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.

瑞香狼毒（Stellera chamaejasme L.）对高寒草甸系统生长和非生长季节碳氮循环的影响及其花粉化感效应

瑞香狼毒（Stellera chamaejasme L.）是瑞香科（Thymelaeaceae）狼毒属的一种多年生野草，有毒。据调查，从20 世纪60 年代开始至今，狼毒在青藏高原东缘的高寒草甸上不断蔓延、密度不断变大，在一些地段甚至成为优势物种。有关狼毒在高寒草甸蔓延的生态系统效应的研究尚未见报道。本文从系统碳、氮循环的角度，分别研究狼毒在生长和非生长季节对高寒草甸生态系统的影响。同时，从花粉化感的角度，深入研究狼毒对当地同花期物种有性繁殖的影响。系统地研究高寒草甸生态系统物质循环过程，特别是非生长季节微生物和土壤碳氮库的动态变化，有助于揭示狼毒在系统物质循环方面的“物种效应”以及这种效应的季节变化，为丰富有关高海拔生态系统，特别是其非生长季的物质循环的科学理论做出贡献。同时，碳氮循环和花粉化感的研究还有助于深刻地理解狼毒作为一种入侵性很强的杂草的特殊的蔓延机制，从而为狼毒的有效防治、高寒草甸的科学管理提供依据。 针对狼毒在青藏高原高寒草甸上蔓延的生态系统碳氮循环方面的影响，开展以下2 方面的研究：（1）在生长季，研究松潘县尕米寺附近（北纬32°53'，东经103°40'，海拔3190 m）的两种地形（平地和阳坡）条件下狼毒对土壤碳氮循环影响及可能的原因。狼毒和其它几个主要物种（圆穗蓼（Polygonummacrophyllum D. Don var. Macrophyllum），草地早熟禾（Poa pretensis L.），四川嵩草（Kobresia setchwanensis Hand.-Maizz.），鹅绒委陵菜（Potentilla anserina L.var. anserine）和鳞叶龙胆（Gentiana squarrosa Ledeb.）的地上凋落物产量以及地上凋落物和根的化学组成被测量。在有-无狼毒斑块下，各种土壤的库（比如，铵态氮、硝态氮、无机磷和微生物生物量）和周转率（包括净矿化、净硝化、总硝化、反硝化和微生物呼吸速率）被测量和比较。（2）在非生长季节，尤其是春季冻融交替期，选取了两个研究地点——尕米寺和卡卡沟（北纬32°59'，东经103°41'，海拔3400 m），分别测定有狼毒和无狼毒斑块下土壤微生物生物量碳和氮、可溶性有机碳和氮以及铵态氮和硝态氮的动态变化。同时，分别在上述两个地点有-无狼毒的样地上，研究6 个主要物种（狼毒、圆穗蓼、草地早熟禾、四川嵩草、鹅绒委陵菜和鳞叶龙胆）从秋季开始、为期1 年的凋落物分解过程。 针对狼毒花粉化感对同花期其它物种可能的花粉化感作用开展以下工作：在实验室中，用一系列浓度的狼毒花粉水浸提液对与它同花期的其它物种以及自身花粉进行测试，测定花粉萌发率；在野外自然条件下的其它物种的柱头上施用上述浓度的狼毒花粉水浸提液，观测种子结实率，同时，观察狼毒花粉的种间花粉散布数量。 生长季节的研究结果表明，狼毒地上凋落物氮含量比其它几个主要物种更高，而木质素-总氮比更低。狼毒显著地增加其斑块下表层土壤中有机质的含量，而有-无狼毒的亚表层土壤在有机碳和总磷方面没有显著差异。狼毒表土中硝态氮含量在平地和阳坡比无狼毒土壤分别高113%和90%。狼毒表土中微生物生物量碳和氮量显著高于无狼毒表土。无论是平地还是阳坡，狼毒土壤的总硝化和微生物呼吸速率显著高于无狼毒土壤；而它们的反硝化速率只在平地有显著的差异。狼毒与其它物种间地上凋落物的产量和质量的差异可能是导致有-无狼毒土壤碳氮循环差异的原因。我们假设，狼毒可能通过增加贫氮生态系统土壤中的有效氮含量提高其入侵能力。 非生长季的研究结果表明，在青藏高原东缘的高寒草甸上，土壤微生物生3物量在11 月的秋-冬过渡期达到第一个峰值；在春季的冻融交替期，微生物生物量达到第二个峰值后又迅速降低。无机氮以及可溶性有机碳氮与微生物生物量有相似的变化过程。微生物碳氮比呈现显著的季节性变化。隆冬季节的微生物生物量碳氮比显著高于生长旺季的微生物碳氮比。这种变化可能暗示冬、夏季微生物的群落组成和对资源的利用有所不同。有-无狼毒斑块下土壤微生物和土壤碳、氮库一般只在秋-冬过渡期有显著差异，有狼毒土壤微生物生物量和土壤碳、氮库显著高于无狼毒土壤；而在之后的冬季和春季没有显著差异。所有6 个物种凋落物在非生长季分解率为24%-50%，均高于生长季的10%到30%。其中在秋-冬过渡期，凋落物开始埋藏的两周时间内，分解最快，达10%-20%。不同物种凋落物全年的分解率和分解过程有显著差异。圆穗蓼在全年的分解都较缓慢（非生长季26%，生长季15%），草地早熟禾和四川嵩草等全年的分解速率比较均匀（非生长季和生长季均为30%，非生长季略高），而狼毒在非生长季分解较快（约50%），而在接下来的生长季分解变得缓慢（约12%）。所有物种的凋落物氮含量在非生长季下降，而在随后的生长季上升。 实验室的花粉萌发试验证明，狼毒花粉对自身花粉萌发没有自毒作用，而其它受试的所有物种（圆穗蓼，秦艽（Gentiana macrophylla Pall. var. fetissowii），湿生扁蕾（Gentianopsis paludosa (Hook. f.) Ma var. paludosa），鳞叶龙胆，椭圆叶花锚（Halenia elliptica D. Don var. elliptica），蓝钟花（Cyananthus hookeri C. B.Cl. var. grandiflorus Marq.），小米草（Euphrasia pectinata Ten.），川西翠雀花（Delphinium tongolense Franch.），高原毛茛（Ranunculus tanguticus (Maxim.)Ovcz. var. tanguticus）和鹅绒委陵菜）的花粉萌发率随着狼毒花粉浸提液浓度的增加呈显著的非线性降低。大约3 个狼毒花粉的浸提液就可以抑制受试的多数物种的50%的花粉萌发。在鳞叶龙胆和小米草柱头上狼毒花粉的数量分别为5.76 个和3.35 个。狼毒花粉散布数量的差异最可能的原因在于是否有共同的传粉昆虫。花的形状（辐射对称VS 左右对称）、植株或花的密度以及花期重叠性可以部分解释这种差异。在野外试验中，我们发现6 个物种（秦艽、湿生扁蕾、鳞叶龙胆、椭圆叶花锚、蓝钟花和小米草）的种子结实率随狼毒花粉浸提液浓度的增加呈显著的非线性降低。鳞叶龙胆和小米草柱头上狼毒花粉的数量（分别是5.76 个和3.35 个）分别达到了抑制它们63%和55%种子结实率的水平。因此，狼毒对鳞叶龙胆和小米草可能存在明显的花粉化感抑制作用。狼毒周围的物种可能通过花期在季节或昼夜上的分异避免受到狼毒花粉化感的影响或者通过无性繁殖来维持种群繁衍，因此狼毒通过花粉化感作用对其周围物种繁殖的影响程度还需要进一步地研究。如果狼毒的花粉化感抑制作用确实存在，那么它可能成为一种自然选择压力，进而影响物种的进化。 Stellera chamaejasme L., a perennial toxic weed, has emerged and quicklydominated and spread in the high-frigid meadow on the eastern Tibetan Plateau ofChina since the 1960s. In the present study, effects of S. chamaejasme on carbon andnitrogen cycles on the high-frigid meadow on the eastern Qinghai-Tibetan Plateau ingrowing and non-growing season, and its pollen allelopathic effects on the sympatricspecies were determined. The present study that focused on carbon and nitrogencycles, especially on microbial biomass and pools of carbon and nitrogen innon-growing season, could profoundly illuminate plant-species effects on carbon andnutrient cycles and its seasonal pattern and help to understand spread mechanism ofS. chamaejasme as an aggressive weed. The present study also contributed to furtherunderstand carbon and nutrient cycles on alpine regions in non-growing season andprovide a basis on weed control of S. chamaejasme and scientific management in thehigh-frigid ecosystem. Effects of S. chamaejasme on carbon and nitrogen cycles on the high-frigidmeadow on the eastern Qinghai-Tibetan Plateau were determined. The study couldbe divided into two parts. (1) In the growing season, we quantified the effects of S.chamaejasme on carbon and nitrogen cycles in two types of topographic habitats, theflat valley and the south-facing slope, where S. chamaejasme was favored to spreadlitter and root were measured to explain the likely effects of S. chamaejasme on soilcarbon and nutrient cycles. The sizes of various soil pools, e.g. nitrite, ammonium,inorganic phosphorus and microbial biomass, and turnover rates including netmineralization, gross nitrification, denitrification and microbial respiration weredetermined. (2) In the non-growing season study, microbial biomass carbon andnitrogen, soluble organic carbon and nitrogen, ammonium and nitrate weredetermined through the non-growing season, especially in the processes offreeze-thaw of spring in two high-frigid sites, i.e. Kaka valley and Gami temple, onthe eastern Qinghai-Tibetan Plateau. Meanwhile, litter decomposition of six commonspecies, including Stellera chamaejasme L., Polygonum macrophyllum D. Don var.Macrophyllum, Poa pretensis L., Kobresia setchwanensis L., Potentilla anserina L.var. anserine and Gentiana squarrosa Ledeb., were also examined under theabove-mentioned experimental design through one whole-year, which began in theautumn in 2006. In the study of pollen allelopathy, several work, including in vitro study oneffects of extract of pollen from S. chamaejasme on sympatric species and pollenfrom itself, field experiments on effects of pollen extract with the same regime ofconcentrations on seed set and field observation on heterospecific pollen transfer ofS. chamaejasme to six of those sympatric species has been