羽叶铁线莲杂交起源的初步研究

通过野外观察，发现羽叶铁线莲Clematis pinnata Maxim.在形态上相似于短尾铁线莲C. brevicaudata DC.及大叶铁线莲C. heracleifolia DC.，推测羽叶铁线莲为后二者杂交产生。本文从形态学、解剖学、孢粉学、分子生物学等方面寻找证据，为这一推测寻找证据： 1． 形态学 通过野外观察结合标本馆工作，对这三个种的外部形态性状及其变异进行全面的分析，以探讨羽叶铁线莲C. pinnata在形态上与二假定亲本的异同及其产生原因。发现花、叶、习性等性状，在二假定亲本种种内稳定，但种间差异很大，在羽叶铁线莲中，性状变异的幅度正是处于二假定亲本差异之间。 2．解剖学 在光学显微镜及扫描电镜下，观察了这三种铁线莲的叶表皮特征，结果表明：叶片上表皮均不具有气孔器，下表皮气孔器类型均为无规则型;表皮细胞多为不规则形，仅在短尾铁线莲C. brevicaudata的上表皮中为近多边形;上表皮角质膜均具细条纹，下表皮角质膜均具环状或放射状波状嵴。 在扫描电镜下对瘦果形态及宿存花柱进行观察，结果表明：瘦果均密被长柔毛，并有蜡质颗粒状附属物。 在三个种植物之间叶表皮和瘦果的特征均无明显差别，对解释所讨论的问题意义不大。 3．孢粉学 在扫描电镜下对这三种铁线莲的花粉粒进行观察，结果表明：短尾铁线莲的花粉粒具三沟;大叶铁线莲的杂性株中花粉高度败育，说明其杂性株其实即为雌株;羽叶铁线莲的花粉也高度败育，但本种均为两性株，支持本种为杂交种的推测。 4．等位酶研究 利用莽草酸脱氢酶SKD和苹果酸脱氢酶MDH对采自3个居群的羽叶铁线莲大叶铁线莲和短尾铁线莲进行等位酶试验，结果显示在短尾铁线莲与大叶铁线莲中分别出现种内一致的谱带，而羽叶铁线莲则显示出二者杂合的带型。这一结果有力的支持了羽叶铁线莲是由短尾铁线莲和大叶铁线莲杂交产生的推测。 5．居群遗传结构分析 利用随机扩增多态性DNA标记（RAPD）分别检测三个种的遗传多样性和居群遗传结构。15个随机引物对这三个种共160个个体 （短尾铁线莲C. brevicaudata 五个居群73个个体;大叶铁线莲Clematis heracleifolia五个居群75个个体;羽叶铁线莲Clematis pinnata三个居群12个个体。）进行分析，总共得到123个用于分析的条带。我们对这三个种扩增出的条带按照居群进行了聚类分析，结果表明这三个种在聚类树上能够很好的区分开，但是种内居群间没有表现出地理分布式样方面的信息;个体水平上聚类结果表明，百花山采到的7株无法确定的铁线莲幼苗有6株是短尾铁线莲C. brevicaudata另外一株是羽叶铁线莲Clematis pinnata，这说明在幼苗形态上二者差别不大;我们对这三个种分别进行了居群遗传结构的分析，结果表明：短尾铁线莲C. brevicaudata的多态条带比率为90.65％，遗传变异主要分布在居群内，居群间分化较小，而大叶铁线莲Clematis heracleifolia的多态条带比率为90.76％，居群间遗传变异占30.52％，居群间有明显分化。羽叶铁线莲Clematis pinnata的遗传多样性很低，有可能是由于营养繁殖造成的。 6．DNA序列分析 选取叶绿体基因组的trn L－F序列和核基因组的ITS序列进行了测序分析。发现从三个种十个个体得到的trn L－F序列之间几乎没有差异，在640 bp中只得到了3个信息位点。对ITS测序过程中发现短尾铁线莲可直接用PCR产物测序，而对大叶铁线莲和羽叶铁线莲必须进行克隆测序，由于时间关系，我们得到的序列不足以进行进一步分析。 基于以上研究结果，我们初步认为羽叶铁线莲是通过短尾铁线莲和大叶铁线莲的杂交而起源，并根据我们观察到的变异式样对羽叶铁线莲系的一些种类进行了分类学处理。

