空间培养箱中实时观察GFP标记开花基因表达方法与技术

绿色荧光蛋白（Green Fluorescent Protein,GFP）已被广泛用作一种强有力的生物发光报告蛋白.但是,其在空间植物培养箱中应用尚存很大困难.本文提出一种在空间植物培养箱中利用LED作为激发光源,对拟南芥中开花基因启动子控制下GFP基因表达的观察与分析方法,为在空间生物学实验中研究基因表达模式提供了新的方法和技术.      

微重力对拟南芥长势影响的代谢网络流平衡分析

微重力作为典型的空间环境因素,对植物生长发育的影响机制是空间生命科学的研究热点。微重力环境直接或间接影响植物代谢,并引起许多生理适应。 随着系统生物学的发展,代谢网络模型使微重力环境下的植物代谢建模成为可能。采用流平衡分析方法对模式植物拟南芥不同组织的代谢网络进行分析,研究微重力对拟南芥生长发育的影响机制。通过比较空间与地面条件下拟南芥的生物质产量,发现空间条件下拟南芥黄化幼苗、幼苗、芽、根、下胚轴的生物量分别下降了33.00%,51.52%,6.89%,12.53%,11.70%,与空间环境下拟南芥的长势变化趋势一致。代谢通路富集分析发现,微重力使得拟南芥的碳固定等通路下调,而磷酸戊糖途径上调,初步解析了微重力对拟南芥生长发育的影响机制,也验证了流平衡方法用于微重力生物学效应研究中的可行性。      

微重力环境下植物培养箱设计

基于空间微重力下植物的生物学效应及其微重力信号转导研究需要,在微重力条件下培养拟南芥,获得经微重力条件生长的拟南芥样品.在空间实验过程中实时采集、存储和传输植物样品的数字图像,并根据生物样品的生长周期对生物样品进行低温固定和储存,再由返回式卫星带回地面,开展微重力植物生物学效应研究.      

太阳风高速流能流分布

根据Ylysses观测,比较完整地计算了高纬行星际空间太阳风能流分布．计算表明,行星际空间的高速流能流密度约为2．1×10-3J·m-2·s-1（日心距离r＝1AU）,主要来自于太阳风离子所携带的动能流（占58％）和克服太阳引力的势能流（占39％）．要驱动高速流,需要在日冕底部高速流源区（日心距离r=1Rs）向外输出到太阳风的能流密度为7．1×102J·m-2·s－1·分析表明,由日冕底部向外可能输出两种形式的能流,其中一种具有较短的耗散长度,被耗散在很短的空间区域（～1—2Rs）,使日冕温度迅速提高。另一种储藏在连续向外传播的太阳风中,不断耗散用以加速太阳风（＞2Rs）．     

基于LED光源的运动式太阳模拟器控制系统

为了解决编码式太阳敏感器装星后现场的功能测试,研制了一种基于LED光源的运动式太阳模拟器,为该测试提供模拟的太阳光信号和太阳光矢量信号.基于太阳模拟器组成与工作原理,对光源辐亮度和矢量运动的控制系统进行研究.根据太阳光信号要求,通过光源选取与功率计算确定LED型号和个数,并采用压控恒流源驱动技术对光源辐亮度进行线性调节.根据太阳光矢量信号要求,通过负载扭矩与功率计算选取GUS-60型超声电机,采用16位绝对式编码器对运动角度进行测量,以数字信号处理器为主要器件对电机进行闭环反馈控制.测试结果表明,光源控制系统能够实现辐亮度在0～527.4W·m-2内线性可调,矢量运动装置在-15°～40°内的运动角度控制精度优于±0.01°,满足编码式太阳敏感器的测试要求.      

受控生态生保系统内红萍供氧特性研究

研究红萍载人供O₂特征, 为红萍生物部件进行系统总体地面模拟试验及空间应用奠定基础,构建了受控生态生保系统密闭试验舱和红萍栽培装置, 在“红萍-鱼-人”共存情况下, 测定密闭舱内O₂, CO₂浓度的变化. 试验结果显示, 单位重量的鱼耗O₂量. 0805～0.0831 L·kg-1·h-1, 排放CO₂量为0.0705～0.0736 L·kg-1·h-1; 试验志愿者耗O₂量19.71 L·h-1, 呼吸释放CO₂量18.90 L·h-1. 人工光照保持7000～8000 lx条件下, 红萍的光合作用与人和鱼的呼吸作用相辅相成, 舱内O₂, CO₂浓度趋于平衡. 密闭舱内CO₂浓度升高对促进红萍群体净光合效率有明显效果, 红萍光合放O₂能力很强, 能有效促使密闭舱内O₂, CO₂浓度朝着有利于人生存的环境方向平衡, 进而验证了红萍的空间应用前景.      