done. The results in the growing season showed that aboveground litter of S.chamaejasme had higher tissue nitrogen and lower lignin: nitrogen ratio than thoseco-occurring species. S. chamaejasme significantly increased topsoil organic matter,whereas no significant differences were found for organic C and total P in subsoilbetween under-Stellera and away-Stellera locations. The nitrate in Stellera topsoilwas 113% and 90% higher on the flat valley and on the south-facing slope,respectively. Both microbial biomass C and N were significantly higher in Stelleratopsoil. Gross nitrification and microbial respiration were significantly higher inStellera topsoil both on the flat valley and on the south-facing slope, whereassignificant differences of denitrification were found only on the flat valley. Thedifferences in the quantity and quality of aboveground litter are a likely mechanismresponsible for the changes of soil variables. We assumed that S. chamaejasme couldenhance their spread by increasing nutrient availability in N-deficient ecosystems. The results in the non-growing season showed that microbial biomass achievedthe first summit in late autumn and early winter on the eastern Qinghai-TibetanPlateau. In the stages of freeze-thaw of spring, microbial biomass firstly achieved thesecond summit and subsequently sharply decreased. Inorganic nitrogen, solubleorganic carbon and nitrogen had a similar dynamics with that of microbial biomass.Ratio of microbial biomass carbon and nitrogen had an obviously seasonal pattern.The highest microbial C: N were in the non-growing season, which weresignificantly higher than those in the growing season. The seasonal pattern inmicrobial biomass C: N suggested that large changes in composition of microbialpopulation and in resources those used by microbes between summer and winter.Generally, microbial biomass and pools size of carbon and nitrogen in Stellera soilwere significantly higher than those under adjacent locations in late autumn andearly winter, but there were not significant differences in winter and in spring. Litterof all the focal species (Stellera chamaejasme L., Polygonum macrophyllum D. Donvar. Macrophyllum, Poa pretensis L., Kobresia setchwanensis Hand.-Maizz.,Potentilla anserina L. var. anserine and G. squarrosa Ledeb.) decomposed about24%-50% in the non-growing season, which were higher than those in the growingseason (ranged from 10% to 30%). Litter decomposed 10%-20% within the first twoweeks in late autumn and early winter. Significant differences in the whole-yeardecomposition rate and in the processes of decomposition were found among species.Polygonum macrophyllum decomposed slowly through the whole year (26% and15% in the non-growing season and in the growing season, respectively). Certainspecies, such as P. pretensis L. and K. setchwanensis, decomposed at a similar rate(30% both in the non-growing and in the growing season, slightly higher in the8growing season than those in the growing season), whereas S. chamaejasmedecomposed more rapidly (about 50%) in the non-growing season and subsequentlydecomposition became slow (about 12%) in the growing season. Litter nitrogencontents of all the focal species firstly decreased in the non-growing season and thenincreased in the growing season. In vitro experiments of pollen allelopathy, the results showed that pollen from S.chamaejasme was not autotoxic, whereas pollen germination in all the sympatricspecies (Polygonum macrophyllum D. Don var. Macrophyllum, Gentianamacrophylla Pall. var. fetissowii, Gentianopsis paludosa (Hook. f.) Ma var. paludosa,Gentiana squarrosa Ledeb., Halenia elliptica D. Don var. elliptica, Cyananthushookeri C. B. Cl. var. grandiflorus Marq., Euphrasia pectinata Ten., Delphiniumtongolense Franch., Ranunculus tanguticus (Maxim.) Ovcz. var. tanguticus andPotentilla anserina L. var. anserina) decreased nonlinearly as the increasingconcentrations of extract of pollen from S. chamaejasme. Pollen Extract of threepollens from S. chamaejasme generally inhibited 50% pollen germination of most ofthe focal species. 5.76 and 3.35 pollens from S. chamaejasme were observed in fieldon stigmas of G. squarrosa and E. pectinata, respectively. Differences inheterospecific pollen transfer of S. chamaejasme could be attributed to the primaryreason whether they shared common pollinators. Flower morphology (e.g.zygomorphic or actinomorphic), plant or floral density and concurrence in floweringphonologies could explain, in part, the differences in heterospecific pollen transfer.In field experiments, the results showed that seed set in six sympatric species(Gentiana macrophylla Pall. var. fetissowii, Gentianopsis paludosa (Hook. f.) Mavar. paludosa, Gentiana squarrosa Ledeb., Halenia elliptica D. Don var. elliptica,Cyananthus hookeri C. B. Cl. var. grandiflorus Marq. and Euphrasia pectinata Ten.)decreased nonlinearly as the increasing concentrations of extract of pollen from S.chamaejasme. According to the nonlinear curves, the amounts of pollens from S.chamaejasme on stigmas of G. squarrosa and of E. pectinata (i.e. 5.76 grains and3.35 grains, respectively) could reduce 63% and 55% seed set of G. squarrosa and ofE. pectinata, respectively. Thus, allelopathic effects of S. chamaejasme on G.squarrosa and E. pectinata could be realistic. The sympatric species of S.chamaejasme could avoid pollen allelopathy of S. chamaejasme to sustainthemselves. This highlights the need to study how much pollen allelopathy in S.chamaejasme influences the sympatric species through divergence in seasonal ordiurnal flowering phonologies or through asexual reproduction. If pollen allelopathyin S. chamaejasme was confirmed, it could be as a pressure of natural selection andthus play an important role in species evolution.

