三种兰科植物的保护遗传学研究

本研究在野外调查的基础上，采用随机扩增多态DNA (RAPD)分析和形态学方法，研究了我国三种珍稀濒危兰科植物硬叶兜兰（Paphiopedilummicranthum）、麻栗坡兜兰（P. malipoense）和独花兰（Changnienia amoena）的遗传多样性与群体遗传结构，主要结果如下： 1．采用1 2个引物对分布于我国云贵地区的4个硬叶兜兰群体共161个体进行RAPD扩增和分析，得出物种水平的多态条带百分率（PPB）为71.6%，Nci的基因多样度(h)为0.217，Shannon多样性指数（1)为0.3301;4个群体的平均多样性水平为PPB=45.2%，h=0.1457，1= 0.2204：低于远交兰花的平均水平。分子方差分析(AMOVA)表明，在总遗传变异中，群体间遗传变异占20.31%．群体内占79.69%;POPGENE给出的基因分化系数 （Gst）为0.2958;遗传分化略高于远交物种的平均水平。空间自相关分析表明，所检测的两个群体中存在明显的空间结构，基因型在群体中以不同的小斑块存在。遗传距离和空间距离不存在相关关系。 2．用于麻栗坡兜兰的RAPD引物同上，但取样范围只有贵州的2个群体共10个个体。就所研究的个体柬看，麻栗坡兜兰的遗传多样性明显低于远交兰花物种的平均水平。物种水平上，多态条带百分率(PPB)为49.5%。Nei的基因多样度（h）为0. 1174， Shannon多样性指数(I)为0.1764：在群体水平上，上述三个指标的平均值则分别为12. 75%、0.0486和0.0712，均大大低于硬叶兜兰。然而，尽管作了种种努力，麻栗坡兜兰的取样个体数量仍很少，因此所得结果可能会有误差。 3．用16个引物对分布于河南、湖北、湖南、江西4个省11个独花兰群体共216个体进行了RA PD扩增和分析，独花兰在物种水平PPB=80. 7%，h=0.197．1=0. 3116;在群体水平，上述三个指标的平均值则分别为40. 9%、0.1247和0. 1902，均低于远交兰花的平均水平。AMOVA分析表明，11个独花兰群体间的遗传变异占43.48%，群体内的占56.52%：在神农架和新宁地区内部，群体间的遗传变异分别占13.68%和49.3g%(AMOVA)。POPGENE给出的11个群体的基因分化系数(Gst)为0.3580．神农架和新宁地区内的Gst，值分别为0.1194和0.2597。可见，群体间的遗传分化明显高于远交物种的平均水平。空间自相关分析表明，独花兰的遗传变异在群体内不存在明显的空间结构。群体之间的遗传距离和空间距离不存在相关关系。 4．对独花兰7个群体形态性状的分析发现，12个形态性状在群体内均有较高的变异性，cv值变动于0.022-0.30O。庐山群体(LS)在所有性状上的平均值均为最高。营养性状和花部性状的变异性基本一致。除花葶长和花距直径与某些花部性状之间没有显著的相关关系外，各性状之间均有显著的相关性。对XN4群体的统计没有发现假磷茎数目与其他性状之间存在显著相关性。 根据以上对硬叶兜兰、麻粟坡兜兰和独花兰遗传多样性和群体遗传结构韵研究，结合其他方面的资料;对三种兰花的濒危机制进行了初步的分析。首先，人为采挖和破坏是导致这些兰花物种濒危的直接原因，尤其是麻栗坡兜兰。其次， 适宜兰花生存的生境正在只益萎缩、退化和片段化。这两方面因素的共同作用导致上述兰花群体的数目和规模日益下降，由此引发的遗传多样性降低和遗传结构的改变进一步加剧其濒危状况。对于独花兰而言，较低的繁殖能力又使其生存状态雪上加霜。针对三个物种不同的繁殖特性和遗传学状况，提出如下保护措施。(1)硬叶兜兰由于繁殖能力较强、现存个体尚多，遗传多样性损失不甚严重，因此以保护其所在的生境为基础、实施原位保护，是比较合适的保护策略。(2)麻粟坡兜兰目前受破坏程度非常严重;所剩个体很少，遗传多样性较低，已经很难进行有效的原位保护。因此;应利用迁地保护手段抢救目前尚存的个体。(3)独花兰的繁殖能力较弱，因此在保护生境和严禁采摘的基础上，可采用人工授粉等方式，提高结实率、增加繁殖效率，促使其复壮：在进行迁地保护时，则应注意不同群体间存在较大遗传变异而群体内多样性较低这一现实。

