HPLC测定不同海拔小叶金露梅17种氨基酸含量

目的:建立高效液相色谱法测定小叶金露梅中17种氨基酸含量的方法。方法:采用N,N-二硝基氟苯柱前衍生氨基酸后测定;色谱柱为Phenomenex Gemini 5μC18,流动相为醋酸钠(pH=6.40)和50%乙腈,采用梯度洗脱,检测波长为360nm,外标法计算含量。结果:17种氨基酸线性关系、精密度、稳定性、重复性良好,加样回收率为97.4%～102.8%(RSD为1.21%～2.5%)。各海拔的氨基酸总量范围是10%～14.79%,在不同海拔小叶金露梅中氨基酸平均质量分数顺序为亮氨酸>天冬氨酸>异亮氨酸>组氨酸>丝氨酸>精氨酸>丙氨酸>苯丙氨酸>缬氨酸>谷氨酸>脯氨酸>甘氨酸>赖氨酸。小叶金露梅含有7种必需氨基酸,它们的含量均大于48%。本方法简便、准确,为小叶金露梅药理提供理论依据。     

金露梅幼苗对高温胁迫的生理生化响应

通过测定金露梅2年生幼苗在连续6d高温胁迫(每日9:00~15:00为42℃,其余时间30℃)下及1d恢复期间(对照,每日6:00~18:00为30℃,其余时间20℃)的叶片生理生化指标的变化,探讨金露梅耐热的生理生化机制。结果显示:随高温胁迫时间的延长,细胞膜透性以及丙二醛(MDA)、可溶性糖和游离脯氨酸含量均呈上升的趋势,超氧化物歧化酶(SOD)活性呈下降趋势,过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性呈先上升后下降趋势,且除细胞膜透性与对照无显著差异外,其它指标均显著高于对照;恢复1d后,各项指标水平均与对照持平。研究表明,在高温胁迫条件下,金露梅幼苗能通过提高渗透调节物质可溶性糖、脯氨酸含量来增强对高温逆境的适应性,同时还通过增强POD、CAT、APX活性清除因胁迫积累的H2O2以及活性氧,以维持细胞内活性氧产生和清除的动态平衡,保护细胞膜的稳定性,进而提高其对高温逆境的抗性。     

珠穆朗玛峰自然保护区小叶金露梅灌丛群落数量分类和排序及其多样性垂直格局

根据野外样方调查数据,采用双向种指示分析(TWINSPAN)和典范对应分析(CCA),对珠穆朗玛峰国家级自然保护区小叶金露梅灌丛群落进行分类和排序,并分析物种多样性沿海拔梯度的分布格局。结果表明:(1)该区域24个样地中,记载的维管束植物共有23科45属80种,出现频度较高的种有小叶金露梅(Potentilla parvifolia)、高山嵩草(Kobresia pygmaea)、木根香青(Anaphalis xylorhiza)、垫状点地梅(Androsace tapete)、藏沙蒿(Artemisia wellbyi)、垫状雪灵芝(Arenaria pulvinata)和柴胡红景天(Rhodiola bupleuroides)等。(2)经TWINSPAN等级分类将该区域小叶金露梅灌丛24个样地划分为10个群丛类型。(3)样地和物种CCA二维排序结果表明,海拔和坡位是影响该区域小叶金露梅灌丛群落和物种分布格局的主要环境因子。(4)该区域小叶金露梅灌丛群落物种丰富度、Shannon-Wiener指数和Simpson指数均随海拔升高呈下降的趋势,而Pielou指数呈上升的趋势。(5)样地中优势种小叶金露梅的盖度和高度沿海拔梯度呈显著下降趋势。     

