摘 要：
为了构建猪MEF2C基因的RNA干扰重组质粒,并筛选出最佳干扰效率的序列,本试验采用RNAi技术,设计并合成了3条针对猪MEF2C基因的RNA干扰序列,构建了RNA干扰重组质粒(命名为siRNA-1,siRNA-2,siRNA-3),将其转染到小鼠C2C12细胞,并用实时定量PCR检测细胞中MEF2C基因mRNA的表达,Western blot测定细胞中MEF2C基因蛋白的表达。结果表明:重组质粒经PCR扩增后显示产物大小与预期一致,产物经双酶切及测序鉴定该序列与NCBI提供的序列一致。重组质粒转染C2C12细胞后,MEF2C基因mRNA表达受到不同程度抑制,抑制率分别为(33.67±2.51)%、(21.00±3.60)%、(71.67±4.04)%。Western blot结果表明MEF2C基因蛋白表达受到不同程度抑制,抑制率分别为(47.33±0.05)%、(38.25±0.16)%、(67.27±0.12)%。本试验成功构建猪MEF2C基因干扰重组质粒,最终确定干扰质粒(siRNA-3)具有最佳干扰效果,证实MEF2C基因的干扰质粒能有效抑制目的基因mRNA和蛋白的表达,为探讨该基因与肌肉肉质性状的关系提供理论基础。
译 名：
Construction of RNA interference plasmid targeting of porcine MEF2C gene and interference efficiency determination
作 者：
ZENG Yangyang;ZHU Hongyan;TIAN Yumin;SU Yuhong;College of Animal Husbandry and Veterinary Medicine,Liaoning Medical University;
关键词：
MEF2C;;C2C12;;recombinant plasmid;;transfection
摘 要：
The present study was conducted to construct a RNA interference plasmid targeting of porcine MEF2 C and identify the highly efficient interference sequence. Three lines of RNA interference sequence targeting MEF2 C gene were designed and synthetized by using RNAi technology and construction of RNA interference plasmids( named siRNA- 1,siRNA- 2,siRNA- 3),they were subsequently transfected into C2C12 cells. The expression status of mRNA of MEF2 C in cells was determined by real- time PCR and the expression status of protein of MEF2 C in cells determined by western blot. Results indicated that the products of recombinant plasmids were identical to expectation after dual- enzyme digestion and being sequenced of these plasmids,these sequences were identical to sequences that provided by NCBI after PCR amplification. After recombinant plasmids being transfected into C2C12 cells,the expression of mRNA of MEF2 C was inhibited at different degree by real- time PCR,the inhibition rates on mRNA of MEF2 C were 33. 67 ± 2. 51%,21. 00% ± 3. 60% and 71. 67% ± 4. 04%. The expression of protein of MEF2 C was inhibited at different degree by western blot and the inhibition rates on protein of MEF2 C were 47. 33% ± 0. 05%,38. 25% ±0. 16% and 67. 27% ± 0. 12%. Together,RNA interference plasmid targeting of porcine MEF2 C gene had been successfully constructed and siRNA- 3 had been defined the highly efficient interference plasmid. The interference plasmid had inhibition on expression of mRNA and protein of MEF2 C,providing theoretical basis for studying relations between MEF2 C and the quality of meat.
