摘要
目的 有效地提取铁皮石斛高质量的RNA,为研究环境胁迫对铁皮石斛生长与代谢影响的分子表达机制奠定基础.方法 建立声波处理的刺激组与对照组,改良苯酚-氯仿法制备的RNA,分光光度法检测其量,1.0%变性琼脂糖凝胶电泳检测其完整性;所得的总RNA经过RT PCR扩增,6%变性聚丙烯酰胺凝胶电泳和银染检测显示其差异cDNA片段.结果 提取的总RNA A260/A230为3.16,A260/A280为1.96,产量达0.27μg/mg,电泳条带清晰,完整性良好,可筛选出明显的差异cDNA片段,分子质量在240~600 bp.结论 用该方法能高质高量地提取富含多糖的药用植物总RNA.
Objective The high quality RNA from the plantlet of Dendrobium candidum was isolated efficiently. It laid a foundation to study the mechanism of molecular expression on its growth and metabolism under environmental stress. Methods The groups stimulated by sound wave and the control groups were established. Total RNA was prepared by a modified phenol-chloroform method. The content was determined by a spectrophotometer. The integrity of RNA was detected by electrophoresis with 1.0% denatured agarose gel. The total RNA obtained was amplified by RT-PCR. The differential cDNA bands were displayed on 6% denatured polyacrylamide gel by electrophoresis and silver staining. Results The ratios of total RNA A260/A230 and A260/A280 were 3.16 and 1.96, respectively. The yield was 0.27 μg/mg fresh weight. The bands were clear on agarose gel and the integrity of RNA was good. The differential eDNA bands were screened with molecular weight of 240-600 bp. Conclusion The method of modified phenol-chloroform can be used to isolate RNA from D. candidium with high quality and high yield.
出处
《中草药》
CAS
CSCD
北大核心
2006年第4期585-588,共4页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金(30470431)
重庆市科委自然科学基金(CSPC2005BB1094)
重庆大学优秀博士论文风险基金资助(200301)
关键词
铁皮石斛
RNA提取
声波
RT-PCR
Dendrobium candidum Wall. ex Lindl
RNA extraction
sound wave
RT-PCR