| Quantitative real-time reverse transcription PCR(qRT-PCR)is one of the most powerful quantification methods for gene expression analysis,for accurate and reliable gene expression results,normalization of qRT-PCR data is required against suitable reference genes,which should be validated prior to use across samples and treatments.These genes are stably expressed in all samples and processing.Currently,few statistically validated reference genes have been reported in melon.In order to identify an ideal reference gene as candidate for gene expression studies in melon,we systematically evaluated 10 commonly used reference genes in a set of 26 melon samples under different growth and developent processes,and evaluated 11 commonly used reference genes in a set of 27 melon samples under biotic and abiotic stresses.In order to acquire the reference gene which is the most suitable one for the analysis of muskmelon's gene expression,we use geNorm,NormFinder and BestKeeper to analyze the stability of all reference genes.The results are as follows:1.This experiment adopts an RNA extractinion method of guanidine isothiocyanate.W-e have made some improvement on the extractinion method in the experiment proess likeshortening the time of water bath,etc.And obtained more complete RNA of non-degradation and high purity,thus completely meeting the requirements of follow-up e-xperiments.2.As is shown in the result of geNorm analysis,in the nutritive organs of melon,the m ost stably expressed genes in total samples,root,stem and leaf samples are(actin,actin3),(actin,actin3),(actin1,actin5);In accordance with NormFinder analysis,actin3 turns to be the most stably expressed gene in total samples,root,stem and leaf samples of the nutritive organs,while the BestKeeper analysis showed that the most stable genes respectively were actin3,actin3 and actin2 in total samples,root,stem and leaf samples of the nutritive organs.All things considered,the most suitable reference gene in the root samples is actin3,the most suitable reference gene in the stem and leaf samples is actin1,and the most suitable reference gene in the nutritive organs of melon is actin3.In the reproductive organs of melon,the analyzing results of three kinds of soft-ware are slightly different.The results of geNorm analysis shows that the most stably expressed genes in total samples,female and male flowers,fruits and total samples are(actin,actin3),(actin1,UBI-ep),(actin,actin4),respectively;The results of NormFinder analysis show that the most stable genes respectively are actin3,actin3,CYC in the total samples,female and male flowers,fruit seeds samples;The results of BestKeeper analysis show that the most suitable reference genes in the total samples,female and male flowers,fruit seeds samples respectively are ctin3,actin and 18s rRNA.Combined with the three methods,we recommend actin,actin,actin3 respectively as the most suitable reference genes in the total samples,female and male flowers,fruit seeds samples to use in developent process.4.In biotic and abiotic stresses,the results of geNorm analysis show that the most stable reference genes respectively are(actin,actin3),(actin,actin3),(actin4,EFla),(actin4,actin5)in the total samples,different hormone stress,high/low temperature stress,P.cubensis and salt stress;The results of NormFinder analysis show that the most stable reference genes are TUA,actin5,actin4,TUA in the total samples,different hormone stress,high low temperature stress,P.Cubensis and salt stress,Respectively;The results of BestKeeper analysis show that the most stable reference genes respectively are actin5,actin1,actin,actin2 in the total samples,different hormone stress,high/low temperature stress,P.cubensis and salt stress.Overall consideration,we suggest the reference genes in biotic and abiotic stresses are actin 3,actin4,CYC,TUA.5.Overall consideration,Our data support ACT3 as the most relevant reference genes for total samples. |
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