Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

逆转录定量PCR（Reverse Transcription quantitative PCR，RT-qPCR）凭借其高灵敏度与良好的重复性，是基因表达谱分析的关键技术之一。然而，其结果的可靠性高度依赖于数据标准化流程——即通过将目的基因的表达谱与组成型表达的内参基因的表达谱进行比对来完成标准化。尽管该技术已广泛应用于果实采后实验，但针对此类实验条件下内参基因的转录稳定性研究仍不够充分。为此，我们针对此类实验条件，对三种常用内参基因——肌动蛋白（ACTIN，MdACT）、蛋白质二硫键异构酶（PROTEIN DISULPHIDE ISOMERASE，MdPDI）以及泛素结合酶E2（UBIQUITIN-CONJUGATING ENZYME E2，MdUBC）——以及两种新型候选内参基因——组蛋白H1（HISTONE 1，MdH1）和核小体组装蛋白1（NUCLEOSSOME ASSEMBLY 1 PROTEIN，MdNAP1）——的转录谱进行了检测。我们共开展五组实验以分析这些基因的表达谱：其中三组覆盖采后阶段，另外两组分别涵盖发育阶段与组织空间分布阶段。我们采用四款不同的软件工具：BestKeeper、NormFinder、geNorm与DataAssist，对基因的转录稳定性进行了比较分析。除BestKeeper外，其余三款软件得到的基因转录稳定性排序结果高度一致。经典内参基因MdUBC在所有受试实验条件下均位列转录稳定性最高的基因行列。新型候选内参基因MdH1的转录本积累谱在所有受试条件下均保持稳定，尤其在采后相关实验中表现更为突出。综上，本研究为苹果采后实验提供了一款新型内参基因，同时也强调了在目标实验条件下验证内参基因转录谱的重要性。