Terpenoid Metabolites Distribution In Different Tissues Of Chrysanthemum Indicum L And Functional Characterization Of TPSs

Objective:The dried flower bud of Chrysanthemum indicum L.is one of the sources of traditional Chinese medicine Chrysanthemi indici flos.It has the functions of clear heat and detoxify,relieve fire and calm liver.Treatment of boils,carbuncle,red eyes,swelling and pain,headache and dizziness.Because of its antioxidant,anti-inflammatory,anti-viral and antibacterial benefits and bioactivity,it is widely used in medicine,tea,cosmetics industry and food additives.This tissue studyed and compared the distribution of terpenoids(mainly volatile terpenoids),the main active ingredient,in different tissues of the C.indicum.At the same time,functional characterization of two monoterpene synthases CiTPS 1,CiTPS2,sesquiterpene synthase CiTPS3 and bifunctional synthase CiTPS4 was carried out in order to further understand the distribution and synthesis mechanism of secondary metabolites in C.indicum,provide a theoretical basis for the synthesis of medicinal active ingredients such as terpenoids in synthetic biology and expansion of effective parts of C.indicum.This paper intends to study the composition and distribution of volatile terpenoid metabolites in different tissues of C.indicum,the expression of corresponding terpene enzyme genes,and the correlation between terpenoid metabolites and terpene enzyme gene expression.The level of metabolic pathway gene reveal the diversity of different tissue parts of C.indicum,and Provide scientific theoretical support and technical solutions for the synthesis of terpenoid medicinal active ingredients and the diversity of medicinal parts of C.indicum.Methods:1.1 Expression analysis of CiTPSsRNA extracted from root,stem,leaf,flower and flower bud of C.indicum and reverse transcript to cDNA.Analyze the expression patterns of CiTPSs in different tissues by quantitative reverse transcription polymerase chain reaction(qRT-PCR).1.2 Distribution of volatile terpenoids in different tissues of C indicumFresh roots,stems,leaves,flowers and flower buds of C.indicum as materials,extract at constant temperature with hexane,and volatile terpenoid metabolites in C.indicum detected by GC-MS.Select monoterpene standard substance a-pinene and sesquiterpene standard substance β-caryophyllene to make standard curve for quantitative analysis.1.3 CiTPSs cloning and sequence alignmentCiTPSs cloned using specific primers and select the cDNA from different tissues of C.indicum as templates.The PCR products were cloned into pLB vectors and sequencing of the recombinant cloning vector.Bioinformatics analysis of the protein sequence were using DNAMAN7.0,MEGA and ITOL tools.1.4 Prokaryotic expression vector construction of CiTPSsThe fμLl-length CiTPSs were cloned by specific primers and select the recombinant plasmids pLB-CiTPS1,pLB-CiTPS2,pLB-CiTPS3 and pLB-CiTPS4 as templates.Were used to construct the corresponding expression vectors pET-28a(+)and pMAL-C5X of the recombinant proteins and transformed into E.coli Rosetta(DE3)cells to prokaryotic.1.5(-tp)CiTPS cloning and prokaryotic expressionTargetP 1.1 analyze the potential chloroplast TPs(Transit peptides)of CiTPS1 and CiTPS2.Two(-tp)CiTPS were cloned by specific primers and select the recombinant plasmids pLB-CiTPS1 and pLB-CiTPS2 as templates.The PCR products of(-tp)CiTPSs were subcloned into a pET-28a(+)expression vector and were transformed into E.coli Rosetta(DE3)cells.1.6 Purification and enzyme assay of CiTPSs proteinprotein expression induced by 0.1 mM isopropyl-β-D-1-thiogalactopyranoside.Purify the recombinant protein pMAL-C5X-CiTPSs with Dextrin Beads 6FF column(with MBP tag),and purify the recombinant protein pET-(-tp)-CiTPSs and pET-(-tp)-CiTPSs with NiNTA column.CiTPSs were incubated with substrates GPP and FPP,and terpenoid products detected by GC-MS.Results:2.1 Expression analysis of CiTPSs in different tissues