栀子高质量总RNA的提取

Isolation of High Quality Total RNA from Gardenia jasminoides Eills

利用改进的高盐高pH值法和EASYspin植物RNA快速提取试剂盒,从富含次生代谢产物的栀子叶片和果实中提取总RNA。用紫外分光光度法和1%非变性琼脂糖凝胶电泳检测总RNA质量,0.7%非变性琼脂糖凝胶电泳检测反转录产物质量,利用RT-PCR反应检测反转录产物用于基因扩增的有效性。结果表明,高盐高pH值法提取的栀子果实总RNA,试剂盒法提取的栀子叶片总RNA产量均较高,28S和18S条带清晰,28S条带亮度是18S条带的2倍,且A260/A280在1.9~2.0;以提取的RNA为模板反转录的cDNA长度范围较广（250~5000bp）;上述反转录获得的cDNA均能够成功用于60S核糖体蛋白L18A基因片段的扩增。综上所述,利用高盐高pH值法提取栀子果实总RNA、利用EASYspin植物RNA快速提取试剂盒提取栀子叶片总RNA,效果最好。本研究成功建立了从栀子果实和叶片中提取高质量总RNA的方法。

To establish an efficient method for high quality total RNA extraction from leaves and fruits of G. jasminoides, total RNA were extracted with a high salt, low pH method and an EASYspin Plant RNA Mini Kit. The quality of the RNA was estimated by UV spectrophotometric analysis, detected by 1% non-denaturing agarose gel electrophoresis. And the products reverse-transcribed from the total RNA were also checked by 0.7% non-denaturing agarose gel electrophoresis. Both the RNA extracted from fruits with the high salt, low pH method and the RNA extracted from leaves with the kit had given a satisfactory yield. When the above two total RNA were run on a 1% denaturing agrose gel, sharp and clear 28S rRNA and 18S rRNA bands were observed, and the robust of 28S rRNA band was twice as intense as the 18S rRNA band, respectively. The ratio A260/A280 for the two total RNAs were from 1.9 to 2.0, and cDNA products reverse transcribed from the two intact RNA had exhibited expected sizes from 250 bp to 5000 bp. Using these cDNA above as templates, fragment amplification of 60S ribosomal protein L18A gene was carried out successfully. Therefore, high quality total RNAs of G. jasminoides could be extracted from fruits with a high salt, low pH method, and extracted from leaves with an EASYspin Plant RNA Mini Kit.