甘蓝型油菜（Brassica napus L.）花粉和柱头的发育及其与自交不亲和性的关系

作者用扫描电镜、透射电镜和组织化学方法研究了甘蓝型油菜(Brassica napus L．)花粉和柱头的发育及相互作用,得到了如下结果： 一．甘蓝型油菜具有典型的同型孢子体自交不亲和性(Homomorphic and sporophytic self-incompatibility)。自花授粉后，部分花粉粘住在乳突细胞表面，随后萌发出花粉管，花粉管生长受阻于乳突细胞，花粉管表现出各种异常形态，缠绕卷曲(Coiled pattern),顶端膨大成基足状(pad-Like swelling),花粉管二叉分枝（dichotomous branching Pollen tubes），花粉管相互连接(connections of pollen tubes），有的花粉粒还形成两个花粉管。 二．不同时期的乳突细胞的扫描电镜观察表明： 开花前6—7天 乳突细胞壁内陷，柱头中央有一沟槽; 开花前4—5天 乳突细胞被有孔的块状物覆盖; 开花前2—3天 覆盖物消失，壁表面只剩下波状纹或小凹; 开花前l - 2天 乳突细胞充分吸水膨胀，呈指状，排列疏松，细胞壁上有一些小颗粒。此时的乳突细胞已发育成熟。 三.不亲和授粉时，花粉和乳突细胞均有强烈的胼胝质荧光。 四.乳突细胞的组织化学特点：缺乏淀粉积累，细胞壁和细胞质中有过氧化物酶活性。 五.乳突细胞的超微结构特点：细胞壁分为三层，蜡质层、角质层和纤维素层。粗面内质网成群分布在细胞壁附近，并以及泡形式向细胞壁分泌物质。缺乏质体，细胞核位于乳突细胞基部，细胞中央为大液泡占据。 六.花粉的超微结构发育特点：单核花粉已发育出内壁和外壁，外壁内层不明显。细胞核位于中央。细胞质浓厚，缺乏层膜结构而积累大量淀粉粒的质体存在于细胞质中。其他细胞器不发达。两细胞花粉时期，花粉壁接受乌氏体转运的孢粉素和含油体转运的脂类物质。生殖细胞没有壁，悬浮在营养细胞的细胞质中。细胞核大，细胞质稀薄，只有一些嵴不明显的线粒体。营养细胞的核显著，细胞质浓厚，线粒体发达，质体内部的淀粉消失，转变成嗜饿小体。内质网短而粗，遍布于细胞质中，高尔基体缺乏。 七.绒毡层积极参与了花粉外壁的建成。首先，它通过分泌作用把物质（可能是蛋白质）转移到单核花粉的腔隙中或在它后期滚解后，由分布在二细胞花粉间的粗面内质网合成蛋白质，转移到花粉壁内。其次，绒毡层细胞的质体层膜形成许多造油小体，至二胞花粉时，造油小体进入壁的柱状层，参与花粉鞘形成。第三，绒毡层细胞形成许多乌氏体，花粉发育后期，乌氏体与花粉外壁接触，将孢粉素转移到花粉外壁上。

