铁线莲属萼片荧光定量PCR内参基因的筛选和评价

Selection and Evaluation of Reference Genes for Quantitative Real-time PCR in Sepals of Different Clematis Varieties

以不同花色的铁线莲属(Clematis L.)栽培品种‘恬美’(C.‘Sympatia’)、‘罗兰’(C.‘Rooran’)、‘仙女座’(C.‘Andromeda’)、‘阿拉贝拉’(C.‘Arabella’)、‘普鲁吐斯’(C.‘Proteus’)以及‘路易斯罗维’(C.‘Louise Rowe’)初花时期的萼片为研究材料,利用RT-qPCR技术检测Atpb、Arp7、Ubc34、Wit1、Cxip4、Cyp71a1和Cyp35a1等7个候选内参基因的mRNA表达情况,并利用软件geNorm、NormFinder、BestKeeper和RefFinder对7个候选内参基因的稳定性进行综合评估。结果表明,内参基因表达差异显著;利用geNorm软件计算结果为基因Atpb与Cxip4最稳定;利用NormFinder软件计算结果为基因Cxip4最稳定;而BestKeeper软件计算出的最稳定的候选内参基因为Arp7,其次为Atpb;按这3个软件的计算结果,Cyp71a1与Cyp35a1的稳定性均最差,不适合作为内参基因;RefFinder软件分析的最佳的候选内参基因为Arp7。经综合分析,这几个铁线莲品种萼片基因表达的适宜内参基因是Arp7和Atpb。

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 m RNA expression of 7 candidate reference genes,including Atpb,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 Atpb 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 Atpb.According 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.