摘要
为了准确分析尼罗罗非鱼(Oreochromis niloticus)髓样分化因子(myeloid differentiation factor88,MyD88)在天然免疫反应中的作用,根据MyD88EST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倍系列稀释的尼罗光罗非实鱼时肝定脏量cPDCNRA检样测品方构法建。标应准用曲2线-ΔΔ并Ct分进析行方融法解初曲步线检分测析了,建尼立罗尼罗罗非罗鱼非肝鱼脏M、脾yD脏88、的血S液Y和BR肌G肉re等en不Ⅰ荧同组织中的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组织成肝单脏一、特脾异脏峰和,血Tm液值中分高别丰为度78表.5达℃和。77.5℃。定量分析结果显示。
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.
出处
《中国农学通报》
CSCD
2012年第5期92-97,共6页
Chinese Agricultural Science Bulletin
基金
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"罗非鱼髓样分化因子的克隆和表达分析"(2011JBFA04)
