麻花艽和管花秦艽( 龙胆科) 之间自然杂交类型的分子验证

野外考察发现麻花艽( Gentiana straminea Maxim. ) 和管花秦艽( G. siphonantha Maxim . ex Kusn. ) 同域分布时存在大量形态位于二者之间的个体。经形态变异研究后发现它们可能是这两个物种之间的杂交后代。对两个亲本种以及假设杂交群体共55 个个体的核糖体ITS 序列和叶绿体trnS-G 序列的分析结果表明:中间形态个体是麻花艽和管花秦艽的自然杂交后代。此外分析了两个亲本种以及杂交群内个体间trnS-G和ITS 序列的变异状况以及分子标记结果与形态鉴定不一致的可能原因; 指出可能是杂交诱导的叶绿体基因组重组以及早期物种分化中的谱系筛选不彻底等原因造成了亲本种群体内序列变异的多样化。     

藏药麻花艽愈伤组织的诱导及植株再生

1植物名称麻花艽（Gentiana straminea Maxim．）,又名麻花秦艽,藏药中称为“解吉嘎保”。 2材料类别幼嫩叶子。     

管花秦艽的传粉生态学研究——兼与同域分布近缘种的比较

在青藏高原东北部连续两年观察了晚秋开花植物管花秦艽Gentiana siphonantha的传粉生态学特征,并在此基础上进一步比较分析了与该物种同域分布且亲缘关系较近、但开花较早的麻花艽G straminea之间的传粉生态学特征.管花秦艽的花发育过程表现出雌雄异熟和雌雄异位的特点,不存在花内的自花传粉,套袋隔离的花不结实也支持这一结论;株内自交的高结实率表明该物种是自交亲和的.盛花期每植株平均有15朵开放的花,雄性和雌性阶段的花比例为1.2:1;自然条件下产生种子必须依赖传粉媒介;苏氏熊蜂是最有效的传粉昆虫,且访花过程中埘雄性和雌性阶段花不具明显的偏向性;株内连续访花的频率高达87.8%,从而导致同株异花传粉自交的广泛存在.与同域分布的麻花艽相比,管花秦艽的单花花期、雄性和雌性期持续时间缩短.但盛花期开花数量明显增加.令人感兴趣的是尽管两个近缘种的花形态特征存在显著差异,但都是由同一种熊蜂传粉.这一特点与过去认为花颜色和花管长度是物种分化过程中与不同传粉昆虫协同进化导致牛殖隔离的假说不相符合.管花秦艽单花的访花频率和同株异花连续访花的比例都明显高于麻花艽.两个物种不同花序设计导致访花昆虫行为的改变可能是造成这一差异的主要原因.两个物种具有不同的开花时间,但仍然存在一定的花期重叠,表现出不完全的传粉生殖隔离状态.     

西宁和海北麻花艽净光合速率和叶绿素荧光参数的日变化比较

以青藏高原药用植物麻花艽为材料,研究了西宁和海北两个地区麻花艽叶片的净光合速率和叶绿素荧光参数的日变化进程。结果表明：在中午太阳辐射较强时两地麻花艽叶片的净光合速率（Pn）均下降,下午随日间光强的减弱逐渐上升,形成双峰曲线;海北麻花艽叶片的净光合速率（Pn）及其日变幅均低于西宁。随日间光强的增加麻花艽叶片的PSⅡ最大光化学效率（Fv／Fm）、PSⅡ的潜在活性（Fv/Fo）下降,非光化学猝灭系数（NPQ）则上升,黄昏各参数都恢复到接近早晨的水平,表明未发生光合机构的破坏;一天中海北麻花艽叶片的Pn、Fv／Fm、Fv／Fo均低于西宁,表明随海拔的升高、光强的增加,海北麻花艽热耗散增多,午间光抑制加重。     

麻花艽叶片光合色素含量及厚度对UV-B辐射的响应

以中国科学院海北高寒草甸生态系统定位站自然生长的麻花艽(Gentiana straminea Maxim.)为材料,进行了不同月份和草盛期不同天数的短期增补和过滤UV-B辐射试验,比较分析叶片光合色素含量和叶片厚度等的变化.结果表明:(1)生长季内麻花艽叶片叶绿素a+b含量呈波动变化的趋势,7月份含量均较高;草盛期不同天数处理时,UV-B辐射对麻花艽叶片叶绿素a+b含量的影响不大.(2)生长季内麻花艽叶片类胡萝卜素含量也是7月份较高,短期增补UV-B辐射有降低其含量的趋势.(3)增加UV-B辐射能够降低Chl a/b值;自然UV-B辐射下Car/Chl比值能维持一个较高水平,是对强辐射的适应.(4)随处理时间延长,麻花艽叶片厚度有降低趋势,其叶缘出现一些发黄、变黑、变透明等受害症状,叶片能通过增加叶片厚度来适应增强的UV-B辐射.可见,生长于高海拔地区的植物麻花艽虽然对UV-B辐射表现出诸多的生理适应特性,但依然不可避免地受到其损伤.     

