水稻受精前后胚囊内钙调素分布的变化：免疫金电镜观察

用胶体金免疫电镜技术观察了水稻 (Oryzasativasubsp .japonica)受精前后胚囊内钙调素的分布变化。授粉后 ,卵细胞、助细胞和中央细胞内的钙调素较授粉前均有所增加。中央细胞内钙调素的增加要比卵细胞中约早 2h ,退化助细胞与宿存助细胞之间的钙调素含量无明显差异。授粉到受精期间 ,钙调素的主要分布形式由分散的单颗粒转变为聚集颗粒 ,受精完成后再变为分散的单颗粒形式。胚囊壁及珠心细胞的细胞壁和胞间隙中也观察到钙调素的分布和数量变化。初步讨论了胞内和胞外钙调素在水稻受精与合子形成中的作用。     

受精前后烟草卵细胞内玉米素和三类酸性植物激素分布的免疫金电镜观察

用胶体免疫金电镜技术观察了受精前后烟草（Nicotianatabacumvarmacrophylla）卵细胞内玉米素（t－Z）、GA7与GA4、（＋）ABA与IAA分布的变化。受精前卵细胞内有大量t－Z,主要位于细胞核、内质网与线粒体上;与卵细胞相邻的助细胞合点端及中央细胞珠孔端亦有较多t-Z。受精后,合子与宿存助细胞t－z显著减少,正加厚的合子细胞壁中有t－Z分布。未受精卵细胞内GA7与GA4及（＋）ABA减少,IAA更少。合子内GA7与GA4及（＋）ABA略有增加,IAA仍然很少。初步讨论了上述植物激素与受精作用的关系。     

烟草受精前后胚囊中钙调素的免疫细胞化学定位

应用辣根过氧化物酶标记抗体的光镜免疫细胞化学技术和胶体金标记抗体的电镜免疫细胞化学技术定位烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍｖａｒ．ｍａｃｒｏｐｈｙｌａ）胚囊成员细胞中的钙调素,比较了不同类型细胞之间钙调素的分布特点,并探讨了受精前后钙调素在胚囊中分布的变化规律。     

受精前后烟草卵细胞内玉米素和三类酸性植物激素分布的免疫金电？…

用胶体免疫金电镜技术观察了受精前后烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ ｖｅｒ．ｍａｒｏｐｈｙｌｌａ）卵细胞内玉米素（ｔ－Ｚ）、ＧＡ７与ＧＡ４、（＋）ＡＢＡ与ＩＡＡ分布的变化。受精前卵细胞内有大量ｔ－Ｚ,主要位于细胞核、内质网与线粒体上;与卵细胞相邻的助细胞合点端及中央细胞珠孔端亦有较多ｔ－Ｚ。受精后,例子与宿存助细胞ｔ－Ｚ显著减少,正加厚的合子细胞壁中ｔ－Ｚ分布。未受精卵细胞内ＧＡ７与ＧＡ４     

蚕豆保卫细胞中钙调素的免疫电镜定位

以蚕豆横切和平切气孔为材料,对钙调素进行了免疫胶体金电镜定位的结果表明:在蚕豆保卫细胞的细胞核、细胞质、细胞膜、叶绿体、液泡、高尔基体、细胞壁中都有金颗粒分布,在线粒体上的分布较少.     

钙调素mRNA在受精前后分离的烟草胚囊中的定位

建立了一种新的ｍＲＮＡ原位杂交方法,适用于微量材料的整体观察。应用这一方法定位烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．ｃｖ．Ｗ３８）受精前后胚囊成员细胞中的钙调素ｍＲＮＡ（ＣａＭｍＲＮＡ）。结果显示成熟胚囊中的ＣａＭｍＲＮＡ主要分布于珠孔极的卵器和合点极的反足细胞;中央细胞中较少。受精前后胚囊中ＣａＭｍＲＮＡ的分布发生显著变化,特别是授粉后到受精前极核与卵器之间出现一条暂时的钙调素ｍＲＮＡ条带。受精前不久该带消失,ＣａＭｍＲＮＡ扩展为占据胚囊珠孔端的扇形区域。受精后胚囊中钙调素ｍＲＮＡ主要集中于伸长的合子和原胚的合点端。讨论了钙调素ｍＲＮＡ表达与受精的关系。     

