青杨组(Populus Section Tacamahaca Spach)不同种对非生物胁迫的响应差异

土壤是人类赖以生存的自然环境和农业生产的重要资源，目前土壤受到干旱和盐胁迫的危害越来越严重。杨树具有适应性强、生长快和丰产等特性，本论文以青杨组杨树为模式植物，研究杨树对土壤干旱和盐胁迫的生态生理及蛋白质组学反应，研究成果可为我国干旱半干旱地区营造人工林、防止沙漠化提供理论依据，也为恢复与重建盐污染地区退化生态系统提供科学指导。主要研究结果如下： 1 青杨不同种对逐步干旱胁迫的响应差异 将来自喜马拉雅山东缘高海拔的康定杨和低海拔的青杨枝条扦插在温室中，用来检测它们对逐步干旱胁迫的响应。研究结果表明来自不同海拔的杨树对逐步干旱胁迫的适应性反应是不一样的。株高、叶片发育、叶片相对含水量、丙二醛、过氧化氢等指标的显著性变化在青杨中比在康定杨中来得早些，而且随着干旱胁迫程度的增加，这些参数的变化越来越明显，尤其是当青杨受到严重干旱胁迫的时候；而可溶性蛋白、可溶性糖、游离脯氨酸、抗氧化酶活力变化在康定杨中来得早一些。与青杨相比，在干旱胁迫下，康定杨仍能保持较好的植株生长和叶片发育；康定杨也能在逐步干旱条件下积累更多的可溶性蛋白、可溶性糖、游离脯氨酸及抗氧化酶活力，但是在丙二醛和过氧化氢含量方面增加的更少些。而且，我们的研究结果表明高海拔的康定杨有更强的耐干旱能力，杨树对干旱胁迫的适应能力与干旱发生的速度、强度、持续时间及两种杨树的海拔有关。 2 干旱胁迫下青杨不同种的蛋白质组学分析 来自青杨和康定杨雌株的枝条扦插在温室中，用来研究它们对干旱胁迫的蛋白质组学反应。采用TCA-丙酮/酚提取法提取总蛋白，并进行双向电泳分析。在每个处理的重复图像中都能检测到1,000 个以上的蛋白点。在青杨中有58 个蛋白在干旱处理后发生显著变化，其中22 个蛋白通过肽指纹图谱成功鉴定。康定杨中有69 个蛋白的表达量发生了显著变化，其中有25 个蛋白通过肽指纹图谱成功鉴定。这些被鉴定的蛋白主要参与了光合作用、氧化还原平衡、信号传导、能量代谢、蛋白质合成等过程。尽管被鉴定的蛋白只占叶片总蛋白的很少一部分，但这些被鉴定的干旱响应蛋白可能对维持植株内部平衡方面有重要作用。 3 青杨的盐胁迫响应 青杨植株分别用 0、50 和100 mM NaCl 溶液进行处理。叶片相对含水量、叶绿素a、b 含量、CO2 同化速率和气孔导度的降低表明叶绿体受到了盐胁迫的影响。过氧化氢、丙二醛含量及电导率的升高表明细胞受到了伤害。可溶性糖、游离脯氨酸含量及抗氧化酶含量的上升增加了植株耐盐胁迫的能力。在每个处理的重复图像中都能检测到1,000 个以上的蛋白点。其中有38 个盐响应蛋白被成功鉴定，有16 个蛋白(点4、10、11、14、15、21、24、26、27、28、33、34、35、36、37 和38)出现在盐胁迫的植株中；3 个蛋白(点10、11 和35)只出现在重度盐胁迫处理中；而1 个蛋白(点1)只出现在对照处理中。2 个蛋白(点1 和2)表达量下降，其余蛋白点表达量都增加。被鉴定的蛋白一部分参与了生理生化反应，而另一部分则在信号传导、蛋白质合成等方面有重要作用。盐胁迫下的生理生化变化及蛋白质组学的联合研究有利于青杨对盐胁迫的适应性分析。 Soil is the indispensable environment for human survival and important resource for agriculture development. Nowadays soil is threatened by drought stress and salt stress. Poplars (Populus spp.) possess some characters such as strong acclimilation, fast growth and great production of biomass. In this study, different species of Populus section Tacamahaca spach were used as model plants to investigate the ecophysiological and proteomic responses to drought stress and salt stress. Our results can provide theoretical evidence for the afforestation and prevention of desertification in the arid and semi-arid areas, and also can supply scientific direction for the reconstruction and rehalibitation of ecosystems contaminated by salinity. The results are as follows: 1 Adaptive responses to progressive drought stress in two contrasting poplar species originating from different altitudes Cuttings of Populus kangdingensis C. Wang et Tung and Populus cathayana Rehd., originating from high and low altitudes in the eastern Himalaya, respectively, were examined during one growing season in a greenhouse to determine the effects of progressive drought stress. The results manifested that the adaptive responses to progressive drought stress were different in these two species from different altitudes. Significant changes in height increment, leaf development, relative water content (RWC), malondialdehyde (MDA) and hydrogen peroxide (H2O2) appeared earlier in P. cathayana than in P. kangdingensis, whereas changes in soluble protein, soluble sugar, free proline and antioxidant enzymes appeared earlier in P. kangdingensis. In addition, changes in these parameters became more and more significant when the drought stress progressed, especially under severe drought stress in P. cathayana. Compared with P. cathayana, P. kangdingensis was able to maintain a superior height increase and leaf development under drought stress. Also, P. kangdingensis possessed greater increments in soluble protein, soluble sugar, free proline and antioxidant enzymes, but lower increments in MDA and H2O2 than did P. cathayana when the cuttings were exposed to progressive drought stress. Our results suggest that P. kangdingensis originating from the high altitude has a better drought tolerance than does P. cathayana originating from the low altitude. Furthermore, this study manifested that acclimation to drought stress are related the rapidity, severity, duration of the drought event and the altitude of two contrasting species. 2 Proteomic responses to drought stress in two contrasting poplar species originating from different altitudes The cuttings from a female clone of P. kangdingensis and P. cathayana were used to determine proteomic response to drought stress, respectively. Total proteins of the leaves were extracted by a combination of TCA-acetone and phenol, and separated by two-dimensional gel electrophoresis. More than 1,000 protein spots were reproducibly detected on each gel. 58 differentially expressed spots were detected under drought stress in P. cathayana and 22 drought-responsive proteins were identified by peptide mass fingerprint. 69 differentially expressed spots were detected under drought stress in P. kangdingensiss and 25 drought-responsive proteins were identified by peptide mass fingerprint. The identified proteins are involved in several processes, i.e., signal transduction, protein processing, redox homeostasis, CO2 fixation and energy metabolism. Although the proteins identified in this investigation represent only a very small part of the poplar leaf proteins, some of the novel drought-responsive proteins identified here may be involved in the establishment of homeostasis in response to drought stress in the woody plants. 3 Responses to salt stress in P. cathayana Cuttings from a female clone of P. cathayana were treated by Hoagland’s solution: 0, 50, 100 mM NaCl, respectively. Salinity significantly decreased the relative water content of leaves, the contents of chlorophyll a and chlorophyll b, CO2 assimilation rate (A) and stomatal conductance (gs) in both salt stress treatments，which suggested the chloroplast was affected by salt stress. The observed increases of H2O2 and malondialdehyde contents and electrolyte leakage suggested that salinity caused cellular damage, whereas the increases in compatible solutes and in the activities of antioxidant enzymes enhanced the salt tolerance. More than 1,000 protein spots were reproducibly detected on each gel, and 38 salt-responsive proteins were successfully identified by peptide mass fingerprint (PMF). 16 spots (spot 4, 10, 11, 14, 15, 21, 24, 26, 27, 28, 33, 34, 35, 36, 37 and 38) absent in the control sample were induced by the salt treatment, and three spots (spot 10，11 and 35) were present only in the severely salt-stressed treatment. The %vol of the differentially expressed proteins generally increased with progressing salt stress, except for the decreased %vol of two proteins (spot 1 and 2) under salt stress and the presence of spot 1 only in the control sample. Some of the novel salt-responsive proteins identified here may be involved in physiological, biochemical response to salt stress in P. cathayana, the other identified proteins play a role in numerous cellular functions, including signal transduction and protein processing. An integrated physiological, biochemical and proteomic approach was used here to systematically investigate salt acclimation in poplar.

