Genes From Biosynthetic Pathways In Tripterygium Wilfordii Cloning And Expression Analysis

A new putative gene encoding1-deoxy-D-xylulose5-phosphate reductoisomerase（designated as TwDXR）, which catalyses the second step in the1-Deoxy-D-xylulose5-phosphate （DXP） biosynthetic pathway, which converts DXP to2-C-Methyl-D-erythritol4-phosphate （MEP）, as an important role in regulating the MEP pathway was isolated fromyoung leaves of T. wilfordii by rapid amplification of cDNA ends （RACE） for the first time.The full-length cDNA of the putative TwDXR was1,709bp containing a1,410bp openreading frame （ORF） encoding a polypeptide of469amino acids. Comparative andbioinformatic analyses revealed that TwDXR showed extensive homology with DXRs fromother plant species. Tissue expression pattern analysis revealed that the putative TwDXR wasconstitutively expressed in all the tested tissues and strong in leaf, moderate in root and stem,weak in hairy root, which was in contrast to TwDXR in adventitious root. TwHMGR wasexpressed strongly in adventitious root, moderate in hairy root and leaf, weak in stem. Theputative TwDXR and TwHMGR were found to be an elicitor-responsive gene, which could beinduced by exogenous elicitors, including AgNO₃（Ag⁺） and methyl jasmonate （MJ）. Theseresults will help in understanding the role of DXR in isoprenoid biosynthesis in T. wilfordii.1) After induced by AgNO₃（Ag⁺）, the transcription of TwDXR level reached the highestlevel after4d of the treatment,4.2-fold of the control level. TwHMGR transcription levelreached the highest level after2d of the treatment,4.3-fold of the control level.2) After induced by methyl jasmonate （MJ）, TwDXR transcription level reached thehighest level after2d of the treatment,3.9-fold of the control level. TwHMGR transcriptionlevel reached the highest level after2d of the treatment,2.3-fold of the control level.3)After induced by AgNO₃（Ag⁺）,,the highest content of metabolites triptolide andwilforine were28.61μg/g （2.7-fold of control） and100.36μg/g （3.3-fold of control）,respectively.4) After induced by methyl jasmonate （MJ）, the highest content of triptolide andwilforine were36.32μg/g （2.6-fold of control） and149.20μg/g （4.1-fold of control）,respectively. Preliminary results demonstrated terpenoids biological metabolic pathways oftripterygium wilfordii were regulated by DXR and HMGR gene. The results createdfoundation for bioengineering the secondary metabolics and improving contents of terpenoidsin tripterygium wilfordii in the future...