The Primary Study On Genetic Diversity And Chemical Constituents Of A Medicinal Plant, Gastrodia Elata B1

Gastrodia elata BI (Tian Ma in china), belonging to orchid family, is mainly distributed in Shichuan Province,Yunnan Province,Guizhou Province and northwest of china, northeast of china etal..It is also distributed extensively in Japan and Korea. Gastrodia elaba BI is a very important Chinese herbal medicine,which comprise the important medical component named. In this study,the genetic diversity was investigated by AFLP and ISSR in order to make further exploitation on Gastrodia elata B1. Moreover the chemical ingredient of Gastrodia elata B1 was analyzed by HPLC and GS-MS methods. The results arc as follows:Amplified fragment length Polymorphism (AFLP) was used to evaluate the genetic diversity of 27 accessions of Gastrodia B1 elata from 5 provinces of china. A Total of 548 bands ranging in size from 100bp to 2500bp were generated from 16 primer combinations screened from 64 primer combinations and 428 of these bands showed polymorphism. The polymorphic ratio (PP) is up to 78%. Results showed that some genetics diversity existed among gastrodia elata B1 accessions from the different regions and AFLP can be used to investigate effectively genetic diversity of gastrodia elata B1. The analysis of genetic variation indicated that the genetic distant among 27 accessions ranged from 0.54 to 0.018, with an average of 0.2199, which implied that the genetic discrepancy of gastrodia elata B1 from different sources was not high and the genetic diversity of gastrodia elata B1 was limted. However the genetic variance of gastrodia eIata B1 accessions from Guizhou was more evident than that from other areas, which indicated that there was abundant gene resource of gastrodia elata B1 in Guizhou province. Therefore, the accessions from Guizhou can become significant germplasm resources in the genetic improv.ement of gastrodia elata B1. The investigation on different forms of Gastrodia elata B1 from the same sources using AFLP analysis illustrated that the variance of intra-specific level was morphologic variation. Whether the classification of Gastrodia elata B1 forms was correct still needed farther investigation.ISSR (inter-simple sequence repeat) markers were used to analyze the genetic diversity and patterns of population structure within and among 9 populations of Gastrodia elata B1, a Chinese traditional herb, in Guizhou Province. Using twelve ISSR primers, 120 discernible DNA bands ranging in size from 200bp to 1600bp were generated with 97 (80.83%) being polymorphic, indicating considerable genetic variation at the species level. In contrast, ISSR diversity within population was lower than that among population (at species level: PPB=80.83%, H_E=0.2083, H_O=0.3326; at the population level: PPB=17.5%～40.83%, there were relatively low levels of polymorphism at population level with the percentage polymorph bands (PPB) ranging from 17.5% to 40.83%, averaging 25.56%. Based on Nei's Gst value calculated by POPGEN1.31, genetic diversity (He) ranged from 0.042 to 0.153, averaging 0.073, Shannon information index was 0.3326±0.2241 at species level. The genetic differentiation (Gst) among population was 0.6377, which indicated that there was 63.77% of the variance among populations and 36.33% of the variance within populations. Restricted gene flow (Nm = 0.2841) among population was a plausible reason for the high genetic differentiation observed for the species. Limited gene flow may result from several factors, such as seed dispersal, poor seed viability and isolation of population.To compare the Gastrodia elata B1 from different producing areas and their quantities of trace elements, 17 samples, wild and artificially planted, were gathered from 14 distributing areas. Their gastrodins were extracted by means of the method from codex. The quantities of gastrodins were mensurated with HPLC to be compared. The quantities of gastrodins were found in an extent between 0.1603% and 2.5513%. The quantities of gastrodins of wild Gastrodia elata B1 are evidently larger than those of ones artificially planted. The average quantity of wild Gastrodia elata B1 is 1.702%. At the same time, that of Gastrodia elata B1 artificially planted is 0.567%. There existed a difference between the quantities of gastrodins of "Gastrodia elata B1 wild and artificially planted from same producing area. An appropriate planting means is propitious to increasing the quantity of gastrodin. The growth condition can be an important factor to influence the quantity of gastrodin. Results of our study reveal also that the quantities of gastrodin of wild Gastrodia elata B1, as well as the Gastrodia elata B1 artificially planted, exceed the standard of Chinese Codex, so they both may be used as medicine. Furthermore, the quantities of N, P, K, Ca, Fe, Mn, Cu, Zn, and heavy metals Pd and Cd, were also mensurated in our study. The result can enter into support to evaluate the quality of Gastrodia elata B1. The results indicate that the quantity of N is between 4.63～14.52 g·kg^-1, that of Pd 0.24～1.73 mg·kg^-1, that of Cd 0.19～0.80 mg·kg^-1, the average quantity of P is 1.11 g·kg^-1, and that the quantity of Fe differs largely. The analysis of pertinence of multi factors, which was carried out with SPSS software, indicates that element N gives a benign pertinence with gastrodin among the 11 elements (r=0.632420; p=0.085643). But the others don't give any pertinence with gastrodin.For the first time, the volatile components of secondary protocorm were extracted by the improved vapor distillation. The compounds were determined, and their relative quantities were calculated by GC-MS. The test to inhibit bacterium was carded out bycylinder-plate assay. We got 48 components from Gastrodia elata B1.f.viridis Mak (29 known), accounting for 63.189% total content; 36 from Gastrodia elata Bl.f.glauca S.Chow (20 known), accounting for 76.392%. The main components of Gastrodia elata B1.f. elata are 2,3,5,6-tetramethyl pyrazine (25.331%), 2-pentyl-furan (11.974%) and E,E-2,4-decadienal (8.66). The main components of Gastrodia elata B1.f.viridis Mak are ethyl linoleate (15.444%), styrene (10.352%) and ethyl palmitate (10.666%). The main components of Gastrodia elata Bl.f.glauca S.Chow are 4-methyl-phenol (20.409%), styrene (12.607%) and 1-methylethyl ester-hexadecanoic acid (8.842%). There are 6 common components in the three kinds of Gastrodia elata B1, which may be chemical dactylograms in determination of Gastrodia elata B1. The test to inhibit bacterium with Gastrodia elata B 1.f.glauca S.Chow indicates that the volatile components of Gastrodia elata B1.f.glauca S.Chow shows an activity to inhibit Aspergillus oryzae, Aspergillus flavus, Penicillium sp, Rhizoctonia cerealis and Pestalotiopsis theae. Our study establishes a primary foundation to make more use of Gastrodia elata B1.