青藏高原东部植物生态特征在季节性雪厚度梯度上的变化

本文以青藏高原东部的高山草甸为研究对象，设置早融、中间及晚融三个融雪部位，采用实验室测量、野外测量、野外样方调查相结合的 方法，从个体、种群和群落的水平上比较研究了高山雪场植物在同一雪场样地中不同融雪梯度上的特征变异及适应，结果表明： 从早融到晚融的梯度上，随着融雪时间的逐渐推迟，表土日温差降低，冻融交替的强度减弱，土壤水份逐渐增加，总N、总P、总K 以及 可溶性的N、P 和pH 变化不明显，土壤有机质及可溶性的K 和Ca 逐渐降低。冻融交替强度上的差异以及土壤水分差异被认为是融雪梯度上 影响植物生长的主要原因。 从早融到晚融的梯度上，伴随着生态因子的改变，几种常见植物的个体特征也发生相应的变化。首先，物候期推迟。植物开始生长的时间 一般要推迟将近二十天，但同一种植物在不同的融雪部位上的衰老期趋于一致，这预示着在晚融部位同一植物的生长期要缩短。其次，个体生 长特性发生改变。黑褐穗苔草（Carex atrofusca subsp. minor (Boott) T.Koyama）和西北黄芪（Astragalus fenzelianus Pet.-Stib.）的个体生长（株高、单株叶数、单叶面积和地上生物量）表现为逐渐增加的趋势；斑唇马先蒿（Pedicularis longiflora Rudolph var. tubiformis (Klotz.) Tsoong）和川西小黄菊（Pyrethrum tatsienense (Bur. et Franch.) Ling ex Shih.）则表现为逐渐降低的趋势；长叶火绒草（Leontopodium longifolium Ling）在融雪梯度上的变化趋势不明显。再次，从繁殖特性来看，大卫马先蒿（Pedicularis davidii var. pentodon Tsoong）的单株花数、单花种子数、种子千粒重及种子萌发率随融雪的推迟呈现为逐渐增加的趋势；圆穗蓼（Polygonum macrophyllum D.Don）的种子（小坚果）千粒重和萌发率也表现为逐渐增加，其余繁殖特征变化不明显。 在种群层次上，几个常见物种的分布格局随着融雪的推迟都发生一定的变化，基本上表现为从早融的集群分布到中间或晚融部位的随机分布。物种间的联结性也发生较大的变化，由早融部位的总体上的正关联逐步过度到晚融部位上的总体上的负关联。特定种对间的联结性也发生较大的变化。恶劣环境条件（如剧烈的冻融交替）的影响以及对恶劣条件适应被认为是分布格局及种间联结性发生变化的主要原因。 在群落层次上，物种多样性的变化表现为单峰曲线的格局，即在中间部位多样性最高。早融部位强烈的冻融交替和晚融部位缩短的生长季是早融及晚融部位物种多样性不高的重要原因。几乎所有的只出现在一个融雪部位（雪深级别）上的物种都发生在中间融雪部位。这说明，中等的雪深更有利于许多高山植物的存活，而过浅过深的积雪都不利于植物的生存。另外，相距较近的融雪梯度之间的物种相似性较大，而相距较远的梯度之间物种的替代率较高，物种的相似性较小。在群落的生物量方面，地上生物量随融雪的推迟而升高，地下生物量随融雪的推迟而下降，地上与地下生物量之总和随着融雪的推迟而下降，地下生物量与地上生物量之比随着融雪的推迟而下降。早融部位的地上生物量主要集中于地上0-10cm 的范围内，表明在早融部位植物地上部分有变矮的趋势；早融部位的地下生物量在土壤各深度分布相对较均一，而晚融部位地下生物量则主要集中于地下0-10cm 的范围内。生物量的变化趋势主要与雪场中各部位的土壤水分含量及地表日温度差异有关，是植物适应特定环境的结果。 To detect the plants’ responses to snow-cover gradients in an alpine meadow of eastern Tibetan plateau, laboratory method and field sample plot method were employed, and three gradeients （early-, medium and late-melting）were established in a natural snowbed. The measurements were carried out for two years and was done on three levels——individual, population and community. The results are shown as follows : From early- to late-melting gradients, daily ground temperature difference between day and night decreased, amplitude of freeze-thaw alternation weakened, soil organic matter contents and soluble K and Ca decreased, while soil water content increased. Total N, total P, total K，pH soluble N and soluble P kept constant from early- to late-melting portions. Among these factors, the changes of intense freeze-thaw alternation and soil water contents were considered as main factors affecting plants’ growth. From early- to late-melting portions, all phenological phases postponed, e.g. phase of plant emergence postponed almost twenty days. However, the same species’ individuals at different portions withered in step, which implied that the individuals at late-melting portion possessed shorter growing season length. Along the same gradient, both Carex atrofusca subsp. minor (Boott) T. Koyama and Astragalus fenzelianus Pet.-Stib. increased their individual growth, whereas Pedicularis longiflora Rudolph var. tubiformis (Klotz.) Tsoong and Pyrethrum tatsienense (Bur. et Franch.) Ling ex Shih. decreased their individual growth. Unlike the four plants mentioned above, Leontopodium longifolium L. did not show any evident change. As to reproductive charateristics, the flowers per individual, the number of seeds per flower, the thousand seed weight and the seed germination rate of Pedicularis davidii var. pentodon showed an increasing trend; and Polygonum macrophyllum D.Don also increased its thousand seed weight and seed germination rate along the same gradient. However, the other reproductive charateristics of Polygonum macrophyllum D.Don did not change significantly. At population level, the distribution pattern of several selected species changed from cluster pattern to random pattern as the snowmelt postponed. Overall association among the species changed from positive to negative along the same gradient. Further, interspecific association also changed evidently. Adverse circumstances such as intense freeze-thaw alternation were considered as primary factors resulting in changes of population distribution pattern and interspecific association. At the level of community, species diversity showed a pattern of a unimodal trend, i.e. the highest diversity occurred at medium snow depth，perhaps because of intense freeze-thaw alternation at early-melting portions and the shortest growing season at late-melting portions. Almost all species that only appeared at one snowmelt portion occurred at medium portion, indicating that medium snow depth was more suitable for many species’ survival. Species replacement from one snowmelt portion to its neighboring portion seldom took place. However, while distance between two portions became farther, species replacement between the two portions occurred more frequently. As for biomass, aboveground biomass increased from early- to late-melting portions, whereas belowground biomass, total biomass and the ratio of belowground to aboveground all decreased along the same snow gradient. A majority of aboveground biomass distributed in a height range of 0-10 cm, suggesting that height of plants inhabiting early-melting portion be shorter compared with other portions. In addition, belowground biomass at early-melting portion was evenly distributed at different soil depth in comparison with aboveground biomass, whereas belowground biomass at late-melting portion concentrated 0-10cm soil layer below ground. The changing trend of biomass was also related to two factors. One was soil water content, and the other topsoil temperature difference between day and night.

