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核桃不同时期叶片及不同组织RNA提取方法筛选

Methods of Total RNA Extraction from Different Juglans regia Tissues and Leaves

    摘要
  • 摘要:
    目的 针对核桃组织中富含多酚、多糖及黄酮类物质含量较高的特点，筛选经济高效、适合核桃不同发育时期叶片及不同组织总RNA的提取方法。
    方法 以核桃幼嫩、成龄和衰老叶片为材料，分别采用改良硼砂—CTAB法、改良硼砂—CTAB—异丙醇法、改良SDS法、改良Trizol法Ⅱ以及试剂盒法进行总RNA的提取。以筛选与优化的改良硼砂—CTAB法、改良硼砂—CTAB—异丙醇法分别提取核桃胚、叶柄、韧皮部、雄花的总RNA，并对RNA产量、纯度、电泳图谱和RT-PCR进行分析。
    结果 5种方法都可获得核桃总RNA，其中，改良硼砂—CTAB法能够从幼嫩叶片、成龄叶片、叶柄和雄花中获得质量高、完整性好的总RNA，28S rRNA亮度约为18S rRNA的2倍，A260/A280介于1.9～2.1之间，A260/A230大于2.0，且总RNA质量浓度可达1 185.2 ng·μL−1；改良硼砂—CTAB—异丙醇法则适合衰老叶片、胚和韧皮部的提取，总RNA质量浓度可达1 019.0 ng·μL−1。通过RT-PCR在不同组织中均能扩增出核桃18S rRNA和翻译起始因子（JreIF1A）片段。
    结论 改良硼砂—CTAB法和改良硼砂—CTAB—异丙醇法相比其他3种方法更易排除蛋白质、酚类等杂质干扰，对于不同时期不同组织均有更好的适用性，提取所获得的总RNA纯度和浓度完全符合后续的分子生物学试验要求。

     

    Abstract:
    Objective To find the efficient methods of extracting total RNA contents in Juglans regia tissues and young and mature leaves.
    Method The young and mature leaves were used as the materials, and the total RNA was extracted by improved borax-CTAB method, improved borax-CTAB-isopropanol method, improved SDS method, improved TrizonⅡ method, and Plant kit method. The concentration, purity, electrophoregram and RT-PCR of the RNA were analyzed, which extracted by the improved borax-CTAB method, improved borax-CTAB isopropyl alcohol method from the total RNA of J. regia embryo, petiole, phloem and male flower.
    Result The total RNA were extracted successfully by the 5 methods mentioned above. The high concentration and integrity of the total RNA was extracted by the improved borax-CTAB method. The brightness of 28S rRNA was 2 times that of 18S rRNA. The ratios of A260/A280 and A260/A230 were approximately 1.9-2.1 and higher than 2.0. The concentration was up to 1 019.0 ng·μL−1. In different tissues, 18S rRNA and translation initiation factor (JreIF1A) fragments were amplified by RT-PCR.
    Conclusion Compared with the other three methods, the improved borax-CTAB method and the improved borax-CTAB-isopropanol method have better applicability for RNA extraction from different tissues of J. regia. They can avoid interference from impurities. And the purity and concentration of RNA extraction are in line with the experimental requirements.

     

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