斑地锦RT-qPCR内参基因的筛选

实时荧光定量PCR(RT-qPCR)的前提条件之一是具有合适的内参基因。为筛选斑地锦(Euphorbia maculata)合适的RT-qPCR内参基因,该文利用同源克隆法克隆斑地锦GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP等基因片段,RT-qPCR检测7个候选内参基因在斑地锦不同生长期根、茎、叶和果实中的表达情况,并用geNorm、NormFinder和BestKeeper等生物学软件对各候选基因表达稳定性进行评价。结果表明:(1)克隆的GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP基因片段为729、808、753、422、233、656、313 bp,分别编码242、269、250、140、77、218、103个氨基酸,与其他植物相应氨基酸序列的最高同源性均在85%以上。(2)综合3个分析软件分析内参基因表达稳定性得出,表达稳定性排名为UBQ>EF-1α>TUB-α>eIF-4A>GAPDH>CYP>act。因此,可以选取UBQ作为斑地锦RT-qPCR分析的内参基因,用于不同生长期基因组织特异性表达研究。

Screening of reference genes for RT-qPCR in Euphorbia maculata

The suitable reference genes is a prerequisite for real-time quantitative PCR(RT-qPCR).In order to find a suitable reference gene for gene expression analysis using RT-qPCR in Euphorbia maculata,GAPDH,EF-1α,act,UBQ,TUB-α,eIF-4A,and CYP gene fragments from roots,stems,leaves and fruits at different growth stages were cloned with the method of homologous cloning.Subsequently,the expression patterns of the seven candidate reference genes were obtained by RT-qPCR in E.maculata,and the expression stability was assessed by geNorm,NormFinder,and BestKeeper.The results were as follows:(1)The fragment sequences of GAPDH,EF-1α,act,UBQ,TUB-α,eIF-4A and CYP contained 729 bp(encoding 242 amino acids),808 bp(encoding 269 amino acids),753 bp(encoding 250 amino acids),422 bp(encoding 140 amino acids),233 bp(encoding 77 amino acids),656 bp(encoding 218 amino acids),and 313 bp(encoding 103 amino acids),respectively.And the seven amino acid sequences shared over 85%identity with other GAPDH,EF-1α,act,UBQ,TUB-α,eIF-4A,CYP by BlAST in GenBank.(2)The order of expression stability was UBQ>EF-1α>TUB-α>eIF-4A>GAPDH>CYP>act by geNorm,NormFinder,and BestKeeper.Therefore,UBQ can be selected as a reference gene for RT-qPCR in E.maculata using for gene expression analysis in different plant tissues at different growth stages.