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.

青藏高原栽培青稞遗传多样性研究

青稞，是我国藏区居民对裸大麦的称谓，它不仅是藏民的主要食粮、燃料和牲畜饲料，而且也是啤酒、医药和保健品生产的原料；青稞不仅为藏区人民的健康和经济发展做出了很大的贡献，而且对人类健康和社会经济的可持续发展都有重要的意义。青藏高原是我国及世界上青稞分布和种植面积最大的地区，资源极其丰富。虽然从经典遗传直到分子标记对我国大麦遗传多样性都有研究，但研究手段、数量仍然不够深入，对我国大麦资源遗传多样性研究的信息非常有限，不能很好地满足大麦遗传研究和育种应用的需要，尤其是对西藏栽培大麦的遗传多样性的研究还只是刚刚开始，关于栽培青稞多态性的研究报道很少。本研究采用SSR标记和蛋白质电泳两类技术，从SSR标记位点、单体醇溶蛋白、B组醇溶蛋白和淀粉粒结合蛋白（SGP）等四个方面对我国青藏高原栽培青稞的遗传多样性进行了综合评价。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究采用SSR标记分析了64份青藏高原栽培青稞的遗传多样性，同时评估SSR标记在我国大麦育种和品种鉴定中的应用潜力。选择了30个已知作图位点SSR标记，其中25个标记与重要性状的控制位点连锁紧密。选择的30个SSR标记，5个未得到很好的扩增产物，3个无多态性。22个多态性SSR标记位点中，每位点检测出等位基因2~15个，共检测出等位基因132个，平均每位点6.0 个。各多态位点检测出基因型为2~11种，位点HVM33的基因型最多。各多态位点的多态信息指数为0.16~0.91, 平均为0.65。根据PIC值选择了13个SSR标记用于我国青藏高原栽培青稞基因型鉴定，这些标记的PIC值为0.6以上。结合PIC值和基因型差异，选择了8个多态信息含量高的SSR标记，构建了高效指纹图谱，此图谱能把64份材料完全区分。 贮藏蛋白电泳分析是研究相关编码蛋白基因多态性的非常有效的方法。大麦单体蛋白与小麦醇溶蛋白相对应，具有丰富的多态性，可用于大麦遗传多样性、品种鉴定和群体进化等研究。本研究通过A-PAGE电泳技术研究了84份青藏高原栽培青稞的单体醇溶蛋白多态性。大麦单体醇溶蛋白图谱与小麦醇溶蛋白电泳图谱类似，所分离的蛋白清晰地分为ω－，γ－，β－和α－四个部分。青藏高原栽培青稞单体醇溶蛋白具有丰富的多态性，84份青稞材料中存在43条不同的蛋白带，75种组合带谱；其中67种为单一材料所独有，另8种则分别包含了2-3份材料。每份材料中拥有醇溶蛋白带为6－16条，含有6-10条单体醇溶蛋白带材料较多。西藏和四川材料群体单体醇溶蛋白多态性不同，具有区域特异性。西藏材料中发现了40条不同蛋白带，3条特异带，46 种蛋白组合；四川材料中出现了40种不同蛋白带，26种条带组合, 3条特异带。基于单体蛋白多态性的聚类与材料的来源有一定的相关性。A-PAGE单体蛋白具有丰富的多态性，可作为遗传研究和品种鉴定的标记。 大麦醇溶蛋白（hordein）是大麦籽粒的主要贮藏蛋白，与大麦的营养品质和加工品质密切相关，而且具有丰富的多态性，广泛用于品种鉴定、种质筛选、遗传多样性和亲缘关系研究。B组醇溶蛋白是主要的醇溶蛋白组份，约占总醇溶蛋白的80%，而且具有丰富的多态性。本研究采用SDS-PAGE分析了72份青藏高原栽培青稞B组醇溶蛋白的遗传多样性。青藏高原栽培青稞B组醇溶蛋白具有丰富的多态性，72份青稞材料中存在15种蛋白带，30种组合带谱，其中15种为单一材料所独有，另15种则分别包含了2-10份材料。每份材料中B组醇溶蛋白条带数为4-8条，含5、6条的材料较常见。不同来源的群体材料间B组醇溶蛋白组成存在差异，西藏青稞含有26种蛋白组合带谱，其中有19种特异带谱；四川群体中共发现11种蛋白组合带型，其中有4种特有带谱。两群体中都存在稀有条带。聚类分析将材料分成三组，材料聚类与材料来源地没有明显的相关性。 淀粉粒蛋白（Starch granule proteins, SGPs）是一类与淀粉粒结合的微量蛋白，一些淀粉粒蛋白具有淀粉生化合成中主要的酶蛋白功能，其变异会影响淀粉含量和特性，从而影响淀粉的应用。关于我国大麦淀粉粒组成研究还未见报道。本实验首次开创了我国大麦淀粉粒结合蛋白的研究工作。采用SDS-PAGE电泳技术研究了青藏高原栽培青稞的SGP组成，并分析了不同SGP组合间淀粉含量的差异，初步探索了所分离的SGP蛋白与淀粉合成的关系。66份青稞材料中分离了10种主要的SGP，其表观分子量为40-100KD，低于60KD的SGP带有7条，共有16种组合带谱；各SGP蛋白和组合带谱出现的频率存在差异，青藏高原青稞的SGP组成存在多态性。西藏青稞和四川青稞的SGP组成有很大差异，SGP组成具有地域差异性，西藏青稞含有12种蛋白组合带谱，其中有9种特异带谱；四川群体中共发现7种蛋白组合带型，其中有4种特有带谱；两群体中仅有3种共同的蛋白组合带谱。SGP蛋白特性将66份青稞分为三组, 即Ⅰ、Ⅱ、Ⅲ，材料聚类与材料来源具有一定的相关性。不同组合带谱材料间淀粉含量差异显著性检验结果显示，不同带谱间材料的总淀粉含量、直链淀粉含量和支链淀粉含量有差异，带谱2（SGP1+3+7+9+10）和8（SGP1+2+4+6+8）的总淀粉含量及支链淀粉含量显著大于组合带谱3（SGP1+3+7+10）的总淀粉含量。组合带谱7（SGP1+2+6+8）的直链淀粉含量显著低于带谱11（SGP1+5+8）的直链淀粉。带谱SGP2、3、4、5、6、7、8、9、10可能参与淀粉合成，SGP9可能与高支链淀粉的合成相关。 SSR标记位点、单体醇溶蛋白、B组醇溶蛋白、淀粉结合蛋白等四个方面的研究结果表明青藏高原SSR标记多态性、单体醇溶蛋白多态性、B组醇溶蛋白多态性和SGP多态性都非常丰富，与青藏高原是栽培青稞的多样性分布中心的观点一致。 青藏高原栽培青稞的SSR标记、单体醇溶蛋白、B组醇溶蛋白和SGP多态性表现出很大差异。SSR标记覆盖了整个基因组，多态性非常高。单体蛋白、B组醇溶蛋白、SGP蛋白是育种中非常关注的性状，他们只是代表基因组中的某一区域或位点，多态性相对较低。但单体蛋白多态性很高，84份材料中检测出43条不同蛋白带，75种不同的组合带谱。SSR标记技术和单体蛋白技术都是遗传多样性研究的有力工具，但单体蛋白技术不仅多态性高，而且经济、操作简便，是种质鉴定的理想方法。 对不同标记的多态性材料数据进行聚类，聚类图能为我们提供各材料间的遗传相似信息，为材料选择提供参考。但材料聚类与材料来源的地理区域的相关性表现不一致。SSR聚类和B组醇溶蛋白聚类与材料的来源地无相关性，而单体醇溶蛋白和SGP聚类与材料来源地有一定相关性，即西藏群体和四川群体分别有集中类群，这可能是人为选择的附加效应。 不同来源的群体材料的遗传多样性不同，具有区域特异稀有基因，加强不同地区间资源的交换和配合使用，有利于增加群体遗传多样性和新品种培育。 青藏高原栽培青稞的麦芽浸提性状、淀粉性状、病虫及裸粒等重要农艺性状控制位点存在丰富的变异，遗传基础宽广，可能蕴藏着多种不同的等位基因，是研究重要性状遗传特性、基因资源挖掘和遗传育种的宝贵资源库。 