基于叶绿体基因组的藏药“解吉”DNA分子鉴定研究

The origin of Tibetan herb Jie-Ji is from the plants of the Section Cruciata, Genus Gentiana (Gentianaceae). According to our field study and market survey carried out with Tibetan Traditional Medical College, there is confusion about the identification of original plants and practical application, and it is necessary to make molecular identification. In our previous study, we amplified and sequenced the regions of ITS and 9 chloroplast segments of 10 original plants used as Jie-Ji. However, It is not effective at identifying some closely related species. In this study, the complete chloroplast genomes of G. robusta, G. straminea, G. crassicaulis and G. tibetica will be sequenced, and different loci will be detected. Also, we will explore if the complete chloroplast genomes are useful for identifying related species within Gentianaceae. Based on comparison and analysis of the four complete chloroplast genome sequences, segments with high efficiency of identification will be found. The segments of all the specimens of Section Cruciata will be sequenced, and the most useful segments will be selected to be used as the standard segments. All the standard segments will be helpful to identify original plants of Jie-Ji with high efficiency. This study also could provide new methods and ideas for identifying multi-source traditional Tibetan medicine.

藏药“解吉”来源于龙胆科龙胆属秦艽组多种植物。通过前期和西藏藏医学院合作进行的考察和调研，发现“解吉”在植物鉴定和应用方面存在较大的混乱，亟需进行整理工作。我们采集了“解吉”的10种基原植物，应用ITS，psbA-trnH等10个片段进行条形码筛选及分子鉴定研究，结果表明多个片段组合可以对部分物种进行鉴定，但对其中一些近缘种的鉴定效果不理想。为寻找更高效的鉴别序列，本研究从基因组水平进行探讨，对麻花艽等4个秦艽组近缘种的叶绿体全基因组进行测序，在基因组水平上寻找鉴别位点，初步评价叶绿体基因组在龙胆科中的鉴别意义。通过对所有叶绿体基因、间隔区序列进行比较分析，初步筛选出对秦艽组具有较高鉴别效率的特异性片段，并对所有样品进行分析，最终确定特异性鉴定片段或片段组合，并获得所有相关物种的标准鉴定序列，以更高效的对“解吉”基原植物进行准确鉴定，同时为多基原藏药的近缘种鉴定提供思路和方法。

藏药“解吉”为多来源药材，基原植物涉及于龙胆科龙胆属Gentiana秦艽组Sect. Cruciata多种植物。本课题在品种整理的基础上，对秦艽组叶绿体基因组结构进行分析，并结合核基因组相关序列，确定“解吉”基原植物的分子鉴定方法。.前往西藏、云南、贵州、四川等省区进行野外考察与采集，结合前期工作，共采集鉴定解吉基原植物秦艽组10个物种（麻花秦艽Gentiana straminea，粗壮秦艽G. robusta，小秦艽G. dahurica，粗茎秦艽G. crassicaulis，西藏秦艽G. tibetica，长梗秦艽G. waltonii，管花秦艽G. siphonantha，黄管秦艽G. officinalis，全萼秦艽G. lhassica，大叶秦艽G. macrophylla）472份及龙胆科外类群5个物种37份，总计509份样品。完成样品鉴定与标本制作。.基于麻花秦艽、粗壮秦艽、西藏秦艽和粗茎秦艽，及龙胆科外类群轮叶獐牙菜的叶绿体全基因组序列，阐明秦艽组的叶绿体基因组结构，并通过序列比较，获得物种间热点变异区。.通过筛选叶绿体DNA序列，结合核基因组ITS序列，对10个秦艽组物种进行有效分子鉴定。分别选用稳定鉴别位点，片段组合，及基因分型统计方法，构建藏药“解吉”基原植物的DNA鉴定体系。.以粗茎秦艽为切入点，进一步评价叶绿体基因组在秦艽组种内居群间的鉴别意义。将高通量测序技术与Sanger法测序相结合，获得粗茎秦艽9个居群的叶绿体基因组序列，分别筛选叶绿体基因组差异片段及SSR位点，并进行验证，可对粗茎秦艽不同居群（产地）进行有效区分，初步构建其居群条形码。.基于叶绿体基因组及核基因组ITS克隆序列的系统学分析，西藏秦艽为一杂合物种，粗茎秦艽为其母系来源支，其父系来源支可能来自于麻花秦艽或粗壮秦艽。粗茎秦艽群体间基因流较大，物种经历过扩张过程。.本工作可为藏药“解吉”基原植物的有效鉴定、遗传多样性分析、道地性研究，及龙胆科龙胆属的系统学研究等提供基础资料。