【摘要】：旨在進一步揭示MSTN在綿羊成肌細胞中的調(diào)控機制,制備可有效失活MSTN基因的工具,為通過RNA干擾技術(shù)提高綿羊產(chǎn)肉量提供方法和理論依據(jù)。本研究以綿羊成肌細胞為試驗材料,構(gòu)建特異靶向綿羊MSTN基因的shRNA干擾質(zhì)粒載體,將干擾效果好的質(zhì)粒進一步包裝為重組腺病毒,轉(zhuǎn)染細胞后采用qRT-PCR和Western blot檢測MSTN基因以及生肌調(diào)節(jié)因子和干擾素反應基因的表達。結(jié)果表明,質(zhì)粒ShR218和ShR511干擾MSTN基因效率分別達到35%和48%,雙元干擾質(zhì)粒ShR3+4干擾效率最高達到85%。成功包裝shRNA重組腺病毒載體Sh511和Sh3+4,病毒滴度達到1×10~8 pfu·mL~(-1),對成肌細胞的感染效率達到90%以上。Sh511和Sh3+4對MSTN基因mRNA的表達抑制分別達到53%和76%,對蛋白表達抑制分別達到55%和64%。MSTN基因沉默后,伴隨著Myf5、MyoD、MyoG、Myf6基因mRNA水平的極顯著性下調(diào)(P0.01),但只引起MyoG蛋白水平極顯著升高(P0.01),未引起Myf5、MyoD、Myf6蛋白水平的顯著變化;腺病毒感染成肌細胞未引起OAS1基因mRNA水平的顯著變化,但引起IFNGR1基因mRNA水平的極顯著升高(P0.01),對二者蛋白水平均無顯著影響。本研究成功構(gòu)建靶向MSTN基因的shRNA腺病毒載體,能有效抑制成肌細胞MSTN的mRNA和蛋白表達,并影響生肌調(diào)節(jié)因子Myf5、MyoD、MyoG、Myf6基因和干擾素受體基因IFNGR1表達。
[Abstract]:The aim of this study was to further reveal the regulatory mechanism of MSTN in sheep myoblast and to prepare a tool for inactivating the MSTN gene effectively, and to provide a method and theoretical basis for increasing the meat yield of sheep by RNA interference technique. In this study, sheep myoblasts were used as experimental materials to construct shRNA interference plasmid vector specifically targeting sheep MSTN gene. The plasmid with good interference effect was further packaged as recombinant adenovirus. After transfection, the expression of MSTN gene, myogenic regulatory factor and interferon response gene were detected by qRT-PCR and Western blot. The results showed that the interference efficiency of plasmid ShR218 and ShR511 reached 35% and 48%, respectively, and the interference efficiency of plasmid ShR3 4 reached 85%. The shRNA recombinant adenovirus vectors Sh511 and Sh3 4 were successfully packaged. The viral titer reached 1 脳 10 ~ (8) pfu mL~ (-1). The infection efficiency of myoblasts was over 90%. Sh511 and Sh3 4 inhibited the mRNA expression of MSTN gene by 53% and 76%, respectively. After 55% and 64%.MSTN gene silencing, The mRNA level of Myf5,MyoD,MyoG,Myf6 gene was significantly down-regulated (P0.01), but only the level of MyoG protein was significantly increased (P0.01), but no significant change of Myf5,MyoD,Myf6 protein level was caused by adenovirus infection of myoblast, but no significant change of mRNA level of OAS1 gene was found in adenovirus infected myoblasts. However, the mRNA level of IFNGR1 gene was significantly increased (P0.01), but had no significant effect on both protein levels. In this study, shRNA adenovirus vector targeting MSTN gene was successfully constructed, which can effectively inhibit the expression of mRNA and protein in myoblasts MSTN, and affect the expression of Myf5,MyoD,MyoG,Myf6 gene and IFN- receptor gene IFNGR1.
【作者單位】： 河北農(nóng)業(yè)大學動物科技學院;河北省畜牧獸醫(yī)研究所;
【基金】：國家肉羊產(chǎn)業(yè)技術(shù)體系資助項目(CARS-39)
【分類號】：S826

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