ABSTRACT
Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.
Subject(s)
Aconitum , Gene Expression Profiling , Genes, Plant/genetics , Real-Time Polymerase Chain Reaction , Reference Standards , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Objective To establish an acute yunaconitine poisoning rat model with a single oral administration and to determine the contents of yunaconitine in rat tissues by UPLC-MS/MS method, then investigate the distribution of yunaconitine in rats. Method The rats were randomly divided into three groups and were intragastrically administered a single dose of 2.2mg/kg,1.1mg/kg,0.7mg/kg yunaconitine, respectively.. The rats were killed 2h later, the stomach tissue, intestine tissue, liver tissue, pancreas tissue, kidney tissue, lung tissue, spleen tissue, heart tissue, bladder tissue, testis tissue, brain tissue and heart blood samples were collected. The contents of yunaconitine in the biological materials were determined by UPLC-MS/MS method after the biological samples extracted by liquid-liquid extraction. Result A rat model of the yunaconitine poisoning was made with a single dose of 1.1mg/kg, the concentrations of yunaconitine displayed in the organs with the following order:stomach, small intestine, liver, pancreas, kidney, lung, spleen, heart, bladder, testis, heart blood and brain. Conclusion Yunaconitine was widely distributed in rats, especially the levels in the stomach, small intestine and liver were the highest. The conclusion provides a basis for the selection of test materials for the poisoning of Aconitum vilmorinianum Kom.