摘要
从香蕉果实在中分离高质量的RNA是从分子水平上研究香蕉果实成熟软化过程中相关基因表达的重要前提。实验表明,用已报道的相关RNA的提取方法,即便利用已报道的从其它果实中成功提取出高质量RNA的方法,也不能从香蕉果实中分离出高质量的RNA。这主要是由于香蕉果实富含多糖、多酚和一些其它次生代谢物,在RNA提取过程中这些物质会与RNA共同沉淀,从而影响RNA的产量和质量。到目前尚无商业化的RNA抽提试剂盒适用于香蕉果实RNA的提取。因此,探索出从香蕉果实中提取高质量RNA的方法就具有十分重要的意义。本文所报道的RNA提取的简便方法,可从以香蕉果肉和果皮中成功提取高质量的RNA,产量可达48￣72μg·g-1·FW-1,且整个提取过程可在一天完成;通过测定其A240/260和A260/280的比值表明,该该方法可有效降低RNA提取过程中多糖、酚类物质和其它次级代谢物以及蛋白质的污染,提取方法所提取的RNA纯度较高;在1%琼脂糖凝胶电泳,EB染色后结果表明RNA在整个的提取过程中结构完整,未发生明显降解;利用RT-PCR技术,从所提RNA中成功克隆出了β-半乳糖苷酶基因cDNA片段,这表明其质量完全可满足进一步分子生物学研究的要求。
High-quality RNA isolation from banana fruit is a prerequisite to the study of gene expression at the molecular level of banana fruit ripening and softening. However, Previous attempts to extract high-quality total RNA from banana fruits 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 banana fruit is difficult due to banana fruit containing large amounts of polysaccharides, polyphenol, which often co-precipitate and contaminate the RNA during the extraction, thereby affecting both the quality and quantity of RNA when using conventional protocols. Until now, no commercial kit has been developed specifically for the isolation of high-quality RNA from banana fruit. So optimized protocols for RNA isolation from banana fruit are necessary. Here we describe a simple procedure for RNA isolation from banana fi＇uit. Using this protocol we obtained good RNA yield （48-721μg·g^-1 ·FW^-1） from banana fruit pulp and peel with low levels of polysaccharides, polyphenolic compounds and protein contaminants as determined by A240/260 and A260/280, respectively. Furthermore, this RNA was not degraded, as was demonstrated by visualizing the ribosomal RNA of the samples on 1% agarose gel electrophoresis. RT-PCR analysis of actin in these RNA extracts also demonstrated that there are no contaminants that interfere with reverse transcription or PCR reactions. All of above demonstrated that the banana RNA isolation protocol presented in this paper can be used to efficiently isolate high quality RNA with low levels of contaminants suitable for related molecular researches.
出处
《分子植物育种》
CAS
CSCD
2006年第1期143-146,共4页
Molecular Plant Breeding
基金
广东省重点科技攻关项目资助.
