长白山阔叶红松林与大气间CO2及H2O交换的过程与模拟

在全球变化背景下，森林生态系统CO2和H2O交换过程已经成为国际研究的前沿问题。本研究以长白山阔叶红松林为研究对象，利用基于生理生态学过程的多层模型，考虑了混交林中不同树种的生物量、冠层高度和厚度，对冠层及生态系统尺度的碳水通量进行了详细模拟及分析，同时用涡动相关实测数据对模拟结果进行检验，并模拟和预测了阔叶红松林生态系统碳水收支对气候变化的响应，以期为我国研究区域甚至更大尺度碳水平衡的时间和空间格局特征提供模型储备。本文的主要结论如下： 1）长白山阔叶红松林主要优势树种红松、紫椴、水曲柳、色木槭和蒙古栎的生理生态学参数（初始量子效率α、光饱和时的最大净光合作用速率Pmax、最大Rubisco催化反应速率Vcmax、CO2饱和时的最大净光合作用速率Jmax）有着明显且不同的季节变化。6、8月的α值较大，Pmax、Vcmax和Jmax的最大值也出现在6—8月，而5、9月的各项参数值均较小。 2）对阔叶红松林冠层结构的观测，发现各树种在冠层中所处位置有明显差异。假设各树种水平分布均匀，以各树种的冠高、冠厚和叶片生物量为依据，将冠层垂直分为20层，模拟出各层各树种的CO2和H2O通量。这与传统的假设各树种垂直均匀分布相比，更加符合阔叶红松林的实际群落结构。 3）长白山阔叶红松林的能量平衡比EBR为0.800，居于国际同类观测的中上水平，涡相关观测数据较为可靠。CO2和H2O通量有明显日变化，夜间值较小且变化平缓，白天值呈单峰形的日变化。对2003—2007年生长季的模拟结果分析表明，CO2和H2O通量模拟值与涡相关实测值的回归线斜率分别为0.935和0.875，截距为-0.0136 mg•m-2•s-1和13.7 W•m-2，相关系数为0.655和0.622（n=30107）；CO2通量模拟和实测的平均值分别为-0.138和-0.134 mg•m-2•s-1，模型高估了2.99%；H2O通量模拟和实测的平均值分别为88.5和85.4 W•m-2，模型高估了3.63%，模拟效果较好。从季节变化来看，生长季初、末期（5月和9月）CO2和H2O通量较小，生长旺盛期（6—8月）通量值较高。CO2和H2O通量受环境因子的综合影响，其中，辐射和气温是主要限制因子。 4）对模型的主要参数和环境因子的敏感性分析表明，CO2通量对a1 (气孔导度的参数)、Vcmax、Ca (大气CO2浓度)变化的响应较强，而H2O通量对a1、LAI (叶面积指数)、Ta (气温)变化的响应较强。CO2通量对Ca的变化最为敏感，而H2O通量对其它环境因子的响应程度均高于CO2通量。与将冠层分为20层的方法相比，5层方法（Gaussion五点积分法）得到的碳吸收量和蒸发散量分别低估了25.3%和11.1%。这两种分层方法产生的差异，主要来自于不同层的辐射吸收和权重分配。

L-异亮氨酸高产菌定向育种与代谢控制发酵

以L-异亮氨酸产生菌A_(41-3)为出发菌株，经过定向育种和与L-缬氨酸生物合成突变株原生质体融合，从中选育出一株抗α-氨基-β-羟基戊酸、S-（-2-氨基乙基-）-半胱氨酸、乙硫氨酸、异亮氨酸羟肟酸、2-噻唑丙氨酸和红霉素的亮氨酸生物合成缺陷型CN_(69-1)，通过对L-异亮氨酸生物合成代谢控制发酵条件的研究，在含葡萄糖11%，生物素100 μg/L，硫酸铵4.5%，磷酸二氢钾0.3%，硫酸镁0.02%，硫胺素800 μg/L，硫酸亚铁0.004%，硫酸锰0.004%，碳酸钙2%，L-亮氨酸0.02%，pH6.8-7.0培养基中，32 ℃摇瓶发酵72h，可产生L-异亮氨酸23.6g/L。通过定向筛选缬氨酸代谢活性菌株CN_(69-8)，使缬氨酸生物合成量由8.73g/L下降至1.81g/L，L-异亮氨酸生物合成量保持在24g/L左右。在2.000L发酵罐上进行了pH值、通气量、补料等培养条件优化的研究，在适宜条件下，菌株CN_(69-8)发酵44h，L-异亮氨酸生物合成量可达20g/L以上。测定了有关菌株L-异亮氨酸生物合成关键酶苏氨酸脱氢酶、乙酰羟酸合成酶和分枝链氨基酸转氨酶性变化，结果表明，有关关键酶的活性均比出发菌株为高。通过生长谱法确定L-亮氨酸生物合成途径所缺失的酶是异丙基苹果酸脱氢酶。采用732阳离子交换树脂从发酵液中分离提取L-异亮氨酸。研究了发酵液酸化pH值、吸附体积、不同浓度洗脱剂以及单柱吸咐与双柱串联吸咐对L-异亮氨酸分离提取的影响。确定酸化pH值为2.0，以双柱串联吸咐，用0.2mol.L~(-1)氯化铵-0.1 mol.L~(-1)氨水复合洗脱分离的工艺条件，提取总收率可达45%以上。发酵产品经红外光谱，纸层析，比旋光度等项测定证明明是L-异亮氨酸。发酵中试分离提取的总收率可达40%以上，产品经检验确定为L-异亮氨酸，其质量符合《中华人民共和国药典》标准。

