【作者】 顾蔚；

【导师】 王喆之；

【作者基本信息】 陕西师范大学 ， 植物学， 2010， 博士

【摘要】 华中五味子(Schisandra sphenanthera Rehd. et Wils.)是我国常用药材,近年来其药用价值重新受到重视,市场需求逐年增加,致使野生资源急剧下降,目前已被我国纳入《国家重点保护野生药材物种名录》,属于三级保护野生药材物种,即资源严重减少的主要常用野生药材物种。由于对华中五味子种质资源研究甚少,有关其遗传多样性水平和遗传结构等问题仍不清楚,也缺乏不同种群间表型性状、药用成分以及遗传变异的比较与分析。因而开展对华中五味子种质资源的收集、保存与分析以及遗传多样性研究更为迫切,弄清其种群遗传结构及分布格局,为种质资源保护与可持续开发利用奠定基础。本研究采用形态学、植物化学及分子标记技术,以我国河南、山西、甘肃、四川、重庆、浙江、安徽、陕西8个省市12个华中五味子种群为材料,对采集的238份种质进行表型多样性分析,并以其中的85份种质进行木脂素含量和AFLP分子标记研究,从表型、木脂素含量和DNA三个层面对我国华中五味子种质资源的遗传多样性进行分析,获得的主要研究结果如下：1.华中五味子20个表型性状在各种群间和种群内存在极显著差异(P<0.01),以陕西汉中、商洛、宝鸡、甘肃、重庆及河南种群的变异较大,具有丰富的遗传多样性。其遗传多样性主要存在于种群内(71.04%),果部表型性状具有丰富的多样性,特别是单果重、小浆果纵径、横径以及百粒鲜重在种群内变异大,而聚合果果形指数、聚合果纵径、聚合果果柄长和单果小浆果数在种群间变异大。主成分分析显示,不同种群间性状差异比较大的有小浆果纵径、横径、百粒鲜重、聚合果纵径、聚合果果形指数、新枝和老枝叶长和叶宽等。UPGMA聚类将12个华中五味子种群聚为4类,其中四川、浙江和安徽各自聚为一类，其余9个种群聚为一大类。2.华中五味子果部表型变异与地理气候因子关系密切,经度与聚合果果柄长、果形指数、纵径呈极显著正相关,而与单果小浆果数、单果重呈极显著负相关。纬度主要与小浆果纵径、横径、单果重、单果小浆果数呈极显著或显著正相关,与聚合果果形指数和纵径呈极显著负相关。年降雨量、年均温度以及平均海拔对果实性状影响较大。3.华中五味子木脂素中五味子酯甲与五味子甲素呈极显著正相关,而五味子甲素与五味子乙素呈极显著负相关。85份华中五味子种质中五味子酯甲(除4份未检测到)含量均高于国家药典标准,同时均检测到五味子乙素,而且含量较高,五味子醇甲(甘肃除外)含量很低。4.陕西商洛、渭南、河南及陕西安康种群的五味子酯甲(平均0.724%)和五味子甲素(平均0.812%)含量较高；陕西汉中、宝鸡、重庆及四川种群的五味子乙素(平均1.085%)含量较高,而五味子酯甲平均为0.428%；山西、浙江和安徽种群的五味子酯甲(平均0.310%)、甲素(平均0.262%)和乙素(平均0.421%)含量处于中等水平,介于其他种群之间；甘肃华中五味子种群的五味子醇甲(平均0,492%)和乙素(平均1.081%)含量高。12个华中五味子种群依据4种木脂素的欧式距离聚为4类。5.8对选择性引物对12个种群85份种质进行AFLP扩增,共检测到1384个位点,其中1380个为多态位点,多态百分率达99.711%,每对引物平均扩增173个位点,所有种质具有4条共有带(E-ACT/M-CTC-96.5、E-ACG/M-CAA-150.5、E-ACG/M-CAA-270.5和E-ACG/M-CAC-90.5)。6.62.35%的华中五味子种质具有丰富的特异位点,共获得183个,主要存在于陕西汉中、河南、甘肃、重庆、浙江以及陕西宝鸡种群,各种群具有特异带也提示种群间存在着遗传差异。7.本研究筛选出的4个引物组合(E-ACT/M-CTC、E-ACA/M-CTC、E-AGG/M-CTA和E-ACA/M-CAA)扩增出较多的特异带,该引物组合可应用于华中五味子种质指纹图谱研究。8.采用AFLP标记Nei’s遗传距离可将12个种群分为5个类群,河南、甘肃、四川和山西聚为一类,第二类中含有重庆、安徽和陕西宝鸡,浙江和陕西渭南各自聚为一类,陕西汉中、商洛和安康聚为一类。9.分子方差分析(AMOVA)揭示了华中五味子种群遗传结构较为丰富,特别是甘肃、陕西汉中、商洛及河南种群。PhiST系数(ΦST)为0.135,进一步说明华中五味子遗传变异主要集中在种群内,与表型性状多样性分析结果一致。由于种群间的基因交流受到一定限制,在保护和开发利用上,特别要重视扩大对种群内优良单株的选育。综合形态、木脂素含量和分子标记研究,华中五味子种群遗传变异主要存在于种群内,以陕西商洛、汉中、河南及甘肃种群变异较大。因此,在对其种质研究中首先要选择遗传多样性丰富的种群,注重对种群间变异较大性状的筛选,如聚合果果形指数、聚合果纵径、聚合果果柄长和单果小浆果数等；其次要扩大种群内样本量,加大对种群内优良单株的选择,特别注意小浆果的纵径、横径、百粒鲜重、单果重等性状的筛选。以上研究在形态学、植物化学及分子水平上揭示了华中五味子种质资源的遗传多样性,为其种质资源研究积累了数据,对合理开发利用具有重要意义。更多还原

【Abstract】 Schisandra sphenanthera Rehd. et Wils. is a frequently-used Chinese herb plant, Due to renewed emphasis on its medicinal value in recent years, its market demand has been rising year by year, resulting in a sharp decline of its wild resources. Currently, it has been incorporated into our National Key Protected Wild Medicinal Species List, and it is the third class conservation of wild medicinal species, namely, the severely reduced resource and meanwhile the major common species of wild medicinal herbs. As there is very little information about S. sphenanthera, their genetic diversity and genetic structure are still not clear, besides, there is also a lack of comparison and analysis about phenotypic traits, pharmaceutical composition and genetic variation among different populations. Thus, it has been more urgent to make effort to collect, preserve and