低温弱光胁迫下茄子幼苗实时荧光定量PCR内参基因的筛选与评价

    Screening and Evaluation of Reference Genes in Eggplant Seedlings by Real-time Fluorescence Quantitative PCR Under Low Temperature and Weak Light Stress

    • 摘要: 为了探索茄子Solanum melongena L.的基因表达模式,利用qRT-PCR方法分析了茄子ACT、EF1α、TUA、TUB、GAPDH、eIF、UBQ、UBI3、PP2A、CYP、RPL28、L25、SAND、TBP、DNAJ、APRT、EXP、CAC、HSP20CysPro 20个候选内参基因mRNA的表达差异情况,并运用GeNorm、NormFinder和BestKeeper 3种计算方法评价茄子20个候选内参基因在低温、弱光、低温弱光叶片样品中的表达稳定性。结果表明:茄子20个候选内参基因荧光定量引物扩增效率(E)数值在96.8%~117.6%,相关系数(R2)介于0.968 8~0.999 9,扩增效率良好,扩增反应具有高度的专一性,引物能特异性扩增,特异性好,均为单峰,样品间扩增曲线重复性强,可用于qRT-PCR扩增。茄子20个候选内参基因CT值表达丰度分析,发现茄子20个候选内参基因平均CT值在19.76~31.20。3种计算方法的综合评价分析结果显示,TBP基因在所有样本中为最稳定的内参基因。研究可为茄子低温、弱光和低温弱光胁迫下基因特异性表达研究提供了合适的内参基因。

       

      Abstract: In order to explore the gene expression pattern of Solanum melongena L., the qRT-PCR method was used to analyze the expression differences of mRNA in the 20 candidate reference genes of eggplant, including ACT, EF1α, TUA, TUB, GAPDH, eIF, UBQ, UBI3, PP2A, CYP, RPL28, L25, SAND, TBP, DNAJ, APRT, EXP, CAC, HSP20 and CysPro. The three calculation methods, including GeNorm, NormFinder and BestKeeper, were used to evaluate the expression stability of 20 candidate reference genes in eggplant samples at low temperature, weak light and low temperature and weak light stress. The results showed that the fluorescence quantitative primer amplification efficiency (E) values of 20 candidate reference genes in eggplant ranged from 96.8% to 117.6%, and the correlation coefficient (R2) was between 0.968 8−0.999 9, indicating that the amplification efficiency was good, and the amplified reaction was highly specific. The primers could be specifically amplified, with good specificity, all of which were unimodal. The amplification curves among the samples had strong repeatability, which could be used for qRT-PCR amplification. By analyzing the expression abundance of CT values of 20 candidate reference genes in eggplant, it was found that the average CT values of 20 candidate reference genes in eggplant ranged from 19.76 to 31.20. The comprehensive evaluation results of the three calculation methods showed that the TBP gene was the most stable reference gene in all the samples. The study could provide a suitable reference gene for the study of gene-specific expression in eggplant under low temperature stress, weak light stress and low temperature and weak light stress.

       

    /

    返回文章
    返回