改良TRIZOL试剂法提取矮牵牛衰老花瓣总RNA

Extracting Total RNA from Petal Senescence of Petunia hybrida Vilm with Improved TRIZOL Method

为分离矮牵牛花瓣ACC氧化酶基因的保守片段,构建矮牵牛ACC氧化酶基因RNAi表达载体,探索了从富含色素等物质的组织中提取RNA的方法。以矮牵牛衰老花瓣为材料,提取RNA时对传统的Trizol试剂法进行了改良,利用紫外分光光度计及普通琼脂糖凝胶电泳技术对总RNA的浓度和完整性进行了检测.结果表明改良Trizol试剂法提取的总RNA用DEPC处理水稀释后,OD260/OD280值为1.75;电泳显示28S和18SrRNA条带清晰可见,亮度比在1.0～2.0之间;经RT-PCR后,电泳检测,出现了清晰的目的条带。这些说明改良Trizol试剂法适合提取矮牵牛衰老花瓣总RNA,能用于RT-PCR等后继分子生物学实验。

In order to isolate the conservative fragments of ACC oxidase gene ofPetunia hybrida Vilm flower petals, to construct ACC oxidase gene RNAi expression vector ofPetunia hybrida Vilm. The total RNA was extracted from petal senescence ofPetunia hybrida Vilm rich in substances such as pigments with improved Trizol method, UV spectrophotometer and ordinary agarose gel electrophoresis was used to detect the concentration and completeness of the total RNA. The results showed that OD260/OD280 value of the extracted RNA was 1.75 after total RNA was diluted with DEPC treated water; the electrophoresis pattern showed the bands of 28S and 18SrRNA were clearly discernible, brightness ratio was 1.0-2.0; After RT-PCR, there was the clear goal band. These suggested the improved Trizol method could be suitable for the extraction of total RNA in petal senescence ofPetunia hybrida Vilm and used in the subsequent molecular biological experiments such as RT-PCR.