不同光周期和温度处理下桂花内参基因的筛选

光周期和温度处理能够影响桂花Osmanthus fragrans的基因表达水平和花芽分化过程，通过实时荧光定量聚合酶链式反应（qRT-PCR）分析基因表达已成为研究这些过程机制的重要手段，而筛选出适合光周期和温度处理的内参基因在研究桂花分子机制中尤为重要。为了得到不同光周期和温度处理下桂花中稳定的内参基因，以桂花品种‘佛顶珠’O.fragrans‘Foding Zhu’当年生枝条的第2或第3对幼叶为材料，以OfACT，OfEF1α，OfIDH，OfRAN1，OfTUB，OfUBC2和Of18S等7个基因作为候选内参基因，利用geNorm，NormFinder和BestKeeper 3个软件对候选内参基因的表达稳定性进行比较分析；以昼夜节律相关基因GIGANTEA（GI）对筛选出的最佳内参基因进行验证。结果表明:在不同处理条件下，桂花叶片中OfRAN1和OfIDH为最佳内参基因，Of18S的稳定性最差（geNorm分析结果显示OfRAN1和OfIDH的稳定值为0.347，而Of18S的稳定值为0.601；NormFinder显示OfRAN1和OfIDH的稳定值为0.135和0.206，而Of18S为0.474；BestKeeper显示OfRAN1的稳定值为0.80，OfIDH为0.133，而Of18S为0.474）；GI基因的相对表达水平分析表明（昼夜节律基因GI的表达模式为在白天累积并在夜间下降），在7个内参基因及OfRAN1和OfIDH的基因组合中，使用OfRAN1和OfIDH的内参基因组合可获得GI基因更为精确的基因表达结果。综上所述，OfRAN1和OfIDH内参基因组合是桂花在不同光周期和温度处理下最佳内参基因组合。这为桂花分子机制研究奠定基础。

Screening reference genes of Osmanthus fragrans with differing photoperiod and temperature treatments

Photoperiod and temperature treatment can affect the gene expression level and flower bud differentiation process of Osmanthus fragrans. Analysis of gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) has become an important means to study the mechanism of these processes and it is especially important to screen for an internal reference gene suitable for photoperiod and temperature treatment. To screen reference genes in O. fragrans using different photoperiod and temperature treatments, the young leaves of O. fragrans 'Foding Zhu' were used as materials. The O. fragrans treatment conditions were 14 h D (light)/10 h N (dark) +26℃, 14 h D/10 h N +19℃, 10 h D/14 h N +26℃ and 10 h D/14 h N +19℃, light intensity 100%, relative humidity 60%-80%. After treatment for 40 days, the second or third young leaves of the current shoots were taken every 4 h from 8:00 in the morning frozen in the liquid nitrogen and stored at -80℃. The seven genes, including OfACT, OfEF1α, OfIDH, OfRAN1, OfTUB, OfUBC2, and Of18S, were chosen as candidate reference genes. qRT-PCR was used to screen the reference genes, and GeNorm, NormFinder, and BestKeeper were used to analyze the stability of the candidate reference genes and screen out the best ones. Finally, the circadian rhythm gene GIGANTEA (GI) was used to verify the best reference genes. Results showed that OfRAN1 and OfIDH had the highest stability(GeNorm showed that the stable value of OfRAN1 and OfIDH were 0.347, while Of18S was 0.601; NormFinder showed that the stable value of OfRAN1 and OfIDH were 0.135 and 0.206, while Of18S was 0.474; BestKeeper showed that the stable value of OfRAN1 was 0.800 and OfIDH was 0.133, while Of18S was 0.474), which meant they were the best reference genes for the different conditions and Of18S had the worst stability(The greater the stability value, the worse the stability). The relative expression level of GI balanced by seven reference genes and the genetic combinations of OfRAN1 and OfIDH meant more accurate expression results(The expression pattern of circadian rhythm gene GI is accumulated during the day and falls at night). In summary, the genetic combinations of OfRAN1 and OfIDH were the best combination of reference genes used to study the molecular mechanism of O. fragrans under different photoperiod and temperature treatments.