| 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).
|
PDF Full Text Request