Molecular Cloning,Protein Expression And Preliminary Functional Study Of C17orf64 Gene

Over the past decade,there are about 15% infertility among couples of child-bearing age and male infertility accounts for about 50%.The main reason is the male sperm development disorder,including weak vitality,oligospermia and no sperm symptoms.At present,hundreds of genes which are associated with spermatogenesis or spermatogenic cell apoptosis have been identified,however,it was not very clear that how these genes interact with each other to regulate the process of spermatogenesis and whether or not that other unknown genes were involved in spermatogenesis.Therefore,cloning of testis-specific novel genes that are related to spermatogenic cells of formation,development and apoptosis is of great significance to understand the spermatogenesis mechanism of physiology and pathology.In this study,a novel testis-specific expressed genes C17orf64 was cloned by combing bioinformatics and molecular biology techniques,its expression pattern in various tissues and in different developmental stages of testis and preli minary function were investigated.Our main results are as follows:1.A new testis specific gene C17orf64 was cloned and analyzed by bioinformatics.The results showed that the total length of mouse C17orf64 c DNA was 975 bp,which was located at 85.14m-85.18 m of qc-qd band of chromosome 11.It contained 5 exons and encoded a 27.12 kd protein composed of 236 amino acids.It was speculated that C17orf64 protein was located in the nucleus.2.C17orf64 gene is a testis specific gene,which is closely related to sperm formation.The results of RT-PCR and q PCR showed that C17orf64 gene was highly expressed in normal mouse testis,no expression was detected in other tissues.These results were further confirmed by Western blotting analysis in protein expression level.RT-PCR and Western blotting showed that C17orf64 gene and its encoded protein were not expressed in testis within two weeks after birth,but increased gradually from the third week to the sixth week,and remained stable at the seventh and eighth weeks,indicating that the gene was closely related to sperm formation.Immunohistochemistry showed that C17orf64 protein was expressed in mouse spermatocytes and spermatids,but not detected in spermatogonia,Sertoli cells and stromal cells,indicating that the gene expression has cell specific expression pattern.Subcellular localization showed that green fluorescence appeared in the nucleus and cytoplasm of GC-1 cells after transfection of p EGFP-C3 / C17orf64 plasmid,indicating that C17orf64 protein was mainly in the nucleus and cytoplasm Expression in plasma.3.C17orf64 gene can promote cell apoptosis and inhibit cell proliferation.The effects of overexpression and interference of C17orf64 gene on the proliferation and apoptosis of GC-1 and MCF-7 cells were analyzed by flow cytometry.The results showed that overexpression of C17orf64 gene promoted the apoptosis and inhibited cell cycle progression.of the two kinds of cells.Cell cycle analysis showed that after overexpression of C17orf64 gene,MCF-7 cells were blocked at G0 / G1 phase.Western blot and q RT-PCR were used to analyze the possible apoptotic pathway of C17orf64 gene.The results showed that the overexpression of C17orf64 gene led to the significant increase of caspase-9 and Caspase-3,suggesting that C17orf64 gene might promote MCF-7 cell apoptosis through mitochondrial apoptotic pathway.These results indicate that C17orf64 gene is related to spermatogenesis and spermatogenic cell apoptosis,and may play an important role in spermatogenic cell apoptosis.