Molecular Cloning And Functional Analysis Of Floral Scent Related Genes From Chimonanthus Praecox L.

Chimonanthus praecox L.(wintersweet) is a traditional fragrant plant that originates from China,and is a well-known winter-flowering deciduous shrub.This plant can be taken as a new source of natural essential oil.Terpenoids(terpenoids and sesquiterpenoids) and benzenoids are the major components of floral scent in C.praecox.In order to investigate the mechanism of biosynthesis and regulation of methyl salicylate and farnesyl pyrophosphate(FPP),this text presents the cloning and characterization of S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase(CpSAMT) gene and farnesyl pyrophosphate synthase(CpFPPS) gene.CpSAMT catalyzes the formation of methylsalicylate,an important floral scent compound in C.praecox,from salicylic acid and S-adenosyl-L-methionine(SAM).CpFPPS catalyzes the formation of FPP,the substrate of sesquiterpenoids,from isopentenyl pyrophosphate(IPP) and dimethylallyl pyrophosphate(DMAPP).The main results were as follows:1.Headspace analysis of floral scent in C.praecoxThe method of HS-SPME-GC-MS was applied to determine the volatile compounds in flowers of 6 C.praecox samples in a same environment.A total of 45 compounds were detected,of which 37 were identified and only 16 common to all 6 samples.The terpenoids and benzenoids dominated the scent for all samples.(E)-β-ocimene,linalool, benzyl acetate and methylsalicylate were the major compound in the scent.The results suggested qualitative and quantitative differences of volatile compounds occurred among samples.2.Molecular clone and functional analysis of CpSAMT gene1) According to the homologous sequences from other plants,degenerate primers were designed to amplify specific cDNA fragment from wintersweet flower.By RT-PCR and RACE,we isolated SAMT gene from wintersweet flower named CpSAMT,with the accession number EU106367.The full-length sequence of CpSAMT cDNA contained a total of 1498 nucleotides.It encodes an ORF of 1140 nt corresponding to a protein of 380 amino acids.Sequence analysis shows that CpSAMT exhibits the features of SAM dependent carboxyl methyltransferase and other plant carboxyl methyltransferase, including the SAM and substrate binding sites.Thus,it is most likely that we have isolated the wintersweet S-Adenosyl-L-methionlne:salicylic acid carboxyl methyltransferase gene. 2) For functional characterization of CpSAMT protein,the coding region of the gene was cloned into a prokaryotic expression vector pET-32a(+),and encoded protein was expressed in ArcticExpressTM(DE3) RP strain and analyzed for CpSAMT activity. Recombinant purified CpSAMT protein catalyzed the formation of methyl salicylate from salicylic acid and SAM.3) To examine the temporal expression of CpSAMT gene,real-time RT-PCR analysis was performed from five developmental stages of the wintersweet flower.The flower of stage 3 contained the highest levels of CpSAMT expression,whereas,in the stage 5,after the flower had been pollinated,these levels sharp dropped to 6%of stage 3.In addition, CpSAMT activity was analyzed by GC.CpSAMT activity increased gradually to achieve a maximum in stage 4,but,the activity remain high(approximately 82%of peak level) in stage 5.The expression pattern of CpSAMT was investigated in healthy leaves and different parts of flower.The highest levels of SAMT expression were observed in the petals,and stamens and healthy leaves had little discernible expression of CpSAMT. Maximal CpSAMT activity was observed in petals and absence in leaves.To examine the expression of C.praecox SAMT in damaged leaves,total RNA was extracted from damaged leaves at intervals of 0,2,4,6,8,12,and 24 h after wounding,and the RNA samples were used as template of real-time RT-PCR analysis.CpSAMT expression levels rose dramatically from 4 to 6 h after wounding,with peak expression evident after 6 h, decreasing afterward.Our results showing the induction of CpSAMT in wounded leaves point to the possible role of such methyltransferases in the defense response of leaves to injury.3.Molecular clone and functional analysis of CpFPPS gene1) The full-length cDNA sequence of FPPS was isolated from wintersweet flower (designated CpFPPS) using RT-PCR and RACE,with the accession number FJ415102, which contained a total of 1277 nucleotides.It encodes an ORF of 1077 nt corresponding to a protein of 359 amino acids.Sequence analysis shows that CpFPPS exhibits the features of prenyltransferase and other plant FPPS,including the high conserved first Asn-rich motif(FARM,DDXX(XX)D) and second Asn-rich motif(SARM,DDXXD). Thus,it is most likely that we have isolated the wintersweet farnesyl pyrophosphate synthase.2) For functional characterization of CpFPPS protein,the coding region of the gene was cloned into a prokaryotic expression vector pCold TF,and encoded protein was expressed in Rosseta gami(DE3) and ArcticExpressTM(DE3) RP strain and analyzed for CpFPPS activity.Recombinant purified CpFPPS protein catalyzed the formation of farnesol as a main product from IPP and DMAPP after the production was hydrolyzed by 4 N HCl.3) Real-time RT-PCR analysis showed that the flower of stage 4 contained the highest levels of CpFPPS expression,then,in the stage 5,after the flower had been pollinated, these levels sharp dropped.The expression pattern of CpFPPS was investigated in healthy leaves and different parts of flower.The highest levels of CpFPPS expression were observed in healthy leaves,in petals the level of CpFPPS was approximately 15% of peak level,and stamens and sepals had little discernible expression of CpFPPS.