| 摘要: |
| 采用RACE方法克隆了半滑舌鳎(Cynoglossus semilaevis)PKR基因(CsPKR), 获得了CsPKR全长cDNA序列为2907bp, 其中包含1959bp的开放阅读框, 100bp的5′非编码区和848bp的3′非编码区。保守结构域分析显示推导的CsPKR氨基酸在N端存在两个特异的dsRBD(dsRNA binding domain dsRBD)结构域, 在C端具有多个保守的激酶活性位点, 包括ATP结合位点和底物配体结合位点, 以及活性环结构A-loop。系统进化树分析显示鱼类、两栖类、鸟类及哺乳类的PKR具有共同的进化起源, CsPKR与牙鲆PKR的亲缘关系最为密切。荧光定量PCR结果显示, CsPKR基因在健康鱼多个组织中广泛表达, 在脑中的表达最高, 头肾中表达最低。经鳗弧菌和淋巴囊肿病毒分别感染后, CsPKR基因在免疫相关组织中呈现上调表达趋势, 其中感染鳗弧菌12h后在血液中表达量较对照组上升了9.28倍, 在感染淋巴囊肿病毒24h后, 在肝脏中的表达达到最大, 为对照组的9.97倍。以上结果暗示CsPKR基因在半滑舌鳎响应细菌和病毒免疫应答中起重要作用。 |
| 关键词: 半滑舌鳎 PKR基因 克隆 病原 基因表达分析 |
| DOI：10.11693/hyhz201304028028 |
| 分类号: |
| 基金项目:国家高技术研究发展计划(863)“海水养殖生物重要功能基因的发掘与研究”课题, 2012AA10A401号; 青岛市科技计划基础研究项目“半滑舌鳎抗病相关基因的鉴定与应用潜力研究”, 10-3-4-11-3-jch号; 国家重点基础研究项目(973)“重要养殖鱼类功能基因组学和分子设计育种研究”, 2010CB126303号。 |
附件 |
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| MOLECULAR CLONING AND EXPRESSION OF PKR GENE IN HALF SMOOTH TONGUE SOLE CYNOGLOSSUS SEMILAEVIS |
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SHA Zhen-Xia1, WANG Qi-Long2, LI Min1, GONG Guang-Ye3, LIANG Zhuo1, CHEN Song-Lin1
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1.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory For Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory For Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture Tengzhou Fisheries Center;3.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory For Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture Shanghai Ocean University, College of Fisheries and Life Science
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| Abstract: |
| In this report, the full length cDNA of half smooth tongue sole (Cynoglossus semilaevis) PKR (CsPKR) was cloned by RACE method. It was of 2907bp, including 100bp 5′ UTR, 848bp 3′ UTR and an open reading frame (ORF) of 1959bp encoding 652 animo acids. The putative CsPKR protein contains two N-terminal conserved dsRNA binding domain (dsRBD) and some active sites ATP binding sites and A-loop structures on the C-terminal. Phylogenetic analysis showed that fish PKR shared a common origin with birds and mammals PKRs. Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsPKR mRNA was constitutively expressed in all examined healthy tissues with the predominant in blood and the lowest in head kidney. After infection with Vibrio anguillarum, CsPKR gene expression had a varying degree of increase in blood, intestine, spleen, head kidney, and liver from 12h to 7d. The expression of CsPKR was up-regulated to 9.28-fold higher than PBS control at 12h in blood. After infection with Lymphocystis disease virus (LCDV), the expression of CsPKR increased gradually and reached a peak at 24h up to 9.97-fold than PBS control in the live. All of the results suggested CsPKR plays a potential role in response to bacterial and virus infections in half smooth tongue sole. |
| Key words: Cynoglossus semilaevis PKR gene molecular cloning pathogens infection gene expression analysis |
