Expression And Function Analysis Of Glucose-6-Phosphate Dehydrogenase Gene Family EcG6PDH In Eucalyptus Camaldulensis

The Oxidative Pentose Phosphate Pathway?OPPP?was an important metabolic pathway to regulate the normal growth,responde to different environmental stresses,and improve plant resistance to the stresses.Glucose-6-phosphate dehydrogenase?G6PDH?was a key rate-limiting enzyme that catalyzed the first step in OPPP to generate NADPH.Since G6PDH had important roles in OPPP,we cloned the four G6PDH genes from Eucalyptus camaldulensis,investigated their expression patterns and identify their functions under stresses.The main results were as follows:?1?Four novel G6PDH genes,EcG6PD1-EcG6PD4 were cloned from the E.camaldulensis.Bioinformatic analyses showed that these four EcG6PDH proteins contained highly conserved G6PD-C domain.The N-terminal of EcG6PD1 did not have signal peptide and belonged to cytosolic G6PDH,while the rest three EcG6PDH proteins belonged to plastidic G6PDH.?2?Transient EcG6PDs expression vector was constructed through Gateway system and transformated into tobacco plants.LCM observation showed that the expressed protein of EcG6PD1 was located in the plasma membrane,and the other three proteins EcG6PD2-EcG6PD4 were located in the chloroplast.?3?Expression patterns of EcG6PDH genes in different tissues and in response to abiotic stresses were analyzed by real-time quantitative PCR.We observed that EcG6PDH genes were ubiquitously expressed in the tissues of roots,stems,leaves and stem tips with variable transcript levels.EcG6PD2 had relatively high expression levels in roots,as compared to the other three tissues.In contrast,EcG6PD1 and EcG6PD3 was abundantly expressed in leaves,stems and stem tips,but not in roots.EcG6PD4 had relatively high expression levels in stem tips.In addition,the expression patterns of EcG6PD1-4 under 4°C,different temperatures,drought stress,NaCl stress and ABA treatments were also analyzed.The results showed that the expression of EcG6PD1-4 gene was significantly induced by low temperature and drought stress.Under NaCl treatment,the expression of EcG6PD3 was slightly up-regulated,while the expression levels of the other three genes were not significantly changed.under ABA treatment,the expression level of cytoplasmic EcG6PD1 did not change significantly,but the plastid type?EcG6PD2,EcG6PD3,and EcG6PD4?expression was inhibited.EcG6PD4 expression began to increase after 6 h,although it was suppressed within 6 h.?4?Plant expression vectors were constructed in order to verify the functions of the four G6PDH genes.Four expression vectors were transformed into Arabidopsis thaliana by Agrobacterium meditation.We compared wild-type A.thaliana?WT?,G6PDH over-expression system in Arabidopsis?G6PD1-4?and G6PDH-inhibited Arabidopsis T-DNA mutants?g6pd1-4?from the growth and physiological indicators.The results demonstrated that the transgenic plants overexpressing EcG6PD1-4 were significantly better than the WT plants and the muatant groups.The mutant g6pd2exhibited short and broad leaves,and oval shape,while the other three mutant groups were not significantly different from the WT plants in phenotypic growth.Compared with WT,the LT₅₀0 of G6PD1-4 plants decreased from-7.6°C to-9.77°C,-9.78°C,-8.99°C,-8.99°C,respectively,while the mutant LT₅₀0 was not significant difference from WT.Except G6PD2,the relative chlorophyll content and Fv/Fm values of the other three groups of G6PDH transgenic plants were higher than those of WT and mutants.The G6PDH enzyme activity of the transgenic line G6PD1-4 was increased to 2.56,2.44,2.59 and 1.83 times to that of WT,and the POD enzyme activity was increased by 61.2%,81.4%,59.7%and 94%respectively,and the SOD enzyme activity was increased by 32.5.%,42.1%,23.5%and 51.8%.Moreover,correlation analysis showed that G6PDH enzyme activity was significantly correlated with POD activity,SOD activity,chlorophyll fluorescence parameters and LT₅₀,indicating that overexpression of EcG6PDs gene could obviously increase plant G6PDH enzyme activity,then enhancing the activity of protective enzymes and photosynthetic capacity,finally enhancing the resistant ability of plants to stresses.In summary,the expression of EcG6PDs was induced by multiple stress conditions.Overexpression of EcG6PDs enhanced the cold resistance of Arabidopsis.The mode of the four genes were different.