Mining And Functional Analysis Of Genes Involved In The Biosynthetic Of Terpenoids Of Chrysanthemum Indicum Var.aromaticum Induced By Methyl Jasmonate

Chrysanthemum indicum var.aromaticum,characterized by a special scent,is a rare source of aroma in genus Chrysanthemum.Our previous studies had shown that the leaves and petals of C.indicum var.aromaticum were densely covered with glandular trichome.Secretions of glandular trichomes were closely related to the aroma components.Terpenoids including monoterpenes,sesquiterpenes,and their oxygen-containing derivatives were identified from the trichome secretions using gas chromatography-mass spectrometer(GC-MS).However,the causes and molecular regulation mechanisms of the formation of aroma of C.indicum var.aromaticum have not been reported so far.In this study,methyl jasmonate(MeJA)was used as an exogenous inducer.C indicum.var.aromaticum was treated with MeJA in different concentrations and time,and the optimum condition for aroma enhancement was preferred.The transcriptome sequencing analysis of C.indicum var.aromaticum under the treatment of MeJA was carried out to reveal the regulation mechanism of terpenoids biosynthesis pathway of C.indicum var.aromaticum at the molecular level and to analyze the functional of terpenoids biosynthetic related genes in the terpenoid metabolic pathway,so as to provide a basis for the further creation of high-terpene aromatic materials by using transgenic engineering technology,and to make it possible to cultivate new fragrant chrysanthemum materials.The main results are as follows:1.The results showed that the density of T-shaped non-glandular trichomes and capitate glandular trichomes in leaves of C.indicum var.aromaticum were increased first and then decreased,and the relative content of terpenoids was increased when the concentration of MeJA and treatment time were increased.Bicyclo[3.1.0]hex-2-ene,4-methylene-1-(1-methylethyl)-and Caryophyllene changed obviously.The relative content of benzene and alcohol increased first and then decreased.At the concentration of 0.5%for 48 h,the content of p-cymene and aromadendrene began to decline,(-)-alpha-Panasinsen,Maaliene,Guaiene and Cis-sabinol were reduced to 0,which indicated that MeJA could induce trichomes and the components of secretions in leaves of C.indicum var.aromaticum,and the best induction effect was found when the concentration of MeJA was 0.25%and the treatment time was 24 h.2.Transcriptome sequencing was performed on leaves of C.indicum var.aromaticum under different induction time of MeJA solution.A total of 176,091 Unigenes were obtained,and 69.94%of Unigenes could be annotated in at least one database.In the MJ4vsCtrl,MJ24vsCtrl,and MJ24vsMJ4 comparison groups,750,650,and 313 differentially expressed genes(DEGs)were obtained,respectively.These DEGs were enriched in 33 secondary metabolic pathways.We analyzed the expression heatmaps of DEGs involved in jasmonate(JA)biosynthesis and signal transduction pathway,terpenoid biosynthesis pathway,phenylpropane metabolism pathway,fatty acid metabolism pathway,differentially expressed post-plant modification enzyme,and transcription factors related to secondary metabolism.It was found that most of DEGs were up-regulated by MeJA.The results of qRT-PCR were basically consistent with those of transcriptome sequencing,which indicated that the gene expression profile data of transcriptome sequencing were reliable.3.The open reading frame(ORF)sequences of GPS and FPS of C.indicum var.aromaticum with lengths of 1278 bp and 1035 bp were cloned by reference transcriptome assembly sequences,which named as CiGPS and CiFPS,respectively.Phylogenetic analysis showed that CiGPS and CiFPS genes were closely related to Artemisia annua and Chrysanthemum×morifolium,respectively.The analysis of physical and chemical properties of the proteins indicated that the proteins encoded by the two genes were hydrophilic proteins.qRT-PCR results showed that CiGPS and CiFPS gene had the highest expression level in leaves,and the MeJA treatment could increase the expression of CiFPS.CiGPS and CiFPS were transformed into tobacco by Agrobacterium-mediated method containing the expression vectors pBI121-CiGPS-GFP and pBI121-CiFPS-GFP.The positive tobacco transformants were selected using MS medium containing kanamycin resistance and PCR and RT-PCR analysis.The three transgenic lines with the highest expression were used for subsequent functional analysis.Compared with control wild type(WT)and empty vector(EV),the CiGPS and CiFPS transgenic tobacco lines showed shorter plant height,darker leaf color,higher chlorophyll and carotenoid content,and increased long glandular trichomes number,while short glandular trichomes only increased significantly on the upper epidermis of leaves of CiGPS transgenic tobacco lines.The relative content of terpenes of CiGPS and CiFPS transgenic tobacco lines increased significantly,and monoterpenoids and sesquiterpenoids were detected in transgenic tobacco without WT and EV lines.The activity of GPS enzyme was increased in CiGPS transgenic tobacco lines,but the activity of FPS in CiFPS transgenic tobaccolines were not significantly,only F6 line was slightly higher than WT and EV.Among CiGPS transgenic tobacco lines,the expression levels of HMGR,DXR,GGPPS,and MTS genes were increased,but EAS decreased.Among CiFPS transgenic tobacco lines,the expression levels of HMGR,DXR,GGPPS,EAS,and CCD genes were rised,but MTS declined.4.The ORF sequences of MYC2 of C.indicum var.aromaticum with length of 1881 bp was cloned by reference transcriptome assembly sequence,and named CiMYC2.Phylogenetic analysis showed that CiMYC2 genes were closely related to Artemisia annua.The analysis of physical and chemical properties of the proteins indicated that the protein encoded by CiMYC2 gene was hydrophilic proteins.The expression level of CiMYC2 gene in leaves was the highest,and CiMYC2 gene could be induced by MeJA.The pBI121-CiMYC2-GFP plant expression vector was constructed and transformed into tobacco by Agrobacterium-mediated method.The subcellular localization analysis of CiMYC2 protein acts on the nucleus.The positive tobacco transformants was selected using MS medium containing kanamycin resistance and PCR and RT-PCR analysis.Three transgenic lines with the highest expression were used for followed functional analysis.Overexpression of CiMYC2 gene in tobacco resulted in shorter plant height,darker leaf color,higher chlorophyll and carotenoid content,and the number of long and short glandular trichomes increased significantly.The relative content of terpenes of CiMYC2 transgenic tobacco lines increased significantly,and terpenes were detected in transgenic tobacco without WT and EV lines.The expression levels of HMGGR,DXR,GGPPS,MTS,and CCD genes were up-regulated in CiMYC2 transgenic tobacco lines,and the EAS gene was down-regulated The expression levels of DXS,GPS,FPS,and EAS genes were up-regulated only in individual transgenic lines.