土圞儿属和旋花豆属的系统学研究

土圞儿属（含8种）和旋花豆属（含2种）隶属于豆科、菜豆族、刺桐亚族。土圞儿属呈东亚-北美间断分布，旋花豆属分布于喜玛拉雅地区。传统上，这两个属被认为系统关系非常近。目前对于土圞儿属中种的划分和分类问题缺乏世界范围内的深入研究，因此该属应予以修订。另外，土圞儿属内种间和土圞儿属与旋花豆属间的系统关系也需要进一步研究。本文对这两个属进行了世界范围内的分类学修订。同时，基于形态学、解剖学、孢粉学特征和分子系统学方面的研究，对土圞儿属内部和这两个属之间的系统发育关系进行了探讨。结果如下： 1. 形态学 在标本室研究和野外考查的基础上，对土圞儿属和旋花豆属植物形态性状的变异式样及其分类学意义进行了分析，发现块茎的形态、小叶的大小和形态、花序的类型和花冠的形态等特征在种内稳定，是比较可靠的分类学性状。通过对花序的研究，认为花序的类型可能反映了土圞儿属内各种之间的演化关系，推测肉色土圞儿可能是土圞儿属中较为进化的种类，而云南土圞儿和纤细土圞儿则可能较为原始。 2. 叶表皮特征 首次在光学显微镜和扫描电子显微镜下，对土圞儿属和旋花豆属全部8种植物的叶表皮进行了观察。发现这两个属叶表皮的性状，特别是表皮细胞的形状、垂周壁的式样和蜡质纹饰的特征，对于理解土圞儿属内和这两个属之间的系统关系有重要意义。叶表皮的特征支持以下结论：纤细土圞儿是一个独立的种，而不是云南土圞儿之下的一个变种;对于土圞儿属进行亚属的划分是不合理的。 3. 孢粉学 在光学显微镜和扫描电子显微镜下，对7种土圞儿属和旋花豆属植物的花粉进行了观察。这两个属的花粉均为三孔沟，三角球形或球形。土圞儿属的花粉可划分为两种类型：肉色土圞儿的花粉外壁为典型的网状纹饰;其他的种花粉外壁较光滑，具颗粒状或短条纹状纹饰。肉色土圞儿花粉外壁纹饰的类型可能反映了其在Apios属中较进化的位置。旋花豆属的花粉特征和肉色土圞儿相似，这也许说明了两者之间的亲缘关系较近。 4. 分子系统学 通过对土圞儿属和旋花豆属6种植物的两个DNA片段ITS和H3D进行简约性分析，构建了这两个属之间的系统关系树。结果如下：土圞儿属和旋花豆属是亲缘关系较近的姐妹群;土圞儿属自身组成一个单系群，是一个自然的类群;北美的土圞儿聚成一支，隐藏在东亚的种里。分子证据和花序类型、花粉形态等重要的形态学证据相吻合。基于上述证据，我们对土圞儿属东亚-北美间断分布的格局及其可能的成因进行了初步探讨。 5. 分类修订 对土圞儿属和旋花豆属进行了分类修订，考证了关于这两个属曾发表过的所有的名称。确认了土圞儿属应包含6种，旋花豆属有2种。排除了5个不包括在土圞儿属内的种名。此外，本文还给出了这两个属各个种的地理分布图、分种检索表、形态描述和插图。

