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

作　　者：余欢[1] 王成栋 李辉[1] 陈友波 石钰仕 赵德鹏 龙霞[1] 谭启松 YU Huan;WANG Cheng-dong;LI Hui;CHEN You-bo;SHI Yu-shi;ZHAO De-peng;LONG Xia;TAN Qi-song(Key Laboratory of Genetic Breeding and Reproduction of Plateau Mountain Animals,Ministry of Education,Guizhou University/Guizhou Provincial Key Laboratory of Animal Genetics and Breeding and Reproduction,Guiyang,Guizhou 550025,China;Bijie Economic Crops Workstation,Bijie,Guizhou 551700,China)

机构地区：[1]贵州大学高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室,贵州贵阳550025 [2]毕节市经济作物工作站,贵州毕节551700

出　　处：《南方农业学报》2024年第1期226-234,共9页Journal of Southern Agriculture

基　　金：贵州省科技支撑计划项目(农业领域重点项目)(黔科合支撑[2022]034);贵州省教育厅科学研究项目(黔教技[2022]061号)。

摘　　要：【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 gro

关 键 词：赤水乌骨鸡 ADSL基因 组织表达 成肌细胞 干扰载体 肉质风味 

分 类 号：S831.2[农业科学—畜牧学]