铁炮百合‘萨莉’鳞茎各发育时期及不同组织qPCR内参基因筛选和验证

Selection and Validation of Reference Genes for qPCR in'Sally'Across Stages and Tissues

为筛选铁炮百合杂交系鳞茎不同发育阶段以及相同发育阶段不同组织中稳定表达的内参基因,本研究以铁炮百合观赏品种‘萨莉’为研究对象,采用实时荧光定量PCR (qPCR)分析百合的6个候选的内参基因(ACT, BHLH, EF1, EIF, GAPDH, lilyact)在‘萨莉’鳞片的不同发育时期以及不同组织中的扩增效率,再使用Normfinder、Bestkeeper、ge Norm、比较ΔCt法以及RefFinder 5种软件或方法对候选内参基因的表达稳定性进行评估。结果表明在所有样品中和在不同发育阶段的鳞茎中都是EIF和BHLH综合稳定性最佳,在不同组织中GAPDH和EIF的综合稳定性最佳,而在所有样品中及不同组织中,ACT的稳定性最差。本研究所得结果为‘萨莉’鳞茎的不同发育阶段以及不同组织的表达量分析提供了合适的内参基因组合,为其后续基因表达量的研究提供了基础。

In order to select the internal reference genes stably exp ressed in different developmental stages of longiflorum hybird bulbs and different tissues at the same developmental stage,this study took longiflorum hybird cultivar'Sally'as the research object,and used quantitative real-time PCR(qPCR)to analyze the 6 levels of lily.The amplification efficiency of two candidate internal reference genes(ACT,BHLH,EF1,EIF,GAPDH,lilyact)in different developmental stages of'Sally'scales and in different tissues,then use Normfinder,Bestkeeper,geNorm,comparativeΔCt method and RefFinder softwares or methods to evaluate the stability of the expression of candidate internal reference genes.The results showed that EIF and BHLH have the best comprehensive stability in all samples and in bulbs at different developmental stages.And in different tissues,GAPDH and EIF are most stable.In all samples and different tissues,ACT has the worst stability.The results of this study provide a suitable combination of internal reference genes for the analysis of the expression levels of different developmental stages and different tissues of'Sally'bulbs,which lays the foundation for the subsequent study of gene expression levels.