Hulless barley, due to its favorable attributes such as high feed value, good human nutrition，rich dietary fiber and ease processing, attracts people,s attention . Hulless barley plays a very important role in Tibetan life, used as essential food crop, main animal feed and important fuel. In addition to tsampa (roasted barley flour), a main food for Tibetan, hulless barley is also made into cake, soup, porridge, recent naked barley liquor and cornmeal. Qinghai-Tibet Plateau is one of a few areas which plant naked barley widely in the world and also has a long growing history. Genetic diversity of the cultivated hulless barley in this region , however, has not been documented. The study of genetic diversity existing within this population is of particular interest in germplasm identification, preservation, and new cultivar development. This study analyzed the genetic diversity of the cultivated naked barley from Qinghai-Tibet plateau through the study of SSR marker loci and monomeric prolamins, B-horden and starch granule proteins. SSRs are present abundantly in genomes of higher organisms and have become a popular marker system in plant studies. SSRs offer a number of advantages, such as the high level of polymorphisms, locus specificity, co-dominance, reproducibility, ease of use through PCRand random distribution throughout the genome. In barley, several hundred SSRs have been developed and genetically mapped and can therefore be selected from specific genomic regions. The genetic diversity of 64 cultivated naked barley from Tibet and Sichuan was studied with 30 SSRs of known map location.Among the selected SSR markers, PCR products of 5 SSR markers were not obtained and 3 SSR marker loci were monomeric. A total of 132 alleles were identified at 22 polyomeric SSR loci. The number of alleles per locus ranged from 2 to 15, with an average of 6.0. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94, with an average of 0.65. 13 SSR markers with the PIC value >0.6 have been selected for discrimination of Qinghai-Tibet naked barley genotypews. A finger Print map was developed through 7 SSR markers with the high PIC value. It could be used as an efficient tool for gene discovery and identification of gernplasm. Hordeins, the main storage proteins of the barley seed, are composed of momomeric and polymeric prolamins and divided into -A, B, C and D groups in order of decreasing electrophoretic mobility. Hordeins show high inter-genotypic variation and have been extensively used as markers for cultivar identification and analyzing the genetic diversity. This study analyzed the genetic diversity of B-hordein in 72 naked barley from Qinqhai-Tibet Plateau. Extensive diversity was observed. A total of 15 different bands and 30 distinct patterns were found. Jaccard's coefficient of similarity was calculated, and the accessions were divided into three　main groups by cluster analysis using UPGMA. Differentiation among