红萍湿养栽培供O2装置研制

红萍作为空间站受控生态生命保障系统中的生物部件, 可望为航天员提供O2和新鲜蔬菜并吸收CO2. 研究红萍湿养栽培供O2装置, 旨在建立地面非生物部件, 满足模拟研究的需要. 介绍了所研发装置及关键部件的结构特点和工作原理. 通过红萍湿养板内湿养栽培介质的结构功能设计, 在蓄水保水基质层内部配置具有毛细作用的渗水管路, 介质始终保持整体湿润而表面无明水状态, 为红萍扎根稳固、营养吸收和生长繁殖创造条件. 水压试验确定了渗水管路的主要技术参数和闭合式红萍培养液输配循环系统的间歇循环周期. 整机产出量试验结果表明, 在层间距125 mm, 整机红萍湿养面积6.3 m2, 超高亮度白色LED人工光源能耗152 Wm 2, 红萍表面的光照强度6000~6500 lx, 整机的红萍湿养产量、红萍放O2量和吸收CO2量相应大幅提高, 装置各项性能指标均达到设计要求.      

非相干散射雷达的空间碎片参数统计分析

采用匹配滤波方法处理了非相干散射雷达的原始采样数据(时长约7h), 共检测到394个空间碎片, 估算了其轨道高度、径向速度、散射截面、等效直径及径向加速度等参数, 统计分析了这些参数的变化特征, 得到穿过雷达 波束的空间碎片流量约为60h-1, 信噪比为10～1000, 空间碎片主要分布在600～1100km和1400～1600km两个高度区间, 散射截面 10-5～10-2m2, 等效直径3～10cm, 径向速度-1.5～1.5km·s-1, 径向加速度20～90m·s-2, 这对于中国的空间碎片探测与研究具有重要参考意义.      

空间高等植物培养装置

空间高等植物培养装置用于中国天宫二号空间实验室开展微重力条件下高等植物生长机理研究.该装置由高等植物培养模块、生命保障模块、实时在线检测模块和返回单元等功能单元组成,可实现高等植物空间长周期培养,在轨启动生物实验,实时在线观察和荧光监测,水分循环利用及营养供给,模拟太阳长短日照周期控制与检测,环境温度测量与控制,CO₂浓度调节,有害气体去除及航天员回收部分样品等功能.      

三维回转器回旋条件下拟南芥种子发育分析

向重力性反应是植物适应地球重力环境的一个重要生理过程, 是植物正常生长发育不可缺少的反应机制, 但是, 微重力是否影响植物种子发育至今尚无一致性结论. 本文研究了三维回转器回旋模拟微重力对拟南芥种子发育(胚胎发育与代谢活动)的影响. 研究结果表明, 三维回转条件下, 拟南芥果荚出现不规则弯曲或扭曲形态, 形成的种子中可溶性糖和淀粉含量明显增加, 盐溶性贮藏蛋白质含量显著降低而碱溶性蛋白质含量显著升高, 球形胚时期的种子对三维回转处理最为敏感. 对球形胚时期的植株进行短期的三维回转处理可抑制胚柄细胞的分裂和伸长, 而胚柄伸长受阻可能会影响到营养物质向胚胎中输送, 进而导致部分胚胎败育. 对种子干燥脱水阶段的植株进行三维回转处理不影响胚胎的发育, 但会导致种子贮藏蛋白质含量下降.      

Evaluation of light emitting diode characteristics for a space-based plant irradiation source.

Light emitting diodes (LEDs) are a promising irradiation source for plant growth in space. Improved semiconductor technology has yielded LED devices fabricated with gallium aluminum arsenide (GaAlAs) chips which have a high efficiency for converting electrical energy to photosynthetically active radiation. Specific GaAlAs LEDs are available that emit radiation with a peak wavelength near the spectral peak of maximum quantum action for photosynthesis. The electrical conversion efficiency of installed systems (micromole s-1 of photosynthetic photons per watt) of high output LEDs can be within 10% of that for high pressure sodium lamps. Output of individual LEDs were found to vary by as much as 55% from the average of the lot. LED ratings, in mcd (luminous intensity per solid angle), were found to be proportional to total photon output only for devices with the same dispersion angle and spectral peak. Increasing current through the LED increased output but also increased temperature with a consequent decrease in electrical conversion efficiency. A photosynthetic photon flux as high as 900 micromoles m-2 s-1 has been produced on surfaces using arrays with LEDs mounted 7.6 mm apart, operating as a current of 50 mA device-1 and at an installed density of approximately 17,200 lamps m-2 of irradiated area. Advantages of LEDs over other electric light sources for use in space systems include long life, minimal mass and volume and being a solid state device.     