鞑靼荞麦（Fagopyrum tataricum Gaertn.）离体再生与遗传转化研究

鞑靼荞麦是我国特有的农业产品，具有抗寒耐旱特性和较高的营养保健功能。荞麦的开花习性及遗传特点导致其人工杂交授粉难以成功，这成为荞麦杂交育种难以获得突破的重要原因。因此利用转基因技术导入有益基因有可能成为荞麦遗传改良的新途径，而再生及转化体系的建立是开展转基因研究的基础。 本文研究了苗龄、外植体、几种激素配比对鞑靼荞麦（Fagopyrum tataricum Gaertn.）离体培养的影响，初步建立了鞑靼荞麦离体再生体系。结果表明，鞑靼荞麦离体再生的最佳取材时间为苗龄6-8d；诱导愈伤组织的最适培养基为MS＋2.0 mg/L 2,4-D＋1.5 mg/L 6-BA，子叶诱愈率达75％左右，下胚轴的可高达86.62％；愈伤组织分化的最适培养基为MS 0.1mg/L IAA＋2.0mg/L 6-BA＋1.0 mg/L KT＋0.5mg/L TDZ，下胚轴的分化率可达9.52%。下胚轴的诱愈率与分化率均高于子叶，更适于离体再生培养。培养基中加入AgNO3后，能有效降低褐化率。生根最适培养基为含有0.5mg/L NAA的1/2MS培养基，生根率在50％左右。TDZ在诱导鞑靼荞麦的愈伤组织分化出芽的过程中起到明显的促进作用，可提高分化率约20%。 在上述研究基础上，本文还对鞑靼荞麦的遗传转化体系进行了探索性研究。分别利用根癌农杆菌（Agrobacterium tumefaciens）介导法和微粒轰击法（基因枪法）对黑水苦荞下胚轴进行遗传转化。 在农杆菌介导的方法中，携带有质粒pCAMBIA2301的农杆菌菌株EHA105用于转化。载体质粒pCAMBIA2301包含有gus和npt-II 基因, 并受35s启动子驱动。研究结果表明，在侵染方式选择上，浸泡方式比吸打方式更有效，根癌农杆菌侵染的较适浓度为OD600＝0.5，共培养3天，恢复培养7天，能检测到gus基因的表达。 基因枪法使用质粒pBI121，同样包含有gus和npt-II基因, 并受CaMV35s 启动子驱动。轰击距离为9cm较合适，甘露醇前处理在本研究中未表现出明显优势。 两种转化方法比较，基因枪法比农杆菌介导法更快速有效。 本研究为进一步的遗传操作研究打下基础。 Tartary buckwheat (Fagopyrum tataricum Gaertn.), the traditional and unique agricultural product of China, is a kind of crop with strong drought and cold tolerance, abundant nutrition and high medical value. Artificial hybridization is hard in buckwheat because of its flowering habits and genetic characteristics, which leads to no breakthrough in tartary buckwheat breeding. However, biotechnological approaches, especially genetic transformation for the direct introduction of good genes into tartary buckwheat for quality improvement, hold great promise. In this study, we established tartary buckwheat regeneration system in vitro. It is the foundation for genetic manipulation of this crop. The effects of seedling age, hypocotyl and cotyledon as explants, and proportions of several growth regulators were tested in tissue culture of tartary buckwheat for establishing its in vitro regeneration system. The results showed that the best seedling age for callus induction was 6 to 8 days. On the MS medium containing 2.0mg/L 2, 4-D and 1.5mg/L 6-BA, the induction rate of callus from hypocotyls was up to 86.62%, while from cotyledons was about 75％. The suitable shooting medium was the MS medium+0.1mg/L IAA+2.0mg/L 6-BA+1.0 mg/L KT+0.5mg/L TDZ, and the shooting rate from hypocotyls was 9.52%. The callus induction and shooting rates were higher from hypocotyls than from cotyledons. Browning reduced when the medium mixed with AgNO3. Half strength MS supplemented with 0.5mg/L NAA was the best for rooting, the rate was around 50% after 30 days culture. TDZ can accelerate the shoot differentiation distinctively, and it could improve the shooting rate nearly 20%. On the base of above, the explorative research of the genetic transformation in tartary buckwheat was done. In the study, hypocotyls from Heishui tartary buckwheat were transformed by Agrobacterium-mediated method and microprojectile bombardment method (gene-gun), comparatively. In Agrobacterium-mediated method, a disarmed Agrobacterium tumefaciens strain EHA105 harboring plasmid pCAMBIA2301 was used. The vector pCAMBIA2301 contains gus and npt-II genes, driven by CaMV35s promoter. The results showed that the appropriate concentration of Agrobacterium tumefaciens for infecting was OD600＝0.5, and co-culture time was 3d. Seven days later after coculture, GUS expression could be tested. In particle bombardment transformation, plasmid pBI121 was used. pBI121 also contains gus and npt-II genes, driven by 35s promoter. Hypocotyls pretreated with mannitol, no effect was observed, and the suitable distance of bombardment is 9cm. Comparing with Agrobacterium-mediated method, gene-gun method is more convenient and effective. All above results could be a basic work for further study in tartary buckwheat transformation.

