Genome-wide Identification Of Ubiquitin Conjugating Enzymes E2 Gene Family And Cloning And Functional Aanlysis Of StUBC9 In Potato

The ubiquitination/26 S proteasome pathway is an important protein turnover system which is capable of degrading and modifying proteins in eukaryotic cells.In most cases,the target protein is rapidly degraded by the 26 S proteasome.This system is responsible for the removal of most abnormal peptides and short-lived cell regulators,thereby participating in the regulation of multiple life processes,including hormone effects,photomorphogenesis,circadian rhythm,organs development,plant defense,leaf senescence,biotic and abiotic stress responses.The E2 enzyme,a key enzyme of ubiquitination,is an important part of ubiquitination of target proteins.The E2 family of Arabidopsis,rice,and tomato has been identified,however,E2 genes family in potato have not been reported till date.In this study,genome-wide identification and expression analysis of E2 ubiquitin-conjugating enzymes in potato were performed to study the E2 gene family;meanwhile,we cloned the StUBC9.The constructed overexpression vector of StUBC9 gene and the interference expression vector were transferred into invitro potato seedlings by Agrobacterium tumefaciens transformation to obtain transgenic potato seedlings,which aims to preliminary study functional of the gene.The main work and results of the experiment are as follows:1.A total of 57 potato E2 family members were identified by Hidden Markov Model Search and BLAST.According to the phylogenetic tree constructed by potato and Arabidopsis,the family was divided into 8 subfamilies and analyzed the bioinformatics of E2 family members.Analysis of gene duplication revealed that there were 13 segmental duplications of the potato E2 family gene and no tandem duplication was found.It suggested that segmental duplication plays a vital role in the process of E2 gene family expansion and functional diversification.2.Structural analysis of potato E2 family members found that the number of introns range from 0 to 9,with the most genes containing 5 introns,similar to the case of corn and tomato.By comparing the motifs of tomato,Arabidopsis and potato E2 family genes,we found that the motifs contained in them were highly similar,which indicated that those motifs are highly conserved during evolution.The tertiarystructure model of the potato E2 family members was analyzed.It found that the potato E2 family members were similar in tertiary structure and the spatial configuration was relatively consistent.The tertiary structure of protein determines the function of the protein,indicating that the potato E2 gene family is functionally conserved.3.In the analysis of cis-acting elements in the promoter region,we found that many cis-acting elements are related to plant growth,abiotic stress and stress response.According to the GO analysis of the E2 gene,the function of protein binding and ligase activity indicated that the E2 gene plays a key role in proteolysis or protein modification.The protein interaction network analysis of the E2 family showed that a total of 143 proteins were involved in the 740 pair's protein interaction network events,including 392 pair's interaction network events that are 57 E2 enzymes interacted with E2 enzymes.The proteins interacting with E2 in this interaction network are primarily involved in ubiquitin-mediated protein hydrolysis,DNA repair,RNA transport,and protein modification.4.According to the sequencing results of the potato genome database,the E2 gene was highly expressed in roots,leaves and tubers,and was up-regulated in ABA,salt stress and high-temperature treatment.The E2 gene family of the genomic database was verified by qRT-PCR.The results showed that 8 StUBCs were significantly up-regulated under salt stress;the expression of 17 StUBCs were significantly up-regulated during heat treatment,and most of the up-regulated StUBCs reached the highest level of expression after 6 hours of treatment.It indicated that E2 genes family are involved in the growth and development of potatoes and respond to abiotic stresses.5.The length of StUBC9 CDS is 486 bp and encodes 161 amino acids.The cis-acting element of the promoter region was predicted by bioinformatics method,indicating that the StUBC9 gene contains cis-acting elements related to abscisic acid,salicylic acid,methyl jasmonate.The GO annotation indicated that the E2 enzyme is involved in catabolism and protein binding;the interaction network of E2 protein indicates interaction with two E3 and four E2;the spatial structure is similar to othermembers of the E2 family.The conserved domains of StUBC9 proteins of different species were compared,and a phylogenetic tree of 13 species with StUBC9 homologous genes was constructed.6.The StUBC9 gene was amplified by PCR and two plant expression vectors were constructed.One of vectors was the overexpression vector pCPB-StUBC9 driven by the strong promoter CaMV35 S,another vector was to amplify the microRNA precursor fragment amiR-StUBC9 of StUBC9 by nested PCR to construct a pCPB121-StUBC9 interference expression vector.Potato transgenic tube seedlings were obtained by Agrobacterium transformation.7.The StUBC9 gene was down-regulated in potato cultivar "Atlantic" tube seedlings when treated with heat,salt,PEG600 and ABA.The expression level of StUBC9 gene was up-regulated in overexpressing plants.The highest expression level of StUBC9 gene was 6.37 times than that of control group.The expression level of StUBC9 gene was down-regulated in the interfering expression plants,and the expression level of StUBC9 gene was 0.53 times lower than that of the control group.