达氏鲟实时荧光定量PCR内参基因的筛选  

Identification of reference genes for qRT-PCR in Dabry’s sturgeon,Acipenser dabryanus

作　　者：武梦斌 叶欢[2] 岳华梅[2] 阮瑞[2] 杜浩[2] 周从利 向浩 李创举[1,2] WU Mengbin;YE Huan;YUE Huamei;RUAN Rui;DU Hao;ZHOU Congli;XIANG Hao;LI Chuangju(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Freshwater Biodiversity Conservation,Ministry of Agriculture and Rural Affairs,Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Wuhan 430223,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]农业农村部淡水生物多样性保护重点实验室,中国水产科学研究院长江水产研究所,湖北武汉430223

出　　处：《中国水产科学》2020年第7期759-770,共12页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(31802282);中国水产科学研究院基本科研业务费项目(2018JBF10).

摘　　要：实时荧光定量PCR(qRT-PCR)是基因表达研究的常用方法,筛选达氏鲟(Acipenserdabryanus)稳定表达的内参基因,是保证该物种基因表达qRT-PCR分析结果准确性的基础。本研究选取了达氏鲟β肌动蛋白(β-actin)、延伸因子1α(EF-1α)、3-磷酸甘油醛脱氢酶(GAPDH)、核糖体蛋白L8 (RPL8)、18S核糖体RNA (18S rRNA)、琥珀酸脱氢酶复合物A亚基(SDHA)和α微管蛋白(α-tubulin)共7个常用内参基因作为候选基因,利用geNorm、NormFinder、BestKeeper和RefFinder 4个软件分析这7个候选基因在达氏鲟成鱼不同组织和不同发育时期胚胎及性腺的表达稳定性。结果表明, 7个候选内参基因的定量PCR引物均可获得特异性扩增产物和理想的扩增效率。在成鱼不同组织中,候选内参基因稳定性由高到低的顺序为EF-1α>β-actin>SDHA>RPL8>18SrRNA>α-tubulin>GAPDH;在不同发育时期胚胎中,候选内参基因稳定性由高到低的顺序为EF-1α>β-actin>SDHA>α-tubulin>18S rRNA> GAPDH>RPL8;在不同发育时期精巢中,候选内参基因稳定性由高到低的顺序为EF-1α>RPL8>β-actin>SDHA>18SrRNA>α-tubulin>GAPDH;在不同发育时期卵巢中,候选内参基因稳定性由高到低的顺序为RPL8>EF-1α>SDHA>β-actin=α-tubulin>18S rRNA>GAPDH。因此,达氏鲟成鱼不同组织、不同发育时期胚胎和精巢最稳定表达内参基因是EF-1α,而不同发育卵巢最稳定表达的内参基因是RPL8。本研究旨在为今后达氏鲟不同组织、不同发育时期胚胎及性腺的基因表达差异研究提供理论基础。In order to identify the most stable reference gene for the real-time quantitative PCR(qRT-PCR)analysis in Dabry’s sturgeon(Acipenser dabryanus),seven commonly used reference genes,including beta actin(β-actin),elongation factor-1(EF-1α),glyceraldehyde 3-phosphate dehydrogenase(GAPDH),ribosomal protein L8(RPL8),18S ribosomal RNA(18S rRNA),succinate dehydrogenase complex subunit A(SDHA),and alpha-tubulin(α-tubulin)were selected as candidates.Their expression stability was evaluated in different adult tissues,and in the tissue of embryos,testes,and ovaries during different developmental stages.We used geNorm,NormFinder,BestKeeper,and RefFinder software to conduct the analyses.Results verified that optimal amplification efficiency was obtained from the primers of these candidate genes.In adult tissues,the stability of these reference genes was as follows:EF-1α>β-actin>SDHA>RPL8>18S rRNA>α-tubulin>GAPDH;during different developmental stages of embryos,the stability of these reference genes was:EF-1α>β-actin>SDHA>α-tubulin>18S rRNA>GAPDH>RPL8;during different developmental stages of testes,the stability of these reference genes was:EF-1α>RPL8>β-actin>SDHA>18S rRNA>α-tubulin>GAPDH;during different developmental stages of ovaries,the stability of these reference genes was:RPL8>EF-1α>SDHA>β-actin=α-tubulin>18S rRNA>GAPDH.Overall,the most stable reference gene in adult tissue,embryos,and testes was EF-1α.For ovary tissue,we found RPL8 to be most stable.This study provides a useful basis for future work examining gene expression in Dabry’s sturgeon.

关 键 词：达氏鲟 荧光定量PCR 内参基因 稳定性 

分 类 号：S917[农业科学—水产科学]