氮磷添加对金露梅叶片化学计量及光合特征的影响

通过三种养分添加处理,氮添加(5、10和15 g??m-2)、磷添加(梯度同氮添加)、氮磷同时添加[(5 g N＋5 g P)??m-2、(10 g N＋10 g P)??m-2、(15 g N＋15 g P)??m-2],对照(无养分添加),探讨养分添加对金露梅叶片性状氮含量(Nmas )、磷含量(Pmas )、氮磷比(N∶P)、比叶重(LMA)、净光合速率(Pn )和光合氮利用效率(PNUE)的影响,以及各性状之间的相互关系.结果表明：在处理水平上,除N或P显著提高金露梅叶片的N∶P外,氮、磷添加对叶片其它性状无显著影响;不同氮、磷处理下添加水平对金露梅叶片的Nmas、N∶P、Pn和PNUE均有显著影响,随着养分水平提高,各性状的变化模式各不相同,叶片Pmas无明显变化,而叶片LMA虽有降低的趋势但不显著.回归分析表明,叶片Pmas与Nmas之间呈显著正相关(R2＝0．347,P＜0．001),叶片Nmas 与N∶P之间也呈显著正相关(R2＝0．018,P＜0．05),而叶片Pmas与N∶P呈显著负相关(R2＝0．505,P＜0．001);叶片LMA与Pn之间显著负相关(R2＝0．02,P＜0．05),而与PNUE之间显著正相关(R2＝0．077,P＜0．001).这表明在一定范围内,环境变化可以改变金露梅叶片的养分保持能力、光合能力以及养分利用效率.     

青海高寒区域金露梅灌丛草甸灌木和草本植物固碳量的比较

以青海海北高寒区域金露梅（PotentillafruticosaLinn.）灌丛草甸为研究对象,分析了6月至9月金露梅灌丛草甸灌木和草本植物不同部位的生物碳量,并据此对灌木及草本植物的年净初级生产碳量进行了比较。结果显示：金露梅灌丛草甸灌木植物地上部和地下部不同层次的生物量和碳含量均有明显差异,根据生物量所占比例确定其地上部和地下部的平均碳含量分别为0.50和0.48。依据不同月份灌丛冠面最大长度、最小宽度和最大高度,采用方程“Wij=e〔aln（A·B·H）＋b〕”计算灌木地上当年新生生物碳量、地上多年累积生物碳量和地下多年累积生物碳量,相关性均极显著（P〈0.01）,表明利用该方程评估金露梅灌丛草甸灌木不同部位的生物碳量是可行的。不同月份金露梅灌丛草甸灌木地上当年新生生物碳量、地上多年累积生物碳量和地下多年累积生物碳量分别为9.36-21.15、78.07-90.12和74.37-101.22g·m-2,差异不明显;其地上部和地下部净初级生产碳量分别为33.20和26.85g·m-2,总计为60.05g·m-2。金露梅灌丛草甸草本植物地上部和地下部净初级生产碳量分别为111.41和445.41g·m-2,总计为556.82g·m-2。如果根据草本和灌木占地面积78%和22%进行加权计算,则金露梅灌丛草甸当年的总净初级生产碳量为447.53g·m-2,其中灌木的净初级生产碳量仅占2.95%,且金露梅灌丛草甸地下部与地上部净初级生产碳量的比值为3.75。研究结果显示：青海高寒区域金露梅灌丛草甸以草本固碳为主,且地下部净初级生产碳量明显高于其地上部。     

放牧影响下不同盖度金露梅灌丛草本植物叶功能性状与土壤因子的关系

植物功能性状与环境因子的研究对认识不同环境梯度下植物群落的形成及对环境的适应机制具有重要意义。该研究以高寒金露梅(Potentilla fruticosa)灌丛草甸为研究对象,分析放牧影响下金露梅灌丛不同盖度梯度(金露梅灌丛盖度为0%、30%、60%和90%的灌草斑块)灌草斑块中草本植物叶功能性状与土壤因子的变化趋势及其相互关系。结果表明:(1)土壤容重(BD)、土壤pH和土壤养分在金露梅灌丛盖度梯度下差异显著,干柴滩金露梅灌丛具有明显的"沃岛效应";随着金露梅灌丛盖度的递增,土壤理化性质变化趋势各有分异。(2)随着金露梅灌丛盖度的增加,比叶面积(SLA)显著上升,叶片厚度(LT)和叶片磷含量(LPC)呈显著下降趋势(P0.05)。(3)比叶面积与叶片厚度呈负相关关系,与叶片氮磷含量(LNC、LPC)呈极显著负相关关系(P0.01),与叶片碳含量(LCC)呈极显著正相关关系(P0.01)。(4)RDA冗余分析表明,土壤因子对叶功能性状变异的平均解释变量达72.25%,影响植物功能性状的主导因子有土壤有机质(SOM)、土壤全氮(TN)、土壤有效氮(AN)、土壤有效钾(AK)和土壤容重(BD),但不同盖度金露梅灌丛群落中影响叶性状的主导因子存在差异。金露梅灌丛盖度较小(30%)时受SOM和TN、AN的影响较大,而金露梅灌丛盖度较大时(60%)时主要受AK和BD的影响。     

