Proteomics Identification Of Mouse Round Spermatids And Construction Of Eukaryotic Expression Vector For High Expression MINCA1Gene

Objectives:1.To identify proteomics of mouse round spermatids by Shotgun strategies with mass spectrometry and to analysing proteins through bioinformatics.2. To construct a eukaryotic expression vector for high expressed mouse testis gene mINCAlin and screen the RNA interference target of mINCA1.Methods:1. Separate round spermatids using gravity sedimentation and then staining the isolated cells by immunofluorescence;2. Extracted total proteins from round spermatids which peptide analysis by automatic2D-Nano-LC-ESI-MS/MS;3. To search proceeds proteins IPI using the bioinformatics database. Then to analyze the function of the protein molecules and participatory biological processes according to the database of DAVID GeneOntology annotation, note the protein metabolism pathway by KEGG annotation.4. The mINCAl gene was amplified from testis tissue of BALB/c mice and and cDNA inserted into eukaryotic expression vector pMSCVpuro to construct recombinant plasmid pMSCVpuro-mINCAl;5. The recombinant plasmid pMSCVpuro-mINCAl was transfected to human embryo kidney(HEK)293Tcells. The mINCAl mRNA transcription and protein expression levels in the cells48h after transfection were determined by RT-PCR and Western blot respectively;6. Four RNA interference plasmids against mINCAl gene were designed and synthesized, then co-transfected to HEK293T cells with pMSCVpuro-mINCAl respectively. The interference target of mINCAl gene was screened by Western blotting.Results:1. The mouse testicular cells had been isolated by gradient method, the purity of sorted round spermatids was assessed via immunofluorescence staining under the microscope.2. We employed a label-free shotgun proteomic technique and LC-MS/MS to identify2,331 proteins of round spermatids and investigate the molecular mechanisms、biological processes and subcellular localization by DAVID analysis.3. The Kyoto Encyclopedia of Genes and Genomes (KEGG) terms were annotated for the identified proteins. As expected,370of the identified proteins were involved in the metabolism pathway,68proteins were annotated to the ribosome pathway,28proteins in the proteasome pathway and40proteins in the liposome pathway.The Pathway Studio revealed that60proteins were annotated to the spliceosome pathway.4.PCR and DNA sequencing proved that recombinant plasmid pMSCVpuro-mINCAl was constructed correctly.5. The mRNA transcription and protein expression of mINCAl were observed in HEK293T cells transfected with plasmid pMSCVpuro-mINCAl.6. PGPU6/GFP/Neo-shRNAl and PGPU6/GFP/Neo-shRNA3decreased the expression level of mINCAl protein significantly.Conclusions:1. The proteome analysis utilized in the current study provided a comprehensive characterization of the protein expression profiles of round spermatids which can provide further information to understand the molecular basis of spermatogenesis.2. The eukaryotic expression vector for mINCAl gene was constructed successfully, and two effective RNA interference targets of the gene were screened, which laid an experimental foundation of further study on the function and action mechanism of mINCAl gene.