欢迎访问《中国农学通报》，
中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (5): 92-97.

所属专题： 生物技术； 水产渔业

• 水产 渔业 • 上一篇    下一篇

尼罗罗非鱼MyD88基因荧光定量PCR检测方法的建立

王倩 邹芝英 李大宇 祝璟琳 肖炜 杨弘   

  • 收稿日期:2011-08-18 修回日期:2011-09-29 出版日期:2012-02-15 发布日期:2012-02-15
  • 基金资助:

    现代农业产业技术体系专项资金;中央级公益性科研院所基本科研业务费专项资金

Development of Real-time PCR for the Detection of Nile Tilapia MyD88 Gene

  • Received:2011-08-18 Revised:2011-09-29 Online:2012-02-15 Published:2012-02-15

PDF (PC)

1043

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。

关键词: 社会化服务体系, 社会化服务体系, 农业, 新型

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-actin gene (GenBank Accession number:AB037865.1). The liver cDNA 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 real-time quantitative PCR was established to examine MyD88 gene expression in 4 tissues (liver, spleen, blood and muscle) of Nile tilapia by the 2?ΔΔCt method. The detection range of CT value of MyD88 and β-actin was 24–35 and 19–30. The amplification efficiency (Evalue) was 100% and 96.7%, and the correlation coefficient (R2 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.