Molecular Cloning?protein Expression And Purification Of Two Novel Testis-specific Expression Genes Spata34 And Mage-k1

Over the past decade,there are about 10-15% infertility among couples of child-bearing age,in which male infertility accounts for about 33.15%,one of the important reasons is that the male impaired spermatogenesis,which results in azoospermia or severe oligozoospermia.At present,several 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 testisspecific novel genes that are related to spermatogenic cells of formation,development and apoptosis,then research of its function,which is of great significance to understand the spermatogenesis mechanism of physiology and pathology.In this study,two novel testis-specific expressed genes Mage-k1 and Spata34 were cloned by combing bioinformatics and molecular biology techniques,their function were preliminarily studied respectively.The main methods and results are as follows:1.Two novel testis-specific expression genes Mage-k1 and Spata34 were cloned,and a comprehensive bioinformatics analysis of Spata34 and Mage-k1 were carried out.The results showed that the full length of rat Spata34 cDNA is 1986 bp,located in the 2q24 region of chromosome 2 and contains 11 exons.It encodes a protein with a molecular weight of 46.47 kD consisting of 415 amino acids,suggesting that Spata34 protein was localized in the nucleus.The full length of mouse Mage-k1 cDNA is 1602 bp,which was located in chromosome XA6 and contains 2 exons.It encodes a protein with a molecular weight of 35.04 kD consisting of 320 amino acids,probably as a cytoplasmic protein.2.The preliminary function research of rat testis spermatogenic cell apoptosisrelated gene Spata34 was conducted.Multiple tissue RT-PCR results showed that in the tested tissues,Spata34 gene was highly expressed in normal rat testis tissues and was weakly expressed in ovarian tissues,while no expression was detected in other tissues.In situ hybridization results showed that Spata34 gene was highly expressed in spermatocytes and spermatozoa of testis tissue and weakly expressed in spermatogonia,suggesting that the Spata34 gene may play an important role in the development of testicular germ cells.The prokaryotic expression vector pET28b/ Spata34 was constructed,Spata34 fusion protein was expressed in E.coli Rosetta.Subcellular localization results showed that after pEGFP-C3/ Spata34 plasmids were transfected into COS-7 cells,green fluorescence appeared in the nucleus and cytoplasm,which indicated Spata34 protein was mainly expressed in nucleus and cytoplasm.3.The preliminary function research of mouse testis-specific expression novel gene Mage-k1 was studied.Multiple tissues RT-PCR showed that Mage-k1 gene was high specificly expressed in normal mouse testes and no expression was detected in other tissues.The RT-PCR results of mouse testis tissue at different development of stages showed that there is a difference for the expression of Mage-k1 gene in testis at different development of stages,Mage-k1 gene was low expressed at 1-4 weeks after mouse birth,afterwards,the expression of Mage-k1 gene increased gradually with the development of testis,and maintained stable expression at sexual maturity.In situ hybridization showed that Mage-k1 gene was highly expressed in spermatocytes and spermatozoa of testis tissue and was not expressed in spermatogonia.The prokaryotic expression vector pGEX-4T-2/ Mage-k1 and pET28b/Mage-k1 were constructed successfully,and Mage-k1 fusion protein was expressed in E.coli BL21,and purified by affinity chromatography,which laid a good foundation for the further function research of Mage-k1 gene.The above experimental results show that Spata34 and Mage-k1 genes are associated with spermatogenesis and spermatogenic cell apoptosis,they may play an important role in spermatogenesis,testicular development and spermatogenic cell apoptosis.Cloning Spata34 and Mage-k1 genes to further research its function,which will contribute to explore the molecular mechanism of spermatogenesis,further provided the new target for the diagnosis and treatment of male infertility patients as well as the development of new contraceptive drugs.