增强UV-B辐射对粗枝云杉(Picea asperata Mast.)和青杨(Populus cathayana Rehd.)的影响

光是植物赖以生存的重要环境因子，但是植物在获得光的同时不可避免的会受到紫外辐射的伤害。尤其是近年来，人类向大气中排放的大量氮氧化合物和氟氯烃类化合物(CFC’s)引起臭氧分子的分解，导致到达地球表面的紫外辐射增加，特别是UV-B辐射增强。而另一方面，植物对UV-B辐射反应的敏感性在种间和品种间存在差异，主要受植物基因型，生态型和生活型的控制。本项目分别以粗枝云杉和青杨组杨树为模式植物，从形态和生理生化方面分别研究了来自不同水分背景下的粗枝云杉种群和来自不同UV-B背景下的青杨种群在增强UV-B下的反应及其反应差异,并探讨了干旱、喷施外源脱落酸(ABA)对它们抗UV-B能力的影响。研究成果可为生态系统的恢复与重建提供理论依据和科学指导。主要研究结果如下： 1. 粗枝云杉的两个种群，湿润种群(来自四川黑水)和干旱种群(来自甘肃迭部)在水分良好和干旱状况下表现出对增强UV-B的不同响应。同时，干旱对粗枝云杉抗UV-B能力的影响也得到研究：两种胁迫共同作用时，干旱表现出在一定程度上减弱了增强UV-B对粗枝云杉的生理特性的影响。 干旱胁迫显著降低了两个粗枝云杉种群的光合同化速率(A), 气孔导度(gs)和PSII的有效光量子产量(Y), 同时，提高了非光化学猝灭效率(qN)和超氧化物歧化酶(SOD)的活性。与湿润种群相比，干旱种群抗旱性更强，表现为干旱种群拥有更高的SOD和干旱进一步加剧了UV-B的胁迫效应。 本研究中，干旱胁迫单独作用时，显著降低了青杨两个种群的生物量积累和气体交换，具体包括A、gs、蒸腾速率(E)和光合氮利用效率(PNUE)，提高了两个种群的瞬时水分利用效率(WUEi)、长期水分利用效率(WUET)、碳同位素组分(δ13C)和氮含量(N)。同时，UV吸收物质和ABA含量也得到积累。另一方面，增强UV-B对青杨两个种群各个指标的影响，同干旱所引起的效应有着相似的趋势。同低海拔种群相比，高海拔种群有着更强的抗旱和抗UV-B能力，具体表现在高海拔种群有着更多的生物量积累，更强的气体交换和水分利用效率及更高水平的ABA和UV吸收物质含量。相比干旱诱导的生物量积累和气体交换的降低，在干旱和增强UV-B两个胁迫同时作用于青杨时，这种降低表现的更为明显。显著的干旱和UV-B的交互作用还表现在WUEi, WUET, δ13C, 可溶性蛋白含量, UV吸收物质含量, ABA, 叶片和茎中的N含量以及C/N比中。 3. 经过一个生长季的试验观察，增强UV-B、外源ABA及两因子共同作用对青杨的生物量积累、气体交换、内源ABA和UV吸收物质含量、抗氧化系统以及碳、氮含量和碳/氮比均产生显著影响。本试验中，青杨的两个种群分别来自中国西南部的不同海拔地区，高海拔种群来自青海大通而低海拔种群来自四川九寨。外源ABA的胁迫为直接喷施ABA到青杨叶片，而增强UV-B胁迫是利用平方波系统分别保证青杨苗暴露于外界UV-B强度和两倍于外界UV-B强度下。 研究结果显示，增强UV-B显著的降低了两个青杨种群的株高、基茎、总叶面积和总生物量等生长指标，同时也导致其A、gs、E和叶片中碳含量的减少。而显著增加了SOD和过氧化物酶(GPx)活性水平，诱导了过氧化氢(H2O2)和MDA的显著增加，促进了UV吸收物质和不同器官中内源ABA含量的显著积累。另一方面，外源ABA引起了青杨光合同化速率的下降，SOD和GPx酶活性的增强，H2O2 和 MDA含量也表现出显著增加，同时，内源ABA含量得到显著累积。同低海拔种群相比，高海拔种群具有更加抗UV-B和外源ABA的特性。显著的UV-B和ABA的交互作用表现在A, E, SOD和GPx活性，以及叶片和根部的内源ABA等一系列指标中。在所有胁迫下，叶片中的碳和氮含量同其在茎和根中的含量显著相关，另外，叶片和茎中的氮含量同茎中的碳含量显著相关。 Sunlight is an indispensable environment factor for plants survival and development. Meanwhile, photosynthetic organisms need sunlight and are thus, inevitably, exposed to UV radiation. Especially for recent years, ultraviolet radiation, especially UV-B reaching the Earth’s surface increased because of depletion of ozone layer resulted from emission of NxO and CFC’s from human activities. On the other hand, the sensitivity of plants to UV-B radiation depends on the species, developmental stage and experimental conditions. In this experiment, two populations of Picea asperata Mast from different water background and two populations of Populus cathayana Rehder from different altitude background were selected as model plants to assess the effects of enhanced UV-B radiation. Morphological and physiological traits induced by enhanced UV-B in each plant species were observed and the different responses were discussed, furthermore the influences of drought and exogenous ABA on responses induced by enhanced UV-B were studied. The study could provide a strong theoretical evidence and scientific direction for the afforestation and rehabilitation of ecosystem. The results are as follows: 1. Different responses of two contrasting Picea asperata Mast. populations to enhanced ultraviolet-B (UV-B) radiation under well-watered and drought conditions were investigated. And the effects of enhanced UV-B on tolerance of drought were also observed in our study that the UV-B exposure may have alleviated some of the damage induced by drought. Two contrasting populations, originating from a wet and dry climate region in China, respectively, were employed in our study. Drought significantly decreased CO2 assimilation rate (A), stomatal conductance (gs) and effective PSII quantum yield (Y), while it significantly increased non-photochemical quenching (qN) and the activity of superoxide dismutase (SOD) in both populations. Compared with the wet climate population, the dry climate population was more acclimated to drought stress and showed much higher activities of SOD and ascorbate peroxidase (APX), and much lower levels of malondialdehyde (MDA) and electrolyte leakage. On the other hand, enhanced UV-B radiation also induced a significant decrease in the chlorophyll (Chl) content in both populations under well-watered conditions, and a significant increase in UV-absorbing compounds in the wet climate population. After one growing season of exposure to different UV-B levels and watering regimes, the increases in MDA and electrolyte leakage, as induced by drought, were less pronounced under the combination of UV-B and drought. In addition, an additive effect of drought and UV-B on A and gs was observed in the wet climate population, and on the activity of APX and qN in the dry climate population. 