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研究选取黄花蒿的Actin,18S rRNA,PAL,GAPDH,CPR作为候选内参基因,设计特异性引物,通过实时荧光定量PCR,利用geNorm,NormFinder,BestKeeper,Delta CT以及RefFinder软件和方法对5种内参基因在不同浓度镉处理下黄花蒿叶片中的表达稳定性进行分析,为黄花蒿基因差异表达研究提供可靠的内参基因,确保黄花蒿基因表达分析结果的可靠性。结果表明在不同浓度镉处理下,黄花蒿叶片中候选内参基因的表达稳定性存在差异,综合分析得出候选内参基因表达稳定性顺序依次为Actin>18S rRNA>PAL>GAPDH>CPR。因此,在黄花蒿基因差异表达分析中,可以考虑选取Actin,18S rRNA,PAL作为内参基因,采用多内参校正结果。此外,研究还发现同浓度镉处理下黄花蒿叶片中的候选内参基因表达稳定性也存有差异,这说明即使在同一处理条件下也有必要进行内参基因的筛选。总之,该研究首次提供了在不同浓度镉处理下黄花蒿叶片中比较理想的内参基因,也为其他条件下黄花蒿的基因表达分析提供了参考。
In the present study, Actin, 18S rRNA, PAL, GAPDH and CPR were selected as candidate reference genes, and specific primers were designed. Real-time quantitative PCR was performed using real-time PCR, geormot, The stability of expression in the leaves of Artemisia annua L.under different concentrations of cadmium was analyzed to provide a reliable internal control gene for the differential expression of Artemisia annua and to ensure the reliability of the gene expression analysis of Artemisia annua. The results showed that there were differences in the expression stability of candidate reference genes in the leaves of Artemisia annua L. under different concentrations of cadmium treatment. The order of the stability of candidate genes was Actin> 18S rRNA> PAL> GAPDH> CPR. Therefore, we can consider Actin, 18S rRNA and PAL as internal reference genes in the differential expression analysis of Artemisia annua, and adopt the results of multiple internal reference calibration. In addition, the study also found that the same concentration of cadmium treatment of Artemisia annua leaf candidate gene expression stability is also different, indicating that even under the same processing conditions are also necessary for the screening of the reference gene. In conclusion, this study, for the first time, provides an ideal reference gene for leaves of Artemisia annua treated with different concentrations of cadmium and also provides a reference for the gene expression analysis of Artemisia annua under other conditions.