罗氏沼虾Rab11基因cDNA克隆及其组织表达分析
MOLECULAR CHARACTERIZATION AND EXPRESSION RESEARCH OF RAB11 FROM GIANT FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII
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摘要: 为了发掘罗氏沼虾(Macrobrachium rosenbergii)免疫相关基因, 研究Rab蛋白(Ras-related proteins inbrain)在罗氏沼虾免疫应答中发挥的作用, 研究采用RACE-PCR技术克隆了罗氏沼虾Rab11基因全长cDNA序列, 记为MrRab11。全长1381 bp, 包括226 bp的5'UTR, 511 bp的3'UTR和645 bp的开放阅读框, 编码214个氨基酸, 含有一个Rab结构域。氨基酸序列比对显示, 罗氏沼虾与冈比亚按蚊(Anopheles gambiae)、蚤状溞(Daphniapulex)、叶蝉(Homalodisca vitripennis) Rab11一致性分别为82%、83%和82%。软件预测, MrRab11编码的蛋白分子量约为23.75 kD, 等电点约为5.34。实时荧光定量表达分析表明, MrRab11基因在罗氏沼虾各组织中都有表达, 肝胰腺中的表达量最高, 其次是肌肉和肠道。在阴沟肠杆菌(Enterobacter cloacae)感染12h后, 罗氏沼虾肝胰腺中MrRab11的表达量上升, 显著高于对照组(P0.05), 推测这是MrRab11对阴沟肠杆菌的应激表达,MrRab11在肝胰腺中参与了罗氏沼虾免疫应答过程。Abstract: Rabs play important role in vesicular transport in all eukaryotic cells. The current study successfully cloned the full-length cDNA of a Rab gene of Macrobrachium rosenbergii (named MrRab11) by the rapid amplification of cDNA ends (RACE) techniques. The full-length of MrRab11 cDNA was 1381 bp, including a 226 bp 5'-terminal untranslated region (UTR), a 511 bp 3'UTR and a 645 bp open reading frame (ORF) encoding a polypeptide of 214 amino acids residues that contain a Rab domain. The calculated molecular mass of deduced MrRab11 polypeptide was 23.75 kD and the isoelectric point was 5.34. BLASTX analysis revealed that MrRab11 had significant homology to Anopheles gambiae, Daphnia pulex and Homalodisca vitripennis with the identity of 82%, 83% and 82%, respectively. Quantitative real-time reverse transcription PCR analysis demonstrated a broad expression of MrRab11 in many tissues with the highest level in hepatopancreas and then muscle and intestine. The expression level of MrRab11 in hepatopancreas was significantly (P0.05) induced by Enterobacter cloacae at 12h. These results suggest that MrRab11 may play a role in the innate immunity of M. rosenbergii.