排除棉酚等干扰提取棉纤维细胞RNA方法的研究  

Isolation of High-quality RNA from Cotton Fiber Cells by Eliminating the Interference of Endogenous Phenolics and Secondary Metabolites

作　　者：徐亚浓[1] 王学德[1] 蒋淑丽[1] 程超华[1] 

机构地区：[1]浙江大学农业与生物技术学院,杭州310029

出　　处：《棉花学报》2002年第3期143-146,共4页Cotton Science

基　　金：国家自然科学基金 (3 9770 473)

摘　　要：棉花纤维细胞含有高的棉酚、多糖和次生代谢物质 ,对其 RNA的提取造成困难。为高质量提取棉花纤维细胞的 RNA,进行了三种提取方法的比较研究。研究表明 ,用高离子、高 p H值提取液提取棉花纤维细胞的 RNA比其它两种提取液TRIZOL提取液和异硫氢酸胍提取液更适合。高离子、高 p H值提取液的特点是 :p H值偏碱性 ,加入脱氧胆酸钠及 NP- 40两类盐类 ,提高其他盐类的浓度 ,并加入 PVP和巯基乙醇两种抗氧化剂。用本方法提取的棉花纤维等组织细胞的 RNA,纯度高 ,不降解 ;如棉纤维细胞 RNA的 A2 6 0 /A2 80 可达 1 .85 ,平均得率为 0 .65 mg· g- 1,棉叶 RNA的A2 6 0 /A2 80 可达 2 .0 2 ,平均得率为 6.35 mg· g- 1,适合于 Northern杂交及 RT-The cotton fiber cells contain a lot of endogenous phenolics, polysaccharides and secondary metabolites that may result in difficulty of RNA isolation from fiber cells. In order to isolate high quality RNA from cotton fiber cells the comparison of three methods of RNA isolation with each other was conducted. The comparison analysis show that the method by using a extraction buffer with high ion density and pH value was better. This extraction buffer with high ion density and ph value was characterized by adding extra reagents include sodium deoxycholate and Nonider 40 (NP 40), enhancing density of other salt compounds, elevating the value of pH , and supplementing polyvinylpyrrolidone (PVP 40) and 2 mercaptoethanol. High quality RNA from cotton fiber and other tissue cells were obtained by using this isolation method, for example, the fiber cell RNA can reach 1.85 of the 260/280 ratio with the yield of 0.65 mg·g 1 and the cotton leaf RNA 2.02 with 6.35 mg·g 1 .It was this method that RNA isolated from cotton tissues, especially from fiber cells, were suitable for the experiments of RT PCR and Northern blot hybridization.

关 键 词：棉花 棉酚 多糖 纤维细胞 细胞提取 RNA 

分 类 号：S562[农业科学—作物学]