Abstract:The identification of reference genes is critical for the establishment of sensitive and reproducible qRT-PCR-based assays. The current study was designed to explore the effects on acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strains (VPAHPND) on the growth of shrimp in the presence of different concentrations of Sanguisorba officinalis L. alcoholic extracts and to select the optimal reference genes suitable for the evaluation of the inhibitory effect of S. officinalis L. on VPAHPND. The expression of six common candidate reference genes (rec A, pvs A, pvu A, gapdh, 16S rRNA, and rpo S) of VPAHPND under stress induced by S. officinalis L. alcoholic extracts were detected by qRT-PCR. Data analysis was conducted using the GeNorm, Norm Finder, Best Keeper, Delta CT, and Ref Finder software packages. The results showed that S. officinalis L. alcoholic extracts had a strong inhibitory effect on V. parahaemolyticus. The amplicons of these six genes had good specificity under the stress induced by different concentrations of S. officinalis L. extract. The lowest variation in Ct value was found for 16S rRNA (CV=3.88), and the highest variation occurred in pvs A (CV=12.53). The stability of the six reference genes judged by the five methods was as follows: The stability order results were: rpo S = 16S rRNA > gapdh > rec A > pvu A > pvs A from GeNorm; gapdh > rpo S > pvu A > 16S rRNA> rec A > pvs A from Norm Finder; 16S rRNA > rpo S > gapdh > rec A > pvu A > pvs A from Best Keeper; and gapdh > rpo S > pvu A > 16S rRNA > rec A > pvs A from Delta Ct. The comprehensive ranking result from by Ref Finder was rpo S > gapdh > 16S rRNA > pvu A >pvs A > rec A. After consideration of the pairwise variations, it is recommended to use both rpo S and gapdh as reference genes in these conditions. It was also revealed that the stability of reference genes differed between different strains and under different experimental conditions. With the improved experimental accuracy requirements, screening and verification of the appropriate reference gene has become an essential part of the experimental methodology. The results provide a foundation to support the study of the inhibitory mechanism of S. officinalis L. on VPAHPND through the perspective of gene expression. It is of great significance for the establishment of AHPND-prevention technology using S. officinalis L. as the core drug.
