Application of inductively coupled plasma mass spectrometry in biological samples analysis

Recent important applications of inductively coupled plasma mass spectrometry in biological samples analysis are reviewed. The sample preparation, sample introduction techniques, interference correction and typical applications are introduced in detail with 154 references.

表面等离子体激元共振与生物分子相互作用分析

对表面等离子体激元共振(surface plasmon resonance, SPR)的原理和在生物学研究方面的应用进行了综述。这种技术可以直接原位、实时地跟踪生物学实验研究系统, 而不需要附加参数如进行标记等手段, 具有高敏感性, 也可以连续监测吸附或解吸附过程。目前有关的应用涉及到生物学结合分析、动力学及亲和力测定、免疫识别研究、结构与活性研究和核酸研究等多个领域。

染色体加倍对忍冬生物学性状及其生态适应性的影响

忍冬（Lonicera japonica Thunb.）属忍冬科忍冬属，是一种重要的药用植物，其花蕾称为金银花（Flos Lonicerae），在我国已有1000多年的药用历史，具有清热解毒、凉散风热等功效。我们利用秋水仙素诱导二倍体“大毛花”品种茎尖选育出同源四倍体“九丰一号”品种，并在生产中发现花蕾产量显著提高，而药效成份含量是否发生变化了呢？四倍体忍冬表现出典型的器官巨大性，这些变化是否与其较强生态适应性之间存在联系呢？染色体加倍增强了忍冬的生态适应性，其生态修复功能如何呢？在本研究中，主要从以下几个方面进行了探讨：（1）染色体加倍对其叶片，茎、花蕾和花蕾产量及其药效成份等生物学性状的影响;（2）染色体加倍后植物对水分胁迫的响应;（3）染色体加倍后植物对热胁迫的响应;（4）除上述两个品种外，增加一个变异品种“红银花”，探讨3个忍冬品种对退化生态系统的修复功能。主要结果如下： 1. 通过测定根尖细胞染色体数目和使用流式细胞仪分析茎尖细胞DNA含量，表明四倍体忍冬（2n = 4x = 36）确实来自二倍体忍冬（2n = 2x = 18）的染色体加倍。四倍体忍冬气孔细胞大小显著大于二倍体，而气孔密度显著低于二倍体。四倍体忍冬没有光合“午休”，而二倍体存在明显的光合“午休”，这可能与其存在抗高温的叶片解剖结构特性有关。四倍体忍冬叶片较二倍体变大、变厚、变浓绿（较高叶绿素含量）。与二倍体相比，四倍体忍冬单位叶面积重量显著增高，表明其具有较强的生态适应性。四倍体忍冬单个花蕾的鲜重和干重均显著大于二倍体。连续3年的花蕾产量调查表明，四倍体显著高于二倍体。染色体加倍使其茎干粗壮、节间变短、新梢上着生花蕾数目增多及单个花蕾变大，这是其花蕾高产的生物学基础。对金银花的药效成份而言，染色体加倍不影响绿原酸的含量，但增加了木犀草苷的含量。结果表明，染色体加倍能增加金银花的产量和药效成份的含量，建议四倍体忍冬在药材生产中推广应用。 2. 水分胁迫显著降低二倍体和四倍体忍冬叶片的净光合速率、气孔导度和蒸腾速率。水分胁迫也降低电子传递速率、光系统II实际量子产量和光化学猝灭，而增加非光化学猝灭、总可溶性糖、脯氨酸和丙二醛的含量。四倍体忍冬对水分胁迫的响应表现为其叶片水势、气体交换、叶绿素荧光和有关代谢物含量的变化程度低于二倍体，并且复水后其恢复能力快于二倍体，表明染色体加倍增强了忍冬的抗旱能力。分析其主要原因，可能是由于四倍体植株总叶面积减少、单位叶面积重量增加、叶片表皮细胞和栅栏组织增大以及叶片表皮毛较浓密等形态解剖结构特性有关。结果表明，染色体加倍能增加忍冬植物的抗旱能力，而使其具有较强的生态适应性。 3. 二倍体和四倍体忍冬受48 ºC热胁迫处理6 h和恢复10 h，以及离体叶片45 ºC，50 ºC，55 ºC 水浴热胁迫3 min，应用叶绿素荧光成像系统研究了它们对热胁迫的响应。热胁迫显著降低了两个品种叶片的最大光化学效率、电子传递速率、光系统II实际量子产量和光化学猝灭，降低了四倍体的非光化学猝灭，而增加了二倍体的非光化学猝灭。热胁迫增加了两个品种叶片的总可溶性糖、脯氨酸和丙二醛的含量。四倍体受热胁迫的叶绿素荧光参数和有关代谢物的响应程度低于二倍体，以及其恢复程度快于二倍体，表明染色体加倍提高了抗热性。此外，叶绿素荧光成像的异质性也表明，四倍体的抗热能力大于二倍体。进一步的叶片解剖结构分析表明，四倍体叶片的表皮细胞变大、栅栏组织增厚、表皮毛较浓密等特点，是其抗热性强的主要原因。 4.根据以上的研究结果，通过四倍体忍冬生态修复功能的野外试验证明染色体加倍后其生态适应性变化。在本研究中，针对北京市门头沟区大面积不同类型的废弃地急需恢复植被和景观的问题，在恢复生态学理念的指导下，综合运用集成技术，将3个忍冬品种植物用于这些退化生态系统的恢复。在王平镇的公路下边坡（以碎石和矿渣为主）、煤矿、石灰窑和采石场4种类型废弃地建立生态修复的试验示范区。结果表明，在4个废弃地类型上引进的3个金银花品种，具有使示范区快速复绿、当年成景和群落快速形成的潜力，并具有对不同退化迹地的适应能力和恢复效果，其中四倍体忍冬效果更好些，这主要与其形态解剖结构和较强的生态适应性有关。