the populations from different collecting regions based on the polymorphism of B-hordein was investigated. Monomeric prolamins show high inter-genotypic variation and have been used as molecular markers for cultivar identification, analyzing the genetic diversity in collections and investigating the evolution processes and structure of populations However, the cultivated hulless accessions from Qinghai-Tibet Pateau in China have never been examined with respect to monomeric prolamins. This study analyzed the genetic diversity of monomeric prolamins (protein fraction corresponding to wheat gliadins) using the Acid -PAGE technique in eighty-four cultivated hulless barley from Qinqhai-Tibet Plateau in China. Extensive diversity was observed. A total of 43 different bands were found, of which 21 different bands were in the region of ω group, 8 in the region of γ, 8 in the region of β, and 6 in the region of α group. Among the 86 accessions, 75 distinct patterns were identified. The number of bands ranged from 6 to 16, depending on the variety. Jaccard’s coefficient of similarity was calculated, and the lines were grouped by cluster analysis using UPGMA. A dendrogram was obtained from the analysis of the groups and five main clusters were identified. No relationship between the distribution in the dendrogram and growth habits and origins of the cultivars could be detected. Starch is the major constituent of the cereal endosperm, comprising approximately 65% of the dry weight of the mature wheat grain. The starch formed in all organs of plants is packaged into starch granules, which vary widely between species and cultivars in size and shape. Wheat endosperm starch granules contain about corresponding to the main biosynthase of starch. This report firstly dealed with intraspecific variation of the major SGPs in cultivated naked barley from Qinghai-Tibet plateau. A total of 10 major SGPs were observed in the range of 40KD-100KD and 16 types of patterns were found. Based on the variation of SGPs, accessions studied were classified into 3 groups. A geographical cline of electrophoregram was observed. In addition, significance test of the difference of starch content among groups and types of patterns were done, and the results indicated those SGPs could be related to the content of starch. Diagram obtained through cluster analysis exhibited a structuration of diversity and genetic relationship among cultivated hulless accessions. In breeding program, parents with genetically distant relationship for hybridization will increase genetic diversity of progenies. In conclusion, cultivated naked barley from Qinghai-Tibet Plateau in China presents a high variability with respect to monomeric prolamins，SSR markers , B- hordeins and SGPs. The result of this study supports Qinghai-Tibet Plateau is the center of cultivated hulless barley and the cultivated naked barley is considered to be a gene pool with large diversity and could be applied to breeding for cereal.