青藏高原高山植物麻花艽的传粉生态学研究

对青藏高原东部麻花艽 (Gentiana straminea)海北居群的传粉生态学进行了 3a连续的观察和实验。试验表明自然去雄、人工自交和杂交处理均结实 ,而人工去雄套袋和自然套袋不结实。麻花艽自交亲合 ,但必须依赖传粉媒介才能完成授粉过程 ,不存在无融合生殖。野外捕捉到 14种访花昆虫 ,它们分别属于 2个纲 ,7个目 ,8个科。观察和分析了各种昆虫的访花行为后 ,认为苏氏熊蜂 (Bombus sushikini)是麻花艽有效而稳定的传粉者。测量表明麻花艽花蜜通道的深度和苏氏熊蜂的舌长基本吻合。苏氏熊蜂的访花频率在 10 :0 0～ 12 :0 0 ,13:0 0～ 15 :0 0和 16 :0 0～ 18:0 0时间段没有差别 ,单花的访花频率为 0 .0 0 5次 / (花· min)。和其它高山植物相比 ,青藏高原高山植物麻花艽的访花频率较高。熊蜂传粉和高频率的访花维持了麻花艽在极端寒旱的青藏高原环境下的有性生殖。此外 ,高频率的访花对于维持该地区高山植物的生殖保障具有重要的现实意义 ,但是否具有普遍性 ,还有待研究更多的高山代表类群。     

高寒草甸麻花艽和美丽风毛菊的光合速率午间降低现象

在中国科学院海北高寒草甸生态系统定位研究站地区,用便携式光合蒸腾测定仪（CI－301PS）和液相极谱氧电极（SP－2）观测到,全晴天2种高山植物麻花艽和美丽风毛菊叶片的净光合速率（Pn),光合放氧速率和表观量子效率（AQY）有明显的午间降低现象,遮光实验表明,这种现象是由高原地区当地太阳正午时前后较强的太阳辐射造成的。     

粗茎秦艽与西藏秦艽(龙胆科)种间的形态学、染色体与分子界定

粗茎秦艽Gentiana crassicaulis Duthie ex Burk和西藏秦艽G. tibetica King ex Hook. f.的主要区别是前者花冠小、檐部蓝色, 后者花大、檐部紫褐色; 但二者在西藏东南部地区的形态界限不清楚, 具有较多的中间形态过渡类型。本文以该地区居群作为主要研究对象, 并以云南和西藏其他地区的典型形态类群为对照, 比较研究了两个物种间的形态学、染色体和分子标记界定。共研究了10个居群69个个体的trnS-G和ITS序列、6个居群26株植株的染色体倍性和54份标本的形态变异。结果表明, 花冠大小与染色体倍性密切相关。云南等地分布的粗茎秦艽均为二倍体(2n=26), 花冠较小; 西藏西部的西藏秦艽均为四倍体(2n=52), 花冠较大。而西藏东南部的形态过渡类型也为四倍体, 花冠较大, 且其中的部分植株花颜色变浅, 类似西藏秦艽。但母系与双亲遗传背景的分子标记都表明西藏东南部居群与典型形态特征上的粗茎秦艽聚为一支, 应属于粗茎秦艽; ITS序列上也未发现杂合位点。这些分子证据都不支持近期杂交渗入造成形态过渡性的假设。综合分析表明多倍化造成了粗茎秦艽种内形态特征的变异, 其四倍体个体与四倍体西藏秦艽在形态特征上的趋同, 最终导致了二者在西藏东南部地区形态界限的模糊。但是, 分子证据可以很好地区分这种多倍化造成的形态趋同。我们的研究结果还表明, 综合利用分子、染色体和广义形态证据来解决近缘种之间种范围的界定具有更大的优势。此外, 还讨论了粗茎秦艽不同染色体倍性的分布以及该高山物种可能的起源与扩散途径。     

内蒙古2种白刺间自然杂交类型的分子验证

本实验对齿叶白刺(Nitraria roborowiskii Kom.)和唐古特白刺(N.tangutorum Bobr.)以及疑似杂交个体共48个个体的叶绿体trnL-F序列和核糖体ITS序列进行分析.trnL-F结果显示,齿叶白刺和唐古特白刺在15个位点上存在差异,杂交个体在其中11个位点上与齿叶白刺相同,4个位点...     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morpho-logy always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermedi-ate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence compar-ison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) inter-genic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we exam-ined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species.     

