【作者】 周珏；

【导师】 王喜军；

【作者基本信息】 黑龙江中医药大学 ， 中药学， 2007， 博士

【摘要】 目的：本文旨在建立刺五加种质资源的化学及分子生物学的评价方法，对来源于不同产地、不同性别的刺五加种质资源药材的质量进行系统研究，最终找到刺五加道地药材及其种质资源的遗传学特性，并为对优良种质进行强化奠定基础。方法：利用高效液相色谱(HPLC)法对来源于不同产地刺五加种质资源药材进行化学评价，分别测定刺五加根和茎中刺五加苷B、绿原酸、异嗪皮啶和刺五加苷D四种活性成分的含量，并研究其与生态因子关系；对不同刺五加种质资源进行分子生物学评价，应用随机扩增多态性分子标记(RAPD)方法找出不同产地刺五加种质资源及不同性别刺五加种质资源的遗传学差异，并对雌性刺五加的特异分子标记进行克隆和测序，运用序列特异性扩增区域(SCAR)的方法对雌性刺五加样品进行扩增。结果：1．刺五加种质资源药材的化学评价：刺五加不同产地种质资源药材根据化学成分含量分为三个类型。化学型Ⅰ：东方红，五常，绥棱。化学型Ⅱ：密山，亚布力。化学型Ⅲ：清河，伊春。化学型含量顺序为：化学型Ⅰ＞化学型Ⅱ＞化学型Ⅲ；其中伊春产刺五加化学成分含量最高，根、茎中含量分别为2.031±0.667mg及1.934±0.667mg。2．刺五加种质资源药材的生态学评价：分析了六个生态因子包括经度、纬度、年降水量、平均温度、年积温及平均海拔与刺五加四种活性成分刺五加苷B、绿原酸、异嗪皮啶和刺五加苷D含量的相关性。结果表明，年积温与刺五加苷B、绿原酸、异嗪皮啶和刺五加苷D含量的相关系数分别为-0.4562，-0.5049，0.6507，0.6819，比其他生态因子与活性成分含量的相关系数均大。根据刺五加不同产地的生态资料将刺五加种质资源分为三个生态型，生态型Ⅰ：东方红(年积温为2900℃左右)；生态型Ⅱ：绥棱、密山、亚布力(年积温为2500℃左右)；生态型Ⅲ：伊春、清河、五常(年积温为2200℃左右)。生态型中年积温顺序为：生态型Ⅰ＞生态型Ⅱ＞生态型Ⅲ。3．刺五加种质资源的分子生物学评价：结果表明刺五加不同产地即密山、绥棱、东方红、清河、亚布力、伊春、五常种质资源样品间遗传相似度平均值分别为0.8540、0.8927、0.8792、0.7541、0.8488、0.7194、0.8057；Shannon信息多样性指数各产地密山、绥棱、东方红、清河、亚布力、伊春、五常分别为0.2300、0.2155、0.2486、0.1809、0.2111、0.1811、0.2771。不同产地种质资源样品在1100bp和800bp均有条带，但亮度不同。刺五加七个不同产地种质资源样品根据Dice相似性系数被分为三个基因型，基因型Ⅰ：密山，绥棱，亚布力，东方红；基因型Ⅱ：五常。基因型Ⅲ：清河，伊春；遗传距离顺序为：基因型Ⅰ＜基因型Ⅱ＜基因型Ⅲ。4．本研究在947bp处首次获得了雌性及雄性刺五加种质资源的差异条带。不同性别刺五加种质资源SCAR研究表明，刺五加雌性、雄性种质资源在不同性别间均有一定的分化，刺五加雌性种质资源的遗传多样性明显高于雄性种质资源。序列同源性分析结果表明，所获得的雌性刺五加种质资源特异片段与含谷氨酰胺合成酶类(Glutamine synthesize gene)(GS)蛋白家族成员之间的同源性较高。结论：本研究结合化学、生态学、分子生物学三方面对不同产地、不同性别刺五加种质资源药材进行评价。将不同产地刺五加种质资源药材分为化学型、生态型和基因型。1．三个化学型包括化学型Ⅰ：东方红，五常，绥棱。化学型Ⅱ：密山，亚布力。化学型Ⅲ：清河，伊春。化学型的特点为：刺五加苷B为四个成刺五加苷B、绿原酸、刺五加苷D、异嗪皮啶中的主成分。三个化学型(除异嗪皮啶含量在密山产刺五加种质药材中略有不同外)剌五加苷B、绿原酸、刺五加苷D、异嗪皮啶含量含量顺序为：化学型Ⅰ＞化学型Ⅱ＞化学型Ⅲ。结果显示七个不同产地四个成分含量差异较大。以刺五加苷B、绿原酸、刺五加苷D、异嗪皮啶的含量为指标，伊春产刺五加各成分的含量明显高于其它产地。2．三个生态型包括生态型Ⅰ：东方红；生态型Ⅱ：绥棱、密山、亚布力；生态型Ⅲ：伊春、清河、五常。年积温与刺五加中活性成分相关性最高。生态型中年积温顺序为：生态型Ⅰ＞生态型Ⅱ＞生态型Ⅲ。年积温与刺五加苷B、绿原酸含量呈负相关，与异嗪皮啶和刺五加苷D含量呈正相关。年积温为不同产地刺五加种质资源药材活性成分含量的主要影响因素。3．三个基因型包括基因型Ⅰ：密山，绥棱，亚布力，东方红。基因型Ⅱ：包括五常。基因型Ⅲ：包括清河，伊春。基因型遗传距离顺序为：基因型Ⅰ＜基因型Ⅱ＜基因型Ⅲ。其中伊春产刺五加种质资源药材遗传多样性最高。4．化学型、生态型和基因型三者之间的关系如下刺五加苷B含量高的刺五加种质资源药材与生态型Ⅲ和基因型Ⅲ的分类相吻合。刺五加苷B含量高的刺五加种质在年积温较低、年平均温度较低而降雨量较高的区域出现频率较高；且其遗传多样性较高。刺五加苷B为刺五加活性成分中的主成分，故可推断环境因素为年积温较低、年平均温度较低而降雨量较高是优质刺五加种质资源药材的适宜环境参数。绿原酸含量高的刺五加种质资源与生态型Ⅱ和基因型Ⅱ的分类相吻合，此种类型刺五加种质资源具有高绿原酸含量，中等遗传多样性，各环境因素都较温和的地区为绿原酸含量高的刺五加种质资源药材的适宜产区；刺五加苷D、异嗪皮啶的含量和环境因素分布趋势尚不明确，有待于进一步分研究。5．本研究对雌性及雄性刺五加种质资源进行研究，首次获得了雌性刺五加种质资源所特异片段。进行BlastX分析证明该雌性刺五加种质资源所特异片段与对植物氮代谢起决定作用之一的谷氨酰胺合成酶类蛋白家族成员之间的同源性较高。更多还原