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.

青藏高原青稞淀粉资源的评价

青稞（Hordeum vulgare L.var．nudum Hook．f.），即裸大麦，是兼食用、饲用和酿造于一体的作物，有着重要的利用价值。淀粉是青稞籽粒中含量最多、最重要的碳水化合物，淀粉含量、直支淀粉比将会直接影响淀粉的功能特性，进而影响淀粉的应用领域。我国青藏高原青稞的栽培和食用历史悠久，特色青稞资源极其丰富。目前关于青藏高原青稞淀粉特性的报道还不多见，筛选和培育特色淀粉青稞利于拓展青稞的应用领域, 从而提高其经济价值。 本研究以114份青藏高原青稞品种(系)为实验材料，通过SDS-PAGE对材料的胚乳淀粉颗粒结合蛋白（SGAPs）进行分离，确定各蛋白的分子量大小、组合类型和多态性等。然后按照国标法测试材料的籽粒总淀粉含量和直链淀粉含量，通过微型糊化粘度仪分析相应的淀粉糊化特性，最后使用显微镜观察比较了青稞的淀粉颗粒形态特征。主要结果如下： 1、114种青稞中共分离出20种不同的SGAP条带，条带分子量为35.00～112.39 KDa，分布频率为12.28～97.37%。材料含有的SGAPs条带数从10到14不等，超过一半的材料含11种SGAP条带。20种条带形成16种组合类型，其中西藏地区青稞包含所有16个组合类型，四川地区青稞包含其中12个组合类型。青藏高原青稞籽粒淀粉颗粒结合蛋白的差异很大，遗传多样性丰富。 2、114份青稞的总淀粉含量、直链淀粉含量、直支淀粉比、峰值粘度、糊化温度和峰值温度的变幅分别为51.26～66.70%、14.64～29.74%、0.17～0.42、194～1135BU、58.8～65.2℃和81.4～92.4℃，相应的平均值分别为59.82%、23.60%、0.31、722.30BU、62.1℃和88.8℃。群体在总淀粉含量、直链淀粉含量、直支淀粉比、峰值粘度、糊化温度和峰值温度上的分布具有明显的正态性；所有胚乳淀粉体的淀粉粒都呈复粒结构。对西藏和四川的材料进行了分组比较， 两地区的青稞在直链淀粉含量和直支淀粉比上的差异达到显著水平。 3、筛选出18份具有特殊淀粉特性的青稞品种，其中5份材料的总淀粉含量超过65%，包括NB63-1、NB67、甘孜白六棱、98221-1和NB63；3份材料的直链淀粉含量大于29%，包括藏青85、藏青3号和喜马拉6号；8份材料的直支淀粉比小于0.25，包括99033-6、春青稞、阿坝330、Jan-03、米麦114、396、NB63-1和92013；7份材料的糊化温度低于60℃，同时材料的峰值粘度大于1000BU,并且峰值温度低于90℃，包括足捉春、Jan-03、阿坝330、米麦114、春青稞、20003和阿青5号。 4、各淀粉特性间存在高度相关性。直链淀粉含量和直支淀粉比与糊化温度成极显著正相关，与峰值粘度成极显著负相关，与A型淀粉粒数量和大小呈负相关。不同SGAPs组合的品种之间，淀粉含量和淀粉糊化特性间差异均达显著水平。SGAP2、SGAP5、SGAP6和SGAP7可能对籽粒直链淀粉含量、直支淀粉比和糊化温度有正向效应；SGAP3、SGAP9∼SGAP20可能对峰值粘度有正向效应。 本研究对青藏高原青稞淀粉资源进行了较为全面的评价，对该区青稞淀粉特性有了系统的认识。研究筛选出的特殊青稞品种可作为青稞育种和青稞淀粉工业应用的潜在资源，淀粉特性差异巨大的众多青稞品种也为拓宽青稞应用领域提供了丰富的资源保障。本研究对部分SGAPs在性质上的鉴定和功能上的初步推断为青稞材料的筛选提供了指导，也为品质育种提供了理论参考。 Hulless barley (naked barley, Hordeum vulgare L.) is a short- season, early maturing crop with a wide range of adaptation. It has been attracting more and more attention due to its superior nutrition and extensive industrial applications. Starch is the main ingredient in hulless barley seeds which makes up 65 percent of hulless barley’s dry weight. The ratio of the amylose/amylopectin and the size, shape, distribution of starch granules can affect the physico-chemical and functional properties of starch, which may turn affect its utilizations. The Qinghai-Tibet Plateau, which is located in southwestern China, is a typical area of vertical agricultural ecosystem and one of the barley origin centers with abundant hulless barley resources. There are little reports about hulless barley in Qinghai-Tibet Plateau at present. To screen and cultivate some characteristic hulless barley can improve its value. An improved SDS-PAGE was used to identify SGAPs combination of 114 hulless barley varieties. Starch content (total starch and amylose starch) was determined according to the standard methods GB5006-85 and GB/T 15683 using PerkinElmer M341 Precision Automatic Polarimeter and UV spectrophotometer 755B respectively. The pasting properties were measured by BRABENDER Micrio Visco-Amylo- Graph 803201. The morphology of starch granules were observed and compared with Axioplan 2 Imaging light microscopy. The following were the results obtained: 1. There were 20 major SGAPs presented in 114 varieties, with the molecular weight ranged from 35.00 to 112.39 KDa, and the frequencies ranged from 12.28% to 97.37%. The number of SGAP bands in each accession varied from 10 to 14, more than half of the population had 11 bands. There were 16 distinct SGAP patterns in the 114 varieties, the Tibet hulless barley had all of the 16 types and the Sichuan hulless barley had 12 types. The results indicated the Qinghai-Tibet Plateau hulless barley had a polymorphism of the SGAPs. 2. The ranges of the total starch content, amylose content, Am/Ap, peak viscosity, pasting temperature and peak temperature of the 114 hulless barley were 51.26～66.70%,14.64～29.74%,0.17～0.42,194～1135BU,58.8～65.2 and 81.4℃～92.4, with an average of ℃59.82%, 23.60%, 0.31, 722.30BU, 62.1 and 88.8,℃℃ respectively. The distributions of the total starch content, amylose content, Am/Ap, peak viscosity, pasting temperature and peak temperature were visibly normal school. All of the amyloplasts in endosperm of varieties showed bimodal size distributions.The main starch properties of hulless barley from Tibet and Sichuan were separated and compared, the differences on amylose content and Am/Ap were obvious. 3. Eighteen accessions which had special starch properties were screened out. Five accessions with total starch content beyond 65%, including NB63-1, NB67, Ganzibailiuleng, 98221-1 and NB63; three accessions, Zangqing85, Zangqing3 and Ximala6, with the highest amylose content (>29%); five accessions with Am/Ap less than 0.25, including 99033-6, Chun Qingke, A Ba 330, Jan-03, Mi Mai114, 396, NB63-1 and 92013; seven accessions had a pasting temperature under 60, ℃meanwhile their peak viscosity beyond 1000BU and their peak temperature under 90℃，including Zu Cuochun, Jan-03, A Ba 330, Mi Mai 114, Chun Qingke, 20003 and A Qing 5. 4. There were high correlations between starch properties. Amylose content and Am/Ap were positively correlated to pasting temperature, negatively correlated to peak viscosity, negatively correlated to the number and granule size of A-type granule. Different SGAP combinations caused significant diversities in starch content and pasting properties. SGAP2, SGAP5, SGAP6 and SGAP7 may have positive effect on amylose content, Am/Ap and pasting temperature; SGAP3, SGAP9∼SGAP20 may have positive effect on peak viscosity. Our research made a comprehensive evaluation on the hulless barley starch from the Qinghai-Tibet Plateau, we can get a systemic understanding. Some special accessions were screened out can be used on the hulless barley breeding lines and industries utilization.The combination of the SGAPs may become a criterion to evaluate the hulless barley endosperm starch quality. Consequently, the results will be good information for further studies on the hulless barley.

