Molecular Cloning And Foundational Researching Of A Novel Human Testis-specific Gene Septin 12 Transcript Variant 2

About 10-15%of couples suffer from infertility,the causes of infertility owe half to male.The hereditary factor plays an important role in infertility of male,gene deletion,mutation or translocation was often observed in infertile patients.The researching of spermatogenesis-related genes is significant to the diagnosis and therapy for male infertility.Spermatogenesis is a very complicated process which is precisely controlled, the process consists of mitosis phase,meiosis phase and postmeiotic phase.Researching indicated that over 2000 genes involve in the process of spermatogenesis,while the proteins involving in the process are much more than the genes.With the executing of the human genome project(HGP)and the rapid development of biotechnology,the data of human genomic sequence increases enormously,various biology databases are set up,it promotes the development of bioinformatics remarkably.Cloning and functional researching of novel gene by bioinformatics has become the hot spot of biology,many new genes were cloned by this mean,including some spermatogenesis-related genes.We try to discover another new gene related to spermatogenesis by bioinformatics,analyzing it's function, learning the role of it in the process of spermatogenesis,this does some help to reveal the molecular mechanism of spermatogenesis.Partâ… cloning of a novel testis-specific geneWe compared the testis-derived cDNA libraries and other tissuederived cDNA libraries of human,searching the contig which was expressed in testis much higher than in other tissues,and the contig might represented a new gene,it was chosed for further researching.Assembling and extending the ESTs of the contig by the program BLAST,finaly we obtained a extended sequence,reverse transcription polymerase chain reaction(RT-PCR)was performed to identify the reality of the novel gene and the sequence of open reading frame(ORF). By the method of Digital Differential Display(DDD),we discovered a contig Hs.126780,which expression was significant different between testis-derived cDNA libraries and other tissue-derived cDNA libraries, bioinformatics analysis indicated it was a testis-specific gene.Hs.126780 consisted of 39 ESTs,assembling the ESTs,a novel elongated homology sequence was obtained,it was 1524bp in length and contained a 1077bp open reading frame(ORF),encoded a protein of 358 amino acid,the new gene consisted of 10 exons.The results of PCR showed that it was really expressed in the testis tissue and the sequence was right.Cloning novel testis-specific gene by bioinformatics was a feasible and high efficient method.We had cloned a novel testis-specific gene by this mean,the new gene was named septin 12 transcript variant 2 by Human Gene Nomenclature(HGNC),and the accession number of GenBank was DQ517531.Partâ…¡Bioinformatics analysis of the novel gene and proteinwe analyzed the new gene and protein of septin 12 transcript variant 2 by bioinformatics before functional researching,this would provide some useful information for late study.Similarlity analysis was performed with the program BLAST.Restriction maps were constructed with the program BioEdit 7.0.1.Comparing with the human genome sketch,we could located the gene in chromosome 16p13.3.Subcellular location was predicted by the software PSORTâ…¡.Physico-chemical property,motif searching,transmembrane regions and protein orientation analysis were finished by the tools of ExPASy.Analysis of bioinformatics indicated septin 12 transcript variant 2 belongs to the gene family of septin,it did not have restriction site of EcoR I,SalI and XhoI.The new gene encoded a protein of 358 amino acids with a theoretical molecular weight of 40747D and isoelectric point of 6.67.There were no transmembrane regions,signal peptide and hydrophobie structure in the protein.Several phosphorylation sites and other Post-translational modification sites were found in it.The protein might locate in mitochondria(56.5%)or cytoplasm (17.4%). Partâ…¢The preliminary functional researching of septin 12 transcript variant 2The ultimate purpose of gene researching was to learn it's function. we had performed preliminary functional researching of the novel gene by methods of multi-tissue RT-PCR,Northern Blots and in situ hybridization. Eukaryotic expression vector pcDNA3.1(+)/septinl 2 and PEGFPC3/septin12 were constructed for late study.All the results displayed that septin 12 transcript variant 2 was a testis-specific gene in adult male,it was expressed in few tissue of fetus,the expression was weak in foetal testis,strongest in adult testis,and down regulated in testis of the aged,and there was no expression in cryptorchidism,only one type of septin 12 transcript was expressed in testis.The results of in situ hybridization displayed the novel gene located in spermatogonium and spermatocyte. Septin 12 transcript variant 2 was a developmental stage-specific and tissue-specific gene,it might concerned with spermatogenesis and probably involved in division,development and apoptosis of germ cell.Conclusion:We had cloned a testis-specific gene septin 12 transcript variant 2 successfully.Multi-tissue RT-PCR,Northern Blots and in situ hybridization were used to research the expression character and function of it,all the results demonstrated that septin 12 transcript variant 2 might be a spermatogenesis-related gene,and play an important role in spermatogenesis.