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

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.