大鲵不同发育阶段及组织中内参基因稳定性评价

Stability evaluation of reference genes in tissues of Andrias davidianus at different development stages

【目的】研究18S核糖体RNA(18SrRNA)、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,NormFinder,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SDHA,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。

【Objective】The expression stability of five reference genes including 18 SrRNA,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 3years old health Chinese giant salamanders and skin of 3years,2years,1year,9months and 1week old health Chinese giant salamanders,and skin of Chinese giant salamanders 3,5,and7 days after Aeromonas hydrophilainfection.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-αand SDHA,the most stable reference genes in skin in different development and in skin after Aeromonas hydrophilainfection were EF1-αand GAPDH.【Con-clusion】The most stable reference gene was EF1-α,which was the first choice for qRT-PCR.The expressions of SDHA,GAPDHandβ-actin were strongly influenced by physiological state,and the selection as a second reference gene should depend on purpose.SDHAshould be used for comparing quantitative PCR in different tissues,whereas GAPDH should be used in different physiological conditions.