Analysis Of Ubiquitin Conjugating Enzyme Gene Expression In Response To Mulberry Vein Bandingassociated Virus Infection And Screening Of Its Interacting Proteins In Mulberry

The E2 ubiquitin conjugating enzyme is one of the important enzymes in the ubiquitin proteasome system(UPS),which is involved in physiological activities such as growth and development,abiotic stress,and immune response in plants.In our previous study,three differentially expressed Ma UBC10,Ma UBC20,Ma UBC C were obtained by analyzing the transcriptome of mulberry(Morus atropurpurea)infested with mulberry vein binding associated virus(MVBa V).To further investigate the function of the E2 s,we validated their differential expression of in MVBa V-infected and healthy mulberry leaves,and cloned the E2 s to obtain bioinformatics analysis and subcellular localization,followed by Quantitative Real-time PCR(RT-PCR)to determine the expression of the E2 s in response to exogenous hormone(ABA,GA,Me JA,SA)and abiotic stress(low temperature,high temperature,high salt,drought)treatments.Prokaryotic expression vectors for Ma UBC10,Ma UBC20,Ma UBC C were constructed to obtain the proteins,the polyclonal antibodies of Ma UBC10,Ma UBC20,Ma UBC C were prepared for co-immunoprecipitation(CO-IP)screening of proteins interacting with them and verified using yeast two hybrid(Y2H).The main research results are as follows:1.The expression of Ma UBC10,Ma UBC20,Ma UBC C in MVBa V-infected and healthy mulberry leaves showed that Ma UBC10,Ma UBC C were significantly up-regulated and Ma UBC20 was down regulated compared to healthy mulberry leaves.2.The c DNA sequences of Ma UBC10,Ma UBC20 and Ma UBC C were cloned.Ma UBC10 has a full length of 802 bp,including a coding sequence of 447 bp,a 5’ untranslated region(UTR)of 114 bp and a 3’ UTR of 241 bp,which can encode 148 amino acids;Ma UBC20 has a full length of 1015 bp,including a coding sequence of 546 bp,a 5’UTR of 204 bp and a 3’ UTR of 265 bp,which can encode 181 amino acids;The coding sequence of Ma UBC C is 567 bp long,and the 3′ UTR is 163 bp long,encoding 188 amino acids.Ma UBC10,Ma UBC20 and Ma UBC C all contain ubiquitin conjugating(UBC)domains.3.Subcellular localization results showed that Ma UBC10,Ma UBC20,Ma UBC C were expressed in cytoplasm and nucleus.4.The expression pattern analysis results showed that Ma UBC10,Ma UBC20,Ma UBC C were expressed in the roots,stems,and leaves of mulberry.All the E2 s produced different response patterns under the treatment of abiotic stress,exogenous hormones.Ma UBC10 expression was significantly induced by SA,with the highest expression at 24 h being 15.36 times of that at 0 h;Ma UBC20 expression was suppressed by all abiotic stress treatments,with a minimum of 5%of normal expression at 6 h of high temperature treatment;the expression of Ma UBC C was significantly induced by ABA and Me JA,and the expression at 24 h was 30.26,8.70 times higher than that at 0 h,respectively.5.A total of 117 Ma UBC10,Ma UBC20 and Ma UBC C interacting proteins were screened by CO-IP,including 115 mulberry proteins and 2 MVba V proteins.E3 ligase Ma RGLG4,MVBa V NSm and MVBa V NSs were selected for verification of interaction with E2,and the results showed that Ma UBC20 interacted with Ma RGLG4.This study demonstrates that Ma E2 s,in response to hormonal signals and stress of adversity,participates in multiple physiological activities when MVBa V infests mulberry,providing a theoretical basis to investigate the mechanism of UPS in mulberry immunity.