羊草繁殖生物学特性的研究

羊草(Leymus chinensis (Trin.) Tzvel. )又称碱草，隶属禾本科，赖草属，因其营养价值高，富含蛋白质，适口性好，抗旱，耐盐碱，耐贫瘠，抗逆性强，适应性广等优点，对我国发展草原畜牧业和退化草地、荒漠化治理方面具有举足轻重的作用。近年来，由于自然环境变劣，荒漠化加剧，以及过度放牧等不利影响，加之羊草本身固有的“三低”问题（即结实率低、出苗率低、产草量低）已对羊草生物多样性维持构成了严重的威胁，严重限制了我国人工草地建设和天然草地的改良和沙化治理的步伐。加强羊草生物学研究，开展羊草种质生物多样性保护，成为当前研究的紧迫课题。经对国内外相关文献的查新发现：国外发表的文献匮乏，国内的报导大多集中在草原生态等宏观领域，在羊草繁殖生物学方面缺乏系统的研究。本文以本课题组从国内外收集的羊草资源为材料，从以下几个方面进行了初步探索： 一、羊草繁殖性状与遗传多样性分子标记指标的相关分析。利用分子标记与形态标记对随机抽取的17份羊草种质进行了种质评估的比较研究。结果表明：两种方法均在17份供试材料中鉴别出9份羊草种质,说明分子标记方法用于羊草种质资源鉴定是可行的，并具有快速、准确、不受环境条件限制等优点。在40个10 Mer的随机引物当中筛选出21个有效引物，以之对9份羊草材料进行RAPD 分析，共扩增出115条带，其中95条带表现出多态性，多态比率82.61%，并筛选出S1213-900，S1213-1700，S1215-5500， S1396-1370，S1384-900，S1202-5180，S1220-2200，S1381-1580，S1211-1300，S1211-800为羊草种质所具有的10个特异性标记，据此可将羊草种质与披肩草、赖草加以区分。同时在羊草种内亦发现13条可区分供试羊草种质的特有标记。形态标记与分子标记相关性分析结果显示：羊草种质的小穗数，种子千粒重，叶色，有性繁殖量和结实率5个形态学指标与遗传多样性指标---特有带百分率及遗传距离之间，存在一定相关性。同时对羊草种质资源在收集和评价过程中存在的问题进行了探讨。 二、羊草不同基因型无性繁殖特性比较研究。以本课题组从吉林、内蒙古等省份收集的10个基因型羊草为供试材料，在相同的生态因子作用下，以吉生1号羊草为对照，对10个基因型羊草的叶数增量、芽数增量、芽高度、芽间距、芽重量、根量六个无性系形态性状指标进行测评。结果表明：基因型的差异也是影响羊草无性系生长发育的重要影响因子。因此，在今后的羊草无性繁殖生物学研究中，应综合考虑环境因子和基因型因子对羊草无性繁殖生长发育的影响。在所测评的10个基因型中，各基因型的形态性状指标差异很大，栽3基因型较其他基因型优于对照吉生1号，此结论可为今后培育羊草新种提供重要资料。 三、羊草幼穗离体培养方法的建立。其方法是取羊草幼穗为外植体，经0.1%升汞溶液表面消毒后，接种到含2mg/L的2,4-D的MS培养基上，置于恒温25℃条件下诱导愈伤组织。在加有1mg/L2,4-D的MS培养基上继代2次后，转移到含1mg/L KT和0.5mg/LNAA的MS培养基上分化培养得到再生芽。在除去激素后的基本培养基上获得了生根的试管苗。试管苗移栽到温室后生长正常。羊草试管苗的分化因基因型和外源激素条件的不同而异。 四、羊草有性生殖特性的研究。在自然条件下进行了羊草自交、异交结实性实验，采用FDA染色法检测羊草小孢子活性，并观测羊草雌蕊、雄蕊发育的时空特点。结果表明：在大田中羊草异交结实率远大于其自交结实率;成熟花药中有活性的花粉达到92.2%以上;同时，在发育时间顺序和空间结构上，羊草的雌蕊、雄蕊并不妨碍自体授粉。因此，初步结论认为羊草具自交不和性。

