OPN基因序列分析及其在产蛋后期鸡胫骨中的表达研究

试验旨在研究鸡骨桥蛋白（osteopontin，OPN）基因的结构与功能，并探索其在蛋鸡产蛋后期骨骼组织中的表达特性。采集产蛋后期海兰灰蛋鸡胫骨组织进行RNA提取，根据GenBank数据库中公布的中国原鸡OPN基因序列（登录号：NC_006091.5），利用Primer Premier 3.0在线软件设计引物，利用RT-PCR扩增OPN基因编码区（coding sequence，CDS）并对其进行生物信息学分析，实时荧光定量PCR检测海兰灰产蛋后期胫骨组织中OPN基因mRNA的表达。RT-PCR及测序结果显示，鸡OPN基因CDS区长795 bp，共编码264个氨基酸。应用Mega 6.0软件构建系统发育树发现，鸡与鹌鹑的亲缘关系最近，同源性为100%，OPN基因在禽类进化的过程中具有高度保守性。生物信息学分析表明，OPN为不稳定的水溶性蛋白，含有信号肽，无跨膜结构域，有27个O-糖基化位点、1个N-糖基化位点和34个磷酸化位点，亚细胞主要定位于细胞质（44.4%）和线粒体（33.3%），二级结构主要是无规则卷曲和α-螺旋。实时荧光定量PCR结果表明，OPN基因mRNA在胫骨中的表达存在个体差异，但与骨骼强度无关。结果表明，蛋鸡产蛋后期OPN基因表达与骨断裂强度无显著相关，为进一步研究OPN基因在产蛋后期蛋鸡骨代谢中的作用机制提供依据。

Sequence and Expression Analysis of OPN Gene in Tibia of Layer During Late Period of Laying

To investigate the structure and function of osteopontin (OPN) gene and explore its expression characteristics in bone metabolism of layer during late period of laying.The tibia of Hyline Grey layer at late period of laying was selected for RNA isolation.The specific primers were designed based on OPN gene sequence of Gallus gallus in GenBank (accession No.:NC_006091.5) using Primer Premier 3.0 online software.The coding sequence (CDS) of OPN gene was cloned by RT-PCR and analyzed by bioinformatics.Real-time PCR technology was used to detect the expression of OPN gene mRNA in tibia.The results showed that the full-length of chicken OPN gene CDS was 795 bp, encoding 264 amino acids.The phylogenetic tree constructed by Mega 6.0 software showed that the relationship was the closest between chicken and carp, with 100% similarity.The OPN gene was highly conserved in avian evolution.The bioinformatics analysis suggested that OPN was an unstable and hydrophilic protein with 27 O-glycosylation sites, 1 N-glycosylation site and 34 phosphorylation sites.The subcellular localization of OPN was mainly localized in cytoplasm (44.4%) and mitochondria (33.3%).The secondary structure was mainly composed of random coil and alpha helix.The difference of OPN gene mRNA expression among individuals was not related to bone strength.The results indicated that OPN gene mRNA expression was not related to the bone strength, which provided a reference basis for further study on the mechanism of OPN gene in bone metabolism of layer during late period of laying.