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题名: 拟球状念珠藻(葛仙火)形态建成与发育及其生理学研究
作者: 李敦海
答辩日期: 2000
导师: 刘永定
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 念珠藻 ; 形态发育 ; 细胞化学 ; 藻殖段分化 ; 信号转导 ; 胁迫 ; 叶绿素荧光 ; 生理学
摘要: 本研究以拟球状念珠藻(Nostoc sphaeroides Ktitzing,俗称葛仙米)为材料,研究了念珠藻的形态建成、发育和生理学,其中着重研究了念珠藻的生活史、藻殖段分化和抗性生理学,并对不同发育时期的藻体的细胞化学和光合生理进行了比较。以藻殖段和丝状体为生活史的起点,观察了葛仙米的发育过程。在光合自养条件下,藻殖段和丝状体都经过失去伪空泡、形成胶鞘、分化出异形胞和细胞横裂形成非丝状体期群体等过程,最终形成具丝状体的群体。在异养条件下,藻殖段可发育至非丝状体期,但丝状体的发育却比较慢,甚至不能进行细胞分裂,发育最终仍停止在丝状体阶段。实验表明,异形胞的分化能够被氨态氮所抑制,而不能被硝态氮所阻止。若异形胞分化受阻,则最终导致葛仙米群体发育受阻。对细胞化学变化的定性研究发现,胞外蛋白质、负高电荷多糖、酸性多糖和硫化多糖的变化都与发育过程紧密相关。脂质和孢粉质只是胞外的少量成分。定量测定表明,藻体的总糖和还原性多糖的量均随发育过程而变化。藻体热水提取多糖中硫化多糖由于含量太低而无法定量测定,酸性多糖含量则在发育过程中出现一定的变化趋势。文中对多糖与发育的关系进行了讨论。从营养、光和信号转导三方面探讨了藻殖段分化与三者的关系。藻殖段分化与预培养时培养基的磷、钾元素和渗透效应并没有直接的关系,而分化与否则于藻体所处在的时期有关:对数生长期的藻体,无论其预培养的培养基如何,均有藻殖段的分化;而静止期的藻体,则不能诱导出藻殖段的产生。藻殖段的释放却与藻体的预培养有关,预培养时的不完全培养基则有利于藻殖段的释放。对藻殖段分化的光控制的研究发现,在白光、红光或绿光下,藻殖段分化率达到最大时的光强范围为 5-10 μmol.m~(-2).s~(-1)。白光对藻殖段的诱导作用最有效,达到100%;绿光次之,约有80%;红光效率最低,只有0-10%。黑暗条件下藻殖段不能分化,若有蔗糖和葡萄糖的存在,则藻殖段的分化率能够达到l00%。具有同等渗透势的NaCl则不能诱导藻殖段的分化,这说明藻殖段的分化是一个需能的过程,而非由外界的渗透势改变而引起。实验表明,红光与绿光在藻殖段分化过程中并非起到相反的作用的。另外藻殖段的分化亦有光合电子传递链氧化-还原态和蛋白质重新合成的参与。以钙调素抑制剂和钙离子螯合剂实验证明,葛仙米藻殖段的分化有钙调素和钙离子信号转导途径的参与。虽然NO可以大大降低藻殖段的分化率,但是NOS抑制剂NNA却可抑制藻殖段的分化;外源氧自由基的加入也抑制了藻殖段的分化,但清除了氧自由基也使得藻殖段分化完全受抑制。因此,作为某些高等动植物信号转导因子的NO和氧自由基,在葛仙米藻殖段分化中是否参与了信号转导,还有待进一步研究。从盐度、水分和温度几个方面研究了这些因子对葛仙米群体和(或)藻殖段的生理和生化方面的影响。葛仙米的叶绿素荧光、光系统活性和光合放氧均随盐度升高而降低,而呼吸作用的变化则不大。实验还表明,脯氨酸并非葛仙米的渗透调节剂。藻体的膜脂过氧化产物丙二醛则由于盐胁迫加重而上升,SOD的活性则呈现出“V”形变化。藻殖段对盐胁迫的忍耐力有限,在NaCl浓度高于0.4mol/L的BG-11_0培养基中,处理24h足可以是藻殖段的叶绿素荧光参数Fv/Fm降至为零。在失水过程中,群体和藻殖段以及藻殖段发育培养物的Fv/Fm变化趋势相似,都有一个下降-上升-下降的过程。干燥群体吸水后Fv/Fm的恢复对光和蛋白质的重新合成具有依赖性,且其恢复速率与贮藏时间有关。温度处理实验表明,低温有利于葛仙米的存活,较高温度下生长的继续可能是其逐渐失去生理活性的主要原因。通过测定光合放氧、叶绿素的室温和低温(77K)荧光以及直接测定PSI和PSII的活性发现,除了表观放氧速率外,群体的光合能力要高于藻殖段。这些差别的出现,一方面取决于两者的形态学差别,另一方面也因为营养丝体在分化为藻殖段的过程中,细胞的光合色素含量和比例发生了变化,同时,伪空泡的形成也影响了藻殖段的光合能力。群体和藻殖段在对不断上升的光强下PSII(Fv/Fm)活性的变化说明,藻殖段较强的耐受不良环境的能力,这是它作为繁殖单位所必须具备的特性之一。
英文摘要: The macrocolonies of pure culture of Nostoc sphaeroides Kutzing were gained by choosing appropriate medium and culturing methods in laboratory. And a simple description of the species was given at morphological and ultrastructural levels. By using hormogonia and filaments from homogenized colonies as the starting developmental stage, the life histories of Nostoc sphaeroides were studied. When the culture was bubbled with air or not, all the hormogonia would develop in the following pattern: initially the gas vesicles disappeared, then followed by formation of sheath-enclosed filament; after the heterocysts differentiated, the cells start to divide with a plane perpendicular to longitudinal or polar cell axis and formed aseriate colonies, eventually reached to the filamentous colony stage. Under heterotrophic conditions, all the hormogonia could form aseriate colonies; however, only part of the cells remained the ability of dividing when cultured in glucose-containing medium, the others could not divide and lost the red autofluorescence. The developmental behavior of filaments from homogenized colonies was somewhat different from that of hormogonia when cultured in the above mentioned media. The former formed winding trichomes after cell division and the cell packs were slightly loosening when compared with those from hormogonia; thus the colonies formed by filaments were longer than those by hormogonia. The heterotrophic development of filaments from homogenized colonies was very slowly, even cultured in sucrose-containing medium, there was almost no cell division occurred. In BG-11 medium (containing fixed nitrogen NaNO_3), the developmental behavior of hormogonia was the same as when they were cultured in BG-11_0 medium, but the development process of the former was faster than that of the latter. In both NH_4Cl and NaNO_3-containing BG-11 medium (the sum of mole concentrations of NH_4Cl and NaNO_3 was equal to that of NaNO_3 in BG-11 medium), the higher ratio of NH_4Cl to NaNO_3, the worse effect on development of heterocysts. With the addition of NaCl in the culture, the developmental process became slower or ceased, meanwhile, the shape of nucleolus turned into more irregular, and the cells lost the red autofluorescence little by little. The cytochemical changes during the developmental process of Nostoc sphaeroides were studied. It was observed that the shape of the nucleolus remained almost no alternation during the developmental process. The changes of extracellular proteins, high charged polysaccharides, acid polysaccharides (AP) and sulfated polysaccharides (SP) appeared to closely relate with the developmental process. Lipids and sporopollenin were minor extracellular components. As shown by quantification method, the total content of saccharides increased during the development process and reached maximum in filamentous colony stage. In contrast, the content of reducing saccharides was highest in hormogonia stage but it dropped quickly once the development begins. In hot water extracted saccharides, SP couldn't be detected. The content of AP in hormogonia was the lowest, the highest in old colonies and no apparent differences in the aseriate colonies, the young and the mature filamentous colonies. The contents of AP in same amounts of saccharides from different stages were also measured. In the light of these findings, the implications of polysaccharides in development and evolution was discussed. The relationships between hormogonia differentiation and nutrients, light condition and signal transduction were studied respectively. After the colonies of Nostoc sphaeroides in exponential phase of growth were transferred to fresh BG-11_0 medium, the hormogonia differentiated no matter which type of preculture was used. This provided the evidence that the differentiation was not directly related to the phosphorus, potassium status and the osmotic effect in the media. In contrast, all the cultures in stationary phase of growth had no hormogonium differentiation after transferred to fresh medium. However, the incomplete media for preculture seemed in favor of the liberation of hormogonia. Under white light, red light or green light, the differentiation rate of hormogonia reached maximum at the light intensities ranged from 5 to 10 μmol.m~(-2).s~(-1), in the order of 100%, 80% 0-10%, respectively. No hormogonia differentiation occurred when cultured the colonies in the dark. However, if sucrose or glucose exists, the differentiation rate could reach 100%; when sucrose or glucose was substituted with NaCl of the same osmotic potential as the organic counterpart, no differentiation occurred. The results suggested that hormogonia differentiation was a process that needs energy and was not induced by osmotic potential changes in the media. Some experiments also suggested red light and green light didn't play a reversed role in the process of hormogonia differentiation. The result showed that the state of electron transport in the photosynthetic electron chain and the de novo synthesis of proteins were quite possibly involved in the differentiation process. Both W-7 and FPZ, the inhibitors of calmodulin (CaM), could inhibit hormogonia differentiation in Nostoc sphaerorides. As shown by the fluorescence of PFZ, cells being treated with inhibitors of CaM had less CaM distribution as compared with normal ones. EGTA also can prevent the differentiation of hormogonia. To use chlorotetracycline as the fluorescent indicator, it was observed that the membrane-bounded calcium was greatly decreased in cells treated with EGTA. But other tests suggested that the normal differentiation of hormogonia was the result of effect of several metal ions. Nitric oxide (NO) could greatly decrease the rate of hormogonia