Accurate Reflection of Hepatopancreas Antioxidation and Detoxification in Procambarus Clarkii During Virus Infection and Drug Treatment: Reference Gene Selection, Evaluation and Expression Analysis

Hepatopancreas as the main organ detoxifying xenobiotics with higher detoxifying and antioxidant enzymes activities, could best reflected the status of antioxidant defence against virus infection in the organisms. Catalase (CAT) and glutathione S-transferase (GST) were two important enzymes associated with hepatopancreas antioxidation and detoxification. To obtain the accurate expression levels of CAT and GST gene which reflect the status of hepatopancreas antioxidation and detoxification, two or more reference genes expressed stably under different conditions should be selected for RT-qPCR analysis. In this study, eight candidate reference genes were investigated using RefFinder program to screen the most stable reference gene during virus (white spot syndrome virus, WSSV) infection and drug (matrine) treatments in Procambarus clarkii, the relative expression levels and the activities of CAT and GST in hepatopancreas were detected and compared to verify the validity of selected reference genes. The results showed that taken different tissues together, EIF and EF1 alpha were the most stable and unstable reference genes respectively in P. clarkii no matter under different WSSV infection periods (0, 24, 48, 72 h) or different matrine treatment methods according to RefFinder analysis. The optimal number of reference genes in hepatopancreas was two using geNorm analysis, i.e. EIF and TBP, which were top two most stable reference genes in hepatopancreas. Moreover, based on the comparison between the relative expression levels and the activities of CAT and GST, EIF and TBP as reference genes were proved to be necessary and practicable to evaluate hepatopancreas antioxidation during WSSV infection and matrine treatments. Besides, the optimal number of reference genes in other P. clarkii tissues was also two genes, i.e. EIF and TUB in intestine, EIF and EF1 alpha in muscle, EIF and GAPDH in gills, EIF and TBP in brain, TBP and UB in gonads. Our study was helpful to acquire the accurate data of antioxidation and detoxification-related and immune-related gene expression during virus infection and drug treatments in crayfish, especially in hepatopancreas tissues