金毛狗(Cibotium barometz)是一种高经济价值药用蕨类植物，其粗大的根状茎是制备中药狗脊的原材料。从野生植物资源保护和中药资源开发利用考虑，宜加强对金毛狗的科学研究，帮助缓解野生资源保护与中药产业发展之间的矛盾。荧光定量PCR技术是研究植物基因功能常用的方法，而合适的内参基因是该技术的关键之一。目前，金毛狗相关的基因功能研究较少，没有合适的内参基因可用。该研究从前期的金毛狗转录组数据中筛选了12个常用的植物内参基因，通过计算扩增效率，确定8组可用的内参基因引物。根据8组内参基因针对金毛狗10个组织部位的Ct值，采用RefFinder和Normfinder软件筛选出最佳的内参基因是CbUBC4和CbEF1A。若研究金毛狗不同发育时期根状茎中基因的表达变化，CbEF1A和CbUBC4是较合适的内参基因。机械损伤和水淹条件下推荐使用内参基因CbUBC4和CbUBC28，通过检测机械损伤和水淹胁迫条件下一些快速响应基因的表达量变化，进一步证实CbUBC4和CbUBC28的稳定性。该研究为金毛狗不同组织部位基因功能的研究提供了一些合适的内参基因，助力金毛狗分子生物学相关领域的研究工作。

INTRODUCTION: Cibotium barometz is a medicinal fern species with high economic value, Chinese people produce the traditional medicine herb “Gou-ji” using its robust rhizome. To balance the conservation of wild plant resources with the sustainable development of herbal medicine, much more effort is needed in scientific research on C. barometz, which might be crucial to mitigate conflicts between wild resource protection and the development of the traditional medicine industry. Real-time quantitative PCR (RT-qPCR) is a commonly used method for characterizing plant gene molecular function, and an appropriate reference gene is pivotal for this technique. Currently, limited literature focuses on the gene function studies for C. barometz, and little validated reference genes is available. 

RATIONALE: Previous studies have demonstrated that the expression stability of reference genes is influenced by multiple factors, including species, tissue type, developmental stage, stress treatment, and sequence specificity. Consequently, distinct reference genes are required for different research subjects. When employing RT-qPCR to analyze gene expression pattern in C. barometz tissues, it is essential to pre-validate the stability of reference genes within the novel experimental system. This validation process ensures the accuracy and reliability of experimental results by maintaining proper data normalization. 

RESULTS: In this study, 12 commonly used plant reference genes were identified as candidate genes from transcriptome data of C. barometz rhizome. The amplification efficiency of candidate reference genes was calculated by template gradient dilution method, and eight pairs of reference gene primers with high amplification efficiency were screened out. The Ct values of these eight reference genes in ten tissues of C. barometz were measured using RT-qPCR method; the software RefFinder and Normfinder determined that the best reference genes are CbUBC4 and CbEF1A, respectively. If studying the gene expression patterns of C. barometz at different developmental stages, it was suggested that CbEF1A and CbUBC4 should be used as optimal reference genes. The reference genes CbUBC4 and CbUBC28 were recommended under mechanical injury and waterlogging conditions. 

CONCLUSION: CbUBC4, CbEF1A and CbUBC28 were validated as stable reference genes in C. barometz across distinct experimental contexts, demonstrating their suitability for normalization in gene expression studies for RT-qPCR assay. From a methodological rigor perspective, we recommended that the stability of reference genes should be evaluated prior to conducting qRT-PCR experiments, particularly when analyzing samples from diverse tissues or distinct developmental stages of a specific tissue.