牦牛性激素结合蛋白基因克隆及组织表达研究

Cloning and Tissue Expression of Sex Hormone-binding Globulin Gene of Yaks

试验旨在阐明牦牛性激素结合蛋白(SHBG)基因CDS序列及其在母牦牛生殖轴中的表达特点,为探讨该基因在牦牛繁殖中的调控作用奠定基础。试验分别采集5头健康成年母牦牛和母黄牛的下丘脑、脑垂体前叶、输卵管、卵巢和子宫组织,根据NCBI公布的黄牛SHBG基因设计特异性扩增引物,分别采用RT-PCR技术、生物信息学方法和实时荧光定量PCR技术进行牦牛SHBG基因克隆、序列分析和组织表达检测。结果显示,牦牛SHBG核苷酸序列的编码区(CDS)全长为1 206 bp,共编码401个氨基酸,其中,亮氨酸(L)、甘氨酸(G)、脯氨酸(P)和丝氨酸(S)较多,含量分别为16.5%、9.7%、8.7%和9.0%。蛋白质分子式为C 1944 H 3061 N 535 O 566 S 10,分子质量为43.30 ku,理论等电点5.63,与黄牛核苷酸和氨基酸同源性分别为99.0%和97.5%。SHBG蛋白为亲水不稳定蛋白,存在跨膜结构和信号肽;二级结构中主要包含无规则卷曲和延伸链,与三级结构分析一致。系统进化树表明牦牛与黄牛亲缘关系最近。SHBG基因在牦牛输卵管的表达量显著高于其他组织(P<0.05),其他组织之间差异不显著(P>0.05)。SHBG基因在黄牛输卵管表达量极显著高于牦牛(P<0.01),在黄牛卵巢表达量显著高于牦牛(P<0.05),两物种的其他组织中表达量差异不显著(P>0.05)。本研究探讨了牦牛SHBG基因序列及其在生殖轴的表达特性,为进一步研究SHBG基因对牦牛繁殖调控作用提供了一定理论依据。

The aim of this study was to investigate the potential role of sex hormone-binding globulin(SHBG)in regulating yak reproduction by clarifying its CDS sequence and expression characteristics in the reproductive axis of female yak(Bos grunniens).Tissue samples of hypothalamus,anterior pituitary,oviduct,ovary and uterus were collected from five adult and health female yaks and cattle,respectively.The SHBG gene-specific amplification primers of yak were designed according to bovine SHBG gene in NCBI.Cloning,bioinformatics analysis and tissue expression of yak SHBG gene were performed by RT-PCR,bioinformatics methods and Real-time PCR,respectively.The result showed that the coding region(CDS)of yak SHBG was 1 206 bp,encoding a 401 amino acids protein which contained a high proportion of leucine(16.5%),glycine(9.7%),proline(8.7%)and serine(9.0%).The formula of SHBG protein was C 1944 H 3061 N 535 O 566 S 10 and its molecular weight and theoretical isoelectric point were 43.30 ku and 5.63,respectively.Yak SHBG had the highest homology with cattle in nucleotide sequence(99.0%)and amino acid sequence(97.5%).It was a hydrophilicity instability protein contained a signal peptide and a transmembrane region.Random coil and extend chain were mainly in the secondary structure of SHBG,which was consistent with the result of the tertiary structure analysis.The phylogenetic tree showed the closest genetic relationship between yak and cattle.The expression of SHBG gene in yak oviduct was significantly higher than that in other tissues(P<0.05),and the expression among other tissues was not significantly different(P>0.05).The expression of SHBG gene in cattle oviduct was extremely significantly higher than that in yak(P<0.01),and the expression in cattle ovary was significantly higher than that in yak(P<0.05).The expression of SHBG gene in other tissues was not significantly different between yaks and cattle(P>0.05).The research on the SHBG gene sequence and its expression in the reproductive axis of the yak provided a theoretical basis for further studying on the regulation of SHBG on the reproduction in yak.