青稞B组醇溶蛋白基因与上游调控区的克隆及基因表达

高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源，也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白（hordeins），占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点，大麦醇溶蛋白被划分为三种类型：富硫蛋白亚类(B，γ-hordeins)、贫硫蛋白亚类（C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白，它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明，大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H（5）上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白（B-hordein）。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织，探明Hor2位点基因表达的发育调控机制，最终达到改良禾谷类作物籽粒品质的目的，本研究以青藏高原青稞为材料，采用同源克隆法，分别克隆B-hordein基因和启动子，通过原核生物表达验证B-hordein基因功能，并利用实时定量PCR探索B-hordein基因表达时空关系，取得如下研究结果： 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料，根据GenBank中三个B-hordein基因序列（GenBank No. X03103, X53690和X53691）设计一对引物，通过PCR扩增，获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明，所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子，推测这11个克隆可能是假基因。推测的氨基酸序列分析表明，所有大麦B-hordein具有相似的蛋白质基本结构，均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构，更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析，结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族，来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族，表明这两个亚家族的成员存在显著差异。此外，我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征：即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组（除BXQ053，BZ09-1，BZ26-5分别单独聚为一类外）。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类，避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物，以青稞Z09和Z26的基因组DNA为模板，采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列（命名为Z09P和Z26P）。序列分析表明，推测的TATA box位于–80 bp，CAAT–like box位于–140 bp处。此外，Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box，EB)，在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构，预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节，推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中，将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高；3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物，同时，选择大麦α-微管蛋白基因（GenBank no. U40042）为看家基因并设计特异引物，利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达，荧光定量结果显示：两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录，而Z26开花4天后就有低水平B-hordein的表达，这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外，在4个不同的胚乳发育时期中，Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中，Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期，Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053，BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.

