梨果实细胞分裂期内参基因表达稳定性分析

【目的】果实细胞分裂期是果实品质调控的关键时期,qRT-PCR技术在果实品质研究中发挥着重要作用,qRT-PCR技术中内参基因的选择关系到检测结果的准确性。为了筛选出适合分析梨果实细胞分裂期基因表达水平的内参基因。【方法】分别以杜梨、香梨和鸭梨梨花露红期、盛花期的花托和授粉后10、20、30、40 d的梨果肉为研究材料,应用qRT-PCR技术,对TUB2、TUB1、TUB3、Actin1、Actin2、UBI、EF1α和GAPDH这8个内参基因在不同梨品种果实细胞分裂期的表达情况进行了分析,利用geNorm和NormFinder对候选内参基因的稳定性进行了分析评价;以FWL1为目标基因,验证了候选基因在同一花期不同梨品种之间表达的稳定性。【结果】Actin1在花期花托中的表达稳定性最高,TUB2在果期的表达稳定性最高,TUB2在总样本中的表达稳定性最高。以FWL1为目的基因对表达稳定性不同的TUB2、Actin1、Actin2和TUB1这4个候选基因的验证结果表明,以TUB2、Actin1和Actin2为内参基因时,FWL1在同一时期不同梨品种之间的表达模式基本一致,使用TUB1作为内参基因得到的FWL1在杜梨、香梨和鸭梨中的表达模式与应用TUB2作为内参基因时的表达模式完全不一致。【结论】Actin1是研究梨花期花托基因表达分析的首选内参基因;TUB2是研究果期果肉基因表达分析的首选内参基因,也是梨果实细胞分裂期基因表达分析的首选内参基因。

Analysis on expression stability of internal reference genes at cell division stage of pear fruits

【Objective】Fruit cell division stage is a key period for fruit quality regulation.qRT-PCR technology plays an important role in fruit quality research.Selection of internal reference genes in qRT-PCR technology is related to accuracy of detection result.The purpose of the study is screening out some suitable internal reference genes for gene expression analysis in fruits at cell division stage of pear fruit.【Method】Using tori(at preliminary blooming stage and full blooming stage),and sarcocarp(10 days,20 days,30 days and 40 days after pollination)of Pyrus betulifolia,P.sinkiangensis and P.bretschneideri as test materials,expression status of eight candidate internal reference genes(TUB2,TUB1,TUB3,Actin1,Actin2,UBI,EF1αand GAPDH)in different cultivars of pear fruits were analyzed at cell division stage of pear fruit by the aid of qRT-PCR technology,and then their stability was evaluated by using GeNorm software and NormFinder software.Expression stability of candidate genes during the same blooming stage of different pear cultivars was verified by using FWL1 as target gene.【Result】Actin1 is the most stable reference gene in torus at blooming stage,TUB2 is the most stable reference gene at fruit stage,and TUB2 is the most stable reference gene in total samples.Very results of expression stability of four candidate reference genes(TUB2,Actin1,Actin2 and TUB1)with FWL1 as target gene show that expression patterns of FWL1 in different pear cultivars during the same stage are basically coincident with TUB2,Actin1 and Actin2 as internal reference genes.While expression patterns of FWL1 in P.betulifolia,P.sinkiangensis and P.bretschneideri with TUB1 as internal reference gene are completely different from that with TUB2 as internal reference gene.【Conclusion】Actin1 is the preferred internal reference gene for gene expression analysis in pear torus at blooming stage.TUB2 is the preferred internal reference genes for gene expression analysis in sarcocarp at pear fruit stage and fruit cell division stage.