【摘要】 金毛狗(Cibotium barometz)是一种经济价值较高的药用蕨类植物,其粗大的根状茎是制作中药狗脊的原材料。加强对金毛狗的科学研究可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的常用方法,而选择合适的内参基因是该技术的关键环节之一。目前,金毛狗相关基因功能研究较少,无合适的内参基因可用。该研究从金毛狗转录组数据中筛选出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组内参基因对金毛狗10个组织部位的Ct值,利用RefFinder和Normfinder软件筛选出最佳内参基因CbUBC4和CbEF1A。对于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。研究机械损伤和水淹条件下基因的表达变化推荐用CbUBC4和CbUBC28作为内参基因,通过检测机械损伤和水淹条件下一些快速响应基因的表达量变化,进一步证实了CbUBC4和CbUBC28的稳定性。研究结果为金毛狗不同组织部位基因功能研究提供了合适的内参基因。更多还原

【Abstract】 INTRODUCTION: Cibotium barometz is a medicinal fern species with high economic value, and Chinese people produce the traditional medicinal 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. Quantitative real-time PCR(qRT-PCR) 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 few validated reference genes are 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 qRT-PCR to analyze gene expression patterns in C. barometz tissues, it is essential to prevalidate the stability of reference genes within the novel experimental system. This validation process ensures the accuracy and reliability of the 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 the template gradient dilution method, and eight pairs of reference gene primers with high amplification efficiency were screened out. The cycle threshold values of these eight reference genes in ten tissues of C. barometz were measured using the qRT-PCR method; the software RefFinder and Normfinder determined that the best reference genes were CbUBC4 and CbEF1A, respectively. 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 qRT-PCR assays. 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.更多还原