Light intensity and production parameters of phytocenoses cultivated on soil-like substrate under controlled [correction of controled] environment conditions.

To increase the degree of closure of biological life support systems of a new generation, we used vermicomposting to involve inedible phytomass in the intra-system mass exchange. The resulting product was a soil-like substrate, which was quite suitable for growing plants (Manukovsky et al. 1996, 1997). However, the soil like substrate can be regarded as a candidate for inclusion in a system only after a comprehensive examination of its physical, chemical, and other characteristics. An important criterion is the ability of the soil-like substrate to supply the necessary mineral elements to the photosynthesizing component under the chosen cultivation conditions. Thus, the purpose of this work was to study the feasibility of enhancing the production activity of wheat and radish crops by varying the intensity of photosynthetically active radiation, without decreasing the harvest index. The increase of light intensity from 920 to 1150 micromoles m-2 s-1 decreased the intensity of apparent photosynthesis of the wheat crops and slightly increased the apparent photosynthesis of the radish crops The maximum total and grain productivity (kg/m2) of the wheat crops was attained at the irradiance of 920 micromoles m-2 s-1. Light intensity of 1150 micromoles m-2 s-1 decreased the productivity of wheat plants and had no significant effect on the productivity of the radish crops (kg/m2) as compared to 920 micromoles m-2 s-1. The qualitative and quantitative composition of microflora of the watering solution and substrate was determined by the condition of plants, developmental phase and light intensity. By the end of wheat growth under 1150 micromoles m-2 s-1 the numbers of bacteria of the coliform family and phytopathogenic bacteria in the watering solution and substrate were an order of magnitude larger than under other illumination conditions. The obtained data suggest that the cultivation of plants in a life support system on soil-like substrate from composts has a number of advantages over the cultivation on neutral substrates, which require continual replenishment of the plant nutrient solution from the system's store to complement the macro- and micro-elements. Yet, a number of problems arise, including those related to the controlling of the production activity of the plants by the intensity of photosynthetically active radiation. It is essential to understand why the intensity of production processes is limited at higher irradiation levels and to overcome the factors responsible for this, so that the soil-like substrate could have an even better chance in the competition for the best plant cultivation technology to be used in biological life support systems.     

Red light-induced suppression of gravitropism in moss protonemata.

Moss protonemata are among the few cell types known that both sense and respond to gravity and light. Apical cells of Ceratodon protonemata grow by oriented tip growth which is negatively gravitropic in the dark or positively phototropic in unilateral red light. Phototropism is phytochrome-mediated. To determine whether any gravitropism persists during irradiation, cultures were turned at various angles with respect to gravity and illuminated so that the light and gravity vectors acted either in the same or in different directions. Red light for 24h (> or = l40nmol m-2 s-1) caused the protonemata to be oriented directly towards the light. Similarly, protonemata grew directly towards the light regardless of light position with respect to gravity indicating that all growth is oriented strictly by phototropism, not gravitropism. At light intensities < or = l00nmol m-2 s-1, no phototropism occurs and the mean protonemal tip angle remains above the horizontal, which is the criterion for negative gravitropism. But those protonemata are not as uniformly upright as they would be in the dark indicating that low intensity red light permits gravitropism but also modulates the response. Protonemata of the aphototropic mutant ptr1 that lacks a functional Pfr chromophore, exhibit gravitropism regardless of red light intensity. This indicates that red light acts via Pfr to modulate gravitropism at low intensities and to suppress gravitropism at intensities < or = 140nmol m-2 s-1.     

The effect of gravity on surface temperature and net photosynthetic rate of plant leaves.

To clarify the effects of gravity on heat/gas exchange between plant leaves and the ambient air, the leaf temperatures and net photosynthetic rates of plant leaves were evaluated at 0.01, 1.0, 1.5 and 2.0 G of 20 seconds each during a parabolic airplane flight. Thermal images of leaves were captured using infrared thermography at an air temperature of 26 degrees C, a relative humidity of 15% and an irradiance of 260 W m-2. The net photosynthetic rates were determined by using a chamber method with an infrared gas analyzer at an air temperature of 20 degrees C, a relative humidity of 50% and a photosynthetic photon flux of 0.5 mmol m-2 s-1. The mean leaf temperature increased by 1 degree C and the net photosynthetic rate decreased by 13% with decreasing gravity levels from 1.0 to 0.01 G. The leaf temperature decreased by 0.5 degree C and the net photosynthetic rate increased by 7% with increasing gravity levels from 1.0 to 2.0 G. Heat/gas exchanges between leaves and the ambient air were more retarded at lower gravity levels. A restricted free air convection under microgravity conditions in space would limit plant growth by retarding heat and gas exchanges between leaves and the ambient air.     

