基于实时荧光定量PCR对镉处理下黄花蒿内参基因稳定性的分析  

Stability analysis of reference gene based on real-time PCR in Artemisia annua under cadmium treatment

作　　者：周良云[1] 莫歌 王升[1] 唐金富[1] 岳红[2] 黄璐琦[1] 邵爱娟[3] 郭兰萍[1] 

机构地区：[1]中国中医科学院中药资源中心,北京100700 [2]长春医学高等专科学校,吉林长春130031 [3]中国中医科学院中药研究所,北京100700

出　　处：《中国中药杂志》2014年第5期777-784,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81130070;81072989);国家中医药管理局行业科研专项(201107009)

摘　　要：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 this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and Ref- Finder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The resuhs showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of can- didate reference gene was Actin 〉 18S rRNA 〉 PAL 〉 GAPDH 〉 CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and muhiple internal control genes were adopted for results cali- bration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concen- trations of Cd were observed,which suggested that the screening of candidate reference genes was needed even under the same treat- ment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.

关 键 词：实时荧光定量PCR 黄花蒿 镉 内参基因 稳定性分析 

分 类 号：R284[医药卫生—中药学]