The Expression Analysis Of Fancd2os Gene In Mouse And Construction Of Fancd2os Eukaryotic Expression Vector

Objective:Mammalian spermatogenesis is a complicated and highly ordered process. Many genes highly or specifically expressed in the testis are involved in spermatogenesis.According to the previous data in the lab, the study choose gene Fancd2 os to investigate the basic information using bioinformatics methods, to investigate the specific expression during mouse spermatogenesis and its location by molecular biology techniques, to further construct Fancd2 os eukaryotic expression recombinant plasmid, which lay the foundation for the function research of gene Fancd2 os.Methods:(1)Bioinformatics methods and related databases were used to analyze the structural of Fancd2 os gene and its encoded protein, including homology, phylogenetic tree, physical and chemical properties of protein, the signal peptide, hydrophilicity, transmembrane domain, localization, post-translational modification sites, phosphorylation sites, antigenic epitope sites, protein secondary structure, interaction, EST and GEO(Gene Expression Omnibus).(2)Semi-quantitative PCR(RT-PCR) was applied to analyze the tissue-specific expression of Fancd2 os m RNA.(3)Western blotting was used to detected the tissue-specific expression of Fancd2 os protein.(4)The stage-specific expression of Fancd2 os m RNA inspermatogenesis was examined by real-time quantitative PCR.(5)Western blotting was used to utilized stage-specific expression of Fancd2 osprotein.(6)Immunohistochemical stain was applied to identify the cellular location of Fancd2 os in mouse seminiferous tubules.(7)Molecular cloning techniques was used to construct eukaryotic expression recombinant plasmid p3 × FLAG-CMV-14-Fancd2 os and identify.(8)The recombinant plasmid was transfected to HEK293 T cells, and the Fancd20 s m RNA and protein expression levels were determined by RT-PCR and Western blotting.Results:(1)Bioinformatics analysis revealed that the mouse Fancd2 os gene was mapped to chromosome 6 E3. The full c DNA length of Fancd2 os was 1358 bp, which encoded a protein product with 178 amino acids. The protein had 13 acidic amino acids and alkaline23 amino acids. Theoretical PI was 9.56. The instability index was computed to be 51.95.The mouse Fancd2 os protein had an amino acid similarity of 97.8%, 93.3%, 92.1% with rat, cattle and human respectively. Fancd2 os protein mainly located in cytoplasm and its secondary structure comprised a large amount of random coil and alpha helices. The protein contained multiple phosphorylation sites, without signal peptide. Phosphorylation site analysis predicted the presence of 8 tryptophan and 5 threonine phosphorylation sites.Using Motif Scan predicted 10 post-translational modification sites, which were a cyclic adenosine monophosphate phosphorylation sites, three casein kinase II phosphorylation sites and six protein kinase C phosphorylation sites. EST(expressed sequence tags)showed that Fancd2 os gene was mostly expressed in testis, weak expression in kidney tissue. GEO data base analysis demonstrated that Fancd2 os gene may be regulated by mitochondria caseinolytic protease(Clp P). Otherwise, there were 95 mi RNA probably interact with Fancd2 os gene according to MGI Interaction Explorer analysis.(2)Semi-quantitative RT-PCR analysis showed that Fancd2 os was highly expressed in mice testis, weak expression in kidney tissue, while other tissues were lack of expression.(3)Fancd2os protein showed obvious tissue-specific expression by Western blotting.(4)Real-time quantitative PCR analysis showed that Fancd2 os had a significant stage-specific expression during mouse spermatogenesis.(5)Fancd2os protein showed stage-specific expression during spermatogenesis by Western blotting.(6)Fancd2os was detected in the cytoplasm of spermatocytes and round spermatids by immunohistochemical staining.(7)The recombinant eukaryotic expression plasmid p3 × FLAG-CMV-14-Fancd2 os was successfully built, and the sequence was correct by PCR, sequencing and restriction analysis.(8)The m RNA and protein expression of Fancd2 os were observed in HEK293 T cells transfected with recombinant plasmid p3×FLAG-CMV-14-Fancd2 os.Conclusion:Fancd2os gene is highly expressed in mouse testis and its expression levels were up-regulated during spermatogenesis, which has a significant stage-specific expression profiles. Combined the bioinformatics analysis results, Fancd2 os may be associated with the development and spermatogenesis process of testicular tissue. Besides, the recombinant plasmid can be further applied to explore the gene function at a cellular level, especially in the specific function of spermatogenesis.