基于大黄叶绿体基因组高变区分析鉴定机理研究

Rhubarb is a commonly used multi-origin herb in China, while its clinical safety and effect have been threatened by the species confusion for the difficult in their identification. In the view of the universal DNA barcodes exhibited limitation on the identification of rhubarb, we intend to resolve the identification problem based on the chloroplast genome using high throughput sequencing technology. Three aspects following will be studied in this project: 1) Firstly, assembling the chloroplast genome sequences of the three rhubarb origin from the data sequenced using Illumina and PacBio, analyzing the variation of the genes and spacer regions, finding out the SNP sites, and investigating their nucleotide varying patterns. 2) Then, assessing the amplification success rate of the candidate sequences using universal primers toward the more than 200 collected samples, screening out the DNA barcode sequence suitable for the identification of rhubarb using Wilcoxon rank sum test, K2P model and Blast1 algorithm, and exploring its identification mechanism on these complex species of Rheum. 3) Finally, establishing a rapid identification technology system for the identification of rhubarb using rapid detection technology. The results of this research can provide a technical support and theoretical basis on the precision medicine, and the identification of authenticity and species origin for multi-origin herbs.

大黄为常用多基原大宗药材，品种混乱，基原物种鉴定困难，给其药用安全及稳定性造成极大威胁。鉴于常用DNA条形码在大黄鉴定上的局限，申请人提出基于高通量叶绿体全基因组测序技术深入分析其高变区变异规律，解决大黄基原鉴定困难问题。本项目将重点研究：①利用Illumina结合PacBio测序技术获得全部大黄基原植物叶绿体全基因组；应用生物信息学方法分析叶绿体基因及间隔区片段变异率；筛查单核苷酸多态性位点，探索大黄叶绿体基因组碱基变化规律。②设计通用引物针对已收集的200余份大黄属样品评估扩增成功率；运用Wilcoxon秩和检验评估序列变异性，应用K2P模型及Blast1算法评估鉴定成功率；统计分析筛选获得高变分子标记，对其在大黄属植物中鉴定机理进行探讨。③建立鉴定大黄基原物种的快速检测技术体系。本研究将为多基原药材真伪鉴别及基原物种准确鉴定提供技术支持，为多基原药材精准用药提供理论依据。

多基原药材物种不同，势必有效成分不同，功效各异，特别是药材毒性差异，极易给患者造成危害，典型多基原药材大黄的三个基原在化学成分上存在较大差别，势必导致功效差异，突破现有鉴定方法局限，准确鉴定其物种，为确保大黄药材更为精确的对症用药，适应中药全球化具有重要意义。. 从全国大黄主产区收集大黄属样品197份，通过高通量测序获得药用大黄叶绿体基因组161,093bp，掌叶大黄叶绿体基因组161,541，唐古特大黄叶绿体基因组161,053bp。利用生物信息技术手段对叶绿体基因组注释分析，三个物种mRNA、tRNA及rRNA基因数量相同，其中7个基因含1个内含子，2个基因含2个内含子，18个基因存在2拷贝，1个基因3拷贝，在叶绿体基因组的四个区域中，SSR数量非常相似，但在不同区域中SSR比例却存在很大差别。虽然这三个物种的基因结构高度相似，但仍存在值得进一步分析的细微差别。. 利用包含大黄基原物种在内的23个物种的叶绿体基因组构建系统进化树发现大黄基原物种各自聚为一支呈现很好单系性，表明叶绿体基因组可作为超级条形码序列有效鉴别大黄基原物种。根据叶绿体基因组差异，针对高变区设计21对特异性引物筛选候选鉴定序列，经大样本实验验证筛选出8对引物可用于大黄基原物种的准确鉴定。结合形态特征阐释了叶绿体基因组高变区鉴定大黄药材基原鉴定机理。针对特定区域设计的高效扩增引物实现大黄药材基原的快速鉴定，解决多基原药材大黄的快速鉴定问题。研究成果发表SCI论文4篇，核心期刊论文3篇，获得4项国家及省级课题资助，纳入湖北省科技进步奖一等奖“湖北中药优势品种质控关键技术与应用”。. 课题研究利用叶绿体全基因组信息开发疑难多基原药材基原准确鉴定分子标记，实现了复杂多基原药材大黄基原的准确鉴定，对现有方法难以鉴定的复杂多基原药材真伪及其基原鉴定提供理论依据，针对中国药典收录的153种多基原药材具有广泛应用前景。