中国蔷薇属芹叶组（Rosa Sect. Pimpinellifoliae DC. ex Ser.）的分类学研究

本研究通过我国CDBI、 KUN、PE、SZ等主要标本馆约3, 500份馆藏标本的研究和野外考察相结合，对我国蔷薇属（Rosa L.）芹叶组(Sect. pimpinellifoliae DC. ex Ser.）植物以及相关组的一些种进行了性状特征、形态和微形态的研究，对该组的一些种的形态特征描述进行了补充，同时给出详细的地理和海拔范围分布图。综合花粉以及种子（瘦果）形态的研究结果重新制订了分种检索表，同时，对该组一些形态相近容易混淆的种进行了对比研究，特别对一直存在争议的绢毛复合体（绢毛蔷薇R. sericea Lindl.和峨眉蔷薇R. omeiensis Rolfe）进行了大量宏观形态特征的研究，并用光学显微镜（LM）和扫描电镜(SEM)对二者的花粉及种子形态、微形态进行对比研究和分析，主要研究内容包括： 1. 芹叶组孢粉研究 对芹叶组的10个种及相关的4个组共17个种（18个样品）的植物花粉进行了光镜和扫描电镜观察和比较研究。研究结果表明：蔷薇属植物花粉粒大小为中等偏小，极轴长23.98[21.82(R. graciliflora Rehd. et Wils.)～29.18(R. tsinglingensis Pax. et Hoffm.)] μm，赤道轴长28.65[24.15（R. graciliflora）～34.70(R. davidii Crép.)] μm；花粉属辐射对称等极单花粉，花粉形态赤道面观呈球形到超长球形；极面观为三裂圆形或近圆形,三孔沟，孔缘加厚,具中部突起的桥状盖。花粉外壁纹饰为条纹状，光镜下形态特征相差不大；在电镜下外壁条纹和脊沟内穿孔的形状、大小和频度等特征，常具组至种水平上的可见变异，可作为组至种水平划分的依据。 根据花粉外壁条纹特征及穿孔形状和数目等特征，本研究将这些植物的花粉归为5个类型，并编制了分组检索表。同时，根据条纹状的清晰度，排列方式、条纹形状、穿孔大小及其频度等方面的差异，各有特点，对该组的10个种编制了分种检索表。 2. 芹叶组种子形态研究 应用光学显微镜和扫描电镜对我国蔷薇属芹叶组14个种及相关组5个组共36种植物的种子宏观形态及种皮微形态特征进行了观察研究。结果显示，蔷薇属种子形态多样,形状分别为肾形、卵形或锥形等；种子颜色以淡棕色、褐色以及土黄色为主；种子大小种间相差悬殊，相对体积为（长×宽×厚）36.66（4.79～114.47) mm3。光镜下，种子宏观形态特征具组内一致性，在扫描电镜下种子表面结构特征因种而异，其纹饰以网纹为主，可分为3种类型，即近平滑型、负网纹型和网纹型。研究结果表明，蔷薇属种子表面纹饰与地理分布关系不大,具有组及种内稳定性。其种子形态、大小、表而纹饰类型等特征可作为蔷薇属组及种水平上的分类依据。 结合蔷薇属花粉形态研究结果，得出蔷薇属种皮微形态特征与花粉外壁纹饰特征相吻合，在代表组及种的特征上具相关性的结论。同时根据种子形态、微形态结构特征的组间区别和种间差异编制了分组及芹叶组14个种的分种检索表。 3. 绢毛蔷薇复合体的研究 通过对大量标本的研究、野外观察以及扫描电镜对绢毛蔷薇复合体的花粉形态和种皮表面结构进行研究，通过对小叶、花粉及种子的形态定量分析结果支持Rowley (1959)的观点，将峨眉蔷薇处理为绢毛蔷薇的一个变种。 综上研究结果得出，蔷薇属植物的小叶片数目、花被基数以及花粉及种子形态等性状是较为稳定的，这些特征可很好的作为分类学依据。 The morphology, pollen exine sculpture and seed coat structure of the species of Rosa sect. Pimpinellifoliae and related sections were studied.About 3,500 herbarium specimens at CDBI, KUN, PE, and SZ were examined. Field work in Sichuan and Yunnan were conducted. Revisions of some species were carried out and a new key to species of sect. Pimpinellifoliae was proposed based on morphology, pollen exine sculpture and seed coat structure, Detailed morphological descriptions, geographical distributions and the altitudinal ranges of some taxa are given. The systematics of the species complex, the Rosa sericea complex (R. sericea Lindl. & R. omeiensis Rolfe), was emphasized. This thesis focused on the following three aspects: 1. Pollen morphology of Rosa sect. Pimpinellifoliae The pollen morphology of 18 samples representing 10 species of the Eurasian Rosa sect. Pimpinellifoliae and 7 additional species of related sections was investigated under LM and SEM. The pollen grains are monadic, actinomorphic, equipolar, medium-sized, spheroidal to perprolate in equatorial view, 3-lobed circular or semi-circular in polar view, crassimarginate, pontoperculate, and with striate exine sculpture. The striate sculpture varies among sections and species. The equatorial axis ranges from 17.97 