【作者】 庞启华；

【导师】 赵树进；

【作者基本信息】 华南理工大学 ， 发酵工程， 2010， 博士

【摘要】 高良姜(Alpinia officinarum Hance)是著名的十大南药之一,在医药、食品和化妆品加工以及蔬果保鲜上有广泛的应用。在高良姜药材长期的使用过程中,出现了替代品和混淆品,造成用药混乱,影响治疗效果。高良姜野生资源日益枯竭,种质资源丢失严重,栽培类型众多,但遗传多样性家底不清,药材的鉴定技术滞后,尤其是分子水平遗传多样性以及分子鉴定的研究仍为空白。本文以高良姜为研究对象,首次从形态表型、挥发油成分和DNA分子三个不同层面较系统地研究了高良姜的理化特性及遗传多样性,探讨它们与地理分布之间的相关性以及和混淆品的亲缘关系,并建立有效的分子鉴定体系,为澄清其遗传背景和亲缘关系,进一步为种质资源保护、优良品种的选育和规范化种植提供参考,且为在优良种质筛选的基础上进行高良姜的细胞培养和借助发酵工程手段进一步开拓高良姜的应用前景打下基础,丰富高良姜的真伪鉴定和质量控制理论数据,研究结果具有较重要的理论与现实意义。主要的研究结果如下:1.采用水蒸气蒸馏法和经优化的超声波辅助石油醚提取法提取不同产地的高良姜及其混淆品大高良姜的挥发油,通过GC-MS检测其挥发油的化学成分,比较挥发油成分的差异,并根据欧几里得距离进行聚类分析。结果显示,不同产地高良姜的挥发油成分有差异,高良姜与大高良姜的挥发油成分差异更显著。广东产的高良姜样品先聚类,然后与海南的样品聚类,最后才与大高良姜聚类,表明高良姜挥发油成分与地理分布有相关性,而且从挥发油成分的差异可以鉴别高良姜的真伪。2.通过株高、每枝叶片数、叶长、叶宽、叶舌长、叶片质量、花序长、花冠裂片长和唇瓣长等形态表型数据分析各地不同群体高良姜的遗传变异,结果发现,高良姜群内和群间都存在差异,高良姜野生种类的叶片性状多样性程度大于栽培种类,高良姜的表型变异主要来源于群内的差异。聚类分析结果表明,高良姜的形态表型与地理分布没有明显的相关性。3.用改良的CTAB法提取各地高良姜不同群体及其混淆品山姜、华山姜、大高良姜等181个个体样品的基因组DNA,用优化的AFLP技术体系分析它们的遗传差异和亲缘关系。利用筛选出来的4对引物对各地不同群体的高良姜的个体样品进行选择性扩增,分析群内的多样性。结果表明,不同群体高良姜的群内多样性的程度有较大的差别,道地产地的高良姜群体的群内多样性程度均较小。用6对引物对各群体的个体混合模板DNA进行扩增,分析群体间的多样性,结果共检测出462个位点,其中418个是多态性位点,多态性百分率达90.5%,群体间呈现了很高的多样性。聚类分析结果显示道地药材产地龙塘镇的3个栽培群体Xj、Xn和XHn的遗传距离非常近,首先聚一起,体现了高良姜道地性的遗传本质,然后与其他产地的群体聚成一类,混淆品华山姜和山姜聚为一类,大高良姜为一类。在AFLP变性聚丙烯酰胺凝胶电泳指纹图谱和毛细管电泳指纹图谱上都能分辨出高良姜与混淆品大高良姜的特征性条带或特征峰组合,对鉴别高良姜的真伪有明显的鉴别效果。4.运用PCR直接测序法测定了不同产地高良姜及山姜、华山姜、大高良姜等混淆品的rDNA ITS序列,获得高良姜rDNA ITS序列802bp,山姜和华山姜的序列皆为800bp,大高良的序列为810bp,其中包括18S和26S的部分序列,以及ITS1、5.8S和ITS2全部序列。除了杂合位点之外,各地高良姜样品的序列完全一致。高良姜与混淆品的rDNA ITS序列中有61个变异位点,其中60个是信息位点,同源性为96.32%。在这些r DNA ITS序列中有11个位点是高良姜和混淆品的鉴别位点。5.用PCR直接测序法测得的高良姜和混淆品的matK基因的部分序列皆为1212bp。不同产地的高良姜除广西的样品有两个变异位点外,其余的高良姜样品的序列完全相同,所有高良姜样品的同源性达99.97%。高良姜与山姜、华山姜、大高良姜等三种混淆品的matK基因的部分序列的比对结果显示有24个变异位点,其中22个是信息位点,同源性为99%。高良姜与三个混淆品之间有一个鉴别位点。6.基于AFLP、rDNA ITS序列和matK基因序列等三种分子标记的系统树基本一致,反映出各地高良姜有很近的亲缘关系,高良姜与山姜和华山姜的亲缘关系比较近,与大高良姜的亲缘关系比较远。从研究结果可以得出以下结论:高良姜遗传背景复杂,存在复杂的引种迁移种植现象;高良姜的挥发油成分受地理分布的影响较大,形态表型次之,基因组DNA相对稳定;高良姜与其混淆品之间存在明显的差别,可以通过理化特性和DNA分子特征加以鉴别。更多还原

