目的
针对三叶半夏成熟叶片化学成分复杂,富含多糖、酚类及其他多种化学物质的特点,选择优化一种简便、经济、高效的半夏成熟叶片高质量总RNA提取方法。
方法
用4种不同的RNA抽提方法如异硫氰酸胍法、皂土法、CTAB-LiCl法和改进的SDS/酚法提取总RNA,通过电泳、紫外分光光度计检测以及RT-PCR验证等对提取结果进行分析比较。
结果
4种方法均能从半夏成熟叶片中提取到总RNA。其中皂土法的提取过程只需3~4h,是其他3种提取方法所用时间的一半。提取1g叶片的成本只有其他3种方法的1/13~1/14。此法提取的总RNA18S、28S带型清晰无弥散,完整性好,无严重的蛋白质和其他杂质污染,A260/A280为1.8~2.0,A260/A230大于2.0,且总RNA的产率高,为365.744μg/g鲜重,提取的总RNA适用于RT-PCR试验。
结论
皂土法提取的RNA完全适用于DDRT-PCR、Northern blotting、cDNA文库构建等分子生物学研究,是一种快速、经济,适于抽提半夏总RNA的好方法。

Objective To optimize a simple,economic.and efficient method for total RNA extraction with high-quality from mature leaves of Pinellia ternata which has plenty of polysaccharides,phenols,andmany other chemical substances.Methods Four methods,i.e.guanidine isothiocyanate,bentonite,CTAB-LiCl,and the modified SDS／phenol,were used to extract the total RNA.Then the results of the extraction were compared and analyzed by electrophoresis,UV spectrophotometer detection,and RT-PCRverification.Results Among all of the four methods,the bentonite method took only about 3-4 h,which was half time of the other three methods,and the COSt of extracting 1 g lear was 1／13-1／14 of theother three.The 1 8S and 28S bands of the total RNA extracted by bentonite method were clear and nodispersion。the integrity of the RNA was well,and there was no obvious contamination with proteins andother impurities in the RNA extracts.The ratios of A260／A280 were equal to 1.8-2.0 and A260／A230 over2.0.the yield of total RNA was higher at 365.744 tLg／g fresh weight.The total RNA was applied to RT-PCR test.Conclusion The bentonite method iS quick,economic,and efficient for the total RNAextraction from the leaves of P.ternata.The RNA iS suitable for DDRT-PCR,Northern blotting,cDNA library construction,and other molecular biology research.

国家农业成果转化资金资助项目(05EFN213400124)