Yang G, Zhou R, Tang T, Shi S
State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou, China.
Prep Biochem Biotechnol. 2008;38(3):257-64. doi: 10.1080/10826060802164991.
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.
黄槿是红树林伴生植物之一，是研究生态适应和耐盐性的理想植物，因为它可能为红树林高度特化的适应综合征的进化途径提供线索。该物种在其各种组织（如叶子和根）中含有极高含量的多糖、次生代谢物，尤其是多酚。因此，从其各种组织中提取RNA极其困难，这阻碍了后续的分子操作以及诸如微阵列和RT-PCR等表达水平的研究。基于CTAB的传统方法可以从特定物种或组织中产生高质量的RNA，但在黄槿中效果不佳。我们在此描述一种基于CTAB的改良方法，使用聚乙烯吡咯烷酮（PVP）和交联聚乙烯吡咯烷酮（PVPP），依次用氯仿和酸性酚提取，然后进行选择性盐沉淀，该方法可以在短时间内持续从黄槿的各种组织中分离出高质量的RNA。通过逆转录和RT-PCR实验评估RNA质量，表明其适用于许多下游目的。我们已经用通过改良方法分离的总RNA构建了一个全长cDNA文库，并成功地将其用于后续的微阵列实验，该实验旨在揭示黄槿的生态适应和耐盐性。此外，我们表明该方法也可以成功应用于锦葵科广义（包括黄花稔属、瓜栗属、苹婆属、梵天花属和秋葵属）中其他含有非常丰富多酚和多糖的植物。
