UPL Gene Family Analysis And Intergrated Investigation Of Transcriptome And Proteome Of Apple Under Abiotic Stress

Ubiquitination widely exists in eukaryotes,participates in diverse cellular processes including the adaptation of abiotic stress.The HECT ubiquitin-protein ligase(UPL),one type of E3 s,is characterized as containing a conserved HECT domain.UPL plays an important role in the protein ubiquitination.Studies on organization and roles of UPLs in tree plants,especially in fruit trees are still limited.In this study,members of MdUPLs were identified in apple genome,and their sequence features,functional domain and abiotic stress response were analyzed.We also performed transcriptome and proteome analysis under drought,salt and cold stress using RNA-seq and i TRAQ technologies on Malus sieversii.These results provided more bases for understanding the molecular mechanisms of abiotic stress tolerance in apple.This study would be significant to the abiotic stress response rule research,plant wild resources protection and plant stress resistant breeding.The main results were showed as the following:(1)13 MdUPLs were identified in apple genome.Two strategies,BLASTp and Hidden Markov Model(HMM)profile were used to obtain 13 UPL genes in apple genome.The 13 MdUPLs were mapped to ten different chromosomes,named MdUPL1-13.The peptides ranged in length from 411 amino acids to 4239.The subcellular location prediction showed that 7MdUPLs were present in the nucleus and 4 were in the plasma membrane.One Md UPL was predicted to be located in chloroplasts and one in cytosol.This result suggested that UPLs may have dissimilar functions in different organelles.(2)Analysis of the functional domains in MdUPLs.Three conserved HECT motifs were identified in the HECT domains of MdUPLs.They all formed alpha helixes and made up a unique spatial structure of HECT domain.A conservative cysteine residue was located in the central of the unique spatial structure of HECT domain.The cysteine residue in the HECT domain played a key role in generating an intermediate of Ub-E3 in the ubiquitination pathway.The 3 motifs and the unique spatial structure may play important role on ubiquitination.5MdUPLs only had a HECT domain.And there were 5 domains were identified in 8 MdUPLs.The Ub-associated(UBA)domain and Ub-interacting motif(UIM)domains were always existed together in 4 MdUPLs.Armadillo-repeat(ARM)domain existed in 3 MdUPLs.F-box domain only existed in the N-terminal region of MdUPL7.Ubiquitin homologues(UBQ)domain existed in MdUPL3 and 7.(3)MdUPLs were involved in abiotic stress response.Several abiotic stress-related cis-acting elements were detected in the promoters of 13 MdUPLs,suggesting MdUPLs may be regulated by corresponding transacting factors on stress response.Almost MdUPLs were induced to varying degrees under abiotic stresses,including drought,salt,or cold.In particular,the relative transcript levels of MdUPL7 and MdUPL9 were both up-regulated bigger than 15-fold,compared with the room temperature control.MdUPL7 and MdUPL9 were also significantly up-regulated under drought treatment.These results suggested that MdUPL7 and MdUPL9 maybe stress-sensitive genes with a potential role in cold and drought acclimation.However,MdUPL4 and MdUPL6,which are homologous to the up-regulated gene AtUPL3 under drought stress,were down-regulated in qRT-PCR assays.This indicates that the expression patterns of UPL genes were different between Arabidopsis and apple.(4)Transcriptome profiling analysis of Malus Sieversii under abiotic stress using RNA-Seq technology.Under drought stress,totally 6625 differential expression genes(DEGs)were generated,among them,2227 genes were up-regulated and 4398 were down-regulated.Under salt stress,totally 7605 DEGs were generated,among them,2033 genes were up-regulated and 5572 were down-regulated.Under cold stress,totally 14963 DEGs were generated,among them,6729 genes were up-regulated and 8234 were down-regulated.About20%DEGs were unknown genes.There were 2639 genes significantly different expressed under each stress treatment.13 DEGs were randomly selected for qRT-PCR assays to analyze the expression levels.The qRT-PCR showed similar results as the RNA-Seq methods,suggesting that the RNA-Seq methods were valid.KEGG pathway analysis revealed that the DEGs under stresses were participated in many pathways,including metabolic pathways,biosynthesis of secondary metabolites,plant-pathogen interaction and plant hormone signal transduction,etc.This result indicated that these pathways play an important role in apple response abiotic stress.Most of the top 10 up-regulated or down-regulated genes under three stress treatments were function unknown.This result suggested that there were many new stress response genes in Malus Sieversii.(5)Proteome profiling analysis of Malus Sieversiiunder abiotic stress using iTRAQ technology.In this study,4155 proteins were identified through iTRAQ.Under drought stress,totally 634 differentially expressed proteins(DEPs)were generated,among them,373 genes were up-regulated and 261 were down-regulated.Under salt stress,totally 471 DEPs were generated,among them,239 genes were up-regulated and 232 were down-regulated.Under cold stress,totally 803 DEPs were generated,among them,466 genes were up-regulated and337 were down-regulated.A total of 100 proteins were all significantly different expressedunder drought,salt and cold stresses.GO functional annotation analysis revealed that the DEPs under stresses were involved in molecular function including oxidoreductase activity,etc.;cell components including chloroplast stroma,thylakoid,plastid part,etc.;biological process including oxidation-reduction process,response to stress,etc.KEGG pathway analysis revealed that the DEPs under stresses were mainly participated in metabolic pathways,oxidative phosphorylation and photosynthesis.(6)Intergrated investigation of transcriptome and proteome under abiotic stress.We performed an intergrated investigation of transcriptome and proteome database.We found that about half of intergrated DEGs had the same differentially expressed trend on both m RNA and protein levels.Under drought stress,there were 52 genes differentially expressed on both mRNA and protein levels,among them,23 genes had the same trend.Under salt stress,there were 47 genes differentially expressed on both mRNA and protein levels,among them,29 genes had the same trend.And under cold stress,there were 141 genes differentially expressed on both mRNA and protein levels,among them,71 genes had the same trend.COG analysis revealed that the intergrated DEGs under stresses were mainly participated in energy metabolism and posttranslational modification.The correlation analysis of all associated DEGs and DEPs found that the correlation was low between the two databases.Therefore,to clear understanding of plant physiological regulation and control mechanism,the credibility of some data need to be further determined through other different ways.