Isolation And Characterization Of A U-Box Gene, GmPUB2, From Soybean

Ubiquitin-26S proteasomesystem (UPS) is one kind of widespread biological activities that identify proteins then degrade them, and influence the plant cell cycle, stress tolerance response, signal transduction, transcriptional regulation, DNA repair, cell apoptosis, secretory pathway and other processes. The Plant U-box protein is an important component of UPS that identify substrates, but we do not know too much about the function of these proteins in soybean. In this study we got a gene with a U-box domain from soybean cultivar 'Dabaima' and we named this gene as GmPUB2.Sequence analysis showed that the full length of GmPUB2 is 1885bp and it contained an open reading frame (ORF) of 1320 bp encoding 439 amino acids. Phylogenetic analysis based on the amino acids sequence of GmPUB2 gene with other species showed that the protein encoded by this gene had the closest relationship with Phvul.008G238500.1 in Phaseolus vulgaris and Medtr5g077510.1 in Medicago truncatula, with 87.5% and 82.0% amino acid identities, respectively. The promoter sequence 2023bp of'Dabaima' was cloned and many biologic and abiotic stress responsive elements related to mesophyll-specific expression, drought, salt, cold stress and virus were found by PLACE Databank. Real time-PCR analysis indicated that GmPUB2 had the higher expression level in the roots, leaves, flowers, pods and seeds than in the stems. GmPUB2 was up-regulated when subjected to the stresses including mechanical injury, salt, and low temperature but not soybean cystnematode infection. While ABA and SA can also cause to increasing expression of GmPUB2. Subcellular localization showed GmPUB2-GFP fusion protein was localized in the nucleus, cytoplasm and plasma membrane.We constructed transgenic lines of Arabidopsis by floral dip method to express GmPUB2 in Arabidopsis. The transgenic Arabidopsis roots grew faster, more sensitive to salt stress and grew brawny by increasing numbers of small-sized cells in the root apical part. The expression analysis in the transgenic Arabidopsis showed that expression levels of several genes related to cell cycle, AtCYCBl; 1, AtPINl, AtBRL3, AtAGL42, were increased. We inferred GmPUB2 possibly affect the cell cycle through regulation of auxin or brassinosteroid pathways, resulting in accelerating cells division in the root apical part.Transgenic Arabidopsis also appeared late bolting and late flowering in long day. The expression analysis of flowering genes in the transgenic Arabidopsis showed that the expression level of FLC which act as an inhibiting factor of flowering was improved while the expression levels of FT, CO, LFY which promote flowering were reduced, suggesting that GmPUB2 may be involved in the photoperiod and vernalization pathways to regulate flowering time.