2. The significant effects of drought, enhanced UV-B radiation and their combination on Populus cathayana Rehd. growth and physiological traits were investigated in two populations, originating from high and low altitudes in south-west China. Our results showed that UV-B acts as an important signal allowing P. cathayana seedlings to respond to drought and that the combination of drought and UV-B may cause synergistically detrimental effects on plant growth in both populations. In both populations, drought significantly decreased biomass accumulation and gas exchange parameters, including A, gs, E and photosynthetic nitrogen use efficiency (PNUE). However, instantaneous water use efficiency (WUEi), transpiration efficiency (WUET), carbon isotope composition (δ13C) and nitrogen (N) content, as well as the accumulation of soluble protein, UV-absorbing compounds and abscisic acid (ABA) were significantly increased by drought. On the other hand, cuttings from both populations, when kept under enhanced UV-B radiation conditions, showed very similar changes in all above-mentioned parameters, as induced by drought. Compared with the low altitude population, the high altitude population was more tolerant to drought and enhanced UV-B, as indicated by the higher level of biomass accumulation, gas exchange, water-use efficiency, ABA concentration and UV-absorbing compounds. After one growing season of exposure to different UV-B levels and watering regimes, the decrease in biomass accumulation and gas exchange, induced by drought, was more pronounced under the combination of UV-B and drought. Significant interactions between drought and UV-B were observed in WUEi, WUET, δ13C, soluble protein, UV-absorbing compounds, ABA and in the leaf and stem N, as well as in the leaf and stem C/N ratio. 3. During one growing season, significant effects induced by enhanced UV-B radiation, exogenous ABA and their combination on biomass accumulation, gas exchange, endogenous ABA and UV-absorbing compounds concentrations, antioxidant system as well as carbon (C) content, nitrogen (N) content and C/N ratio were investigated in two contrasting Populus cathayana populations, originating from high and low altitudes in south-west China. Exogenous ABA was sprayed to the leaves and enhanced UV-B treatment was using a square-wave system to make the seedlings under ambient (1×) or twice ambient (2×) doses of biologically effective UV-B radiation (UV-BBE). Enhanced UV-B radiation significantly decreased height, basal diameter, total leaf area, total biomass, A, gs, E and carbon (C) content in leaves, and significantly increased activities of SOD and guaiacol peroxidase (GPx), hydrogen peroxide (H2O2) and malonaldehyde (MDA) content as well as the accumulation of UV-absorbing compounds and endogenous ABA concentrations among different organs in both populations. In contrast, exogenous ABA showed significant decrease in A and significant increases in activities of SOD and GPx, H2O2, MDA content and the endogenous ABA concentrations. Compared with the low altitude population, the high altitude population was more tolerant to enhanced UV-B and exogenous ABA. Significant interactions between UV-B and ABA were observed in A, E, activities of SOD and GPx, as well as in endogenous ABA in leaves and roots of both populations. Across all treatments, C and N content in leaves was strongly correlated with those were in stems and roots, respectively. Additionally, leaf and stem N content were significant correlated with stem C content.

