牦牛和犏牛Dmrt7基因序列分析及其 在睾丸组织中的表达水平

【目的】研究牦牛和犏牛Dmrt7基因编码区序列和编码蛋白的结构，以及在睾丸组织中mRNA及其蛋白表达水平，探讨Dmrt7与犏牛雄性不育的关系，为揭示犏牛雄性不育的分子机理提供依据。【方法】利用分子克隆技术获得牦牛和犏牛Dmrt7基因编码区序列，并采用生物信息学方法对该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；通过半定量PCR技术检测Dmrt7基因mRNA在牦牛各组织器官中的表达水平；利用实时荧光定量PCR技术检测牦牛和犏牛睾丸组织中Dmrt7基因mRNA表达水平；并通过western blotting检测牦牛和犏牛睾丸组织中Dmrt7蛋白的表达水平。【结果】牦牛和犏牛Dmrt7基因cDNA序列一致，包含一个长度为1 113 bp的开放阅读框，编码370个氨基酸，具有完整的DM功能域，二级结构主要以无规则卷曲、α螺旋和延伸链为主。在牦牛各组织器官中，Dmrt7基因mRNA仅在睾丸组织中特异性表达。牦牛睾丸组织中Dmrt7mRNA和蛋白的表达水平极显著高于犏牛（P＜0.01）。【结论】牦牛睾丸组织中Dmrt7基因mRNA和蛋白表达水平明显高于犏牛，且Dmrt7蛋白表达水平与其mRNA表达水平相一致。

Sequence Analysis and Study on the Expression Level of Dmrt7 Gene in Yak and Cattle-Yak Testis

【Objective】The aim of the study is to investigate the coding region sequences, their structures and the expression levels of Dmrt7 gene in yak and cattle-yak testis, for offering references for the revealing of the molecular mechanism of infertility of cattle-yak.【Method】A coding region sequence of yak and cattle-yak Dmrt7 was cloned by molecular cloning technique in this study. Some characters of the Dmrt7 gene and encoded protein sequences were predicted and analyzed by the methods of bioinformatics in the following aspects as the functional sites and secondary structure. The expression levels of Dmrt7 mRNA in some organs of yak were detected by semi-quantitative PCR. Real-time PCR was employed to examine the expression levels of Dmrt7 mRNA in yak and cattle-yak testis. Western blotting was applied to detect the expression levels of Dmrt7 protein in yak and cattle-yak testis. 【Result】 The coding region sequences of Dmrt7 gene in yak and cattle-yak were consistent，contains a complete ORF (1 113 bp) which encoding 370 amino acids. The protein encoded by yak and cattle-yak Dmrt7 contains a DM domain. The secondary structures were mainly composed of Helix, Coli and Extended strand. Dmrt7 mRNA expression in the testis was detected, but not in any other tissue tested. And the difference of expression level of Dmrt7 mRNA and protein in testis between yak and cattle-yak was extremely remarkable (P＜0.01).【Conclusion】The expression levels of Dmrt7 mRNA and protein in yak testis were significantly higher than cattle-yak testis，and the expression levels of Dmrt7 mRNA and protein were consistent. The results indicate that the expression level of Dmrt7 protein in cattle-yak may be too low to block the meiosis process.