一种可用于分析水稻花药中钙调素蛋白分布的胶体金免疫电镜标记技术

以水稻花药作为实验材料,在前人应用胶体金技术标记钙调素蛋白(Ca M)的基础上,采用Epon812常规包埋方法,并在标记过程及染色体系上做了一些改进,建立了一套标记特异性较强且超微结构保存较好的花药中Ca M的标记方法。     

水稻幼穗中钙调素的免疫金银法定位（简报）

在水稻二枝梗及颖花原基分化期、雌雄蕊原基分化期的纵切面上,GaM分别以二次枝梗、颖片和雌雄蕊中分布最多。在横切面上,CaM又以维管束四周的分布最多。这显示水稻幼穗中CaM的分布与该组织当时的生长发育速率相一致     

多胚水稻品系APⅣ不同类型胚囊的受精及其胚胎形成

通过ＧＭＡ半薄切片技术对ＡＰⅣ不同类型水稻胚囊的受精及其胚胎发育的研究表明,ＡＰⅣ中５－２－１型胚囊的３个卵细胞在少数情况下都可受精并发育形成３个胚;但多数情况中只有１个或２个卵细胞受精发育成１个胚或２个胚。６－２－０型和５－３－０型胚囊多个卵受精频率都很低。由此证明ＡＰⅣ多胚是来自如５－２－１型胚囊的多卵卵器胚囊多个卵细胞都受精的结果,其中３胚来自３个卵细胞受精发育,２胚来自２个卵细胞受精发育。     

水稻雌配子体发生过程中胚胚囊内微管骨架变化的进一步研究

用PEG包埋切片法及荧光抗体标记技术对水稻（Oryza sativaL.)雌配子体发生过程中微管骨架的变化进一步研究。经PEG包埋切片技术处理的胚囊内的微管结构能够保持得比较完整,特别是在一个较大和成熟的胚的胚囊内,效果更佳,微管清晰度高,对雌配子体发生过程中的一些主要时期的微管结构变化作了详细描述和分析（包括：单核、二核、四核、八核和成熟胚囊时期）。发现了一些新的微管结构,如在中央细胞中有纵向微管,这些微管在两个极核移至中央部位时存在,之后当极核移至靠近卵细胞时便消失,显示中央细胞纵向微管与极核的移动和定位可能有关。     

Structure of Embryo Sac Before and After Fertilization and Distribution of Transfer Cells in Ovules of Green Gram

The structure of embryo sac before and after fertilization, embryo and endosperm development and transfer cell distribution in Phaseolus radiatus were investigated using light and transmission electron microscopy. The synergids with distinct filiform apparatus have a chalazal vacuole, numerous mitochondria and ribosomes. A cell wall exists only around the micropylar half of the synergids. The egg cell has a chalazally located nucleus, a large micropylar vacuole and several small vacuoles. Mitochondria and plasrids with starch grains are abundant. No cell wall is present at its chalazal end. There are no plasma membranes between the egg and central cell in several places. The zygote has a complete cell wall, abundant mitochondria and plastids containing starch grains. Both degenerated and persistent synergids migh.t serve as a nutrient supplement to proembryo. The wall ingrowths occur in the central cell, basal cell, inner integumentary cells, suspensor cells and endosperm cells. These transfer cells may contribute to embryo nutrition at different developmental stages of embryo.     