Mapping the shallow-water coral ecosystems of the Freely Associated States: an implementation plan

This Freely Associated States Shallow-water Coral Ecosystem Mapping Implementation Plan (FAS MIP) presents a framework for the development of shallow-water (~0–40 m; 0–22 fm) benthic habitat and possibly bathymetric maps of critical areas of the Freely Associated States (FAS). The FAS is made up of three self-governing groups of islands and atolls—the Republic of Palau (Palau), the Federated States of Micronesia (FSM), and the Republic of the Marshall Islands (RMI)—that are affiliated with the United States through Compacts of Free Association. This MIP was developed with extensive input from colleges, national and state regulatory and management agencies, federal agencies, non-governmental organizations, and individuals involved in or supporting the conservation and management of the FAS’s coral ecosystems. A list of organizations and individuals that provided input to the development of this MIP is provided in Appendix 1. This MIP has been developed to complement the Coral Reef Mapping Implementation Plan (2nd Draft) released in 1999 by the U.S. Coral Reef Task Force’s Mapping and Information Synthesis Working Group. That plan focused on mapping United States and FAS shallow-water (then defined as <30 m) coral reefs by 2009, based on available funding and geographic priorities, using primarily visual interpretation of aerial photography and satellite imagery. This MIP focuses on mapping the shallow-water (now defined as 0–40 m, rather than 0–30 m) coral ecosystems of the FAS using a suite of technologies and map development procedures. Both this FAS MIP and the 1999 Coral Reef Mapping Implementation Plan (2nd Draft) support to goals of the National Action Plan to Conserve Coral Reefs (U.S. Coral Reef Task Force, 2000). This FAS MIP presents a framework for mapping the coral ecosystems of the FAS and should be considered an evolving document. As priorities change, funding opportunities arise, new data are collected, and new technologies become available, the information presented herein will change.

Diet shifts of juvenile red snapper (Lutjanus campechanus) with changes in habitat and fish size

We examined the diets and habitat shift of juvenile red snapper (Lutjanus campechanus) in the northeast Gulf of Mexico. Fish were collected from open sand-mud habitat (little to no relief), and artificial reef habitat (1-m3 concrete or PVC blocks), from June 1993 through December 1994. In 1994, fish settled over open habitat from June to September, as shown by trawl collections, then began shifting to reef habitat — a shift that was almost completed by December as observed by SCUBA visual surveys. Stomachs were examined from 1639 red snapper that ranged in size from 18.0 to 280.0 mm SL. Of these, 850 fish had empty stomachs, and 346 fish from open habitat and 443 fish from reef habitat contained prey. Prey were identified to the lowest possible taxon and quantified by volumetric measurement. Specific volume of particular prey taxa were calculated by dividing prey volume by individual fish weight. Red snapper shifted diets with increasing size. Small red snapper (<60 mm SL) fed mostly on chaetognaths, copepods, shrimp, and squid. Large red snapper (60–280 mm SL) shifted feeding to fish prey, greater amounts of squid and crabs, and continued feeding on shrimp. We compared red snapper diets for overlapping size classes (70–160 mm SL) of fish that were collected from both habitats (Bray-Curtis dissimilarity index and multidimensional scaling analysis). Red snapper diets separated by habitat type rather than fish size for the size ranges that overlapped habitats. These diet shifts were attributed to feeding more on reef prey than on open-water prey. Thus, the shift in habitat shown by juvenile red snapper was reflected in their diet and suggested differential habitat values based not just on predation refuge but food resources as well.

Factors influencing the timing and frequency of spawning and fecundity of the goldlined seabream (Rhabdosargus sarba) (Sparidae) in the lower reaches of an estuary

