Plant Terpenoids Metabolism In The Comparison Of The Key Enzymes And Its Expression In Salvia Miltiorrhiza Analysis

Terpenoids play important roles in plant growth, development and metabolism. Many terpenoids also have good pharmacological activity; they are widely used in medicine and health, industrial and agricultural production, such as taxol, artemisinin and tanshinones. Therefore, studying on identification and characterization of the key enzymes and the encoding genes in the plant terpenoid metabolic pathways is very important. Generally, the biosynthetic pathway of plant terpenoids can be divided into three stages. The first stage leads to the synthesis of the universal isoprene precursor ispentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In the second stage, the intermediate diphosphate precursors, including geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP), are synthesied. The last stage involves the formation of diverse terpenoids. In this paper, the key enzymes and the encoding genes in the first and second stages of plant terpenoid metabolic pathways have been analnyzed by the tools of bioinformatics in the following aspects:the physical and chemical chatacters of amino acid sequences, hydrophobicity or hydrophilicity, transit peptide, transmembrane topological structure, secondary structure of protein, molecular phylogenetic evolution and so on. The conservative and the variation pattern, in particular the commonness and characteristics of the key enzymes in plant terpenoid metabolic pathways have been found. This study will provide useful reference information for the follow-up study of gene function. In addition, the fat-soluble components of Salvia miltiorrhiza Bunge are also terpenoids, and the key biosynthetic enzymes of terpenes are particularly important, so this study also analnyzed expression pattern of HMGR and DXS gene family in S. miltiorrhiza by RT-PCR. The results are as followings:1. The first stage of the plant terpenoid metabolic pathways has four key enzymes, namely:HMGR, DXS, DXR and IPI. HMGR is a hydrophilic and transmembrane protein which lacks of signal peptide. The plant HMGR is synthesized on free ribosomes without protein teansport, directly involved in the biochemical reactions in the cytoplasm. The main motif of predicted seconday structure of HMGR are alpha helix and random coil; HMGR sequences in four functional domains are highly conserved, but the NADPH binding domain lose in rubber HMGR1, may be ralated to the function. DXS protein is a hydrophilic protein, no obvious hydrophobic structure, also transmembrane; DXS protein contains a chloroplast transit peptide, inferred after its synthesis in the cytoplasm, is transported to participate in the related reaction in the chloroplast, this result with the MEP pathway occurs in the plastid consistent. The main motif of predicted seconday structure of DXS are alpha helix and random coil. Multiple sequence alignment, found that its N-terminal peptide chain is not conservative, presumably, the change is caused by the non-conservative nature of the transit peptide. Combination of molecular phylogenetic tree found DXS protein is divided into three categories, the DXS1, DXS2were respectively clustered into two, the Arabidopsis DXS3and Zea mays DXS3for the third branch. Three categories in the conserved region of TPP binding region and the pyrimidine-binding region is conservative, but there are still differences between each type.DXR protein also is a hydrophilic protein, no obvious hydrophobic structure and transmembrane; DXR protein contains a chloroplast transit peptide, expect DXR in Nicotiana tabacum, Catharanthus roseus and Stevia rebaudiana. This inferred that after its synthesis in the cytoplasm, is transported to participate in the related reaction in the chloroplast. DXR protein secondary structure is also mainly composed of alpha helix and random coil, both of them the proportion is similarly. Multiple sequence alignment of DXR protein, the five functions conserved region is highly conserved, the main difference is the occurrence of the N-terminal of the peptide chain, caused by the transit peptide is not conserved. The DXR molecular phylogenetic tree was basically in line with plant taxonomy, gymnosperms and angiosperms are divided into two major categories.IPI is a hydrophilic and non-transmembrane protein with transit peptides, the main motif of predicted secondary structure of IPI are alpha helix and random coil; IPI protein sequence is highly conserved; non-conserved region in its N-terminal, combined with the transit peptide analysis, containing the transit peptide of the species, the IPI protein sequence is longer than the species does not contain the transit peptide, and the transit peptide is not a conservative district, may be associated with recognition specificity, no significant relationship with the secondary structure. The phylogenetic analysis reflects the phylogenetic relationship among the IPI in the different plants, and natural evolution are not the same, but have a certain reference to determine the phylogenetic relationships among different species.2. The second stage of the plant terpene biosynthetic pathway consists of three enzymes, respectively, GPPS, FPS and GGPPS, they are belong to the short-chain prenyl transferase. These three enzymes are hydrophilic and non-transmembrane protein. Transit peptide analysis found that the diverse distribution of the three proteins in the cell, indicating them broad participation in the cells of plant secondary metabolites biosynthesis. The main motif of predicted secondary structure of them are alpha helix and random coil, beta turn and extended strandare spreaded in the whole secondary structure of protein. Short-chain prenyl transfer enzymes contain two aspartate-rich region (DDXXD), the results about multiple sequence alignment, showed that the two aspartate-rich region is highly conserved in FPS and GGPPS, but these two regions in the GPPS less conservative. Arabidopsis FPS1and Ginkgo FPS is not conservative in them N-terminal, and multiple sequence alignment of GGPPS the non-conservation also in N-terminal. The classification results of the phylogenetic tree and biological systems evolution is not consistent, but may determine to provide a reference for the phylogenetic relationships among different species.3. HMGR and DXS expression level are detected by use the real-time quantitative RT-PCR in different orgens. The results showed that HMGR1is significantly higher in roots, and mRNA expression levels has significant different among HMGR1, HMGR2and HMGR3. HMGR2is significantly higher in leaves and stems, and mRNA expression levels has significant different among HMGR2, HMGR1and HMGR3. the data suggest that HMGR1is associated with the synthesis of terpene compounds in Salvia miltiorrhiza roots. In DXS gene family, DXS2is significantly higher in roots and leaves, DXS1is significantly higher in stems, and mRNA expression levels has significant different among others. To speculate that DXS2play an important role in synthesis of terpenoids of Salvia miltiorrhiza.