【作者】 金帅；

【导师】 齐社宁；

【作者基本信息】 兰州大学 ， 人体解剖与组织胚胎学， 2013， 硕士

【摘要】 犏牛是牦牛和普通牛的杂交后代,杂种F1代犏牛具有很强的杂交优势,生产性能明显提高,但是F,代公犏牛不能正常产生精子,无法稳定遗传杂种优势,这成为牦牛杂交改良的一大难题,至今仍未能阐明犏牛雄性不育的分子机理。近年来的研究发现,Dmrt7作为Dmrt (doublesex and mab-3related transcriptional factor)基因家族的一员,在雄性特有的性染色体联会过程中扮演着关键角色。在本研究中,我们采用生物信息学方法对Dmrt7基因及其编码蛋白的功能位点和二级结构等方面进行了预测和分析,发现牦牛和犏牛Dmrt7基因编码区序列一致,编码370个氨基酸,具有完整的DM功能域；利用半定量PCR技术检测出Dmrt7mRNA仅在成年牛睾丸组织中表达,而未见于其它组织器官中；通过实时荧光定量PCR和Western Blot检测Dmrt7基因mRNA和蛋白在黄牛、牦牛和犏牛睾丸组织中的表达水平,发现mRNA与蛋白表达水平相一致,且犏牛睾丸组织中Dmrt7mRNA和蛋白的表达水平显著低于黄牛和牦牛(P<0.01)；同时我们通过H.E染色、免疫组织化学染色等手段对黄牛、牦牛和犏牛睾丸组织形态进行观察和比较,发现Fl代犏牛曲细精管高度滤泡化,仅含有少量生殖细胞,缺乏精子细胞,呈现明显的退化形态,减数分裂后期的生殖细胞出现明显缺失,犏牛的生殖细胞出现大量凋亡情况,生精过程被阻滞在粗线期后期。这些结果表明,Dmrt7作为减数分裂粗线期和双线期之间雄性特有的性染色体联会过渡中的一个关键因子,其表达水平的低下很有可能是犏牛染色体减数分裂障碍的原因之一。更多还原

【Abstract】 Cattle-yak, the first hybrid generation of cattle and yak, shows obvious heterosis. However, F1male cattle-yak cannot produce sperm, which prevents the heterosis from being inherited. The inability of cattle-yak to produce sperm has become a major problem in the crossbreeding of yak, and the molecular mechanism for male cattle-yak sterility is not clear. Dmrt7, a member of the Dmrt (doublesex and mab-3related transcriptional factor) family of genes, plays an important role in male sex chromosome synapsis. In our study, the coding region sequence of yak and cattle-yak Dmrt7was cloned by molecular cloning techniques, and the sequence, conserved domains, functional sites, and secondary and tertiary structures of the Dmrt7-encoded protein were predicted and analyzed°using bioinformatics methods. The coding region sequences of the Dmrt7gene, encoding370amino acids, were consistent in yak and cattle-yak. The protein encoded by yak and cattle-yak Dmrt7contains a DM domain. We detected Dmrt7mRNA expression in the testis, but not in any other tissue tested. Dmrt7mRNA and protein expression was significantly higher in testis of cattle and yak than that in cattle-yak (P<0.01). Histological analysis indicated that seminiferous tubules in male cattle-yak were highly vacuolated and contained primarily Sertoli cells and spermatogonia, while those of cattle and yak contained abundant primary spermatocytes. The male cattle-yak testis contained a significantly larger number of apoptotic cells than those of cattle and yak, as assessed by TUNEL analysis. The accumulation of SCP3-positive spermatocytes indicated the arrest of spermatogenesis at the pachytene stage in the cattle-yak. These results suggest that low levels of Dmrt7expression lead to male sterility in cattle-yak.更多还原