We have studied the reproductive biology of the goldlined seabream (Rhabdosargus sarba) in the lower Swan River Estuary in Western Australia, focusing particularly on elucidating the factors influencing the duration, timing, and frequency of spawning and on determining potential annual fecundity. Our results demonstrate that 1) Rhabdosargus sarba has indeterminate fecundity, 2) oocyte hydration commences soon after dusk (ca. 18:30 h) and is complete by ca. 01:30−04:30 h and 3) fish with ovaries containing migratory nucleus oocytes, hydrated oocytes, or postovulatory follicles were caught between July and November. However, in July and August, their prevalence was low, whereas that of fish with ovaries containing substantial numbers of atretic yolk granule oocytes was high. Thus, spawning activity did not start to peak until September (early spring), when salinities were rising markedly from their winter minima. The prevalence of spawning was positively correlated with tidal height and was greatest on days when the tide changed from flood to ebb at ca. 06:00 h, i.e., just after spawning had ceased. Because our estimate of the average daily prevalence of spawning by females during the spawning season (July to November) was 36.5%, individual females were estimated to spawn, on average, at intervals of about 2.7 days and thus about 45 times during that period. Therefore, because female R. sarba with total lengths of 180, 220, and 260 mm were estimated to have batch fecundities of about 4500, 7700, and 12,400 eggs, respectively, they had potential annual fecundities of about 204,300, 346,100 and 557,500 eggs, respectively. Because spawning occurs just prior to strong ebb tides, the eggs of R. sarba are likely to be transported out of the estuary into coastal waters where salinities remain at ca. 35‰. Such downstream transport would account for the fact that, although R. sarba exhibits substantial spawning activity in the lower Swan River Estuary, few of its early juveniles are recruited into the nearshore shallow waters of this estuary.

中药青蒿紫穗槐二烯合酶基因表达及调控的研究

青蒿素是从我国传统药用植物中药青蒿（Artemisia annua L.）中提取的新型抗疟特效药，其生物合成途径属于植物类异戊二烯代谢途径。目前，青蒿素生物合成的组织部位及其调控机制仍不完全清楚。紫穗槐二烯合酶（amorpha-4， 11-diene synthase, ADS）作为青蒿素生物合成分支途径的第一个关键酶，催化倍半萜化合物的通用前体法呢基焦磷酸环化，生成紫穗槐二烯。本论文通过对ADS 表达特性的分析，研究了青蒿素生物合成的组织特异性及其调控机制，主要研究结果如下： 一．紫穗槐二烯合酶基因启动子功能的研究 从青蒿高产株系001 中克隆得到了2850 bp 的ADS 启动子调控区。通过比较5’RACE 的测序结果与启动子序列，确定转录起始位点位于翻译起始位点上游44 bp，TATA 盒下游27 bp。该启动子序列包含的顺式作用元件有脱落酸应答元件(ABRE )、乙烯应答元件(ERE)、生长素应答元件(AUXRE）等植物激素反应元件，以及低温应答元件（LTRE）、高温应答元件（HSE）等与逆境有关的反应元件，还有与真菌诱导有关的W-box 元件等。将不同长度ADS 启动子与报告基因GUS 融合，构建了植物表达载体，通过农杆菌介导的方法获得稳定整合的转基因烟草。经过组织化学、GUS 荧光活性检测及RT-PCR 分析，发现该启动子的转录活性很低，无法通过GUS 染色进行观察。GUS 荧光活性检测及RT-PCR 结果表明，转录起始位点上游346 bp 是ADS 基础表达所必需的。高温、低温、干旱、水杨酸、茉莉酸甲酯等处理均能促进青蒿中ADS 的表达，而脱落酸和乙烯的作用效果较小，与启动子序列分析的结果并不完全一致。 二．紫穗槐二烯合酶基因表达特性的研究 以青蒿高产株系001 为材料，在基因和蛋白水平揭示了ADS 的表达特性。RT-PCR 和Western 分析结果表明，ADS 在幼叶和花蕾中大量表达，在老叶和完全开放的花中表达量很低，而在青蒿的根和茎中几乎检测不到ADS 的表达。石蜡切片和整体原位杂交的结果表明，ADS 在顶端分生组织、叶原基及分泌腺毛中表达，在非分泌的T 型腺毛中不表达。当叶片完全展开后，ADS 只在分泌腺毛中表达，而且随着叶片的生长和老化，ADS 的表达量逐渐减少。另一个非常有趣的发现是同一叶片上的分泌腺毛，有些有ADS 的表达，有些则没有。用强光、低温、高温和水杨酸等因素处理后，有ADS 表达的分泌腺毛的比例没有明显的变化。 三．外源水杨酸促进青蒿素的生物合成 研究了外源水杨酸对青蒿素生物合成的影响，结果表明：1 mM 水杨酸处理后，青蒿叶片中的游离态水杨酸含量快速增加，处理后4 h 达到 0.79 μg g-1 FW，是对照的3.5 倍。外源水杨酸能够抑制青蒿中过氧化氢酶活性，提高抗坏血酸过氧化物酶活性，并通过对抗氧化酶活性的抑制引起青蒿体内活性氧水平的迅速升高。在处理后4 h，青蒿中H2O2 和O2-的含量分别达到对照的2.1 倍和2.4 倍。青蒿素含量在水杨酸处理后的前8 h 缓慢升高，随后升高的速度增加。外源水杨酸处理后8 h 和96 h，青蒿素含量分别达到9.1 mg g-1DW 和13.9 mg g-1DW，比对照高21.7%和75.8%。处理后8 h，青蒿酸的含量没有明显变化，随后开始增加。处理后16 h，青蒿酸的含量达到3.6 mg g-1DW，比对照高90%， 随后继续升高，至96 h 达到4.98 mg g-1 DW，比对照高127%。二氢青蒿酸的含量在处理后的8 h 内有所下降，随后缓慢升高。处理后8 h，二氢青蒿酸的含量降低了23.3%，随后二氢青蒿酸的含量开始升高，在处理后96 h，达到7.4 mg g-1DW，比对照高72.1%。外源SA 处理提高了青蒿素及其前体的总含量，在处理后1、2、4 天分别比对照提高了1.3、1.5 和1.8 倍。Northern 结果表明，水杨酸强烈诱导了青蒿素生物合成基因HMGR、ADS 的表达，但是对FPS、CYP71AV1 的诱导作用较小。这些研究结果表明，外源水杨酸至少通过两条途径诱导青蒿素的生物合成：一是通过诱导活性氧的产生促进二氢青蒿酸向青蒿素的转化；二是上调部分青蒿素生物合成相关基因的表达。根据这一研究成果，在青蒿田间栽培中，可以在收获前通过喷施水杨酸来快速、有效和低成本地提高青蒿素产量。