【Abstract】 Objective:To explore the good germplasm of A. senticosus (Rupr.et Maxim) Harms, the samples of A. senticosus (Rupr.et Maxim)Harms of different gender and the samples collected in different regions, were analyzed with the chemical, molecular biological, and ecological techniques. These methods could be used to find the genetical character of the good germplasm as a geoherb, which could be used as a basis to strengthen the character.Methods:The samples were collected from seven different places. Each sample was divided into three parts, including fresh leaves, roots and stems. Four active ingredients (eleutheroside B, chlorogenic acid, eleutheroside D, and isofraxidin) in the roots and stems were simultaneously determined with HPLC. The genetical polymorphism was analyzed by the molecular markers using the fresh leaves. Based on the above, a female character region of A. senticosus Rupr. et Maxim. Harms was obtained as a characterstic marker. The characterized amplified region was then cloned and the gene sequence was detected. According to the region, new character primer was designed. The female A. senticosus Rupr. et Maxim. Harms was amplified successfully with the method of SCAR.Results:1. The chemical analysis: According to the ingredient content and the cluster results the seven samples were classified into three groups.2. The ecological analysis: The annual accumulated temperature was the most important factor in all the ecological factors of the contents of the bioactive ingredients in the plant among the data of 0.4562, 0.5049, 0.6707, 0.6819. The samples were divided into three groups based on ecological factors.3. The molecular biological analysis: The index of similarity was respectively 0.85.40, 0.8927, 0.8792, 0.7541, 0.8488, 0.7194 and 0.8057. The Shannon index was respectively 0.2300, 0.2155, 0.2486, 0.1809, 0.2111, 0.1811 and 0.2771. The analysis of different gender with RAPDs functioned as the basis of the SCAR analysis. Dice was designated to distinguish the female plant from the male in A. senticosus Rupr. et Maxim. Harms. The cluster analysis of genetic distance shown that the samples were divided into three groups.4. The research, for the first time, obtained the distinglished band 947bp.Conclusions:1. The chemical analysis: In our research, the sample preparation method, HPLC condition, and the calibration equation were established first. The samples were collected from Mishan, Suiling, Dongfanghong, Qinghe, Yabuli, Yichun, and Wuchang district. The content of the active ingredients eleutheroside B, chlorogenic acid, eleutheroside D, and isofraxidin were simultaneously determined with HPLC. The principal component analysis showed that of the four active ingredients, eleutheroside B was the principal component. According to the ingredient content and the cluster results the seven samples were classified into three groups. The comparison shown that the content of the active components in the roots and the bark were higher than that of the stem and the marrow. This approach is supposed to revolutionize the natural product research and to facilitate the development of scientific based herbal medicine. Of all the samples, the sample from Yichun district was the best germplasm. A. Senticosus (Rupr.Et Maxim) collected from Yichun district appeared the best germplasm.2. The ecological analysis: The result of the cluster analysis of the ecological analysis was the same as that of the chemical analysis. The multiple linear Stepwise regression analysis showed that the annual accumulated temperature was the most important factor in all the ecological factors of the contents of the bioactive ingredients in the plant.3. The molecular biological analysis: The fresh leaves of A. senticosus (Rupr.et Maxim) Harms were used in the genetic analysis. The marker obtained from the RAPD markers distinguished the female from the male. SCAR was proved to be useful in the process of germplasm identification. In addition, it could be potentially correlated with the content of saponin in the herb.4. The cluster analysis of genetic distance shown that the samples were divided into three groups. Group one included the samples collected in Mishan, Suiling, Dongfanghong, Yabuli district. Group two included the sample from Qinghe and Yichun district. Group three included the sample from Wuchang district. In the chemical cluster, the sample from Suiling, Dongfanghong and Wuchang were in the same group. While in the genetic cluster, the sample from Suiling, Dongfanghong and Mishan, Suiling were in the same group. The rest of the result were the same. The difference could be induced by the ecological influence on the genetic factors. The analysis of different gender with RAPDs functioned as the basis of the SCAR analysis. SCAR could be useful for screening female A. senticosus plants even before they reach reproductive maturity, resulting in considerable saving of time and economic resources.5. There existed high similarity of BlastX between the different genes of tha male and female and Glutamine synthesize gene (GS).更多还原