青杨(Populus cathayana Rehd.)对逆境胁迫的反应及lea基因的表达分析

杨树具有分布广、适应性强，在生态环境治理和解决木材短缺方面均占有重要位置。青杨(Populus cathayana Rehd.)是青杨派树种的重要成员之一，也是我国的特有种。本研究通过对不同水分梯度的干旱胁迫下青杨形态和生理生化的反应，不同pH值盐碱胁迫下不同海拔和不同气候地区的四个青杨种群在生理生态上的反应差异，以及在干旱和低温胁迫下青杨lea2, lea3组基因表达差异的研究，从形态、生理、生化和分子生物学水平系统地研究了青杨在不同逆境胁迫下的反应和青杨不同种群在盐碱胁迫下的反应差异。主要研究结果如下： 1. 青杨在干旱胁迫下的反应机制：中度和重度干旱胁迫下植株的生长受到明显抑制。表现在光合系统上青杨的净光合同化速率(A)下降，主要原因是气孔导度(gs)，胞间二氧化碳浓度(Ci)下降。另外最大量子产量(Fv/Fm)、光化学猝灭效率(qP)降低反应了干旱胁迫下光合系统II(PSII)受到严重损伤, 而且非光化学猝灭效率(qN)上升，导致可利用化学能产量下降，叶绿体产生淀粉的量减少。qP降低qN上升导致产生的过量电子对光合系统的伤害造成活性氧以及丙二醛(MDA)的含量增加。超微解剖结构显示，干旱胁迫增强时，叶绿体内淀粉粒的数目减少，而且叶绿体、线粒体等细胞器中嗜锇颗粒的数目增加。为清除细胞内的活性氧，植物一般的反应是抗氧化系统酶活性增加，对青杨来讲超氧化物歧化酶(SOD), 抗坏血酸过氧化物酶(APx)活性的增加远大于过氧化物酶(POD)，这显示了在青杨中SOD、APx酶在清除活性氧的作用上大于POD。另外同工酶研究结果显示这些酶活性的升高主要是由于各条同工酶带表达量的增加，而不是诱导新酶带的产生。另外，75% FC水分处理下有些指标非但没有下降，像A和有效光量子产量(Y)的值都略有增加，而且gs同时增加。另外，100% FC比75% FC细胞内淀粉粒的数目少一些，但有少量的嗜锇颗粒。这证明100% FC土壤水分也许并非最适合青杨生长。 2. 盐碱胁迫对不同海拔地区青杨种群的反应差异：青杨高海拔和低海拔种群的各种生理特性随着pH值上升都受到了很大的影响。两种群叶和根中Na+、K+ 含量, Na+/K+比率随着pH值的上升影响显著。在pH值高于10.4时高海拔种群叶和根中Na+/K+比率急剧下降但是低海拔种群中却一直维持在较高水平。两种群中MDA、脯氨酸(Proline)的含量，抗氧化系统酶的活性都受到了严重的影响，证明两个种群都属于盐碱胁迫敏感类型但是高海拔的种群对盐碱胁迫的耐性要高于低海拔。这主要是由于高海拔种群一般具有耐干旱、低温胁迫的能力，而植物的抗逆机制一般都有共通之处。 3. 盐碱胁迫对不同气候地区青杨种群的反应差异：盐碱胁迫下两种群的光合作用受到明显的抑制，具体表现在叶绿素的含量和A 显著下降。净光合速率的下降主要是由于叶片gs，Ci 值降低引起的。与湿润地区的种群相比盐碱胁迫增强时，干旱地区的种群叶绿素含量和光合能力的升高与K+离子含量增加有关。植物维持细胞质高K+/Na+值对植物的抗盐性有很重要的作用。为清除盐碱胁迫产生的活性氧，抗氧化系统酶活性增加。盐碱胁迫下干旱地区的种群在SOD、CAT 和谷胱甘肽还原酶(GR)等酶的活性均显著上升，而湿润地区种群只有谷胱甘肽氧化酶(GST)的活性明显增加，说明干旱种群的抗氧化酶系统在较高盐碱胁迫下的保护作用要强于湿润种群。这主要是由于植物抗盐碱胁迫与抗干旱胁迫在一些方面的机制是一致的，抗旱种群一般也能抵抗一定程度的盐碱胁迫。 4. 青杨lea2、lea3 基因在干旱和低温胁迫下的表达差异：通过荧光定量PCR 分析，lea2、lea3 组基因在干旱和低温胁迫下在mRNA 水平的瞬时表达量明显升高，说明了两基因在青杨耐干旱和低温胁迫上都起显著的作用。而且两基因在干旱胁迫下，表达量的升高和降低的时间近乎同步，表明两基因在干旱胁迫下对植物应急保护机制的启动都发挥着重要的作用。低温胁迫下lea3 基因在mRNA 水平上表达量显著上升的时间要早于lea2，而且lea3 基因的持续作用时间明显长于lea2 组基因，说明了低温胁迫开始时lea3基因在植物应对逆境的作用上要大于lea2 基因。 Poplars play an important role in lumber supply, and are important components of ecosystems due to their wide distribution and well adaptation. Populus cathayana Rehd., which belongs to Populus Sect. Tacamahaca Spach, is one of the most important resources of poplars and is specialist to china. In this study, different altitudes and climates populations of P. cathayana were used as experiment materials to investigate the adaptability to drought and salt-alkali stresses. And the cultures of P. cathayana were used to analyze the lea2 and 3 group genes expression when exposed to drought and low temperature stresses. The results are as follows: 1. A large set of parallel responses to drought stress: Drought stress caused pronounced growth inhibition. A decreased significantly and was mainly the result of gs and Ci down. Besides, Fv/Fm, qP decreased and that reflected the harmful effects to PSII of drought stress. In accordance with qN increasing, decreased useful energy production caused the starch numbers reduction in chloroplast. The qP up and qN down improved the levels of ROS and MDA. Starch numbers in chloroplast reduced and plastoglobuli numbers increased when soil water content decreased. To reduce ROS, the activities of SOD, APX, CAT and PPO were activated. The isozymes results show that the rising activities of the antioxidant enzymes resulted from certain isoform content increased, and not from the new band produced. Interestingly, morphological results show 100%FC maybe wasn’t the favorite water content for P. cathayana growth. 2. Effect of salt-alkali stress on morphological and physiological changes in two different altitudes populations of P. cathayana: We compared the physiological responses of two populations of Populus cathayana Rehder, originating from altitudes 2,840 m and 1,450 m. Our results demonstated that Na+ and K+ contents, and Na+/K+ ratios in leaves and roots are greatly affected by pH values. At pH 10.4, the Na+/K+ ratios in both leaves and roots sharply dropped in the higher altitude population but were always maintained at higher levels in the lower altitude population. The pH values causing maximum malondialdehyde (MDA) level, free proline content and antioxidant enzyme activities were significantly different in two populations. These results indicated that the higher altitude population exhibits greater tolerance to alkalinity stress than does the lower altitude population. 3. Morphological and physiological changes in two different climates populations of P. cathayana when exposed to salt-alkali stress. Salt-alkali stress caused pronounced inhibition of the growth and especially in photosystem. Pigments content and A decreased significantly and at the same time gs and Ci decreased too. Compared with wet climate population, the Chlorophyll content and A increased in drought climate population as pH value rising was related to the K+ content increasing. It is important to resist salt-alkali stress that the K+/Na+ ratio matained at high level in cytoplasm. To reduce ROS content, the SOD, CAT and GR activities rised significantly in drought population but only GST increased in wet population. The drought population showed higher salt-alkali tolerance than the wet population mainly resulted from the fact that drought tolerance was in accordance with salt-alkali tolerance to some extent. 4. The different expressional model of lea2 and lea3 gene when P. cathayana was exposed to drought and cold stress. RT-PCR results show both lea2 and lea3 suddenly expressed significantly in mRNA level under drought and cold stress. The expression level of two genes reached optimal level at the same time. But under cold stress, the earlier significantly rising expressional time and the longer maintained higher level time in lea3 than lea2 elucidated that lea3 may be more important than lea2 in resisting cold stress in short time in P. cathayana.