μm (R. sikangensis) to 29.18 μm (R. tsinglingensis) with an average of 23.98 μm in length, while polar axis varies from 24.15 μm (R. gracilifolra) to 34.70 μm (R. davidii) with an average of 28.65 μm in length. The pollens can be divided into five types based on striate sculpture and a key to the sections sampled was proposed accordingly. The pollen morphology of species of sect. Pimpinellifoliae is more homogeneous and different from other sections sampled and did not support the two-series subdivisions in sect. Pimpinellifoliae. A key is also provided based on characers of pollen morphology among species in sect. Pimpinellifoliae. 2. Seed coat structure of Rosa sect. Pimpinellifoliae The seed coat structure of 39 samples representing 14 species of Rosa sect. Pimpinellifoliae and 12 additional species of related sections was investigated under LE and SEM. The seed relative volume (Length × width × thickness) ranges from 4.79 to 114.47 mm3 with an average of 36.66. mm3. The seeds are reniform, ovate or oblong in shape, with orange-brown, light brown or deep brown color. Seed coat sculpture was reticulate or striate-like reticulate. There was no difference in sculpture character of various speices under LM, while three types of seed coat sculpture were identified under SEM and a key to species based on the seed coat sculpture was provided. The three types of seed coat sculpture were nearly smooth, areolate and reticulate. The study of the seed coat sculpture of same species sampled from different populations showed that characters on the seed coat are stable, and thus the size, shape and seed coat sculpture can be used in species level identification. Interestingly, characters in the seed coat sculpture and the pollen morphology in sect. Pimpinellifoliae are consistent at in specific or sectional levels. A key to the 14 species sampled was given based on seed coat sculpture. 3. The study on Rosa sericea complex The Rosa sericea complex contains R. omeiensis and R. sericea. They are morphologically similar to one another and the systematic status of R. omeiensis has been controversial. In this study we examined large numbers of herbarium specimens of R. omeiensis and R. sericea and conducted field observations in the Hengduan Mts.. We also performed SEM study of pollen morphology and seed coat structure of R. omeiensis and R. sericea. We further carried out intensive morphometric study on the leaflet, pollen, and seed morphology. Our results showed that R. omeiensis should be sunk to be a variety of R. sericea, just as Rowley’s treatment in 1959. In conclusion, the features in the number of leaflet and petal, and the morphological character on pollen and seed are relatively stable. Therefore these characters are very useful in taxon delimition.

