煙草Nt-syr1基因?qū)崟r(shí)熒光定量PCR檢測方法
A Real-time Fluorescent Quantitative PCR for Detecting Transcripts of Nt-syr1 Gene in Tobacco
-
摘要: 根據(jù)煙草Nt-syr1基因mRNA序列設(shè)計(jì)特異引物,，建立了SYBR Green I實(shí)時(shí)熒光定量PCR反應(yīng)體系，對煙株打頂后葉片Nt-syr1基因進(jìn)行了mRNA轉(zhuǎn)錄水平上的定量分析,，為從分子生物學(xué)水平上研究烤煙鉀素營養(yǎng)調(diào)控機(jī)理提供新的技術(shù)手段,。該方法簡單實(shí)用，獲得的熒光定量PCR擴(kuò)增曲線基線平整,，指數(shù)區(qū)擴(kuò)增明顯,，斜率大；穩(wěn)定性和重現(xiàn)性好,，變異系數(shù)?。谎h(huán)閾值Ct與PCR起始模板量的對數(shù)值之間存在良好的線性關(guān)系,。對基因表達(dá)結(jié)果分析表明,，煙株打頂后1 h，Nt-syr1基因在葉片中強(qiáng)烈表達(dá),，表達(dá)量約是同期不打頂處理的480倍,，隨后逐漸降低。與打頂處理相比,，打頂后涂抹生長調(diào)節(jié)劑可以降低其表達(dá)量,。Abstract: The project aimed to establish real-time fluorescent quantitative PCR detecting Nt-syr1 gene in tobacco and provided a new technique for the mechanism study of potassium regulation at the molecular level. By designing two pairs of amplification primers based the mRNA sequence of Nt-syr1 in tobacco and establishing SYBR Green I reaction system of real-time fluorescent quantitative PCR, quantitative analysis was conducted to detect the changes of Nt-syr1 gene in tobacco leaves after decapitation. The technique showed that the amplification curve had flat baseline, distinct exponential area, large and stable slope, and the coefficient of variation was very little. There was a linear relationship between threshold cycle values at which samples crossed threshold and the logarithmic values of template concentration. The result indicated that the Nt-syr1 gene was strongly expressed in tobacco leaves after decapitation about 1 hour, and the expression was enhanced about 480-fold in comparison to no decapitation tobacco, but using plant growth regulators could decrease the expression caused by decapitation.
下載: