花生种子老化相关蛋白质的初步研究

本文以粤油 116花生(Arachis hypogaea L.)为材料,对不同处理种子的除子叶“种胚”(以下简称“种胚”)的蛋白质进行了研究.实验结果表明,当花生种子活力下降到一定程度时,其“种胚”内出现一种新蛋白质( pI6.2、MW 10 KD),随种子老化程度加深,含量逐渐增多.我们认为该蛋白质与花生种子老化存在着一定的相关关系,可作为该种子老化的标志.     

顽拗性黄皮胚脱水过程中核酸、蛋白质代谢的变化

黄皮种子发育晚期,胚内核酸、蛋白质合成能力增强,而花生胚的核酸、蛋白质合成能力在发育晚期则呈下降趋势。黄皮胚的发育在达到生理成熟后维持着活跃的生理代谢并转入萌发状态;而花生胚的代谢活性逐步降低并转入生理静止状态。脱水处理引起生理成熟期黄皮胚核酸、蛋白质合成能力急剧下降,核酸水解酶活性增强。不同程度脱水的黄皮胚吸胀２４ｈ,核酸、蛋白质的合成能力随脱水程度的加深而降低;生物大分子代谢能力的变化是顽拗性     

不同成熟度花生胚萌发时子叶中贮藏蛋白质的降解

花生(Arachis hypogaea L.)汕油71果针入土20d(20 DAP)的种子剥去种皮后,10％的胚可以萌发,至40 DAP发芽率达98％。不同发育时期的花生胚萌发 10d后子叶盐溶蛋白质和花生球蛋白降解表明,20和32 DAP胚萌发后,子叶中这些蛋白质只有部分降解。随着胚成熟度增加,子叶中降解这些蛋白质的能力不断提高。20～40 DAP胚萌发4d时,子叶的BAPAase和GHE活性较低。50～80DAP胚萌发 4d,子叶中上述两种酶均显示较高的活性。     

花生种子萌发早期胚轴DNA合成与种子活力的关系

随着花生种子萌发率和活力指数的下降,胚轴DNA开始合成的时间推迟,其合成水平也降低。但DNA合成都是先于吸胀的12h(高活力胚)或18h(低活力胚)出现一个峰,然后再持续上升。腐胺预处理明显地促进老化胚轴萌发早期(12～2dh)的DNA合成。钙离子预处理则有一定抑制作用,但两种预处理均能提高种子的活力指数,并能促进吸胀30h以后的DNA合成。     

种子萌发内在条件的探究

种子的萌发需要一定的内在条件和环境条件。根据北师大版生物学教材7年级上册内容,种子萌发的内在条件之一是具有完整的胚。对双子叶植物花生种子缺少部分子叶的不完整胚进行了萌发探究,结果表明花生种子缺少部分子叶的不完整胚仍可以萌发。     

脱落酸对发育中花生胚萌发和贮藏蛋白质合成的影响

发育中的花生胚在无激素固体培养基上高体培养时提前萌发,其发芽力随胚的成熟增加而提高。果针入土后40d胚的发芽率达100％。禹体培养过程中,外源ABA能够阻止花生胚提前萌发和促进胚的发育。胚成熟前期,较低浓度的ABA(10~(-5)mol/L)便抑制胚的萌发;而在成熟中期以后,则要求较高浓度的ABA(10~(-4)mol/L)才能抑制胚的萌发。ABA对成熟前期胚的贮藏蛋白质合成无影响,而对成熟中期至后期胚的贮藏蛋白质合成起促进作用。ABA维持花生胚贮藏蛋白质合成和积累的作用表现在转录水平上。     

在不同气体贮藏下花生种子活力的研究

花生种子(含水量6.21%)在38—40℃下进行人工老化贮藏,N_1和 CO_2空气有延缓种子老化的效果。种子在 N_2或 CO_2气体下老化贮藏26周,发芽率不受影响;而在空气中贮藏的种子,其发芽率几乎丧失。用公式(胚根+下胚轴)mm×发芽率(第5天)来测定花生种子活力。它能够既简单又准确地反映种子老化时品质的变化。花生种子活力与呼吸及幼苗生长有较好的正相关,与浸泡液的电导和糖含量成显著负相关。     

低能钒离子注入花生种子的深度分布

采用鼎瑟福背散射（RBS）和X射线能谱分析法（EDAX）对V元素在花生种子中的深度分布进行了测量,并用扫描电镜对注入前后花生种胚的形貌变化进行观察。结果表明,由于样品表面较粗糙以及其特殊结构,RBS方法不适于测量钒在花生肿胚中的深度分布,trim95也不适合于对注入钒离子在花生种子中的深度进行模拟,而EDAX的测量结果表明V离子的穿透深度可达到15μm。另外,注入前后花生种胚的形貌发生了显著变化。     

