苯酚胁迫下多刺裸腹溞实时定量PCR内参基因的筛选

REFERENCE GENE SELECTION FOR QUANTITATIVE REAL-TIME PCR NORMALIZATION IN MOINA MACROCOPA EXPOSED TO PHENOL

为研究苯酚胁迫下多刺裸腹溞(Moina macrocopa)相关基因的表达情况,筛选用于实时定量PCR分析的最佳内参基因。利用内参基因表达的cycle threshold(Ct值)非参数检验、GeNorm、NormFinder和Best-Keeper四种方法对β-actin、16S rRNA和12S rRNA进行分析,筛选出苯酚胁迫下多刺裸腹溞表达相对稳定的内参基因。结果显示,从Ct值初步判定不同浓度苯酚胁迫后,多刺裸腹溞体内β-actin、16S rRNA和12S rRNA均可稳定表达,且稳定性顺序为:16S rRNA>12S rRNA>β-actin,从GeNorm软件分析显示内参基因的稳定性顺序为:16S rRNA=β-actin>12S rRNA,NormFinder和Bestkeeper分析显示的稳定性顺序均为:16S rRNA>β-actin>12S rRNA。基于以上四种方法对三个候选内参基因的筛选,确定了16S rRNA作为多刺裸腹溞实时定量PCR的最佳内参基因,有助于提高qRT-PCR分析的准确性,为进一步研究苯酚胁迫多刺裸腹溞后目的基因功能的表达提供了基础。

To search suitable reference genes for normalization of quantitative Real-Time PCR(qRT-PCR)in Moina macrocopa,we tested three reference genes ofβ-actin,16S rRNA and 12S rRNA by using four analysis methods:(1)expression level of the genes(cycle threshold value);(2)GeNorm;(3)NormFinder;and(4)BestKeeper.The results showed that the Ct values of theβ-actin,16S rRNA and 12S rRNA genes remained unchanged in M.macrocopa treated with different concentrations of phenol,and the order of the stability was 16S rRNA>12S rRNA>β-actin.Ge-Norm analysis revealed that the order of the stability was 16S rRNA=β-actin>12S rRNA.Both NormFinder and Bestkeeper software analysis demonstrated that the order of the stability was 16S rRNA>β-actin>12S rRNA.These results indicated that 16S rRNA was the best-fit reference gene for qRT-PCR in M.macrocopa,at least under phenol treatment,which provide useful information for future functional investigations of target gene expressions in M.macrocopa in response to environmental stress.