葛属(豆科)的分类学修订

本论文对葛属（Pueraria DC.）进行了形态学、解剖学及植物地理学研究，重点分析了小叶、托叶、花序及花的形态变异规律。首次发现葛属生长习性和种皮纹饰特征在组的划分上具有重要的分类学意义，支持前人运用托叶着生方式、花序类型及每节簇生花的数目等特征划分组的观点。首次在光学显微镜下对葛属植物14种1变种叶表皮性状做了较为全面、深入的观察，发现葛属叶表皮细胞的形状、垂周壁的式样以及气孔大小有一定分类学意义，为葛属的分类修订提供了新资料。首次应用电子扫描显微镜观察了葛属10种1变种和2个外类群广西土黄芪Nogra guangxiensis C. F. Wei和琼豆Teyleria koordersii (Backer ex Koord.-Schum.) Backer的种皮纹饰特征，发现葛属的种皮纹饰有网纹型、断棱型、复网纹型和锐棱型等4种类型，他们与托叶的着生方式具有一定的关联性。托叶背着的类群为网纹型，其中采自中国和越南不同居群的山野葛Pueraria montana (Lour.) Merr.种皮次级纹饰有差别，这显示山野葛分化比较活跃。托叶基着的三裂叶野葛P. phaseoloides (Roxb.) Benth.、大花三裂叶野葛P. phaseoloides (Roxb.) Benth. var. javanica (Benth.) Baker、苦葛P. peduncularis (Graham ex Benth.) Benth.以及云南野葛P. yunnanensis Franch.的种皮纹饰为断棱形，小花野葛P. stricta Kurz种皮纹饰为复网纹型，喜玛拉雅葛藤P. wallichii DC.种皮纹饰为锐棱型。依据种皮纹饰推断，喜玛拉雅葛藤最原始，黄毛葛藤P. calycina Franch.最进化，与形态学特征演化方向相吻合。广西土黄芪种皮纹饰为网纹型，琼豆种皮纹饰为复网纹型，表明它们与葛属亲缘关系较近。这与形态学和分子证据相吻合。本文首次运用广义线形模型（GLM）结合地理信息系统（GIS）对葛属现有地理分布、潜在分布及50年后潜在分布进行了分析。所预测的潜在分布范围与观察到的地理分布范围一致，说明对特化的气候忍耐性是限制葛属物种地理分布的主要因素。温度、降水和海拔高度对葛属地理分布限制作用比较大。未来潜在分布预测显示葛属有向南、北扩散的趋势。 作者在研究了四千余份腊叶标本及野外形态考察的基础上，结合叶表皮及种皮纹饰资料，确认葛属分为3组，共19种3变种。其中，对Sect. Breviramulae Maesen ex Z. F. Le & X. Y. Zhu进行了合格发表，运用形态学、解剖学及种皮纹饰资料恢复了云南野葛种的地位，并为其指定了后选模式，P. pulcherrima (Koord.-Schum.) Merr. ex Koord.-Schum.正确的学名为P. sericans Schum.，对4个名称进行了归并。本文还给出了组和种的检索表、葛属各个种的形态描述、地理分布图及模式标本照片。

钻地风属和冠盖藤属（虎耳草科）的分类学修订

钻地风属（Schizophragma Siebold & Zucc.）和冠盖藤属（Pileostegia Hook. f. & Thomson）隶属于虎耳草科绣球花亚科绣球花族植物，分布于东亚。本论文对钻地风属和冠盖藤属进行了全面的分类学研究：重点分析了钻地风属和冠盖藤属各形态性状的变异式样，确定了二属的性状变异范围;在光学显微镜下，对钻地风属中6种5变种和冠盖藤属中2种共130份材料的叶表皮特征进行了深入、全面的观察和研究;并对二属的叶脉序特征进行了研究。结果显示，苞片存在与否、不育花存在与否、花瓣顶端是否联合、蒴果和种子的形状、叶片质地、生长习性、表皮细胞垂周壁加厚与否、气孔器分布、类型、气孔大小、保卫细胞轮廓、外拱盖是否双层、二级脉、三级脉、高级脉及网眼等特征在属的水平上具有重要的分类学意义，这些性状特征支持将钻地风属和冠盖藤属作为两个独立的属处理。根据形态性状及叶脉序特征推测，冠盖藤属较原始，钻地风属较进化。在钻地风属中，叶片毛被的有无及类型、叶片形状和叶缘的形状等特征可作为可靠的分种依据;叶表皮特征在种及种下等级具有重要的分类学意义;叶脉序多呈现环节曲行羽状脉式样，二级脉具分枝，在种间存在细微差别。根据形态性状及叶脉序特征推测，厚叶钻地风S. crassum Hand.-Mazz.和白背钻地风S. hypoglaucum Rehder可能为钻地风属中较原始的类群，秦榛钻地风S. corylifolium Chun和绣球钻地风S. hydrangeoides Siebold & Zucc.可能较进化。在冠盖藤属中，叶片毛被的有无及类型、叶片形状等特征可作为可靠的分种依据;该属中叶表皮特征相似，2个种间叶表皮特征无差异;叶脉序多呈现环节曲行羽状脉式样，二级脉无分枝，在种间差异极小。 作者在研究了近2000份腊叶标本的基础上，结合叶表皮特征和叶脉序特征，认为钻地风属和冠盖藤属为两个独立的属。首次对钻地风属和冠盖藤属进行了世界性的分类学修订，确认钻地风属含7个种、5个变种，冠盖藤属含2个种。对钻地风属中的5个名称和冠盖藤属中的2个名称进行了归并;为钻地风属中的1个名称和冠盖藤属中的2个名称指定了后选模式;发现被命名为维西钻地风S. crassum Hand.-Mazz. var. hsitaoianum (Chun) C. F. Wei的类群其正确名称为S. crassum Hand.-Mazz. var. ellipticum J. Anthony;恢复了变种小粉绿钻地风S. integrifolium Oliv. var. minus Rehder;重新界定了白背钻地风和小粉绿钻地风的变异范围，澄清了中国文献中记载的粉绿钻地风S. integrifolium Oliv. var. glaucescens Rehder长期存在的名实混乱问题。本论文对各个分类群进行了详细的形态描述，给出了分种检索表，并附分布资料及地理分布图。