青杨(Populus cathayana Rehd)雌雄植株对锈病和干旱胁迫的生理响应及差异蛋白质组比较

雌雄异株植物对环境的不同响应一直是一个有趣而新颖的研究领域，由于雌雄个体不同的繁殖成本及不同的生存策略，使得雌雄植株在生长、存活、生殖格局、空间分布、资源配置等方面已经表现出明显的不同，在生理和分子水平上也表现出明显的性别间差异。干旱是制约农林业发展的环境因子之一，叶锈病是对杨树危害最严重的病害之一，由于长期进化的结果，不同性别的植物必然对生物和非生物胁迫有着不同的响应。本文以雌雄异株的青杨为模式植物，研究雌雄间在生理、生化、亚细胞结构和蛋白质水平上对生物和非生物胁迫的差异响应。主要研究结果如下： （1） 青杨雌雄植株对锈病胁迫的生理生化差异响应 在正常的对照组中，雄株叶片比雌株叶片有着较高的活性氧自由基产生速率、较高的SOD、POD、PPO 和较低的CAT 活性；在锈病感染的早期阶段， SOD、POD、CAT 活性、活性氧自由基产生速率、H2O2 含量、膜脂过氧化程度和细胞膜的电渗率在雌雄株中都增加，而PPO 仅在雄株中增加明显，APX 仅在雌株中增加明显，并且雌株比雄株有着更严重的锈病感染程度、细胞膜的伤害程度和光合系统II 的破坏程度，雌株有更多的净光合速率、气孔导度和叶绿素a 含量的降低，在同工酶变化上，雌雄间对锈病也显示出不同的表达模式。结果显示，雄株比雌株对锈病有着更好的抗性和更有效的ROS 清除系统。 （2） 青杨雌雄植株对干旱胁迫的生理生化及亚细胞结构的差异响应 与较好水分条件相比，干旱下雄株比雌株有着更高的A-Ci 响应参数，如Rubisco 最大羧化速率、光呼吸速率、暗呼吸速率和最大电子传递速率等。干旱显著地增加了膜脂过氧化程度和游离脯氨酸含量，并且雄株比雌株表现出较低的膜脂过氧化程度，较高的总蛋白和游离脯氨酸含量。无论是中度干旱还是极度干旱，除了CAT 外，雄株比雌株表现为较强的抗氧化酶活性，在同工酶谱带上，雌雄间表现出不同的变化模式，并且有些条带是干旱影响应的，而有些条带是性别特异性的，这些性别特异性条带能够作为鉴定性别快速而准确的标记。干旱显著地影响了线粒体、叶绿体和细胞壁的结构，尤其在中度干旱胁迫下，雄株线粒体和叶绿体比雌株呈现出较好的完整性，并且雄株细胞壁要比雌株更厚。因此， 雄株比雌株表现出更强的干旱忍耐性和更高效的抗氧化酶系统。 （3） 青杨雌雄植株对干旱胁迫的蛋白质组差异响应 用双相电泳检测到雌雄间近1000 个蛋白点，通过对比发现对照组雌雄间有54 个差异蛋白点，干旱下雌雄间有108 个差异点，其中102 个被质谱成功鉴定。对照组雌雄间的差异蛋白主要集中在与光合作用相关蛋白、抗氧化酶、胁迫防御蛋白和一些调节基因表达的蛋白；干旱胁迫下雌雄间差异蛋白明显增多，主要有参与信号转导、调节基因表达、蛋白质加工、转录产物的转录翻译后修饰的调节性蛋白蛋白和参与氧化还原平衡、抗胁迫、细胞壁合成、光合作用、能量代谢、氨基酸代谢和脂肪酸代谢等的功能性蛋白。干旱下这些蛋白的表达量在雌雄中有的表现出相同的表达模式，如干旱下雌雄株中Rubisco 激活酶、小热激蛋白等表达都增加，而有的表现出相反的表达模式，如Rubisco 大亚基的降解片段、羰酸酯酶等在雄株中表达量上调而在雌株中却是下调。因此，雌雄间在蛋白质水平上对干旱胁迫响应的差异是显著的，也是复杂的。 It is an interesting and novel topic that dioecious plants possess different responses to environmental stress. As for the different productive cost and different survive strategy, different sexual plants have shown obviously morphological, physiological and molecular differences. Drought is one of the most worldwidely important environmental stress factors that limit plant growth and ecosystem productivity. Rust disease is one of the economically important diseases in many trees. As a result of the long evolutionary process, male and female plants should show different responses to abiotic and biotic stress. In this paper, using a dioeious tree of Populus cathayana Rehd as a model, we study the sexual differences to drought and rust disease stress in physiological, biochemical, sub-cellular and proteomics levels. The main results are follows: (1) The sexual differences in physiology and biochemistry of poplar to rust disease In controls, males showed higher production of superoxide radicals, higher activities of SOD, POD, PPO and lower CAT activity. Under rust disease, the activities of antioxidant, the content of ROS and the degree of cellular member destroyed were increased in both sexes, except for PPO in diseased males and APX in diseased females. However, females showed more seriously disease severity and cellular member and PS II destroyed degrees. Net photosynthesis rate, transpiration rate and chlorophyll a content were decreased more in diseased females than in males. There were also some different changes inantioxidant isozymes under rust disease. The results suggested that male poplar possessed a more effectively antioxidant system and were more resistant to rut disease than females. (2) The sexual differences in physiology and biochemistry of poplar to drought stress Under drought stress, there were higher rates of RuBP-saturated CO2 assimilation, dark respiration, photorespiratory release of oxygen, the max electron transportrate in CO2-saturated and carboxylation efficiency in males than in females. And males showed lower TBARS and higher proline content. Except for CAT, the activities of other antioxidants were higher in males than in females. Meanwhile, there were obviously differences in isozyme changes between teo sexes. Drought stress obviously destroyed the integralities of chloroplasts and mitochondria and the sexual differences in sub-cellular level were obviously under the moderate water stress. Male cell walls were more sensitive to drought stress than did female. The results suggested males were more resistant to drought stress. (3) The sexual differences in proteomics of poplar to drought stress By 2-D and MS analysis, we identified 102 different protein spots between males and females. Under control conditions, the different proteins were mainly in photosynthesis related proteins, antioxidants, stress response proteins and some gene expression related proteins. Under drought stress, the different proteins were focused on (i) regulated proteins such as signaling conduction, kinase, HSP, gene expressional regulation and protein modification, (ii) functional proteins such as photosynthesis, energy metabolism, antioxidant, redox, stress response, lipid metabolism and amino acid metabolism. Some protein showed the same expressional pattern, while some showed contrary expressional pattern. Thus, the results suggested that sexual differences in proteomics were significant and complex.

Assessing genetic identity of sporophytic offspring of the brown alga Undaria pinnatifida derived from mono-crossing of gametophyte clones by use of amplified fragment length polymorphism and microsatellite markers

The haploid stage of gametophytes of the subtidal brown alga Undaria pinnatifida can be vegetatively propagated under favorable conditions. This unique characteristic makes it possible to establish independent gametophyte cell lines that are zoospore-derived. Sporophytic offspring can be generated through hybridizing the male and female gametophytes, which are derived from different cell lines. Accumulated experiences in this and other species in Laminariales demonstrated the applicability of this novel way to breed desired strains for open-sea cultivation. Sporophytic offspring originated from mono-crossing of male and female gametophyte clones were shown to have similar morphological characteristics under identical ambient conditions. However, there has been no report to relate this similarity on molecular levels. In this report, amplified fragment length polymorphism (AFLP) and microsatellite markers were used to analyze the genetic identity of sporophytic offspring of U. pinnatifida originated from two mono-crossing lines (M1 and M2), two self-breeding lines (S1 and S2) and one wild population (W). Totally 318 AFLP loci were revealed by use of 11 primer sets, of which 4.7%, 0.3%, 17.9%, 16.4% and 36.5% were polymorphic in M1, M2, S1, S2 and W, respectively. The pairwise genetic identity among the individuals of the same line was assessed. It was shown that offspring from mono-crossing lines had a higher degree of identity (95.6-100%) than self-breeding lines (87.7-98.4%) and the wild population (81.5-92.1%). Analysis by use of six microsatellite loci also revealed a higher genetic identity among individuals of the mono-crossing line, further confirming the results of AFLP analysis. Results from this investigation support, on molecular levels, the novel way to produce and maintain strains in U. pinnatifida by use of different gametophyte cell lines.