中国青藏高原青稞抗穗发芽资源评价与α-淀粉酶基因的克隆及表达

穗发芽（PHS，preharvest sprouting）是影响禾本科作物生产的重要的灾害之一。收获时期如遇潮湿天气容易导致穗发芽发生。发生穗发芽的种子内部水解酶（主要是α-淀粉酶）活性急剧升高，胚乳贮藏物质开始降解，造成作物产量和品质严重降低。因此，选育低穗发芽风险的品种是当前作物育种工作中面临的重要任务。 青稞(Hordeum vulgare ssp. vulgare)主要分布于青藏高原，自古以来就是青藏高原人民的主要粮食。近年来，由于青稞丰富的营养成分和特有的保健品质、在燃料工业中的潜力以及在啤酒酿造工业中的利用前景，在发达国家日趋受到重视，掀起综合研究利用的热潮。我国拥有占全世界2/3 以上的青稞资源，具有发展青稞产业的得天独厚的条件。然而，由于青稞收获期间恰逢青藏高原雨季来临，常有穗发芽灾害发生，使青稞生产损失巨大。目前对青稞穗发芽研究很少，适用于育种的穗发芽抗性材料相对缺乏，不能很好的满足青稞穗发芽抗性育种的需要。本研究以青藏高原青稞为材料，对其穗发芽抗性的评价指标和体系进行构建，同时筛选青稞抗穗发芽品种并对其抗性进行评价，还利用分子生物学手段对青稞穗发芽抗性的分子机理进行了初步探讨。主要研究结果如下： 1. 本试验以来自于我国青藏高原地区的青稞为材料，对休眠性测定的温度范围进行探讨，并对各种穗发芽抗性测定方法的对青稞的适用性进行评测。通过探讨温度对13 个不同基因型的青稞籽粒发芽和休眠性表达的影响，对筛选青稞抗穗发芽资源的温度条件进行探索，并初步分析了其休眠性表达的机理。在10，15，20，25，30℃的黑暗条件下，选用新收获的13 个青稞品种为材料进行籽粒发芽实验，以发芽指数（GI）评价其休眠性。结果发现，不同品种对温度敏感性不同，其中温度不敏感品种，在各温度条件下均表现很低的休眠性；而温度敏感品种，其休眠性表达受低温抑制，受高温诱导。15℃至25℃是进行青稞休眠性鉴定的较适宜的温度范围。通过对供试材料发芽后的α-淀粉酶活性，发现温度对青稞种子的休眠性表达的影响至少在一定程度上表现在对α-淀粉酶活性的调控上。随后，对分别在马尔康和成都进行种植的34 份青稞穗发芽指数（SI），穗发芽率（SR），籽粒发芽指数（GI）和α-淀粉酶活性（AA）进行了测定和分析，发现它们均受基因型×栽培地点的极显著影响，且四个参数之间具有一定相关性。GI 参数由于其变异系数较低，在不同栽培地点稳定性好，且操作简便，是较可靠和理想的穗发芽评价参数。SI 参数可作为辅助，区别籽粒休眠性相似的材料（基因型）或全面评价材料（基因型）的穗发芽抗性特征。AA 参数稳定性较差，并且检测方法复杂，因此不建议在育种及大量材料筛选和评价时使用。此外，青稞穗发芽抗性受环境影响较大，评价时应考虑到尽可能多的抗性影响因素及其在不同栽培条件下的变异。 2. 对来自青藏高原的青稞穗发芽抗性特征及其与其它农艺性状间的关系进行研究。通过测定穗发芽指数（SI）、籽粒发芽指数（GI）和α-淀粉酶活性（AA），表明113 份青稞材料的穗发芽抗性具有显著差异。SI、GI 和AA 参数的变幅分别为1.00~8.86、0.01~0.97 和0.00~2.76，其均值分别为4.72、0.63 和1.22。根据SI 参数，六个基因型，包括‘XQ9-5’，‘XQ33-9’，‘XQ37-5’，‘XQ42-9’，‘XQ45-7’和‘JCL’被鉴定为抗性品种。综合SI、GI 和AA 参数，可以发现青稞的穗发芽抗性机制包含颖壳等穗部结构的抗性和种子自身的抗性（即种子休眠性），且供试材料中未发现较强的胚休眠品种，除‘XQ45-7’外，所有品种在发芽第四天均能检测出α-淀粉酶活性。穗部结构和种子休眠的抗性机制因基因型不同而不同，在穗发芽抗性中可单独作用或共同作用。农家品种和西藏群体分别比栽培品种和四川群体的穗发芽抗性强，而在不同籽粒颜色的青稞中未发现明显差异。相关性检验发现，青稞的穗发芽抗性，主要是种子休眠性，与百粒重、开花期、成熟期、穗长、芒长和剑叶长呈显著负相关关系，与株高相关性不显著。农艺性状可以作为穗发芽抗性材料选育中的辅助指标。本试验为青稞穗发芽抗性育种研究提供了必要的理论基础和可供使用的亲本材料。 3. α-淀粉酶是由多基因家族编码的蛋白质，在植物种子萌发时高度表达，与植物种子的萌发能力密切相关。在大麦种子发芽时，高等电点α-淀粉酶的活性远大于低等电点的α-淀粉酶。为了研究不同穗发芽抗性青稞品种中编码高等电点α-淀粉酶Amy1 基因结构与抗性间的关系，我们以筛选得到的抗性品种‘XQ32-5’（TR1）、‘XQ37-5’（TR2）、‘XQ45-7’（TR3），易感品种‘97-15’（TS1）、‘9657’（TS2）以及强休眠大麦品种‘SAMSON’（SAM）为材料，对其Amy1 基因的编码区序列进行克隆和结构分析，并对它们推导的氨基酸序列进行比较。结果显示，青稞Amy1 基因具有三个外显子、两个内含子，编码区中有13 个核苷酸变异位点，均位于2、3 号外显子，2 个变异位点位于2 号外显子。SAM 和TS1 分别在2 号外显子相应位置有5 个相同的碱基(GAACT)的插入片段。相应α-淀粉酶氨基酸序列推导发现，所有核苷酸变异中有8 个导致相应氨基酸残基的改变，其余位点为同义突变。青稞Amy1 基因编码区序列品种间相似度高达99%以上，部分序列变异可能与其穗发芽抗性有关。随后，我们又通过SYBR Green 荧光定量技术对该基因在不同发芽时间（1d~7d）的相对表达水平进行了差异性检测。结果发现，7 天内不能检测到SAM 的Amy1 基因表达，5 个青稞品种间的Amy1 基因的相对表达量均随着发芽时间延长而上升，但上升方式有所不同。弱抗品种该基因表达更早，转录本增加速率更大，且在4~5 天可达到平台期。发芽7 天中，抗性品种总转录水平明显低于易感品种。本研究结果表明，青稞Amy1 基因的转录水平是与其穗发芽抗性高度相关。 我国青藏高原青稞，尤其是农家品种的穗发芽抗性具有丰富的变异，蕴藏着穗发芽抗性育种的宝贵资源。本研究为青稞穗发芽抗性育种建立了合理抗性评价体系，筛选出可供育种使用的特殊材料，阐明了农艺性状可辅助穗发芽抗性育种，同时还对穗发芽抗性与α-淀粉酶基因的结构和表达关系进行分析，为青稞穗发芽抗性资源筛选奠定了基础。 Preharvest sprouting (PHS) is a serious problem in crop production. It often takes place when encountering damp, cold conditions at harvest time and results in the decrease of grain quality and great loss of yield by triggering the synthesis of endosperm degrading enzymes (mostly the α-amylase). Therefore, PHS is regarded as an important criterion for crop breeding. In order to minimize the risk of PHS, resistant genotypes are highly required. Hulless barley (Hordeum vulgare ssp. vulgare) is the staple food crop in Qinghai-Tibetan Plateau from of old, where is one of the origin and genetic diversity centers of hulless barley. Recently, interest in hulless barley has been sparked throughout the world due to the demonstrations of its great potential in health food industry and fuel alcohol production. Indeed, hulless barley can also be utilized to produce good quality malt if the appropriate malting conditions are used. In China, overcast and rainy conditions often occur at maturity of hulless barley and cause an adverse on its production and application. PHS resistant genotypes, therefore, are highly required for the hulless barley breeding programs. However, few investigations have been made so far on this issue. The objectives of this study were: 1) to assessment of methods used in testing preharvest sprouting resistance in hulless barley; 2) to evaluate the variability and characteristics of PHS resistance of hulless barley from Qinghai-Tibet Plateau in China; 3) to select potential parents for PHS resistance breeding; 4) to primarily study on the molecular mechanism of PHS resistance of hulless barley. Our results are as followed: 1. We investigated the temperature effects on seed germination and seed dormancy expression of hulless barley, discussed appropriate temperature range for screening of PHS resistant varieties, and analyzed the mechanism of seed dormancy expression of hulless barley. The dormancy level of 13 hulless barley were evaluated by GI (germination index) values calculating by seed germination tests at temperature of 10，15，20，25，30℃ in darkness. There were great differences in temperature sensitivity among these accessions. The insensitive accessions showed low dormancy at any temperature while the dormancy expression of sensitive accessions could be restrained by low temperature and induced by high temperature. The temperature range of 15℃ to 25℃ was workable for estimating of dormancy level of hulless barley according to our data. Analysis of α-amylase activity showed