亚高寒草甸不同坡向金露梅种群的空间分布格局及空间关联

植物种群在不同干扰条件下对环境的适应对策可以通过其空间分布格局及关联性反映出来.金露梅是甘南亚高寒草甸中的优势种,通过野外群落调查,运用空间点格局分析的Ripley K函数,分析甘南不同坡向亚高寒草甸中金露梅种群的空间分布格局及其关联性.结果表明: 金露梅种群在3个坡向的分布具有显著差异,重要值、生物量和盖度在北坡均高于南坡;北坡和西坡金露梅种群Ⅰ、Ⅱ级个体在小尺度上呈显著聚集分布,随着龄级和空间尺度的增加,金露梅种群聚集强度逐渐减弱,趋向于随机分布;南坡由于其生境条件对金露梅生长的抑制,种间竞争激烈,各级金露梅种群的分布格局均以随机分布为主;不同坡向的金露梅种群Ⅰ、Ⅱ级个体之间在一定尺度内表现为负关联,随着尺度增加,关联度降低,Ⅲ、Ⅳ级与Ⅰ、Ⅱ级个体之间的关联性呈显著负关联;较大个体之间无明显关系,表明年龄接近的个体在空间分布上是相互独立的,利于其充分利用环境资源.     

基于静态箱式法和生物量评估海北金露梅灌丛草甸碳收支

高寒灌丛是青藏高原重要植被类型,因特殊生物学性质致使其系统碳功能较难评估。采用静态箱式法测定高寒金露梅(Potentilla fruticosa)灌丛草甸的生态系统呼吸,结合生物量收获法估测生态系统净初级碳量。结果表明,高寒金露梅灌丛草甸生态系统呼吸、土壤呼吸和植物呼吸具有明显的季节动态变化,其年总量分别为886.28、444.93 gC/m2和441.36 gC/m2;灌丛区、草本区以及土壤区的呼吸均与5 cm地温具有极显著的指数关系(R2分别为0.95、0.94和0.83),各区温度敏感系数Q10分别为4.40、4.13和3.16;8a(2003—2010)植被净初级生产力平均为468.55 gC/m2。结合系统土壤呼吸,生态系统年均净固碳量为27.19 gC/m2,即高寒金露梅灌丛草甸生态系统为碳汇。对比涡度相关标准方法连续观测数据表明该方法评估生态系统碳功能具有较大可信度。     

祁连山金露梅灌丛草甸群落结构及主要种群的点格局分析

金露梅灌丛草甸是青藏高原高寒草地生态系统的重要组分之一,采用Ripley的K(t)函数分析方法,对放牧和封育条件下主要种群的分布格局和种间关系及金露梅灌丛群落动态变化进行了研究.结果表明:(1)经过6年的围栏封育,金露梅灌丛草甸主要种群的空间分布格局表现出从均匀或随机分布向随机或聚集分布转变的趋势,聚集强度随围封尺度增大而增加,随放牧的干扰而降低,聚集分布有利于种群发挥功能群整体效应.(2)围栏内不同功能群之间主要呈显著正关联;围栏外的禾草与杂类草、莎草与杂类草之间均呈显著负关联,出现种间资源竞争,且随着尺度的增大负关联逐渐增强,不同物种的生态位差异明显.(3)围栏内外种群点格局分布都具有明显的尺度依赖性,植物间相互作用和外界放牧干扰是影响物种不同尺度下空间格局变化的主要原因,点格局的变化驱动了群落结构的变化.可见,长期夏季放牧抑制了高寒金露梅灌丛草甸禾草和莎草植物的分株和生长,促进了杂类草的生长,金露梅灌丛草甸呈现出退化的趋势,围栏封育有利于草地生态系统的恢复演替.     