北京山区落叶阔叶林优势种水分生理生态研究

本文研究了北京山区落叶阀叶林优势种一辽东栎、大叶白蜡、北京丁香、核桃楸、山杏和荆条等乔灌木的稳定碳同位素比率，长期水分利用效率、植物蒸腾特性和土壤植物体的水分运动特点，并从植物解剖学的角度研究了这些植物叶片特点和其水分生理生态特性的关系。 对北京山区落叶阔叶林生态系统这几种乔、灌木植物叶片中的碳稳定性同位素比率(δ¹³C值)测定结果显示，这些植物叶片的δ¹³C值受多种因子的影响，具有较大的种间差异及时空异质性。主要表现在不同植物种叶片δ¹³C值不同，其排列顺序为山杏(- 24.75±0.85%。>大叶白蜡(- 25,94±1.52%。)=荆条(- 26.01±1.63%。)=辽东栎(一26.07±1.17%。)=北京丁香(-26.46±0.80c70。)>核桃楸(-28.11±1.52%。);生长初期叶片δ¹³C值较生长末期高，尤以核桃楸和辽东栎表现明显其生长初期和末期的叶片δ¹³C值皆相差达3‰;生境条件，特别是土壤水分含量和土层厚度，对植物叶片的813C值的高低有较大的影响，生长在于旱生境中的植物具有较高的δ¹³C值。另外，即使是同一株植物，叶片δ¹³C值也因其在冠层中所处的位置不同而异，冠层项部叶片的δ¹³C值高于林冠内部的叶片。北京山区落叶阔叶林优势种的长期水分利用效率与种的特点有关，山杏最高(4.950±0．l71mmolC0_2•mol-1H_2O)，核桃楸最低(3.760±0.203mmolC0_2•mol-1H_2O)，大叶白蜡、荆条、辽东栎和北京丁香居中(4.346- 4.530 mmolCO_2•mol-1H_2O)，大部分植物长期水分利用效率在春季（5月）较高，秋季较低，荆条由于物候期的特殊性在其生长季初期较低，而后逐渐增高。核桃楸在不存在水分亏缺情况下，树干液流速率受微气候因子的影响，液流速率的最大值达1600g•hour 左右。树干液流速率的日进程和大气相对湿度、温度的日进程具有相当好的生态学同步性。通过对核桃楸夜晚树干液流的分析可以得出其有根压存在的结论。 植物叶片和枝条中自由水和束缚水含量主要决定于植物种的特性，枝条的年龄、生境特点，特别是土壤水分特点。在落叶阔叶林I(样地2)中植物叶片自由水含量的排列顺序是：北京丁香>核桃楸>大叶白蜡>辽东栎;而杂灌丛（样地1）中植物叶片自由水含量的排列顺序是：核桃楸>大叶白蜡>山杏>荆条>北京丁香>辽东栎，可见群落类型对植物自由水含量影响是很大的，植物束缚水含量与其自由水含量的格局完全相反，荆条、山杏等植物含量高，核桃揪含量低。枝条水分含量有与叶片水分含量相类似的特点。 北京山区落叶阔叶林优势种的水分生理生态学特性和其叶片的特点有很大的关系，首先是植物叶片的特点总是和其种的特性相联系，主要表现在叶的类型、叶片上毛、气孔密度、着生方式等，如荆条叶片上下表面都密被披针形毛，气孔小，核桃楸气孔较大且凸出，大叶白蜡叶片上的气孔凹陷，辽东栎的气孔呈椭圆状，保卫细胞上有许多白色蜡质结晶。有一些种有环状的气孔外缘。生境的变化对叶片的形态特征有影响，在全光照条件下叶片小而犀，而在庇荫条件下叶片大而薄，在扫描电镜下可见全光照条件下北京丁香叶片基本无毛，庇荫条件下则有短微毛，全光条件下荆条叶片上毛有小乳头状凸起，庇荫条件下没有。本文所研究的植物种气孔都着生在叶片的下表面，气孔密度的大小排列顺序是：辽东栎>山杏>北京丁香>核桃楸>大叶白蜡。经方差分析显示种闷气孔密度存在极显著性差异。对所研究植物的气孔导度和环境因子、叶片解剖特点进行线性回归分析，得到了总体的和各个种的回归模型，结果表明光照强度、气孔密度等对气孔导度影响显著，但因种的不同相互之间存在差别。 植物的蒸腾速率受多因子的影响，主要有种的特点、微气候因子（光照强度、大气相对湿度、叶面温度、叶室温度等）和土壤水分特点。植物的蒸腾速率日进程和微气候因子日进程有相当好的生态学同步性。对辽东栎的蒸腾速率和光照强度的研究发现二者有很好的线性关系。这些植物蒸腾速率都表现出一定规律的日进程和季节进程，大多数植物蒸腾速率在一天中有数次波动，最高峰一般出现在中午12时之前;在整个生长季中，6月底至8月初的蒸腾速率高于其他月份。

Road damage due to dynamic tyre forces measured on a public road