短柄五加种子结构、后熟作用及其细胞化学研究

短柄五加Eleutherococcus brachypus Harms种子为扁肾形,种皮由一层细胞构成。种子脱落时,胚 为心形胚期,胚周围的胚乳细胞解体形成液样囊腔,并包裹胚,胚细胞中存在较多蛋白质,胚乳细胞贮 存大量蛋白质颗粒和脂类,但两者均未见多糖颗粒。有萌发潜能的种子只占全部种子的9.27％。在试 验地种植条件下饱满种子经18～19个月后萌发,出苗率为1．67％,该过程细胞化学特点是胚细胞中蛋 白质含量逐渐减少,并逐渐积累少量多糖颗粒。种子经变温层积处理6个月即可完成后熟过程,其细胞 化学特点是胚细胞中蛋白质含量逐渐减少,但在心形胚后期即已积累大量多糖颗粒,并一直保持至胚 完全发育成熟。经比较短柄五加与刺五加的种子结构、后熟作用及其细胞化学特点,认为短柄五加种子质量差、自然状态下后熟时间长和出苗率低是短柄五加致濒的重要生殖因素,并提出了相应保护方法。     

成熟脱水对种子发育和萌发的作用

成熟脱水是正常性种子发育的末端事件。种子在成熟时胚的脱水耐性增加;当种子萌发时胚变得不耐脱水。当种子获得脱水耐性时,糖、蛋白质和抗氧化防御系统等保护性物质积累;当脱水耐性丧失时,这些物质被降解。成熟脱水是种子从发育过程向萌发过程转变的“开关”,它降低发育的蛋白质和mRNA的合成,终止发育事件和促进萌发事件。顽拗性种子不经历成熟脱水的发育阶段,对脱水高度敏感。     

Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos

Mouse embryos which were hatched from the zona pellucida in vitro in the presence of mouse hepatitis virus (MHV) or outgrown on coverslips and then exposed to MHV were shown by immunohistochemical staining to have virally infected trophoblast cells. Zona-intact embryos incubated with MHV for 48 h (2-cell embryos) or 1.5 h (blastocysts) were resistant to infection. Morulae and early blastocysts collected from donor mice experimentally infected with MHV were not infected, but the medium in which they were flushed from the uterine horns was contaminated with virus. No virus was detected after embryos were washed through three changes of uncontaminated medium. MHV was transmitted to foster mothers when embryos were transferred in medium contaminated with the virus. Fetal and decidual tissues were not infected. We suggest that embryo transfer is an effective and simple alternative to Caesarian rederivation of MHV-contaminated mice.     

A 30-HZ Pulsed Magnetic Field Can Stop Early Embryonic Development

Chick embryos were exposed in vivo to a 30-Hz magnetic field (MF); the frequency was chosen to produce near-resonance conditions for calcium and sodium ions in the local geomagnetic field. After a 48-hour incubation and exposure to the MF, a morphological analysis of the embryos was performed and they were classified as normal or not normal. The not normals were developed embryos with slight (abnormals) or strong (malformed) abnormalities and non-developed organisms. The results showed that the proportions of normal and not-normal embryos were not modified by the MF. However, significant changes were observed among the not normal embryos: The proportion of abnormals and malformed was decreased, while the non-developed embryos significantly increased. The results support the hypothesis that a resonance-like effect on calcium and/or sodium ions was produced in organisms which already had some developmental defects.     

Can embryo metabolism be used for selecting bovine embryos before transfer?

The quality of in vitro-produced bovine embryos remains variable. The selection of these embryos based only on their morphology does not allow for acceptable gestational rates to be obtained. The use of metabolic markers to select viable embryos before transfer would be of valuable help, both economically and as a research tool. The ideal marker should meet several conditions: it should be able to be evaluated 1) in a totally non-invasive manner, 2) on individual embryos (which necessitates very sensitive techniques), 3) very rapidly (so that it is compatible with the immediate transfer of fresh embryos), and 4) in order to allow viable embryos to be separated from those that are not viable, whatever the production system used. In practice, such a marker does not exist, but certain methods of metabolic evaluation resemble it. The development of a metabolic marker is confronted by the metabolic characteristics of the embryo, notably the evolution of the metabolism during the development of the embryo and its adaptation to the changes in the environment.     

