应用荧光定量PCR方法研究RPL29基因在通城猪和长白猪不同时期胚胎骨骼肌中的表达

Expression Analysis of RPL29 Gene in Embryo Skeletal Muscle at Different Stages from Tongcheng and Landrace Pigs Using Real-time PCR Method

为了研究蛋白合成相关基因RPL29在骨骼肌发育中的作用，采用SYBRGreen染料建立该基因的实时荧光定量PCR（QPCR）分析方法，并分析该基因在猪胚胎骨骼肌中的表达变化。以不同浓度稀释样本为标准品，建立标准曲线方程：y=-3.303X＋43．077（决定系数R^2=0．999）。分析表明：该方法具有较大的检验范围（PCR扩增Q值13～35），很好的扩增效率为100％，熔解曲线显示产物特异的单峰，其Tm值为86．5℃。本研究以H3F3A基因为内参，用所建立的实时定量PCR方法，分析RPL29在妊娠33、65和90d的通城猪和长白猪胚胎骨骼肌中的表达变化。研究发现：RPL29基因在通城猪和长白猪胚胎骨骼肌发育过程中分别呈现下调和上调表达模式；妊娠33d时，通城猪中的表达水平高于长白，而妊娠90d时在长白猪中高表达；妊娠65d时两品种间无显著差异。结果显示RPL29基因可能与猪骨骼肌的生长发育有关，并可能影响产肉性状。

In order to investigate effect of RPL29 gene associated with protein synthesis on skele- tal muscle development, the SYBR-Green quantitative real-time PCR （QPCR） method was established to analyze RPL29 gone. The expression change of RPL29 gone was analyzed in pig embryo skeletal muscle using the QPCR method. Based on standard samples with different concentrations, the standard curve ＂Y=-3. 303X＋43. 077 （determination coefficient, R2 =0. 999）＂ was established. The results indicated the method had large test extension （Ct arranged from 13 to 35 cycle number）, high amplification efficiency （100%） and single peak with unique PCR product in the melt curve. The Tm value was 86.5 ℃. In this study, the H3F3A gone was used for internal control, the expression change of RPL29 gone was analyzed in embryo skeletal muscle at 33, 65 and 90 days post coitus （dpc） from Tongcheng and Landrace using the QPCR method. We found that RPL29 exhibited the down- and up-regulation pattern in Tongcheng and Landrace pigs, respectively. There was higher expression in Tongcheng than that in Landrace at 33 dpc, and opposite result between two breeds at 90 dpc. No significant difference was found between two breeds at 65 dpc. These results indicated that RPL29 gene have potential association with skeletal muscle development and effect on meat production traits in pigs.