Isolation Of Eleutherococcus Senticosus And The Effects Of FPS,SS And SE Methylation On Its Content

Objectives 1 Explore the best extraction method of total saponins of Eleutherococcus senticosus.2 To study the DNA methylation of farnesyl pyrophosphate synthase,squalene synthase and squalene epoxidase gene promoters and their effects on the synthesis and accumulation of saponins.3 Excavation of adaptive evolution sites of the genes of Araliacea farnesyl pyrophosphate synthase,squalene synthase and squalene epoxidase.Methods 1 Ultrasonic extraction of E.senticosus was carried out with ethanol solutions of different concentrations,and the conditions such as ultrasonic time,material ratio and ethanol concentration were investigated by spectrophotometry.2 Determine the methylation site and type of pyrimidine C in the promoter region of the target gene by bisulfite sequencing technology,and determine the different methylation type of the target gene by reverse transcription-polymerase chain reaction method and vanillin-concentrated sulfuric acid color method The expression level and its total saponin content.The difference between high and low DNA methylation groups was analyzed by liquid chromatography-tandem mass spectrometry.3 Using PAML and other methods,adaptive evolutionary analysis was performed on 13 farnesyl pyrophosphate synthase genes,32 squalene synthase genes,and 22 squalene epoxidase genes of Araliaceae.Results 1 Ultrasonic time of 20 min,material ratio of 1:25,ethanol volume fraction of 75%,the highest absorbance,that is the highest yield of E.senticosus total saponins.2 The farnesyl pyrophosphate synthase gene has 19 DNA methylation sites and 8 DNA methylation types;The squalene synthase gene has 9 methylation sites and 2 methylation types;The squalene epoxidase gene has 16 methylation sites and 7 types.The total saponin content in the high DNA methylation group was 1.07±0.12 mg/g,that in the low DNA methylation group was 2.92±0.32 mg/g.Statistical analysis found that the gene expression levels of the three genes were significantly positively correlated with their saponin content(P<0.05),and the methylation ratio was significantly negatively correlated with saponin content(P<0.01).A total of 488 metabolites were detected,100 differential metabolites were screened out.It was found that the content of most metabolites related to the mevalonate pathway in the low DNA methylation group was higher than that in the high DNA methylation group.3 The PAML analysis did not reveal the presence of positive selection sites,while the Datamonkey analysis revealed 21 positive selection sites for squalene synthase and 18 positive selection sites for squalene epoxidase.Conclusions 1 The best solution for extracting total saponins of E.senticosus Ultrasonic time 20 min,material ratio 1:25,ethanol volume fraction 75% 2 The promoter regions of farnesyl pyrophosphate synthase,squalene synthase and squalene epoxidase genes DNA methylation site,and DNA methylation in this region can significantly inhibit the synthesis of mevalonate pathway,thereby reducing the content of E.senticosus in the final product.3 Negative selection plays a major role in the evolution of Araliaceae farnesyl pyrophosphate synthase,squalene synthase and squalene epoxidase genes.Figure 24;Table 13;Reference 86...