条斑紫菜HSP90基因实时荧光定量PCR检测方法的构建  

Establishment of Real-time PCR for HSP90 Gene of Porphyra yezoensis Udea

作　　者：周向红[1] 阎斌伦[1,2] 王萍[1] 易乐飞[1,2] 

机构地区：[1]淮海工学院海洋学院,江苏连云港222005 [2]淮海工学院江苏省海洋生物技术重点建设实验室,江苏连云港222005

出　　处：《淮海工学院学报（自然科学版）》2010年第2期81-84,共4页Journal of Huaihai Institute of Technology:Natural Sciences Edition

基　　金：江苏省海洋生物技术重点建设实验室开放课题(2008HS004);淮海工学院自然科学基金资助项目(Z2007035)

摘　　要：以管家基因18S rRNA为内参,采用SYBR GreenI荧光染料法,构建了条斑紫菜HSP90基因实时荧光定量PCR的检测方法,为条斑紫菜HSP90和18S rRNA基因筛选出定量引物各1对,获得的扩增曲线基线平整、指数区明显、斜率大且固定。两基因的熔解曲线显示单特异峰,Tm分别为88.5和85℃。两基因标准曲线的斜率分别为-3.307和-3.308,扩增效率都约为100.6%,Ct值在13～32范围内有良好的线性关系。构建的实时荧光定量PCR检测方法灵敏度高、特异性强,准确可靠、重复性好,检测周期短,为进一步研究HSP90基因在胁迫下的表达差异奠定了基础。The method of real-time PCR for HSP90 gene of Porphyra yezoensis Udea was established with 18S rRNA as the reference gene,using SYBR Green I. The optimal primers for HSP90 and 18S rRNA gene were selected and amplification curve had flat baseline,distinct exponential area,large and stable slope. The melting curve of the two genes showed a single peak with a Tm of 88.5 ℃ and 85 ℃ respectively.Standard curve＇s slope of the two genes was-3.307 and-3.308 respectively. Amplification efficiency of the two genes was 100.6%. The linear range of standard curve for relative quantification was 13—32. The real-time PCR method developed in this study is highly sensitive,specific,accurate,reproducible and quick,which provides the basis for further investigation of HSP90 expression under stress.

关 键 词：条斑紫菜 HSP90基因 18S RRNA基因 实时荧光定量PCR SYBR GreenI 

分 类 号：Q75[生物学—分子生物学]