两种抗HER2重组免疫毒素的构建与活性研究

HER2／neu基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子。人肿瘤坏死因子（TNF-α）和肿瘤坏死因子相关的凋亡诱导配体（Trail）对肿瘤细胞的杀伤作用使其成为前景看好的抗肿瘤药物，对它们的细胞杀伤机制研究日渐深入。但临床研究发现HER2／neu过度表达的肿瘤细胞抵制TNF-α和Trail的肿瘤杀伤作用，因此经常产生耐药现象。为了增加过度表达HER2／neu的肿瘤细胞对TNF-a的敏感性和提高HER2／neu抗体的肿瘤杀伤效应，我们将抗HER2／neu人源化单链抗体scFvC6.5与人翔F-a融合，构建了免疫毒素scFvC6.5-TNF-α，完成了该重组蛋白在大肠杆菌中的表达，产率为800μg／L菌液。经过亲和层析和柱复性，融合蛋白的纯度达95％以上。ELISA试验表明scFvC6.5-TNF-a能够特异结合HER2／neu阳性卵巢癌细胞SKO从3和乳腺癌细胞MCF-7，而不结合HERZ／neu阴性的黑色素瘤细胞A-375。MTT试验表明scFvC6.5-TNF-a能够选择性的杀伤SKOV-3和MCF-7细胞，而不影响A-375细胞的生长。同时为了增加过度表达HER2／neu的肿瘤细胞对人可溶性肿瘤坏死因子相关的凋亡诱导配体（sTrail）的敏感性和提高HER2／neu抗体的肿瘤杀伤效应，我们构建了scFvC6．5与人sTrail的融合蛋白scFvC6.5-sTrail。重组子经酶切及测序证明序列正确后，在大肠杆菌BL21（DE3）中进行诱导表达。经SDS-PAGE及westem一blot鉴定，获得高水平包含体表达菌株，产率为700雌／L菌液。对表达产物进行变性、复性及纯化，SDS-PAGE结果显示纯度达95％以上。用ELISA法检测纯化后蛋白的结合活性表明融合蛋白scFvC6.5-sTrail能够特异结合HERZ／neu阳性卵巢癌细胞SKO从3、乳腺癌细胞McF-7和Trail敏感菌株MDA-MB-231，而不结合HER2／neu阴性和Trail受体阴性的黑色素瘤细胞A-375。MTT法检测其生物活性显示纯化后的scFvC6.5-sTrail蛋白对SKO从3、MCF-7、MDA-MB-231均具有细胞毒活性，并存在剂量依赖性，但对A-375细胞没有作用。细胞凋亡流式分析表明这两种免疫毒素对SKO从3靶细胞的杀伤作用是通过诱导细胞凋亡所致。提示这两种免疫毒素在抗肿瘤靶向治疗中具有潜在的应用价值。

Electron trapping materials for use in a picosecond infrared streak camera

We describe our research on the employment of an infrared upconversion screen made of electron trapping material (ETM) in combination with the high sensitivity of the S-20 photocathode responsive to visible radiation to produce a streak camera arrangement capable of viewing and recording infrared incident pulses. The ETM-based upconversion screen converts 800-1600 nm infrared radiation to visible light which is viewed or recorded by the S-20 photocathode. The peak values of the upconversion efficiency are located at 1165 nm for CaS:Eu, Sm and 1060 nm for CaS:Ce, Sm. The present experiment showed time resolution was 12.3 ps for a CaS:Eu, Sm screen and 8.4 ps for a CaS:Ce, Sm screen. The minimum detectability is 4.8 x 10(-9) J/mm(2) (minimum detectability of the coupled visible streak camera is 8.3x10(-10) J/mm(2)). Other parameters, such as spatial resolution and dynamic range, have also been measured and analyzed. The results show ETM can be used in the measurement of infrared ultrafast phenomena up to picosecond time domain. In consideration of the limited number of trapped electrons in ETM, the infrared-sensitive streak camera consisting of an ETM-based upconversion screen is suitable to operate in the single shot mode. (C) 1999 American Institute of Physics. [S0034-6748(99)00112-4].

