| 尼罗罗非鱼MyD88基因荧光定量PCR检测方法的建立 |
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| 引用本文： | 王倩,邹芝英,李大宇,祝璟琳,肖炜,杨弘.尼罗罗非鱼MyD88基因荧光定量PCR检测方法的建立[J].中国农学通报,2012,28(5):92-97. |
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| 作者姓名： | 王倩 邹芝英 李大宇 祝璟琳 肖炜 杨弘 |
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| 作者单位： | 1. 中国水产科学研究院淡水渔业研究中心/农业部淡水渔业和种质资源利用重点实验室,江苏无锡214081;南京农业大学无锡渔业学院,江苏无锡214081
2. 中国水产科学研究院淡水渔业研究中心/农业部淡水渔业和种质资源利用重点实验室,江苏无锡,214081 |
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| 基金项目： | 现代农业产业技术体系建设专项资金“罗非鱼产业技术体系”（CARS-49）：中央级公益性科研院所基本科研业务费专项资金“岁非鱼髓样分化因子的克隆和表达分析”（2011JBFA04）. |
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| 摘 要： | 为了准确分析尼罗罗非鱼（Oreochromis niloticus）髓样分化因子（myeloid differentiation factor 88，MyD88）在天然免疫反应中的作用，根据MyD88 EST序列（GenBank登录号：GR679416.1）和β-actin基因序列（GenBank登录号：AB037865.1），分别在保守区域设计并合成引物。实验利用5倍系列稀释的尼罗罗非鱼肝脏cDNA样品构建标准曲线并进行融解曲线分析，建立尼罗罗非鱼MyD88的SYBR GreenⅠ荧光实时定量PCR检测方法。应用2?ΔΔCt分析方法初步检测了尼罗罗非鱼肝脏、脾脏、血液和肌肉等不同组织中的MyD88相对表达量。扩增结果表明MyD88和β-actin基因标准曲线CT值检测范围分别为24~35和19~30，扩增效率分别为100%和96.7%，相关系数分别为0.998和0.995；熔解曲线分析显示产物均形成单一特异峰，Tm值分别为78.5℃和77.5℃。定量分析结果显示，MyD88基因在参与机体免疫的相关组织肝脏、脾脏和血液中高丰度表达。
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| 关 键 词： | 社会化服务体系 社会化服务体系 农业 新型 |
| 收稿时间： | 2011/8/18 0:00:00 |
| 修稿时间： | 2011/9/29 0:00:00 |
Development of Real-time PCR for the Detection of Nile Tilapia MyD88 Gene |
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| Wang Qian
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Zou Zhiying
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Li Dayu
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Zhu Jinglin
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Xiao Wei
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Yang Hong.Development of Real-time PCR for the Detection of Nile Tilapia MyD88 Gene[J].Chinese Agricultural Science Bulletin,2012,28(5):92-97. |
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| Authors: | Wang Qian Zou Zhiying Li Dayu Zhu Jinglin Xiao Wei Yang Hong |
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| Institution: | 1 (Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center/Chinese Academy of Fishery Sciences, Wuxi Jiangsu 214081; Wuxi Fisheries College, Nanjing Agricultural University, Wuxi Jiangsu 214081) |
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| Abstract: | To analyze accurately the role of myeloid differentiation factor 88 （MyD88） during immune response of Nile Tilapia （Oreochromis niloticus）, the specific primers were designed and synthesized in conserved region based on MyD88 EST sequence （GenBank Accession number：GR679416.1） and /β-aetin gene （GenBank Accession number：AB037865.1）. The liver eDNA sample of Nile tilapia was amplified with a series of 5-fold dilution to make standard curve. Specificity of PCR primers was assessed by melting curve analysis of PCR products. With/β-actin as an endogenous gene, a method of SYBR Green I realtime quantitative PCR was established to examine MyD88 gene expression in 4 tissues （liver, spleen, blood and muscle） of Nile tilapia by the 2^AACt, method. The detection range of Cr value of MyD88 and /β-aetin was 24-35 and 19-30. The amplification efficiency （E value） was 100% and 96.7%, and the correlation coefficient （Ba value） was 0.998 and 0.995. The melting curve appeared a single peak, with Tm value of 78.5℃and 77.5℃ respectively. The results showed that MyD88 gene was expressed highly in immune tissues such as liver, spleen and blood. |
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| Keywords: | Nile Tilapia MyD88 /β-aetin realtime quantitative PCR 2^AACt method |
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