Analysis Of Potato Ubiquitin Proteomics And Study Of StPUB27 Gene Function

The ubiquitin-proteasome system,a highly specific protein degradation pathway in cells and an important post translational modification of protein,is involved in many physiological processes including plant abiotic stress response and cell signaling..Drought stress is one of the major factors that affect the productivity of potato.Thus,improving drought resistance in potato is one of the major challenges for potato genetic improvement.The overarching goal of this study was to perform high-throughput screening of ubiquitinated modified proteins in potato and to study the mechanism of U-box ubiquitin ligase gene family and St PUB27 gene involved in drought stress in potato.Potato in vitro plantlets were subjected to osmotic stress using Murashige and Skoog medium supplemented with polyethylene glycol(PEG).The relative expressions of Ub and 26 S genes in osmotic-stressed potato were analyzed using qRT-PCR.Simultaneously,the total protein contained in the potato was hydrolyzed using trypsin followed by the enrichment of the hydrolysate with ubiquitinated modified protein using K-ε-GG antibody.The ubiquitinated modified proteins were identified using LC-MS/MS and were analyzed using bioinformatics.The results showed an increased expression of 26 S gene(p<0.05)in osmotic-stressed potato,which indicated increased level of ubiquitination in the osmotic-stressed potato.A total of 314 ubiquitination sites were detected which were distributed on 200 modified proteins.There were 25 differentially modified sites,including three significant down-regulations and 22 significant up-regulations.RNAseq analysis was performed on the drought stressed potato.Eight commonly used reference genes were selected for further analysis of the most stable internal reference genes under drought stress,osmotic stress,and simulated drought stress conditions.The results showed that EF1α was the most suitable reference gene in potato under drought and simulated drought environments,while sec3 was the most appropriate reference gene under osmotic stress condition.It was observed that the EF1α and sec3 were the most stable reference genes under the three treatments studied.The U-box domain was used to construct the hidden Markov model and to search the potato genomics resource.The U-box genes identified in Arabidopsis and rice were simultaneously used for BLAST search in the potato genomics resource.Finally,the characteristics of potato U-box family members were analyzed.A total of 66 U-box genes were found in potatoes,23 of which contained the ARM domain.Out of 66 genes,37 St PUB genes had two or more exons,while the remaining 29 StPUB genes had only one exon.Phylogenetic analysis showed that the genetic distance between the most StPUB genes and potato U-box genes was closer.There were large numbers of highly homologous genes in the potato U-box family genes were often distributed on the chromosome adjacent to each other.St PUB27 was cloned from two cultivars of potato,Atlantic and Qingshu 9.The sequence alignment and structural analysis of the cloned StPUB27 were performed.The results showed that the CDS(Coding Sequence)length of the StPUB27 gene in different cultivars was 2049 bp and had continuous alignment.The sequence alignment showed that the CDS region and amino acid of St PUB27 gene of the tetraploid cultivars,Atlantic and Qingshu 9 and the diploid cultivar,DM1-3,were highly homologous with 97.66% and 96.33%,respectively.Phylogenetic analysis revealed that the StPUB27 gene was closely related in Solanum pennellii and Solanum lycopersicum.Analysis of signal peptides revealed that the protein of StPUB27 contained no signal peptide.Thus,StPUB27 was a separate protein.Subcellular localization predictions revealed that StPUB27 protein plays a biological role in the mitochondria and is a nuclear encoded protein.There were multiple abiotic stresses and hormone-related cis-acting elements such as CCAAT-box and MRE in the 1000 bp sequence upstream of the StPUB27 gene promoter.When plakophilin 1 was used as a template for homology modeling of spatial structures,it was found that the spatial three-dimensional structure of StPUB27 gene in “Atlantic” and “Qingshu 9” cultivars were highly similar.The StPUB27 gene was incorporated into the constitutive promoter CaMV35S-driven plant overexpression vector and RNAi vector to get CaMV35S-pCPB-StPUB27 and pHellsgate8-stpub27,respectively.The potatoes were transformed and further analyzed for the function of the StPUB27 gene.The results showed that the overexpression of StPUB27 gene could accelerate the weight loss of leaves in in vitro.The leaves of StPUB27 gene over-expressed plants had greater stomatal conductance as compared to the control,but there was no significant difference in the proline content.The results indicated that the StPUB27 gene does not participate in the ubiquitin degradation pathway and is more likely to be involved in ubiquitination modification and regulation of protein activity and function.In summary,this study showed that the ubiquitination pathway is closely related to potato drought response.Among the 66 U-box gene family,StPUB27 was found to be involved in potato stomatal regulation and negatively regulates potato drought tolerance.Moreover,EF1α and sec3 were found to be the most stable reference genes under drought stress,osmotic stress and simulated drought conditions.