中国锦鸡儿属的分子系统发育

测定了中国锦鸡儿属Caragana各属下分类群20个代表种的ITS、trnL-F和trnS-G序列.基于3种DNA片段的单独分析所获得的系统发育树具有相似的拓扑结构;3种片段的合并分析提高了各分支的支持度,并获得了相似的系统发育树.落轴亚属subgen.Caragana的种类构成了一个在系统树上首先分化出来的单系分支,与形态特征和地理分布的研究一致.短齿系ser. Occidentales和长齿系ser. Bracteolatae的代表种构成了1个单独的分支,因此短齿系应被放入长齿系所属刺叶组sect.Longspina 而不是针刺组sect.Spinosae或sect.Pruinosa.分子系统学证据支持依据叶片宽窄在掌叶组sect.Frutescentes中再划分2个系的形态学研究结论;但Ser.Dasyphyllae和针刺系ser.Spinosae的亲缘关系较近,系统发育分析的结果似乎不支持在针刺组中单独划分2个系.宿轴类的物种聚成一个单系的分支,因此应被处理为一个组--鬼箭组sect.Jubatae;荚果里面被毛和无毛的种类各自构成2个小支,支持依据该特征在组下分系.系统树显示Sanczir定义的sect.Tragacanthoides然为多系类群,应将该组中所包含的刺叶组、针刺组、和鬼箭组的种类划分出来.基于ITS的遗传距离表明卷叶锦鸡儿C. ordosica与藏锦鸡儿C, tibetica应该是2个不同的种.     

Floral closure in response to temperature and pollination in Gentiana straminea Maxim. (Gentianaceae), an alpine perennial in the Qinghai-Tibetan Plateau

Floral closure may be induced by pollination and various other factors, but is rarely studied comprehensively. Different kinds of floral closure should have various effects on reproductive fitness of plants. Two contrasting types of floral closure were observed in the flowers of Gentiana straminea Maxim. in the eastern Qinghai-Tibetan Plateau. The first type occurred prior to pollination during both gender phases, in response mainly to decreasing air temperatures. Flowers closed when decreasing temperatures approached 20°C and subsequently began to reopen the following day during mid-morning when air temperatures warmed to approximately 13–15 °C. This kind of floral closure can protect pollen grains on either stamens or stigmas, increasing fitness of both male and female. Following pollination, permanent floral closure occurred, although there was a delay between the dates of pollination and permanent closure, during which flowers continued to show temporary closure in response to low temperature episodes. The time required for permanent, pollination-induced closure varied according to the age of the gender phase, including a prolonged time before closure if pollination occurred early in the female phase. The retaining of permanent closed flowers increased both approaching (to inflorescences) and visiting (to unpollinated flowers) frequencies of individual plants when with fewer open flowers and the persisting corolla is further beneficial for seed sets of these pollinated flowers. Thus, two separate types of floral closure, one in response to environmental cues and the other in response to the age of each gender stage, appeared to have a strong influence on reproductive fitness in this species. These results revealed a different adaptive strategy of alpine plants in the sexual reproduction assurance in addition to the well-known elevated floral longevity, dominant role of more effective pollinators and increased reproduction allocation in the arid habitats.     

白花龙胆花抗炎作用研究

为了研究白花龙胆花的抗炎作用,本文对白花龙胆花水提物和70%乙醇提取物的抗炎活性进行了试验,并测定了水提物和70%乙醇提物中龙胆苦苷的含量.试验采用二甲苯所致小鼠急性耳肿胀试验和二甲苯所致小鼠腹部毛细管通透性试验.将昆明种小白鼠分为7组,分别为空白对照组,白花龙胆花水提物高、中、低剂量组和70%乙醇提取物高、中、低剂量组,灌胃(ig)给药14 d后,用二甲苯分别于小鼠右耳及腹部致炎.测定左右耳重量,计算肿胀度及肿胀抑制率,测定吸光值.结果表明:白花龙胆花水提物和乙醇提取物的中、高剂量组对于二甲苯所致的小鼠急性耳肿胀和小鼠腹部毛细管通透性都有显著的抑制作用.从而表明:白花龙胆花具有显著的抗炎疗效.     

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences

BACKGROUND AND AIMS: The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences. METHODS: Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae. KEY RESULTS: The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya. CONCLUSIONS: The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.     