超临界压力下航空煤油流动与传热特性试验

本文针对超燃冲压发动机再生冷却系统运行条件,实验研究了大庆RP-3煤油在超临界压力下的流动和传热特性,目的在于研究煤油在各种工况下的流动参数变化以及对流传热规律. 煤油通过二级煤油加热/输运系统加热,试验的煤油压力约2.6M~5.0MPa,油温约300~800 K. 相应的壁面热流密度为10~300 kW/m2. 通过油温与壁温的同步测量,结合非定常传热分析,获得了超临界压力下、亚/超临界温度范围内煤油的流动和传热物理参量的变化曲线

电子束蒸发制备YBCO超导薄膜研究

采用电子束沉积制备YBCO超导薄膜,研究了760℃-840℃的不同退火温度下高温热处理对YBCO薄膜双轴织构,表面形貌及超导性能的影响.超导临界电流密度测试,X射线衍射(XRD)和扫描电镜(SEM)的结果表明,退火温度在在800℃时,YBCO薄膜具有良好的织构和平整致密的表面形貌,在77K自场下的临界电流密度J可达4.2×106/cm2

利用可调谐二极管激光吸收光谱诊断超声速流动

本文利用建立的TDLAS测量系统，应用于氢氧燃烧驱动Φ800低密度激波管，进行了高超声速气流诊断实验。由于激波管③区的气流速度，温度和水蒸气组分分压等参数和超燃燃烧台尾喷管出口气体状态较为接近。因此对激波管③区水蒸气的测量模拟了对超燃台尾喷管气流的吸收诊断。

高焓激波风洞研究进展

国际上已发展的高焓激波风洞按驱动方式不同可以分为3种：加热轻气体驱动,自由活塞驱动,和爆轰驱动激波风洞。对不同类型激波风洞的发展历程进行了回顾，对其存在的,以及进一步发展过程中潜在的问题进行了探讨。\newline (1)加热轻气体驱动激波风洞\newline 加热轻气体驱动方式主要采用高比热比的轻气体，再利用加热手段进一步提高气体声速。国际上应用加热轻气体驱动的有NASACalspan-UB研究中心的LENS系列激波风洞和俄罗斯TSNIIMASH中心机械工程研究院的U-12大型激波风洞。LENS系列激波风洞的性能基本覆盖了航天飞机的飞行走廊，Calspan-UB研究中心在LENS风洞上开展了大量的超高速流动研究。\newline (2) 自由活塞驱动激波风洞\newline 自由活塞驱动激波风洞是利用高速运动的自由活塞压缩产生高压驱动气体。主要有澳大利亚国立大学T3, 昆士兰大学T4,日本国家航天实验中心的KEK和HIEST，美国加州理工学院T5,德国DLR的HEG。其中，日本的HIEST以其尺寸最大, 技术成熟,实验时间长而具有代表性。\newline (3) 爆轰驱动激波风洞\newline 爆轰驱动是应用可燃混合气体爆轰产生的化学能来压缩驱动气体的一种驱动方式。爆轰驱动有3种模式：反向驱动,正向驱动和双爆轰驱动。反向驱动在主膜处起爆，应用稀疏波后压力均匀的高压气体作为驱动气体，能够产生稳定的驻室状态，但驱动能力较低。正向驱动在驱动段上游末端起爆，驱动能力强，比反向驱动高一个量级，但受爆轰波后稀疏波的影响，气流品质不稳定。双爆轰驱动模式，通过辅驱动段，可以消除正向爆轰波后面的稀疏波，以获得平稳的试验气流。\newline 中国科学院力学研究所从20世纪60年代起就开展了爆轰驱动技术的系统研究，并于1996年研制成功了JF10爆轰驱动高焓激波风洞。JF10模拟总温高达8000K，总压高达800个大气压，有效试验时间超过6ms。为开展高超声速气动力/热,真实气体效应, 气动物理等问题的研究创造了基本条件。\newline (4)问题与展望\newline 轻气体驱动能够产生稳定的试验气流，运行技术可靠，重复性好，但是大量轻气体的运输,储存,加热与排放是风洞运行的主要困难。自由活塞驱动技术的驱动能力强，风洞尺度容易扩展，但是运行技术复杂，试验气流稳定时间短，是风洞发展的主要限制。爆轰驱动激波风洞近十几年来发展迅速，突破了一些重要的关键技术，产生高焓试验气流的能力强，提供的有效试验时间长，运行成本低，可扩展性好，是一种具有良好发展前途的高超声速激波风洞。\newline 到目前为止，尽管高超声速激波风洞的研制已经取得了重大进展，但是从地面模拟的流动要满足飞行条件所要求的自由来流马赫数, 自由来流雷诺数, 流动速度,飞行高度压力, 来流总焓, 跨过激波的密度比, 试验气体组分,壁温/总温比,化学反应进程等条件来看，还有很大差距。所以，为了获得可靠的试验数据，通过不断改进,完善,提高激波风洞的性能，尽可能"复现"高超声速飞行条件，是今后主要的研究方向。

