一种简便有效的香蕉小分子RNA提取方法  

A simple and efficient method for isolating small RNAs from banana(Musa nana L.)

作　　者：王静毅[1,2] 冯仁军[1] 柴娟[1,3] 陈友[4] 张银东[3] 马子龙[1,2] 武耀廷[4] 

机构地区：[1]中国热带农业科学院热带作物生物技术研究所.农业部热带作物生物学与遗传资源利用重点实验室,海口571101 [2]中国热带农业科学院航天育种研究中心,海口571101 [3]海南大学农学院,海口570228 [4]琼州大学,海南三亚572002

出　　处：《果树学报》2015年第2期335-338,共4页Journal of Fruit Science

基　　金：中央级公益性科研院所基本科研业务费项目(ITBB130201;1630052014006);海南省自然科学基金(312050;312051);国家自然科学基金(31201603);海南省重大科技项目子课题(ZDZX2013023-1)

摘　　要：【目的】提供一种简单、经济、高效的香蕉小分子RNA提取方法,以满足RT-PCR、Northern杂交等分子生物学研究的需要。【方法】以巴西蕉(Musa acuminata L.AAA group,‘Brazilian’)的叶片、雄花、果实和根系为材料,利用改良的CTAB法,结合使用PEG8000分级沉淀DNA和大分子RNA,从而获得小分子RNA。【结果】琼脂糖凝胶电泳显示小RNA带型清晰,无DNA和大分子RNA干扰,说明小RNA质量较好。获得的小RNA经紫外光谱分析其A260/A280的比值在1.872~2.020,产量可达35μg·g-1。以提取的各组织小分子RNA为模板,利用茎环RT-PCR方法在香蕉不同组织小RNA中均检测到mi RNA156a,其扩增片断大小约为70 bp,且测序结果与预测的香蕉mi R156a序列一致。【结论】本实验提供了一种简便高效的香蕉小分子RNA提取方法,可满足RT-PCR、Northern blotting及小RNA文库构建等后续分子生物学研究的需要,为研究人员在实际工作中提供了更多的选择。【Objective】The aim of this study was to provide a simple and efficient method to isolate smallRNAs from banana which is suitable for downstream handling like RT-PCR and northern blot assays.【Methods】Small RNA was extracted from roots,leaves,flowers,and fruits of banana(Musa acuminataL. AAA group,‘Brazilian') by the modified CTAB method with PEG precipitation step.【Results】The re-sults indicated that high quality small RNA was obtained from each tissue. The ratio values of A260/A280 ranged from 1.872-2.020 and the yield of small RNA isolated by our method were more than 35 μg·g-1.To evaluate further the application of small RNA isolated by our method,stem-loop RT-PCR was per-formed for detecting banana conserved mi RNA156 a using small RNA isolated from roots,leaves,flow-ers,and fruits respectively. The expected PCR products(70 bp) were detected on 2.5% agarose gel andwere verified by sequencing.【Conclusion】A simple,quick and efficient small RNA extraction methodwas provided for the purpose of RT-PCR,Northern blotting and library construction assays.

关 键 词：香蕉 小分子RNA 提取 

分 类 号：S668.1[农业科学—果树学]