Expression Analysis Of Soybean SUMO System Under Salt,Heat And ABA Stress And Substrates Prediction

The small ubiquitin-like modifier(SUMO)proteins of approximately 100-115 amino acid,which are structurally similar to ubiquitin.SUMOylation is one of the important regulatory mechanisms in the process of post-translational modification of proteins.SUMO proteins reversibly bind to specific target substrates to regulate multiple processes in eukaryotic cells.SUMO has shown to regulate various nuclear processes,including transcriptional control,subcellular trafficking,and regulation of the cell cycle.SUMO conjugating was reported to be essential in Arabidopsis,and an increasing number of studies have demonstrated that SUMOylation has significantly role in plant growth and development,hormone signal transduction,fungi resistance and defense,and response to abiotic stress.However,the important mechanism of SUMOylation in the major legume crop,soybean,remains obsure.In this study,the amino acid sequences of the known pathway genes from Arabidopsis were used as queries to search members of SUMO system in soybean genome.Homologous proteins alignment,characteristics sequences,functional domains and SUMO interaction sites were analyzed to further identification of soybean genes related to SUMOylation process.In order to predicted the evolution and the relationship among species,multiple sequence alignment of SUMO pathway components in soybean,Arabidopsis,polar,rice,maize,tomato,human and yeast were carried out using clustalW software and phylogenetic trees were constructed by MEGA.The cis-acting elements analysis revealed that 1500 bp upstream of SUMO pathway genes contained different combinations of stress and development-related cis-acting elements.Tissue-specific expression patterns of SUMO pathway genes were analyzed using RNA-Seq data.qRT-PCR was performed to analyze the expression profiles of key SUMO pathway members under NaCl,heat and ABA stimuli during the 24 h period of treatment.Furthermore,SUMOylation dynamics in soybean roots under abiotic stress treatment were analyzed by western blot,which were characterized by regulation of SUMOylated proteins.In addition,the soybean SUMOylation substrates were predicted and the possible biological processes were analyzed.Besides,soybean SUMOylation system was reconstructed in the E.coli,and SUMOylation of candidate SUMO substrate protein in soybean was validated in vitro.Main results of this study are as follows:1.Using homolog searches and domain confirmation,a total of 40 genes encoding SUMO pathway members were found in soybean.The six family groups include SUMO,activating enzyme E1,conjugating enzyme E2,ligase enzyme E3,SUMO protease and SUMO chain binding protein.Besides,functional domain and protein characteristic analysis showed that the members of each family were relatively conserved.Furthermore,subcellular location prediction of these proteins proved that most of the members were located in the nucleus,which was consistent with the functional localization of SUMOylation process.2.Promoter analysis showed that soybean SUMOylation genes have cis-acting elements that respond to abiotic stress and plant hormone signal,and the plant growth and development related elements.In order to study the expression regulation of these SUMOylaiton members,RNA-Seq data at different developmental stages of soybean was analyzed.The results showed that SUMO system members exhibited different tissue-specific expression and most of the members had high expression levels in roots.Therefore,the root tissues under stress treatments were selected as plant materials in the subsequent experiments.3.qRT-PCR was used to study the expression changes of 13 SUMO members in soybean roots after treated with NaCl(150 mM),heat(42?)and ABA(100 ?M)stress for different time points.The results indicated that the members of the soybean SUMO system responded to abiotic stress treatment in the transcriptional level and most of them were up-regulated under treatments.Western blot analysis of SUMO conjugates using anti-AtSUMO1 antibody showed that the SUMO conjugates of soybean roots increased significantly under high salt,heat and ABA treatment at the early stages..And the SUMO conjugates gradually decreased to the initial levels with time.4.679 unique proteins were identified as SUMO substrate candidates after in silico analysis.These proteins were involved in transcription,related to stress and defence and signaling process etc.Key SUMOylation members and candidate substrate proteins in soybean were expressed using E.Coli system.The conditions of induced expression in vitro were optimized.The competent cells simultaneously expressing GmSUMO,GmSAE1/2 and GmSCE proteins were produced.which the candidate SUMO target protein GmMLP423 were transformed into the competent cells,and using anti-AtSUMO1 and anti-Trx antibody showed it can SUMOylation in vitro.