用双流体模型模拟超声速气固两相流动

采用了双流体模型对JPL(Jet Propulsion Laboratory)喷管中气固两相流动以及超声速射流两相流动进行了数值模拟,并研究了可压缩两相流动中气相与颗粒的相互作用规律.自主开发的一般曲线坐标系下二维轴对称可压缩双流体程序Solve2D,对气相求解Navier-Stokes(N-S)方程组,采用k-ε湍流模型,颗粒相求解Euler方程组.对JPL喷管内的两相流场和湍流两相射流流场进行了数值模拟,研究了不同颗粒质量百分数以及不同颗粒直径时的气固两相流场的流动规律.

中国卧龙自然保护区不同海拔川滇高山栎(Quercus aquifolioides)群体的遗传变异

海拔梯度造成的环境异质性，如崎岖的地形、复杂的植被结构以及花期延迟等可能会极大地影响到物种的形态和遗传变异格局。理解物种形态和遗传变异的海拔格局对于物种多样性的管理和保护是非常重要的。尽管植物群体遗传学是一个飞速发展的研究领域，然而与海拔相关的形态变异、遗传变异及群体间遗传差异的研究却很少。到目前为止，还不清楚遗传变异与海拔之间是否必然的相关性。 川滇高山栎是一种重要的生态和经济型树种，广泛分布于中国西南的四川、西藏、贵州和云南省的高海拔地区，在保持水土、调节气候方面起着十分重要的作用。尽管主要受阳光限制而仅分布于阳坡，但其海拔梯度范围较大，表明川滇高山栎对不同的环境具有很强的适应性。本文通过叶型及生理响应、微卫星分子标记和扩增性片段长度多态性方法，试图探索川滇高山栎叶沿海拔梯度的形态和生理响应及其沿海拔梯度的遗传变异格局，为川滇高山栎的保护和利用提供进一步的遗传学理论依据和技术指导。 对叶形、含氮量及碳同位素的试验结果表明，平均比叶面积、气孔密度、气孔长度和气孔指数等气孔参数随海拔的升高呈非线性变化。在海拔大于2800 m时，川滇高山栎的比叶面积、气孔长度和气孔指数都随海拔升高而降低，但是在海拔小于2800 m时，这些指标都随海拔的升高而增大。相对而言，单位叶面积的含氮量和碳同位素则表现出相反的变化模式。另外，比叶面积是决定碳同位素沿海拔梯度变化的最重要参数。本研究结果表明，海拔2800 m附近是川滇高山栎生长和发育的最适地带，在这里生长的植物叶片厚度更薄、气孔更大、叶碳同位素值更小。 利用六对微卫星引物对五个不同海拔川滇高山栎群体遗传多样性进行研究，结果表明，群体内表现出较高的遗传多样性，平均每位点等位基因数11.33个，平均期望杂合度达0.820。群体间差异较小，分化仅为6.6％。聚类分析也并没有显示出明显的海拔格局。然而低频率等位基因却与海拔呈显著性正相关(R2=0.97, P < 0.01)，表明在高海拔处，川滇高山栎以更多的稀有基因来适应恶劣的环境条件。本试验结果表明由海拔梯度形成的选择性压力对川滇高山栎群体的遗传变异影响并不明显。 为了进一步探讨川滇高山栎群体遗传变异与海拔之间的相互关系，我们还对其进行了扩增性片段长度多态性分析。结果表明：(1)随海拔的升高(从群体WL2到群体WL5)，群体内遗传变异降低，而群体间遗传差异增加；(2)低海拔群体WL1表现出最低的遗传变异性(HE = 0.181)，同时与其余四个群体间呈现出最大的遗传差异性(平均FST = 0.0596)；(3)在除去低海拔群体WL1后，Mantel检测表明群体间遗传距离与海拔距离之间表现出正相关性。另外，研究结果还表明，遗传变异受生境条件(过度的湿热环境)及人为干扰(火烧、砍伐和放牧)的影响，这一点至少在低海拔群体WL1上发生了作用。 通过叶形态、生理及DNA分子水平的研究，结果表明叶形态特征和碳同位素与海拔紧密相关，与海拔之间呈非线性变化，海拔2,800 m附近是川滇高山栎生长和发育的最适地带。海拔梯度在一定程度上会影响到川滇高山栎群体的遗传变异结构，但在这样一个狭窄的地理分布区域里，这种影响并不足以导致群体间较大的遗传分化。同时生境条件及人为干扰也是影响遗传变异的限制性因子，不容忽视。 Altitudinal gradients impose heterogeneous environmental conditions, such as rugged topography, a complex pattern of vegetation and flowering delay, and they likely furthermore markedly affect the morphological and genetic variation pattern of a species. Understanding altitudinal pattern of morphological and genetic variation at a species is important for the management and conservation of species diversity. Although plant population genetics is a fast growing field of research, there are only few recent investigations, which analyzed the genetic differentiation and changes of intra-population variation along altitudinal gradients. At present, it is still unclear whether there are some common patterns of morphological and genetic variation with altitude. Quercus aquifolioides Rehder & E.H. Wilson, which is an important ecological and economical endemic woody plant species, is widely distributed in the Yunnan and Sichuan provinces, Southwest China. Its large range of habitat across different altitudes implies strong adaptation to different environments, although it is mainly restricted to sunny, south facing slopes. It plays a very important role in preventing soil erosion, soil water loss and regulating climate, as well as in retaining ecological stability. In this paper, we tried to understand the altitudinal pattern of morphological and genetic variation along altitudinal gradients through the experiments of leaf morphological and physiological responses, microsatellite analysis and AFLP markers. In leaf morphological and physiological responses experiment, we measured leaf morphology, nitrogen content and carbon isotope composition (as an indicator of water use efficiency) of Q. aquifolioides along an altitudinal gradient. We found that these leaf morphological and physiological responses to altitudinal gradients were non-linear with increasing altitude. Specific leaf area, stomatal length and index increased with increasing altitude below 2,800 m, but decreased with increasing altitude above 2,800 m. In contrast, leaf nitrogen content per unit area and carbon isotope composition showed opposite change patterns. Specific leaf area seemed to be the most important parameter