枸杞花药发育过程中钙的分布

在枸杞花药发育过程中,用焦锑酸钾沉淀的钙颗粒显示出了一个与花药发育事件有关的分布特征：在孢原细胞时期的花药中钙颗粒很少。在造孢细胞到小孢子母细胞时期,花药中钙颗粒增加。当花粉母细胞进行减数分裂时,花药中的钙颗粒进一步增加,尤其是在小孢子母细胞的胼胝质壁中。在小孢子发育早期,花药药隔部位的绒毡层细胞质中钙颗粒也明显增加并特异性地分布在其内切向壁上。当小孢子被释放出后,钙颗粒开始特异性积累在正在形成的花粉外壁中,尤其在萌发孔的部位聚集了大量的钙颗粒。当小孢子形成大液泡时,其细胞质中的钙颗粒明显减少。在小孢子分裂形成二胞花粉后,在二胞花粉的大液泡中又特异性地出现许多细小钙颗粒。随着二胞花粉的大液泡完全消失,其细胞质中又出现了许多钙颗粒。接近开花时的成熟花粉粒细胞质中,细小的钙颗粒主要分布在营养细胞和生殖细胞中。枸杞花药发育过程中钙的分布特征反映了其参与调控花粉发育过程。     

水芹花药发育中多糖和脂滴积累规律研究

运用组织学和组织化学方法详细研究了水芹(Oenanthe javanica D.C) 花药发育过程中,造孢细胞时期、小孢子母细胞时期、四分体时期、小孢子早期、小孢子晚期、二胞花粉早期和二胞花粉晚期(成熟花粉时期)的多糖以及脂滴分布特征和变化特点.结果显示:花药作为营养物质吸收的"库",在特定的时间(二胞花粉早期)和特定的部位(二胞花粉的营养细胞)积累特定的营养物质(先出现多糖颗粒,后出现脂滴物质).通过分析不同发育阶段花药中糖类和脂类分布变化,确定水芹发育花药中营养物质的运输与转换规律.     

TA29—Barnase嵌合基因导入对烟草花药绒毡层及花粉发育的影响

比较研究了烟草(Nicotiana tabacum L.)TA29-Barnase转基因不育植株和正常植株的花药绒毡层及花粉发育的全过程。研究表明,外源基因在花药中特异表达导致绒毡层细胞的提前降解,这种降解一般在减数分裂早期开始,至四分体时期完成,而正常花药绒毡层的降解发生在二细胞雄配子体初期,至花粉发育的后期方才完成。转基因植株花药绒毡层的降解在细胞结构上表现为:最初发生细胞的液泡化,然后细胞核凝聚,最后整个细胞溃解。转基因植株的花粉母细胞则在减数分裂过程中逐渐降解、退化,只有少数花粉母细胞能够顺利完成减数分裂发育成小孢子。观察结果还表明外源基因在花药中的表达是不均一的。对转基因不育和自然败育在细胞结构上的不同表现进行了讨论。     

金线莲花药发育中的钙离子分布特征

该研究以金线莲不同发育时期的花药为材料,采用电子显微镜观察花粉块中的钙离子分布,以揭示钙离子在金线莲花药发育中的相关生理功能。结果发现:(1)在造孢细胞时期,较多的钙沉淀颗粒出现在花药表皮和药室内壁细胞的液泡中,暗示钙离子与植物细胞的液泡发生和形成有关。(2)在减数分裂前期,小孢子母细胞核中聚集了较多的钙沉淀颗粒,当小孢子母细胞分裂时,在二组染色体之间有大量的钙沉淀颗粒,显示钙离子与细胞分裂有关。(3)在合成淀粉的质体表面覆盖了较多的钙沉淀颗粒,显示钙离子与质体中的糖代谢有关。研究表明,开花时在花粉块表面的花粉外壁上和成熟花粉中仍保持有大量的钙沉淀颗粒,为花粉萌发所需钙离子做好了储备。     

小麦不同发育时期花药对离体培养的反应

花药的发育时期是决定花药培养时诱导频率高低的重要因素。据文献报道,绝大多数植物只在花药中小孢子处于单核期至二核期时才对离体培养有反应(即产生愈伤组织),只有少数植物能从四分体时期的花药获得花粉胚,如烟草、毛地黄、油菜、大麦、野生二粒小麦、玉米     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加, 同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少,伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后,小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡,体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大波泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

四环素诱导组织特异性表达系统的构建

如果获得一种可以特异调控基因表达的系统,对于研究某一基因在各个不同发育时期的功能会有很大的帮助.为了控制外源基因在特定的时间和组织内在转基因植物中表达,将四环素诱导元件和花药绒毡层特异表达启动子TA29结合,构建了一个新的系统.通过基因枪将该系统转入烟草(Nicotiana tabacum L. cv.Wisconsin 38)组织,GUS瞬时表达结果表明,四环素诱导前,没有任何烟草组织染上蓝色,表明该系统不表达;而四环素诱导仅30min后,花药就染上明显蓝色,而且集中在花药绒毡层.其对照子房在四环素诱导前和诱导后均未染上蓝色.这些结果表明,该系统既是四环素诱导又是花药绒毡层特异表达的.     

