Study On The Clone Expression Of Mice New Genes Dnajcb5 And The Phenotype Detection In Rcet1 Knockout Mice

Mice Dnajc5b and Rcet1 are the new genes that we clone in mice testis and reproductive field of specific expression respectively.The Dnajc5 b is the homologue of DNAJ(HSP40).It is in the second place of subfamily C in Member 5,which is named Dnajc5 b.Dnajc5b is a new gene which has a complete reading frame and which is gained by electronic cloning.Dnajc5 b is the third chomosome of mice,which contains five exons.The total length is 947 bp and the open reading frame is 597 bp which codes 199 amino acids in the protein.Using RT-PCR to testify Dnajc5b's specific expression in the testis of mice.,mice show different expression in different period of testis tests.Before three weeks,Dnajc5 b shows weak expression.After three weeks,it enhances its expression gradually,till the eighth week,it gets its peak.Simultaneously,we use situ hybridization to detect the expression of the gene in mice testicular tissue,which shows that Dnajc5 b gene is specifically expressed in mice spermatogonia.After studying the reconstruction of eukaryotic expression vector of PEGFP-C3/Dnajc5 b,we find that Dnajc5 b is in cytoplasm.At the same time,we find the recombinant protein of 25 KD,Dnajc5b prokaryotic expression by using His Antibody and the dictation correctly tested by Western Blot.After separating and Purificating Dnajc5 b recombinant protein,we get the purified Dnajc5 b protein,and the 6His testing is right.Rcet1 is the new cloning gene of this laboratory,and we construct the mice model of knockout Rcet1,which shows that the male mice being deleted is complete sterility,the testis and epididymis can not develop normally,and the testicular atrophies and the epididymis gets cyst.Besides,spermatocyte becomes bigger in the seminiferous tubule,,and the sperm is a serious shortage.The lumen of epididymal duct becomes larger,the columnar cells fall off and the number of sperm is very few.The gene research of Dnajc5 b shows that its specific expression in testicular tissue,and its expression quantity relates to testicular development period.Basing on the facts that Dnajc5 b has Dnajc structural domain and molecular chaperone structural domain,its sub-cellular localization is in cytoplasm.Therefore,we can speculate that Dnajc5 b has the function of molecular chaperone or heat shock protein.Deleting mice's Rcet1 shows that mice's testis and epididymis can not develop normally in the condition of lacking Rcet1.It shows few sperms oreven no sperms,which indicates male mice's sterility.The research results of Dnajc5 b and Rcet1 are aiming at laying the foundation of studying molecular mechanism in the process of the sperm production.