绿豆总RNA的分离及环氧水解酶基因的克隆

Isolation of Total RNA and Cloning of Epoxide Hydrolase Gene from Vigna Radiata

目的研究分离绿豆总RNA的方法,并从中克隆环氧水解酶基因。方法采用UNIQ-10柱式法、Trizol法和改良Trizol法等3种方法分离绿豆种子、胚芽和豆芽总RNA,鉴定RNA的产率、纯度及完整性,根据环氧水解酶的保守序列扩增绿豆环氧水解酶基因。结果 UNIQ-10柱式法分离的RNA降解,Trizol法获得的RNA有杂质污染,改良Trizol法得到的RNA产率、纯度及完整性最好;RT-PCR后仅从绿豆胚芽中成功扩增到环氧水解酶基因保守序列。结论改良Trizol法简便易行,能有效地去除多糖、多酚等次生物质的干扰,最适于绿豆胚芽总RNA的分离。

Objective To obtain the method suitable for extracting high quality RNA from Vigna radiata and clone epoxide hydrolase gene from RNA.Methods Total RNA was isolated from seed,germ and sprouts of Vigna radiata by UNIQ-10 column,Trizol and modified Trizol methods,respectively.The yield,purity and integrity of RNA were identified,and the epoxide hydrolase gene was amplified.Results RNA isolated by UNIQ-10 column method was partly degraded and RNA isolated by Trizol method was contaminated by impurities,while RNA isolated by modified Trizol method showed the highest yield,purity and integrity.Conserved region sequence of epoxide hydrolase was only successfully amplified by RT-PCR of germ of Vigna radiata.Conclusion Modified Trizol method is simple and feasible,and is the best method to extract total RNA from germ of Vigna radiata.