睾丸特异表达新基因Spata34和Mage-k1的克隆及蛋白表达和分离纯化

发布时间：2018-03-13 11:13

本文选题：Spata34基因　切入点：Mage-k1基因　出处：《湖南理工学院》2017年硕士论文　论文类型：学位论文

【摘要】：近十年来,育龄夫妇中约有10-15%的不育,而男性不育约占33.15%,其中一个重要的原因是男方生精障碍,表现为无精或严重少精。多个与精子发生或生精细胞凋亡相关的基因已被鉴定,但这些已知的基因如何与其它的基因相互作用来调控精子的生成过程,以及是否存在其它未知的基因参与精子的发生目前尚不完全清楚。因此,发现和克隆新的睾丸生精细胞发生、发育、凋亡特异基因并研究其功能对阐明精子发生的生理和病理机制具有十分重要的意义。在本研究中,应用生物信息学和分子生物学技术相结合的方法克隆了两个睾丸特异表达新基因Spata34和Mage-k1,并分别对其功能进行了初步研究。主要方法及实验结果如下:1.克隆了两个睾丸特异表达新基因Spata34和Mage-k1,并对其进行了系统的生物信息学分析。结果表明大鼠Spata34的cDNA全长为1986 bp,定位于2号染色体2q24区,含有11个外显子,编码由415个氨基酸组成的分子量为46.47 kD的蛋白质,推测Spata34蛋白定位于细胞核。小鼠Mage-k1基因cDNA全长为1602 bp,定位于X号染色体XA6区,含有2个外显子,编码由320个氨基酸组成的分子量为35.04 kD的蛋白质,可能为一种胞浆蛋白。2.对大鼠生精细胞凋亡相关基因Spata34进行了初步的功能研究。多组织RT-PCR结果显示:在已检测组织中,Spata34基因在正常大鼠睾丸组织中高表达,在卵巢组织中有微弱表达,而在其他组织中未检测到表达。原位杂交结果显示:Spata34在睾丸组织的精母细胞、精子中高表达,而在精原细胞中弱表达,提示Spata34基因可能在睾丸生殖细胞的发育过程中扮演着重要的角色。构建了原核表达载体pET28b/Spata34,并诱导Spata34基因在大肠杆菌Rosetta中表达融合蛋白。亚细胞定位结果显示:pEGFP-C3/Spata34质粒转染COS-7细胞后,绿色荧光出现在胞核和胞浆,表明Spata34蛋白主要在胞核和胞浆中表达。3.对小鼠睾丸特异表达新基因Mage-k1进行了初步的功能研究。多组织RT-PCR结果显示:Mage-k1基因在正常小鼠睾丸组织中特异性高表达,而在其他组织中未检测出表达。小鼠不同发育时期睾丸组织RT-PCR结果显示:Mage-k1基因在睾丸中的表达呈发育阶段特异性,其在小鼠出生后1-4周低表达,此后随着睾丸的发育其表达量逐步上升,并在性成熟时保持稳定表达。原位杂交结果显示:Mage-k1基因在睾丸组织的精母细胞、精子中高表达,而在精原细胞中不表达。成功构建出原核表达载体pGEX-4T-2/Mage-k1及pET28b/Mage-k1,并诱导了Mage-k1基因在大肠杆菌BL21中表达融合蛋白,用亲和层析法纯化了蛋白质,为后续Mage-k1基因功能的深入研究奠定了良好的基础。以上实验结果表明Spata34和Mage-k1基因与精子发生及生精细胞凋亡有关,可能在精子发生、睾丸发育以及生精细胞凋亡中起重要作用。克隆Spata34和Mage-k1基因并研究其功能,将有助于探明精子发生的分子机制,并为男性不育患者的的诊治以及新型避孕药物的研制提供了新的靶标。
[Abstract]:In the past ten years, couples of childbearing age in about 10-15% of infertility, male infertility accounted for about 33.15%, one of the important reasons is the dyszoospermia, manifested as azoospermia or severe oligozoospermia. Multiple and spermatogenesis or spermatogenic cell apoptosis related genes have been identified, but these known genes to the interaction with other genes to regulate sperm generation process, and whether there are other unknown genes involved in sperm are still unclear. Therefore, the discovery and cloning of new spermatogenic cells, development, apoptosis gene and study its function has very important significance to elucidate the physiological and pathological mechanism spermatogenesis. In this study, using methods of bioinformatics and molecular biology technology combined with the cloned two testis specific expressed gene Spata34 and Mage-k1 respectively, and the main functions of the Main research method and the experimental results are as follows: 1.. Cloning of two testis specific expressed gene Spata34 and Mage-k1, and has carried on the analysis of biological information system science. The results showed that rat cDNA full-length Spata34 was 1986 BP, located on chromosome 2 2q24, contains 11 exons, molecular weight by encoding 415 amino acids of 46.47 kD proteins, suggesting that Spata34 protein was localized in the nucleus of mouse Mage-k1 gene. The full-length cDNA was 1602 BP, located at X on chromosome XA6, contains 2 exons, encoding the molecular weight of 320 amino acids of 35.04 kD protein may be a cell plasma protein.2. for further research on rat spermatogenic cell apoptosis related gene Spata34. RT-PCR results showed that: in detected tissues, high expression of Spata34 in testicular tissue of normal rats, in ovarian tissue is weak Expression in other tissues was not detected. In situ hybridization results showed that Spata34 in the testis spermatocyte, high expression of sperm, and weak expression in spermatogonia, suggesting that the Spata34 gene may play an important role in the development of testicular germ cells. To construct prokaryotic expression vector pET28b/Spata34, and induce the expression of the fusion protein of Spata34 gene in Escherichia coli Rosetta. The results showed: the subcellular localization of pEGFP-C3/Spata34 plasmid transfected COS-7 cells, green fluorescence appeared in the nucleus and cytoplasm, showed that Spata34 protein was mainly expressed in the nucleus and cytoplasm of mouse testis specific expression of.3. Mage-k1 gene for further research many organizations. RT-PCR results showed that the high expression of Mage-k1 gene in the testis of normal mice, while in other tissues did not detect the expression in different development period. Testicular tissue RT-PCR results showed that the expression of Mage-k1 gene in the testis in a developmental stage specific in mice 1-4 weeks after birth, low expression, then with the development of the testes of the expression level gradually increased, and remained stable in the mature expression. In situ hybridization results showed that Mage-k1 gene in spermatocytes of testis. The high expression of sperm, but not expressed in spermatogonia. Successfully constructed the prokaryotic expression vector pGEX-4T-2/Mage-k1 and pET28b/Mage-k1, and induced expression of Mage-k1 fusion protein in Escherichia coli BL21. The proteins were purified by affinity chromatography, and laid a good foundation for further in-depth study of Mage-k1 gene function. The experimental results show that the above Spata34 and Mage-k1 gene and spermatogenesis and apoptosis of spermatogenic cells, probably in spermatogenesis, testis, spermatogenic cell apoptosis plays an important role in the cloning of Sp. The function of ata34 and Mage-k1 genes will help to identify the molecular mechanism of spermatogenesis, and provide a new target for the diagnosis and treatment of male infertility and the development of new contraceptive drugs.

【学位授予单位】：湖南理工学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R698.2

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