that the temperature effects on seed germination and the expression of seed dormancy be achieved probable via regulating of α-amylase activity. Furthermore, we evaluated the differences in sprouting index (SI), sprouting rate (SR), germination index (GI) and α-amylase activity (AA) between Maerkang and Chengdu among 34 accessions of hulless barley from Qinghai-Tibetan Plateau in China. These PHS sprouting parameters were significantly affected by accession×location, and they had correlation between each other. GI was the most reliable parameter because of its low CV value, good repeatability and simple operation. SI could assist in differentiating between accessions of similar dormancy or overall evaluation of the resistance. AA was bad in repeatability and had relatively complex testing method, therefore, not appropriate for breeding and evaluation and screening of PHS resistant materials. Besides, since PHS resistance of hulless barley was greatly influenced by its growth environment, possibly much influencing factors and variations between cultivated conditions should be considered. 2. In this study, large variation was found among 113 genotypes of hulless barley (Hordeum vulgare ssp.vulgare) from Qinghai-Tibetan Plateau in China, based on the sprouting index (SI), germination index (GI) and α-amylase activity (AA) which derived from sprouting test of intact spikes, germination test of threshed seeds and determination of α-amylase activity, respectively. The range of SI, GI and AA was 1.00~8.86, 0.01~0.97 and 0.00~2.76，the mean was 4.72, 0.63 and 1.22 espectively. Six resistant genotypes, including ‘XQ9-5’, ‘XQ33-9’, ‘XQ37-5’, ‘XQ42-9’, ‘XQ45-7’ and ‘JCL’, were identified based on SI. Integrating the three parameters, it was clear that both hulls and seeds involved in PHS resistance in intact spikes of hulless barley and there was no long-existent embryo dormancy found among the test genotypes. All the genotypes, except ‘XQ45-7’, had detectable α-amylase activity on the 4th day after germination. There was PHS resistance imposed by the hull and seed per se and the two factors can act together or independent of each other. Besides, landraces or Tibet hulless barley had a wider variation and relatively more PHS resistance when compared with cultivars or Sichuan hulless barley. No significant difference was found among hulless barley of different seed colors. The correlation analysis showed PHS resistance was negatively related to hundred grain weight, days to flowering, days to maturity, spike length, awn length and flag length but not related to plant height. This study provides essential information and several donor parents for breeding of resistance to PHS. 3. Alpha-amylase isozymes are encoded by a family of multigenes. They highly express in germinating seeds and is closely related to seed germination ability. In barley germinating seeds, the activity of high pI α-amylase is much higher than low pI α-amylase. The aim of this study was to determine the relationship between preharvest sprouting resistance of hulless barley and the gene structure of Amy1 gene which encodes high pI α-amylase. The coding region and cDNA of Amy1 gene of three resistant accessions, including ‘XQ32-5’ (TR1), ‘XQ37-5’ (TR2), ‘XQ45-7’ (TR3), two susceptible accessions ‘97-15’ (TS1), ‘9657’ (TS2) and one highly dormant barley accession ‘SAMSON’ (SAM) was cloned. Analysis of their DNA sequences revealed there were three exons and two introns in Amy1 gene. Thirteen variable sites were in exon2 and exon3, 2 variable sites were in intron2. SAM and TS1 had a GAACT insert segment in the same site in intron2. Only 8 variable sites caused the change of amino acid residues. There were 99% of similarity between the tested hulless barley and some of the variable sites might be related with preharvest sprouting resistance. Then, we investigated the expression level of Amy1 gene in the 7-day germination test. Results of quantitative real-time PCR indicated that the relative expression trends of Amy1 gene were the same but had significant differences in the increase fashion between hulless barleys and no detectable expression was found in SAM. Susceptible accessions had earlier expression and faster increase and reached the maximum on day 4 ~ day 5. Besides, total transcripts level was found lower in resistant accessions than susceptible accessions. This study indicated that α-amylase activity was highly related to the transcription level of Amy1 gene which not correlated to missense mutation sites. In conclusion, hulless barley, especially the landraces from Qinghai-Tibetan Plateau in China possesses high degree of variation in PHS performance, which indicates the potential of Tibetan hulless barley as a good source for breeding of resistance to PHS. This study provides several donor parents for breeding of resistance to PHS. Our results also demonstrate that agronomic traits may be used as assistants for PHS resistance selection in hulless barley. Besides, analysis of high pI α-amylase coding gene Amy1 revealed the relative high expression of was Amy1 one of the mainly reason of different PHS resistance level in hulless barley.

