Selection and Evaluation of Reference Genes for Quantitative Real-time PCR in Sepals of Different Clematis Varieties
The sepals at the first flowering stage of 6 Clematis varieties with different colours(C.'Sympatia',C.'Rooran',C.'Andromeda',C.'Arabella',C.'Proteus'and C.'Louise Rowe')were used as the research materials.RT-qPCR was used to detect the mRNA expression of 7 candidate reference genes,including A tpb,Arp7,Ubc34,Wit1,Cxip4,Cyp71a1 and Cyp35a1,and the software geNorm,NormFinder,BestKeeper and RefFinder were used to comprehensively evaluate the stability of expression of the 7 candidate reference genes.The results showed that the expressions of internal reference genes were significantly different;the results of geNorm software showed that A tpb and Cxip4 were the most stable genes.The most stable gene Cxip4 was calculated by NormFinder software.The most stable candidate reference gene calculated by BestKeeper software was Arp7,followed by A tpb.Accord-ing to the calculation results of these three softwares,Cyp71a1 and Cyp35a1 have the worst stability and are not suitable as reference genes.The best candidate reference gene analyzed by the RefFinder software was Arp7.After comprehensive analysis,the appropriate internal reference genes for the expression of sepal genes in these Clematis cultivars were Arp7 and Atpb.
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