Identification And Characterization Of Genes Encoding Key Enzymes In Soybean Non-Mevalonate Pathway

Soybean is one of the most important economic crops in the world. However, various diseases and insect pests significantly effect the soybean production. Terpenoids play important roles in ecosystem, including in strengthening the disease-resistance of plants, helping plants resist insect pests and their enemies. In plants, there are two distinct and independent biosynthetic pathways for the synthesis of terpenoids: one is the classic cytosolic acetate/mevalonate (MVA) pathway; the other is the plastid 2C-methyl-D-erythritol-4-phosphate (MEP) pathway (also known as the mevalonate-independent pathway). Genes involved in MVA pathway have already been identified, whereas little is known about that of MEP pathway. In this study, we focus on the identification and characterization of the MEP pathway genes from soybean. The successful cloning of MEP pathway genes may provide valuable information for fully understanding terpenoids biosynthetic pathway and its molecular mechanisms; such information could also be of critical importance in genetic manuscription of the terpenoids biosynthetic pathway. The results were listed as follows:1. 1-deoxy-D-xylulose-5-phosphate synthase (DXS) catalyses the first committed step of the MEP pathway. In this work, a DXS1-like cDNA (GmDXS1) was isolated from soybean. Sequence alignment showed that GmDXS1 had high homology to known DXS proteins from other plant species and contained the conserved N-terminal plastid transit peptide, the N-terminal thiamine binding domain and pyridine binding DRAG domain. Phylogenetic analysis indicated that GmDXS1 belonged to the plant DXS1 cluster. Southern blot analysis indicated that a single copy of GmDXS1 gene existed in soybean genome. Tissue expression analysis revealed that GmDXS1 expressed in all photosynthetic tissues except pod walls and roots. Moreover, GmDXS1 gene expression could be induced by temperature. Green fluorescence analysis with the fusion protein 35S:GmDXSl:GFP suggested that GmDXS1 was localized in plastid. The relatively higher photosynthetic pigment content in transgenic tobacco leaves compared to the control implied that GmDXS1 catalyzed the first potential regulatory step in photosynthetic pigment biosynthesis via the MEP pathway.2. 1-Deoxy-D-xylulose5-phosphate reductoisomerase (DXR) is the second enzyme in the MEP pathway. We have identified two distinct DXR-like cDNAs designated GmDXR1 and GmDXR2 from soybean. The deduced GmDXRs showed extensive sequence similarities to that of other plant DXRs and both of them contained the conserved N-terminal plastid transit peptide, the proline-rich region, two NADPH-binding sites and two DXR-binding sites. However, both GmDXRs shared 74% identity at the amino acid levels. Phylogenetic analysis suggested that GmDXR1 and GmDXR1 clustered to different branches. DNA gel blot analysis revealed that both GmDXRs contained two copies in soybean genome. Semi-quantitative analysis showed that the two GmDXRs genes are differentially expressed in various tissues. GmDXR1 could be expressed in all tissues except pod walls, whereas GmDXR2 expression could not be detected in any tissues. Therefore, we suggest that GmDXR2 expression could be induced by stress. Compared to GmDXR1, high temperature can induce expression of GmDXR2. Green fluorescence analysis with the fusion protein 35S:GmDXR1:GFP and 35S:GmDXR2:GFP suggested that both GmDXRs were localized in plastid. And the photosynthetic pigment content and GA content in transgenic tobacco that overexpressing GmDXR1 and GmDXR2 are much higher compared to that of wild-type plants (p<0.05), while the ABA content is significantly surpressed by the GmDXRs overexpression..3. We isolated and characterized a terpene synthase gene GmTPS from soybean. The deduced amino acid sequence possessed characteristic amino acid residues DDxxD motif, which are highly conserved among terpene synases, and N-terminal plastid transit peptide. Phyogenetic tree analysis showed that GmTPS had closest relationship with monoterpene synthases that from snapdragon, indicating that GmTPS belonged to TPS-g subfamily. Southern blot analysis indicated that a single copy of GmTPS gene existed in soybean genome. Tissue expression pattern analysis revealed that GmTPS expressed strongly in leaves, stems and pod walls, no expression could be detected in roots, flowers and seeds. Expression profiles under different elicitor treatments such as high temperature, cold, mechanical wounding, SA and S. litura were compared, and the results revealed that cold, mechanical wounding and SA can induce GmTPS gene transiently expression. However, high temperature and S. litura did not result in significant induction. Gateway system was used to make the plant overexpressing construct. Green fluorescence analysis with the fusion protein 35S:GmTPS:GFP suggested that GmTPS was localized in plastid. Bacteriostatic and insect-resistant studies reviewed that transgenic plants inherit the GmTPS gene could significantly inhibit the growth of microbes, especially fungi and S. litura larvae. Furthermore, we discussed the proposed ’thermotolerance hypothesis’, which explains the role of isoprene emission for the plant is to prevent leaf metabolic processes from thermal.