Studies On The Quality Formation Mechanism Of Prunellae Spica Under Salt Stress Based On Omics

Prunella Spica,a dicotyledonous plant of Labiatae,is the dried ear of Prunella vulgaris L.,which is full used in Taiwan.It's pungent,bitter and cold.Return to liver and gallbladder meridian.In order to clear the liver,clear the eyes and eliminate swelling,traditional Chinese medicine is often used.It is also one of the dual-use resources of medicine and food listed by the Ministry of health."Shennong Materia Medica" records:"bitter,pungent and cold,heat and scrofula,rat fistula,head injury,disease breaking,gall scattering,Qi formation,swelling of feet,wet arthralgia,light body",which is listed as inferior.In the Song Dynasty,"the meaning of the original grass" recorded:" Prunella Spica is also recorded in the food materia medica of Ming Dynasty:" Prunella Spica after the tender seedlings have been lifted,the bitterness is removed and the oil and salt are mixed".Modern clinical research shows that Prunella Spica mainly through triterpenoids,flavonoids,phenolic acids and coumarins and other secondary metabolites to control blood sugar,reduce blood pressure,anti-inflammatory,anti allergic,antiviral and other functions.Due to the extensive use of Prunella Spica in clinic and diet,the contradiction between supply and demand is increasing.Although there are many planting bases in China,due to the change of ecological environment?salt stress is one of the important factors in abiotic stress?,the accumulation of active components of Prunella Spica is also changed,and its quality control and quality evaluation are facing new challenges.In this paper,according to the rule that salt stress induces the differential expression of transcriptome and proteome and affects the synthesis and accumulation of secondary metabolites,starting with the link between salt stress and secondary metabolites,and with the help of the advantages and characteristics of system biology,the differential expression proteins and transcriptome sequencing are screened by means of plant metabonomics technology,combined with proteomics technology The possible mechanism of the difference of secondary metabolites of Prunella Spica under different salt stress was discussed.It is of great significance to further study the molecular mechanism and quality formation mechanism of the effect of salt stress on the synthesis and accumulation of secondary metabolites in medicinal plants.The main research contents and innovative results are as follows:1.Analysis of Multiple Active Components in Prunella Spica The contents of nine phenolic acids,three coumarins,eight flavonoids and one pentacyclic triterpenoid in Prunella Spica from different habitats and growing periods were determined simultaneously by high performance liquid chromatography tandem quadrupole mass spectrometry?HPLC-QTRAP-MS/MS?.The total contents of 21 components in different habitats and growing periods were 986.04-2276.99 ?g/g and 805.22 ?g/g-1806.9?g/g,respectively.Among them,the order of growth stage was:wilt stage>apoptosis stage>flowering stage>germination stage.According to the content of twenty-one components,the quality of Prunella Spica was evaluated by the methods of HCA and GRA.The results showed that the quality of Prunella Spica planted in Anhui Province was better than that of other samples.2.Effects of Salt Stress on Physiological Indexes and Active Components of Prunella Spica The changes of photosynthetic pigments?chlorophyll and carotenoids?,osmotic balance substances?soluble sugar,protein and proline?,lipid peroxidation products?malondialdehyde?,antioxidant enzymes?superoxide dismutase,catalase,peroxidase?and ascorbic acid of Prunella Spica under different concentrations of salt stress?0,50,100 and 150 mM?were studied by plant physiological analysis.The results showed that the high salt?150 mM?treatment significantly inhibited the development of the plant,and the survival rate decreased under the medium and high concentration.In the three groups of low,medium and high concentrations,photosynthetic pigment decreased continuously.Soluble sugar and protein increased compared with the control group,and reached the highest value in the middle concentration salt treatment group.Proline,malondialdehyde,superoxide dismutase,catalase and peroxidase all increased with the increase of salt concentration.Ascorbic acid was the highest in medium concentration,but decreased in high salt stress.HPLC-QTRAP-Ms/MS and multivariate statistical methods were used to detect and analyze the changes of multiple active substances in Prunella Spica under salt stress.The quality order