Isolation And Characterization Of Uridine Diphophate Glucose Dehydrogenase Gene From Larix Gmelinii

Dahurian larch,Larix gmelinii(Rupr.),is a deciduous tree of Pinaceae,which is the major forest tree species for afforestation in Great Khingan.L.gmelinii has extensive application in building,decoration,papermaking and chemical fiber industry owing to its high-quality timber.UDP-glucose dehydrogenase(UGDH)is the key enzyme in polysaccharide metabolic pathway,which catalyzes UDP glucose(UDP-Glc)oxidation generating UDP-glucuronic acid(UDP-G1cA).UDP-G1cA is the direct precursor of UDP-galacturonate,UDP-Dxylose,UDP-arabinose and UDP-apiose,which are glycosyl donor substrates for hemicellulose and pectin biosynthesis.UGDH expression abundance influence biosynthesis of cell walls,which affect plant growth and wood formation.Therefore,the study on expression regulation mechanism of the UGDH gene has important significance.In the study,the ortholog of UGDH was isolated from L.gmelinii,referred to as LgUGDH2,using degenerate-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE),and function and expression characteristics of the gene was analyzed.The results showed that the 1795 bp of the LgUGDH2 cDNA contains 1449-bp of open reading frame(ORF),108 bp of 5'-untranslated region(UTR)and 238 bp of 3,-UTR,which encodes a protein of 482 amino acids.Real-time fluorescent quantitative-PCR(real time Q-PCR)analysis showed that the LgUGDH2 expressed in all organs of the larch plants with higher expression in stems than in leaves or roots.For further investigating the expression mechanism of the LgUGDH2,2091 bp of LgUGDH2 promoter sequence was cloned using the tail-PCR,LgUGDH expression patterns was analyzed using the GUS reporter gene.Sequence analysis revealed that the LgUGDH2 promoter region contains some elements responding to plant hormones including gibberellin,ethylene,auxin,cytokinins and abscisic acid,photoinducible elements and abiotic-stress response elements such as low temperature and drought.GUS staining experiments indicated that the LgUGDH majorly expressed in the vascular tissues of stem and leaf,corresponding to its function.In order to further analyze functions and heterologous expression features of the LgUGDH2,pBl101-LgUGDH2 was constructed,and transgenic Arabidopsis were obtained via agrobacterium-mediated genetic 'transformation.The results of RT-PCR and enzyme activity detection confirmed the stable expression of LgUGDH2 in transgenic A.thaliana.Phenotypic analysis showed that the excessive expression of LgUGDH2 enhanced the vegetative growth,increased the contents of soluble sugars,and promoted the accumulation of hemicellulose and pectin in transgenic plants.The research results show that growth and development of plants,as well as timber improvement,can be regulated through the genetic modification of UGDH,the key enzyme in the synthesis pathways of hemicellulose and pectin,These results provided basis for further understanding the expression and regulation mechanism of LgUGDH gene,and will likely be useful for timber improvement of forestry trees.