Molecular Cloning, Characterization And Functional Analysis Of The New Genes Involved In The Biosynthetic Pathway Of Terpenoid Indole Alkaloids From Rauvolfia Verticillata

Plant secondary metabolites which are not essential of growth and development for plant were a large group of micromolecular organic compounds all that play irreplaceable important roles in improving autoprotection and competitiveness to survive,and coordinating plants with environment. Alkaloids have multiple physiologic functions and are effective constituents in medicinal plants, such as anti-tumor camptothecin,antihypertensive reserpine,anticholinergic sopolamine and so on.There are two upper stream pathways for synthesis terpenoid indole alkaloids,shikimic acid pathway and methylerythritol phosphate（MEP）pathway.To research the molecular mechanism and provide targets for metabolic engineering of Rauvolfia verticillata,we cloned and characterized the MECS,HDR and TDC genes involved in the biosynthetic pathway of terpenoid indole alkaloids.2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase（MECS,EC:4.6.1.12）,the fifth enzyme of MEP pathway,converts CDP-MEP to ME-cPP.The important function of MECS has been confirmed in cantharanthus roseus culture cells.We cloned the cDNA via RACE technology, bioinformatics and tissue expression pattern analysis.The new cDNA was named as RvMECS and submitted to GenBank（R）to be assigned with an accession number EU034700.The full-length cDNA of RvMECS was 1011 bp containing 717 bp open reading frame encoding a polypeptide of 238 amino acids.Comparative and bioinformatics analysis showed RvMECS was highly homology with MECSs from other plant species.The active site residues in MECS from Escherichia coli,such as D8,H10,and H42(Zn²⁺ligands),and E135(Mg²⁺or Mn²⁺-binding site)were conserved in RvMECS and also found in all aligned plant MECSs.Subcellular localization predicted that RvMECS contain 57 aa plastid transport peptides.Phylogenetic analysis revealed that MECSs divided into two groups including plant and bacterial;RvMECS belonged to angiosperm MECS family.The structural modeling result showed that the 3-D structure of RvMECS was composed of a large four-strandedβ-sheet,a small two-strandedβ-sheet and four a-helices and contains three domains:zinc-binding site,CDP-binding sites and homotrimer interaction site.Tissue expression pattern analysis showed that RvMECS expression could be detected in leaves and flowers but could not in stems,roots and fruits,and the expression level in leaves was highest,followed by in flowers.1-Hydroxy-2-methyl-2-（E）-butenyl 4-diphosphate reductase（HDR,EC 1.17.1.2）,the last enzyme of MEP pathway,converts 1-hydroxy-2-methyl-2-（E）-butenyl 4-diphosphate to isopentenyl diphosphate（IPP）and dimethylaUyl diphosphate（DMAPP）at fixed ratio 5:1.IPP and DMAPP are the common precursors for natural terpenoids.We cloned the cDNA via RACE technology, bioinformatics and tissue expression pattern analysis.The new cDNA was named as RvHDR and submitted to GenBank（R）to be assigned with an accession number EU034699.The full-length cDNA of RvHDR was 1702 bp containing 1389 bp open reading frame（ORF）encoding a polypeptide of 462-amino-acids.Comparative and bioinformatics analysis showed RvHDR was highly homology with HDRs from other plant species.The RvHDR included four conserved cysteine domains. Subcellular localization predicted that RvHDR contain 34 aa plastid transport peptides.Phylogenetic analysis revealed that HDRs divided into two groups including plant and bacterial;RvHDR belonged to angiosperm HDR family.Tissue expression pattern analysis showed that RvHDR expression could be detected in leaves,fruits and stems but could not in flowers and roots.The expression level in leaves was highest,followed by in fruits and stems.After transformation with RvHDR,the E.coli ispH mutant strain MG1655 ara<>IspH was able to grow on LB media containing 0.2%Glc;as a control,with the empty vector（pQE）alone,the resulting strain was not able to grow on media containing 0.2%Glc.The results confirm that the RvHDR protein has the HDR enzymatic activity.Tryptophan decarboxylase is an important enzyme of shikimic acid pathway,catalysing tryptophan to tryptamine which is one of the important precursors.This catalytic reaction is a crucial step in YIAs biosynthesis.We cloned the cDNA using RACE technology,bioinformatics and tissue expression pattern analysis,and functional analysis.The new cDNA was named as RvTDC and submitted to GenBank（R）to be assigned with an accession number EU684270.The full-length cDNA of RvTDC was 1802 bp containing 1500 bp open reading frame encoding a polypeptide of 499 amino acids.Comparative and bioinformatics analysis showed RvTDC was highly homology with TDCs from other plant species.Tissue expression pattern analysis showed that RvTDC expression could be detected in leaves,fruits,flowers and roots but could not in stems.The expression level in fruits was highest,followed by in roots and leaves,the lowest in flowers.Expressive levels of TDC were increasing to some extent in callus cells after being treatment by elicitors（methyljasmonate, acetylsalicle acid and abscisci acid）and UV.Reconstructed TDC enzyme in Prokaryotic bacteria （about 55 KD）was purified using affinity chromatography.The reconstructed TDC enzyme can convert tryptophan to tryptamine,so the reconstructed TDC has enzymatic activity.Alkaloids extracted from five various tissues including roots,stems,leaves,flowers and fruits, and then detected the reserpene content via HPLC.The content of reserpine in leaves was highest, followed by flowers but could not in stems.The cloning,characterization and functional analysis of RvMECS,RvHDR,and RvTDC will be helpful to understand more about the roles involved in R.verticillata TIAs biosynthesis at the molecular level and provides a candidate genes for metabolic engineering of the TIAs pathway in R. verticillata.