高质量提取银杏种仁总RNA的改良方法  

An Improved Method for RNA Isolation from Seeds of Ginkgo biloba L

作　　者：王亚红[1] 刘缙[2] 王玉国[2] 

机构地区：[1]山西农业大学信息学院,山西太谷030800 [2]山西农业大学农学院,山西太谷030801

出　　处：《中国农学通报》2010年第15期48-52,共5页Chinese Agricultural Science Bulletin

摘　　要：提取高质量的RNA是获取银杏（Ginkgo bilobaL.）种仁药用蛋白基因以及从基因表达水平研究林木良种银杏种子发育和后熟的必要条件。现有提取方法难以获得高纯度的银杏种仁总RNA。旨在改良现有提取方法,以满足抽提富含蛋白质、多糖、多酚等特殊材料高质量RNA的需要。文中将改进的CTAB法和Trizol一步法相结合,优化了试剂组合并改进实验细节,从银杏种仁中获得了高质量的总RNA。通过琼脂糖凝胶电泳、紫外分光光度计检测,提取的总RNA具有清晰的28SrRNA、18SrRNA条带,亮度满足2：1比例关系。OD260/OD280比值在1.85~2.00之间。将提取的总RNA用于Northern杂交分析可检测到清晰的信号,而用于RT-PCR和5＇-RACE也可获得清晰的目的条带。说明这种方法获得的总RNA完整度和纯度很高,完全可以满足下游分子生物学实验要求。Isolation of high-quality RNA from ginkgo （Ginkgo biloba L.） kernels is a prerequisite to acquire the pharmaceutical protein genes and study the development and maturation of ginkgo seeds at the gene expression level. Ginkgo seeds are rich in protein, polysaccharide and phenolic compounds, which increases the difficulties in isolating RNA from them. In this study, modified CTAB method and Trizol single-step method were combined and the extraction conditions were optimized for RNA extraction. The quality of total RNA was analyzed with agarose gel electrophoresis and UV spectrophotometer. The bright and clear strip of 28S rRNA and 18S rRNA were shown in the RNA electrophoresis, and the luminance signal ratio was approximate 2：1. The value of OD 260 /OD 280 was 1.85 to 2.00. It is indicated that the method could isolate high quality and high integrity total RNA. When the acquired total RNA was used in the molecular biology experiment, sharp target hybridization signals were obtained from Northern blotting, and the target gene fragments were also successfully amplified by RT-PCR and 5＇-RACE. These results also demonstrated that the total RNA isolated by this method was of sufficient quality for subsequent molecular applications.

关 键 词：银杏种仁 总RNA提取 

分 类 号：S188[农业科学—农业基础科学]