that determined the carbon isotope composition along the altitudinal gradient. Our results suggest that near 2,800 m in altitude could be the optimum zone for growth and development of Q. aquifolioides, and highlight the importance of the influence of altitude in research on plant physiological ecology. Genetic variation and differentiation were investigated among five natural populations of Q. aquifolioides occurring along an altitudinal gradient that varied from 2,000 to 3,600 m above sea level in the Wolong Natural Reserve of China, by analyzing variation at six microsatellite loci. The results showed that the populations were characterized by relatively high intra-population variation with the average number of alleles equaling 11.33 per locus and the average expected heterozygosity (HE) being 0.779. The amount of genetic variation varied only little among populations, which suggests that the influence of altitude factors on microsatellite variation is limited. However, there is a significantly positive correlation between altitude and the number of low-frequency alleles (R2=0.97, P < 0.01), which indicates that Q. aquifolioides from high altitudes has more unique variation, possibly enabling adaptation to severe conditions. F statistics showed the presence of a slight deficiency of heterozygosity (FIS=0.136) and a low level of differentiation among populations (FST=0.066). The result of the cluster analysis demonstrates that the grouping of populations does not correspond to the altitude of the populations. Based on the available data, it is likely that the selective forces related to altitude are not strong enough to significantly differentiate the populations of Q. aquifolioides in terms of microsatellite variation. To further elucidate genetic variation pattern of Q. aquifolioides populations under sub-alpine environments, genetic variation and differentiation were investigated along altitudinal gradients using AFLP markers. The altitudinal populations with an average altitude interval of 400 m, i.e. WL1, WL2, WL3, WL4 and WL5, correspond to the altitudes 2,000, 2,400, 2,800, 3,200 and 3,600 m, respectively. Our results were as follows: (i) decreasing genetic variation (ranging from 0.253 to 0.210) and increasing genetic differentiation with altitude were obtained from the WL2 to the WL5 population; (ii) the WL1 population showed the lowest genetic variation (HE = 0.181) and the highest genetic differentiation (average FST = 0.0596) with the other four populations; (iii) the positive correlation was obtained using Mantel tests between genetic and altitude distances except for the WL1 population. Our results suggest that altitudinal gradients may have influenced the genetic variation pattern of Q. aquifolioides populations to some extent. In addition, habitat environments (unfavorable wet and hot conditions) and human disturbances (burning, grazing and felling) were possible influencing factors, especially to the low-altitude WL1 population. The present study shows that there were close correlations between morphological features and carbon isotope composition in our data. This indicates that a coordinated plant response modified these parameters simultaneously across different altitudes. Around 2,800 m altitude there seems to be an optimum zone for growth and development of Q. aquifolioides, as indicated by thinner leaves, larger stomata and more negative d13C values. All available evidence indicates altitudinal gradients may have influenced the genetic variation pattern of Q. aquifolioides to some extent. Decreasing genetic variation and increasing genetic differentiation with altitude was obtained except for the WL1 population. And the environment of habitats and human disturbances were also contributing factors, which impact genetic variation pattern, especially to the low-altitude WL1 population.