Preparations for CELSS flight experiments with wheat.

We are planning a short-term experiment with Superdwarf wheat on the U.S. Space Shuttle and a seed-to-seed experiment on the Russian Space Station Mir. The goals of both experiments are to observe effects of microgravity on developmental steps in the life cycle and to measure photosynthesis, respiration, and transpiration by monitoring gas exchange. This requires somewhat different hardware development for the two experiments. Ground-based research aims to understand plant responses to the environments in the space growth chambers that we will use (after some modification): the Plant Growth Unit (PGU) on the shuttle and units called Svet, Svetoblock 2, or Oasis on Mir. Low irradiance levels (100 to 250 micromoles m-2 s-1 at best) pose a particular problem. Water and nutrient supply are also potentially limiting factors, especially in the long-term experiment. Our ground-based studies emphasize responses to low light levels (50 to 400 micromoles m-2 s-1); results show that all developmental steps are delayed by low light compared with plants at 400 micromoles m-2 s-1. We are also testing various rooting substrates for the shuttle experiment. A 1:1:1 mixture of peat:perlite:vermiculite appears to be the best choice.     

Effects of air velocity on photosynthesis of plant canopies under elevated CO2 levels in a plant culture system.

To obtain basic data for adequate air circulation for promoting plant growth in closed plant production modules in bioregenerative life support systems in space, effects of air velocities ranging from 0.1 to 0.8 m s-1 on photosynthesis in tomato seedlings canopies were investigated under atmospheric CO2 concentrations of 0.4 and 0.8 mmol mol-1. The canopy of tomato seedlings on a plug tray (0.4 x 0.4 m2) was set in a wind-tunnel-type chamber (0.6 x 0.4 x 0.3 m3) installed in a semi-closed-type assimilation chamber (0.9 x 0.5 x 0.4 m3). The net photosynthetic rate in the plant canopy was determined with the differences in CO2 concentrations between the inlet and outlet of the assimilation chamber multiplied by the volumetric air exchange rate of the chamber. Photosynthetic photon flux (PPF) on the plant canopy was kept at 0.25 mmol m-2 s-1, air temperature at 23 degrees C and relative humidity at 55%. The leaf area indices (LAIs) of the plant canopies were 0.6-2.5 and plant heights were 0.05-0.2 m. The net photosynthetic rate of the plant canopy increased with increasing air velocities inside plant canopies and saturated at 0.2 m s-1. The net photosynthetic rate at the air velocity of 0.4 m s-1 was 1.3 times that at 0.1 m s-1 under CO2 concentrations of 0.4 and 0.8 mmol mol-1. The net photosynthetic rate under CO2 concentrations of 0.8 mmol mol-1 was 1.2 times that under 0.4 mmol mol-1 at the air velocity ranging from 0.1 to 0.8 m s-1. The results confirmed the importance of controlling air movement for enhancing the canopy photosynthesis under an elevated CO2 level as well as under a normal CO2 level in the closed plant production modules.     

Toluene removal from air by Dieffenbachia in a closed environment.

Higher plants are likely to play a major role in bioregeneration systems for food, air and water supplies. Plants may also contribute by the removal of toxic organic substances from the air of a closed environment. Dieffenbachia amoena plants were exposed to 0 to 1.2 x 10(6) micrograms toluene m-3 at light intensities of 35 and 90 micromoles m-2 s-1 in sealed chambers. Toluene removal, photosynthesis and respiration were measured. An increased light intensity increased the rate of toluene removal five-fold over the rate at the lower intensity; the kinetics suggest active regulation by the plant. The removal rate saturated at 2700 micrograms toluene h-1 at the lower intensity and failed to saturate at the higher intensity. Toluene exposure inhibited photosynthesis and respiration only transiently and without correlation to toluene concentration. These plants can act as efficient scavengers of toluene in a contaminated environment.     

Gravity-dependent reactions of the moss Pohlia nutans protonemata.

