茶树叶片总RNA提取方法的比较研究

Comparison of Methods for Total RNA Extraction from Camellia sinensis

针对茶树叶片中富含多酚类物质的特点,以陕西地方茶树代表种质紫阳槠叶种的叶片为材料,比较了Trizol法、异硫氰酸胍法和改良SDS-酸酚法三种不同的总RNA提取方法。结果表明改良SDS-酸酚法能够从新鲜叶片、冷冻叶片和干燥叶片中提取到质量高、完整性好的总RNA,经检测28SrRNA亮度为18SrRNA的2倍,A260/A280值介于1.83～2.01之间。以鲜叶、冻叶和干燥叶片的总RNA为材料,进一步用于DDRT-PCR分析,均可获得清晰稳定的多态性结果。说明改良的SDS-酸酚法能抑制茶树叶片中多酚类物质对总RNA提取的影响,是一种适于茶树叶片总RNA提取的有效方法。

To develop an improved protocol to isolate total RNA in good yield and integrity from Camellia sinensis leaves containing high levels of tea polyphenols, three different total RNA extraction techniques, the Trizol method, the isothiocyanate guanidine method and improved SDS-phenol method were compared. Modified SDS-phenol method can be used to extract the high quality and integral total RNA from fresh leaves, frozen leaves and dry leaves. The testing result showed that the brightness of 28S rRNA was two times of 18S rRNA, and the value of A260/A280 was between 1.83 and 2.01, and further DDRT-PCR experiment also acquired clear and distinguished polymorphism results. So the modified SDS-phenol method is an effective way to extract total RNA for tea plants, meanwhile can inhibit the polyphenol influence on total RNA extraction.