牦牛DBI基因的分子特征及其在乳腺组织中的表达与定位

Molecular characterization of yak(Bos grunniens)DBI gene and its expression and localization in mammary tissue

地西泮结合抑制因子(Diazepam binding inhibitor,DBI)与酰基辅酶A具有高亲和力,在动物组织中广泛存在,与脂肪酸代谢、类固醇激素合成密切相关。为研究DBI基因的分子特征及该基因在乳腺发育中的作用,对牦牛DBI基因编码区进行克隆,进行生物信息学分析;采用实时荧光定量PCR(Quantitative real-time PCR,qPCR)、蛋白免疫印迹技术(Western blotting,WB)和免疫组织化学(Immunohistochemistry,IHC)方法对牦牛泌乳前期、泌乳期和干乳期的乳腺组织中DBI的相对表达量和表达部位进行研究。DBI序列分析显示:牦牛DBI基因编码区序列长264 bp,编码87个氨基酸残基,与牛的同源性高达99.62%;qPCR数据表明:牦牛泌乳前期乳腺组织中DBI基因的相对表达量显著高于泌乳期和干乳期(P<0.05);WB结果显示:牦牛泌乳前期乳腺组织中DBI蛋白的表达量最高,干乳期次之,泌乳期最低(P<0.05);IHC结果表明:不同发育时期的牦牛乳腺组织中DBI的表达部位并无明显差异,主要表达于乳腺腺泡上皮细胞、导管上皮细胞及小叶间质细胞。DBI在不同发育时期牦牛乳腺组织中的相对表达量具有明显差异(P<0.05),揭示DBI可能参与牦牛乳腺发育的过程,这为进一步探究DBI基因在生物体中的作用提供相应的理论参考。

Diazepam binding inhibitor(DBI)has a high affinity with acyl-CoA and is widely expressed in animal tissues,which is closely related to fatty acid metabolism and steroid hormone synthesis.In order to study the molecular characteristics of DBI gene and its role in the development of mammary gland,the coding region of DBI gene in yaks was cloned and bioinformatics analysis was performed.Quantitative Real-time PCR(qPCR),Western blotting(WB)and Immuno-histochemistry(IHC)assays were used to explore the relative expression level and expression sites of DBI in the mammary tissues of yak in the early lactation,lactation,and dry milk periods.DBI sequence analysis showed that the coding region of yak DBI gene is 264 bp long,encoding 87 amino acids,and the homology with Bos taurus is 99.62%.qPCR data showed that the relative expression level of DBI gene in the mammary tissue of yak in early lactation was significantly higher than during lactation and dry periods(P<0.05).WB results showed that the expression of DBI protein in the mammary tissue of yak was the highest in early lactation,followed by dry period,and the lowest in lactation(P<0.05).IHC results showed that there was no significant difference in the expression sites of DBI in different development stages of yak mammary gland,and it is mainly expressed in acinar epithelial cells,ductal epithelial cells,and lobular mesenchymal cells.There were significant differences in the relative expression levels of DBI in different development stages of yak mammary tissue(P<0.05),suggesting that DBI may be involved in the development of yak mammary gland,which provides a theoretical reference for further exploration of the role of DBI gene in the organism.