卧龙自然保护区不同海拔的中国沙棘(Hippophae rhamnoides subsp. sinensis)天然群体的遗传多样性分析

中国沙棘是一种雌雄异株、风媒传粉的灌木或乔木，在中国西南的卧龙自然保护区有广泛的分布。本研究以采集于四川卧龙自然保护区5 个海拔(1800 m、2200 m、2600 m、3000 m、3400 m)梯度的中国沙棘天然群体为材料，以ISSR 和AFLP 标记技术研究其遗传多样性水平及其遗传结构，旨在了解卧龙地区中国沙棘天然群体的遗传多样性水平以及遗传多样性在群体间、群体内以及雌雄亚群体间的分布和特征，为中国沙棘树种的遗传改良及种质资源保存提供遗传研究背景与实验依据。同时探讨ISSR、AFLP 和RAPD三种标记对中国沙棘天然群体的遗传变异水平和群体间遗传结构的评估能力和各自的优缺点。研究得出以下主要结论： 1. ISSR和AFLP分析都表明卧龙自然保护区的中国沙棘群体拥有较高的遗传变异水平(h = 0.249，HT = 0.305)。出现这种结果的主要原因可能与卧龙自然保护区多变的气候条件和生境的异质度大有关。 2. ISSR 和AFLP 都揭示出卧龙自然保护区中国沙棘群体的遗传多样性随着海拔的增加发生显著的变化，表现为中海拔群体(2200 m 和2600 m)比高海拔群体(3000 m 和3400 m)和低海拔群体(1800 m)有更高的遗传多样性的趋势。出现这种趋势的可能解释是低海拔群体处在相对高温和相对干旱的环境，高海拔群体受到低温和紫外线胁迫，而中海拔群体存在中国沙棘生长的适宜环境。 3. ISSR 和AFLP 分析都表明：卧龙自然保护区中国沙棘的遗传结构遵循分布范围广、交配系统以异交为主的木本植物的通常模式，即大多数的遗传变异存在于群体内，只有少部分的遗传变异存在于群体间。 4. 经Mantel 检测表明，卧龙自然保护区中国沙棘群体间的海拔距离和对应遗传距离之间存在显著的正相关关系，即随着垂直海拔距离的增加，群体间的遗传距离也随之增加。Mantel 检测结果以及聚类分析将卧龙自然保护区5 个不同海拔的中国沙棘群体分为低、中、高海拔群体三组的研究结果都表明，海拔很可能是限制群体间基因交流的主要因素。 5. ISSR 分析发现同一海拔的雌雄亚群体首先聚类的研究结果表明，同一海拔的雌雄亚群体在遗传上最相似。方差分析结果表明只有3.8%的总遗传变异存在于雌雄亚群体间，这可能与雌雄植株间的交配和遗传物质的混合有关。 6. ISSR、AFLP 和RAPD 分析都表明卧龙自然保护区不同海拔的中国沙棘天然群体的遗传多样性水平较高。它们的分析结果估算得到的Nei's 平均基因多样度(h)分别为0.249、0.214 和0.170。从该结果可以看出ISSR 和AFLP 比RAPD 检测到更多的遗传多态性,这很可能是不同标记检测的基因组的位点不同所致。 7. 依据对不同标记系统的比较分析，认为ISSR、AFLP 和RAPD 三种分子标记系统都能成功地用于调查卧龙自然保护区不同海拔的中国沙棘群体的遗传变异水平及遗传变异结构，提供关于中国沙棘天然群体多态性水平和遗传变异分布的有用信息。在三者中，AFLP 具有最高效能指数和标记指数，在确定种间分类关系或鉴别个体方面是一种比较理想的标记。 Hippophae rhamnoides subsp. sinensis, a dioecious and deciduous shrub species,occupies a wide range of habitats in the Wolong Nature Reserve, Southwest China. Ourpresent study investigated the pattern of genetic variation and differentiation among fivenatural populations of H. rhamnoides subsp. sinensis, occurring along an altitudinal gradientthat varied from 1,800 to 3,400 m above sea level in the Wolong Natural Reserve, by usingISSR and AFLP markers to guide its genetic improvement and germplasm conservation. And,comparative study of ISSR, AFLP and RAPD was performed to detect their capacity toestimating the level and pattern of genetic variation occurring among the five elevationpopulations of H. rhamnoides subsp. sinensis, and to discuss their application to the study onplant genetics. The results were list following: 1. The ISSR and AFLP analysis conducted for the H. rhamnoides subsp. sinensispopulations located in the Wolong Natural Reserve of China revealed the presence of highlevels of genetic variation (h = 0.249, HT = 0.305). Besides such features as relatively widedistribution, dominantly outcrossing mating system, and effective seed dispersal by small animals and birds, it is sometimes argued that hard climatic conditions and heterogeneous habitats may also contribute to high levels of diversity. 