In darkness, protonemata of Pohlia nutans (Hedw.) grew negatively gravitropically (upwards). However, not all filaments became gravitropic immediately after transfer to darkness. Some of them (~20%) for several days grew in different directions with respect to gravity. The apical cells of those protonemata predominantly contained multiple chloroplasts. The intensity of chlorophyll fluorescence rapidly decreased in the apical cells of such protonemata while starch content increased in comparison with upright growing protonemata. Light, especially in the red and blue part of the spectrum, inhibited protonemal gravitropism. Red light induced stronger inhibitory effects than blue light. Red light of 1.0 to 1.5 micromoles m-2 s-1 intensity induced bud differentiation in apical cells on almost all side branches of main protonemal filaments. Bright fluorescence of F-actin bundles in the tip of apical protonematal cells and a delicately fluorescing network enclosing plastids basal to the tip in a sedimentation zone were visualized. Bright fluorescence of actin as local patches and fine prominent axially oriented bundles was observed in cells of gametophore buds.     

Volatile metabolites of higher plant crops as a photosynthesizing life support system component under temperature stress at different light intensities.

The effect of elevated temperatures of 35 and 45 degrees C (at the intensities of photosynthetically active radiation 322, 690 and 1104 micromoles m-2 s-1) on the photosynthesis, respiration, and qualitative and quantitative composition of the volatiles emitted by wheat (Triticum aestuvi L., cultivar 232) crops was investigated in growth chambers. Identification and quantification of more than 20 volatile compounds (terpenoids--alpha-pinene, delta 3 carene, limonene, benzene, alpha- and trans-caryophyllene, alpha- and gamma-terpinene, their derivatives, aromatic hydrocarbons, etc.) were conducted by gas chromatograph/mass spectrometry. Under light intensity of 1104 micromoles m-2 s-1 heat resistance of photosynthesis and respiration increased at 35 degrees C and decreased at 45 degrees C. The action of elevated temperatures brought about variations in the rate and direction of the synthesis of volatile metabolites. The emission of volatile compounds was the greatest under a reduced irradiation of 322 micromoles m-2 s-1 and the smallest under 1104 micromoles m-2 s-1 at 35 degrees C. During the repair period, the contents and proportions of volatile compounds were different from their initial values, too. The degree of disruption and the following recovery of the functional state depended on the light intensity during the exposure to elevated temperatures. The investigation of the atmosphere of the growth chamber without plants has revealed the substances that were definitely technogenic in origin: tetramethylurea, dimethylsulfide, dibutylsulfide, dibutylphthalate, and a number of components of furan and silane nature.     

Plant experiments with light-emitting diode module in Svet space greenhouse

Light is necessary for photosynthesis and shoot orientation in the space plant growth facilities. Light modules (LM) must provide sufficient photosynthetic photon flux for optimal efficiency of photosynthetic processes and also meet the constraints for power, volume and mass. A new LM for Svet space greenhouse using Cree® XLamp® 7090 XR light-emitting diodes (LEDs) was developed. Monochromic LEDs emitting in the red, green, and blue regions of the spectrum were used. The LED-LM contains 36 LED spots – 30 LED spots with one red, green and blue LED and 6 LED spots with three red LEDs. Digital Multiplex Control Unit controls the LED spots and can set 231 levels of light intensity thus achieving Photosynthetic Photon Flux Density (PPFD) in the range 0–400 μmol m⁻² s⁻¹ and different percentages of the red, green and blue light, depending on the experimental objectives. Two one-month experiments with plants – lettuce and radicchio were carried out at 400 μmol m⁻² s⁻¹ PPFD (high light – HL) and 220 μmol m⁻² s⁻¹ PPFD (low light – LL) and 70% red, 20% green and 10% blue light composition. To evaluate the efficiency of photosynthesis, in vivo modulated chlorophyll fluorescence was measured by Pulse Amplitude Modulation (PAM) fluorometer on leaf discs and the following parameters: effective quantum yield of Photosystem II (Φ_PSII) and non-photochemical quenching (NPQ) were calculated. Both lettuce and radicchio plants grown at LL express higher photochemical activity of Photosystem II (PSII) than HL grown plants, evaluated by Φ_PSII. Accelerated rise in NPQ in both LL grown plants was observed, while steady state NPQ values were higher in LL grown lettuce plants and did not differ in LL and HL grown radicchio plants. The extent of photoinhibition process in both plants was evaluated by changes in malonedialdehyde (MDA) concentration, peroxidase (POX) activity and hydrogen peroxide (H₂O₂) content. Accumulation of high levels of MDA and increased POX activity correlating with decreased H₂O₂ content were observed in both HL grown plants. These biochemical indicators revealed higher sensitivity to photodamage in HL grown lettuce and radicchio plants. LL conditions resulted in more effective functioning of PSII than HL when lettuce and radicchio plants were grown at 70% red, 20% green and 10% blue light composition.