Comparative study on CO2 emissions from different types of alpine meadows during grass exuberance period

Potentilla fruticosa scrub, Kobresia humilis meadow and Kobresia tibetica meadow are widely distributed on the Qinghai-Tibet Plateau. During the grass exuberance period from 3 July to 4September, based on close chamber-GC method, a study on CO2 emissions from different treatments was conducted in these meadows at Haibei research station, CAS. Results indicated that mean CO2emission rates from various treatments were 672.09+152.37 mgm-2h-1 for FC (grass treatment); 425.41+191.99 mgrn-2h-1 for FJ (grass exclusion treatment); 280.36+174.83 mgrn-2h-1 for FL (grass and roots exclusion treatment); 838.95+237.02 mgm-2h-1 for GG (scrub+grass treatment); 528.48+205.67 mgm-2h-1for GC (grass treatment); 268.97 ±99.72 mgm-2h-1 for GL (grass and roots exclusion treatment); and 659.20±94.83 mgm-2h-1 for LC (grass treatment), respectively (FC, FJ, FL, GG, GC, GL, LC were the Chinese abbreviation for various treatments). Furthermore, Kobresia humilis meadow, Potentilla fruticosa scrub meadow and Kobresia tibetica meadow differed greatly in average CO2 emission rate of soil-plant system, in the order of GG＞FC＞LC＞GC. Moreover, in Kobresia humilis meadow,heterotrophic and autotrophic respiration accounted for 42% and 58% of the total respiration of soil-plant system respectively, whereas, in Potentilla fruticosa scrub meadow, heterotrophic and autotrophic respiration accounted for 32% and 68% of total system respiration from G-G; 49% and 51%from GC. In addition, root respiration from Kobresia humilis meadow approximated 145 mgCO2m-2h-1,contributed 34% to soil respiration. During the experiment period, Kobresia humilis meadow and Potentilla fruticosa scrub meadow had a net carbon fixation of 111.11 grn-2 and 243.89 grn-2,respectively. Results also showed that soil temperature was the main factor which influenced CO2 emission from alpine meadow ecosystem, significant correlations were found between soil temperature at 5 cm depth and CO2 emission from GG, GC, FC and FJ treatments. In addition, soil moisture may be the inhibitory factor of CO2 emission from Kobresia tibetica meadow, and more detailed analyses should be done in further research.

Effects of vegetation control on ecosystem water use efficiency within and among four grassland ecosystems in China

Through 2-3-year (2003-2005) continuous eddy covariance measurements of carbon dioxide and water vapor fluxes, we examined the seasonal, inter-annual, and inter-ecosystem variations in the ecosystem-level water use efficiency (WUE, defined as the ratio of gross primary production, GPP, to evapotranspiration, ET) at four Chinese grassland ecosystems in the Qinghai-Tibet Plateau and North China. Representing the most prevalent grassland types in China, the four ecosystems are an alpine swamp meadow ecosystem, an alpine shrub-meadow ecosystem, an alpine meadow-steppe ecosystem, and a temperate steppe ecosystem, which illustrate a water availability gradient and thus provide us an opportunity to quantify environmental and biological controls on ecosystem WUE at different spatiotemporal scales. Seasonally, WUE tracked closely with GPP at the four ecosystems, being low at the beginning and the end of the growing seasons and high during the active periods of plant growth. Such consistent correspondence between WUE and GPP suggested that photosynthetic processes were the dominant regulator of the seasonal variations in WUE. Further investigation indicated that the regulations were mainly due to the effect of leaf area index (LAI) on carbon assimilation and on the ratio of transpiration to ET (T/ET). Besides, except for the swamp meadow, LAI also controlled the year-to-year and site-to-site variations in WUE in the same way, resulting in the years or sites with high productivity being accompanied by high WUE. The general good correlation between LAI and ecosystem WUE indicates that it may be possible to predict grassland ecosystem WUE simply with LAI. Our results also imply that climate change-induced shifts in vegetation structure, and consequently LAI may have a significant impact on the relationship between ecosystem carbon and water cycles in grasslands.

