Melaleuca bracteata F. Muell. is among the most significant aromatic plants belonging to the Myrtaceae family, and its essential oil has various biological activities. However, the key enzymes involved in essential oil biosynthesis and their expression patterns are not clearly understood. Appropriate reference genes are required for quantitative real-time polymerase chain reaction (qRT-PCR), sensitive for gene expression studies, to obtain reliable results. In this study, 14 candidate genes, namely ACT, 18S, α-TUB, β-TUB, GAPDH, EF1-α, UBQ, UBC, CYP, EIF-4A, DNAJ, PP2A, TIP41, and UBX, were selected from the M. bracteata transcriptome datasets for analysis under abiotic stresses and hormonal stimuli. The stability of these genes was analyzed using three widely used software programs (geNorm, NormFinder, and BestKeeper). Their results were integrated into a comprehensive ranking by geometric mean. According to the study results, TIP41 and CYP were the most stable genes for abiotic stresses, while TIP41 and UBQ were the most stable genes for hormonal stimulation. Further, TIP41 and CYP were the most stable reference genes across all tested samples, whereas ACT was the least stable. Based on the expression of two target genes (COMT1 and COMT2, which are involved in methyleugenol biosynthesis), the suitability of the selected reference genes for qRT-PCR normalization was further verified. The study results could offer a foundation for gene expression analysis and functional studies in M. bracteata and other Melaleuca species.

Melaleuca bracteata F. Muell。是桃金娘科最重要的芳香植物之一，其精油具有多种生物活性。然而，参与精油生物合成的关键酶及其表达模式尚不清楚。定量实时聚合酶链反应 (qRT-PCR) 需要适当的参考基因，对基因表达研究敏感，以获得可靠的结果。本研究筛选出14 个候选基因，即 ACT、18S、α-TUB、β-TUB、GAPDH、EF1-α、UBQ、UBC、CYP、EIF-4A、DNAJ、PP2A、TIP41 和UBX 。大苞米用于在非生物胁迫和激素刺激下进行分析的转录组数据集。使用三个广泛使用的软件程序（geNorm、NormFinder 和 BestKeeper）分析了这些基因的稳定性。他们的结果按几何平均数综合成一个综合排名。根据研究结果，TIP41和CYP是非生物胁迫最稳定的基因，而TIP41和UBQ是激素刺激最稳定的基因。此外，TIP41和CYP是所有测试样本中最稳定的参考基因，而ACT是最不稳定的。基于两个靶基因（COMT1和参与甲基丁香酚生物合成的COMT2 ），进一步验证了所选参考基因对 qRT-PCR 标准化的适用性。该研究结果可为M. bracteata和其他Melaleuca物种的基因表达分析和功能研究提供基础。