杉科植物的表皮结构研究及古环境重建

全球古近纪和新近纪气候波动明显，很多科学家对古近纪和新近纪生物演化和气候演变规律的定量研究给予了相当的重视。杉科植物有长期发展的历史、少数的现存种和丰富的化石记录，成为指示古环境的理想植物之一。利用杉科植物重建古环境，首先要对化石植物进行正确的分类鉴定。杉科各属枝叶形态特征变异幅度较大，且枝叶排列和形态特征近似，有时各属之间存在交叉特征。杉科化石标本通常仅保存枝叶形式，且多个属的化石标本经常发现于同一地层，因而分类鉴定比较困难。植物的叶表皮结构是压型植物化石细胞信息的重要来源，是属种分类鉴定重要依据之一。本文在以往研究的基础上，完整分析了杉科9属现生植物的表皮特征，建立了杉科化石植物分类鉴定的现生植物的表皮特征参照系。 杉科植物的叶多为条形、钻形、鳞形或披针形，同种植物有1种、2种甚至3种叶型。其中水杉属的叶交互对生，其它属的叶螺旋状互生。水杉属的多数表皮细胞垂周壁明显弯曲，落羽杉属和杉木属有时微呈波状，其他属的表皮细胞垂周壁直。多数植物的叶片近轴面和远轴面的气孔数量和分布不同。一般来说，条形叶和披针形叶的远轴面气孔分布状况和气孔数量稳定，远轴面的中部最稳定。条形叶远轴面的气孔分布于中脉两侧，形成纵向的气孔带。条形叶近轴面气孔分布状况和气孔数量变化大，近轴面的气孔数量有时与远轴面近似，但多数情况下比远轴面少，有时整个叶片的近轴面仅少数几个气孔或没有气孔分布。钻形叶的近轴面和远轴面的气孔数量近似，或叶片近轴面的气孔数量比远轴面的气孔数量多，气孔分布范围也比远轴面气孔分布范围广。气孔椭圆形，落羽杉属植物和水松鳞形叶的气孔长轴方向与叶片长轴垂直或斜向排列，柳杉属植物的气孔多斜向排列，水松的条形叶和条状钻形叶的气孔多平行向排列。落羽杉属和柳杉属以外的杉科植物的气孔长轴多数与叶片长轴平行。在扫描电子显微镜下的密叶杉属植物叶片角质层的内表面，副卫细胞和表皮细胞的垂周壁与叶表面的角度一致，且副卫细胞的形态与表皮细胞类似，这种特殊气孔器被称为A-型气孔器。杉科植物中水杉属、落羽杉属和杉木属的气孔器近似密叶杉的这种A-型气孔器。台湾杉属、柳杉属、水松属、红杉属和巨杉属植物的气孔器与这种A-型气孔器不同，这几属的副卫细胞垂周壁表皮细胞的垂周壁方向不同，且副卫细胞的形态与表皮细胞的形态明显不同。因而气孔所有副卫细胞组成呈明显的圆盘状，保卫细胞在盘子的中央。综合分析植物的枝叶形态和表皮特征可以区分杉科各属。 本文研究了采自抚顺始新世的杉科植物标本，经枝叶特征、表皮结构和球果鳞片等特征分析为水松属的欧洲水松。论文分析了中国东北地区古温度定量研究结果与杉科植物的相关性。抚顺植物群发现有化石水杉M. occidentalis、中华红杉和欧洲水松，根据这三种化石植物的年均温度和年降水量，通过共存分析，推测抚顺始新世的年均温为12.1-17℃，年降水量为1199.6-2231mm。

