地榆醇提物对对虾致病菌VPAHPND生长影响及最适内参基因的筛选

Effects on the Growth of Shrimp Pathogen VPAHPND and Selection of Suitable Reference Genes Under Different Concentrations of Sanguisorba officinalis L. Alcoholic Extracts

中文关键词: 急性肝胰腺坏死病(AHPND)  副溶血弧菌  地榆  内参基因  qRT-PCR

英文关键词: Acute hepatopancreatic necrosis disease (AHPND)  Vibrio parahaemolyticus  Sanguisorba officinalis L.  Reference genes  qRT-PCR

中文摘要:

      为筛选适用于研究地榆(Sanguisorba officinalis L.)对对虾急性肝胰腺坏死病致病株副溶血弧菌(Acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus, VPAHPND)抑制机理的内参基因，本文探究了地榆醇提物胁迫下VPAHPND生长的变化，并利用qRT-PCR技术探究了该条件下VPAHPND中 6种常见内参基因(rec A、pvs A、pvu A、gapdh、16S rRNA和rpo S)的表达情况，利用GeNorm、Norm Finder、Best Keeper、Delta CT以及Ref Finder这5种方法对其表达稳定性进行了评估和筛选。结果显示，地榆醇提物能抑制VPAHPND的生长，且抑制效果与浓度呈正相关。在不同浓度地榆醇提物处理条件下，这 6种内参基因扩增产物特异性良好，其中，Ct值变化差异最小的为16S rRNA (CV=3.88)，变化差异最显著的为pvs A (CV=12.53)。5种方法对6种内参基因稳定性排序略有差异，其中，GeNorm给出的稳定性排序为rpo S = 16S rRNA > gapdh > rec A> pvu A > pvs A；Norm Finder结果为gapdh > rpo S > pvu A > 16S rRNA > rec A > pvs A；Best Keeper结果为16S rRNA > rpo S > gapdh > rec A > pvu A > pvs A；Delta Ct结果为gapdh > rpo S > pvu A > 16S rRNA > rec A > pvs A；Ref Finder综合排名结果为rpo S > gapdh > 16S rRNA > pvu A > pvs A > rec A。本研究根据配对变异系数结果，最终推荐同时使用rpo S和gapdh作为该条件下内参基因。本研究为以地榆为核心药物的AHPND防控技术的建立和从基因表达角度研究地榆对VPAHPND的作用机理提供了基础。

英文摘要:

      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.