牛奶子实时定量PCR分析中内参基因的评价与验证(英文)

Evaluation and Validation of Potential Reference Genes for Quantitative Real-Time PCR Analysis in Elaeagnus umbellata

【目的】实时定量PCR结果的精确性在很大程度上取决于选择的内参基因的稳定性。通过评估候选管家基因的表达稳定性,筛选出用于牛奶子研究的最佳内参基因。【方法】设计简并引物从牛奶子中克隆12个潜在的内参基因片段,包括14-3-3、18S核糖体RNA(18SrRNA)、β-actin(Actin)、延长因子1-α(EF1-α)、真核起始因子4A(e IF4A)、3-磷酸甘油醛脱氢酶(GAPDH)、RNA聚合酶-Ⅱ(RPⅡ)、60S核糖体蛋白(RPL7)、翻译延长因子2(TEF2)、泛素连接酶E2(UBCE)、泛素(UBQ)和泛素延伸蛋白5(UBQ5)。采集牛奶子5个不同器官(根、茎、叶、花和红果)、4个成熟期的果实(绿果、黄果、深粉果和完全成熟的红果)、2种激素(脱落酸、赤霉素)处理4个时间点的绿果、热处理4个时间点的离体叶片、幼苗盐胁迫2个时间点的根茎叶,应用实时荧光定量PCR技术检测12个内参基因在各样品中的表达情况,采用基于CT值的ge Norm、Normfinder和Best Keeper及CT比较4种算法评价这些内参基因的稳定性,最终的排名则由Ref Finder算法产生。【结果】器官组稳定性好的前2位内参基因为UBCE和RPL7,果实成熟期组排名前2的为e IF4A和UBCE,激素处理组排名前2的为e IF4A和UBCE,非生物胁迫组排名前2的则为Actin和EF1-α,综合分析所有样品排名前3位的是e IF4A、RPL7和UBCE。分别用筛选出的稳定的e IF4A、RPL7、UBCE和不稳定的RPⅡ作为内参基因评价目的基因八氢番茄红素合成酶基因Eut Psy在果实成熟过程中的表达,结果显示分别以3个稳定的内参基因为单内参基因与以e IF4A同UBCE组合做内参基因所得到的结论一致,而RPⅡ给出的则不同。【结论】在应用荧光实时定量PCR技术研究牛奶子基因转录表达时,通常情况下e IF4A、RPL7和UBCE相对于其他9个候选内参基因更为可靠。研究结果为牛奶子及胡颓子属其他物种的基因表达分析奠定基础。

[Objective]The accuracy of quantitative real-time polymerase chain reaction （ qRT-PCR） analysis strongly depends on transcript normalization using stably expressed reference genes. The present study was conducted to evaluate the stability of candidate housekeeping genes and identify the most reliable gene or a set of genes to be used as reference genes in qPCR analysis of Elaeagnus umbellata.[Method]Twelve potential reference genes were selected among the most common reference genes reported in literature and their fragments were cloned by degenerate primers from E. umbellata, including 14-3-3 ,18 S ribosomal RNA gene （ 18 SrRNA ） ,β-actin （ Actin ） ,elongation factor 1-α （ EF1-α） ,eukaryotic initiation factor 4A （eIF4A）,glyceraldehyde-3-phosphate dehydrogenase （GAPDH）,RNA polymerase-Ⅱ （RPⅡ）,60S ribosomal protein L7 （ RPL7 ） ,translation elongation factor 2 （ TEF2 ） ,ubiquitin-conjugating enzyme E2 （ UBCE ） , ubiquitin （UBQ） and ubiquitin extension protein 5 （UBQ5）. Samples were collected from five types of organs （root, stem,leaf,flower and red fruit）,fruits at four different ripening stages （green,yellow,dark yellow and fully matured red fruit） ,green fruits at four time points after hormone ABA or GA3 treatments,detached leaves at four time points under 40 ℃,and three types of organs （ root,stem and leaf） of the seedlings treated with salt stress at three time points. The expression stability of these 12 genes was evaluated based on the CT values using four statistical algorithms including geNorm,Normfinder,BestKeeper,and the comparative ΔCT. Overall ranking of four sets aforementioned was generated using RefFinder software.[Result]UBCE and RPL7 were ranked as the two best reference genes for organ set,eIF4A and UBCE for fruit-ripening samples,eIF4A and UBCE for hormone treatment set,and Actin and EF1-α for abiotic stress set. When including the data obtained from all the 29 samples into the analysis,eIF4A,RPL7,and UBCE were identified as the top three reference candidates. The expression levels of phytoene synthase （ EutPsy） were further assessed during fruit ripening by using the top three reference genes in comparison to the worst one RPⅡ. When the three stable reference genes eIF4A,UBCE and RPL7 or the combination of top two eIF4A and UBCE were used for normalization,the trend of the relative transcript abundance of EutPsy were consistent. However,when the worst reference gene RPⅡwas employed for normalization,the expression profile was different.[Conclusion]eIF4A followed by RPL7 and UBCE were found to be more reliable than other nine genes for normalization purposes in measuring gene expression of E. umbellata. This was the first systematic analysis for the selection of superior reference genes for qRT-PCR in E. umbellata under different conditions,which benefits future studies on gene expression in E. umbellata and other species of the genus Elaeagnus.