适用于基因全长克隆的灰毡毛忍冬不同器官总RNA提取方法筛选

摘要

Objective To screen of total RNA extraction methods from different organs in Lonicera Macranthoides Hand.-Mazz (L. Macranthoides). Methods The total RNA of leaf, stem and flower buds from L. macranthoides, respectively, were isolated by SDS, Trizol, improved CTAB and polysaccharide polyphenols kit methods, and the results were compared. Results SDS was not suitable for this experiment; Trizol only could isolate total RNA from its leaf. Improved CTAB was the best way to isolate total RNA from its stem; Polysaccharide polyphenols kit method could isolate total RNA from its flower buds, and obtained specific bands by RT-PCR. Conclusion Trizol can be used to isolate the total RNA from leaf of L. macranthoides. Improved CTAB was the best method to isolate the total RNA from its stem; Polysaccharide polyphenols kit method could be used to isolate the total RNA from its flower buds. The total RNA by the three methods could meet the request of full-length gene cloning and temporal and spatial expression analysis.%目的 获取灰毡毛忍冬不同器官总RNA的最佳提取方法. 方法 以灰毡毛忍冬叶、茎、花蕾为材料,分别采用SDS法、Trizol法、改良CTAB法、多糖多酚试剂盒法提取总RNA,比较分析实验结果. 结果 SDS法不适用于三种器官样品总RNA的提取;Trizol法仅适用于叶总RNA的提取; 改良CTAB法对茎总RNA提取效果最佳; 而多糖多酚试剂盒法提取的花蕾总RNA质量高、 完整性较好, 且通过RT-PCR扩增能获得特异性条带. 结论 Trizol法最适于灰毡毛忍冬叶总RNA提取; 改良CTAB法最适于茎总RNA提取;多糖多酚试剂盒法最适于花蕾总RNA提取;3种方法提取出来的总RNA均能满足灰毡毛忍冬中相关基因全长克隆及时空表达分析的要求.