【Abstract】 Alpinia officinarum Hance is one of the ten famous medical materials in south China, which is extensively utilized in medicine, food and cosmetics industry. The succedaneums and adulterants appear in the long history of application of A. officinarum, so result in drug confusion and therapeutic dfficacy loss. The wild resource of A. officinarum is increasingly exhausted and the germplasm resource lost severely. On the other hand, the cultivated form of A. officinarum is multitude but the genetic diversity is not known clearly, and the identification techniques are hysteresis, especially, the study on genetic diversity and identification on molecular levels are blank. In this paper, we use the medical plant A. officinarum as research object, for the first time, to study the physico-chemical property and genetic diversity of A. officinarum on three levels such as morphological phaenotype, volatil oil components and DNA with a set material, investigate their correlation with geographical distribution and genetic relationship with adulterants, reveal the genetic essence of geoherbalism of A. officinarum, and establish efficient identification system, so as to clarify the genetic background and genetic relationship and to move forward a single step to provid information for germplasm resource conservation, breed selection and standardized growth offer theory data for true-and-false authentication and quality control of A. officinarum. This work has theoretical and practical importance.The main results are as follow:(1)The wet distillation and ultrasonic-petroleun ether extraction were used to extract the volatile oils from A. officinarum and its adulterant A. galangal (L.) Willd from different places, and the volatile oils component were detected by GC-MS. The differences of volatile oils components were compared among A. officinarum and its adulterant A. galangal (L.) Willd from various places, and their volatile oil componets were made clustering analysis according to euclidian distance. The resuts indicate that A. officinarum volatil oils components from various places are diverse. The volatile oil components are extremely obvious defferent betwin A. officinarum and A. galangal (L.) Willd. The samples form Guangdong firstly cluster and subsequently cluster with the sample from Hainan and definitively cluster with A. galangal (L.) Willd. The volatile oil component is relative to geographic distribution, and by analyzing volatile oil component, we could authenticate A. officinarum and A. galangal (L.) Willd.(2)We analyzied the the genetic diversity of A. officinarum of different population from defferent habitat by statisticsing morphological phaenotype data such as trunk height, branches number, leaf length, leaf width, leaf ligula length, leaf mass, inflorescence length, corollalobe length and flabs length, etc..The results discover that there are distinctions in intra-group and inter-group, and the diversity degree of leaf blade characters in A. officinarum wild specides is larger than cultivated species, moreover, the origin of phenotype variation mainly exsits in intro-group. Cluster analysis result indicates that morphous phenotype of A. officinarum has no obvious correlation with geographic distribution.(3)181 genome DNA samples were extracted by modified CTAB method, and these DNA samples were of differet populations of A. officinarum and its adulterant as follow: A. japonica (Thunb.) Miq.)、A. chinensis (Retz.) Rosc.and A. galanga (L.) Willd, and optimized AFLP technique was applied to analyze their genetic diversity and genetic relationship. 4 pairs of primers were used to amplificate DNA samples of A. officinarum from differet populations. The result told us that the intro-group diversity was of large dissimilar among different populations, and the populations from famous region had less diversity. Using 6 pairs of primers to amplificate the mixed DNA template of each population individual and detected out 462 sites in all, 418 sites were polymorphic site of the total, and the polymorphic percent was 90.5%, which showed high degree inter-group diversity. The clustering analysis result discovered that the 3 cultural populations from famous region Longtang such as population Xj, Xn and XHn had closer genetic distance, and firstly cluster together, which suggested the genetic essence of genuine A. officinarum, subsequently they cluster with the other A. officinarum populations, simultaneously adulterant A. japonica (Thunb.) Miq. and A. chinensis (Retz.) Rosc. clustered together and two A. galanga (L.) Willd samples clustered for a group. Both the AFLP denaturing polyacrylamide gel electrophoretic fingerprint and capillary electrophoresis fingerprint present mark bands or mark peak group for A. officinarum and A. galanga (L.) Willd, so provides significant.marker for authentication of A. officinarum .(4)By applying PCR direct sequencing to detect rDNA ITS sequences for A. officinarum , A. japonica (Thunb.) Miq.)、A. chinensis (Retz.) Rosc.and A. galanga (L.) Willd from different habitats, we obtained 802bp sequences of A. officinarum, 800bp sequences of A. japonica (Thunb.) Miq. and A. chinensis (Retz.) Rosc. and 810bp sequences of A. galanga (L.) Willd., which include partial sequences of 18S and 26S and total sequences of ITS1, 5.8S, and ITS2. Except heterotic sites, the other rDNA ITS sequences of all A. officinarum samples are the same. There are 61 mutation sites in rDNA ITS sequences among A. officinarum and its adulterants, and 60 sites of them are information sites, with a homology of 96.32%. 11 sites in the rDNA ITS sequences are the marker sites for authentication of A. officinarum and its three adulterants.(5)Partial matK gene sequences are detected by PCR direct sequencing, and these sequences are the same to be 1212bp for all samples of A. officinarum and its three adulterants. 2 mutation sites are found in the sampl of Guangxi and the other samples of A. officinarum from different habitats have the same sequences. The homology of all A. officinarum samples is 99.7%. There are 24 mutation sites among A. officinarum and its three adulterants, 22 sites of them are information sites, with a homology of 99%. 1 site in the partial matK gene sequences are the marker sites for authentication of A. officinarum and its three adulterants.(6)The phylogenetic trees based on AFLP, rDNA ITS and matK gene sequences are similar, which indicates that A. officinarum from differet habitats have very close genetic relationship and A. officinarum has closer genetic relationship with A. japonica (Thunb.) Miq. and A. chinensis (Retz.) Rosc. and has far genetic relationship with A. galanga (L.) Willd..According to the results we can make a conclusion: A.officinarum has complex genetic backgrounds, and there is complex immigration growth phenomenon. The valotil oil component of A.officinarum is influenced comparatively large by geographic distribution, the morphous phaenotype is influenced smaller and the genome is relatively stably. The geoherbalism of A.officinarum principally display in germplasm DNA that was formed under the special environ for a long time. A.officinarum distinguishes to its adulterants, and they can be authenticated by physico-chemical property and DNA molecular character.更多还原