Ultracytochemical Localization of Calcium in Micropyle and Embryo Sac of Brassica napus Before and After Pollination

Potassiam antimonate was used to localize Ca2+ in the micropyle and embryo sac of Brassica napus L. before and after pollination. To identify the nature of the pyroantimonate deposits, energy-dispersive X-ray microanalysis (EDXA) was employed and the deposits were proved to contain calcium pyroantimonate. Image processing system was employed to measure the volume density and the diameter of the deposits. Before and after pollination, calcium was more abundant in the exostome and endostome as compared with the other regions of the integuments, and was concentrated at the apoplast system, i.e. the intercellular matrix of the micropyle canal and the cell wall. Before pollination, each of the two sister synergids accumulated more calcium than the other embryo sac cells. Although the mean diameter of the deposits in the synergid was only two-thirds as that in the egg cell and central cell, the volume density of the deposits in the synergid was about 2.5 times and 1.9 times as that in the egg cell and the central cell respectively. The filiform apparatus and the nucleus had the most abundant calcium within a synergid. After pollination both sister synergids degenerated conspicuously and were characterized by much more deposited calcium (about 2.4 times more than before); and the diameter of the deposits decreased dramatically, which was less than one-third as before. The relationship between calcium distribution and synergid degeneration as well as its functions was discussed.     

Distribution of Trans zeatin,GA7/4, (+)ABA and IAA in Tobacco Egg Cells Before and After Fertilization: Immunogold Electron Microscopic Observations

Changes in distribution of trans-zeatin (t-Z), gibberellin A7 and A4(GA7/4), ( + )abscisic acid [( + )ABA] and indoleacetic acid (IAA) in the egg cells of Nicotiana tabacum var. macrophylla before and after fertilization were studied with immunoelectron microscopy. The ovules just at pollination or 96 h after pollination were fixed with 2% EDC [ 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] and then with the mixed paraformaldehyde and glutaraldehyde for acidic phytohormones (or only with the aldehydes for t-Z), then slightly posffixed in 0.5% OsO4 solution for 30 min. After etched in 1% H2O2 for 10 min, the ultrathin sections embedded in Epon 812 resin were immunostained with rabbit anti-t-Z (and t-ZR) polyclonal antibody (PAB), anti-lAA methyl ester PAb, mouse anti-GA7 and GA4 methyl esters monoclonal antibody(MAb), or anti-( + ) ABA methyl ester MAb, respectively. Protein A- or sheep anti-mouse IgG-colloidal gold (Φ 10 nm) were used to indicate rabbit PAbs or mouse MAbs respectively. In the model system of nitrocellulose membrane via immunogold-silver enhancement, the authors ascertained that immunostaining results at the basis of 1 ng per phytohonnone (t-Z, IAA, GA4, or ( + )ABA) were comparable among the four-kind phytohormones and that t-Z riboside was far less fixed than t-Z with aldehydes. So the anti-t-Z PAb mainly recognized t-Z in aldehyde-fixed tissues. Immunogold electron microscopic observations showed that t-Z was rich in the egg cells before fertilization. In contrast the amounts of GA7/4 and ( + )ABA were lower in egg cells before fertilization but slightly increased after fertilization. Less IAA in egg cells was found either before or after fertilization, t-Z in unfertilized egg cells appeared to concentrate on the nucleus, endoplasmic reticulnm and mitochondria, t-Z is rarely observed in the nuclei of synergids before fertilization but is abundant in the chalazal end of synergids and micropylar end of the central cell adjacent to the unfertilized egg cell. After fertilization, t-Z decreased bviously in the zygotes and the persistent synergids, but appeared in the thickened walls of the zygotes.     

利用激光扫描共聚焦显微术对同源四倍体水稻胚囊形成与发育的进一步观察

应用改进的整体染色透明激光扫描共聚焦显微术(WCLSM),对同源四倍体水稻PDER-2B-4x胚囊的形成与发育过程进行观察。发现其胚囊的形成发育过程与二倍体的一致,可以清楚地划分为8个发育时期,即孢原细胞形成期、大孢子母细胞形成期、大孢子母细胞减数分裂期、功能大孢子形成期、单核胚囊形成期、胚囊有丝分裂期、八核胚囊发育期和成熟胚囊期。除正常发育的过程外,大孢子发育的各个过程均出现一些异常现象,包括:细胞退化、核位置异常、核数目异常和细胞分化异常等。这些异常可能最终导致多种结构异常成熟胚囊的形成。     

