Function Research Of DNA Damaged Binding Protein 1(Es-DDB1) In Spermatogenesis In The Chinese Mitten Crab

Chinese mitten crab(Eriocheir Sinensis)belongs to Arthropoda,class Crustacea,Decapoda.Because of being high in nutrition as well as economic value,it is one of the main varieties in aquaculture.Damaged DNA-binding protein 1(DDB1)is a multifunctional protein that recognizes UV-induced DNA lesions and activates nucleotide excision repair process,and could also be a linker protein for Cullin4 in ubiquitination to regulate cell cycle,transcription,signal transduction and damage repair process.In recent years,there were some studies have found that DDB1 attended to embryonic development and spermatogenesis,however,there are few studies of DBB1 in crustaceans,In this study,a cDNA representing the DDB1 gene from Eriocheir sinensis(Es-DDB1)was cloned.The full-length Es-DDB1 cDNA comprises 4871 nucleotides,a 3414bp open-reading frame(ORF),which encodes 1137 amino acid residues and had a calculated molecular mass of 125 kDa.There are an MMS1_N domain and a CPSF_A domain,3-D protein structure model consisted of seven-bladed ? propellers(referred to as BPA to BPC)and a C-terminal helical domain(CTD).Bioinformatics' analysis showed that both domain and structure were highly conserved during evolution.Quantitative real-time PCR and western blotting showed high expression in the testis compared with the other tissues,thus we predicted Es-DDB1 involved in spermatogenesis of Eriocheir sinensis.Then we found the highest level of Es-DDB1 in different testis development stages was in spermatocyte stage,and gradually decreased in the spermatids and spermatozoa stages,implied Es-DDB1 might participate in regulating early stage of spermatogenesis.A co-immunoprecipitation assay showed that Es-DDB1 could bind Es-Cul4 in the testis of Eriocheir sinensis.Immunofluorescence assays showed that Es-DDB1 was mainly distributed in the cytoplasm in the early and middle developmental stages.These results all indicated that Es-DDB1 might bind Es-Cul4 to be a complex which play a key role in spermatogenesis of E.sinensis.