Cloning And Functional Analysis Of UDP-glucose Pyrophosphorylase Gene Family From Populus Euphratica And Populus Pruinosa

UDP-glucose pyrophosphorylase （UGPase EC2.7.7.9） is one of the most important enzymes involved in sugar metabolism in organisms. It exists extensively from prokaryotes （eg:E.coli） to eukaryotes（eg:fungi plants and animals）. It catalyzes the reversible synthesis of UDP-glucose which serves as precursors for biosynthesis of sucrose and cell wall in plant cells.In this study, we blasted three other homologous genes respectively in Populus euphratica and P. pruinosa genomes （data from our lab） using a positively selected UGPase-function-like gene in the genus. Through designing primers in the upstream and downstream of the Untranslated Regions, we used callus tissues and fresh leaves of Populus euphratica and Populus pruinosa as material for CDS cloning of above-mentioned four genes. They are named UGP1, UGP2, UGP3 and UGP4.After cloning RNA sequences from different individuals of two species, we found that there are no stable amino acid differences in the UGP1/UGP2 between P. euphratica and P. pruinosa. These two genes are highly homologous and they encode two proteins with same molecular size. In addition, UGP2 alternatively splice two introns compared to the CDS sequence annotated from genome. Three different RNA haplotypes of UGP3 were cloned by two different pairs of Primers. Among them, haplotypes UGP3Y₁ and UGP3Y₂ have no stable amino acid between P. euphratica and P. pruinosa while UGP3W has five stable amino acid differences between P. euphratica and P. pruinosa. There is a site deletion mutation in UGP3Y₂, which leads to shifting of open reading frame and ends up with early termination of protein translation. UGP3Y₁ have an alternative splicing in the 3’CDS which results in ten amino acid differences in the C-terminus compared to UGP3W. There are two RNA haplotypes of UGP4 in both P. euphratica and P. pruinosa. One haplotype has an alternative splicing of an intron which includes a termination codon. This alternative-splicing haplotype has a shorter protein than the other haplotype.After constructing a phylogenetic tree with the sequences of UGP gene family of P. euphratica, P. pruinosa, P. trichocarpa and Arabidopsis, we found out that the UGP1/UGP2 derived from the independent duplications within the genus Populus. The UGP3 genes are totally specific to the genus and no closely related homologous genes were found in Arabidopsis. It may have developed after genome duplication of the genus Populus or even earlier. Neofunctionalization or subfunctionalization may have occurred for this gene.We simulated the protein 3D structure of three RNA haplotypes of UGP3 using the crystal 3D-structure of AtUGPase-A （PDB ID:2ICY） as template. The common N-terminal structure was predicted to be a ribosomal protein S2, which is involved in the formation of translation initiation complex. The conserved central catalytic domain was found in all these three different haplotypes. These three haplotypes differ in the C-terminal domain. UGP3Y₂ lacks the entire C-terminal domain when UGP3W differs slightly in the last ten amino acids in C-terminus compared with UGP3Y₁. Previous studies have already demonstrated that the C-terminal domain is closely related to the dimerization and multimerization of UGPase and the monomer serve as the active form of UGPase in plants. We speculate that in P. euphratica and P. pruinosa, the activity of UGP3 proteins is improved by regulating the relative contents of these three different transcriptional haplotypes.For better understanding the relationship of UGP gene family and salt tolerance of P.eu. We picked up nine one-year-old P. euphratica seedlings, and applied salt stress to them to three different concentrations of Nacl for three weeks:control （OmM Nacl）, low salt stress （150mM Nacl） and high salt stress （250mM Nacl）. We collected the leaves, shoots and roots of these seedlings as materials for Real-time PCR. After specific RT-primer design for four UGP genes respectively, we conducted Real-time PCR reaction for each one of them.UGP1 and UGP2 genes are found to have similar response model to salt stress:transcriptional levels up-regulated in shoots and leaves and down-regulated in root. Three different RNA haplotypes of UGP3 react differently in different tissues, with UGP3W up-regulated in shoots and roots, UGP3Y₁ down-regulated in shoots and roots and UGP 3 Y 2 up-regulated in leaves and down-regulated in shoots and roots. They may be involved in salt tolerance of P. euphratica in different tissues. UGP4 show no obvious transcriptional change in leaves, shoots and roots. It may not be involved in salt tolerance in P. euphratica. Moreover, the relative transcriptional level of four UGP genes in different tissues in P. euphratica （data obtained from transcriptome data in our lab） show that UGP1 and UGP2 mRNA are much more abundant than that of UGP3 and UGP4. Given the high homology and similar response model to salt stress of UGP1 and UGP2, we speculate that UGP1 and UGP2 intercoordinate and together promote the normal growth and development of P. euphratica.