天祝白牦牛HSP60基因的克隆及其在下丘脑-垂体-睾丸中的表达定位研究

发布时间：2018-06-17 01:24

本文选题：HSP基因 + 天祝白牦牛　；参考：《核农学报》2017年07期

【摘要】：热休克蛋白60(HSP60)不仅在细胞保护方面发挥作用,也参与雄性动物生殖生理的调控。为研究HSP60基因序列特性及其在天祝白牦牛下丘脑-垂体-睾丸轴(HPT-axis)上的表达情况,探寻其在白牦牛睾丸发育、精子发生过程中的作用机制,本研究采用RT-PCR和RACE技术获得白牦牛HSP60基因全长cDNA序列,采用生物信息学方法分析HSP60蛋白的理化性质、结构及不同物种之间的同源性等,并利用qPCR、Western blotting、免疫组化法分析HSP60在白牦牛下丘脑-垂体-睾丸轴中的表达及定位。结果表明,白牦牛HSP60基因cDNA全长为2 300 bp,开放阅读框为1 722 bp,编码572个氨基酸;其理论分子量为60.977k Da、等电点为5.69,编码的蛋白为非跨膜可溶性蛋白。氨基酸序列比对结果显示,白牦牛HSP60氨基酸序列与牛、瘤牛、绵羊、藏羚羊、骆驼、白犀牛、兔和黑猩猩的氨基酸序列同源性高、进化水平相近。HSP60基因及蛋白在白牦牛的下丘脑、垂体及睾丸组织中均有表达,其中下丘脑及垂体组织表达量显著高于睾丸,下丘脑和垂体之间差异不显著。免疫组化结果显示,HSP60蛋白定位表达于白牦牛下丘脑组织的室旁核大细胞、室旁核小细胞和神经角演网、垂体组织的腺细胞、睾丸组织的精原细胞、精母细胞、支持细胞和间质细胞,而精子细胞中表达较弱。通过对健康成年白牦牛HSP60在下丘脑-垂体-睾丸轴的表达与定位检测,推断HSP60参与雄性白牦牛生殖轴调控,并参与睾丸发育与精子发生。本研究结果为进一步研究HSP60对雄性生殖调控奠定了基础。
[Abstract]:Heat shock protein 60 (HSP60) not only plays a role in cell protection, but also participates in the regulation of reproductive physiology in male animals. To study the sequence characteristics of HSP60 gene and its expression in the hypothalamus-pituitary-testicular axis of Tianzhu white yak, and to explore the role of HSP60 gene in the development of testis and spermatogenesis of white yak. In this study, the full-length cDNA sequence of HSP60 gene of white yak was obtained by RT-PCR and race. The physicochemical properties, structure and homology of HSP60 protein were analyzed by bioinformatics. The expression and localization of HSP60 in the hypothalamus-pituitary-testicular axis of white yak were analyzed by using qPCRX Western blotting and immunohistochemistry. The results showed that the full length of HSP60 gene cDNA of white yak was 2 300 BP, the open reading frame was 1 722 BP, the theoretical molecular weight was 60.977 K Da, the isoelectric point was 5.69, and the protein was non-transmembrane soluble protein. The results of amino acid sequence alignment showed that the amino acid sequence of HSP60 in white yak had high homology with cattle, sheep, Tibetan antelope, camel, white rhinoceros, rabbit and chimpanzee. HSP60 gene and protein were expressed in hypothalamus, pituitary and testis of white yak, and the expression of hypothalamus and pituitary was significantly higher than that of testis, but there was no significant difference between hypothalamus and pituitary. Immunohistochemical results showed that HSP60 protein was localized in the large paraventricular nucleus cells, paraventricular nucleus small cells and neural keratinocytes, pituitary gland cells, testicular spermatogonia and spermatocytes in the hypothalamus of white yak. Sertoli cells and interstitial cells were less expressed in sperm cells. By detecting the expression and localization of HSP60 in the hypothalamus-pituitary-testicular axis of healthy adult white yaks, it was inferred that HSP60 was involved in the regulation of reproductive axis, testicular development and spermatogenesis of male white yaks. The results laid a foundation for further study on the regulation of HSP60 on male reproduction.
【作者单位】：甘肃农业大学动物医学院;甘肃农业大学动物科技学院;
【基金】：“十二五”农村领域国家科技计划项目(2013AA02505-3)
【分类号】：Q78;S823.85

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