Isolation And Characterization Of UDP-Glucose Pyrophosphorylase And UDP-Glucose Dehydrogenase Genes From Larix Gmelinii

Dahurian larch, Larix gmelinii (Rupr.), is a deciduous tree of Pinaceae, which presents a large proportion of coniferous forests in northeastern China, including Inner Mongolia forest region, and alpine region of northwest and southwest China. As one of three major timber conifers in northeast China, which has high wood density, strong mechanical properties, and a clear wood texture, the larch showed important economic value in the construction, decoration, paper production, and chemical fiber industries, because of long fiber cells and low content of nonfiber cell. Therefore, it has an important significance to develop the wood formation-related genes and to improve growth and timber qualities by controlling expression of wood formation-related genes of L. gmelinii.The cell wall consists of a large part of different polysaccharides, including cellulose, hemicellulose, pectin and callose. UDP-glucose pyrophosphorylase (UGPase), an important regulatory enzyme in cellulose and callose biosynthesis, catalyzes the reversible production of glucose1-phosphate (Glc-1-P) and the conversion of uridine triphosphate to uridine diphosphate glucose (UDP-Glc) and pyrophosphate. UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-Glc to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of hemicellulose and pectin. As the key eneymes in cellulose and hemicellulose biosynthesis, UGPase and UGDH affect wood formation of trees by regulating polysaccharide meatablism.In the study, the orthologs of UGPase and UGDH were isolated from larch (L. gmelinii) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), referred to as LgUGPase and LgUGDH, respectively. LgUGPase, contains1443-bp open reading frame, encodes a protein of480amino acids with a predicted molecular weight of53.7kDa and isoelectric point of5.41. The1904bp full-length cDNA of LgUGDH encodes a protein of480amino acids with a predicted molecular weight of52.62kDa and isoelectric point of5.74. Phylogenetic analysis indicated that LgUGPase and LgUGDH belong to a clade of gymnosperms, which showed striking sequence homology with UGPase and UGDH of Picea sitchensis, respectively. Semiquantitative RT-PCR analysis shows that LgUGPase and LgUGDH were expressed in all organs of the larch plant with higher expression in stems than in leaves or roots. Southern blot analysis indicated that LgUGPase and LgUGDH are encoded by two genes in the L. gmelinii genome, respectively.To investigate the function and expression characteristics of LgUGPase and LgUGDH, homozygous lines for transgenic arabidopsis were obtained by the simplified in-plant infiltration method. Phenotype analysis of transgenic arabidopsis indicated that overexpression of LgUGPase and LgUGDH enhanced vegetative growth and increased the contents of soluble sugars and cellulose in transgenic arabidopsis. Histological observation showed that the parenchyma cell walls were thickened in leaf veins and stems of transgenic Arabidopsis. In addition, overexpression of LgUGPase and LgUGDH increased the contents of cellulose and hemicellulose in transgenic arabidopsis, respectively.These results showed that overexpression of LgUGPase and LgUGDH, the key eneymes in cellulose and hemicellulose biosynthesis, affect contents of soluble carbohydrates, cellulose and hemicellulose, growth and timber quality of transgenic plants by regulating polysaccharide meatablism, which would provide theoretical and practical proof for devoloping genetic resources correlate to wood formation, and for improving growth and timber qualities by controlling expression of wood formation-related genes of L. gmelinii, including LgUGPase and LgUGDH.