氮磷复合肥添加对高寒草甸金露梅灌丛中豆科植物的影响

以高寒草甸金露梅灌丛为研究对象,通过添加不同量的氮磷复合肥NH₄H₂PO₄(0、40、80、120g·m^-2),测定了花苜蓿(Medicago ruthenica Linn.)、异叶米口袋(Gueldenstae dtiadiversifolia Maxim)、甘肃棘豆(Oxytropis kansuensis Bunge.)的地上生物量、叶片氮磷含量和土壤氮磷含量,探讨了添加氮磷之后豆科植物的叶片氮磷含量、N:P比值在处理间和种间的差异以及它们与土壤养分之间的关系。结果表明,添加氮磷使花苜蓿和异叶米口袋叶片中的磷含量和N:P比值发生显著变化,而对叶片氮含量的影响不显著。氮磷的添加对甘肃棘豆的氮磷含量无显著影响。就总的豆科植物而言,不论种内还是种间,平均可变性都是磷变化最大,氮变化最小,N:P比值居中。植物叶片的氮磷含量以及N:P比值与土壤速效氮磷没有显著相关性。比较不同处理下的数据可知,添加氮磷复合肥主要增加了豆科植物对磷素的养分吸收。三个物种对氮磷的吸收能力不同,叶片的氮磷含量比N:P比值更能反映植物对氮磷的吸收利用情况。一次性施用氮磷复合肥不能提高第二年的氮供应能力,但是会提高土壤的有效磷供应。为保护豆科牧草的产量,对于金露梅灌丛草地施用磷酸二氢铵的量不应超过80g·m^-2。     

The role of shrub (Potentilla fruticosa) on ecosystem CO2 fluxes in an alpine shrub meadow

Aims Recent studies have shown that alpine meadows on the Qinghai-Tibetan plateau act as significant CO₂ sinks. On the plateau, alpine shrub meadow is one of typical grassland ecosystems. The major alpine shrub on the plateau is Potentilla fruticosa L. (Rosaceae), which is distributed widely from 3 200 to 4 000 m. Shrub species play an important role on carbon sequestration in grassland ecosystems. In addition, alpine shrubs are sensitive to climate change such as global warming. Considering global warming, the biomass and productivity of P. fruticosa will increase on Qinghai-Tibetan Plateau. Thus, understanding the carbon dynamics in alpine shrub meadow and the role of shrubs around the upper distribution limit at present is essential to predict the change in carbon sequestration on the plateau. However, the role of shrubs on the carbon dynamics in alpine shrub meadow remains unclear. The objectives of the present study were to evaluate the magnitude of CO₂ exchange of P. fruticosa shrub patches around the upper distribution limit and to elucidate the role of P. fruticosa on ecosystem CO₂ fluxes in an alpine meadow.Methods We used the static acrylic chamber technique to measure and estimate the net ecosystem productivity (NEP), ecosystem respiration (R e), and gross primary productivity (GPP) of P. fruticosa shrub patches at three elevations around the species' upper distribution limit. Ecosystem CO₂ fluxes and environmental factors were measured from 17 to 20 July 2008 at 3 400, 3 600, and 3 800 m a.s.l. We examined the maximum GPP at infinite light (GPP max) and maximum R e (R emax) during the experimental time at each elevation in relation to aboveground biomass and environmental factors, including air and soil temperature, and soil water content.Important findings Patches of P. fruticosa around the species' upper distribution limit absorbed CO₂, at least during the daytime. Maximum NEP at infinite light (NEP max) and GPP max of shrub patches in the alpine meadow varied among the three elevations, with the highest values at 3 400 m and the lowest at 3 800 m. GPP max was positively correlated with the green biomass of P. fruticosa more strongly than with total green biomass, suggesting that P. fruticosa is the major contributor to CO₂ uptake in the alpine shrub meadow. Air temperature influenced the potential GPP at the shrub-patch scale. R emax was correlated with aboveground biomass and R emax normalized by aboveground biomass was influenced by soil water content. Potentilla fruticosa height (biomass) and frequency increased clearly as elevation decreased, which promotes the large-scale spatial variation of carbon uptake and the strength of the carbon sink at lower elevations.     

Phylogeography of Potentilla fruticosa (Rosaceae) in the Qinghai-Tibetan Plateau revisited: a reappraisal and new insights

Background: Most empirical studies to date have suggested that alpine plants in the central Qinghai-Tibetan Plateau (QTP) originated from the south-eastern QTP. However, previous phylogeographcial analysis of Potentilla fruticosa suggested that it diversified in the central QTP, which has also been assumed to have been a glacial refugium, and expanded to the north-eastern regions during the mid-Holocene period.

Aim: We reappraise this claim in the light of information acquired from sampling, haplotyping and analysing additional populations.

Methods: We sequenced the chloroplast trnT-L intergenic spacer of 346 individuals collected from 52 populations. We reconstructed phylogenetic relationships among haplotypes through Bayesian and Network analysis, and used several methods for demographic analysis.

Results: Some common and highly divergent haplotypes were distributed in the central, western and south-eastern QTP. Mismatch and other population genetic analyses applied to our data suggested that a distinct range expansion had occurred between approximately 30,000 and 325,000 years ago (kya).

