赤水乌骨鸡ADSL基因的组织表达特征及其干扰载体构建

09Expression characteristics of ADSL gene in pectoral muscle tissue of Chishui black-bone chicken and the construction of ADSL gene interference vector

【目的】探究赤水乌骨鸡胸肌组织中腺苷琥珀酸裂解酶基因(ADSL)表达特征及其目的基因的干扰载体构建,为深入研究畜禽组织肌肉中肌苷酸(IMP)的表达规律及ADSL基因影响畜禽风味的机制提供理论参考。【方法】以不同月龄(3、4、5和6月龄)的赤水乌骨鸡(公鸡和母鸡各半)为试验材料,运用实时荧光定量PCR检测不同月龄及不同性别赤水乌骨鸡胸肌组织中ADSL基因的表达情况;同时根据GenBank中鸡ADSL基因mRNA序列(NM_205529.2)设计4对短发夹RNA(shRNA)干扰序列(sh1-ADSL、sh2-ADSL、sh3-ADSL和sh4-ADSL)和1对阴性对照序列(sh-NC)与pGPH1/GFP/Neo载体连接后进行测序,将构建成功的ADSL基因干扰载体转染至成肌细胞,检测并筛选出干扰效率最高的干扰载体。【结果】ADSL基因在不同月龄赤水乌骨鸡胸肌组织中的相对表达量排序为5月龄>4月龄>6月龄>3月龄,赤水乌骨母鸡3月龄母鸡的ADSL基因相对表达量极显著高于公鸡(P<0.01,下同),4月龄母鸡ADSL基因的相对表达量显著高于公鸡(P<0.05),6月龄母鸡ADSL基因的相对表达量极显著低于公鸡;ADSL基因质粒的测序结果显示干扰质粒构建成功,质粒转染后干扰组及阴性对照均可见绿色荧光,空白对照不发光,说明各重组质粒成功转染赤水乌骨鸡成肌细胞;ADSL基因沉默结果显示,sh2-ADSL沉默效果最佳,干扰效率为90.30%。【结论】赤水乌骨鸡胸肌中ADSL基因的相对表达量随着月龄增加表现为先上升后下降的变化趋势;3、4和5月龄赤水乌骨母鸡ADSL基因的相对表达量高于公鸡,随着月龄增加ADSL基因的相对表达量增加,公鸡和母鸡间的差异减小。成功分离提取赤水乌骨鸡成肌细胞,并筛选出RNA干扰效果最佳载体sh2-ADSL,为后续研究ADSL基因调控鸡肉肌苷酸及风味的分子机制提供参考。

【Objective】This experiment was carried out to investigate the expression characteristics of adenylosucci-nate lyase gene(ADSL)in the pectoral muscle tissue of Chishui black-bone chicken and to construct the interference vec-tor of the target gene,.which provided a theoretical reference for an in-depth study of the expression pattern of inosine mo-nophosphate acid(IMP)in the muscles of livestock tissues and the mechanism by which the ADSL gene affected the fla-vour of livestock and poultry.【Method】Chishui black-bone chicken(half male and half female)of different ages(3,4,5 and 6 months old)were used as experimental materials.The expression of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken at different ages was detected by real-time fluorescence quantitative PCR.Meanwhile,four pairs of shorthairpinRNA(shRNA)interference sequences(sh1-ADSL,sh2-ADSL,sh3-ADSL and sh4-ADSL)and one pair of negative control sequences(sh-NC)were designed according to the mRNA sequence of chicken ADSL gene in Gen-Bank(NM_205529.2).They were sequenced after ligated with pGPH1/GFP/Neo vector.The constructed ADSL gene inter-fering vectors were transfected into myoblasts,and the most efficient interference vector was detected and screened.【Result】The relative expression level of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken at diffe-rent months age was in the order of 5 months>4 months>6 months>3 months.The relative expression level of ADSL gene in the pectoral muscle tissue of Chishui black-bone chicken was extremely significantly higher in 3-month-old females than in males(P<0.01,the same bellow).The relative expression level of ADSL gene was significantly higher in 4-month-old females than in males(P<0.05).The relative expression level of ADSL gene was extremely significant lower in 6-month-old females than in males.The sequencing results of the ADSL plasmid showed that the interference plasmid was successfully constructed and the green fluoresce was observed in the interference group and the negative control after plas-mid transfection,and the blank control showed no fluoresce,indicating that each recombinant plasmid successfully trans-fected the myoblasts of Chishui black-bone chicken.The ADSL gene silencing results showed that sh2-ADSL silencing effect was the optimal,with an interference efficiency of 90.30%.【Conclusion】The relative expression level of ADSL gene shows a trend of increasing and then decreasing with the increase of month age.The relative expression level of ADSL gene in the pectoral muscle tissue of females at 3,4 and 5 months old higher than that of males.The relative expres-sion level of ADSL genes increases with increasing month age,and the differences between males and females decrease.The experiment is successful in isolating and extracting myoblast from Chishui black-bone chicken,and screening out the sh2-ADSL vector with the best RNA interference effect.This study provides a reference for further research on the molecu-lar mechanism of ADSL gene regulation of meat inosinate and flavor.