羊草生殖生物学研究

羊草 (Leymus chinensis (Trin.) Tzvel. ) ，隶属禾本科赖草属，是欧亚大陆草原区东部的重要草原建群种之一。羊草是牧草之王，属于我国有比较优势的战略性生物资源，对我国北方畜牧业发展以及保护生态环境均具有重要作用。 羊草较弱的有性生殖特性限制了其应用，本文从实验生物学角度，研究了羊草有性生殖的基本特点，并试图通过现代分子生物学手段探讨自交不亲和性的有关机理。本论文的主要结果如下： 1. 实验发现羊草具有自交不亲和性。以6 个羊草居群为材料，测定得知开放授粉时的结实率在6.5% - 56.7%之间，自交时结实率为0.6% - 4.3%，差异极其显著; FDA 染色法检测结果显示羊草成熟花药中有活性的花粉达到92.2% 以上;在发育时间顺序和空间结构上，羊草雌蕊、雄蕊适于异花和自花授粉;花粉柱头亲和性实验表明，自交花粉只有5.5%-11.7% 是亲和的，杂交花粉亲和率达到了60.0%-84.8%，说明自交花粉在柱头上萌发受到抑制，其次，荧光显微镜还观察到“不亲和花粉”在进入柱头后生长缓慢，或停止延伸。 2. 初步确定羊草自交不亲和性具有配子体型遗传特点。以不同居群羊草杂交后的姊妹系作为实验材料，观察到自交组合的亲和率变幅为0 % - 6.9 %，杂交组合的亲和率具有连续性变异和变幅较宽的特点（47.5％ - 96.0 %），且正反交结果具有一定的一致性（88.2％），表现出配子体遗传特性。 3. 羊草居群内结实率存在一定变异。以羊草单株为单位分别进行自交、随机互交和开放授粉，结果显示三者的平均结实率分别为4.6%，18.1% 和35.7%，株间的变异系数分别为33.4%，21.2%和17.1%，这些株间的变异均达到统计上的显著差异;同时羊草自交、杂交和开放授粉之间具有一定的相关性，显示羊草的这种株间差异与株系本身的生理特性相关。 4. 分离了羊草硫氧化还原蛋白 H 基因(ThioLc)并对其功能进行了分析。克隆了ThioLc全长和cDNA序列。序列分析结果显示，DNA全长2257 bp，包括3 个内含子和4 个外显子，与水稻Thio h 的cDNA 序列相比，具有 32.0% 的同源性;Southern 杂交显示 ThioLc 在羊草基因组中是单拷贝;Northern 杂交显示 ThioLc 在羊草根、茎、叶和幼小的雌蕊中没有表达, 在成熟雌蕊和幼小的花粉中微量表达, 在成熟花粉中大量表达，说明分离的羊草硫氧化还原蛋白H 基因具有花粉特异表达特点。 5. 原核表达的ThioLc 蛋白具有较高的催化活性。构建了ThioLc 基因的原核表达载体，检测证明ThioLc基因在大肠杆菌中正常表达;提取表达蛋白，纯化，用胰岛素和二硫苏糖醇反应体系进行硫氧化还原蛋白的催化作用反应，结果表明表达的蛋白具有催化活性。这一结果为进一步搜寻靶向蛋白和研究该蛋白的结构、功能和作用方式奠定了基础。

入侵植物紫茎泽兰遗传变异及克隆多样性研究

现存植被与其花粉分布的关系可作为利用化石花粉图谱重建古植被时的参照，因此，理解现存表土花粉分布格局对解释花粉图谱十分关键。提高花粉鉴定精度是孢粉学和古生态学家一直努力想要解决的关键问题。目前的孢粉学方法将表土花粉鉴定到属的水平都十分困难，因此造成依据花粉图谱进行植被重建的时候出现无法矫正的误差。本研究用分子生物学方法将藜科植物表土花粉鉴定精度从科的水平提高到种的水平。表土花粉样品取自新疆中部样带，利用样带内所有出现的藜科植物共19个种建立藜科植物核基因内转录间隔1区（ITS1）序列库，然后通过巢式聚合酶链式反应（PCR）进行单粒表土花粉的ITS1序列扩增和测序，与序列库中的序列进行比对，从而确定单粒花粉来自哪种藜科植物。这种相对简单的以PCR为基础的方法可以将表土花粉鉴定到种，使在种的水平解释植被与花粉分布的关系成为可能。 紫茎泽兰（Eupatorium adenophorum）是广泛分布在中国西南的入侵植物。以无融合生殖为主，其压条性克隆繁殖方式经常被忽视，本文以紫茎泽兰为例，研究无融合生殖和压条性克隆繁殖为主的入侵种群遗传变异与克隆多样性，分析无融合生殖入侵种群具有高度竞争力和适应能力的分子机制。实验用AFLP分子标记检测了包括缅甸一个种群在内的17个种群的基因组多样性，结果表明，紫茎泽兰是多克隆植物，有丰富的基因型数量，遗传多样性水平低（He＝0.0439），大部分（73.59%）遗传变异存在于种群内，种群间存在显著遗传分化。紫茎泽兰克隆生长策略为游击型，倒伏性克隆繁殖有助于小范围内种群的扩张。各种群基因型组成存在很大差异，但基因型间相似性很高，有相当多的基因型可能来自遗传重组。紫茎泽兰的有性繁殖比例可能比以往所推测的高得多，有助于保持基因型的多样性。

