【作者】 关萍；

【导师】 陈放；

【作者基本信息】 四川大学 ， 植物学， 2006， 博士

【摘要】 天麻(Gastrodia elata B1)是兰科(Orchidaceae)多年生共生草本植物，广泛分布于我国吉林和辽宁东部、内蒙古、河南西北部、云南、四川、贵州、西北等地，在日本、韩国、尼泊尔、不丹也有广泛分布，主要生长于海拔400-3200米的疏林下、林中空地、林缘和灌丛边缘，是中国传统的名贵中药。实验证明天麻素是天麻的主要活性成分之一。本研究运用AFLP和ISSR分子标记对不同分布区天麻遗传多样性和居群的遗传结构进行分析，为天麻资源合理保护利用及优良种质资源的选育奠定基础。同时，运用HPLC等方法对不同产地天麻中的天麻素及无机元素含量进行比较；运用GC-MS分析了天麻的挥发性成分。主要结果如下：在AFLP研究中，从贵州、云南、四川、陕西和辽宁收集了27份天麻样品，从64对AFLP选择性引物组合中筛选出16对引物组合，对不同分布区天麻及天麻不同变型的遗传多样性进行分析。16对引物共产生548条大小在100-2500bp的谱带，其中多态性带428条，多态性比率(PP)达78％。该结果说明天麻中存在较为丰富的遗传多样性，且AFLP可以有效地进行天麻遗传多样性的分析。遗传差异分析结果表明，27个样品的遗传距离在0.54～0.018之间，平均为0.2199，表明不同分布区天麻个体间存在一定的遗传差异。四个省区的样品中来自贵州的样品遗传差异相对较大，遗传距离最大的是大方(6号)与施秉(8号)之间，其遗传距离为0.5934，遗传距离最小的是桐梓(11号)与梵净山(10号)之间，其遗传距离为0.0338。表明贵州蕴藏着相对丰富的基因资源，因而可以作为天麻遗传改良的重要种质资源。对同一来源不同变型的天麻的AFLP分析结果表明，天麻种内的变型仅仅是表型上的变异，遗传距离在0.0224～0.0570之间，表现出较小的遗传差异，这表明分类学上的几种变型仅仅是表型上的变异，没有在分子水平上发生的根本变异。这可能是同一基因型由于环境条件的改变在表型上发生的变化，尚未在遗传上固定下来。对天麻几个变型的划分是否成立还有待进一步研究。把包括海拔高度、经度、纬度、年均降雨量和年均气温在内的环境因子划分为不同的范围，用POPGEN软件分别分析不同环境因子范围下天麻居群的遗传多样性，结果表明处于不同环境因子下的天麻呈现出遗传多样性一定的差异，但遗传样性与上述环境因子的之间并无明显的相关性。环境因子对天麻群体间产生的遗传变异仅占总变异的9.47％至26.41％，而73.59％～90.53％的变异来自于各居群内。利用ISSR(inte-simple sequence repeat)分子标记技术，对分布于贵州(Gastrodia elata B1．)9个居群的天麻遗传多样性水平和遗传结构进行分析。12条ISSR引物共扩增出120条大小在200bp-1600bp清晰的谱带，其中97条具有多态性，总的多态位点百分率为80.83％，表明在物种水平上有较高的遗传变异。居群水平的多态性相对较低，多态百分率在17.5％～40.83％之间，平均25.56％。用POPGENE软件计算各居群间和居群内的遗传参数，结果表明：物种水平上的Nei’s遗传多样性(H_E)为0.042-0.153，平均0.073。Shannon信息指数为0.3326±0.2241。Nei’s基因分化系数(Gst)为0.6377，表明居群间的遗传变异占总变异的63.77％，而居群内的遗传变异占总变异的36.33％，这与有限的基因流有关。天麻居群间的基因流较为有限(Nm=0.2841)，明显低于异花传粉植物的平均值，这表明天麻的基因流不足。种子生活力低可能降低了因种子传播而产生的基因流。加之，传播出去的天麻种子需要吸收小菇属真菌的营养才能萌发菌，这也造成天麻居群间较低的基因流动。基因流不足和种子的传播距离、种子极低的生活率以及居群间的地理隔离和营养繁殖等因素可能是导致天麻居群遗传分化的主要原因。在不同产地天麻和无机元素含量的比较研究中，从14个不同分布区采集了17个野生和栽培的天麻样本，采用药典天麻素的提取方法，提取天麻素，并采用高效液相色谱法对其含量进行了测定。并比较各产地栽培和野生天麻中主要药用成分天麻素的含量。结果表明，14个分布区的17个天麻样品天麻素含量在0.1603％到2.5513％之间。野生天麻中的天麻素含量明显高于栽培天麻，野生天麻的天麻素平均含量为1.702％，而栽培天麻的天麻素平均含量为0.576％。同一产地的野生和栽培天麻其天麻素的含量也有差异，说明良好的栽培措施有利于天麻素的提高，而不同生长环境也是影响天麻素的含量的重要因素。实验结果也显示，野生天麻和人工栽培天麻的天麻素含量都超过《中国药典》的标准，符合国家用药标准。此外，本研究还对天麻中的N、P、K、Ca、Mg、Fe、Mn、Cu、Zn以及重金属元素Pb、Cd的含量进行测定，以便为天麻的质量评估提供依据。结果表明，N的含量在4.63-14.52g／kg，磷的含量平均为1.11g／kg，铁的含量差异最大，重金属元素铝和镉的含量在0.24-1.73 mg／kg和0.19-0.80mg／kg。SPSS软件多因子相关性分析结果表明，11种元素中与天麻素含量相关性较为明显的元素是氮元素(r=0.632420；p=0.085643)，其余元素含量与天麻素的含量无相关性。运用水蒸汽蒸馏法首次从天麻次生块茎(箭麻)中提取挥发性成分，并用气相色谱质谱法(GC-MS)对化学成分进行分离鉴定，并用面积归一化计算各成分相对含量。杯碟法进行抑菌实验。结果显示：从红天麻中共有42个成分，其中已知成分有14种，其质量占总质量的63.189％。绿天麻共有48个成份，确定成分的有29种，质量占总质量的84.313％。乌天麻有36个成份，20种成分已确定，占76.392％。红天麻中的主要成分是2，3，5，6-四甲基吡嗪(25.331％)、2-戊基呋喃(11.974％)和E，E-2，4-癸二烯醛(8.66％)。绿天麻挥发油的主要成分是亚油酸乙酯(15.444％)、苯乙烯(10.352％)和棕榈酸乙酯(10.066％)。乌天麻中主要包括4-甲基-苯酚(20.409％)、苯乙烯(12.607％)、1-甲乙醚十六烷酸(8.842％)。比较三种天麻的挥发油成分，有6种共有成分，这6种共有成分可以作为鉴定天麻的化学指纹成分。而其它组成和含量都有较大差异。用乌天麻进行的抗菌试验表明：乌天麻挥发性成分对包括米曲霉、黄曲霉、青霉、小麦纹枯病和茶轮斑病在内的部分植物病源真菌有一定的抑菌活性。为天麻挥发油进一步开发利用提供了初步的实验基础。更多还原

【Abstract】 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.更多还原