Effect of light period on egg-discharge of gametophyte clones of Undaria pinnatifida (Phaeophyta)

A sporeling culture method using gametophyte clones developed in the early 1990s led to egg discharge occurring in the dark 5 min after the start of the dark period under growth under a 11:13 L-D photoperiod. The course of egg discharge could be disturbed by light, with irradiance as low as 5-6 mu mol photon m(-2) s(-1) causing 75-80% of the discharged eggs to detach from the oogonia and consequently to die within several hours. In order to enhance outgrowth rate of young sporophytes, a study was conducted to test the effect of controlling darkness in the period 2-3 h after dusk. When the slides were transferred from the standard 11:13 L-D regime to continuous light, eggs were discharged 5 min after the end of the light phase and peaked 5-l5 min later on first day after transfer, indicating that the female gametes "remember" the light-dark regime. This suggests the existence of an endogenous circadian rhythm. During the second and third days, very few eggs were discharged throughout the 11 h of the normal light phase of the L-D regime, indicating the inhibitory effect of continuous light and that the rhtyhm is easily damped by light.

Mass culture of Undaria gametophyte clones and their use in sporeling culture

Undaria pinnatifida (Harv.) Sur. is one of the three main seaweed species under commercial cultivation in China. In the mid-1990s the annual production was about 20000 tons dry. The supply of healthy sporelings is key to the success of commercial cultivation of Undaria. Previous studies demonstrated that instead of the zoospore collection method, sporelings can be cultured through the use of gametophyte clones. This paper reports the experimental results on mass culture of clones and sporeling raising in commercial scale. Light had an obvious effect on growth of gametophyte clones. Under an irradiance of 80 mumol m(-2) s(-1) and favorable temperature of 22-25degreesC, mean daily growth rate may reach as high as 37%. Several celled gametophyte fragments were sprayed onto the palm rope frame. Gametogenesis occurred after 4-6 days. Juvenile sporeling growth experiments showed that nitrate and phosphate concentrations of 2.9 10(-4) mol 1(-1) and 1.7 10(-5) mol 1(-1) were sufficient to enable the sporelings to maintain a high daily growth rate. Sporelings can reach a length of 1 cm in a month. Since 1997, extension of the clone technique has been carried out in Shandong Province. Large-scale production of sporelings for commercial cultivation of 14 and 31 hectares in 1997 and 1998 had been conducted successfully.

A new method of Laminaria japonica strain selection and sporeling raising by the use of gametophyte clones

One-celled female and male gametophytes of three Laminaria japonica strains were isolated, cultured and gametophyte clones were formed. A technique combining strain selection with sporeling raising by the use of these female and male gametophyte clones was studied. Experiments on 9 different crossing combinations was conducted in November of 1997 in Qingdao, P. R. China. The main economic characteristics, frond length and fresh weight, of sporophytes of different crossing combinations were measured. F-1 sporophytes of No. 2 showed a higher fresh weight and longer length, therefore, No. 2 (Wh860 + x Lid) was selected as a good combination. Its parental female and male gametophyte clones are being mass cultured for sporeling production. By this method, the time needed for strain selection was shortened from 5-6 to 2 years. As compared with the routine method of sporeling raising by the collection of zoospores, the time of sporeling raising of this method decrease by 50%, and the production cost is also reduced by 50%. It is believed that this method will be labour and time saving and a more economic way for strain selection and sporeling raising in L. japonica cultivation industry.

Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.

Characterization of eastern oyster (Crassostrea virginica Gmelin) chromosomes by fluorescence in situ hybridization with bacteriophage P1 clones

Chromosome identification is an essential step in genomic research, which so far has not been possible in oysters. We tested bacteriophage P1 clones for chromosomal identification in the eastern oyster Crassostrea virginica, using fluorescence in situ hybridization (FISH). P1 clones were labeled with digoxigenin-11-dUTP using nick translation. Hybridization was detected with fluorescein-isothiocyanate-labeled anti-digoxigenin antibodies and amplified with 2 layers of antibodies. Nine of the 21 P1 clones tested produced clear and consistent FISH signals when Cot-1 DNA was used as a blocking agent against repetitive sequences. Karyotypic analysis and cohybridization positively assigned the 9 P1 clones to 7 chromosomes. The remaining 3 chromosomes can be separated by size and arm ratio. Five of the 9 P1 clones were sequenced at both ends, providing sequence-tagged sites that can be used to integrate linkage and cytogenetic maps. One sequence is part of the bone morphogenetic protein type 1b receptor, a member of the transforming growth factor superfamily, and mapped to the telomeric region of the long arm of chromosome 2. This study shows that large-insert clones such as P1 are useful as chromosome-specific FISH probes and for gene mapping in oysters.

农杆菌对毛白杨的转化及完整转化植株的获得

本文报道农杆菌转化毛白杨的高效遗传转化系统的建立。所用农杆菌菌株为：1.发根农杆菌R1000，含有Ri质粒pRiA4b。2.发根农杆菌R1000(pTVK85)，是菌株R1000中除含有pRiA4b外，并兼容一个带有超致病区(Supervirulent region)的质粒pTVK85。3.根癌农杆菌C58C1(pBZ693)，其质粒pBZ693是改建过的Ti质粒，载有T-DNA的基因1和基因2。将毛白杨外植体分别与上述菌株在MS+0.5ppm激动素培养基上先培养2天后，转移至MS+500ppm氨噻肟头胞霉素的培养基上。一个星期后即有根从外植体上产生。根癌农杆菌诱导的根形态明显与发根农杆菌诱导的根不同。R1000(pTVK85)诱导生根的外植体可占供试外植体总数的59%。转化的根有的可自发地形成不定芽或愈伤组织。通过培养基中激素的调整，可使转化的根系统100%再生出不定芽，并可由这些不定芽得到完整植株。转化植株的各克隆之间表型差异很大。有的地上部形态正常，仅根系与未转化植株有所不同。有的节间短、叶片多、顶端优势弱、根系发达而多发枝、多根毛。但所有转化植株皆无皱叶现象，其叶片形态与正常植株无异。普遍地有根生于植株的培养基平面以上部分的现象。取三个克隆的植株进行Southern杂交，其中两个为杂交阳性，表明确已被转化;另一个克隆为杂交阴性。