枸杞Lb14-3-3b基因的表达分析及转烟草研究

在宁夏枸杞(Lycium barbarum L.)品种宁杞1号花药发育差异蛋白质组学研究的基础上,克隆了一个花药发育相关基因Lb14-3-3b,证实该基因在花器官中优势表达。本试验进一步利用荧光定量PCR技术分析枸杞Lb14-3-3b基因在花药发育过程中不同时期的表达特征,并通过构建植物过表达载体Lb14-3-3b-pCambia1305. 1-35s,经根瘤农杆菌介导法转化模式植物烟草,探究该基因在植物生长发育中的功能。结果表明,在枸杞花药发育的各个时期,Lb14-3-3b都有表达,在花药二核花粉时期表达量最高。与野生型烟草相比,转Lb14-3-3b基因烟草生长发育迟缓,植株矮小,花器官畸变,花瓣缺少致使花型趋于四角化,雄蕊及花丝数目缺少且发育异常。推测枸杞Lb14-3-3b基因在烟草生长发育及花器官发育中起调控作用,研究结果为进一步探讨该基因在枸杞生长发育中的调控作用提供参考依据。     

胡萝卜花药发育的组织化学研究

胡萝卜四分体时期的花药在药室内壁和绒毡层细胞中积累淀粉粒,随着花药的发育,花粉先出现大液泡,同时药室内壁和绒毡层细胞中淀粉粒消失;以后花粉中的大液泡消失,在花粉细胞质中出现淀粉粒。伴随着花粉的发育,绒毡层细胞退化,在细胞中积累较多的脂类物质,同时花粉中脂类物质含量也明显增加。胡萝卜成熟花粉粒的储存物主要为脂滴,也有少部分淀粉颗粒。胡萝卜花药在特定时间和特定部位积累营养储存物的过程也是其发育的一个特征。     

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.     

水稻花药发育过程中腺苷三磷酸酶的分布

水稻花粉母细胞中的ATP酶反应颗粒很少,主要分布在细胞核中。组成花药药壁的4层细胞中只有绒毡层细胞核中有较多的ATP酶。减数分裂后,绒毡层细胞质中分化出许多内质网片层,但ATP酶反应颗粒仍很少,其它3层药壁细胞中质膜ATP酶明显增加。在花粉内、外壁中形成了大量的ATP酶反应颗粒,但花粉外壁在小孢子时期形成,ATP酶反应颗粒来自绒毡层细胞的鸟氏体。花粉内壁在二胞花粉时期形成,其中的ATP酶反应颗粒来自花粉营养细胞。二胞花粉的营养细胞比生殖细胞含有更多的ATP酶反应颗粒。     

白菜核雄性不育系可育和不育花药中Ca2+的分布

研究了白菜(Brassica campestris L. ssp.chinensis Makino)细胞核雄性不育系花药中Ca2 的分布特征.在可育花药发育过程中,减数分裂后花药壁细胞中钙颗粒明显增加.早期小孢子开始积累钙颗粒并特异性地附在小液泡膜上.小孢子分裂后,大液泡消失过程中又伴随着许多钙颗粒附在小液泡膜上,显示出Ca2 与花粉中液泡的形成和分解有关.在不育花药中,最早出现的钙颗粒异常分布是在小孢子母细胞的胼胝质壁中积累了较多的钙颗粒.然而,在小孢子细胞质中钙颗粒一直很少,也不形成大液泡,最后通过细胞质收缩的方式败育.这是首次发现Ca2 参与调控花药发育过程,其异常分布与花粉败育密切相关.     

维生素A酸受体γ基因表达及其对ES细胞分化和凋亡的影响

We have constructed pSG5-RAR gamma-neo plasmid containing mouse retinoic acid receptor gamma (RAR gamma) gene and neo gene, and introduced it into embryonic stem ES-5 cells, by calcium phosphate mediated transfection. Some G418-resistant clones were isolated and from RNA dot blot analysis of these clones, a clone overexpressing RAR gamma gene was established, designated as ES-gamma cell line. Northern blot hydridization and Southern blot hydridization analysis of ES-gamma cells (Fig 3, 4) demonstrated that ES-gamma cells overexpressed exogenous RAR gamma mRNA and the exogenous RAR gamma cDNA integrated into the genome of ES cells. ES-gamma cells retained undifferentiated morphology and positive alkaline phosphatase activity (Plate I, Fig. 1, 2), so it resembled ES-5 cells in terms of stem cell characteristics. When ES-gamma cells were subcutaneously inoculated into nude mouse and differentiated in vivo, tumorous nodules containing various tissue structures were obtained, demonstrating their pluripotent properties just like parent ES-5 cells. Contrasting with ES-5 cells, the histological features of tumors showed no cartilage tissues, but abundant muscle tissues and keratinized cyst like structures constituted by stratified squamous epithelia (Plate I, Fig. 3). Differentiating in vitro by hanging drop culture methods, ES-gamma cells differentiated mostly into fibroblast-like cells, (Plate II, Fig. 1-5). The above results indicated that overexpression of RAR gamma gene changed the cell type of ES cells differentiating in vivo and in vitro. During the differentiation of ES-5 cells induced by RA, a large number of cells rounded up, detached from the dish and tended to die. We suspected that this phenomenon may be apoptosis. The ultrastructure appearance of the dying cells displayed typical apoptotic changes including chromatin condensation and nuclear fragmentation (Plate I, Fig. 4, 5). Detection of DNA fragments using agarose gel electrophoresis showed characteristic laddered patterns of apoptotic DNA fragments (Fig. 5). The above results indicated that RA induced apoptosis of ES-5 cells in the course of differentiation. The percentage of apoptosis of ES-5 cells increased accordingly, with the increase of RA concentration (Fig. 6). With the same concentration of RA 10(-7) mol/L, the percentage of apoptotic of ES-gamma death was roughly one times more than that of ES-5 cells (Fig. 7), a fact indicating that RAR gamma may mediate the apoptotic signal transduction of ES cells by RA.     

