水稻、玉米、小麦成熟种子总RNA的快速提取

A Method of Rapid Isolation of Total RNA from Mature Seeds in Rice,Maize,and Wheat

本研究以水稻、玉米、小麦的成熟种子为试验材料,使用液氮分别进行研磨,研磨后的样品先用10%SDS和50 mmol/L Tris-HCl处理,再使用TRIzol试剂盒提取,并将提取的总RNA分别进行了电泳、浓度测定、反转录与Real-time PCR分析。结果显示:18S r RNA和28S r RNA条带清晰;A260/A280比值介于1.95~2.08之间;小麦的得率高于200μg/g,水稻的得率高于300μg/g,而玉米的得率低于230μg/g。Real-time PCR结果表明:0.1μmol/L或10μmol/L GA处理后,水稻种子中SLRL1基因的表达水平显著升高。研究结果表明,本方法提取的水稻、玉米、小麦成熟种子总RNA质量较好,能满足成熟种子中基因表达的检测。

Mature seeds from maize, rice, and wheat were treated with liquid nitrogen, respectively, then were grinded. Subsequently, the grinded samples were transferred into RNase-free tube, and added to 10% SDS and50 mmol/L Tris-Hcl solutions. The total RNA isolation was carried out using TRIzol solution. The gel electrophoresis, RNA concentration, and real-time PCR were performed to determine RNA quality. The results showed that the total RNA displayed clear bands of 28 S r RNA and 18 S r RNA on 1%(w/v) agarose gels; the value of A260/A280 was between 1.95 and 2.0; RNA content in wheat and rice is over 200 μg/g and 300 μg/g, respectively, but is low230 μg/g in maize. The real-time PCR analysis demonstrated that the m RNA level of SLRL1 in mature seeds of rice was significantly increased under GA treatment. Our results confirmed that the quality of total RNA isolated from mature seeds of rice, maize, and wheat is good, and is available for further research.