牦牛ERBB2基因的克隆及其在不同发情周期输卵管中的表达分析

Cloning of ERBB2 from yaks and analysis of its expression in oviducts during different phases of an oestrous cycle

摘要:

本研究旨在探究表皮生长因子受体2 (erb-b2 receptor tyrosine kinase 2, ERBB2)基因的分子结构特征及其在牦牛不同发情周期输卵管不同部位中的表达情况。采集牦牛输卵管组织样品，对牦牛ERBB2基因编码区序列(coding sequence, CDS)进行克隆，分析ERBB2的理化性质等；应用实时荧光定量PCR (real-time fluorescence quantitative PCR, RT-qPCR)检测ERBB2基因在牦牛发情周期不同时期输卵管各段中的表达情况；应用蛋白免疫印迹法(Western blotting, WB)和免疫组织化学法(immunohistochemistry, IHC)检测发情周期不同时期输卵管各部位中ERBB2蛋白的表达。结果显示，牦牛ERBB2基因编码区片段大小为3 768 bp，共编码1 255个氨基酸，与黄牛的亲缘关系最近，与骆驼亲缘关系最远。牦牛ERBB2蛋白属于亲水性蛋白，ERBB2基因编码蛋白分子式为C₆₄₆₄H₁₀₁₄₅N₁₈₁₅O₁₉₃₁S₈₁，相对分子质量146 kDa，其二级结构以不规则卷曲为主。该基因在卵泡期和黄体期这2个时期的输卵管各段中的表达存在差异，可能参与精卵结合以及胚胎发育等生殖过程。本研究为进一步研究ERBB2在雌牦牛繁殖中的功能和分子机制提供了理论依据。

Abstract:

The aim of this study was to characterize the molecular structure of the erb-b2 receptor tyrosine kinase 2 (ERBB2) gene and its expression in different parts of the oviducts of yaks in different phases of an oestrous cycle. We collected yak oviduct tissue samples, cloned the coding sequence (CDS) of ERBB2, and analyzed the physicochemical properties of ERBB2. The expression of ERBB2 in different parts of oviducts of yaks in different phases of an oestrous cycle were determined by RT-qPCR. The expression of ERBB2 protein in different parts of oviducts of yaks in different phases of an oestrous cycle was detected by Western blotting and immunohistochemistry. The results showed that the CDS of ERBB2 from yaks was 3 768 bp in size, encoding 1 255 amino acid residues. This gene was most closely related to that from yellow cows and most distantly related to that from camels. ERBB2 was a hydrophilic protein, with the molecular formula of C₆₄₆₄H₁₀₁₄₅N₁₈₁₅O₁₉₃₁S₈₁ and the relative molecular mass of 146 kDa. Its secondary structure was dominated by random coils. The gene was differentially expressed in different parts of the oviducts in follicular and luteal phases, which indicated that it may be involved in reproductive processes such as sperm-egg binding and embryonic development. The findings provide a theoretical basis for further research on the functions and molecular mechanisms of ERBB2 in female yak reproduction.