Screening and Stability Analysis of Internal Reference Genes for qRT-PCR in Haloxylon ammodendron
The aim of this study is to find out the reference genes stably expressed in various abiotic stres-ses of Haloxylon ammodendron,so as to lay a foundation for the subsequent study on the function of genes related to stress resistance of H.ammodendron.In this study,real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression changes of 14 candidate internal reference genes,including GAPDH,EF-1α,UBC,RPL32,ALB,50S-1721,50S-1063,RP Ⅱ,H3,PP2A,SOD,HSC70,TUA,and TUB,from the transcriptome database of H.ammodendron,under heat,drought,salt,abscisic acid(ABA)and circadian rhythms.By using geNorm,NormFinder,BestKeeper and RefFinder software,the stability of candidate internal reference genes of H.ammodendron was evalu-ated,and suitable internal reference genes were finally selected.Furthermore,the expression of phos-phoenolpyruvate carboxylase(PEPC)gene was analyzed to verify the influence of different reference genes on the experimental results.Significant differences were found in the optimal internal reference genes ana-lyzed by the four software programs,and the comprehensive ranking analysis on the RefFinder website showed that ALB was the optimal internal reference gene under ABA treatment and circadian rhythms,the RPⅡ gene was most stably expressed in leaves under drought stress,TUB and RP Ⅱ were most suit-able internal reference genes under salt stress,and the H3 gene was most stably expressed under heat stress.The most suitable internal reference genes under all stresses were ALB and RPL32.By calculating the geometric mean,the comprehensive stability ranking of 14 candidate internal reference genes was ob-tained,of which the top two genes were SOD and RPL32,respectively.In conclusion,SOD and RPL32 can be used as internal reference genes for qRT-PCR standardization.
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