Selection Of Reference Genes For Quantitative Real-time PCR In P.yezoensis

Real time fluorescent quantitative PCR is an effective technique for the study of geneexpression.We need housekeeping genes as reference genes to get accurate gene expressionstatus. P. yezoensis is the representation model species of intertidal algae, along with tidalaction, laver have to undergo opposite environmental conditions changes between sea and airperiodically, and faces many environmental factors change stress which will meet stress fromenvironmental factors change at low tide; at the same time, Life history of P. yezoensis is aheterogenesis life history which existes thallus and filamentous morphology with significantdifference between the generations, so it is a good material to study large algae-resistantmechanism and generation development. Reference gene selection for P. yezoensis will laythe Foundation to study gene expression pattern and reveal the biological character of P.yezoensis.This study gives different stress treatments to P. yezoensis, including temperature andlight intensity changes，losing water. The experiment selected six common housekeepinggenes including actin(ACT3), ubiquitin binding protein(UBC), glycerol-3-phosphatedehydrogenase(GAPDH), translation initiation factor4A(eIF4A), elongation factor(EF)andalpha tubulin(TUA) as candidate genes. By real-time fluorescence quantitative PCRtechnology, we research genes expression in different environment factor and developmentgeneration and then use NormFinder, GeNorm and Best-keeper Software to filters the moststable expression gene.Best-keeper analysis shows that in different environmental stress, ACT3is the best geneused together with other genes as reference, followed by eIF4A and EF. If we used only onereference gene, GAPDH is the best, followed by the UBC. In different developmental stages, eIF4A is the best gene used together with other genes as reference, followed by EF.Best-keeper analysis shows that in different environmental stress, ACT3, eIF4A, EFare appropriate for selection of a single internal reference, or more than one internalreference, GAPDH is suitable for one internal reference selected separately. In differentdevelopment stages, eIF4A, EF is selection of a single internal reference, or more than oneinternal reference, UBC is suitable for one internal reference selected separately.NormFinder software analysis indicated that in different environmental stress, ACT3isthe most stable genes, followed by eIF4A and EF; but at different development period, themost steady expression gene is UBC, followed by EF and GAPDH；TUA is the most unstablegene and can not be used as a reference gene.With GeNorm software analysis we find that both in the environmental stress and thedifferent developmental stages, eIF4A and EF is the right pair of internal reference gene,which is match with the analysis of best-keeper and NormFinder.Therefore, UBC can be applied to research gene expression of P. yezoensis at differentdevelopmental stages, and actin can be used to study gene expression of thallus in differentenvironmental stresses. If we used a pair of genes as a candidate gene, eIF4A and EF is themost appropriate.