| 水稻种子总RNA提取质量问题的探究 |
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| 引用本文： | 谢月兰,崔欢,王长龙,陈志强,肖武名.水稻种子总RNA提取质量问题的探究[J].中国农学通报,2020,36(29):37-46. |
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| 作者姓名： | 谢月兰 崔欢 王长龙 陈志强 肖武名 |
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| 作者单位： | 华南农业大学国家植物航天育种工程技术研究中心,广州 510642 |
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| 基金项目： | 国家自然科学基金“cl7.1调控水稻耐淹萌发机制的研究”(31872885);广东省自然科学基金“水稻粒长新基因gl的克隆及功能研究”(2018A030310207) |
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| 摘 要： | 水稻种子总RNA提取在水稻分子遗传基础实验中起着非常重要的作用,然而,高质量水稻种子RNA的获取难度高,极大阻碍了cDNA文库构建、RNA-Seq和基因表达分析等相关研究工作的开展和深入。为了探讨水稻种子总RNA提取的相关影响因素,寻找合适的水稻种子总RNA提取方式,本研究通过比较Trizol法、GeneMark试剂盒法和艾德莱试剂盒法3种不同的提取方式、不同的耗材处理方法、研磨方式等不同因素改进水稻种子总RNA的提取效果。不同的检测结果显示,艾德莱多糖多酚提取试剂盒能够提取到28S、18S条带完整、降解少和纯度高的水稻种子总RNA;不同耗材处理方法中,蛋白酶K处理过的耗材提取的总RNA比另外两种方法处理过的耗材提取的总RNA降解更少,28S和18S条带更清晰和完整、总RNA质量最高、效果最好;而研磨方式上,液氮冷冻研磨法能够有效抑制RNA酶对RNA的降解。本研究对水稻种子RNA提取过程相关因素的分析和改进,为促进以水稻种子为研究对象的分子实验奠定了一定的技术基础。
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| 关 键 词： | 水稻种子 RNA提取 完整性 提取方式 耗材处理 |
| 收稿时间： | 2019-09-12 |
Total RNA Extraction of Rice Seeds: Quality Discussion |
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| Xie Yuelan,Cui Huan,Wang Changlong,Chen Zhiqiang,Xiao Wuming.Total RNA Extraction of Rice Seeds: Quality Discussion[J].Chinese Agricultural Science Bulletin,2020,36(29):37-46. |
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| Authors: | Xie Yuelan Cui Huan Wang Changlong Chen Zhiqiang Xiao Wuming |
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| Institution: | National Plant Space Breeding Engineering Technology Research Center, South China Agricultural University, Guangzhou 510642 |
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| Abstract: | Total RNA isolation from rice seeds plays an important role in the molecular genetic basic experiments of rice. However, it is difficult to obtain high-quality rice seed RNA, which hinders the construction of cDNA library, RNA Seq and gene expression analysis. In order to explore the relevant impact factors of total RNA isolation from rice seeds and find the appropriate isolation methods, this study compared three different isolation methods, including Trizol method, GeneMark Kit Method and Adlaid kit method, different consumable treatment methods, grinding methods and other different factors to improve the effect of total RNA isolation from rice seeds. Different tests results showed that the Aidlad Polysaccharide Extraction Kit isolated total RNA from rice seeds with complete 28S, 18S bands, low degradation and high purity. Among the different consumables treatment methods, the total RNA isolated by the proteinase K-treated consumables degraded less than the other two methods. Besides, the 28S and 18S bands were clearer and more complete, and the total RNA had the highest quality and the best effect. In the grinding methods, the liquid nitrogen freeze-grinding method could inhibit the degradation of RNA by RNase effectively. In this study, the analysis and improvement of the relative factors of rice seed RNA isolation process lays a certain technical foundation for promoting the molecular experiment of rice seed as the research object. |
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| Keywords: | rice seeds RNA isolation integrity isolation methods lab consumables processing |
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