Prolonged cardiac allograft survival in presensitized rats after a high activity Yunnan-cobra venom factor therapy

Complement-dependent antibody-mediated acute humoral rejection is the major obstacle of clinical transplantation across ABO incompatibility and human leukocyte antigen presensitization. We previously demonstrated that Yunnan-cobra venom factor (Y-CVF) cou

Increasing the genetic uniformity of bighead carp [Aristichthys nobilis (Richardson)] by means of spontaneous diploidization of gynogenetically activated eggs

Three groups of gynogenetic diploid bighead carp were successfully obtained by means of artificial gynogenesis. The activation rates of gynogenesis varied from 75.9% to 98.8%, and the frequency of spontaneous diploidization was around 0.4%. Over 2000 normally gynogenetic diploid fry were obtained in three gynogenetic groups. The haploid karyotype consisted of nine metacentric, 12 submetacentric, three subtelocentric chromosomes and 45 arms. The chromosome number was 48 from gynogenetic diploid. The results showed that the genetic material of offspring was maternal. The aneuploid hybrid embryos of bighead carp and Xingguo red common carp with chromosome numbers ranging from 28 to 73 did not survive post hatch, likely the result of incompatibility between the nucleus and the cytoplasm of two parents. Sixty RAPD primers from three groups were used for total DNA amplification of gynogenetic offspring, maternal and 'paternal' fish. A total of 451 bands were amplified from three kinds of samples above. From maternal bighead carp, 256 bands were amplified; however, there were 251 shared bands between maternal and gynogenetic bighead carp. From artificial gynogenetic offspring, two 'paternal' DNA segments without an expression function were found. An UPGMA tree showed that gynogenetic offspring were closely clustered and the genetic identity among them was very high (0.956).

Microphase-Separated Brushes on Square Platelets in PS-b-PEO Thin Films

In polystyrene-block-poly(ethylene oxide) thin square platelets can be obtained via fast solvent evaporation by controlling the tethering density (0.08 < sigma < 0.11). The tethering density of the brushes is proportional to the thickness of the PEO crystal and increases with increasing initial solution heating temperature (T-i). When T-i < T-m, where T-m is the melting point of PEO, brushes with microphase-separated structures are observed. The formation of microphase-separated brushes depends on two factors: the strong incompatibility between PS and noncrystalline PEO chains (attached to the crystalline PEO) and the weak interaction between PS-PS brushes.

Hydrogen Bonding and Crystallization in Biodegradable Multiblock Poly(ester urethane) Copolymer

The hydrogen bonding and crystallization of a biodegradable poly(ester urethane) copolymer based on poly(L-lactide) (PLLA) as the soft segment were investigated by FTIR. On slow cooling from melt, the onset and the progress of the crystallization of the urethane hard segments were correlated to the position, width, and relative intensity of the hydrogen-bonded N-H stretching band. The interconversion between the "free" and hydrogen-bonded N-H and C=O groups in the urethane units in the process was also revealed by 2D correlation analysis of the FTIR data. The crystallization of the PLLA soft segments was monitored by the ester C=O stretching and the skeletal vibrations. It was revealed that the PLLA crystallization was restricted by the phase separation and the urethane crystallization, and at cooling rates of 10 degrees C/min or higher, the crystallization of the PLLA soft segments was prohibited.