人氟斑牙釉质表面结构及漂白后改变的电镜观察

目的:目前国内外对氟斑牙的研究主要集中在流行病学调查和临床疗效分析,对其漂白后釉质表面脱矿程度的研究却鲜有报道.观察不同程度人氟斑牙电镜下微观结构的特点,了解冷光美白对不同程度人氟斑牙釉质表面的影响,探讨冷光美白对重度氟斑牙的安全性.方法:选择因牙周病拔除的完整无龋坏的正常牙10颗,人各型氟斑牙50颗,选取颊侧典型区域制备标本.氟斑牙标本按Dean氏分类法分为轻、中、重度3组,正常牙标本作为对照组.各组标本随机分为4个亚组,A组:不做任何处理;B组:釉质表面蚀刻;C组:釉质剖面蚀刻;D组:釉质表面冷光美白,扫描电镜下观察标本的表面形貌.结果:轻度氟斑牙釉柱间隙增宽,少量晶体排列紊乱,晶间隙增宽.重度氟斑牙釉质表面凹凸不平,呈弹坑状或蜂窝状,釉柱轮廓不清,晶体排列紊乱甚至消失.中度氟斑牙居于二者之间.冷光美白后,正常牙和氟斑牙釉质表面低倍镜下均未见明显改变,高倍镜下散在浅碟状凹坑;中重度氟斑牙还伴有大量粟粒状小孔.结论:随着氟斑牙严重程度的加重,釉质表层损害程度加重.冷光美白造成釉质表层的轻微脱矿,氟斑牙稍重于正常牙.从结构和漂白后釉质表面脱矿程度方面进一步探讨氟斑牙的表面结构特点,从而为氟斑牙的临床治疗提供基础数据.     

Microarray Analysis Reveals Similarities and Variations in Genetic Programs Controlling Pollination/Fertilization and Stress Responses in Rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

Previously, we identified 253 cDNAs that are regulated by pollination/fertilization in rice by using a 10K cDNA microarray. In addition, many of them also appeared to be involved in drought and wounding responses. To investigate this relationship, we obtained their expression profiles after dehydration and wounding treatments in this study. Venn diagram analysis indicated that 53.8% (136/253) and 21% (57/253) of the pollination/fertilization-related genes are indeed regulated by dehydration and wounding, respectively, and nearly half of the genes expressed preferentially in unpollinated pistils (UP) are responsive to dehydration. These results indicated that an extensive gene set is shared among these responses, suggesting that the genetic programs regulating them are likely related. Among them, the genetic network of water stress control may be a key player in pollination and fertilization. Additionally, 39.5% (100/253) cDNAs that are related to pollination/fertilization appear not to be regulated by the stress treatments (dehydration and wounding), suggesting that the existence of additional genetic networks are involved in pollination/fertilization. Furthermore, comparative analysis of the expression profiles of the 253 cDNAs under 18 different conditions (various tissues, treatments and developmental status) revealed that the genetic networks regulating photosynthesis, starch metabolisms, GA- and defense-responses are involved in pollination and fertilization. Taken together, these results provided some clues to elucidate the molecular mechanisms of pollination and fertilization in rice. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s11103-005-3958-4     

Monitoring of Gene Expression Profiles and Isolation of Candidate Genes Involved in Pollination and Fertilization in Rice (Oryza Sativa L.) with a 10K cDNA Microarray

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.     

人工授精平角涡虫早期胚胎和幼虫的扫描电镜观察

为探明平角涡虫(Planocera reticulata)胚胎发育规律,采用人工授精方法,获得不同发育阶段的无卵外胶膜胚胎,并运用扫描电镜技术,观察了受精卵早期胚胎发育和幼虫发育.结果表明,从第3次卵裂开始表现出螺旋式卵裂的特征.在囊胚时期和原肠时期在动物极顶端有几个卵裂球向内下陷形成一凹陷.浮游幼虫期的幼虫利用体表纤...