2. Genetic diversity of H. rhamnoides subsp. sinensis populations was found to varysignificantly with changing elevation, showing a trend that mid-elevation populations (2,200m and 2,600 m) were genetically more diverse than both low-elevation (1,800 m) andhigh-elevation populations (3,000 m and 3,400 m). H. rhamnoides subsp. sinensis is thoughtto be stressed by drought and high temperature at low elevations, and by low temperature athigh elevations. The high genetic variability present in the mid-elevation populations of H.rhamnoides subsp. sinensis is assumed to be related to a greater plant density in the middlealtitudinal zone, where favorable ecological conditions permit its continuous distributioncovering the zone from 2,200 m to 2,600 m above sea level. 3. The genetic structure of H. rhamnoides subsp. sinensis revealed by ISSRs andAFLPs followed the general pattern detected in woody species with widespread distributionsand outcrossing mating systems. Such plants possess more genetic diversity withinpopulations and less variation among populations than species with other combinations oftraits. 4. In the present study, Mantel tests showed positive correlations between altitudinaldistances and genetic distances among populations or subpopulations. The observedrelationship between altitude and genetic distances, and the result of the cluster analysisincluding populations or male subpopulations and classifying the groups into three altitudeclusters suggest that altitude is a major factor that restricts gene flow between populationsand subpopulations. 5. The analysis of molecular variance showed that only 3.8% of the variability residedbetween female and male subpopulations. Such a very restricted proportion of the totalmolecular variance between female and male subpopulations is due to common sexuality andmixing of genetic material between females and males. 6. The analysis based on ISSRs, AFLPs and RAPDs all revealed relatively high levelsof genetic variation among different altitudinal populations of H. rhamnoides subsp. sinensisin Wolong Natural Reserve of China. Their estimates of mean Nei’s gene diversity is equal to0.249, 0.214 and 0.170 respectively, suggesting the higher capacity of detecting geneticvariation of ISSR and AFLP than RAPD. It might be ascribed to their distinct sensitivity todifferent type of genetic variation. 7. Based on the coparative study on ISSR, AFLP and RAPD, we drew a conclusion thatthey all successfully reveal some useful information concerning the level and pattern ofgenetic vatiation occurring among different elevation populations of H. rhamnoides subsp.sinensis. AFLP is a ideal tool to taxonomic study and individual identification for theirhighest efficiency index and marker index among the three marker systems.

