番茄果实总RNA提取与cDNA-AFLP

从番茄果实中提取高质量的RNA是对调控番茄果实品质、生长发育和代谢等相关基因进行分子生物学研究的基础。实验表明，已报道的相关RNA的提取方法，即使利用已经报道的从其它果实中成功提取出高质量RNA的方法，也不能从番茄果实中分离出高质量的RNA。这主要是由于番茄果实中多糖、多酚类物质及其他次生代谢，在RNA提取过程中与RNA共同沉淀，从而影响RNA的产量和质量。目前，商业化的RNA抽提试剂盒无法直接用于番茄果实RNA的提取。笔者采用4种方法对番茄果实总RNA进行提取，筛选出一种最佳的改进方法。可以从不同时期番茄果实中成功提取高质量的RNA，产量可达401.6--840.2μg/g，通过测定A260/A230和A260/A280 的比值表明，该方法排除了番茄果实中蛋白、多糖、以及多酚等次生物质的干扰，提取到了高质量番茄果实总RNA；1.2%琼脂糖电泳结果表明RNA在整个提取过程中结构完整，未发生明显降解；所提取的RNA用于cDNA-AFLP等到了清晰的表达图谱，这表明其质量完全可满足下一步分子生物学研究。

Isolation of total RNA from fruit of tomato and cDNA-AFLP

High quality RNA isolation from tomato fruit is a basically molecular biology studies of relative gene controlling quality、development and metabolism of tomato fruit. However, previous attempts to extract high quality RNA from tomato fruit using published protocols have proven to be unsuccessful, even the use of protocols developed for the extraction of RNA from other fruit tissue. RNA isolation from tomato fruit is difficult due to tomato fruit containing large amounts of polysaccharides, polyphenol which often coprecipitate and contaminate, RNA during the extraction，thereby affecting both the quality and quantity of RNA when using conventional protocols. Now some commercial kits have not been developed directly for the isolation of high quality RNA from tomato fruit. Here four methods were used to isolate functional RNA from tomato fruit, and the best one was screened out. Using this protocol we obtained good RNA yield (401.6--840.2μg/g) from different stage tomato fruit with low levels of polysaccharides, polyphenolic compounds and protein contaminants as determined by A260/A230 and A260/A280 respectively. Furthermore，this RNA was not degraded，as was demonstrated by visualizing the ribosomal RNA of the samples on 1.2％ agarose gel electrophoresis. The RNA is suitable for cDNA-AFLP, which demonstrated that RNA isolation protocol modified in this paper can be used to related molecular researches．