The Effect Of Ammonium Citrate And Methyl Jasmonate On The Metabolites Of Gentiana Macrophylla

The secondary metabolites in medicinal plants are the material basis for clinical efficacy and an important indicator of the quality of medicinal materials.The main secondary metabolites of Gentiana macrophylla are the secoiridoid glycosides represented by gentiopicroside.Gentiana macrophylla is mainly produced in Shaanxi and Gansu areas,and the supply has been mainly wild which usually takes 4-6 years to become useful.Exogenous elicitors stimulated the accumulation of metabolites in the short term.Therefore,this study first to selected a elicitor(ammonium citrate)from varieties which can improve the secondery metabolism,and further screening the best induction concentration.Comparative transcriptome and metabolite analysis to explore the similarities and differences between ammonium citrate and methyl jasmonate regulation.The main results are obtained.1.The elicitor prepared from the Trichoderma fungus fermentation broth,0.92 mM AgN03(Ag+),200 ?M CuSO4(Cu2+),10 mg/L Arachidonic acid(AA),200 ?M Ammonium citrate(AC)and 200 ?M salicylic acid(SA)were used to spraying the leaves of Gentiana macrophylla.The effects of treatment factors on the induction of secoiridoid glycosides compounds were different.The time and response intensity were different.It was further confirmed that the different inducing agents had different effects on plant induction.The content of loganic acid was up to 12.33 mg/g for the third day,induced by arachidonic acid which was 225.5%higher than that of the control.The highest content of swertiamarin was 20.88 mg/g,which induced by salicylic acid was 357.9%higher than that of the control.Ammonium citrate induced could significantly increase the content of gentiopicroside in Gentiana macrophylla at 6 days,which was 43.4%higher than that of control,and the content was up to 184.99 mg/g.Indicating that the different metabolites,response time and intensity were different with kinds of elicitors.2.Construction of high performance liquid chromatography(UPLC)for the determination of secondary metabolites of Gentiana macrophylla,which can be used to identify metabolites quickly and efficiently.By comparing the effects of different concentrations of ammonium citrate on metabolites,it was found that low concentration of ammonium citrate(50 ?M)could more effectively enhance gentiopicroside,and high concentration(1000 ?M)inhibited the accumulation of gentiopicroside.Different concentrations of ammonium citrate did not significantly promote the accumulation of swertiamarin.Indicating that the response intensity of metabolites is related to the concentration of the elicitor.3.The sequencing data of 21.7 G bp were obtained by three samples(including methyl jasmonate,ammonium citrate and control)of Gentiana macrophylla by Illumina HiSeq 2000.The number of assembly sequences obtained from mixed assembly was 129,208 and N50 was 1393bp.Database comparison results show that there are 40 269 sequence alignments to the uniprot and Pfam databases,compared to 31.17%.Three samples were compared between the two pairs,and about 1073 differentially expressed genes were obtained.Differential genes are mainly related with plant metabolic pathways.The metabolic pathway of terpenoid compounds of Gentiana macrophylla is clear from the MVA pathway and the MEP pathway,but it is not clear to the downstream pathway.In this study,the metabolic pathway enzyme genes were mining,including three IS,four IO,eleven 7DLGT and three 7DLH.These sequences greatly enriched the downstream metabolic pathway,but the synthesis pathway of secologanin to gentiopicroside still unknown.4.The stability of the internal reference gene determines the real-time quantitative analysis of the reliability of gene expression results.A large number of studies have shown that there are differences in species and histological differences.The work of internal reference screening is limited because lack of genetic data.In this study,ten genes(UBC21,GAPC2,EF-1a4,UBQ10,UBC10,SAND1,FBOX,PTB1,ARP and Expressed1)were found in the transcriptome database,and stability analysis was performed in 16 different tissues.Screening SAND1 and EF-1a4 can be used as a reliable internal reference gene for gene expression of Gentiana macrophylla.Studies on the key enzyme genes of the terpenoid metabolic pathway found that the key enzymes expression in leaves higher than in the roots.This may be related to the secondary metabolite of Gentiana macrophylla,which is mainly produced in the leaves and accumulation in the roots.5.Ammonium citrate and methyl jasmonate could increase NO in root of Gentiana macrophylla.Addition of NO inhibitor L-NAME and sodium tungstate could significantly reduce the synthesis of secondary metabolites,indicating that NO was involved in the metabolic process.The addition of L-NAME resulted in a decrease in fluorescence intensity compared with that of sodium tungstate.It was found that the nitric oxide synthase-related gene was up-regulated and the nitrate reductase was down-regulated when Gentiana macrophylla was induced by the elicitor.It is speculated that the production of secondary metabolites after induced is related to NOS and NR pathways,and more dependent on the NOS pathway.These results suggest that NO plays an important role in plant response to the elicitor.6.Ammonium citrate and methyl jasmonate all reduced the amino acid content to varying degrees decreased,but the content of serine,proline and tyrosine increased.Compared with methyl jasmonate,ammonium citrate has a weak effect on amino acids,which is a more affordable and effective elicitor.Combination with transcriptome and metabolomics data,it is presumed that one of the reasons for the reduction in most of the amino acid content may be associated with a decrease in the metabolic flux entering the tricarboxylic acid cycle.The expression of glyceraldehyde-3-phosphatedehydrogenase was downregulated,and glyceraldehyde-3-phosphate was briefly accumulated so that the sugar metabolic pathway was "partially blocked" to glyceraldehyde-3-phosphate,thereby reducing the primary carbon skeleton of primary metabolite synthesis.At the same time,fumaric acid reductase gene expression down,so that fumaric acid reduced to succinic acid,because the reaction is irreversible,may thus break or reduce the TCA cycle,thereby reducing the synthesis and accumulation of primary metabolites.The addition of NO-inhibitor L-NAME increased the content of arginine,isoleucine and leucine,while the contents of proline,alanine and phenylalanine decreased,indicating that NO was not only involved in the synthesis of secondary metabolites also involved in the amino acid metabolism process.7.The decrease of glyceraldehyde-3-phosphate dehydrogenase and fumarate reductase genes was induced,and the content of glyceraldehyde-3-phosphate was accumulated in 3 hours,and the content of pyruvic acid and acetyl-CoA were decreased.The primary enzyme gene of the MEP pathway(starting glyceraldehyde-3-phosphate +pyruvate)was up-regulated and the key enzyme gene of the MVA pathway(initial acetyl-CoA)was generally up-regulated,and these factors could stimulate metabolic flow bias synthesis of secoiridoid glycosides.At the same time,the down-regulation of GGPPS gene expression in the downstream metabolic pathway of terpenoids promoted the decrease of the content of ABA(diterpenoid)and gibberellin(sesquiterpenoids),and then effectively controlled the metabolic flow to the target metabolites such as gentiopicroside.It is suggested that the synthesis process of secondary metabolism of Gentiana macrophylla induced by elicitor is closely related to the pathway of glucose metabolism.Controlling the content of glyceraldehyde-3-phosphate may be one of the key compounds in the pathway of the gentiopicroside.