Study On Quality Change And Molecular Mechanism Of Ephedra Sinica Stapf. Based On The Potassium Nutrition Regulation

Objective To determine the direct effects of different potassium application rates on the pharmacodynamic components of E.sinica;to determine the key genes,metabolic pathways and regulatory pathways affecting the production of pharmacodynamic components of E.sinica under potassium regulation;to reveal the molecular mechanism of the effects of potassium on pharmacodynamic components of E.sinica.Methods?1?Field experiments were conducted to design and cultivate E.sinica with different potassium treatments at random.Potassium sulfate?containing K₂O 52%?was used as potassium fertilizer.After the emergence and stable growth of E.sinica in late April,15treatments were applied in field experiments.Samples were collected from 1 month after potassium fertilizer application,once every 21 days and 6 times;?2?To determine the effects of different potassium treatments on the active ingredients of ephedra.The average samples of ephedrine hydrochloride and pseudoephedrine hydrochloride,total alkaloids,total flavonoids and volatile components in 15 potassium-treated samples were sampled by mixed sampling method.The direct effects of different potassium treatments on the content of effective components of E.sinica were revealed;?3?To determine the molecular mechanism of E.sinica quality response under potassium regulation,high-throughput transcriptome sequencing was carried out,differential genes were screened;functional gene co-expression network related to pharmacodynamic components was constructed;key modules and pivotal gene functions were revealed;metabolic pathways and regulatory pathways of E.sinica quality formation based on potassium regulation were revealed.Results1.The content of ephedrine hydrochloride in Ephedra samples treated with different potassium was 0.069%⁰.699%.The content of ephedrine hydrochloride in treatment group 5,6,7,8,9 and 10 was significantly affected by the amount of potassium applied at666.4¹499.4 g/12m2.The ephedrine content in each treatment group showed a trend of"slow increase period,fast increase period,stable period and decrease period"with time.The content of pseudoephedrine hydrochloride in Ephedra samples treated with different potassium was 0.011%⁰.268%.The content of pseudoephedrine hydrochloride could be significantly increased by controlling the application of potassium at the levels of2165.8²332.4g/12m² in treatment 14 and 15.The change trend of pseudoephedrine content with time was similar in each treatment group.The increase of pseudoephedrine content showed a trend of"slow increase period,rapid increase period and stable period".2.The total alkaloid content of Ephedra under different potassium treatments ranged from 0.177%to 1.220%.The total alkaloid content of treatment group 6,7,8,9,14 and 15could be significantly increased by controlling the potassium application levels at833-1332.8g/12m² and 2165.8-2332.4g/12m².The content of total alkaloids in each treatment group showed a trend of"slow increase period,fast increase period and slow increase period"with time.The potassium application level was positively correlated with the total alkaloid content of the fourth and fifth samples,and positively correlated with the total alkaloid content of the sixth samples.3.The total flavonoids content of Ephedra under different potassium treatments ranged from 0.270%to 2.790%.Compared with the control group,each potassium application level has a significant effect on improving the content of total flavonoids in ephedra.The content of total flavonoids in each treatment group showed a trend of"slow increase period,fast increase period and slow increase period"with time.There was a significant positive correlation between potassium application level and total flavonoids content in each batch.4.45 Ephedra samples were analyzed by headspace solid phase microextraction and gas chromatography-mass spectrometry?HS-SPME-GC-MS?.336 volatile components were identified.The peak area of all peaks in each sample was normalized by peak area normalization method,and the relative contents of 336 volatile components in 45 samples were obtained.Compared with the control group,it was found that potassium application level at 2332.4g/12m² was the most important factor affecting the volatile components in the treatment group 15.The contents of 10 volatile components were significantly increased and9 volatile components were significantly decreased in the treatment group 15.5.In NR annotations,Ephedra sinica was more similar to Quercus variabilis than other species.GO annotation results showed that there were 153077 Unigenes sequences,of which52856,58515 and 41706 were classified into BP,CC and MF respectively.KEGG results showed that there were 135 and 166 unigenes annotated to the phenylalanine biosynthesis and metabolism pathways related to ephedrine,and 172 unigenes annotated to the flavonoid biosynthesis pathways.Twenty-four modules were constructed by running WGCNA software package on R platform.It was found that the concentration of genes in turquoise module was the highest,which was most related to the phenotypic characteristics of alkaloids and flavonoids.Thirty hub genes were screened through further analysis of turquoise module,and10 pathways were enriched,including phenylalanine related to alkaloid synthesis pathway-Tyrosineandtryptophanbiosyntheticpathwaygenes,including3-deoxy-7-phosphoheptulonate synthase,3-dehydroquinate synthase,3-dehydroquinate dehydrataseI,shikimatekinas,shikimatedehydrogenase,3-phosphoshikimate1-carboxyvinyltransferase,chorismate synthase,indole-3-glycerol phosphate synthase,anthranilate phosphoribosyltransferase,tryptophan synthase alpha chain,tryptophan synthase beta chain,chorismate mutase,arogenate/prephenate dehydratase,bifunctional aspartate aminotransferaseandglutamate/aspartate-prephenateaminotransferase,aspartate aminotransferasecytoplasmic,tyrosineaminotransferase,histidinol-phosphate aminotransferase,aromatic-amino-acid transaminase,arogenate dehydrogenase NADP+,phenylalanine-4-hydroxylase,etc.ConclusionsThe effects of different potassium treatments on the chemical constituents of Ephedra sinica were systematically determined.Compared with the non-fertilizer group,potassium application level controlled at 833-1332.8g/12m²?i.e.2-3 times of potassium application in local agricultural production?can significantly increase the content of effective components in Ephedra medicinal materials,which provides a reference for the rational application of potassium fertilizer in Ephedra agricultural production and has certain application value.In addition,the transcriptome high-throughput sequencing technology was used to identify the functional genes related to alkaloid and flavone synthesis in ephedra,determine the key module and pivotal gene functions,and reveal the metabolic pathway and regulatory pathway of Ephedra quality formation under potassium regulation.