硬叶常绿阔叶林建群种-高山栎组的生物生态学研究

栎属(Quercus)高山栎组（Sect．Suber)是我国硬叶常绿阔叶林的主要建群种。本文从地理分布、群落特征、形态解剖特性方而对高山栎的生态适应性进行了研究，讨论了它们的起源与青藏高原隆升的关系。得到如下结论： 1．丽江一木里为现代高山栎的分布中心，高山栎组的种数等值线从这里呈同心园向外扩散。地质历史时期存在于该地的康滇古陆应当是高山栎的起源地。2．高山栎的水平分布不超过北纬32º，即亚热带的北界;垂直分布表现为超地带性的特点，从海拔1200到4300m，随海拔的增高而矮生。3．丽江有硬叶常绿阔叶林8种群落类型，它们的结构、分布各有其特点。4．高山栎组的表皮特征，除气孔器类型外，其他表皮性状受环境修饰，不是稳定的性状。气孔的大小、数目随环境有规律性地变化。5．川滇高山栎为复表皮，表皮附属物、角质膜及叶肉细胞随环境有规律性变化。6．虽然高山栎组的叶片表现为硬叶的特性，但解剖结构差别明显随环境发生变化。

Molecular evolution of the keratin associated protein gene family in mammals, role in the evolution of mammalian hair

Background: Hair is unique to mammals. Keratin associated proteins (KRTAPs), which contain two major groups: high/ultrahigh cysteine and high glycine-tyrosine, are one of the major components of hair and play essential roles in the formation of rigid and

Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells

Generation of homogeneous oligodendrocytes as donor cells is essential for human embryonic stem cell (hESC)-based cell therapy for demylinating diseases. Herein we present a novel method for efficiently obtaining mature oligodendrocytes from hESCs with high purity (79.7 +/- 6.9%), using hepatocyte growth factor (HGF) and G5 supplement(containing insulin, transferrin, selenite, biotin, hydrocortisone, basic fibroblast growth factor and epidermal growth factor) in a four-step method. We induced hESCs into neural progenitors (NP) with HGF (5 ng/ml) and G5 (1 x) supplemented medium in an adherent differentiation system. The purified NPs were amplified in suspension as neurospheres for 1 month, and terminal oligodendrocyte differentiation was then induced by G5 supplement withdrawal and HGF treatment (20 ng/ml). The cells generated displayed typical morphologies of mature oligodendrocytes and expressed oligodendrocyte markers O4 and myelin basic protein (MBP). Our result revealed that HGF significantly enhanced the proliferation of hESC-derived NPs and promoted the differentiation as well as the maturation of oligodendrocytes from NPs. Further studies suggest that HGF/c-Met signaling pathway might play an important role in oligodendrocyte differentiation in our system. Our studies provide a means for generating the clinically relevant cell type and a platform for deciphering the molecular mechanisms that control oligodendrocyte differentiation. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Bryodrilus fuscistriatus, a new enchytraeid species (Oligochaeta : Enchytraeidae) from northeastern China

Bryodrilus fuscistriatus, a new enchytraeid species from Mt. Changbaishan, Jilin Province, north-eastern China, is described. It is characterized by brown epidermal glands, 7 pairs of preclitellar nephridia, poorly-developed clitellar glands, spermatheca with 2 sessile globular diverticula, and a long sperm funnel with a very broad collar. It is similar to the Alaskan B. tunicatus Dozsa-Farkas & Christensen, 2002 in possessing two diverticula in the spermathecal ampulla and the origin of the dorsal vessel, and the Chinese B. longifistulatus and B. macrotheca Xie et al., 2000c in body size, long sperm funnel and undeveloped clitellar glands, but it differs from B. tunicatus by the presence of brown-striped epidermal gland cells in III-V, a poorly-elevated clitellum, the absence of copulatory glands in XIII-XIV, the regular outline of coelomocytes, and 7 pairs of preclitellar nephridia; from B. longifistulatus and B. macrotheca by the shape of spermatheca, the color of epidermal gland cells, the position of the first pair nephridia, and the origin of dorsal vessel.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp beta-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

