不同光强条件下紫花地丁RT-qPCR内参基因的筛选及验证  

Screening and validation of RT-qPCR reference genes of Viola yedoensis under different light intensities

作　　者：杨培芳 史红专[1] 郭巧生[1] 赵坤 刘丽[1] YANG Pei-fang;SHI Hong-zhuan;GUO Qiao-sheng;ZHAO Kun;LIU Li(Institute of Chinese Medicinal Materials,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学中药材研究所,江苏南京210095

出　　处：《中国中药杂志》2021年第21期5620-5626,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(41471243)。

摘　　要：筛选紫花地丁在不同光强下稳定表达的内参基因,为紫花地丁相关分子研究提供参考。该实验通过RT-qPCR技术检测不同光强处理的紫花地丁不同组织中11个候选内参基因表达量,利用Ge Norm、NormFinder和Best Keeper软件以及RefFinder网站综合评估内参基因,通过CAT1验证内参基因的稳定性,最终确定理想内参基因。结果表明,CYP、Actin、SAMDC基因Ct变化范围最小,表达最稳定;GeNorm算法认为CYP、SAMDC、Actin为最适内参基因;NormFinder显示α-TUB表达最稳定;Best Keeper计算CYP、Actin、SAMDC为最佳内参基因;RefFinder评估SAMDC、CYP、α-TUB、Actin稳定性最好,而GAPDH稳定性最差。紫花地丁CAT1在以SAMDC、CYP、Actin为内参基因时表达趋势相同,以GAPDH为内参时表达趋势有较大差异。综上,SAMDC、CYP、Actin最适合作为紫花地丁在不同光强下的理想内参基因。Screening the reference genes that were stably expressed under different light intensities for Viola yedoensis could provide reference for the related molecular research. In this study, 11 candidate reference genes were detected by RT-qPCR for tissues of V. yedoensis treated with different light intensities. Ge Norm, Norm Finder, Best Keeper, and Ref Finder website were used to comprehensively evaluate the reference genes, and verify the stability of the reference gene based on CAT1. Finally, the ideal reference gene was determined. The results showed that CYP, Actin, and SAMDC had small Ct value ranges and stable expression. Ge Norm demonstrated that CYP, SAMDC, and Actin were suitable reference genes. Norm Finder showed that the expression of α-TUB was the most stable. Best Keeper recommended CYP, Actin, and SAMDC as reference genes. Ref Finder assessed that SAMDC, CYP, α-TUB, and Actin had better stability, while GAPDH had the worst stability. The expression trend of CAT1 gene was consistent when calibrated with SAMDC, CYP, and Actin, while it was quite different from that calibrated with GAPDH. In summary, SAMDC, CYP, and Actin can be used as ideal reference genes for the gene expression profiling of V. yedoensis under different light intensities.

关 键 词：紫花地丁 实时荧光定量PCR 内参基因 Ge Norm Norm Finder 

分 类 号：R284[医药卫生—中药学]