大鲵不同发育阶段及组织中内参基因稳定性评价

摘要

[Objective] The expression stability of five reference genes including 18S rRNA,EF1-α,GAPDH,β-actin,and SDHA in tissues of Chinese giant salamander (Andrias davidianus) and in skin at different development stages was evaluated to provide guidance for future post-genomic studies of giant salamander.[Method] Samples were collected from liver,intestines,stomach,pancreas,kidney,muscle,skin,spleen and other tissues of 3 years old health Chinese giant salamanders and skin of 3 years,2 years,1 year,9 months and 1 week old health Chinese giant salamanders,and skin of Chinese giant salamanders 3,5,and 7 days after Aeromonas hydrophila infection.RNA was extracted and the expression level was detected using real-time fluorescent quantitative PCR.Then the expression stability was evaluated using three analysis tools (geNorm,NormFinder,and BestKeeper).[Result] The most stable reference genes in eight tissues of Chinese giant salamander in health were EF1-α and SDHA,the most stable reference genes in skin in different development and in skin after Aeromonas hydrophila infection were EF1-α and GAPDH.[Conclusion] The most stable reference gene was EF1-α,which was the first choice for qRT-PCR.The expressions of SDHA,GAPDH and β-actin were strongly influenced by physiological state,and the selection as a second reference gene should depend on purpose.SDHA should be used for comparing quantitative PCR in different tissues,whereas GAPDH should be used in different physiological conditions.%[目的]研究18S核糖体RNA (18S rRNA)、延伸因子1-α(EF1-α)、甘油醛-3-磷酸脱氢酶(GAPDH)、β-肌动蛋白(β-actin)和琥珀酸脱氢酶A亚基(SDHA)等5种常见内参基因在大鲵不同组织、不同发育阶段及嗜水气单胞菌感染下的稳定性,为大鲵后基因组学研究提供指导.[方法]采集3龄健康大鲵的肝脏、肠、胃、胰腺、肾脏、肌肉、皮肤、脾脏等组织,3龄、2龄、1龄、9月龄、1周龄健康大鲵的皮肤组织及嗜水气单胞菌感染大鲵后第3,5,7天的皮肤组织,提取其RNA,进行实时荧光定量PCR检测,应用3种内参基因稳定性评价分析工具(geNorm,NormFind-er,BestKeeper)分析5种内参基因在大鲵不同组织、不同发育阶段以及不同病理状态下的稳定性.[结果]5种内参基因在大鲵不同组织中表达稳定性较好的是EF1-α和SD HA,而在不同发育阶段及嗜水气单胞菌感染大鲵的皮肤组织中,5种内参基因的表达稳定性不同于在不同组织中的表达,稳定性较好的是EF1-α和GAPDH.[结论]EF1-α表达最为稳定,是大鲵定量PCR首选内参基因,而SDHA、GAPDH以及β-actin表达稳定性受生理状态的影响较大,作为第二内参基因时应视试验目的而定,在不同组织中进行定量PCR比较时应选用SDHA,而在不同生理状态下则应选用GAPDH为第二或备用内参基因.