Metabolomes Of Multi-tissues Of Patchouli And Preliminary Study On Metabolites And Proteins Responsed To MeJA-induced In Leaves

Objective:1.Volatile components and metabolome studies on different tissues of Pogostemon cablin were carried out to reflect the differences in metabolic networks of different tissues of Pogostemon cablin.2.Study on differentially expressed proteins and volatile metabolites in response to MeJA in leaves to illuminated the reflective changes of Pogostemon cablin after induction of methyl jasmonate.3.The PatPTS and PatFPPS promoter binding proteins were screened by DNA Pull-Down technique and the interactive proteins of PatPTS and PatFPPS were obtained by GST Pull-Down technique.Methods:1.Using GC-MS and LC-MS methods,the metabolites were analysed in the three different tissues(Stem,Leaf,Flower)of Pogostemon cablin.Then we used partial least squares discriminant analysis(PLS-DA),fold change analysis(FCA),T test and hierarchical clustering to analyze the difference in secondary metabolism among tissues.2.The metabolites of the leaves of Pogostemon cablin were analyzed by GC-MS under the induction of methyl jasmonate.The proteome was analyzed by iTRAQ method.After detailed analysis by mass spectrometry data,the differentially expressed proteins was identified,and the detailed annotations were made with Gene Ontology(GO)and protein structure.Domain,KEGG pathway,and subcellular localization,protein complexes,etc.were analyzed.3.The nucleoprotein and total protein of the leaves of Pogostemon cablin sinensis induced by methyl jasmonate were extracted.We designed the chain biotin primers of the Pogostemon cablin synthase PatPTS and PatFPPS promoters,and amplify the chain biotin promoter fragment by PCR.After recovery,the Pogostemon cablin nucleoprotein was incubated with the streptavidin promoter for 60 minutes,and the promoter-binding proteins were eluted by DNA Pull-Down technique and identified by LC-MS.The PatPTS and PatFPPS genes were ligated into the PGEX-6P-1 vector to obtain the cloning strain of the fusion expression,and the plasmid transformed expression strain was extracted.The expressed protein was induced to obtain the GST-tagged PatPTS and PatFPPS fusion protein,and the total proteins from the Pogostemon cablin leaves were obtained.The total protein and the captured binding protein were subjected to SDS-PAGE gel electrophoresis,and the protein was recovered by in-gel digestion and the protein was determined by LC-MS.Results:1.In this study,twenty-three volatile compounds were identified from the aerial parts of Pogostemon cablin using GC-MS,many of which were sesquiterpenes.The highest level of Pogostemon cablin was found in leaf tissue and was much lower in stem and flower tissues.Using UHPLC-QTOF-MS,we analyzed the chemical composition of flower,leaf and stem tissues,with the greatest degree of metabolite variation between flower and leaf tissue.Based on the flower,stem-pair comparison of 112,77 and 83 different metabolites,metabolite biomarkers were identified and functional pathways were elucidated.The flowers were found to accumulate more lipids and amino acids,including proline,lysine,arginine,asparagine,threonine and tryptophan.Higher levels of terpenoids,vitamins and flavonoids accumulate in the leaves.Stem tissue was found to contain higher levels of guanidine compounds and carbohydrates.2.After MeJA induction treatment of Pogostemon cablin seedlings,CK group as a control group,by GC-MS analysis,found that the content of patchouli alcohol increased after MeJA induction.The proteome was analyzed by iTRAQ.It was found that after treatment with MeJA,a total of 254 significantly different proteins were obtained,of which 140 were significantly up-regulated and 114 were significantly down-regulated.After KEGG pathway analysis,up-regulated differential proteins are mainly enriched in photosynthesis and photosynthesis-antenna protein pathways,and down-regulated proteins are significantly enriched in steroid skeleton biosynthesis,thiamine metabolism,circadian rhythm-plant,flavonoid biosynthesis And biosynthesis of secondary metabolites on five pathways.254 significant differences in protein showed that the nusk sinensis had significant changes in photosynthesis,steroidal anabolism and defense responses under MeJA induction.3.Using the DNA Pull-Down,the PatPTS and PatFPPS promoter binding proteins in the Pogostemon cablin nucleoprotein extract were harvested,and 12 candidate binding proteins were screened from the results.Yeast one-hybrid assay showed that 9592 basic helix-loop-helix transcription factor(9592 helix factor)and zinc finger BED domain-containing protein RICESLEEPER 2-like(BED-R)may be homologous to PTS in P.The promoter has binding activity.GST Pull-Down technology yields proteins that interact with PatPTS and PatFPPS.Gonelusion:1.The accumulation and exchange of metabolites in different tissues of Pogostemon cablin were studied,which provided a potential network of metabolic pathways for the exploration and metabolic regulation of metabolites in Pogostemon cablin.2.MeJA induction can increase the content of patchouli alcohol of the Pogostemon cablin.3.The preliminary binding proteins of PatPTS and PatFPPS promoters of Pogostemon cablin and the potential interaction proteins of two synthase enzymes of PatPTS and PatFPPS were obtained,which provided references for further research.