analyze its germplasm resource. We also should make clear its genetic structure and its distribution patterns, so that we can protect the germplasm resource and make full use of it.In this study, S. sphenanthera germplasms from Henan, Shanxi, Gansu, Sichuan, Chongqing, Zhejiang, Anhui, Shaanxi, and in 12 populations in China are analyzed by morphological, phytochemical and molecular marker technology. Phenotypic diversities of 238 collected germplasm are analyzed, and lignins contents and AFLP molecular of 85 samples are marked. From morphology, lignins contents and DNA, genetic diversity of S. sphenanthera germplasm in China are analyzed. The main results are as follows:1.Among and within the twenty phenotypic traits of S. sphenanthera populations, there exist a highly significant difference (P<0.01),Hanzhong,Shangluo, Baoji, Gansu, Chongqing and Henan populations varied greatly, with the rich genetic diversity. Genetic diversity is mainly within populations (71.04%).The traits of phenotypic fruit are rich in diversity, especially signal fruit weight (SFW), berry longitudinal diameter (BLD), berry horizontal diameter (BHD) and 100 berries fresh weight (BFW) and other traits in the great variation within populations, while the fruit shape index (FSI), longitudinal diameter of fruit (LDF), fruiting pedicels length (FPL) and berry numbers of signal fruit (BNSF) in large within population variation. Principal component analysis revealed that, BLD, BHD, BFW,and LDF, FSI, leaf length of new branch (LLNB), leaf width of new branch (LWNB), leaf length of old branch (LLOB), leaf width of old branch (LWOB) and so on, are the main factors of the phenotypic variation. UPGMA cluster devides 12 kinds of clustering into 4 types, Sichuan, Zhejiang and Anhui is one type, and the remaining nine kinds of cluster are a category.2.The phenotypic variation of S. sphenanthera fruits and climatic factors are closely related. Longitude and FPL, FSI, LDF show evidently positive correlation, however, longitude and BNSF, SFW show evidently negative correlation. Primarily, latitude and BLD, BHD, SFW, BNSF are evidently positively correlated, but latitude and the FSI and LDF show observably negative correlation. The annual rainfall (AR), annual mean temperature (AMT), and mean altitude (MA) have a greater impact on fruit characters.3.Lignans of S. sphenanthera in schisantherin A and deoxyschizandrin show highly positive correlation, while the deoxyschizandrin and y-schizandrin show highly negative correlation. The schisantherin A content of 85 germplasm (except 4 are not detected) is higher than the national pharmacopoeia standards, and higher levels of y-schizandrin have been found, but schisandrin (other than Gansu) content is very low.4. The contents of schisantherin A (average 0.724%) and deoxyschizandrin (an average of 0.812%) of Shangluo, Weinan, Henan and Ankang populations are high;the contents of y-schizandrin (an average of 1.085%) of Hanzhong, Baoji, Chongqing and Sichuan populations are high and average content of schisantherin