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中国农学通报

中国农学通报 ›› 2016, Vol. 32 ›› Issue (30): 103-107.doi: 10.11924/j.issn.1000-6850.casb16040154

所属专题： 烟草种植与生产

• 生物技术科学 • 上一篇    下一篇

3种烟草RNA高效提取方法的比较

张震彪1,2,宋希云1,郭新梅1   

  1. (1青岛农业大学农学与植物保护学院,山东青岛 266109；2中国农业科学院烟草研究所,山东青岛 266101）
  • 收稿日期:2016-04-26 修回日期:2016-06-18 接受日期:2016-06-24 出版日期:2016-10-31 发布日期:2016-10-31
  • 通讯作者: 郭新梅
  • 基金资助:
    烟草行业烟草基因资源利用重点实验室开放课题资助。

Three High Effective Extraction Methods for RNA in Tobacco

Zhang Zhenbiao1,2, Song Xiyun1, Guo Xinmei1   

  1. (1College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao Shandong 266109;2Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao Shandong 266101)
  • Received:2016-04-26 Revised:2016-06-18 Accepted:2016-06-24 Online:2016-10-31 Published:2016-10-31

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摘要： 为了寻求烟草叶片RNA高效提取方法，为烟草分子生物学研究提供理论基础，试验以烟草叶片为材料，采用试剂盒法、Trizol法、RNAiso法提取RNA，凝胶电泳和分光光度计检测确定高效、经济的RNA提取方法。结果表明：试剂盒法操作简单，但提取量较少；Trizol法方便快捷，但有部分蛋白质污染，并出现RNA降解现象；相比以上2种方法，RNAiso法提取的RNA浓度在100~500 ng/μL之间，A260/A280在1.8~2.0之间，条带清晰明亮，无RNA降解，且28 S条带亮度约是18 S的两倍；RT-PCR检测结果显示，RNAiso法提取的RNA可以扩增出目的基因，大小约2500 bp，与参考序列大小吻合，可用于后续的功能验证。因此，在烟草RNA提取方法比较中，RNAiso法较适合烟草叶片RNA提取，并已在很多实验室推广利用。

关键词: 烟草黑胫病, 烟草黑胫病, 拮抗细菌, 复配, 生物防治

Abstract: In order to find an effective way to extract RNA in tobacco leaves and provide theoretical basis for tobacco molecular biology research, tobacco leaves were used as material, 3 methods including RNA isolation kit, Trizol method and RNAiso method were used to extract RNA, then RNA were detected by gel electrophoresis and spectrometer. The results indicated that the operation of RNA isolation kit was very easy, but the extraction was the least; Trizol method was convenient and efficient, but there were proteins and a little degradation in RNA; in contrast, the concentration of RNA extracted by RNAiso method was very high and ranged from 100 to 500 ng/μL, A260/A280 was from 1.8 to 2.0, RNA bands were clear and bright without degradation, and the brightness of 28S band was about twice that of 18S band; RT-PCR result showed that 2500 bp target gene consistent with the reference sequence was obtained, and it could be used in functional verification. Hence, RNAiso method was the best way to extract total RNA in tobacco leaves and had been used in many laboratories.

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