A Global Crisis for Seagrass Ecosystems

Seagrasses, marine flowering plants, have a long evolutionary history but are now challenged with rapid environmental changes as a result of coastal human population pressures. Seagrasses provide key ecological services, including organic carbon production and export, nutrient cycling, sediment stabilization, enhanced biodiversity, and trophic transfers to adjacent habitats in tropical and temperate regions. They also serve as “coastal canaries,” global biological sentinels of increasing anthropogenic influences in coastal ecosystems, with large-scale losses reported worldwide. Multiple stressors, including sediment and nutrient runoff, physical disturbance, invasive species, disease, commercial fishing practices, aquaculture, overgrazing, algal blooms, and global warming, cause seagrass declines at scales of square meters to hundreds of square kilometers. Reported seagrass losses have led to increased awareness of the need for seagrass protection, monitoring, management, and restoration. However, seagrass science, which has rapidly grown, is disconnected from public awareness of seagrasses, which has lagged behind awareness of other coastal ecosystems. There is a critical need for a targeted global conservation effort that includes a reduction of watershed nutrient and sediment inputs to seagrass habitats and a targeted educational program informing regulators and the public of the value of seagrass meadows.

Coupling of seagrass (Cymodocea nodosa) patch dynamics to subaqueous dune migratio

The coupling between patch dynamics - described by the patch growth (horizontal and vertical), patch mortality, and life-history of Cymodocea nodosa (Ucria) Aschers., and the disturbance caused by the migration of subaqueous dunes over the plants was examined in a shallow NW Mediterranean bay (Alfacs Bay) where this species maintains a patchy cover. C. nodosa shoots survived substantial burial rates (up to 2.4 mm/day) by growing vertically at rates proportional to, albeit four-fold slower than, burial rates. Patch death was caused by erosion as large subaqueous dunes migrated pass the plant patch. Patch growth was fastest over the progressing slope of the dunes ( similar to 2.5 m year super(-1)) and flowering was also stimulated by sand accretion. The time interval between the passage of consecutive dunes, which sets the time window available for patch development, ranged between 2 and 6 years. This time interval allowed C. nodosa to recolonize bare substrata, with patch formation occurring about half a year after the disturbance, and also allowed established shoots to complete their life-cycle and produce seeds and thus enable subsequent recolonization. The time windows available for patch development also set an upper limit to patch size of about 26 m. Significant cross correlations between dune topography and patch dynamics and plant flowering frequency provide evidence that the spatial heterogeneity in the vegetation is closely associated with the disturbance imposed by the migration of sand dunes. The migration of subaqueous dunes maintains C. nodosa in a continuous state of colonization involving spatially asynchronous patch growth and subsequent mortality, which is ultimately responsible for the characteristic patchy landscape of this Bay. 

Seed-bank development, germination and early seedling survival of two seagrass species from The Netherlands: Zostera marina L. and Zostera noltii hornem

Flowering and seed-bank development of annual Zostera marina L. and perennial Z. noltii hornem. were studied in the Zandkreek (S.W. Netherlands). Flowering of Z. noltii started at the end of June and continued until the end of September. A maximum of ca. 1000 flowering shoots (11% of the total amount of shoots per square metre) occurred in early August. Flowering of Z. marina started at the end of July and continued throughout October. Seed banks of both species appeared to be annual. Actual seed densities of Z. noltii were much lower than predicted on the basis of the amount of inflorescences.Germination was studied in the laboratory in relation to temperature (10, 20 and 30°C), salinity (1.0, 10.0, 20.0, 30.0 and 40.0‰) and stratification (at 4°C). Both species showed a maximal germination at 30°C and 1.0‰ salinity, decreasing with higher salinities and lower temperatures. Stratification stimulated germination only at salinities 20.0‰. Desiccation and anaerobia were lethal to Z. marina seeds. Seedlings of Z. marina survived best at 10°C and 10.0–20.0‰ salinity and those of Z. noltii survived best at 10°C and 1.0‰ salinity. Overall, seedlings of Z. marina survived better than those of Z. noltii.

施肥与品种及其种子大小对冬小麦光合及叶绿素荧光特性的影响

以土垫旱耕人为土为供试土样,采用4个冬小麦品种、3种播种方式和4种施肥方式进行盆栽试验,研究施肥、品种和种子大小对小麦叶片光合和叶绿素荧光特性的影响.结果表明:(1)在开花期和灌浆期,旗叶叶绿素相对含量(SPAD值)均为NP配施最高,其平均值分别比对照显著增加17.83%和13.01%;NP配施条件下,在开花期和灌浆期SPAD均为大粒单播较高并显著高于小粒单播;开花期和灌浆期SPAD分别以远丰998和咸农39最高并显著高于其它品种,平均比最低的白芒麦分别高17.68%和18.75%.(2)对旗叶净光合速率来说,开花期的NP配施处理比对照略有下降,而单施N和P分别比对照显著降低13.03%和23.17%,灌浆期的平均值以NP配施最高且比对照显著提高6.95%;小偃6号在开花期显著高于其余品种4.01%~6.19%(P<0.05),而白芒麦和咸农39则在灌浆期具有较明显优势,均分别显著高于其余品种约16.60%~26.91%;在开花期和灌浆期,2种单播方式平均值相近且显著高于混播方式.(3)就叶片Fv/Fm和Fv/Fo值而言,NP配施明显高于其它施肥处理,且NP均衡供应时远丰998和混播方式具有明显优势.可见,氮...

土壤水分与氮肥对玉米根系生长的影响

利用田间小区试验研究了不同土壤水分条件下N肥对根系生长(根长、根重和根冠比)的影响。结果表明,玉米拔节期和开花期无论水分条件如何,施N肥可增加其总根长、表层根长和根重并使根冠比下降;灌浆期施N肥可增加总根长和表层根长,但正常水分条件下N肥使根重和根冠比下降,而干旱条件下N肥对根重和根冠比则无影响。不同处理根冠比大小与N吸收和分配有很大关系。

不同生育期干旱对冬小麦产量及水分利用效率的影响

用盆栽对冬小麦不同生育阶段进行不同程度水分调亏试验结果表明 :拔节—孕穗期、抽穗—扬花期和灌浆—成熟 3个阶段内 RW上限为 4 0 %、5 0 %、60 %的水分亏缺均引起了产量的极显著下降 ,而且水分亏缺越严重 ,产量降低越大。在 3个生育阶段内进行 RW上限为 4 0 %的水分调亏减产幅度都很大 ,而且 3个生育阶段之间差异不明显 ;进行 5 0 %、60 %水分调亏 ,其减产程度则与生育期有关。灌浆—成熟期的减产程度大于前二个时期 ,这可能与前二个阶段复水后作物的补偿生长有关。不同生育期水分亏缺对冬小麦产量构成因素的影响也不同 ,拔节—扬花期水分亏缺主要减少了穗粒数 ,灌浆—成熟阶段的水分亏缺主要减少了千粒重

栽培模式、施氮和品种对冬小麦冠层结构和产量的影响

在黄土高原南部半湿润易旱区土垫旱耕人为土上进行大田试验,研究氮肥、品种和栽培模式对冬小麦叶面积指数(LAI)和透光率(DIFN)及产量的影响。结果表明,小麦生长过程中LAI先增大后减小,在开花期最大,成熟期最小,施氮能够显著增加LAI;DIFN的变化规律与LAI相反。不同品种间LAI和DIFN差异显著。全程覆膜和覆膜150 d的LAI极显著大于常规栽培、集雨面栽培和三密一稀栽培模式,DIFN与此相反;施氮后籽粒产量极显著增加。在不同栽培模式下,以全程覆膜、覆膜150 d和集雨面栽培模式的产量较高,但从维持土壤质量角度考虑,则覆膜150 d和集雨面2种栽培模式优于其它几种栽培模式;从冠层结构特征分析,这2种栽培模式更有利于增加群体光合能力。

Developing Porphyra/salmon integrated aquaculture for bioremediation and diversification of the aquaculture industry

FUNCTIONAL-FORM GROUPS; RED ALGAE; ATLANTIC SALMON; NEW-HAMPSHIRE; NITROGEN; PHOSPHORUS; RHODOPHYTA; TEMPERATURE; NUTRIENTS; KJELLMAN