Bibliometric analysis of biological invasions research during the period of 1991 to 2007

The objective of this study is to conduct a bibliometric analysis of all biological invasions-related publications in the Science Citation Index (SCI) from 1991 to 2007. The indicator citation per publication (CPP) was used to evaluate the impact of articles, journals, and institutions. In the 3323 articles published in 521 journals, 7261 authors from 1905 institutions of 100 countries participated. As the most productive country of biological invasions research, the US will benefit from more collaboration between institutions, countries, and continents. In addition, analysis of keywords was applied to reveal research trends.

Genetic analysis of all-fish growth hormone gene transferred carp (Cyprinus carpio L.) and its F-1 generation

Recombinant "all-fish" growth hormone gene (GH) was microinjected Into the fertilized eggs of carp. A comparison between the growth traits of transgenics and non-transgenics was carried out, and the transgenic individuals with significant "fast-growing" effect were successfully gained. A comparison on the reproductivities was also given out between the transgenics and their non-transgenic siblings, and showed that the reproductive capacity of transgenics was substantially equivalent to those of the non-transgenics. On the other hand, the genetic separation and the characteristic distribution of the F-1 generation were genetically analyzed, which gave solid evidence for the hypothesis that 2-3 chromosomes are integrated with transgene. In addition, the distinct biological effects for multisite-integrated transgenes were further discussed. The present study opens a door for the breeding of "fast-growing" transgenic fish.

Quantitative traits correlative analysis and growth comparison among different populations of bay scallop, Argopecten irradians

The shell traits and weight traits are measured in cultured populations of bay scallop, Argopecten irradians. The results of regression analysis show that the regression relationships for all the traits are significant (P < 0.01). The correlative coefficients between body weight, as well as tissue weight with shell length, shell height and shell width are significant (P < 0.05). But the correlative coefficients between the anterior and posterior auricle length with body weight as well as tissue weight are not significant (P > 0.05). The multiple regression equation is obtained to estimate live body weight and tissue weight. The above traits except anterior and posterior auricle length are used for the growth and production comparison among three cultured populations, Duncan's new multiple range procedure analysis shows that all the traits in the Lingshuiqiao (LSQ) population are much more significant than those of the other two populations (P < 0.01), and there is no significant difference between the Qipanmo (QPM) and Dalijia (DLJ) populations in all traits (P > 0.05). The results indicate that the LSQ population has a higher growth rate and is expected to be more productive than the other two populations.

Identification of monozygotic twin chimpanzees by microsatellite analysis

Zygosity determination is important for epidemiological, biological, obstetric, and prognostic studies in both human and nonhuman primates. In this study, microsatellite loci were used to screen a pair of chimpanzee (Pan troglodytes) twins and their parents. The twins share identical alleles at all loci tested. The probability of dizygotic origin is estimated to be 2.9 x 10(-11). Even after excluding linkage of loci on the same chromosome, the probability is still low enough (3.7 x 10(-9)) to exclude dizygotic origin. MHC typing was also done on Patr-DRB and Patr-DQB loci and the twins share identical alleles at both loci, consistent with the microsatellite results. Together these results demonstrate a monozygotic origin for the chimp twins. Our results suggest that microsatellite analysis is a powerful method for zygosity determination, which can be screened reliably and efficiently. Am. J. Primatol. 52:101-106, 2000. (C) 2000 Wiley-Liss, Inc.