Summer diet of two sympatric species of raptors Upland Buzzard (Buteo hemilasius) and Eurasian Eagle Owl (Bubo bubo) in alpine meadow: problem of coexistence

Summer diets of two sympatric raptors Upland Buzzards (Buteo hemilasius Temminck et Schlegel) and Eurasian Eagle Owls (Bubo bubo L. subsp. Hemachalana Hume) were studied in an alpine meadow (3250 m a.s.l.) on Qinghai-Tibet Plateau, China. Root voles Microtus oeconomus Pallas, plateau pikas Ochotona curzoniae Hodgson, Gansu pikas O. cansus Lyon and plateau zokors Myospalax baileyi Thomas were the main diet components of Upland Buzzards as identified through the pellets analysis with the frequency of 57, 20, 19 and 4%, respectively. The four rodent species also were the main diet components of Eurasian Eagle Owls basing on the pellets and prey leftovers analysis with the frequency of 53, 26, 13 and 5%, respectively. The food niche breadth indexes of Upland Buzzards and Eurasian Eagle Owls were 1.60 and 1.77 respectively (higher value of the index means the food niche of the raptor is broader), and the diet overlap index of the two raptors was larger (C-ue = 0.90) (the index range from 0 - no overlap - to I - complete overlap). It means that the diets of Upland Buzzards and Eurasian Eagle Owls were similar (Two Related Samples Test, Z = -0.752, P = 0.452). The classical resource partitioning theory can not explain the coexistence of Upland Buzzards and Eurasian Eagle Owls in alpine meadows of Qinghai-Tibet Plateau. However, differences in body size, predation mode and activity rhythm between Upland Buzzards and Eurasian Eagle Owls may explain the coexistence of these two sympatric raptors.

Response of chlorophyll fluorescence to dynamic light in three alpine species differing in plant architecture

A study was carried out to examine the effect of dynamic photosynthetically active photon flux density (PPFD) on photoinhibition and energy use in three herbaceous species, prostrate Saussurea superba, erect-leaved S. katochaete, and half-erect-leaved Gentiana straminea, from the Qinghai-Tibet Plateau. Chlorophyll fluorescence response was measured under each of three sets of high-low PPFD combinations: 1700-0, 1400-300, and 1200-500 mu mol m(-2) s(-1), illuminating in four dynamic frequencies: 1, 5, 15, and 60 cycles per 2 h. The total light exposure time was 2h and the integrated PPFD was the same in all treatments. The highest frequency of PPFD fluctuation resulted in the lowest photochemical activity, the highest level of non-photochemical quenching, and the greatest decrease of F-v/F-m (maximal photochemical efficiency of PSII). The 5 and 15 cycles per 2h treatments resulted in higher photochemical activity than the 1 cycle per 2h treatment. The 1700-0 PPFD combination led to the lowest photochemical activity and more serious photoinhibition in all species. S. superba usually exhibited the highest photochemical activity and CO2 uptake rate, the lowest reduction of F-v/F-m,F- and the smallest fraction of energy in thermal dissipation. With similar fractions of thermal dissipation, S. katochaete had relatively less photoinhibition than G. straminea owing to effective F-o quenching. The results suggest that high frequency of fluctuating PPFD generally results in photoinhibition, which is more serious under periods of irradiation with high light intensity. (c) 2005 Elsevier B.V. All rights reserved.

Effect of burrowing activity of root vole (Microtus oeconomus Pallas) on plant diversity in alpine meadow

Herbivory and burrowing activity of mammals may influence the species composition and diversity of plant communities. The effect of corridors and holes systems constructed by root vole (Microtus oeconomus Pallas) on the plant species diversity was studied in the habitat of high - mountain meadow (3250 in a.s.l in Qinghai-Tibet Plateau, China). By using grid method, these disturbances were studied on 16 plots (100 cm x 100 cm) distributed in 4 transects in studied area, in August 2000 and 2001. The disturbance intensity index, D, was calculated as the percent of the ground surface disturbed by voles in the study area. Plant species were identified and counted on the same plots. In total 46 plant species were identified - 39% of this number was considered as sensitive to the vole disturbances as their occurrence and/or abundance decreased along the disturbance intensity. Generally, a significantly negative correlation (r = - 0.911 P < 0.01) between vole aboveground disturbances and plant species diversity (H') was found. The results suggest that root vole ground disturbances, especially in the form of actively utilized holes and corridors have significantly negative influence on plant species diversity in high-mountain grassland habitat.

Local warming about 1.3 degrees C in alpine meadow has no effect on root vole (Microtus oeconomus L.) population during winter

The influence of air and soil warming on root vole (Microtus oeconomus L.) population was studied in winter period in top open chambers (OTC) (0.8-1.8 m(2) warmed by conical fiberglass material and situated in alpine meadow (3250 m) at Qinghai-Tibet Plateau, China. The OTCs were distributed on an area of 30 x 30 m of experimental warming site; another site of the same area was a control one. The root vole population was investigated on two pairs of sites in "low-grazing" and "high-grazing" (by sheep) parts of the meadow; mark-recapture method was used. The winter-season averaged air and soil temperature inside of the chambers were 1.3 degrees C higher than the temperature outside the chambers. The warming in the chambers had no statistically significant effect on root vole numbers, on average body mass of individual, and on average body mass of males and females. In conclusion, as small as 1.3 degrees C warming of soil and air introduced locally and on small (several m(2)) scale, in the alpine meadow habitat in winter period, has possibly no effect on root vole numbers and biomass.

