The Study On Identification And Characteristics For NADP-Malic Enzyme Gene Family Of Populus Trichocarpa

NADP-malic enzyme(NADP-ME)exists in a wide range of metabolic pathways in plants,animals bacterial. NADP-ME catalyzes the oxidative decarboxylation of malate:L-malate+NADP+→pyruvate+CO2+NADPH+H+in the presence of a bivalent meatl ion (Mg2+,Mn2+). However, the analysis of the characteristic and function on woody plant gene family NADP-MEs are very few. In our research, we analyze NADP-ME gene family in Populus trichocarpa, a C3 tree and its expression patterns.1,Homologous searches of NADP-ME suggests that five PtNADP-ME genes exist in the genome of P. Trichocarpa. RT-PCR and DNA sequencing show that five PtNADP-MEs are all expressed genes in P. Trichocarpa. Construction of phylogenetic tree displays that five PtNADP-ME members belong to three evolutional branches of plant NADP-ME family.2,Semi-RT-PCR analysis suggests that all of PtNADP-ME genes have been expressed in stem,leaf and other tissues, but there is no tissue-specific. Under dark condition,PtNADP-ME1 and PtNADP-ME4 transcription levels present no change, and PtNADP-ME5 gene expression is slightly influenced by darkness. PtNADP-ME2 gene expression is increased by mechanical damage,and its level continues to rise. PtNADP-ME1 and PtNADP-ME5 have no obvious expression changes. However, there are observable differences in transcriptional expression of PtNADP-ME genes response to NaCl, PEG and mannitol stresses, respectively. PtNADP-ME4 and PtNADP-ME5 trascription levels are both increased under NaCl stress, whereas their transcription levels are all declined during mannitol stress. Compared with the control, their contents are increased several times.3,Through the E. coli original nuclear expression, we have been expressed and purified PtNADP-ME3 and PtNADP-ME5 fusion protein. Two GST-NADP-ME fusion protein optimal pH value, enzyme activity, enzyme kinetics constant and data analysis show that PtNADP-ME3 fusion protein optimal pH 7.7, PtNADP-ME5 fusion protein optimal pH 7.8. Different concentrations of malic acid and NADP highly influences the activity of PtNADP-ME3 and PtNADP-ME5 protease, the result shows that high levels of malic acid and NADP inhibit the activity of PtNADP-ME3 and PtNADP-ME5 fusion protease. Different metabolites make an diverse effect on the activity of PtNADP-ME3 and PtNADP-ME5 fusion protease. OAA, citric acid, strongly inhibits the activity of PtNADP-ME3 and PtNADP-ME5 enzymes. AcetylCoA, CoA, glucose 6-phosphate-3 and fumaric acid salt also slightly enhance PtNADP-ME3 enzyme activity. So PtNADP-ME3 and PtNADP-ME5 enzymes metabolic reactions function may be controlled by a variety of metabolites TCA cycle.