% through Sh511 and Sh3＋4, respectively. The knockdown of MSTN gene was accompanied with expression downregulation of myogenic regulatory factor MyfS, MyoD, MyoG and My f6 at mRNA level（P〈0.01）, but at protein level only MyoG was significantly increased（P〈0.01）, no significant changes in Myf5, MyoD and Myf6. Adenovirus infecting myoblasts did not cause significant changes at OAS1 mRNA level, but caused significant increase at IFNGR1 mRNA level （P〈0.01）, while had no significant effect on their protein levels. In this study, shRNA adenovirus vector targeting to MSTN gene was successfully constructed, which can effectively inhibit the mRNA and protein expression of MSTN in myoblasts, and affect the expression of MyfS, MyoD, MyoG, My f6 and IFNGR1 genes.