【摘要】 为探究虹鳟(Oncorhynchus mykiss)脂肪酸结合蛋白10(fatty acid binding protein 10,fabp10)基因序列信息及其所编码蛋白的结构和功能等，本试验根据Gen Bank数据库公布的河鳟(Salmo trutta)fabp10基因的CDS序列信息设计引物，通过RT-PCR获得fabp10基因片段，将目的片段与p MD-18-T载体连接转化DH5α感受态细胞，构建p MD-18-T-fabp10克隆载体，双酶切回收基因片段，构建pc DNA3.1-fabp10真核表达载体，分析fabp10基因在虹鳟不同组织中的表达，经双酶切、测序鉴定构建成功。将表达载体转染至EPC细胞，分别于24h、36 h、48 h后收集细胞进行荧光定量PCR检测。结果显示：虹鳟fabp10基因CDS区与Gen Bank数据库中Salmo trutta fabp10基因CDS区同源性高达100%。生物信息学分析发现，fabp10基因编码区全长378 bp，编码126个氨基酸。Fabp10蛋白主要分布在细胞质中，存在23个潜在磷酸化位点。转染pc DNA3.1-fabp10可显著提高EPC细胞中fabp10 m RNA的表达(P<0.05)。RT-q PCR结果显示：fabp10基因在肝脏中表达丰度最高，随之为肾脏、肌肉、肠、脾脏、心脏，在脑中表达量最低。本试验成功扩增出fabp10基因CDS区并构建了真核表达载体，成功预测分析了其结构和功能，为研究虹鳟机体脂质代谢过程提供了基础。更多还原

【Abstract】 In order to understand the bioinformation of the rainbow trout(Oncorhynchus mykiss) fatty acid binding protein 10(fabp10)gene and the structure and function of the encoded protein, fabp10 eukaryotic expression vector was constructed accompanying with the its tissue distribution analysis, thereby exploring the effects on lipid metabolism. In the present study, primers were designed based on the CDS sequence of the river trout(Salmo trutta)fabp10 gene published in the GenBank database, and the obtained fabp10 gene fragment by RT-PCR was connected with pMD-18-T vector to transform DH5α competent cells for constructing pMD-18-T-fabp10 cloning vector. A pcDNA3.1-fabp10 eukaryotic expression vector was constructed after verifying with the double enzyme digestion and sequencing. The expression vector was transfected into EPC cells, and the cells were collected after 24 h, 36 h,and 48 h for real-time quantitative PCR analysis. The results showed that the CDS region of fabp10 was 378 bp in length, encoding 126amino acids, containing 23 phosphorylation sites, which had 100% homology with the CDS region of Salmo trutta fabp10 gene published in the GenBank database. Sub-cellular localization result showed that Fabp10 protein was mainly distributed in cytoplasm. Compared with the control group, the expression of fabp10 gene was extremely increased after transfection into EPC cells(P <0.01).RT-q PCR results showed that fabp10 gene was most abundant expression in liver, followed by kidney, muscle, intestine, spleen, heart,and brain. In conclusion, the rainbow trout pcDNA3.1-fabp10 eukaryotic expression vector was successfully constructed, and its structure was successfully predicted. The results provided the significant theoretical basis for studying the proceeding of lipid metabolism in rainbow trout.更多还原