非生物胁迫下多年生黑麦草qRT-PCR分析中内参基因的选择  

Reference Genes for qRT-PCR in Perennial Ryegrass (Lolium perenne L.) Under Various Abiotic Stresses

作　　者：严海东[1] 蒋晓梅[1] 张新全[1] 黄琳凯[1] 

机构地区：[1]四川农业大学动物科技学院草业科学系,雅安625014

出　　处：《农业生物技术学报》2014年第12期1494-1501,共8页Journal of Agricultural Biotechnology

基　　金：四川省"十二五"饲草育种攻关项目(No.2011YZGG-11);国家现代牧草产业技术体系(No.CARS-35-05);2013年国家及省级大学生创新计划(No.201310626002)

摘　　要：提高实时荧光定量PCR(Real-time quantitative PCR,q RT-PCR)准确性的先决条件是选择表达稳定性较高的内参基因。本研究以多年生黑麦草(Lolium perenne L.)为材料,利用q RT-PCR技术检测翻译起始因子4A(eukaryotic initiation factor 4 alpha,e IF4A)、TNF结合蛋白Ⅰ(Tat binding protein-1,TBP-1)、泛素连接酶(ubiquitin-conjugating enzyme,E2)、多聚泛素酶基因(ubiquitin,UBQ)、生物钟受体蛋白(zeitlupe,ZTL)和甘油醛三磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)6个候选内参基因在不同非生物胁迫下的表达情况。运用ge Norm(ver.3.5)、Normfinder(ver.0.953)、Best Keeper(ver.1.0)、Delta Ct和Ref Finder软件综合分析表明,在干旱、盐、热胁迫及ABA处理下,e IF4A基因表达稳定性最高;在涝胁迫下,UBQ基因表达稳定性最高。因此,e IF4A和UBQ候选基因可作为不同胁迫下的内参基因,为进一步开展多年生黑麦草基因表达的定量研究了提供了一定的理论依据。The selection of high stablely expressed reference genes is an important prerequisite for improving the accuracy of Real-time quantitative PCR （qRT-PCR）. We investigated the expression of 6 candidate reference genes including eukaryotic initiation factor 4 alpha （eIF4A）, Ti-binding peptide-1 （TBP-1）, ubiquitin-conjugating enzyme （E2）, ubiquitin （UBQ）, zeitlupe （ZTL） and glyceraldehyde-3-phosphate dehydrogenase （GAPDH） under various abiotic stresses in perennial ryegrass （Lolium perenne L.）. The analysis with geNorm （ver. 3.5）, Normfinder （ver. 0.953）, BestKeeper （ver. 1.0）, Delta Ct and RefFinder revealed that under drought, salt, heat and ABA stresses, eIF4A gene was the most stable reference gene;under waterlogging stress, UBQ gene was the most stable reference gene. As a result, eIF4A and UBQ genes were selected as reference genes for perennial ryegrass under different abiotic stresses, which provides a foundation for gene expression investigation of perennial ryegrass.

关 键 词：多年生黑麦草 QRT-PCR 内参基因 非生物胁迫 

分 类 号：Q945.78[生物学—植物学]