Immunization of rainbow trout fry with Ichthyophthirius multifiliis sonicate: protection of host and immunological changes

Rainbow trout fry (10 weeks post hatch) were immunized (injection or immersion) with sonicated formalin-killed trophonts of the fish parasitic ciliate Ichthyophthirius multifiliis. Challenge infections 22 days after immunization showed a relative protection represented by significantly fewer established parasites and lower prevalence in the immunized groups compared to the controls. Associations between the obtained protection and changes in differential leukocyte counts, haematocrit values, anti Ichthyophthirius multifiliis antibodies, mucous cell density and some epidermal cell markers were investigated. No changes in antibody titers, haematocrit values and mucous cell counts were associated with the response; however, a minor change in peripheral blood neutrophils and epidermal cell markers were found.

Fridericia nanningensis, a new terrestrial enchytraeid species (Oligochaeta) from southwestern China

Fridericia nanningensis, a new species from wetland soil of Nanhu Park, Nanning city, the capital of Guangxi Zhuang Autonomous Region in southwest China, is described. It is characterized by 2-4 chaetae per bundle, poorly-developed clitellar glands, slender, unbranched peptonephridia, and spermathecae with 2 ampullar diverticula, a deep constriction in the middle of the ampulla and one large ectal gland. It is closely related to the European species, F. alata Nielsen & Christensen, 1959 and the East European species, F. tubulosa Dozsa-Farkas, 1972 by the shape of peptonephridia and the undeveloped clitellar glands. It differs from F. alata by its shorter body length and fewer chaetae per bundle, its type of coelomocytes (type "c"), its deep constriction in the middle of the spermathecae ampulla and a larger ectal gland, and it differs from F. tubulosa by its pale epidermal glands, its more anterior origin of the dorsal vessel, a deep constriction in the middle of the spermathecae ampulla, shorter ectal duct, and only one ectal gland at the spermathecal orifice.

P-选择素CR结构域功能的模拟预测

<正>选择素(selectin)与其配体间相互作用介导的细胞粘附在炎症级联反应、肿瘤转移和淋巴细胞归巢等病理、生理过程中起重要作用。其结构组成从远离细胞表面的N-末端到胞内C-末端,依次为:钙型凝集素功能区(Calcium-type lectin domain,Lec),类上皮生长因子功能区(Epidermal Growth Factor-like module,EGF),具有粘附补体蛋白作用的多个重复序列(Consensus Repeats,CRs),跨膜区(Transmembrane,TM)和胞内区

