Expression Pattern And Functional Analysis Of RBX In Spermatogenesis Of Chinese Mitten Crab Eriocheir Sinensis

RING-box protein is an important part of Cullin-RING E3 ubiquitin ligase,it participates in the regulation of intracellular protein degradation through the ubiquitination-proteasome pathway.This ubiquitination plays important roles in cell cycle,growth and development,signal transduction,DNA replication and repair.It's also very important in spermatogenesis of Eriocheir sinensis,while the RBX protein have not been studied yet.According to the genome date and transcriptome data of E.sinensis,this study cloned two RBX genes.The full length EsRBX1 cDNA comprises 741 nucleotides,and encodes 124 amino acid residues,EsRBX2 cDNA comprises 1325 nucleotides,and encodes 110 amino acid residues.Bioinformatics analysis showed the domains and structure of RBX proteins are highly conserved during evolution.The results of phylogenetic analysis consistent with the traditional evolutionary classification.The expression of RBX was detected by fluorescence quantitative PCR and immunoblotting.The results showed RBX1 high express in testis,and regular changes at different stages of spermatogenesis;while RBX2 did not show significant changes in male reproductive system.Furthermore,we studied the subcellular localization of RBX1,it mainly distributed in the nucleus,and transferred with the nucleus in cell division,it may associate with karyokinesis and cell division.Co-immunoprecipitation showed RBX1 could bind Cullin4.RBX1 may combine with Cullin and form Cullin-RING ligase then work in ubiquitination.These results indicated that EsRBX1 might play a key role in spermatogenesis of E.sinensis though forming CRL4 complex.