Monte Carlo simulation on symmetric ABA/AB copolymer blends in confined thin films

The effects of blend composition on morphology, order-disorder transition (ODT), and chain conformation of symmetric ABA/AB copolymer blends confined between two neutral hard walls have been investigated by lattice Monte Carlo simulation. Only lamellar structure is observed in all the simulation morphologies under thermodynamic equilibrium state, which is supported by theoretical prediction. When the composition of AB diblock copolymer (phi) increases, both lamellar spacing and the corresponding ODT temperature increase, which can be attributed to the variation of conformation distribution of the diblock and the triblock copolymer chains. In addition, both diblock and triblock copolymer, chains with bridge conformation extend dramatically in the direction parallel to the surface when the system is in ordered state. Finally, the copolymer chain conformation depends strongly on both the blend composition and the incompatibility parameter chi N.

Surface morphology evolution of poly (styrene-block-4-vinylpyridine) (PS-b-P4VP)(H+) and poly(methyl methacrylate)-dibenzo-18-crown-6-poly(methyl methacrylate) (PMCMA) supramolecular film

Well-ordered nanostructured polymeric supramolecular thin films were fabricated from the supramolecular assembly of poly(styrene-block-4-vinylpyridine) (PS-b-P4VP)(H+) and poly(methyl methacrylate)-dibenzo-18-crown-6-poly(methyl methacrylate) (PMCMA). A depression Of cylindrical nanodomains was formed by the block of P4VP(H+) and PMCMA associates surrounded by PS. The repulsive force aroused from the incompatibility between the block of P4VP(H+) and PMCMA was varied through changing the molecule weight (M-w) of PMCMA, the volume fraction of the block of P4VP(H+), and annealing the film at high temperature. Increasing the repulsive force led to a change of overall morphology from ordered nanoporous to featureless structures. The effects of solvent nature and evaporation rate on the film morphology were also investigated. Further evolution of surface morphologies from nanoporous to featureless to nanoporous structures was observed upon exposure to carbon bisulfide vapors for different treatment periods. The wettability of the film surface was changed from hydrophilicity to hydrophobicity due to the changes of the film surface microscopic composition.

Studies on water-swellable elastomers .2. Thermal properties and crystalline behavior of amphiphilic graft copolymers with poly(ethylene glycol) side chains

The thermal properties and crystalline structure of the amphiphilic graft copolymers CR-g-PEG600, CR-g-PEG2000, and CR-g-PEG6000 using chloroprene rubber (CR) as the hydrophobic backbone and poly(ethylene glycol) (PEG) with different molecular weights as the hydrophilic side chains were studied by DSC and WAXD. The results showed that a distinct phase-separated structure existed in CR-g-PEGs because of the incompatibility between the backbone segments and the side-chain segments. For all the polymers studied, T-m2, which is the melting point of PEG crystalline domains in CR-g-PEG, decreased compared to that of the corresponding pure PEG and varied little with PEG content. For CR-g-PEG600 and CR-g-PEG2000, T-m1, which is the melting point of the CR crystalline domains, increased with increasing PEG content when the PEG content was not high enough, and at constant PEG content, the longer were the PEG side chains the higher was the T-m1. The crystallite size L-011 of CR in CR-g-PEGs increased compared to that of the pure CR and decreased with increasing PEG content. (C) 1997 John Wiley & Sons, Inc.

