Xu Bi-Xia, Guo Qiao-Sheng, Zhu Zai-Biao, Zhang Jun-Xia
Institute of Chinese Medicinal Materials,Nanjing Agricultural University Nanjing 210095,China.
Zhongguo Zhong Yao Za Zhi. 2021 Feb;46(4):938-943. doi: 10.19540/j.cnki.cjcmm.20201120.104.
Amana edulis is a traditional Chinese medicinal plant with low propagation coefficient. In recent years, the increasing demands of A. edulis lead to a shortage of its wild resources. In order to analyze the expression of related functional genes in A. edulis, the selection of suitable internal reference genes is crucial to improve the accuracy of experimental results. Eight genes(ACT, TUA, CYP, GAPDH, UBQ, UBI, EF1a, UBC)were chosen as candidate reference genes based on the RNA-Seq. Real-time fluorescence quantitative technique was used to detect the expression level of candidate internal reference genes in different organs(bulb, leaf, flo-wer) and stolons at different development stages of A. edulis. Then GeNorm, NormFinder, BestKeeper softwares and RefFinder website were used for a comprehensive analysis of the expression stability of the candidate genes.The results showed that among the 8 candidate reference genes, the variation range of Ct value of UBC was the smallest, and the expression level was stable, which was suitable for an reference gene. GeNorm and NormFinder software analysis showed that UBC and UBI were the optimal reference genes. BestKeeper analysis showed that CYP and UBC expression were relatively stable. Comprehensive evaluation of RefFinder website showed that UBC and UBI were the most stable genes, and ACT displayed the lowest stability in all software evaluation, indicating UBC and UBI were suitable for reference genes. Additionally, the most stable UBC, UBI and the most unstable ACT were used as internal reference genes to detect the expression of GBSS gene in A. edulis, and expression pattern of GBSS gene was the same under the calibration of UBC and UBI. The expression data of GBSS gene confirmed that UBC and UBI genes were reliable for A. edulis qRT-PCR as internal reference genes. The results would benefit future studies on related gene expression of A. edulis.
天麻是一种繁殖系数低的传统中药材。近年来，天麻需求的不断增加导致其野生资源短缺。为分析天麻中相关功能基因的表达，选择合适的内参基因对于提高实验结果的准确性至关重要。基于RNA-Seq选择了8个基因（ACT、TUA、CYP、GAPDH、UBQ、UBI、EF1a、UBC）作为候选内参基因。采用实时荧光定量技术检测候选内参基因在天麻不同器官（鳞茎、叶片、花）及不同发育阶段匍匐茎中的表达水平。然后利用GeNorm、NormFinder、BestKeeper软件和RefFinder网站对候选基因的表达稳定性进行综合分析。结果表明，在8个候选内参基因中，UBC的Ct值变化范围最小，表达水平稳定，适合作为内参基因。GeNorm和NormFinder软件分析表明，UBC和UBI是最佳内参基因。BestKeeper分析表明，CYP和UBC的表达相对稳定。RefFinder网站综合评价表明，UBC和UBI是最稳定的基因，ACT在所有软件评价中稳定性最低，表明UBC和UBI适合作为内参基因。此外，以最稳定的UBC、UBI和最不稳定的ACT作为内参基因检测天麻中GBSS基因的表达，在UBC和UBI的校正下GBSS基因的表达模式相同。GBSS基因的表达数据证实，UBC和UBI基因作为天麻qRT-PCR的内参基因是可靠的。该结果将有助于未来天麻相关基因表达的研究。