川西亚高山人工针叶林下11种植物对两种光生境的适应性研究

青藏高原东缘的亚高山针叶林是长江上游重要的生态屏障，经过近六十年的采伐后，取而代之的是大量人工种植的云杉纯林。目前，这些人工林已经表现出树种单一，结构层次简单等生态问题，其物种多样性及生态效益与同地带天然林相比差距较明显。如何丰富该地区物种多样性，完善人工林生态系统的生态功能是一个十分重要的课题。林下植物是人工林群落的重要组成部分，对维持群落的生物多样性及完善生态系统功能具有明显的作用。因此，研究该地区人工针叶林的林下植被对不同生境的适应性对于理解人工林生态系统物种多样性的形成和维持机制都具有重要的意义。 本文以青藏高原东部亚高山针叶林的主要森林类型----云杉人工林为研究对象，选择林下11种具有不同喜光特性的常见植物，分别设置人工林林冠下及成熟林窗为研究样地，通过对各种植物叶片形态与物质分配特征、叶片解剖学特征、叶片光合生理特性、植物自然分布特征等方面的比较分析，研究林下植物对不同光生境的适应策略及其适应能力，揭示不同物种对人工林生境的适应共性，为西南亚高山地区植被恢复及人工林的经营管理提供科学依据。具体研究结果如下： 在叶片形态和物质分配特征方面：在林窗光生境中，11种林下植物叶片比叶重(LMA)显著高于林下光生境的同种植物。同时，林窗下生长的植物叶片叶片厚度及栅栏细胞长度显著增加，这是影响叶片比叶重变化的直接原因。而多数植物叶重比在两种生境中无明显变化。说明在长期适应自然生境之后，植物可能更多地采取调节叶片组织细胞水平（即叶片功能细胞形态）及叶片器官水平（即单个叶片形态）特征的策略来适应各类生境，而非整株水平上的叶片总比重的增减。 在叶片解剖结构特征方面：多数阔叶物种栅栏组织厚度(PT)、栅栏组织厚度/海绵组织厚度(PT/ST)、栅栏细胞层数及近半数种的气孔密度(SD)在林窗生境中更大或更多，而叶片表皮细胞厚度(UET、LET)气孔长径(SL)及海绵组织厚度(ST)受两种生境影响不大。喜光特性相似的物种在生境适应策略上具有一定的趋同性。 在光合生理特征方面：在林窗生境中多数种植物的最大光合速率(Amax)、暗呼吸速率(Rd)及喜光植物光补偿点(LCP)显著或极显著高于林内生境同种植物。且在同一生境条件下，多数深度耐荫植物比喜光及轻度喜光植物有稍低的Rd和LCP。各植物在林内低光生境中具有更大的内禀光能转化效率，并在中午12:00～14:00之间光强最大的时刻发生了的最深程度的光抑制。多数种能通过调节自身某种光合素含量或色素之间的比例来适应不同的光生境，即通过增加叶绿素含量或降低Chla/b值来适应林内弱光生境，通过提高类胡萝卜素含量或单位叶绿素的类胡萝卜素含量降低强光带来的伤害。绝大多数物种并不采取调节叶片C、N含量的策略来适应不同的光生境。总之，植物部分光合参数(Amax、Rd、LCP)受生境的影响与其自身喜光特性有关，但另一些参数（Fv/Fm日变化、色素含量及比例、叶氮相对含量）受生境影响与其自身喜光特性无明显关联。 在表型可塑性方面：在叶片各表型参数中，器官水平及细胞水平的形态特征参数平均可塑性大于整株水平形态和物质分配特征参数可塑性；叶片光合组织的可塑性大于非光合组织可塑性；反映植物光合能力的参数可塑性大于叶片色素含量参数可塑性。植物叶片形态和物质分配、解剖学特征参数平均可塑性大小与其自身喜光特性基本吻合，即喜光种及轻度耐荫种各参数可塑性最高，深度耐荫种可塑性最小，而这种规律并未在光合生理参数的可塑性大小上体现出来。但是叶片形态和物质分配参数、光合生理参数的平均可塑性水平却大于叶片解剖学参数。 在植物自然分布特征方面：喜光物种云杉幼苗及歪头菜在林内生境中分布密度明显降低，深度耐荫种疏花槭却恰恰相反，更多数物种（7种植物）在两种生境中密度变化趋势不明显。从分布格局来看，7种植物在两种生境中均为聚集分布，但聚集强度为林窗>林内；少数物种桦叶荚迷、直穗小檗、冰川茶藨、黄背勾儿茶在林窗中为聚集型，在林内生境中的分布型发生改变而成为随机型，说明光生境的差异能影响到植物种群的分布特征。但这种影响程度与植物自身的喜光特性无关，同时与各物种叶片表型平均可塑性的大小也无明显关联。 The subalpine coniferous forest area in eastern Qinghai-Tibet Plateau is important ecology-barrier of upriver Yangtze. In past sixty years, those forests had been cut down and replaced with a lot of spruce plantations. At now, there are many ecology problems presenting to us such as singleness species, simple configuration, lower species diversity and ecological benefit than natural forests at the same belt. How to restore the species diversity and enhance the eco-function of the plantations is a very important issue. The understory plants are important part of plantation community, which improved the bio-diversity and eco-function distinctly of forests. So, it is very significance to study the adaptation of understory plants to different environment in plantation, and this study would helping us to understand how plantations to develop and remain their biodiversity. This study was conducted in a 60a spruce plantation in Miyaluo located in western Sichuan, China, and spruce plantation is major types of subalpine coniferous forest in eastern Qinghai-Tibet Plateau. In this paper, the leaf morphological and biomass-distributed characteristics, the anatomical characteristics, the photosynthetic characteristics and the distribution patterns characteristics of eleven different light-requirement understory species grown in two different environments (forest gaps and underneath close canopy) were studied and compared. The purpose of this study was to analyze the adaptation of this forest understory plants, to show up the commonness of these different light-requirement understory species in light acclimation, and to provide some scientific reference to manage and restore the vegetation of subalpine plantation of southwest China. The results were as follows: The leaf morphological and biomass-distributed characteristics: These eleven species in forest gaps had significantly higher dry weight per leaf area (LMA) than those under close canopy. The palisade parenchyma cells of the broad-leaved species in gaps were significantly longer than those grown under the canopy, which been a directed factor for the change of leaf mass per unit area (LMA) in different environment. But the leaf weight ratio (LWR) of most plants species were not evidently changed by the contrasted environments in our study. It was shown the morphological characteristics changing been adopted