板栗实时定量PCR内参基因的筛选与验证

Selection and validation of reference genes for quantitative RT-PCR analysis in Castanea mollissima

实时荧光定量PCR技术具有高灵敏性和高特异性,被广泛应用于基因表达分析,选取合适的内参基因是利用该技术准确分析目的基因表达变化的前提。本文以板栗的7种不同组织器官(根、茎、叶、种子、花芽、雌花和雄花)为材料,采用qRT-PCR技术分析了板栗中6个候选内参基因(EF1α、TUA、TUB、UBQ、18S rRNA和Actin)的表达趋势,运用geNorm、NormFinder和BestKeeper 3种软件对候选内参基因的表达稳定性进行了综合分析,并利用目的基因CmSPL9对筛选出的内参基因进行验证。结果表明:Actin和EF1α在板栗不同组织器官中稳定性最佳, Actin、EF1α或Actin+EF1α可作为板栗的内参基因进行后续试验研究。

Quantitative RT-PCR(qRT-PCR)has been widely used in gene expression analysis because of its sensitivity,specificity,and reproducibility.Application of suitable reference genes to normalize qRT-PCR data is critical in analyzing PCR results.In this study,seven different tissues and organs(roots,stems,leaves,seed,flower buds,female flowers,male flowers)of Castanea mollissima were used as materials.The expression trends of six candidate internal reference genes(EF1α,TUA,TUB,UBQ,18 S rRNA,Actin)in chestnut were analyzed by RT-qPCR.The geNorm,NormFinder and BestKeeper software were used to analyze the expression stability of candidate internal reference genes.The selected internal reference gene was verified by the target gene CmSPL9.The results showed that Actin and EF1αhad the most stability in different tissues and organs of C.mollissima.Actin,EF1αor Actin+EF1αcan be used as reference test for C.mollissima internal reference genes.