中国沙棘(Hippophae rhamnoides subsp. sinensis)天然群体的遗传变异分析

近十年，植物群体遗传学的研究飞速发展，然而与海拔相关的植物群体遗传结构和遗传变异研究却相对较少。到目前为止，还不清楚遗传变异与海拔之间是否有一个通用的格局。在山区，各种生态因子，如温度、降水、降雪、紫外线辐射强度以及土壤成分都随海拔梯度急剧变化，造成了即使在一个小的空间区域，植被类型变化显著，这种高山环境的异质性和复杂性为我们研究植物群体遗传结构和分化提供了方便。沙棘(Hippophea)属于胡颓子科(Elaeagnaceae)为多年生落叶灌木或乔木，雌雄异株，天然种群分布极为广泛。中国沙棘(H. rhamnoides subsp. sinensis)是沙棘属植物中分布较广的一个亚种，种内形态变异非常丰富，加之其具有独特的繁育系统和广泛的生态地理分布，是研究沙棘属植物遗传变异和系统分化的理想材料。本文从1,800 m 到3,400 m 分5 个海拔梯度进行取样，用RAPD 和cpSSR 分子标记研究了卧龙自然保护区中国沙棘天然群体的遗传结构和遗传变异。5 个取样群体依次标记为A、B、C、D 和E，它们分别代表分布在海拔1,800，2,200，2,600，3,000 和3,400 m 的5 个天然群体。RAPD实验用11 条寡核苷酸引物，扩增得到151 个重复性好的位点，其中143 个多态位点，多态率达94.7%。在5 个沙棘群体中，总遗传多样性值(HT)为0.289，B群体内的遗传多样性值为0.315，这完全符合沙棘这种多年生、远交的木本植物具有高遗传变异的特性。5 个群体内遗传多样性随海拔升高呈低－高－低变异趋势，在2,200 m海拔处的B群体遗传多样性达最大值0.315，3,400 m海拔处的E群体则表现最小仅0.098。5 个群体间的遗传分化值GST＝0.406，也即是说有40.6%的遗传变异存在于群体间，1,800 m海拔处的A群体与其它群体的明显分离是造成群体间遗传分化大的原因。UPGMA聚类图和PCoA散点图进一步确证了5 个群体间的关系和所有个体间的关系。最后，经过Mantel检测，遗传距离与海拔表现了明显的相关性(r = 0.646, P = 0.011)。cpSSR 实验中，经过对24 对cpSSR 通用引物筛选，11 对引物能扩增出特异性条带，只有2 对引物(ccmp2 和ARCP4)呈现多态性。4 个等位基因共组合出4 种单倍型，单倍型Ⅰ出现在A 群体的所有个体和B 群体的8 个个体中，C、D、E 三个群体均不含有，而单倍型Ⅱ出现在C、D、E 三个群体的所有个体及B 群体的18 个个体中，A 群体不含有。另外两种单倍型Ⅲ和Ⅳ为稀有类型，仅B 群体中的4 个个体拥有。这种单倍型分布模式和TFPGA 群体聚类图揭示了，C、D、E 群体可能来源于同一祖先种，而A 群体却是由另一祖先种发展起来的，B 群体则兼具了这两种起源种的信息，这可能是因为在历史上的某一时期，在中国沙棘群体高山分化的过程中，B 群体处某个或者某些个体发生了基因突变，具备了适应高海拔环境的能力，产生了高海拔沙棘群体的祖先种。 In recent ten years, studies about population genetics of plants developed rapidly,whereas their genetic structure and genetic variation along altitudinal gradients have beenstudied relatively little. So far, it is uncleared whether there is a common pattern betweengenetic variation and altitudinal gradients. In the mountain environments, importantecological factors, e.g., temperature, rainfall, snowfall, ultraviolet radiation and soil substratesetc., change rapidly with altitudes, which cause the vegetation distribution varying typically,even on a small spatial scale. The mountain environments, which are heterogeneous andcomplex, facilitate and offer a good opportunity to characterize population genetic structureand population differentiation.The species of the genus Hippophae L. (Elaeagnaceae) are perennial deciduous shrubs ortrees, which are dioecious, wind-pollinated pioneer plants. The natural genus has a widedistribution extending from Northern Europe through Central Europe and Central Asia toChina. According to the latest taxonomy, the genus Hippophae is divided into six species and12 subspecies. The subspecies H. rhamnoides ssp. sinensis shows significant morphologicalvariations, large geographic range and dominantly outcrossing mating system. Thesecharacteristics of the subspecies are favourable to elucidate genetic variation and systemevolution. To estimate genetic variation and genetic structure of H. rhamnoides ssp. sinensisat different altitudes, we surveyed five natural populations in the Wolong Natural Reserve at altitudes ranging from 1,800 to 3,400 m above sea level (a.s.l.) using random amplifiedpolymorphic DNA markers (RAPDs) and cpSSR molecular methods. The five populations A,B, C, D, and E correspond to the altitudes 1,800, 2,200, 2,600, 3,000 and 3,400 m,respectively.Based on 11 decamer primers, a total of 151 reproducible DNA loci were yielded, ofwhich 143 were polymorphic and the percentage of polymorphic loci equaled 94.7%. Amongthe five populations investigated, the total gene diversity (HT) and gene diversity within population B equaled 0.289 and 0.315, respectively, which are modest for a subspecies of H.rhamnoides, which is an outcrossing, long-lived, woody plant. The amount of geneticvariation within populations varied from 0.098 within population E (3,400 m a.s.l.) to 0.315within population B (2,200 m a.s.l.). The coefficient of gene differentiation (GST) amongpopulations equaled 0.406 and revealed that 40.6% of the genetic variance existed amongpopulations and 59.4% within populations. The population A (1,800 m a.s.l.) differed greatlyfrom the other four populations, which contributes to high genetic differentiation. A UPGMAcluster analysis and principal coordinate analyses based on Nei's genetic distances furthercorroborated the relationships among the five populations and all the sampling individuals,respectively. Mantel tests detected a significant correlation between genetic distances andaltitudinal gradients (r = 0.646, P = 0.011).Eleven of the original 24 cpSSR primer pairs tested produced good PCR products, onlytwo (ccmp2 and ARCP4) of which were polymorphic. Four total length variants (alleles) werecombined resulting in 4 haplotypes. The haplotype was present in all individuals of Ⅰpopulation A and 8 individuals of populations B, the other three populations (C, D and Epopulations) did not share. The haplotype was present in all individuals of populations C, D Ⅱand E and 18 individuals of populations B, population A did not share. The other twohaplotypes and were rare haplotypes, which were only shared in 4 individuals of Ⅲ Ⅳpopulation B. The distribution of haplotypes and TFPGA population clustering map showedthat the populations C, D and E might be origined from one ancestor seed and population Amight be from another, whereas population B owned information of the two ancestor seeds. Itwas because that gene mutation within some individual or seed in the location of population Bwas likely to happen in the history of H. rhamnoides, which was the original ancestor of thehigh-altitude populations.