褐牙鲆和夏牙鲆杂交的遗传学研究

在进行褐牙鲆(Paralichthys olivaceus)和夏牙鲆(P. dentatus)的杂交及回交的基础上，利用染色体计数、AFLP、线粒体DNA和核基因部分序列等分析方法对褐牙鲆和夏牙鲆正反交和回交子代进行遗传学研究，探讨了褐牙鲆和夏牙鲆正反交不对称的遗传学基础及其生殖隔离现象，主要结果如下： 1. 褐牙鲆和夏牙鲆正反交的活力是不对称的，褐牙鲆♀×夏牙鲆♂的正交杂种活力正常，能够正常存活、生长和发育，而反交夏牙鲆♀×褐牙鲆♂的杂种体态畸形，孵出后不久死亡。染色体计数发现正交个体的染色体核型与父母本一致，均为48条端部着丝粒染色体；而反交杂种比亲本缺失了两条染色体，仅为46条端部着丝粒染色体，这表明反交杂种为非整倍体。进一步利用AFLP方法对遗传物质从亲本到子代的传递进行了分析，结果显示正反交遗传物质的传承方式存在很大差异。几乎所有亲本的AFLP位点(97.71%)均传递到正交子代。然而，仅有86.64%的AFLP位点从亲本传递到反交子代，反交子代中亲本位点的丢失比例显著高于正交子代和亲本种内交配子代的比例 ( P < 0.05)，这可能与反交杂种染色体丢失有关。进一步分析发现，杂交组中的偏分离标记高于对照组，尽管经2检验发现其差异并不显著 (P > 0.05)。 2. 对于可以成活的正交杂种进行培育达到性成熟后，利用褐牙鲆和夏牙鲆的精液分别与雌性杂交鲆的卵子进行母本回交实验。通过统计受精率、孵化率及杂交适合度值（CFM，受精率和孵化率相乘获得的结果）评估褐牙鲆和夏牙鲆的杂交可适度，结果表明正交及各回交组中的CFM值均显著低于褐牙鲆自交(P < 0.05)。同时，利用AFLP对回交子代基因组的变化进行了分析，发现回交中不仅存在亲本位点的丢失(褐牙鲆回交子代-回交1, 3.96%; 夏牙鲆回交子代-回交2, 6.03%)的现象，也存在非亲位点(回交1, 5.63%; 回交2, 3.28%)的现象。而且，两回交组合分别有27.40%和31.18%的AFLP标记偏离孟德尔遗传。 3. 利用线粒体DNA 16S rDNA、COⅠ基因及核基因rag1的部分序列对正反交及回交子代的线粒体及核DNA的传承进行分析，发现正反交子代的16S rDNA和线粒体DNA片段的同源性和母本一致，各回交组中16S rDNA和COⅠ基因片段与褐牙鲆的同源性较高 (98%)，这表明褐牙鲆和夏牙鲆杂交及回交遵循母性遗传规律。但在回交子代中发现16S rDNA和COⅠ基因具有多种单倍型。褐牙鲆和夏牙鲆的rag1基因具有高度的保守性，但在正交子代中发现rag1多种单倍型。 4. 进一步利用线粒体DNA的16S rDNA、COⅠ基因的部分序列对8种重要海水养殖鱼类的系统进化分析，计算了其种间的遗传距离。根据这几种鲆鲽鱼的杂交是否可行的试验结果，评价种间遗传距离与杂交可适度的关系，结果表明，这8种鲆鲽鱼类的种间遗传距离与杂交可适度呈显著的负相关 (r2 = 0.805，P < 0.01)，即种间遗传分化越大，杂交成功的可能性越小，这表明鲆鲽鱼类中可能存在物种进化的不亲和钟 (Incompatibility clock)。

LABORATORY HYBRIDIZATION BETWEEN CRASSOSTREA ARIAKENSIS AND C. SIKAMEA

TO understand possible reproductive interaction between Crassostrea ariakensis (Fujita, 1913) and C. sikamea (Amemiya, 1928), which coexist ill estuaries of China and Japan, we conducted 2 X 2 factorial crosses between the two species. Asymmetry in fertilization success was observed where C. sikamea eggs can be fertilized be C. ariakensis the receprocal cross resulted in no fertilization. Fertilization Success ill C.sikamea female X C. ariakemvis male (SA) crosses was lower than that in the two intraspecific crosses and produced larvae that had similar growth the rate as their maternal species during the first nine days because of maternal effects. After that, genome incompatibility casted negative effects on the growth and survival of the hybrid larvae. Most hybrid larvae died during metamorphosis. but a small number of spat survived. Genetic analysis revealed that the survived SA spat contained DNA from both species and were the hybried. This study demonstrates that hybridization between C. ariakensis and C. sikamea is possible in one direction.