橡胶灵芝菌内参基因筛选与分析

Selection of Reference Genes in Ganoderma pseudoferreum for Studying a Rubber Tree Disease

【目的】分析评价不同胁迫处理的橡胶灵芝菌候选内参基因的稳定性,为探索橡胶灵芝菌基因的功能及其侵染橡胶树的分子机制等提供参考。【方法】以5个非生物因子(温度、盐、氧化、pH、干旱)和1个生物因子(生防细菌)胁迫下的橡胶灵芝菌作为材料,提取RNA并反转录为cDNA,采用实时荧光定量PCR技术,扩增6个候选内参基因(UBC、ACT、RPL6、β-TUB、APT、28s),利用分析软件geNorm、NormFinder、BestKeeper和RefFinder根据基因表达的稳定性对基因进行排序,选择最适合不同胁迫的内参基因组合。【结果】所有样品RNA均为清晰的两条带,且6个候选内参基因的熔解曲线为明显单一峰。结合geNorm、NormFinder、Bestkeeper和RefFinder对其进行表达稳定性分析发现,温度胁迫下基因表达稳定性为UBC>ACT>RPL6>β-TUB>28s>APT,盐胁迫下基因表达稳定性为ACT>RPL6>UBC>APT>β-TUB>28s;氧化胁迫下基因表达稳定性为UBC>ACT>28s>APT>β-TUB>RPL6;pH胁迫下基因表达稳定性为RPL6>UBC>APT>ACT>β-TUB>28s,干旱胁迫下基因表达稳定性为ACT>UBC>β-TUB>RPL6>APT>28s,生物胁迫下基因表达稳定性为UBC>ACT>RPL6>28s>APT>β-TUB。【结论】结合所有候选基因稳定性和胁迫条件,推荐基因ACT和UBC作为在干旱、氧化、温度和生物胁迫下的内参基因,基因ACT和RPL6为盐胁迫下的内参基因,UBC和RPL6为pH胁迫下的内参基因。本研究结果为不同胁迫下橡胶灵芝菌相关基因的表达研究提供了合适的内参基因。

【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 reversetranscribed into cDNA.RT-qPCR was applied to amplify 6 candidate reference genes,i.e.,UBC,ACT,RPL6,β-TUB,APT,and28s.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.