Simple and Efficient Isolation of High-Quality Total RNA from Hibiscus tiliaceus, a Mangrove Associate and Its Relatives
Simple and Efficient Isolation of High-Quality Total RNA from Hibiscus tiliaceus, a Mangrove Associate and Its Relatives
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DOI：
10.1080/10826060802164991
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发表时间：
2008-06
影响因子：
2.9
通讯作者：
Guili Yang;Renchao Zhou;Tian Tang;Suhua Shi
中科院分区：
文献类型：
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作者：
Guili Yang;Renchao Zhou;Tian Tang;Suhua Shi
摘要木槿属是红树林的伴生植物之一，是研究红树林生态适应和耐盐性的理想植物，它可能为红树林高度专门化适应综合征的进化提供线索。该种在叶和根等各种组织中含有极高的多糖、次生代谢物和特别是多酚类物质。因此，从其各种组织中提取RNA是极其困难的，这阻碍了后续的分子操作和表达水平的研究，如微阵列和RT-PCR。传统的基于CTAB的方法可以从特定的物种或组织中提取高质量的RNA，但它们在丁香鱼身上并不奏效。本文介绍了一种改进的基于CTAB的方法，采用PVP和PVPP连续提取氯酚和酸性苯酚，然后选择性盐析，可以在短时间内不断地从鱼腥草的各种组织中提取高质量的RNA。通过逆转录和RT-PCR实验对RNA质量进行了评估，表明它可以适合于一些下游目的。我们已经通过改进的方法构建了总RNA的全长cDNA文库，并成功地将其用于后续的基因芯片实验，旨在揭示丁香的生态适应和耐盐性。此外，我们还发现该方法也可以成功地应用于锦葵科其他植物(包括锦葵科植物Sida、Pachira、Sterculia、Urena和Abelmoschus)，这些植物中含有非常丰富的多酚和多糖。
Abstract Hibiscus tiliaceus, one of the mangrove associates, is an ideal plant for the study of ecological adaptation and salt tolerance as it may give clues to the evolutionary pathway by which the highly specialized adaptive syndrome of mangrove has been achieved. This species has extremely high contents of polysaccharides, secondary metabolites and especially polyphenolics in its various tissues, such as leaves and roots. So it is extremely difficult for RNA extraction from its various tissues, which prevents following molecular operations and expression-level studies like microarray and RT-PCR. Traditional methods based on CTAB can produce high-quality RNA from specific species or tissue, but they don't work well in H. tiliaceus. We describe here a modified CTAB-based method using PVP and PVPP with sequential extraction of chlorophorm and acid phenol and then selective salt precipitation, which can constantly isolate high-quality RNA from various tissues of H. tiliaceus within short time. RNA quality was assessed through reverse transcription and RT-PCR experiments, indicating that it could be suitable for a number of downstream purposes. We have already constructed a full-length cDNA library with total RNA isolated through the modified method and successfully used it in the following microarray experiments, which is aimed to unravel ecological adaptation and salt tolerance of H. tiliaceus. Moreover, we showed that this method could also be successfully applied to other plants in Malvaceae sensu lato (including Sida, Pachira, Sterculia, Urena, and Abelmoschus) with very abundant polyphenols and polysaccharides.