| The material basis for the efficacy of the traditional Chinese medicine is mainly from secondary metabolites in plant.Secondary metabolites in plant are as an important indicator for the evaluation of medicinal quality.Many of the active ingredients in botanicals are produced and accumulated under stress conditions.Currently,more studies focus on the effect of environmental factors on the accumulation of the medicinal ingredient.However,it is still difficult to clarify the mechanism of the quality formation of medicines.UV-B irradiation increased the accumulation of active ingredient in medicinal plants.However,the mechanisms of UV-B effect on gene regulation and enzyme activity involved in pathways of secondary metabolites,are not comprehensively explored.As a systematical approach,metabolomics,transcriptomics and proteomics were integrated to reveal the response mechanism of the Clematis terniflora DC.under high intensity UV-B and dark treatment.qRT-PCR and enzyme activity experiments were used to illustrated the biosynthetic pathway of coumarins in C.terniflora.Finally,a reasonably biosynthetic process of coumarins was assumed.This study not only revealed the relationship between components in C.terniflora under treatment,but also explored the response mechanism of secondary metabolism to UV-B irradiation with dark treatment.The results in this study are as follows:(1)Metabolomic analysis of leaves of C.terniflora under high level UV-B irradiation with dark treatment were performed using NMR,UPLC-MS and GC-MS technologies.5 secondary metabolites were significantly increased and 2 secondary metabolites were significantly decreased in response to high level UV-B irradiation.This result indicates that the influence of high level UV-B irradiation was on the primary metabolism of C.terniflora.38 secondary metabolits which were mainly related to fatty acids,lactones,polyphenols,benzoic acids,coumarins,and stilbenes.Furthermore,19 secondary metabolites were specifically synthesized in the stress period.More secondary metabolites accumulated under additional dark treatment than that under UV-B irradiation,indicating that the dark incubation caused a more intense disturbance on the secondary metabolism of leaves of C terniflora.(2)Transcriptomics,which changed the research model of single gene analysis,takes rapid development to genomics research.Transcritpome of leaf of Clematis terniflora DC.induced by UV-B irradiation with dark treatment was analyzed.103091 unigenes were obtained by de novo assemble.Among them,29875 unigenes were longer than 500bp.Expression profile of genes were analyzed that the number of unigenes was gradually increased under the UV-B irradiation and the additional dark treatment.Based on KEGG annotation,unigenes invoved in secondary metabolism were mainly related to phenylpropanes,stilbenes,flavonoids/flavonols,flavonoids tropane,and isoquinoline alkaloids.54.4%of unigenes related to phenylpropanes were upregulated and 58.3%of them were upregulated under UV-B induction and additional dark treatment,respectively.These results indicated that the phenylpropanoid metabolic pathway positively responsed to the high level UV-B irradiation with dark treatment.(3)To systemically investigate the response of C.terniflora to high level UV-B irradiation with dark treatment,a integrate approach using transcripteomic,proteomic,and metabolomic analyses were performed.Proteomic and metabolomic analysis revealed that the content of metabolites and abundance of proteins related to amino acid metabolism were significantly increased;the abundances of S-adenosylmethionine synthetase,cysteine synthase,dihydrolipoyl dehydrogenase,and glutamate dehydrogenase as well as the content of gamma-aminobutyric acid were significantly increased in response to high level UV-B irradiation with dark treatment.These results indicate the reactive oxygen species scavenging system and gamma-aminobutyric acid shunt pathway were activated in leaf of C.terniflora in response to high level UV-B irradiation with dark treatment.Proteomic and transcriptomic analysis revealed that the genes and proteins related to protein metabolism were largely changed in posttranslational modification,ubiquitin proteasome,and ribosomal protein.The expression of NADP-dependent malic enzyme and the abundance of NADP-malate dehydrogenase were upregulated and increased,respectively.The activities of these two enzymes were also enhanced.These results suggest that the secondary metabolism pathway and tricarboxylic acid cycle might be activated in leaf of C.terniflora in response to high level UV-B irradiation followed by dark treatment.(4)The results show that the effect of UV-B irradiation with dark treatment on Clematis terniflora DC.is the formation of coumarins including linear pyran coumarins and other simple coumarins.Primary metabolism preceded secondary metabolism to respond to the UV-B radiation and provided precursors and energy for the accumulation of secondary metabolites.Clematis terniflora DC.reduced carbon assimilation but strengthened the catabolism to supply more energy for the metabolism downstream and enhanced catabolism producing more metabolites to take part in secondary metabolic pathways.The increased shikimate pathway facilitated the metabolic flux from primary to secondary metabolism.In our experiment,Clematis terniflora DC.was subjected to binary,short-term stress in the form of UV-B radiation and dark cultivation,which led to the emergence of a series of coumarins.Thus,C.terniflora proved to be an ideal model for the study of the biosynthetic process of coumarins.(5)Analysis of the chloroplast genome can help to understand the retulation relationship between the nuclear genome and chloroplast genome and clarify the activated mechanism of secondary metabolism at molecular level.The large-scale sequencing of chloroplast genome of C.terniflora was performed.The results show that the complete chloroplast genome of Clematis terniflora is 159,528bp,which contains a typical quadripartite structure with a pair of inverted repeats(31,045 bp)separated by large(LSC,79,327 bp)and small(SSC,18,lllbp)single-copy regions.The chloroplast genome of C.terniflora encodes 137 unique genes.Compared to other species belonging to Ranuculaceae,C.terniflora has the smallest S SC region,Comparative analysis of five Ranunculaceae species shows that coding region in chloroplast genome of C.terniflora was more conservative than non-coding region.During five chloroplast genome sequences of Ranunculaceae species,three inverted regions presences in LSC area of C.terniflora.The results also show that JLB and JLA regions have big expanision in the chloroplast genome of Clematis and Mahonia. |
PDF Full Text Request