| 摘要: |
| [目的]探索快速高效的RNA提取方法,建立高效率的mRNA差异显示体系.[方法]以萝卜幼嫩薹叶及花蕾为材料,比较了酸性酚-异硫氰酸胍-氯仿提取法(异硫氰酸胍法)与BIOZOL试剂法提取RNA的效果,并对DDRT-PCR体系中Taq酶、Mg2+ 、dNTPs,引物及cDNA用量进行了优化.[结果]BIOZOL试剂提取的RNA量大质好,经纯化后,其OD260/OD280值为1.8～2.1,表明杂质少,质量浓度大于1 μg/μL,符合mRNA差异显示的要求;确定的最佳DDRT-PCR反应体系(20μL)为:10×Buffer 2.5 mmol/L,Mg2+ 2.8 mmol/L,dNTPs 0.25 mmol/L,Taq酶1.5 U,锚定引物2.5 μmol/L,随机引物1.25 μmol/L,cDNA用量0.5 μL。mRNA差异显示结果表明,高分辨率的片断为100～1 000 bp.经验证,该体系也适用于大白菜和油菜mRNA差异显示研究.[结论]通过两种RNA提取方法比较和DDRT-PCR体系优化,获得了萝卜最佳RNA提取方法及mRNA差异显示反应体系. |
| 关键词: 萝卜 RNA提取 DDRT-PCR 体系优化 |
| DOI： |
| 分类号: |
| 基金项目:国家“863”计划项目“植物分子与细胞高效育种技术与品种创制”（2006AA100108-4-7）；陕西省科技厅陕西省13115工程项目“优质多抗专用蔬菜新品种选用技术”(2007ZDKG-05) |
|
| mRNA differential display technique in radish(Raphanus sativus) |
|
|
| Abstract: |
| 【Objective】 The study was to obtain high qualitative RNA applied in later experimentation to establish high efficient mRNA differential display system.【Method】 The effects of acidic phenol guanidine thiocyanate-chloroform method and BIOZOL reagent on RNA extraction with tender leaf and bud of radish were compared,and the main factors of DDRT-PCR such as concentration of Taq enzyme,Mg2+,dNTPs,primer and cDNA were optimized.【Result】 It showed that the RNA extracted by BIOZOL reagent was better than the RNA extracted by acidic phenol-guanidine thiocyanate-chloroform method,its OD260/OD280 reach 1.8-2.1 after RNA was purified,which meant that the impurity in it was very little and it was up to the requirement of mRNA differential display;An optimal DDRT-PCR system was determined (20 μL),which was made up of 1×Buffer，Mg2+ 2.8 mmol/L，dNTPs 0.25 mmol/L,Taq enzyme 1.5U,Anchor Primer 2.5 μmol/L,Random Primer 1.25 μmol/L,cDNA 0.5 μL.The high differentiating amplification of DDRT PCR was between 100-1 000 bp.This system is also suitable for the mRNA differential display of Chinese Cabbage and Rape.【Conclusion】 By comparing the effects of acidic phenol-guanidine thiocyanate chloroform method and BIOZOL reagent on RNA extraction and optimizing DDRT-PCR system,high qualitative RNA method and optimal mRNA differential display system were obtained. |
| Key words: Raphanus sativus RNA extraction DDRT-PCR system optimizing |
