Selection of Reference Genes in Ganoderma pseudoferreum for Studying a Rubber Tree Disease
[Objective]Stability of the reference genes of Ganoderma pseudoferreum under stresses was determined for studying the gene functions and mechanism of the red root rot disease on rubber trees.[Methods]RNA of G.pseudoferreum mycelia under a stress of temperature,salt,oxidation,pH,drought,or a biocontrol bacteria invasion were isolated and reverse-transcribed into cDNA.RT-qPCR was applied to amplify 6 candidate reference genes,i.e.,UBC,ACT,RPL6,β-TUB,APT,and 28s.Software geNorm,NormFinder,BestKeeper,and RefFinder were used to determine the stability of the genes under stresses.[Results]The RNA of all samples displayed two clear bands and melting curves with a single peak.Analyses by geNorm,NormFinder,Bestkeeper,and RefFinder collectively ranked the expression stability of the genes under a temperature stress as UBC>ACT>RPL6>β-TUB>28s>APT;that under a salt stress,ACT>RPL6>UBC>APT>β-TUB>28s;that under an oxidative stress,UBC>ACT>28s>APT>β-TUB>RPL6;that under a pH stress,RPL6>UBC>APT>ACT>β-TUB>28s;that under a drought stress,ACT>UBC>β-TUB>RPL6>APT>28s;and that under the biotic stress,UBC>ACT>RPL6>28s>APT>β-TUB.[Conclusion]It appeared that ACT and UBC to be the choice reference genes for normalizing the gene expression under drought,oxidation,temperature,and biotic stresses,while ACT and RPL6 under a salt stress,and UBC and RPL6 under a pH stress.
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