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

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。

Cloning and Localization of Heat Shock Protein 60 in Hypothalamus-pituitary-testis Axis of Tianzhu White Yak

Heat shock protein 60 (HSP60) not only plays a role in cell protection,also participates in the physiological regulation of male reproduction.At present study,to explore the mechanism of testis development and spermatogenesis,the sequence characteristics and expression level of HSP60 gene in hypothalamus-pituitary-testis (HPT) axis of tianzhu white yak were carried out.The full length cDNA of HSP60 was cloned by RT-PCR and RACE.The physical and chemical properties,protein structure and the homology between different species of HSP60 were analyzed by bioinformatics methods.The qPCR,Western blotting and immunohistochemical method were employed to analyze the expession and location of HSP60 in HPT-axis.The results showed that the full length cDNA of the HSP60 was 2 300 bp,with a ORF length of 1 722 bp,which encoded 572 amino acids.The HSP60 protein was predicted to be a type of soluble and non-transmembrane protein,and the theoretical molecular weight and isoelectric point of the protein were 60.977kDa and 5.69,respectively.The amino acid sequence similarity analysis indicated the high homology and near evolutionary level among the White yak and Bos taurus,Bos indicus,ovis aries,Pantholops hodgsonii,Camelus ferus,Ceratotherium simum simum,Oryctolagus cuniculus,Pan troglodytes.Moreover,the qPCR and western blotting indicated that HSP60 expressed broadly in hypothalamus,pituitary,testis.The expression of HSP60 in hypothalamus and pituitary were significantly higher than testis,but no significant difference between the hypothalamus and pituitary.In addition,the immunohistochemical staining analysis revealed that HSP60 expressed broadly in paraventricular nucleus magnocellular,paraventricular nucleus parvocellular and neurokeratin network in White yak hypothalamus,glandular cells in pituitary,spermatogonium,spermatocyte,sertoli cells and leydig cells in testis.According to HSP60 gene expression in HPT-axis,It was speculated that HSP60 participated in the development of testis and spermatogenesis,which laid a basis for further studying of its roles in White yak reproductive regulation.