从富含淀粉的水稻胚乳组织中提取功能总RNA和克隆wee1基因片段的方法研究

A Novel Protocol to Isolate the Functional Total RNA and Clone wee1cDNA from Rice Endosperm Tissues Containing High Levels of Starch

水稻胚乳中含有大量的淀粉等物质,用传统的方法提取总RNA难度很大.改良了一种SDS/苯酚法,可以从水稻胚乳中提取高质量的总RNA,解决了RNA易降解、易被污染以及由于总RNA与淀粉等物质共沉淀所造成的低产量等问题.通过分光光度计测量OD值以及变性胶电泳,可以检测出所提取的总RNA质量较高,从1.5g水稻胚乳中可提取到800μg左右的总RNA.提取的总RNA已成功用于RT-PCR克隆目的基因.

Isolation of the intact,functional total RNA from rice endosperms cont aining high levels of starches was extremely difficult by means of t he traditional methods.A modified S DS /phenol method was described that could be used to isolate the total RNA from rice endosperms.This method so lved the problems of RNA degradation,contamination,and lo w yield due to RNA binding and co-precipitation with starches.By this pro-tocol the total RNA of 800μg was routinely obtained from 1.5g rice endosperm and undegraded with hig h quality as being determined by spectrophotometric analysis and denatur ing agarose gel electrophovesis.This protocol has been successfully used for RT-PCR to clone the target genes.