ITS2二级结构的系统发育信息在药用植物DNA条形码中的应用价值研究

Internal transcript spacer 2 (ITS2) is one of the broadly used standard core barcodes and also the only nuclear barcode in identification of Chinese traditional medicine. Although the DNA barcode method based on ITS2 is popular and has been included in Chinese Pharmacopoeia, its low discriminatory efficiency is still a problem to its extensive application. Therefore, further study is still necessary to explore its phylogenetic information for medicinal plants identification. In cells, ITS2 activity is based on its secondary structure. The secondary structures are particularly useful in phylogenetic analysis because they include information not found in the primary sequence. Recently, the methods of RNA secondary structure prediction, alignment and transformation are becoming more and more robust with the development of bioinformatics, which makes it possible to utilize its phylogenetic information. In this study we will introduce ITS2 secondary structure into DNA barcode study, developing the research approach that increasing the discriminatory power about ITS2 without the addition of nucleotides. In addition, we will also explore the utility of its conservative structure feature for divergent sequences alignment, which may have significant values for the identification of multiple origin herbs. Based on our previous works, this study will expand samples and use comparative analysis of a large dataset to evaluate its general utility and the scope of application for the identification of a large range of herbs. Finally, we will integrate the analysis methods of secondary structure and DNA barcoding to form a perfect system and thus further promote the application of DNA barcoding in Chinese traditional medicine.

ITS2是中药鉴定的核心DNA条形码之一，也是目前唯一的核基因条形码。虽然基于ITS2的中药鉴定方法已被纳入《药典》，但其分辨力不足仍是制约其推广应用的主要因素，因此需要进一步挖掘其有助于中药鉴定的遗传信息。ITS2以二级结构形式发挥功能，它蕴含的丰富的遗传信息是核酸序列所无法体现的。随着生物信息学的发展，RNA二级结构的预测、比对和编码技术逐渐成熟，为进一步利用二级结构的系统发育价值提供了可能。本项目将ITS2二级结构信息引入到DNA条形码研究中，可在不增加DNA 片段长度的情况下提高其分辨率。此外，本项目利用ITS2二级结构的保守性探讨其对远缘序列比对的意义，这对于具有多来源的中药鉴定具有重要价值。在前期工作基础上本项目拟扩大取样，基于大数据分析，进一步探讨ITS2二级结构信息在中药鉴定中的普适性，最终将ITS2二级结构分析方法与目前的DNA条形码分析方法融合，形成完善的中药鉴定体系。

本项目在ITS2二级结构获取、结构-序列比对、二级结构信息编码、建树分析和碱基补偿替换(compensatory base change, CBC)计算等流程创新了分析方法，构建了完善的ITS2二级结构系统发育分析体系，形成了 “二维分子系统学”研究特色，在解决基原复杂、DNA易降解的中药材等特殊植物材料的分子鉴定方面发挥了重要价值。主要结果如下：（1）在APG框架下从GenBank获取100属进行序列-结构分析表明：加入ITS2二级结构编码信息可增加31%的遗传信息、46%的变异位点和45%的信息位点，在特定类群使物种分辨率平均提升8%，最高达25%；对防风属(Saposhnikovia)及其近缘属种扩大采样后实例分析表明：ITS2序列-结构编码信息与ITS序列全长相当，支持其作为中药鉴定的“超微条形码”。（2）对70属进化模型检测表明RNA模型均优于DNA模型，在RNA模型进化树中有66%的属其高度支持的支系数量减少，43%的属物种鉴定率下降，表明使用ITS2分子鉴定不可忽视二级结构中的CBC效应。（3）重建了紫堇属（Corydalis）CBC替换过程，证实了CBC在ITS2中真实存在，发现CBC对浅层系统发育关系影响小于深层系统发育关系，提出CBC对不同分类阶元的ITS2系统发育分析影响不同。（4）使用80属模拟了CBC替换的具体过程，发现了以AC碱基对作为中间体的CBC替换新途径，估算了其发生的相对频率和速率，为研发新的ITS2-RNA进化模型提供了理论基础和数据支撑。

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