Function Analysis Of The Post Translation Modification Related Gene SUMO And Ubc9 Of Nosema Bombycis

Microsporidia is a class of obligate intracellular parasites that can infect both vertebrates and invertebrates,and is widely distributed and diverse.The microsporidia（Nosema bombycis,Nb）,which causes p?brine disease in silkworms（Bombyx mori）,is a major threat to sericulture industry.As an important post-translational modification method of proteins,sumoylation is widely involved in biological processes such as transcriptional regulation,immunomodulation,apoptosis,chromosomal isolation and signal transduction.Also,SUMO modification has been shown to be associated with the infection process and virulence of some fungi,but the function of the genes associated with sumoylation in microsporidia has not been reported.In this study,two sumoylation-related genes,Nb SUMO and Nb Ubc9 of Nb were cloned,and their characteristics and role in Nb infection were analyzed.The main contents are as follows:（1）Nb SUMO length is 279 bp,contains 93 amino acids,and the predicted molecular weight is about 10.25 KDa.The similarity of its amino acid sequence to human SUMO1-4 is relatively low,only 27-29%,and the similarity of SUMO sequence to silkworm is about 34%.The Nb Ubc9 gene is 468 bp long,contains 156 amino acids with predicted molecular mass of about 17.2 KDa.Amino acid sequences of Nb Ubc9 have the highest identity（56%and 52%,respectively）with Nosema apis（Western Honeybee）and Nosema ceranae（Oriental Honeybee）.And with 32%identity with silkworm Ubc9.The results of the constructed evolutionary tree show that Nb SUMO and Nb Ubc9 are clustered on the same large branches as all homologous genes derived from the genus Microsporidium,and the highly conservation during evolution suggests that they may can serve as a basis for the classification of microsporidia.（2）QRT-PCR was performed to detect the expression of SUMO and Ubc9genes of silkworms and Nb in the midgut of silkworms after Nb infection with silkworms.It was found that the expression of Nb SUMO and Nb Ubc9 increased significantly,while the expression of endogenous SUMO and Ubc9 in silkworms did not change significantly after Nb infection,or even decreased slightly,indicating that Nb mainly used its own expressed SUMO molecules to regulate host and pathogenic cellular activities.（3）The recombinant baculovirus v Bm^{EGFP-Nb SUMO} and v Bm^{EGFP-Nb Ubc9} were constructed by using the Bac-to-Bac baculovirus expression vector system,and the Nb SUMO and Nb Ubc9 proteins were found to be distributed in the cytoplasm and nucleus of Bm N cells by observing the position of fusion expressed fluorescence signals,but mainly in the cytoplasm.With PULL-DOWN and LC/MS/MS mass spectrometry,65 silkworm proteins and 10 Nb proteins were found to be binded by Nb SUMO,and 46 silkworm proteins and 29Nb proteins were likely to be bound by Nb Ubc9.Gene Ontology analysis found that Nb SUMO-bound proteins are involved in ubiquitination modification,transcriptional regulation,DNA replication and other processes,and Nb Ubc9 binding proteins are mainly involved in signal transduction,translation,protein folding and transport,de-ubiquitination and other functions.（4）To analysis the role SUMO modification system in Nb proliferation,the SUMO pathway was blocked with inhibitors or si RNA,respectively,and the effect on Nb replication was detected.The results showed that the inhibitors（ML-792 and 2-D08）reduced the Nb production in cells by 40%and 23%,respectively.After knocked down of Nb SUMO with RNAi,the Nb genome copy number decreased significantly,indicating that Nb SUMO is critical for Nb replication.（4）Finally,altered gene expression of the Bm N cell line with stable expressed Nb SUMO was analyzed by RNA-seq,and 3628 genes were found to be significantly differently expressed,of which3065 were upregulated and 563 were down-regulated.Go enrichment analysis results showed that differentially expressed genes are involved in metabolic process,cellular process,biological regulation,regulation of biological process,localization,response to stimulus and other biological processes,molecular functions mainly include binding,catalytic activity,transporter activity,molecular function regulator,etc.KEGG pathway of the differential gene showed that the significant differential expression of genes was related to the Wnt signaling pathway,oxidative phosphorylation,and cellular senescence.In summary,by the analysis of the genetic characteristics,the binding protein and the effect of RNAi on Nb replication of Nb SUMO and Nb Ubc9,our results has proved that the Nb SUMO modification system plays an important role in Nb infection,but its specific mechanism needs further studies.