Chronological appearance of spontaneous and induced apoptosis during preimplantation development of rabbit and mouse embryos

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope. Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.     

Differences on the microsporogenesis and tapetal development of male fertile and cytoplasmic male sterile pepper (Capsicum annuum L.)

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line pepper were studied using transmission electron microscopy. The results showed that CMS line anthers appeared to have much greater variability in developmental pattern than male fertile maintainer line ones. The earliest deviation from normal anther development occurred in CMS line anthers at prophase I was cytomixis in some microspore mother cells (MMCs), and vacuolisation in tapetal cells. Then, MMCs in CMS line anthers developed asynchronously and a small part of ones at the different stage degenerated in advance appearing to have typical morphological features of programmed cell death (PCD). Most MMCs could complete the meiosis, but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei, and some degenerated ahead of time as well. Tapetal cells in CMS line anther degenerated during meiosis, and were crushed at the tetrad stage, which paralleled the collapse of pollens. Pollen abortion in CMS line anthers happened by PCD themselves, and the premature PCD of tapetal cells were closely associated with male sterility.     

烟草花发育过程中花药和花粉IAA分布规律的研究

以烟草（Nicotiana tabacumL.）花药为材料,通过4’,6-二脒基-2-苯基吲哚（DAPI）染色详细观察花粉发育过程,获得了花药发育时期与花蕾大小的对应关系;通过吲哚乙酸（IAA）单克隆抗体、免疫组织化学技术以及DR5∶∶GUS转基因植株的GUS活性对花药和花粉发育过程中生长素的分布规律进行了研究。免疫酶标记结果表明,在不同的花药发育时期IAA水平呈现出明显的差别。小孢子母细胞时期,IAA在整个花药中均有分布,并且在小孢子母细胞发育晚期,IAA信号集中在小孢子母细胞的细胞核中;随着小孢子母细胞减数分裂后形成四分体,IAA信号逐渐减弱,四分体中几乎没有信号;单核花粉期的花药中IAA信号进一步减弱,仅存在于花药壁中;待小孢子继续发育为成熟二核期时,花粉和整个花药组织中均出现较强的IAA信号。GUS活性检测结果表明,烟草DR5∶∶GUS转基因植株中花药和花粉粒的GUS信号与IAA免疫酶定位结果基本一致。总的来说,IAA在烟草花药和花粉中的积累呈现出由强到弱、再由弱到强的分布规律,暗示IAA在被子植物花药和花粉发育过程中可能起着较为重要的作用。     

Programmed cell death in Xenopus laevis spinal cord, tail and other tissues, prior to, and during, metamorphosis

Programmed cell death is necessary for the shaping and remodelling of nervous and non-nervous tissues during development. Amphibia, whose body undergoes profound modifications during metamorphosis, are particularly useful models for studying the relationship between cell death in muscles and other non-nervous tissues on the one hand, and in the nervous system connected with these tissues on the other hand. We checked the occurrence of apoptotic cells (identified by TUNEL labelling) in different organs and regions from hatching (stages 35-36) to climax (stages 63-64) in the African Clawed Frog Xenopus laevis. Some organs (e.g., skin and digestive tract) contained apoptotic cells during the entire period studied. In transitory organs (cement gland and gills), a single wave of cell death occurred during the regression of these tissues. In order to compare the timing of cell death in the spinal cord with that of tail regression, we counted the number of TUNEL-positive cells in spinal cord sections taken from animals between stages 54 and 64. Three-dimensional reconstructions using confocal microscopy of vibratome slices immunostained for the detection of c-Jun-like protein accumulated in the cytoplasm of apoptotic cells showed numerous cells at various degrees of degeneration. Many of these cells still presented the morphological characteristics of neurones. The peak of apoptosis was found at stage 58, preceding tail regression. This suggests that neural cell death is not a consequence but rather an element upstream in the chain of events leading to tail degeneration.