果树组织中总RNA提取的新方法

New Methods for Total RNA Extraction in Fruit Trees

本研究设计了一种新的RNA提取方法 ,解决了RNA提取时容易被降解和污染这一关键问题。通过加入Rnase抑制剂 ,消除了同外源RNase对RNA的降解 ,结合DNA难呈低盐溶液(140mmol·L -1NaCl)的原理 ,去除了DNA对RNA提取液的污染 ;先后使用酚和氯仿 ,有效地去除了蛋白质和酚类物的污染 ,利用抗氧化剂PVP和巯基乙醇 ,消除了内源酚类物质氧化变色对病毒RNA逆转录的影响。采用上述方法可以在4～5h内得到纯度高、含量大、完整性好的果树总RNA ,并获得了逆转录活性较强的病毒RNA ,同时使提取RNA的成本降低。这些方法对苹果、葡萄、桃、樱桃等果树总RNA的提取均适用。

A highly efficient extraction method was developed according to the principle of RNA extraction and purification. In order to efficiently extract reverse-transcriptable RNA from the tissues of fruit trees, the procedure of extraction of RNA was studied. The other methods for RNA extraction published were chosen and one of them was improved. The results showed that the method N, method B and method C are suitable for RNA extraction of the leaves and phloems in apple, peach cherry and grapevine. With the three methods, the reverse-transcritable RNA can be obtained in 4～5 hours. The cost accounting of RNA extration by method A and Method B is much lower than that by method C. The three methods are suitable for total RNA extration in apple, peach, cherry and grapevine.