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| Cloning and Expression Analysis of 1-deoxy-D-xylulose 5-phosphate Reductoisomerase Gene (DXR) in Tripterygiun wilfordii |
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Abstract 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the key enzyme of terpenoid metabolic pathway in 2-C-Methyl-D-erythritol 4-phosphate (MEP) pathway in plant, and its expression level affects the contents of terpenoid. A new putative gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase (designated as TwDXR), which catalyses the second step in the 1-Deoxy-D-xylulose 5-phosphate (DXP) biosynthetic pathway, which converts DXP to 2-C-Methyl-D-erythritol 4-phosphate (MEP), as an important role in regulating the MEP pathway was isolated from young leaves of Tripterygiun wilfordii by RT-PCR (reverse transcription polymerase chain reaction) and rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of the putative TwDXR was 1 674 bp containing a 1 410 bp open reading frame (ORF) encoding a polypeptide of 469 amino acids (GenBank accession No. KJ174341). Comparative and bioinformatic analysis showed that TwDXR had extensive homology with DXRs from other plant species, such as Populus trichocarpa. Tissue expression pattern analysis revealed that the putative TwDXR and 3-hydroxy-3-methylglutaryl-CoA reductase gene (TwHMGR) which catalyzed the conversion HMG-CoA to mevalonate (MVA), were constitutively expressed in all the tested tissues. The expression of TwDXR was strong in leaf, and TwHMGR was strong in adventitious root. The putative TwDXR and TwHMGR was found to be an elicitor-responsive gene, which could be induced by AgNO3(Ag+) and methyl jasmonate (MeJA). TwDXR was highest on 4th day, and TwHMGR was highest on 2nd day after inducing by 30 mmol/L AgNO3(Ag+). TwDXR and TwHMGR were highest on 2nd day after inducing by 50 mmol/L methyl jasmonate (MeJA).The highest accumulation of the triptolide was on 4th day and and wilforine was on 6th day after 30 mmol/L AgNO3(Ag+), respectively. The triptolide and wilforine were induced and accumulated highest content on 6th day after 50 mmol/L MeJA. These results will help for understanding the role of DXR and HMGR in isoprenoid biosynthesis, especially in promoting the content of active secondary metabolites in T. wilfordii.
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Received: 16 September 2013
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