Duplication and independent selection of cell-wall invertase genes GIF1 and OsCIN1 during rice evolution and domestication

Background: Various evolutionary models have been proposed to interpret the fate of paralogous duplicates, which provides substrates on which evolution selection could act. In particular, domestication, as a special selection, has played important role in crop cultivation with divergence of many genes controlling important agronomic traits. Recent studies have indicated that a pair of duplicate genes was often sub-functionalized from their ancestral functions held by the parental genes. We previously demonstrated that the rice cell-wall invertase (CWI) gene GIF1 that plays an important role in the grain-filling process was most likely subjected to domestication selection in the promoter region. Here, we report that GIF1 and another CWI gene OsCIN1 constitute a pair of duplicate genes with differentiated expression and function through independent selection. Results: Through synteny analysis, we show that GIF1 and another cell-wall invertase gene OsCIN1 were paralogues derived from a segmental duplication originated during genome duplication of grasses. Results based on analyses of population genetics and gene phylogenetic tree of 25 cultivars and 25 wild rice sequences demonstrated that OsCIN1 was also artificially selected during rice domestication with a fixed mutation in the coding region, in contrast to GIF1 that was selected in the promoter region. GIF1 and OsCIN1 have evolved into different expression patterns and probable different kinetics parameters of enzymatic activity with the latter displaying less enzymatic activity. Overexpression of GIF1 and OsCIN1 also resulted in different phenotypes, suggesting that OsCIN1 might regulate other unrecognized biological process. Conclusion: How gene duplication and divergence contribute to genetic novelty and morphological adaptation has been an interesting issue to geneticists and biologists. Our discovery that the duplicated pair of GIF1 and OsCIN1 has experiencedsub-functionalization implies that selection could act independently on each duplicate towards different functional specificity, which provides a vivid example for evolution of genetic novelties in a model crop. Our results also further support the established hypothesis that gene duplication with sub-functionalization could be one solution for genetic adaptive conflict.

Combined effects of polyfluorinated and perfluorinated compounds on primary cultured hepatocytes from rare minnow (Gobiocypris rarus) using toxicogenomic analysis

Polyfluorinated and perfluorinated compounds (PFCs) are used in numerous commercial products and have been ubiquitously detected in the environment as well as in the blood of humans and wildlife. To assess the combined effects caused by PFCs in mixtures, gene expression profiles were generated using a custom cDNA microarray to detect changes in primary cultured hepatocytes of rare minnows exposed to six individual PFCs (perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluorododecanoic acid, perfluorooctane sulfonate, and 8:2 fluorotelomer alcohol) and four formulations of the PFCs mixtures. Mixtures as well as individual compounds consistently regulated a particular gene set, which suggests that these conserved genes may play a central role in the toxicity mediated by PFCs. Specifically, a number of genes regulated by the mixtures were identified in this study, which were not affected by exposure to any single component. These genes are implicated in multiple biological functions and processes, including fatty acid metabolism and transport, xenobiotic metabolism, immune responses, and oxidative stress. More than 80% of the altered genes in the PFOA- and PFOS-dominant mixture groups were of the same gene set, while the gene expression profiles from single PFOA and PFOS exposures were not as similar. This work contributes to the development of toxicogenomic approaches in combined toxicity assessment and allows for comprehensive insights into the combined action of PFCs mixtures in multiple environmental matrices. (C) 2009 Elsevier B.V. All rights reserved.

Applications of factor-criteria system reconstruction analysis in the reproduction research on grass carp, black carp, silver carp and bighead in the Yangtze River

The natural reproduction of grass carp, black carp, silver carp, and bighead will be affected adversely by the Three Gorges Project in the Yangtze River. One of the methods to save the fish is to regulate the water levels, keeping them suited for the species to spawn. Nine factors associated with the scale of larvae-flood of the four species are classified into five levels, and the ranges of these factors producing larvae-floods are given by using the "factor-criteria system reconstruction analysis" method. Moderate beginning water levels and flow, with high daily increases in the rate of water level and flow, and a long duration of water level rising are important for the production of a large larvae-flood.

簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.