九寨沟湖泊湿地植物群落与植物地理研究

九寨沟湖泊湿地在维持九寨沟的生态平衡中起着重要的作用，在旅游产业的发展下，湿地生态系统及生物多样性面临着较大的威胁。尽管九寨沟湿地具有重要的生态价值，但目前对其研究尚比较薄弱。湿地植物群落和植物地理研究可以为湿地资源的可持续利用和监测保护提供科学依据。作者从2004年8月到2007年11月对九寨沟湿地的植物物种组成、地理分布、优势植物群落的结构、生长动态、湿地土壤种子库进行了调查研究。主要结果如下： 1. 九寨沟湿地物种组成、地理分布特点及湿地植物群落特点 九寨沟湿地共有苔藓植物8科13属16种，维管植物为48科107属199种。九寨沟湿地植物的地理成份较为丰富，维管植物在科级水平上有7种地理分布型（变型），在属级水平上有13种地理分布型（变型）， 在种级水平上共有29种地理分布型（变型）。九寨沟湿地植物以温带成份和我国特有成份为主，同时兼有热带、亚热带成份和环极—高山成份。九寨沟湿地植物的分布表现出明显的垂直地带性和水平地带性。湿地植物群落可划分21个群落类型，不同植物群落类型的物种多样性及物种组成存在较大的差异。九寨沟湿地植物的物种多样性和群落多样性以及较高的生产力特征，是维持其湿地生态景观多样性和稳定性的基础。 2. 土壤、水环境、海拔等对湿地植物的分布及生物多样性的影响 九寨沟湿地土壤、水等环境因子存在较大的差异。帕米尔苔草和宽叶香蒲等群落的凋落物较多，土壤有机碳、土壤总磷较高，可能是九寨沟湿地的重要土壤碳库。 九寨沟湿地植物沿水环境梯度的分布规律表现为：沉水植物（轮藻—篦齿眼子菜，水苦荬，杉叶藻）——挺水植物（水木贼，芦苇，宽叶香蒲）——湿生草本（苔草、节节草、披散木贼）——湿生灌木（柳灌丛，小檗灌丛）等。海拔也影响湿地植物的物种组成。 水深对物种多样性有影响，水深与物种丰富度负相关。随着水深的增加，水木贼、芦苇、杉叶藻、宽叶香蒲等群落的物种多样性下降；在长期淹水和季节性淹水的地方，水木贼群落物种多样性存在显著差异。土壤总氮与水木贼群落物种丰富度正相关。 3. 土壤营养元素、水环境对植物生长的影响 水深影响湿地植物生物量的分配。芦苇无性系分株在47 cm水深的环境中单株平均生物量最大；在干滩地中（地面水深0 cm），叶生物量百分比最大，而茎生物量百分比最小，茎的生物量百分比和生长速率随水深的增加而增加；在较干的滩地生境中，开花率、花序的生物量百分比明显大于水较深的生境。 水深与水木贼地上生物量负相关，但水木贼地上生物量在长期淹水和季节性淹水的地方没有显著的差异。在水浅的地方，杉叶藻、水木贼、芦苇等植物群落中，其他伴生物种的生物量占样方总生物量的百分比较大。 土壤有机碳、土壤总氮、土壤总磷等对湿地植物生物量的影响比较大：宽叶香蒲地上生物量与土壤总磷正相关；水木贼地上生物量与土壤总氮正相关；杉叶藻地上生物量与土壤有机碳正相关。 水深、土壤营养成分对湿地植物高度、密度等有影响。水木贼的平均高度在季节性淹水的地方比长期淹水的地方低，平均密度在长期淹水的地方比季节性淹水的地方低；除了5月份，其他观察月份水木贼的密度都与水深负相关，同时与土壤有机碳正相关。另外，芦苇密度与土壤有机碳含量正相关，宽叶香蒲密度与水深负相关，帕米尔苔草高度与土壤有机碳负相关。 4. 优势植物群落的动态变化 在优势植物群落中，优势种的高度、密度、盖度、生物量等在群落中占绝对优势。除五花海，水木贼群落的物种组成、高度、生物量在两年间没有显著的变化。芦苇群落的物种丰富度在近两年有所增加。 湿地植物生长表现为明显的季节动态，生长的峰值大多在7月－8月。优势植物群落的物候与水文周期有关。湿地植物群落的物种组成和密度，可以作为对湿地监测和保护的生物指示。 5. 九寨沟湿地土壤种子库特征及其在湿地生物多样性恢复中的作用 水深和现存植被物种丰富度可以解释湿地土壤种子库的变化。水深可以解释表层物种丰富度45%的变化。现存植被物种丰富度可以分别解释10 cm土层、2－5 cm土层及5－10 cm土层土壤种子库45%、48%和25%的变化。 湿地土壤种子库的密度为0－15945粒m-2, 种子库中共发现23个物种。现存植被优势物种和种子库优势物种不同。各层土壤种子库密度和物种丰富度并不存在显著的差异，但第二层土壤种子库密度最大。海拔、现存植被优势种盖度、土壤总磷、土壤总氮、土壤有机碳对湿地土壤种子库的密度和垂直结构没有影响。土壤种子库物种丰富度小于地上植被物种丰富度。湿地土壤种子库与地上植被的相关性不大。在浅水区域，湿地土壤种子库在湿地植被恢复中有一定作用。但在深水区域，保护现存植被更重要。 The lakeshore wetlands are valuable ecological units of the Jiuzhaigou lakes. Pressure for travel industry development pose a continuing and severe threat to the biodiversity-support function of the wetland system. Despite the ecological importance of wetlands in Jiuzhaigou, they are so far poorly studied. Both general plant communties and biogeographical studies are needed in order to attain basis for sustainable use the wetland resources and adequate protection of these areas. The present study was undertaken to examine aquatic plants distribution and the species compositon, structure and growth dynamics of their communities with variations of environmental factors along altitudes, water depth and soil properities gradients in Jiuzhaigou. Analysis of field survey data collected during August 2004 and November 2007 in lakeshore wetlands in Jiuzhaigou National Nature Reserve, Sichuan, China. The results were as following: (i) Species composition and biogeography in wetland vegetation 8 families, 13 genus, 16 species of moss and 48 families, 107 genus and 199 species of vascular plants in Jiuzhaigou wetlands were found. The floristic compositions were abundunt. Ten geographical distribution types at family level, 13 geographical distributions types at generic level and 29 geographical distribution types at specific level in vascular plants were found. Most species in Jiuzhaigou wetlands are temperate elements and Chinese endemic elements, with a few of tropical and subtropical and some circumarctic elements. And the plant distributions show clear vertical and horizontal patterns. There were 21 major wetland plant community types. Species composition and species richness in different plant communities are different. The species diversity and plant community diversity and their high biomass are the basis for the diversity and stability of wetland landscapes in Jiuzhaigou. (ii) Water depth, soil nutrients and altitudes influence on the species diversity and plant distribution. Total phosphorous and organic cabon in soil were higher in C. pamiernensis and T. latifolia communities, where are important cabon reservoirs in Jiuzhaigou wetlands. Along gradients of water depth, among populations of the dominant plant species present: submerged macrophytes (Chara vulgaris, Potagemonton pectinatus, Veronica anagalis-aquatica，Hippuris vulgaris), emergent macrophytes (Equisetum fluviatile, Phragamites australis, Typha latifolia), helophytes (Carex pamirensis )and shrubs (Salix sp., Berberis sp. ). Altitudes influence on the assemblage of plant communities. Water depth negatively correlated with species richness. Specie richness showed differences between permanently flooded sites and seasonally flooded sites in E. fluvatile communities. And total nitrogen in soil was negatively correlated with species richness in E. fluviatile communities. Altitudes show no significant influence on species richness, but in fact, through our analyses, they do have influence on the assemblage of wetland plants. (iii) Water depth, soil nutrients influence on the plant growth Water depth influences the biomass allocation in Phragmities australis. The average aboveground biomass of a single ramet (4.2 g) was the largest in the habitat with water level 47 cm above the soil surface. At the habitat with water level under soil surface 15 cm (－15 cm), the leaf biomass percentage (of the total ramet biomass) was the largest (46.1%), and the height and percentage of ramose ramets ( with branches on stem )(of the total ramets in a plot) were found obviously different. The deeper in water, the larger the biomass percentage and growth rate of stems were. The flowering rate and biomass of panicles were greater in shallow water than those in deep water. Water depth negatively correlated with aboveground biomass of E. fluviatile. However, above-ground biomass of E. fluviatile showed no significant difference between permanently flooded sites and seasonally flooded sites. But in shallow water, more biomasses of accompanying species were found in dominant plant communities such as H. vulgaris communities, E. fluviatile communities and P. australis communities. Water depth, soil nutrients influence on shoot density and shoot length of wetland plants. The shoot density of E. fluviatile was correlated to water depth in all growth months. Annual average density was significantly lower at permanently flooded sites than at seasonally flooded sites. But the annual average shoot length was significantly lower at seasonally flooded sites than at permanently flooded sites. (iv) Growth dynamics of dominant communities in Jiuzhaigou wetland The shoot length and shoot density, coverage and biomass of domiant species were dominated in plant communities. The species composition increased in P. australis communities in recent two years. The species richness in E. fluviatile communities showed no difference between 2005 and 2007. The above-ground biomass and shoot density in Five－flower Lake from July 2005 to July 2007 were significantly different, while in other sites, the differences were not significant. Shoot height, shoot density and above-ground biomass showed significant seasonal changes in all sites. Growth dynamics correlated with the cycle of water levels in lakes. Most plants growth parameters peaked at July or August. The biomass of T. latifolia peaked in August. But the shoot length of T. latifolia in deeper water peaked in July. The shoot length of E. fluviatile increased significantly from May to August except in seasonally flooded sites in Arrow-bamboo Lake. The species composition of communities and shoot density can be used as bioindicators in Jiuzhaigou wetland. (v) Soil seed bank in Jiuzhaigou wetland and its role in vegetation restoration Seed density in all soil layer samples was negatively correlated to water depth. Water depth can explain 45% variance of species richness in surface layer in sediment. Species richness in extant vegetation can explain 45%, 48%, 25% variance of species richness in total 10 cm and in 2－5 cm and 5－10 cm layer sediment respectively. Mean seed densities in wetlands ranged from 0 to 15945 m–2. A total of 23 species germinated in seed bank. The dominant species in seed bank and extant vegetation showed great difference. The total number of species and seedlings that germinated in different layers was not significantly different. But the second layer had the greatest seed density. In shallow water, seed bank can contribute to vegetation restoration, while in deeper water, protection of extant vegetation may be a better strategy.

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.

delta C-13 variation of C-3 and C-4 plants across an Asian monsoon rainfall gradient in arid northwestern China

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