of Prunella Spica under different concentrations of salt was:100 mM>150 mM>50 mM=0 mM.The results of comprehensive physiological indexes and the content of multiple active components showed that the quality of Prunella Spica was the best under 100 mM salt stress.3.Transcriptomics Analysis of Prunella Spica under Salt Stress RNA-seq was used to analyze the transcriptome changes of Prunella Spica under salt stress.Using Trinity software to assemble the reads of all libraries,118664 unigenes are obtained.After database screening,68119 sequences with functional annotations were found.Compared with the control group,3857 differentially expressed genes were screened,of which 2456 were up-regulated and 1401 down regulated.Go analysis showed that salt stress related genes were mainly enriched in"catalytic activity","binding","metabolic process" and "cell process".KEGG analysis showed that genes related to salt stress and multiple active components were mainly enriched in biosynthesis and transcription related pathways involved in transport,oxidoreductase activity,environmental adaptation,secondary metabolism.The expression level of 10 differentially expressed genes was detected by real-time quantitative PCR,and the results were consistent with RNA-seq data.4.Proteomic Analysis of Prunella Spica under Salt Stress The proteomics of Prunella Spica under different salt stress was analyzed by iTRAQ.To analyze the difference of proteome expression of Prunella Spica under different salt stress,GO,STRING and KEGG were used to analyze the different proteins.The results showed that 1937 proteins were detected,66 up-regulated,132 down regulated in 50 mM group,80 up regulated,98 down regulated in 100 mM group,142 up regulated and 202 down regulated in 150 mM group.Bioinformatics analysis showed 83 differentially expressed proteins under salt stress.Based on the analysis of GO,STRING and KEGG,35 differentially expressed proteins related to salt tolerance and secondary metabolite formation mechanism of Prunella Spica were screened.The results of functional analysis showed that the metabolism of protein and carbohydrate was weak,calcium transport was high,photosynthesis was strong,and the synthesis of protein and secondary metabolites was faster than that under normal conditions.5.Metabolomics Analysis of Prunella Spica under Salt Stress Ultra highperformance liquid chromatography quadrupole time of flight mass spectrometry?UHPLC-QTOF-MS?was used to study the metabolism of Prunella Spica under different salt stress.Compared with the control group,146,175 and 159 different expression metabolites were screened out in the 50,100 and 150 mM salt stress groups.The up-regulated and down-regulated metabolites were 48/97,76/99 and 55/10,respectively.The most significant up-regulated metabolites were 3,4-dimethoxycinnamic acid,creatine,trimethylamine N-oxide/Tyr Leu,deoxyribose,2'-deoxyadenosine 5'-monophosphate?cAMP?;norformamidinone,DL phenylalanine,lysine phenylalanine/cis-9-palmitoleic acid,myristic acid,allopurinol nucleoside;pyridoxamine?PM?,trimethylamine oxide,n-alpha-acety l-l-arginine/3,4-methylenedioxymethylphenylalanine,1-fucose-l-phosphate and ketleucine.Further identification of metabolites and comprehensive analysis of metabolic pathways showed that under salt stress,differential metabolites were mainly enriched in alanine,aspartic acid glutamate metabolism,phenylalanine metabolism and purine metabolism.6.Omics correlation analysis of Prunella Spica under salt stress Using the Rstudio platform and spearman correlation analysis algorithm,correlation analysis of Prunella Spica transcriptomics,proteomics and metabolomics was carried out.It was found that Prunella Spica responds to salt stress through three levels,through amino acid biosynthesis,carbon metabolism,etc.to adjust salt stress to cause osmotic changes,and through changes in ABC transporter and photosynthesis to adjust ion toxicity and oxidative stress caused by salt stress.It also responds to salt stress through more complex pathways affected by the metabolism of lysine,arginine,phenylalanine,aspartic acid,and glutamic acid.The key axis regulated by LYC₆8t000004 was found through correlation analysis.LYC 68t000004 was negatively correlated with Solyco9g0145202,psbC,psbD and GSCOC_T00027397001 proteins,among which psbC and psbD were the key proteins that regulate photosynthesis.The KEGG correlation analysis of various omics found that the metabolic pathway?ath01100?and the secondary metabolite biosynthesis pathway?ath01110?are two basic pathways in response to salt stress.Esi 0003 0214 may be a key enzyme in the biosynthesis of umbelliferone.