| Eucommia ulmoides Oliv. is a tertiary relic species,which is endemic to China andtraditionally utilized as a rare medicinal and high quality temperate rubber-producing tree.Terpeniods in E. ulmoides represented by the iridoids and gutta-percha have been plenty ofeconomic value and widely used in industry as well as to people's daily life. MEP pathway isone of the two elucidated upstream biosynthetic routes modulating the terpeniods biosynthesisin plant. Full length cDNA of high expression genes of completed set enzymes in MEPpathway were systematically isolated based on the transcriptome sequencing data of E.ulmoides leaves,afterwards the sequence structure and gene function were characterized andpredicted through bioinformatics techniques in the study,which will be conducive to dig uppotential key genes,excavate rate-limiting step in the pathway and to provide fundamentalinformation referring to metabolic engineering and gene improvement breeding of E. ulmoides.Two gene members coding the DXS enzyme which was identified as the first keyregulatory point in MEP pathway were separated from E. ulmoides leaves and designated asEuDXS1and EuDXS2. Respectively with highest gene sequence similarity to Mitragynaspeciosa (81%)and to Hevea brasiliensis(82%),EuDXS1full-length cDNA was2805bpincluding5'non-coding region of218bp and3'non-coding region of448bp and encoded712amino acids,EuDXS2was2552bp including5'non-coding region of73bp and3'non-codingregion of337bp and encoded713amino acids. Representative conserved motifs and functionalsites of plant DXS protein containing transit peptide sequence(A1-A18；A1-A34),TPP bindingmotif(A106-A360；A108-A362),pyrimidine binding motif (A397-A553；A399-A555)and TKC-terminal motif (A571-A676；A573-A678)were found in the deduced coding sequence ofEuDXS1and EuDXS2. Loop/coil mainly constituted the secondary structure of the predictedprotein with proportion of EuDXS1to48.74%and EuDXS2to49.37%. The calculated proteintertiary structure of EuDXS1and EuDXS2were both composed of two subunits. Evolutionary relationship of EuDXS1was closest to Ricinus communis DXS1protein,while EuDXS2toHevea brasiliensis DXS2protein.Two gene members coding the DXR enzyme which was identified as the secondrate-limiting step in MEP pathway were separated from E. ulmoides leaves and designated asEuDXR1and EuDXR2. Respectively with highest gene sequence similarity to Populustrichocarpa(81%)and to Vitis vinifera(81%),EuDXR1full-length cDNA was1814bpincluding5'non-coding region of126bp and3'non-coding region of478bp and encoded251amino acids, EuDXR2was1779bp including5'non-coding region of163bp and3'non-coding region of251bp and encoded472amino acids. Representative conserved motifsand functional sites of plant DXR proteins containing transit peptide sequence(A1-A51；A1-A43),two DXR binding moti(fA227-A236,A297-A307；A221-A230,A291-A301),two NADPH bindingmotif (A88-A94,A113-A119；A82-A88,A107-A113)and N terminal proline-rich motif (A61-A69；A55-A63)were discovered in the deduced coding sequence of EuDXR1and EuDXR2.Loop/coil mainly constituted the predicted protein secondary structure with proportion ofEuDXR1to47.91%and EuDXR2to45.34%. The calculated protein tertiary structure ofEuDXR1and EuDXR2were both composed of two subunits,which in space displaying"V"shape. Evolutionary relationship of EuDXR1was closest to Oryza latifolia DXR1protein,while EuDXR2protein to Zea mays DXR2protein.A gene coding the MCT enzyme which catalyzed the third enzymatic reaction in MEPpathway was separated from E. ulmoides leaves and designated as EuMCT. With highest genesequence similarity to Vitis vinifera(82%),the full-length cDNA of EuMCT was1435bpincluding5'non-coding region of223bp and3'non-coding region of252bp and encoded319amino acids. The transit peptide sequence(A1-A75)and multiple conserved functional sitesA100,A102,A103,A104,A105,A106,A114,A170,A171,A173,A176,A195,A196,A198,A228,A244,A250,A252,A300)of plant MCT protein were found in the deduced coding sequenceof EuMCT. Secondary structure of EuMCT protein was predicted with proportion of α-helix to23.82%,β-sheet to18.18%and loop/coil to57.99%.The calculated protein tertiary structure of EuMCT was composed of two subunits,which contained two special P-loop constitution.The evolutionary relationship of EuMCT protein was cloest to Vitis vinifera MCT protein.A gene coding the CMK enzyme which catalyzed the hydroxyl phosphorylate reaction inMEP pathway was separated from E. ulmoides leaves and