This paper describes a series of tests conducted on a UK trunk road, in which the dynamic tyre forces generated by over 1500 heavy goods vehicles (HGVs) were measured using a load measuring mat containing 144 capacitive strip sensors. The data was used to investigate the relative road damaging potential of the various classes of vehicles, and the degree of spatial repeatability of tyre forces present in a typical highway fleet. Approximately half the vehicles tested were found to contribute to a spatially repeatable pattern of pavement loading. On average, air suspended vehicles were found to generate lower dynamic load coefficients than steel suspended vehicles. However, air suspended vehicles also generated higher mean levels of theoretical road damage (aggregate force) than steel suspended vehicles, indicating that the ranking of suspensions depends on the pavement damage criterion used.

The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia : Anura): a new gene order in the vertebrate mtDNA

The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNA(Thr), tRNA(Pro), tRNA(Leu) ((CUN))) a tandem duplication of tRNA(Mer) gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNA(Thr)/tRNA(Pro)/tRNA(Leu)/tRNA(Phe). The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNAM(Met) and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNA(Met) were first observed in the vertebrate mitochondrial genomes. (c) 2004 Elsevier B.V. All rights reserved.

Benthic macro-biota in Gaula River - Kumaon Himalaya

The density of benthic macro-biota in number over weight of biomass at three sampling stations was 29 units m²/0.614 g m² to 171 units m²/11.346 m² at Station I; 22 units m²/0.410 g m² to 155 units m²/8.717 m² at Station II; 15 units m² 10.321 g m² to 122 units m²/6.793 g m² at Station III. The Caddis fly larvae were the most dominant component and contributed 52.41% in the macro-biota. These animals were abundant in Gaula River when benthic algae were abundant; water is well oxygenated, alkaline and contains sufficient nutrients but observed to be less abundant during high velocity of water, high river depth and higher turbid waters.