as a strategy of light acclimation for plants wasn’t on whole plant level (leaf weight ratio) but cellular level (the function cells morphological characteristics) and organic level (the leaf morphological and biomass-distributed characteristics) mostly. The leaf anatomical characteristics: Most broad-leaved plants in gaps increased palisade parenchyma thickness (PT), the palisade parenchyma cell layers and the ratio of palisade to spongy parenchyma (PT/ST). So did as almost about half species in this study in stomatal density (SD). No significant differences in thickness of leaf epidermal cells (UET, LET), stomatal length (SL) and spongy parenchyma (ST) between two environments of most species were observed. The results suggested that species with light-requirement approximately had convergent evolution on adaptation to light condition. The leaf photosynthetic characteristics: The dark respiration rate (Rd) of most plants species, the light compensation point (LCP) of light-demanding plants species in gaps were significantly increased than under close canopy in this study. In a same habitat, most deep-shade-tolerant plants had lower Rd and LCP than those light-demanding plants and slight-shade-tolerant plants. Each species has bigger inherent electron transport rate under close canopy than in gaps, and the greatest photoinhibition happened during 12 to 14 in the daytime. Most species could adapt different light environment by the way of changing their photosynthetic pigments content or the ratio of pigments content. For example, some plants under close canopy increased chlorophyll (Chl) or reduced the values of the ratio Chla/b to adapted the low light condition, some plants in gaps increased carotenoid (Car) or reduced the weight ratio CarChl to avoid been hurt in high light. For most plants, changing the content of C and N in leaf wasn’t a strategy of light acclimation. In conclusion, the variation of some leaf photosynthetic parameters in different light environment such as Fv/Fm, pigments, C and N in leaf related with the light-requirmnet of species, but the others such as Amax, Rd, LCP did not. The leaf plasticity indexes: Among those leaf plasticity indexes, the leaf morphological and biomass-distributed parameters on cellular and organic level were greater than on whole plant level for same species, and the photosynthetic parenchyma parameters were greater than non-photosynthetic parenchyma parameters in same leaf, and photosynthetic capability parameters were greater than photosynthetic pigments content parameters for same species. The average plasticity indexes of leaf morphological and biomass-distributed and anatomical parameters were accordant with plants’ light-requirement approximately: those light-demanding plants and slight-shade-tolerant plants had bigger plasticity indexes than deep-shade-tolerant plants. But this regular wasn’t observed in physiological plasticity indexes for most plants, though the average leaf plasticity indexes of leaf morphological and biomass-distributed, photosynthetic characteristics parameters was greater than the anatomical characteristics parameters. The distribution patterns characteristics: Oppositely to the deep-shade-tolerant specie Acer laxiflorum Pax., the density of light-demanding species Picea asperata Mast. and Vicia unijuga A. Br. in gaps was bigger than under close canopy. Each of the other species has the approximately density in two different environment. The spatial patterns of seven species were aggregated distribution in two environments, but the trend of aggregation of population under close canopy was decrease from in gaps. A few species such as Viburnum betulifoium Batal., Berberis dasystachya Maxim., Ribes glaciale Wall. and Berchemia flavescens Brongn. were aggregated distribution in gaps while random distribution under close canopy. It was shown that the difference between two light environments could affect the distribution pattern of plant population, and the effect didn’t relate with the light-requirement or plasticity indexes of species.

簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.