Abstract：
Trichomonas gallinae, a globally distributed protozoan parasite, significantly affects the pigeon-breeding industry. T. gallinae infection mainly causes yellow ulcerative nodules on the upper respiratory tract and crop mucosa of pigeons, impeding normal breathing and feeding and ultimately causing death. Real-time quantitative PCR (qPCR) is a crucial technique for gene-expression analysis in molecular biology. Reference-gene selection for normalization is critical for ensuring this technique\'s accuracy. However, no systematic screening or validation of T. gallinae reference genes has been reported. This study quantified the transcript levels of ten candidate reference genes in T. gallinae isolates with different genotypes and culture conditions using qPCR. Using the geNorm, NormFinder, and BestKeeper algorithms, we assessed these reference genes\' stabilities and ranked them using RankAggreg analysis. The most stable reference gene was tubulin beta chain (TUBB), while the widely used reference genes TUBG and GAPDH demonstrated poor stability. Additionally, we evaluated these candidate reference genes\' stabilities using the T. gallinae TgaAtg8 gene. On using TUBB as a reference gene, TgaAtg8\'s expression profiles in T. gallinae isolates with different genotypes remained relatively consistent under various culture conditions. Conversely, using ACTB as a reference gene distorted the data. These findings provide valuable reference-gene-selection guidance for functional gene research and gene-expression analysis in T. gallinae.
摘要：
鸡毛滴虫,一种全球分布的原生动物寄生虫，显著影响鸽子养殖业。鸡感染主要导致鸽子上呼吸道和作物粘膜上的黄色溃疡性结节，阻碍正常呼吸和进食，最终导致死亡。实时定量PCR（qPCR）是分子生物学中基因表达分析的关键技术。用于标准化的参考基因选择对于确保该技术的准确性至关重要。然而,目前还没有系统筛选或验证鸡木霉内参基因的报道。这项研究使用qPCR定量了具有不同基因型和培养条件的鸡木霉分离物中十个候选参考基因的转录水平。使用geNorm，NormFinder,和BestKeeper算法，我们评估了这些参考基因的稳定性，并使用RankAggreg分析对它们进行排名。最稳定的内参基因是微管蛋白β链(TUBB),而广泛使用的参考基因TUBG和GAPDH表现出较差的稳定性。此外,我们使用T.gallinaeTgaAtg8基因评估了这些候选参考基因的稳定性。在使用TUBB作为参考基因时，TgaAtg8在具有不同基因型的鸡木霉分离株中的表达谱在各种培养条件下保持相对一致。相反，使用ACTB作为参考基因扭曲了数据。这些发现为鸡鸡的功能基因研究和基因表达分析提供了有价值的参考基因选择指导。