Developmental changes in inhibitory effects of arsenic and heat shock on growth of pre-implantation bovine embryos

Although sensitive to various disrupters, pre-implantation embryos possess some cellular cytoprotective mechanisms that allow continued survival in the face of a deleterious environment. For stresses such as heat shock, embryonic resistance increases as development proceeds. Present objectives were to determine whether (1) arsenic compromises development of pre-implantation bovine embryos, (2) developmental changes in embryonic resistance to arsenic mimic those seen for resistance to heat shock, and (3) developmental patterns in induction of apoptosis by arsenic are correlated with similar changes in resistance of embryos to inhibitory effects of arsenic on development. Bovine embryos produced by in vitro fertilization were exposed at the two-cell stage or at day 5 after insemination (embryos > or = 16-cells in number) to either sodium arsenite (0, 1, 5, or 10 microM) or heat shock (exposure to 41 degrees C for 0, 3, 4.5, 6, or 9 hr). Arsenic induced apoptosis and increased group 2 caspase activity for embryos at the > or = 16-cell stage, but not for embryos at the two-cell stage. In contrast to these developmental changes in apoptosis responses, exposure to arsenic reduced cell number 24 hr after exposure for both two-cell embryos and embryos > or = 16-cells. Similarly, the percentage of embryos that developed to the blastocyst stage at day 8 after fertilization was reduced by arsenic exposure at both stages of development. Heat shock, conversely, reduced development to the blastocyst stage when applied at the two-cell stage, but not when applied to embryos > or = 16-cells at day 5 after insemination. In conclusion, arsenic can compromise development of bovine pre-implantation embryos, the temporal window of sensitivity of embryos to arsenic is wider than for heat shock, and cellular cytoprotective responses that embryos acquire for thermal resistance are not sufficient to cause increased embryonic resistance to arsenic exposure. It is likely that despite common cellular pathologies caused by arsenic and heat shock, arsenic acts to reduce development in part through biochemical pathways not activated by heat shock. Moreover, the embryo does not acquire significant resistance to these perturbations within the time frame in development examined.     

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos.

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.     

Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status

Summary Objectives were to characterize developmental changes in response to heat shock in the preimplantation mouse embryo and to evaluate whether ability to synthesize glutathione is important for thermal resistance in mouse embryos. Heat shock (41° C for 1 or 2 h) was most effective at disrupting development to the blastocyst stage when applied to embryos at the 2-cell stage that were delayed in development. Effects of heat shock on ability of embryos to undergo hatching were similar for 2-cell, 4-cell, and morula stage embryos. The phenomenon of induced thermotolerance, for which exposure to a mild heat shock increases resistance to a more severe heat shock, depended upon stage of development and whether embryos developed in vitro or in vivo. In particular, induced thermotolerance was observed for morulae derived from development in vivo but not for 2-cell embryos or morulae that developed in culture. Administration of buthionine sulfoximine to inhibit glutathione synthesis did not increase thermal sensitivity of 2-cell embryos or morulae but did reduce subsequent development of 2-cell embryos at both 37° and 41° C. In summary, changes in the ability of 2-cell through morula stages to continue to develop following a single heat shock were generally minimal. However, 2-cell embryos delayed in development had reduced thermal resistance, and therefore, maternal heat stress may be more likely to cause mortality of embryos that are already compromised in development. There were also developmental changes in the capacity of embryos to undergo induced thermotolerance. Glutathione synthesis was important for development of embryos but inhibition of glutathione synthesis did not make embryos more susceptible to heat shock.     

Exposure to external environment of low ion concentrations is the trigger for rapid wound closure in Xenopus laevis embryos

Wounds in Xenopus laevis embryos close rapidly, as previously described. In this study, we examined the dependence on extracellular Na(+) and/or Cl(-) ion concentrations of the closure of wounds in Xenopus embryos inflicted by thermal injury. Wound closure did not occur in normal amphibian medium (100% NAM), while wound areas remarkably decreased either in 10-50% NAM or in 100% NAM lacking Na(+) or Cl(-). Similarly, wound areas did not change in a set of Na(+) and Cl(-) ion concentrations equivalent to those of the humoral fluids of intact Xenopus embryos, but rapid wound closure was induced by decreasing the concentration of either of the two ions. A tangential accumulation of actin cytoskeleton along the wound edge was associated with wound closure. However, a similar actin alignment formed even under the 100% NAM condition, in which wounds did not close, as stated above. The epidermis around the wound edge exhibited ellipse-shaped hypertrophy, and the marginal cells centripetally elongated during wound closure. On the other hand, no distinct morphological changes occurred in 100% NAM, although the epidermis was somewhat thickened. Thus, the morphological changes in the epidermis specific to the low ionic environment most likely play active roles in the wound closure of Xenopus laevis embryos, whereas the tangential actin alignment alone may be insufficient. Taken together, we propose that the wound closure in Xenopus embryos is triggered by a decline in either the extracellular Na(+) or Cl(-) ion concentration, and that this process is required for the abovementioned changes in the shape of the marginal cells.