differentiation. Although have no knowledge of exists of NO synthetase (NOS) in Nostoc sphaeroides, the inhibitor of NOS could prevent the differentiation. The exogenous reactive oxygenic intermediates (ROIs) also could inhibit this process, but the scavenger ofROIs, catalase, could inhibit this process too. The above results suggested the differentiation of hormogonia was a complicated signal transductioin process. The effects of salinity, water and temperature on the physiological and biochemical aspects of colonies and/or hormogonia of Nostoc sphaeroides were studied. And a comparison of photosynthetic competence between colonies and hormogonia was performed in order to understanding their role in the natural habitat. When the colonies were treated in medium containing NaCl, the photosynthetic oxygen evolution rates, Fv/Fm and PSI and PSII activities were negatively related with NaCl concentrations, whereas the respiration rates were influenced little by NaCl. No evidence showed that the exogenous proline could relieve the stress of the salt-treated colonies. The endogenous proline contents of the colonies didn't increase with the increased treating salinity, whereas MDA increased when the salt stress became more serious. In addition, the SOD activities in the colonies have a typical "V" shape with treating salinity, presumably the changing was related to the real stress that the colonies experienced. The tolerance of hormogonia to salt stress was sinite. At NaCl concentration over 0.4 mol/L, 24h's treatment was harmful enough to lower the Fv/Fm of hormogonia to zero. Under the artificially designed and natural water losing stress, the changes of Fv/Fm of colonies had a same trend: at first decreasing, then increasing and decreasing again at last. The recovery of Fv/Fm of dry colonies after rewetting depended on light and the de novo synthesis of proteins, and the recovery rate was related with drought periods that the colonies experienced. When suffered by natural water losing, the patterns of Fv/Fm changes in hormogonia, lday's and 10day's cultures of hormogonia in BG-11_0 medium bubbled with air were the same as that of mature colonies. On transferring Nostoc sphaeriodes grown at 30 ℃ to conditions at lower temperature, the activity of the colonies was able to retain. At higher temperatures, the Fv/Fm of colonies decreased with the prolonged culturing. The activity could be partly recovered when the high temperature treated colonies were transferred to low temperature environment. On the contrary, the activity decreased when transferring the low temperature treated colonies to the relatively high temperature environment. The results might infer that low temperature was in favor of the survival of Nostoc sphaeroides, while the growth at relatively high temperature maybe the main reason that disrupt the activities. By comparison, the photosynthetic activities of colonies were higher than that of hormogonia. The differences might be caused by two reasons. First, the morphological differences between two developmental stages and the second, both the contents and the ratio of photosynthetic pigments were changed during the process of hormogonia differentiation; at the same time, the formation of gas vesicles in hormogonium might affect its photosynthetic competence. The changes of PSII (Fv/Fm) activities of colonies and hormogonia under the condition of increasing light intensities inferred that hormogonia have a stronger capacity to survive adverse environment than colonies. That's one of the important features it should have when as a propagulum.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12552
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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拟球状念珠藻(葛仙火)形态建成与发育及其生理学研究.李敦海[d].中国科学院水生生物研究所,2000.20-25
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