大鲵不同发育阶段及组织中内参基因稳定性评价

发布时间：2018-04-12 18:24

本文选题：大鲵 + 嗜水气单胞菌　；参考：《西北农林科技大学学报(自然科学版)》2017年10期

【摘要】：【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。
[Abstract]:[Objective] to study the 18S ribosomal RNA (18SrRNA), elongation factor 1- alpha (EF1- alpha), glyceraldehyde -3- phosphate dehydrogenase (GAPDH), beta actin (beta -actin) and succinate dehydrogenase A subunit (SDHA) of 5 kinds of common reference genes in different tissues in different developmental stages and the stability of giant salamander, Aeromonas hydrophila under the infection, to provide guidance for the study of genome. [Methods] after the giant salamander collected 3 old healthy giant salamander's liver, intestine, stomach, pancreas, kidney, muscle, skin, spleen, 3 age, 2 age, 1 age, 9 month old, giant salamander 1 week old healthy skin tissue and hydrophila article 3,5,7 days after infection of Aeromonas davidianus skin tissue, RNA extraction by real-time fluorescence quantitative PCR detection, the application of 3 kinds of reference gene stability evaluation analysis tools (geNorm, NormFinder, BestKeeper) analysis of 5 reference genes in different groups of giant salamander weave, different developmental stages and different pathological conditions Stability. [results] 5 reference genes in different tissues in the expression of giant salamander with good stability is EF1- alpha and SDHA, and in different developmental stages of Aeromonas hydrophila and giant salamander infected skin tissues, the expression stability of 5 kinds of reference genes in different expression in different tissues, better stability of EF1- alpha [Conclusion] EF1- and GAPDH. expression of the most stable, giant salamander is the best reference genes and quantitative PCR, SDHA, GAPDH and beta -actin expression affects the stability is affected by the physiological status of the larger, second reference genes should be regarded as objective and quantitative test, PCR should be used in SDHA in different tissues, and in under different physiological conditions should be selected for the GAPDH second or alternate reference gene.

【作者单位】：西北农林科技大学动物科技学院;
【基金】：陕西省科技攻关项目(2012K01-18)
【分类号】：S917.4

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