胡杨体细胞再生植株及耐机制的研究

胡杨（Populus euphratica Oliv.）是干旱荒漠风沙前治地区唯一分布的乔木树种，具有极强的抗逆性，突出地表现出较强的耐盐碱能力。由于胡杨在繁殖上存在问题，种子采后极易丧失生活力和无性扦插繁殖难以生根，加之人们对胡杨耐盐抗逆机制缺乏了解，应而极大地制约了这一珍贵抗逆种质资源的开发和利用，现有资源的保存也受到严重危胁。试验首先利用植物细胞工程技术开展了胡杨体细胞再生植株的系统研究，并在分子水平上就愈伤组织的培养和器官发生过程中表达的特异蛋白开展了深入工作。其次，对胡杨耐盐机制进行了研究，分析了胡杨细胞盐胁迫响应蛋白，开展了盐胁迫条件下细胞对离子吸收和分配特性以及与耐盐有关的形态结构的研究。这一工作的开展对于有效地保存、开发和利用胡杨种质资源，对于荒漠化治理，以及深入认识胡杨耐盐性、丰富和发展木本植物耐盐理论，具有十分重要的意义。 研究取得的主要结果如下： 1.较好地解决了胡杨试管培养中黄萎和退化等难以克服的问题，通过全面和系统的比较研究和对培养条件的优化，首次获得了高频率的和成熟的胡杨体细胞再生植株体系。胡杨愈伤组织、离体叶片和离体茎段不定芽再生频率分别可达82.9％、100％和83％，试管苗生根为86.2％。 2.提出了以愈伤组织表达蛋白状况作为判定其器官发生能力的观点，确定了三类愈伤组织和器官发生中三个不同分化阶段的蛋白分子标记。利用SDS-PAGE和IEF－SDS－PAGE对胡杨不同类型愈伤组织和愈伤组织分化不定芽过程的蛋白进行了研究。结果表明：不同类型愈伤组织中表达的蛋白存在着一定差异。在光下和BA／NAA为1诱导产生的具有较强器官发生能力的茎基愈伤组织，其蛋白组分明显地少于其它类型的愈伤组织，表明其分化程度较低。经过黑暗和BA／NAA为0.5的继代培养，愈伤组织产生了特异的24。5KD和58.6KD的标记蛋白，并且也表达了其器官发生时表达的19KD和31KD蛋白。说明愈伤组织经过继代培养其器官发生能力下降是与细胞分化程度增加相关的。茎基愈伤组织在光下和BA／NAA为5的条件下进行器官发生诱导，随着愈伤组织形成分生细胞团块和不定芽原基明显地表达了20KD和55KD蛋白带，并且20KD蛋白中包含有特异的pI为5。5－6.5的蛋白。43KD和pI为6.5-7.5的蛋白为器官发生前期蛋白。本文不愈伤组织表达蛋白状况与器官发生能力间关系进行了讨论。 3.分离和鉴定了胡杨细胞盐胁迫响应蛋白，从蛋白表达上证实盐胁迫对胡杨细胞产生的影响明显地分为渗透胁迫和离子伤害胁迫两种效应。对悬浮培养的胡杨细胞进行NaCL和PEG(6000)胁迫处理，SDS－PAGE分析表明：NaCL和PEG胁迫处理的细胞均明显地表达了28KD和59KD蛋白带，表明28KD和59DK蛋白是与渗透胁迫有关的。66KD和60KD蛋白带仅在高水平盐胁迫细胞中显著表达，应而是与盐胁迫中离子伤害有关的蛋白。进一步证实胡杨细胞中28KD和66KD蛋白带表达受ABA诱导。通过IEF－SDS－PAGE证实，28KD蛋白包含有pI为8.0-9.0的蛋白，渗透胁迫和离子胁迫相关的分离和鉴定为通过蛋白途径克隆与渗透胁迫和离子胁迫相关基因，为深入认识胡杨耐盐机制奠定了基础。 4.通过X－射线细胞微区分析以及与毛白杨细胞比较发现，胡杨细胞对培养介质中高浓度的盐离子具有较强的拒吸作用和一定的忍耐性。胡杨细胞中液泡不具有积聚离子的功能，细胞分室性渗调节作用不明显。胡杨细胞膜对离子进入具有选择功能，表现在培养介质中Na和CL离子进入细胞和由细胞质进入液泡不以等摩尔数形式进行，进入的CL离子比Na离子约高50％，说明了二者通过质膜是由不同机制控制的，是分开进行的，也说明胡杨细胞拒Na离子强于拒CL离子。另外胡杨细胞受到盐胁迫时还表现出比较强的维持细胞内离子平衡的功能。正是由于上述特性，才赋予了胡杨细胞具有较强的耐盐性。 5.利用电子显微镜和光学显微镜中相差和微分干涉等技术，对胡杨细胞和组织结构进行了观察。与毛白杨细胞相比，胡杨细胞中具有较丰富的线粒体和质体，盐胁迫和渗透胁迫均明显地提高了细胞质中线粒体数和质体数，并使质体中内含体增多，细胞质中和液泡内缘出现明显的嗜饿物质。研究还发现，胡杨细胞膜与细胞壁之间呈齿状结合，说明了膜与壁之间结合的牢固性和稳定性，解释了胡杨细胞在胁迫中不易发生质壁分离的原因。胡杨细胞在受到盐或渗透胁迫时，细胞内出现明显的丝状结晚，细胞核变大，核仁明显。在器官和组织结构方面，胡杨根系具有发达的根冠和根内皮层，根毛较多，叶片输导组织不发达等。这些结构的存在与胡杨的抗逆性是密切相关的。文中从形态结构上阐述了胡杨的耐盐碱特性。

盐渍条件下野大豆群体的DNA变异

当前分子生物学的方法以惊人的速度渗透到生命科学研究的各个领域。植物对不断变化的环境逐步适应的过程中，积累了丰富的遗传多样性。与此同时，人类活动空间的不断扩大已经严重威胁到其他生命的生存和繁衍，越来越多的物种以越来越快的速度在我们还没有来得及认识它们时就已经永远地消失了。加快物种鉴定和保护的步伐就必须发展更多能充分揭示物种遗传多样性的实验技术，从具有丰富遗传多样性的野生资源中寻找到更多能够服务于人类可持续发展的基因资源。本文以杨树杂交后代过氧化物同工酶和RAPD分析为基础，论证了我们改进的RAPD方法用于遗传分析的可行性。在前期工作的基础上，进一步测定了野大豆自然群体的耐盐性变异，并且用微卫星和RAPD分析的方法研究分子标记与DXA变异、植株耐盐性之间的关系。对四个可能与抗盐性有关的RAPD片段进行克隆、测序，并进行序列比较。由此得出以下结论： 1、在本文的实验条件下，杨树同工酶和RAPD分析均表明，RAPD标记在亲本及其杂交后代中性状比例符合孟德尔遗传规律，尽管有时也会出现遗传负载等机制引起的基因分布扭曲现象。 2、初步研究了个体发育阶段和环境条件对植株耐盐性的影响。结果表明，植物耐盐性不仅仅与外界的盐度有关，而且受发育阶段和其它环境条件（如，温度）的影响。但也发现了某些个体在各种条件下都具有较高的耐盐性，而且，不易受到其它环境条件的影响。 3、微卫星标记的结果表明，10对引物中的8对引物共检测到时17个等位基因，平均每对引物2.125个等位基因。本文的实验条件下，双核苷酸和三核苷酸的引物对扩增产物都没有出现“ghosts"条带或“打滑”现象。 4、有4个RAPD标记可能与野大豆群体的耐盐性有关，分别是OPCO8460bp、OPCO8213bp、OPCO2690bp、以及OPCO5270bp。测序结果与GenBank中的序列作同源性比较，结果显示，OPCO2_(690bp)与小麦、松树等植物的吉普赛性的逆转录转座子的部分区域（24－－53）有很高的同源性（86－89％）。此外，OPCO2690bp与栽培大豆胞质谷氨酰胺合成酶（gs15）基因的启动子有高达95％的同源性。 5、本文实验条件下，RAPD扩增产物在限制性内切酶消化后，消化产物的多态性未见增大，也没有发现与耐盐性相关的多态位点。 6、野大豆自然群体DNA变异的研究中也可以应用SWAPP方法。

