Teng Tao, Xi Bingwen, Xie Jun, Chen Kai, Xu Pao, Pan Liangkun
Wuxi Fisheries College, Nanjing Agricultural University, Wuxi, 214081, China.
Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, 214081, China.
Fish Physiol Biochem. 2017 Aug;43(4):987-997. doi: 10.1007/s10695-017-0346-3. Epub 2017 Feb 24.
Transferrin (Tf) plays an important function in iron homeostasis and metabolism of organisms. In this study, we identified and characterized the Tf gene in Megalobrama amblycephala and evaluated its expression in basal conditions as well as after iron overload and experimental infection with Aeromonas hydrophila. Furthermore, we studied the iron binding properties of recombinant Tf. The full-length M. amblycephala Tf complementary DNA (cDNA) (GenBank accession no.: KX698308) of 2245 bp was cloned and contained a 1953 bp open reading frame (ORF) encoding 650 amino acid residues and flanked by a 68 bp 5' and a 204 bp 3' untranslated regions (UTR). Predicted conservative structure illustrated that M. amblycephala Tf consisted of two conservative Tf domains. Amino acid sequence alignment revealed that M. amblycephala Tf had high similarity with that of cyprinids deposited in Genbank, and phylogenetic analysis showed that M. amblycephala Tf clustered with Ctenopharyngodon idella and Hypophthalmichthys molitrix. Tissue expression pattern analyses demonstrated that the liver was the main Tf mRNA expressing organ, being significantly higher than other tissues (p < 0.05). In the liver, Tf mRNA expression in fish artificially injected with the pathogenic bacteria A. hydrophila was significantly upregulated, reaching a peak at 12 h post injection (hpi) and then decreasing afterward. The expression in FeCl-injected fish showed a similar tendency, but reached a peak at 8 hpi. Meanwhile, fish serum iron significantly decreased following A. hydrophila injection, but increased to peak at 4 hpi and then decreased in FeCl-injected fish. The recombinant M. amblycephala Tf showed iron binding capacity using CAS analysis. These results are helpful to understand the structure and regulation of expression of Tf, as well as the specific function of Tf for both immune responses and iron homeostasis.
转铁蛋白（Tf）在生物体的铁稳态和代谢中发挥着重要作用。在本研究中，我们鉴定并表征了团头鲂的Tf基因，并评估了其在基础条件下以及铁过载和嗜水气单胞菌实验感染后的表达情况。此外，我们研究了重组Tf的铁结合特性。克隆了全长2245 bp的团头鲂Tf互补DNA（cDNA）（GenBank登录号：KX698308），其包含一个1953 bp的开放阅读框（ORF），编码650个氨基酸残基，两侧分别为68 bp的5'非翻译区和204 bp的3'非翻译区（UTR）。预测的保守结构表明团头鲂Tf由两个保守的Tf结构域组成。氨基酸序列比对显示团头鲂Tf与Genbank中已存的鲤科鱼类具有高度相似性，系统发育分析表明团头鲂Tf与草鱼和鲢鱼聚类在一起。组织表达模式分析表明肝脏是Tf mRNA表达的主要器官，显著高于其他组织（p < 0.05）。在肝脏中，人工注射病原菌嗜水气单胞菌的鱼体内Tf mRNA表达显著上调，在注射后12小时（hpi）达到峰值，随后下降。注射FeCl的鱼体内表达呈现类似趋势，但在8 hpi达到峰值。同时，注射嗜水气单胞菌后鱼血清铁显著下降，但在注射FeCl的鱼体内在4 hpi升高至峰值，随后下降。使用CAS分析表明重组团头鲂Tf具有铁结合能力。这些结果有助于理解Tf的结构和表达调控，以及Tf在免疫反应和铁稳态中的具体功能。