A is 0.428%;Shanxi, in Zhejiang and Anhui populations, schisantherin A (average 0.310%),deoxyschizandrin (an average of 0.262%),and y-schizandrin (an average of 0.421%) content are at the middle level,intermediate between other populations. Schisandrin (mean 0.492%) and y-schizandrin (an average of 1.081%) is the highest in Gansu population.12 populations of S. sphenanthera based on four kinds of lignans Euclidean distance cluster into four categories.5.Eight pairs of selective primers of 85 germplasm in 12 populations are carried on AFLP to expand increases.In total,1384 locus are detected, and 1380 locus are polymorphic, percentage of polymorphic locus (P) is 99.711%.On average, each pair of primers is expanded by 173 locus. All germplasm have four commom bands with E-ACT/M-CTC-96.5,E-ACG/M-CAA-150.5, E-ACG/M-CAA-270.5,and E-ACG/M-CAC-90.5)6.62.35% of the S. sphenanthera germplasm are rich in specific locus, in total, there are 183. They are mainly in populations in Hanzhong of Shaanxi, Henan, Gansu, Chongqing, Zhejiang and Baoji of Shaanxi.A variety of groups with a specific band also indicate that there are genetic differences among populations.7.This study screens out four primer combinations (E-ACT/M-CTC, E-ACA/M-CTC, E-AGG/M-CTA, and E-ACA/M-CAA) amplify more specific bands, and the primer combinations can be applied to germplasm fingerprinting of S. sphenanthera.8.By AFLP markers Nei’s genetic distance can devide 12 populations into five groups; Henan, Gansu, Sichuan and Shanxi together are a class. The second class contains Chongqing, Anhui and Baoji, and Weinan and Zhejiang respectively are two groups, besides, Hanzhong, Shangluo and Ankang cluster together.9. Analysis of molecular variance (AMOVA) reveals the genetic structure of populations to be abundant, especially in Gansu, Hanzhong, Shangluo and Henan population. The PhiST coefficient (ΦST) is 0.135,and it could further explain the genetic variation is mainly within populations, which is accord with phenotypic diversity analysis. As the gene exchanges among populations are subject to certain restrictions, in the protection and development of utilization, special attention should be paid to expand the selection of good single plant within populations.Through morphology, lignins contents and molecular marker studies, the major genetic variation of S. sphenanthera mainly exist within populations. Population variation in Shangluo, Hanzhong in Shaanxi, Henan and Gansu is notable. Thus, to do research of germplasm, the best choice is the population of rich genetic diversity, and to focus on the populations which vary greatly with the selection traits, such as FSI, LDF, FPL and BNSF. Secondly, we should expand the sample sizes within populations and increase the superior choice, and pay special attention to BLD, BHD, BFW, SFW. These researches in morphology, phytochemistry and molecular levels above reveal the genetic diversity of S. sphenanthera, and collect data for germplasm resources, which are significant for its rational development and use.更多还原