中华绒螯蟹铁蛋白基因的克隆及表达分析

Cloning and expression analysis of ferritin gene in Chinese mitten crab Eriocheir sinensis

铁蛋白普遍存在于生物体内,具有铁离子解毒和储存等功能。在构建脂多糖刺激的中华绒螯蟹cDNA文库的基础上,获得了中华绒螯蟹铁蛋白基因EsFer的cDNA序列,其全长为1118bp,包含480bp的开放阅读框,推测编码的蛋白质为159氨基酸,其5′和3′端的非编码区分别为178bp和461bp,预测的分子量为18.3ku,理论等电点为5.81,在15～37aa处有一个跨膜螺旋。在5′非编码区核甘酸序列的135～162的位置有个特殊的结构,即铁反应元件(iron response element,IRE)。通过荧光定量PCR的方法研究了铁蛋白基因在中华绒螯蟹的组织表达分布,以及经脂多糖刺激后在血淋巴、肠和肝胰腺组织中的表达变化,发现EsFer在中华绒螯蟹血淋巴、肌肉、肠、鳃、心脏、性腺、肝胰腺等组织器官中都有表达,在肝胰腺的表达量最高,血淋巴中表达量最低。经脂多糖诱导后EsFer在血淋巴、肠和肝胰腺呈上调表达。

In living organisms, ferritin plays an important role in iron storage and detoxification. The fulllength cDNA sequence of a ferritin（EsFer）was obtained from the cDNA library of the Chinese mitten crab Eriocheir sinensis stimulated with lipopolysaccharide （LPS）. It has 1 118 bp in length and contains a 480 bp open reading frame （ORF） encoding 159 amino acids （aa）. The 5＇-and 3＇-untranslated region （UTR） is 178 bp and 461 bp, respectively. The predicted molecular weight is 18.3 ku and the theoretical isoelectric point 5.81. A transmembrane helix was found at the position of 15 - 37 aa. A complete iron-responsive element （IRE） was found at the 5＇ untranslated region corresponding to the nucleotide sequence at the positions of the 135 - 162. Quantitative real-time PCR was used to assess the mRNA expression of EsFer in organs or tissues and its induced expression in haemolymph, intestine, hepatopancreas of crabs challenged with LPS. The results showed that EsFer was expressed in all tested organs or tissues of the Chinese mitten crab, including haemolymph, muscle, intestine, gills, heart, gonad and hepatopancreas. The highest expression level was observed in hepatopancreas, while the lowest in haemolymph. The expression of EsFer in haemolymph, intestine and hepatopancreas was up-regulated following the challenge with LPS.