Conclusions: Besides the possible postglacial expansion in the north-eastern QTP, our results further suggested that the range of the species radically expanded across the whole Plateau before the last glacial maximum (around 18 kya) and the south-eastern, western and central QTP regions had together provided important refugia during recent glacial stages. These findings, in contrast to the previous conclusions, highlight the importance of adequate sampling strategies in phylogeographical studies.     

Repeated Range Expansion and Glacial Endurance of Potentilla glabra (Rosaceae) in the Qinghai-Tibetan Plateau

To date, little is still known about how alpine species occurring in the Qinghai-Tibetan Plateau (QTP) responded to past climatic oscillations. Here, by using variations of the chloroplast trnJ-L, we examined the genetic distribution pattern of 101 individuals of Potentilla glabra, comprising both the interior QTP and the plateau edge. Phylogenetic and network analyses of 31 recovered haplotypes identified three tentative clades (A, B and C). Analysis of molecular variance (amova) revealed that most of the genetic variability was found within populations (0.693), while differentiations between populations were obviously distinct (Fst = 0.307). Two independent range expansions within clades A and B occurring at approximately 316 and 201 thousand years ago (kya) were recovered from the hierarchical mismatch analysis, and these two expansions were also confirmed by Fu's Fs values and 'g' tests. However, distant distributions of clade C and private haplotypes from clades A and B suggest that they had survived the Last Glacial Maximum (LGM) and previous glaciers in situ since their origins. Our findings based on available limited samples support that multiple refugia of a few cold-enduring species had been maintained in the QTP platform during LGM and/or previous glacial stages.     

Carbon Balance in an Alpine Steppe in the Qinghai-Tibet Plateau

Carbon fluxes were measured using a static chamber technique in an alpine steppe in the Qinghai-Tibet Plateau from July 2000 to July 2001. It was shown that carbon emissions decreased in autumn and increased in spring of the next year, with higher values in growth seasons than in winters. An exponential correlation （Ecarbon = 0.22（exp（0.09T） ＋ In（0.31P ＋ 1））, R^2 = 0.77, P 〈 0.001） was shown between carbon emissions and environmental factors such as temperature （T） and precipitation （P）. Using the daily temperature （T） and total precipitation （R）, annual carbon emission from soil to the atmosphere was estimated to be 79.6 g C/m^2, 46% of which was emitted by microbial respiration. Considering an average net primary production of 92.5 g C/m^2 per year within the 2 year experiment, alpine steppes can take up 55.9 g CO2-C/m^2 per year. This indicates that alpine steppes are a distinct carbon sink, although this carbon reservoir was quite small.     

紫穗槐果实和叶子的紫外光谱指纹图谱研究

本文通过研究紫穗槐的紫外光谱指纹图谱,鉴别紫穗槐及其混淆品。分别取紫穗槐果实和叶子各19个样品以及6个混淆品,应用紫外光谱法对其共有峰和特征峰进行检测。结果显示:紫穗槐果实有2个共有峰,其峰值比为1.330:1;紫穗槐叶有4个共有峰,其峰值比1.45:1:0.71:0.69。     

Isolation and characterization of microsatellite loci for the Potentilla core group (Rosaceae) using 454 sequencing

Microsatellites are valuable markers for the analysis of genetic diversity, linkage mapping or genotyping. The limited availability of microsatellites for the genus Potentilla (Rosaceae) stipulated the isolation of markers from a representative (Potentilla pusilla Host) of the Potentilla core group that constitutes the most species‐rich evolutionary lineage within the genus. Thousand four hundred and seventy‐six simple sequence repeat (SSR) containing candidate sequences were isolated from a single‐type line using 454 sequencing. Seventy‐four functional microsatellite markers were developed from 200 sequences selected for suitable priming sites flanking microsatellite repeats referring to a 37% primer‐to‐marker conversion ratio. Seventy‐two markers were polymorphic. These numbers confirm the increased efficiency of pyrosequencing over traditional isolation techniques in the development of microsatellites. Amplification primer sequences and the sequences of corresponding target fragments are provided for all functional markers, and molecular polymorphisms estimated for four accessions of P. pusilla and among seven core group species represented by 14 individuals are reported. Cross‐species transferability ranged between 86.4% and 97.3% among the studied taxa, and 57, 11 and six of the selected primer pairs amplified fragments of expected size and number in seven, six and five of the species, respectively. Reproducibility of the molecular phenotypes was 97.0%, which was inferred using a replicate sample of P. pusilla.