毛乌素沙地中间锦鸡儿根瘤菌遗传多样性对柠条生态系统功能的影响

生物多样性科学（BiodiversityScience）的国际规划提出生物多样性对生态系统功能的影响是整个研究计划五大核心的核心.生物多样性包括遗传、物种和生态系统三个水平,其中遗传多样性是其它两个水平多样性的基础和最终来源.该文在实验室多年研究毛乌素沙地柠条遗传多样性的基础上,分别从表型（生理生化）、蛋白质、同工酶以及遗传型（rDNA）水平探讨中间锦鸡儿根瘤菌的遗传多样性,并模拟沙地生境,建立人工共生体系,以期发现最有效的共生伙伴关系,这不仅有得提高毛乌素地区农牧业产量,更重要的是在当今沙尘暴肆虐的情况下,发挥柠条防风固沙的能力具有现实意义. 1.毛乌素沙地中间锦鸡儿根瘤菌遗传多样性(1)全细胞可溶性蛋白质谱将供试中间锦鸡儿根瘤菌菌株分为两大类群，其中硬梁覆沙地菌株GH72不同于来自沙丘顶部和底部的菌株，而且中间锦鸡儿根瘤菌独立于参比菌株。酯酶同工酶谱分析表明，中间锦鸡儿根瘤菌与参比菌株仅存在一个等位酶位点差异，其余等位点与参菌株共享，因此，酯酶同工酶反映出中间锦鸡儿根瘤菌的异质性。(2)16SrDNA部分序列与16S-23S rDNA IGS结果表明，所有供试菌株扩增产物均较前人报道的分子量偏高。经16S rDNA PCR-RFLP分析，中间锦鸡儿根瘤菌共形成12种基因型，表现出丰富的遗传多样性，其中属于基因型2的菌株占42.4%。代表菌株GH33 16S rDNA全序列结果显示，与已知的快生型根瘤菌同源性在95%以上。(3）中间锦鸡儿根瘤菌生理生化反应特性B．T．B实验证明所有中间锦鸡儿根瘤菌均产酸，符合快生型根瘤菌的特征．唯一碳源测试显示，95%中间锦鸡儿根瘤菌不利用淀粉，33%菌株不利用乳糖，对其他测试碳源不具有选择性。检洲在不同盐离子浓度、不同酸性梯度以及不同温度条件下菌株生长状况，发现毛乌素沙地中间锦鸡儿根瘤菌具极强的耐盐性．53.8%的菌株可以在9%NaCl的YMA培养基生长．75%的菌株在pH4．O和pHl0,0 环境中仍能生长，66.7%菌株在60℃处理1 0min后仍具有生活力。体现出对于干旱沙地的适应。 2．不同实验共生系统中植物和根瘤菌对生态系统功能的影响14株根瘤菌分与三个柠条种（小叶锦鸡儿，中间锦鸡儿和柠条锦鸡儿）回接，用土壤上覆沙模拟毛乌索沙地景观生态条件，以多石砾贫瘠土壤为对照，比较不同基因型柠条与根瘤菌人工共生体的长和结瘤与生境的关系，初步证明根瘤菌很可能是该生态系统的关键种。寄主植物与共生根瘤菌的遗传多样性对生态系统功能的影响与生态环境有关。实验还表明，选择适当的共生组合对于防治沙漠化有很大潜力。3．银染变性聚丙烯酰胺凝胶电泳检测RAPD遗传模式以85株小钻杨F2代为材料，用本实验室改良的银染变性聚丙烯酰胺凝胶电泳法检测RAPD遗传模式。结果表明，仅用9个引物共扩增到399个位点，其中98个位点表现为多态性，卡方测验显示，79个多态位点符合经典的孟德尔遗传(3：1)，占多态位点80.6%。这种改良的检测RAPD标记的方法必将推动RAPD标汜构建连锁图谱的进程。

北京市土壤--植物系统重金属硫的分布迁移相互间关系和主成份分析

本文选择了北京市香山北京植物园、北京动物园、长安街和北京化工厂4个功能区和国槐（Sophora japonica (L) Beauv)、毛白杨 (populus tomentosa Carr)、丁香(Syringa Oblata)、野牛草(Buohloe dactyloides (Nutt) Engelm)4种植物;采集植物样品105个、土壤样品40个;测定了各样品的镍(Ni)、锌(Zn)、铜(Cu)、镉(Cd)、铅(Pb)、铬 (Cr)、硫(S)7种元素含量;通过对样品元素含量的直观分析、回归分析、主成份分析(PCA)，得出以下结论：各样品各元素含量的最高值或异常值多出现在化工厂内，其次是长安街、动物园、北京植物园无;各功能区各样品各元素含量大小顺序多是：化工厂><长安街>动物园><北京植物园：表明化工厂内土壤、植物受污染的程度较重，其次是长安街，动物园稍轻，北京植物园则较洁净。土壤中7种元素分布特征是：S > Zn > Pb > Cr > Cu > Ni > Cd;植物中7种元素分布特征是：S > Zn >< Cu > Pb > Cr >< Ni >< Cd。7种植物样品对S、Zn、Cu、 Cd具较大的富集系数;对Ni、Pb、Cr具较小的富集系数;说明前者比后者在土壤—植系统中较易迁移。7种植物样品对S均具积累效应;毛白杨叶对zn，毛白杨叶、枝、国槐皮对Cd，野牛草对Cu也都具积累效应。国槐各器官元素含量分配是：皮含量>叶含量>枝含量;毛白杨各器官元素含量分配是：叶含量>枝含量;表明植物各器官中，枝元素含量较低。对元素含量回归分析，发现植物各器官元素含量间具显著性正相关系;植物中元素含量与土壤中元素含量绝大多数呈正相关。前者说明植物各器官生长发育的正相关性;后者说明土壤条件与植物生长发育的正相关性。对元素含量的主成份分析和样品排序，揭示出采自同一功能区的样品大都可形成一集团，但尚存在交叉、重叠和过渡的样品;化工厂集团多处在排序图Z1轴左边，长安街集团多处在Z2轴上方，北京植物园集团多处在Z1轴右边或Z2轴下方。可明确得出，化工厂土壤、植物污染较重（元素含量较高），其次是长安街;动物园污染稍轻，北京植物园较洁净的结论。还可知，化工厂、长安街某些样点污染较轻;而北京植物园、动物园某些样点污染较重。