Interannual fluctuations in floral longevity, pollinator visitation and pollination limitation of an alpine plant (Gentiana straminea Maxim., Gentianaceae) at two altitudes in the Qinghai-Tibetan Plateau

Prolonged floral longevity and bumblebees as dominate pollinators in alpine ecosystem have been suggested to overcome pollination limitation of alpine plants arising from the decrease of pollinator activity with increasing altitude. However, this conclusion has never been examined in the Qinghai-Tibetan Plateau (QTP), the highest and largest plateau in the world. In this study, we intended to test year-to-year correlations between floral longevity, visiting frequency and pollen limitation of this species between two populations (at 3200 m and 4000 m) of Gentiana straminea in this plateau. Pollinator exclusion elongated both male and female phases greatly at both sites, and durations of both male and female phases in natural condition varied greatly over three years. The visiting frequency of bumblebees varied greatly at the higher altitude, but seemed to be stable at the lower altitude. Seed production was pollination limited in both populations in most studied years. The floral durations, pollinator frequency and pollination limitation showed no significant and consistent variations with the increase of altitude. The previous hypothesis that the prolonged floral longevity of alpine plants can compensate for low levels of pollinator visitation therefore could not be confirmed, and our results further suggested that in the QTP platform, the altitude shows no consistent effect on the reproductive performance of this species, despite that the fluctuation of visit frequency intensified at the higher altitude.     

基于核核糖体DNA ITS序列的真藓科系统发育新证据

主要是利用核基因组的核糖体DNA内转录间隔区（nrDNA ITS）序列数据探讨了Buck和Goffinet（2000）限定的真藓科内各属间及属内种间的系统发育关系。ITS序列的最大简约法（MP）及贝叶斯推论（BI）分析结果显示：不支持Cox（1999）利用rps4/trnL-trnF数据分析将丝瓜藓属移至提灯藓科的结论。ITS数据分析与Holyoak（2007）利用trnL-F,trnG和atpB-rbcL序列数据分析将Bryum algovicum、B.caespiticium、B.capillare、B.pseudotriquetrum和Plagiobryum zierii划归Ptychostomum的结论一致,同时,为Spence(2005)依据形态学特征将B.pallescen、B.pallens、B.uliginosum、B.amblyodon和B.lonchocaulon划归Ptychostomum提供ITS分子证据支持。基于以上研究,建议将以上提到的真藓属种和平蒴藓移至Ptychostomum。     

The Use of DNA Barcoding on Recently Diverged Species in the Genus Gentiana (Gentianaceae) in China

DNA barcoding of plants poses particular challenges, especially in differentiating, recently diverged taxa. The genus Gentiana (Gentianaceae) is a species-rich plant group which rapidly radiated in the Himalaya-Hengduan Mountains in China. In this study, we tested the core plant barcode (rbcL + matK) and three promising complementary barcodes (trnH-psbA, ITS and ITS2) in 30 Gentiana species across 6 sections using three methods (the genetic distance-based method, Best Close Match and tree-based method). rbcL had the highest PCR efficiency and sequencing success (100%), while the lowest sequence recoverability was from ITS (68.35%). The presence of indels and inversions in trnH-psbA in Gentiana led to difficulties in sequence alignment. When using a single region for analysis, ITS exhibited the highest discriminatory power (60%-74.42%). Of the combinations, matK + ITS provided the highest discrimination success (71.43%-88.24%) and is recommended as the DNA barcode for the genus Gentiana. DNA barcoding proved effective in assigning most species to sections, though it performed poorly in some closely related species in sect. Cruciata because of hybridization events. Our analysis suggests that the status of G. pseudosquarrosa needs to be studied further. The utility of DNA barcoding was also verified in authenticating ‘Qin-Jiao’ Gentiana medicinal plants (G. macrophylla, G. crassicaulis, G. straminea, and G. dahurica), which can help ensure safe and correct usage of these well-known Chinese traditional medicinal herbs.     

高山植物黄管秦艽cDNA文库构建与表达序列标签（EST）分析

黄管秦艽（Gentiana officinalis）是一种重要的藏药高山植物,本研究构建了该物种开花期的eDNA文库。经检测达到中等cDNA文库水平,文库滴度为1．2×10^7pfu／ml,重组率95．9％,插入片段平均长度大于500bp。对343个随机挑选的重组克隆进行部分测序,获得的ESTs经编辑后共有181条有效序列。经生物信息学方法分析181条表达序列标签（EST）代表144个单克隆序列,其中55个与已鉴定的基因同源,35个序列与未鉴定的EST匹配,54个未找到同源序列;后两者共有89个EST序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现,相关基因主要编码以下蛋白：与蛋白表达相关的占35％;光合作用相关的占笠％;新陈代谢相关的占18％;抗性相关的占11％;质膜运输和细胞分裂相关的分别占5％;染色体变化和细胞信号转导的分别占2％。根据有效EST序列设计引物,通过RT-PCR进一验证了所得EST的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。