| 摘要: |
| 采用 cDNA 文库、RACE、荧光定量 PCR 和 Western blot 技术, 克隆了南移养殖仿刺参铁蛋白基因的全长序列, 并对其表达特征进行了研究。结果表明, 南移养殖仿刺参铁蛋白 cDNA 全长1222bp, 包括 187bp 的 5’UTR; 513bp 的 3’UTR 和编码 173 个氨基酸残基的 522bp 的开放阅读框。 诸如铁结合序列标签和铁调控元件等铁蛋白特征结构基元在南移刺参铁蛋白中也高度保守; 养殖环境条件的改变影响了铁蛋白在某些组织的表达水平, 南方养殖仿刺参肌肉带铁蛋白表达量近乎北方养殖仿刺参的 2 倍, 而在呼吸树中两者表达水平没有明显差异。Western blot 揭示本研究获得的铁蛋白多克隆抗体不仅与重组蛋白起强的特异性反应, 而且还特异性识别肌肉中的天然蛋白, 该抗体可用于仿刺参铁蛋白功能的后续研究。 |
| 关键词: 仿刺参, 铁蛋白, 荧光定量 PCR, 蛋白印迹 Western blot |
| DOI：10.11693/hyhz201104016016 |
| 分类号: |
| 基金项目:国家农业科技成果转化资金项目, 2007GB2C220359 号; 浙江省重大科技专项(优先主题)重大农业项目, 2008C02009-Ⅱ-2号; 宁波市农业科技成果转化资金项目, 2007C30001 号; 宁波市自然科学基金项目, 2011A610013 号; 宁波大学海洋生物学学科项目, xk111087 号 |
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| CLONING AND CHARACTERIZATION OF FERRITIN GENE FROM SOUTH CULTURED STICHOPUS JAPONICUS |
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LI Cheng-Hua1, CUI Jing1, LI Ye1, ZHOU Jun1, SU Xiu-Rong1, LI Tai-Wu1,2
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1.Faculty of Life Science and Biotechnology, Ningbo University;2.Ningbo City College of Vocational Technology
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| Abstract: |
| With the approaches of cDNA library, RACE, fluorescent real-time quantitative PCR and western blot, the full-length ferritin cDNA was identified and characterized in South cultured Stichopus japonicus (denoted as SjFER) in the present study. All these results indicated: 1) The cDNA of SjFER was of 1222bp, consisting of a 5'UTR of 513bp with a putative iron regulatory element (IRE), a 3'UTR of 187bp, and a complete open reading frame of 522bp encoding a polypeptide with 173 amino acid residues. The conserved motifs for ferritin including iron binding signature, H-specific ferroxidase center and phosphorylation site were totally found in the deduced amino acid of SjFER. 2) SjFER was an important molecule in the species environmental adaption. The expression level of SjFER in South sea cucumber was two times higher than that from North in the tissue of muscle, while no difference occurred in tissue of respiratory tree. 3) The ployclonal antibodies generated from the recombinant product of SjFER could be specifically identified not only the recombinant product, but also the native protein from muscles, and should be used for next functional validation. |
| Key words: Stichopus japonicus, Ferritin, Fluorescent real-time quantitative PCR, Western blot |