Phylogenetic analyses of Saussurea sect. Pseudoeriocoryne (Asteraceae : Cardueae) based on chloroplast DNA trnL-F sequences

The genus Saussurea is distributed mainly in the temperate and subarctic regions of Eurasia and consists of about 300 species classified into six subgenera and 20 sections. Sect. Pseudoeriocoryne in the subgenus Eriocoryne comprises four species, and is delimited mainly by acaulescence and an inflorescence with congested capitula surrounded by a rosette of leaves. All of these species are endemic to the and Qinghai-Tibet Plateau. Sequences from the chloroplast DNA trnL-F region were obtained for the four species in this section and 26 other species from four subgenera of Saussurea to resolve phylogenetic relationships among these species and to determine whether the shared characters that define sect. Pseudoeriocoryne are synapomorphic or were acquired by convergent evolution. The resulting phylogenies indicated that Saussurea sect. Pseudoeriocoryne as traditionally defined does not constitute a monophyletic group and that each of its species belongs to separate clades. Furthermore, none of these species showed a close relationship with the other species of subgenus Eriocoryne. Our results further indicated that none of the investigated subgenera are monophyletic, and that species from different subgenera clustered together. All these conclusions are provisional and their confirmation would require stronger phylogenetic support. Two possible explanations are suggested for low sequence divergence, poor resolution of internal clades and clustering of species with the rather distinct morphology of Saussurea detected in the present study. The first is rapid radiation and diversification triggered by fast habitat fragmentation due to the recent lifting of the Qinghai-Tibet Plateau and the Quaternary climate oscillations. This could have led to rapid morphological divergence while sequences diverged very little, and also caused the convergent acquisition of similar characteristics in unrelated lineages due to similar selection pressures. The second possible explanation is that both introgressive hybridization and reticulate evolution might have caused the transferring of cpDNA sequences between morphologically dissimilar species, thus leading to homogenization of sequences between lineages. (C) 2004 Elsevier Ltd. All rights reserved.

Photosynthesis of Saussurea superba and Gentiana straminea is not reduced after long-term enhancement of UV-B radiation

Experiments were conducted in an alpine Kobresia humilis meadow near Haibei Alpine Meadow Ecosystem Research Station (37degrees29'-37degrees45'N, 101degrees12'-101degrees33'E; altitude 3200 m). Effects of enhanced ultraviolet-B (UV-B) radiation on photosynthesis of the alpine plants of Saussurea superba and Gentiana straminea were investigated. Both species were exposed to a UV-B-BE density at 15.80 kJ m(-2) per day, simulating nearly 14% ozone (O-3) reduction during the plant growing season. Neither photosynthetic CO2 uptake rate nor photosynthetic O-2 evolution rate were decreased after a long period of enhanced UV-B radiation treatment. On the contrary, there was a tendency to increase of both parameters in both species. The photosynthetic pigments were also increased, when expressed on a leaf area basis. UV-B absorbing compounds, detected by the absorbance values at 300 mm, had a tendency to increase in both species after enhanced UV-B radiation. After long-term exposure of plants to enhanced UV-B radiation, leaf morphology was also affected. Leaf thickness in both S. superba and G. straminea were increased significantly (P < 0.001). This supports our hypothesis that the increase of leaf thickness in both species after long-term exposure of enhanced UV-B radiation could compensate for the photodestruction of photosynthetic pigments when light passes through the leaf. Therefore, photosynthesis is not reduced in either species when expressed on leaf area basis. (C) 2003 Elsevier B.V. All rights reserved.

Photosynthetic depression in relation to plant architecture in two alpine herbaceous species

The Qinghai-Tibet Plateau is characterized by extremely high radiation, which may induce down-regulation of photosynthesis in plants living in this alpine ecosystem. To clarify whether photoinhibition occurs in the alpine environment and to discern its underlying mechanisms, we examined photosynthetic gas exchange and fluorescence emission in response to the changes in photosynthetic photon flux density (PPFD) and leaf temperature under natural regimes for two herbaceous species: prostrate Saussurea superba and erect-leaved Saussurea katochaete from altitude 3250 m on the Qinghai-Tibet Plateau. S. superba intercepted a higher maximum PPFD and experienced much higher leaf temperature than the erect-leaved S. katochaete. S. superba exhibited a much higher light saturation point for photosynthesis than S. katochaete. Under controlled conditions, the former species had higher CO2 uptake rates and neither species showed obvious photosynthetic down-regulation at high PPFD. Under natural environmental conditions, however, apparent photoinhibition, indicated by reduced electron transport rate (ETR), was evident at high PPFD for both species. After a night frost, the photochemistry of S. katochaete was depressed markedly in the early morning and recovered by mid-day. After a frost-free night, it was high in the morning and low at noon due to high radiation. S. superba did not respond to the night frost in terms of daytime photochemical pattern. In both species, photochemical depression was aggravated by high leaf temperature and the erect species was more sensitive to high temperature. This study suggests that the high radiation on the Qinghai-Tibet Plateau is likely to induce rapidly reversible photoinhibition, which is related closely to plant architecture. Photochemistry in the prostrate species seems able to tolerate higher PPFD, without obvious suppression, than the erect species. (C) 2003 Elsevier Science B.V. All rights reserved.