designated as EuCMK. With highestgene sequence similarity to Lycopersicon esculentum(82%),the full-length cDNA of EuCMKwas1644bp including5'non-coding region of256bp and3'non-coding region of203bp andencoded394amino acids. The transit peptide sequence(A1-A57)and the essentialATP bindingsite (A186-A202)demanded in the catalytic process of plant CMK enzyme were found in thededuced coding sequence of EuCMK. Secondary structure of EuCMK protein was predictedwith proportion of α-helix to32.74%,β-sheet to19.29%and loop/coil to47.97%.Thecalculated protein tertiary structure of EuCMK was composed of two asymmetric subunits.Theevolutionary relationship of EuCMK protein was closest to Vitis vinifera CMK protein.A gene coding the MDS enzyme which catalyzed the fifth enzymatic reaction in MEPpathway was separated from E. ulmoides leaves and designated as EuMDS. With highest genesequence similarity(79%)to Ageratina adenophora,the full-length cDNA of EuMDS was976bp and encoded236amino acids with5'non-coding region of119bp and3'non-coding regionof146bp. The transit peptide sequence (A1-A56)and multiple conserved functional sites(A84,A87,A89,A121,A213,A217,A221,A223,A228)of plant MDS protein were found inthe deduced coding sequence of EuMDS. The secondary structure of EuMDS protein waspredicted with proportion of α-helix to40.25%,β-sheet to13.56%and loop/coil to46.19%.Three subunits which formed a molecular cavity composed of the calculated proteintertiary structure of EuMDS. The evolutionary relationship was more similar than other speciesbetween EuMDS protein and Humulus lupulus MDS protein.A gene coding the HDS enzyme which catalyzed the sixth enzymatic reaction in the MEPpathway was separated from E. ulmoides leaves and designated as EuHDS. With highest genesequence similarity to Vitis vinifera(84%),the full-length cDNA of EuHDS was2786bpincluding5'non-coding region of171bp and3'non-coding region of383bp and encoded743amino acids.The transit peptide sequenc(eA1-A30),PSN moti(fA58-A78),PSI moti(fA354-A620) and three absolutely conserved cysteine site(sA644,A647,A678)of plant HDS protein were foundin the deduced coding sequence of EuHDS. The secondary structure of EuHDS protein waspredicted with proportion of α-helix to37.55%,β-sheet to19.25%and loop/coil to43.20%.The N-terminal domain with an eight-stranded β barrel belonged to the large TIM-barrelsuperfamily and the C-terminal domain consisted of a β sheet flanked on both sides by heliceswere indicated in the calculated protein tertiary structure of EuHDS. The evolutionaryrelationship of EuHDS protein was closest to Vitis vinifera HDS protein.A gene coding the HDR enzyme which was identified as the third rate-limiting step inMEP pathway was separated from E. ulmoides leaves and designated as EuHDR. With highestgene sequence similarity to Camptotheca acuminata(82%), the full-length cDNA of EuHDRwas1653bp including5'non-coding region of82bp and3'non-coding region of188bp andencoded460amino acids. The transit peptide sequence(A1-A33) and multiple conservedfunctional sites(A117,A208,A262,A345)of plant HDR protein were found in the deduced codingsequence of EuHDR. The secondary structure of EuHDR protein was predicted with proportionof α-helix to35.65%,β-sheet to19.78%and loop/coil to44.57%.The calculated proteintertiary structure of EuHDR was composed of a monomer, which in space displayedasymmetrical shamrock-like shape.The evolutionary relationship of EuHDR protein wasclosest to Vitis vinifera HDR protein.A gene coding the IPI enzyme which catalyzed the reversible conversion between IPP andDMAPP and regarded as a hinge point in terpenoid metabolic networks was separated from E.ulmoides leaves and designated as EuIPI. With highest gene sequence similarity toCamptotheca acuminata(84%),the full-length cDNA of EuIPI was1231bp including5'non-coding region of79bp and3'non-coding region of231bp and encoded306amino acids.The transit peptide sequence(A1-A70)and other representative conserved motifs and functionalsites (A154,A175,A107,A119,A156,A196,A206,A208)of plant IPI proteins were found inthe deduced coding sequence of EuIPI. The secondary structure of EuIPI protein was predictedwith proportion of α-helix to22.55%,β-sheet to13.40%and loop/coil to64.05%. The calculated EuIPI protein tertiary structure was constituted by a monomer in space. Theevolutionary relationship of EuIPI protein was closest to Corylus avellana IPI protein.
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