of C.indicumThe expression patterns of CiTPSs in different tissue types were analyzed using RT-qPCR.Among them,CiTPS1 was highly expressed in roots and flower buds,CiTPS2,CiTPS3,and CiTPS4 were all highly expressed in roots.Terpenoid levels produced by CiTPS1,2,3 and 4 in vitro partially consistent with the gene expression patterns of the five parts of the plant.2.2 volatile terpenoids content and distribution in different tissues of C.indicumThe volatile terpenoids in the roots,stems,leaves,flowers and buds of C.indicum were detected through gas chromatography-mass spectrometry(GC-MS).A total of 44 volatile terpenoids in 5 tissues,including 17 monoterpenes and 27 sesquiterpenes.Of the 44 terpenoids identified,24,27,30,29 and 33 compounds were detected in roots,stems,leaves,flowers,and flower buds,respectively.β-farnesene is the main product of 5 tissue parts,and petasitene is only found in root.2.3 Cloning of CiTPSs and sequence analysisIn this paper,a total of 4 TPS unigenes were cloned from C.indicum.The coding region sequence of CiTPS1 is 1812 bp in length(the transit peptide sequence is 34 bp)and encodes an amino acid of 603 aa.CiTPS2 coding region sequence is 1767 bp(including transit peptide sequence 34 bp),coding 588 amino acid.CiTPS3 and CiTPS4 coding region sequence is 1647 bp and 1683 bp and encodes an amino acid 548 aa and 560 aa,respectively.Amino acid sequence alignments of CiTPSs and phylogenetic analysis of four TPSs from C.indicum and some TPSs from other plants showed that CiTPSl and CiTPS2 belong to the TPS-b family and contain the RRX8W conserved domainand with the monoterpene synthases from the other plants,whereas CiTPS3 and CiTPS4 belong to the TPS-a family and contain the RPX8W conserved domain with the sesquiterpene synthase from the other plants.2.4 Purification and enzyme assay of recombinant proteins CiTPSsCiTPSl produced α-pinene after subcloned into pET-28a(+)and pMAL-C5X expression vector.CiTPS2 was only expressed in the pMAL-C5X expression vector but no product was produced.pET-(-tp)-CiTPS1 and pET-(-tp)-CiTPS2 were subcloned into the pET-28a(+)expression system and product α-pinene.Only a pMAL-C5X expression vector successf μLly expressed CiTPS3 and CiTPS4 and have products.CiTPS3 produced 3 sesquiterpenes:petasitene,β-farnesene and α-bisabolene,and CiTPS4 produced 4 monoterpenes and 3 sesquiterpenes:α-pinene,β-myrecene,trans-β-ocimene,α-terpineol,petasitene,trans-α-bergamotene,and β-farnesene.Conclusion:This paper analyzed the composition,distribution and quantity of volatile terpenoid metabolites in fresh roots,stems,leaves,flowers,and flower buds of C.indicum,and according to transcriptome data successf μLly cloned and identified 4 TPS synthase which have catalytic activity to catalyze the production of monoterpenoids or sesquiterpenoids from the substrate GPP or FPP.CiTPS1 belong to monoterpene synthase.Both the fμLl length and truncated transit peptides version of CiTPS1 can produce monoterpene α-pinene,but the truncated version more efficient than the fμL1 length.CiTPS2 is also a monoterpene synthase but is different from CiTPS1,only a small amount of α-pinene can be produced after truncation of the transit peptide,CiTPS3 is a sesquiterpene synthase that can catalyze FPP to generates a mμLti-product with 3 sesquiterpenes,and CiTPS4 is a bifunctional synthase that can catalyze GPP and FPP to produce 4 monoterpenes and 3 sesquiterpenes respectively.Terpenoid levels produced by CiTPSs in vitro partially consistent with the gene expression patterns of the five parts of the plant.The resμLts of this paper provide a basis for extracting the medicinal active ingredients from the roots,stems and leaves of C.indicum,a theoretical basis for the composition,formation and regμLation of terpenoids in C.indicum.Provide scientific theoretical support and technical solutions to improve the medicinal active ingredients of C.indicum from the level of metabolic pathway gene.