周瑶瑶　陈春妍　陈鹏　刁勇　周瑞阳

摘 要：红麻（Hibiscus cannabinus L.）花药和花瓣因含有较多的多糖、酚类等物质而较难提取高质量的RNA，本研究探索一种适用于红麻花药和花瓣高质量总RNA的方法以进行cDNA文库构建等后续试验。通过借鉴植物DNA的CTAB提取法，结合RNA的理化性质，在提取液中加入6%的β-巯基乙醇，并在开始裂解时加入1/10体积的5 mol·L-1 KAc和1/10体积的无水乙醇对去除多糖特别有效。结果显示，利用该方法得到的RNA质量高，条带清晰完整，检测OD 260/280在1.8～2.1之间，OD 260/230在2.0～2.6之间，产量为花药987.3 ng·μL-1，花瓣752.1 ng·μL-1。该方法提取的总RNA可直接用于mRNA纯化，cDNA文库构建及RT-PCR等后续分子试验。

关键词：红麻；花药；花瓣；RNA提取

中图分类号：S563.5 文献标识码：A DOI 编码：10.3969/j.issn.1006-6500.2015.09.004

A Method of Extracting High Quality Total RNA from the Anthers and Petals of Kenaf

ZHOU Yao-yao， CHEN Chun-yan，CHEN Peng，DIAO Yong，ZHOU Rui-yang

（Provincial Key Laboratory of Plant Genetics and Breeding， Guangxi University， Nanning 530005， China）

Abstract：The anthers and petals of Kenaf contain high content polysaccharide and polyphenol. An efficient method for high quality total RNA extraction from the anthers and petals of Kenaf is requisite for the following experiments such as cDNA library construction and so on. Based on the CTAB extraction method for plant DNA and combined with the physicochemical properties of RNA. 6% β-mercapto ethanol was added to extraction buffer and 1/10 volume 5 mol·L-1 KAc with 1/10 volume anhydrous ethanol was used in the initial cell disruption so as to remove polysaccharide from the anthers and petals. The result showed that by this improved method，high quality RNA could be obtained， and the band was clear and complete. A D260/D280 ratio ranged between 1.8 and 2.1. A D260/D230 ratio stand between 2.0 and 2.6. The RNA productivities from the anthers and petals of kenaf were 987.3 ng·μL-1 and 752.1 ng·μL-1 respectively. High quality RNA could be obtained by this improved RNA extraction protocol， and RNA was suitable for RT-PCR， mRNA purification， cDNA library construction and so on.

Key words：kenaf；anther；petal；RNA extraction

红麻（Kenaf）是锦葵科（Malvaceae）木槿属（Hibiscus）一年生韧皮纤维作物，学名为Hibiscus cannabinus L.，别名为洋麻、槿麻等。从红麻中提取高质量的总RNA是进行红麻基因表达研究的重要基础，只有纯度高、完整性好的总RNA才可用于mRNA纯化、Northern杂交、构建cDNA文库及差减文库、Real-time PCR定量分析、蛋白质的体外翻译等进一步研究。而红麻的花药和花瓣富含多糖、多酚、蛋白质和未知次级代谢产物，它们通常与RNA不可逆结合或与RNA共同沉淀，难以提取到高质量、高纯度的RNA；另外，植物组织中存在RNase，在细胞破碎后释放出来的RNA会被其分解，所以要采取有效措施抑制RNase并使之失活[1]。高质量的花药和花瓣总RNA是进行红麻分子育种的基础，也为揭示细胞质雄性不育分子机理提供了重要保证。

目前，有关植物组织中RNA提取方法的报道已有很多，诸如Trizol法[2]、CTAB-PVP法[3]、盐酸胍法[4]，酚-SDS法[5]及CTAB/酸酚法[6]等，这些方法已广泛用于许多植物总RNA的提取。……