Key Laboratory of Forest Genetics & Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, China.
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China.
Genes (Basel). 2021 May 21;12(6):791. doi: 10.3390/genes12060791.
has become one of the main timber afforestation species in subtropical high-altitude areas of China due to its fast growth, good material quality, and strong adaptability, showing broad application prospects. Quantitative real-time PCR (qRT-PCR) is the most accurate and widely used gene expression evaluation technique, and selecting appropriate reference genes (RGs) is essential for normalizing qRT-PCR results. However, suitable RGs for gene expression normalization in have not been reported. Here, we tested the expression stability for 12 RGs in under various experimental conditions (simulated abiotic stresses (cold, heat, drought, and salinity) and hormone treatments (methyl jasmonate, abscisic acid, salicylic acid, and gibberellin) and in different tissues (stems, tender needles, needles, cones, and seeds) using four algorithms (delta Ct, geNorm, NormFinder, and BestKeeper). Then, geometric mean rankings from these algorithms and the RefFinder program were used to comprehensively evaluate RG stability. The results indicated , , , and as good choices for studying gene expression. qRT-PCR analysis of the expression patterns of three target genes ( and ) further verified that the selected RGs were suitable for gene expression normalization. This study provides an important basis for gene expression standardization and quantification.
因其生长迅速、材质优良、适应性强，已成为中国亚热带高海拔地区主要的用材造林树种之一，具有广阔的应用前景。实时荧光定量 PCR（qRT-PCR）是最准确、应用最广泛的基因表达评估技术，选择合适的内参基因（RGs）对于 qRT-PCR 结果的归一化至关重要。然而，尚未报道适用于 基因表达归一化的合适 RG。在这里，我们使用 delta Ct、geNorm、NormFinder 和 BestKeeper 这四种算法，在不同的实验条件（模拟非生物胁迫（冷、热、干旱和盐度）和激素处理（茉莉酸甲酯、脱落酸、水杨酸和赤霉素）以及不同组织（茎、嫩针、针、球果和种子）下，测试了 12 个 RG 在 中的表达稳定性。然后，这些算法和 RefFinder 程序的几何平均值排名用于综合评估 RG 稳定性。结果表明，在研究 基因表达时， 、 、 和 是较好的 RG 选择。对三个靶基因（ 和 ）表达模式的 qRT-PCR 分析进一步验证了所选 RG 适用于基因表达归一化。本研究为 基因表达标准化和定量提供了重要依据。
