黑鲷转铁蛋白基因的分子特征研究

Molecular Cloning and Function Characterization of Transferrin from Acanthopagrus Schlegeli

目的:克隆黑鲷转铁蛋白全基因并分析其分子特征。方法:采用同源克隆的方法,对黑鲷转铁蛋白全基因编码序列进行克隆,在cDNA获得了部分转铁蛋白基因的同源片段。经RACE PCR方法,分别对该基因的3’和5’末端进行扩增,获得的扩增片段经拼接后得到全基因片段。结果:黑鲷转铁蛋白全长2431bp,可编码691个氨基酸,分子量(MW)约为74.3kDa,等电点(PI)为5.63。它与鱼类转铁蛋白的同源性最高,约为65%-89%>;与其它动物(哺乳类、两栖类等)也有一定的相识性。进化分析表明黑鲷转铁蛋白与其它鱼类和哺乳类的转铁蛋白是由早期转铁蛋白共同的祖先进化来的。结论:黑鲷转铁蛋白主要在肝脏中成组成型表达,在大脑等器官中也有少量表达。该基因的表达受病原刺激的影响,表现为经病原刺激后转铁蛋白基因的组织分布显著增多。

Objective： To clone and to analyze the molecular characterization of transferrin from Black seabream（Acanthopagrus schlegeli）. Methods： The full length transferrin（TF） gene from Black Seabream was cloned by homology strategy using a pair of degenerated primers designed according to the conserved regions of other animals＇ TF genes. A part of homologous fragment was obtained by PCR amplification, the fragment was elongated by RACE PCR with the adaptor primer and several gene specific primers to get the full length sequence. Results： The sequence contained 2431 bp including a short 5＇ UTR of 31 bp and 3＇ UTR of 341 bp and an open reading frame of 2072 bp which could code a 691 amino acids peptide with putative MW of 74.3kDa and putative PI of 5.63. The sequence shared high identity and similarity with other animals＇ transferrin, especially with the fish TF genes. There was about 65-89%identities with other fish, and about 40-50% identities with each of the mammalian TFs. Conclusions： Phylogenetic analysis showed that the TF gene evolution from the same ancestor with the others animals＇ transferrin. RT-PCR showed that the Black Seabream TF was constitutively expressed in liver, and it could be regulated by pathogen stimulating, the distribution of TF expression increased significantly after pathogen stimulating.