青藏高原及邻区面波频散反演--岩石圈-软流层体系结构研究

The Qinghai-Tibet Plateau lies in the place of the continent-continent collision between Indian and Eurasian plates. Because of their interaction the shallow and deep structures are very complicated. The force system forming the tectonic patterns and driving tectonic movements is effected together by the deep part of the lithosphere and the asthenosphere. It is important to study the 3-D velocity structures, the spheres and layers structures, material properties and states of the lithosphere and the asthenosphere for getting knowledge of their formation and evolution, dynamic process, layers coupling and exchange of material and energy. Based on the Rayleigh wave dispersion theory, we study the 3-D velocity structures, the depths of interfaces and thicknesses of different layers, including the crust, the lithosphere and the asthenosphere, the lithosphere-asthenosphere system in the Qinghai-Tibet Plateau and its adjacent areas. The following tasks include: (1)The digital seismic records of 221 seismic events have been collected, whose magnitudes are larger than 5.0 over the Qinghai-Tibet Plateau and its adjacent areas. These records come from 31 digital seismic stations of GSN , CDSN、NCDSN and part of Indian stations. After making instrument response calibration and filtering, group velocities of fundamental mode of Rayleigh waves are measured using the frequency-time analysis (FTAN) to get the observed dispersions. Furthermore, we strike cluster average for those similar ray paths. Finally, 819 dispersion curves (8-150s) are ready for dispersion inversion. (2)From these dispersion curves, pure dispersion data in 2°×2° cells of the areas (18°N-42°N, 70°E-106°E) are calculated by using function expansion method, proposed by Yanovskaya. The average initial model has been constructed by taking account of global AK135 model along with geodetic, geological, geophysical, receiving function and wide-angle reflection data. Then, initial S-wave velocity structures of the crust and upper mantle in the research areas have been obtained by using linear inversion (SVD) method. (3)Taking the results of the linear inversion as the initial model, we simultaneously invert the S wave velocities and thicknesses by using non-linear inversion (improved Simulated Annealing algorithm). Moreover, during the temperature dropping the variable-scale models are used. Comparing with the linear results, the spheres and layers by the non-linear inversion can be recognized better from the velocity value and offset. (4)The Moho discontinuity and top interface of the asthenosphere are recognized from the velocity value and offset of the layers. The thicknesses of the crust, lithosphere and asthenosphere are gained. These thicknesses are helpful to studying the structural differentia between the Qinghai-Tibet Plateau and its adjacent areas and among geologic units of the plateau. The results of the inversion will provide deep geophysical evidences for studying deep dynamical mechanism and exploring metal mineral resource and oil and gas resources. The following conclusions are reached by the distributions of the S wave velocities and thicknesses of the crust, lithosphere and asthenosphere, combining with previous researches. (1)The crust is very thick in the Qinghai-Tibet Plateau, varying from 60 km to 80 km. The lithospheric thickness in the Qinghai-Tibet Plateau is thinner (130-160 km) than its adjacent areas. Its asthenosphere is relatively thicker, varies from 150 km to 230 km, and the thickest area lies in the western Qiangtang. India located in south of Main Boundary thrust has a thinner crust (32-38 km), a thicker lithosphere of about 190 km and a rather thin asthenosphere of only 60 km. Sichuan and Tarim basins have the crust thickness less than 50km. Their lithospheres are thicker than the Qinghai-Tibet Plateau, and their asthenospheres are thinner. (2)The S-wave velocity variation pattern in the lithosphere-asthenosphere system has band-belted distribution along east-westward. These variations correlate with geology structures sketched by sutures and major faults. These sutures include Main Boundary thrust (MBT), Yarlung-Zangbo River suture (YZS), Bangong Lake-Nujiang suture (BNS), Jinshajiang suture (JSJS), Kunlun edge suture (KL). In the velocity maps of the upper and middle crust, these sutures can be sketched. In velocity maps of 250-300 km depth, MBT, BNS and JSJS can be sketched. In maps of the crustal thickness, the lithospheric thickness and the asthenospheric thickness, these sutures can be still sketched. In particular, MBT can be obviously resolved in these velocity maps and thickness maps. (3)Since the collision between India and Eurasian plate, the “loss” of surface material arising from crustal shortening is caused not only by crustal thickening but also by lateral extrusion material. The source of lateral extrusion lies in the Qiangtang block. These materials extrude along the JSJS and BNS with both rotation and dispersion in Daguaiwan. Finally, it extends toward southeast direction. (4)There is the crust-mantle transition zone of no distinct velocity jump in the lithosphere beneath the Qiangtang Terrane. It has thinner lithosphere and developed thicker asthenosphere. It implies that the crust-mantle transition zone of partial melting is connected with the developed asthenosphere. The underplating of asthenosphere may thin the lithosphere. This buoyancy might be the main mechanism and deep dynamics of the uplift of the Qinghai-Tibet hinterland. At the same time, the transport of hot material with low velocity intrudes into the upper mantle and the lower crust along cracks and faults forming the crust-mantle transition zone.