对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法
Summary
We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.
Abstract
Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.
Introduction
植物产生的种子，其产生下一代。种子积累大量存储储备，如油，碳水化合物和蛋白质，对于后发芽生长。人类利用种子贮藏储备食品和动物饲料的来源，因此，植物种子是可食用的有机物全球主要供应商之一。提高种子产量是植物科学的一个重要挑战。
因为种子贮藏储量食品和工业材料的商业价值的来源，这些储备的代谢的调节的分子机制已被广泛研究1-6。进一步阐明这些机制将用于提高农作物的种子产量是有用的。发展种子植物子房受精后，他们通过一系列的发展阶段1,6,7的成熟。进一步了解的分子机制基本种子发育需要详细，要生产由一系列发育中的种子的精确基因表达谱。然而，高含量的油，蛋白质，碳水化合物，和多酚类物质在植物的种子使其难以高度纯化的RNA，这排除基因表达的精确分析隔离。
在这里，我们介绍一种从含大量油脂，蛋白质和多酚的油籽RNA提取的有效方法。使用这种方法，研究人员将能够制备高度纯化的RNA。这种RNA将成为监测的关键基因控制种子贮藏储备的开发和成熟的油籽代谢调控转录变化非常有用。
Protocol
Representative Results
Discussion
基因表达谱有助于我们植物生理学的理解;因此，特定的RNA分离方法已经开发了用于每个样品的条件9-12。我们调查了被RNA分离过程中从种子抑制，发现RNA结合硅胶膜是严重抑制的过程。油，蛋白质和多酚的大量抑制RNA分离。我们修改了RNA提取过程以除去这些化合物与的RNA结合到二氧化硅膜上的处理前的裂解溶液。重要的修改包括添加1％PVP（步骤1.2）和从离心裂解液上清液的收集（步骤1.7?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢功能基因组学基金和光谱法和生物成像设备，涅槃核心研究设施，以及植物型研究设施，涅槃生物资源中心的工作人员。
Materials
| RNeasy Plant Mini Kit | QIAGEN | 74904 | |
| polyvinylpyrrolidone | Sigma-Aldrich | P5288-100G | |
| HOMOGENIZER S-303 | AS ONE | 1-1133-02 | |
| NanoDrop Lite | Thermo Scientific | ND-NDL-US-CAN | |
| PrimeScript RT reagent Kit (Perfect Real Time) | TAKARA | RR037A | |
| KAPA SYBR Fast qPCR kit | Kapa biosystems | KK4601 |
Referências
- Hills, M. J. Control of storage-product synthesis in seeds. Curr Opin Plant Biol. 7 (3), 302-308 (2004).
- Li-Beisson, Y., et al. Acyl-lipid metabolism. Arabidopsis Book. 11, e0161 (2013).
- Bates, P. D., Stymne, S., Ohlrogge, J. Biochemical pathways in seed oil synthesis. Curr Opin Plant Biol. 16 (3), 358-364 (2013).
- Santos-Mendoza, M., et al. Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis. Plant J. 54 (4), 608-620 (2008).
- Durrett, T. P., Benning, C., Ohlrogge, J. Plant triacylglycerols as feedstocks for the production of biofuels. Plant J. 54 (4), 593-607 (2008).
- Kanai, M., et al. The Plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth. Plant Cell Physiol. 54 (9), 1431-1440 (2013).
- Kanai, M., et al. Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds. Plant Biotechnol J. 14 (5), 1241-1250 (2016).
- Dekkers, B. J., et al. Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds. Plant Cell Physiol. 53 (1), 28-37 (2012).
- Salzman, R. A., et al. An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol Biol Rep. 17 (1), 11-17 (1999).
- Vicient, C. M., Delseny, M. Isolation of total RNA from Arabidopsis thaliana seeds. Anal Biochem. 268 (2), 412-413 (1999).
- Wang, G. F., et al. Isolation of high quality RNA from cereal seeds containing high levels of starch. Phytochem Analysis. 23 (2), 159-163 (2012).
- Birtic, S., Kranner, I. Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds. Phytochem Analysis. 17 (3), 144-148 (2006).
