Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. Copyright (C) 2004 John Wiley Sons, Ltd.

青杨组(Populus section Tacamahaca Spach)不同种对增强UV-B的反应差异

人类向大气中排放的大量氮氧化合物和氟氯烃类化合物(CFC’s)引起臭氧分子的分解，导致到达地球表面的紫外辐射增加，特别是UV-B辐射增强。本项目以青杨组杨树为模式植物，从形态和生理方面研究了来自不同UV-B背景下的康定杨与青杨在增强UV-B下的反应及其反应差异,并探讨了干旱、施肥对它们抗UV-B能力的影响。杨树具有分布广、适应性强、在生态环境治理和解决木材短缺方面均占有重要位置，研究成果可为生态系统的恢复与重建提供理论依据和科学指导。主要研究结果有以下： 1. 在温室中经过增强UV-B处理，杨树的外部形态及生理活动受到了一定程度的影响。增强UV-B导致康定杨、青杨的生物量、叶面积及节间长度降低，叶片增厚，SOD活性升高，膜伤害增加，而对叶片数目、R/S、叶绿素A、叶绿素B及整个叶绿素含量没有影响。两种杨树对UV-B胁迫的响应存在差异：在增强UV-B条件下，青杨的植株高度、生物量、叶面积、脯氨酸含量、长期用水效率受到的影响大于康定杨，相比而言，康定杨在比叶面积、叶片厚度、可溶性糖含量、UV-B吸收物质的含量及SOD和GPX活性方面增加的程度大于青杨。这些区别说明，来自于高海拔的康定杨比来自于低海拔的青杨对增强UV-B 具有更强的耐性。我们认为二者在叶片厚度、比叶面积、UV-B吸收物质含量及SOD、GPX活性差异是导致对增强UV-B耐性不同的原因。 2. 干旱与增强UV-B对杨树的生长和生理特性均产生了影响，而且两种胁迫共同作用时干旱表现减弱或加剧了UV-B对杨树某些形态和生理特性的影响。 据试验结果，干旱显著地降低了杨树的株高、叶片数目、叶面积，增加了叶片厚度，促进ABA的积累，提高了CAT活性。对于干旱，两种杨树之间也表现出了一定的差异性。可溶性蛋白质和脯氨酸在青杨叶片中得到显著积累，而在康定杨中没有变化。此外，CAT、长期用水效率在康定杨中受到的影响更加明显。长期用水效率的不同变化趋势说明两种杨树对水分胁迫采用了不同的用水策略，康定杨采用的是节水用水策略，提高用水效率，而青杨采用的是耗水的用水策略。根据干旱对叶面积、脯氨酸、ABA含量、CAT活性及长期用水效率等方面的影响，我们认为来自高海拔地区的康定杨比来自低海拔的青杨有更大的耐旱性，这是对生长环境长期适应的结果。在高海拔地区，因霜冻常带来土壤水分不可利用，降低了根系对水分的吸收，树木容易受到的生理性干旱。另外，高海拔的地区低的气温使植株对严寒有较强的耐性，减少了水分的需要。 生长于增强UV-B下的康定杨和青杨植株表现为高度降低，叶面积缩小，比叶面积增加；叶片栅栏组织、海绵组织均受到增强UV-B的影响，其厚度的增加导致整个叶片变厚。增强UV-B还显著提高了杨树的APX活性、UV-B吸收物质含量，而对叶片数目、ABA、可溶性蛋白质含量及CAT活性没有产生影响。试验中也观察到了两种杨树对增强UV-B响应的差异：与康定杨相比，在增强UV-B下青杨株高、叶面积降低的程度更大一些，SOD活性显著提高。另外UV-B吸收物质受到的影响不同。根据这些差别，高海拔的康定杨(3500 m)比来自低海拔的青杨(1500 m)增强UV-B有较强的耐性。 与水分充足情况下UV-B对植株的影响相比，干旱对杨树抗增强UV-B产生了一定的影响，表现为加剧或减弱UV-B对植物的影响，但这种影响与形态、生理指标有关。当干旱与增强UV-B共同作用时，杨树植株的株高、叶面积进一步降低、叶片进一步增厚。就脯氨酸的积累的而言，在没有水分胁迫时，增强UV-B促使它显著增加，而在干旱处理下这种效果变得不明显。干旱对增强UV-B的影响还与杨树的种类有一定的关系。在康定杨中，干旱减弱了增强UV-B对栅栏组织与海绵组织的影响，且在植株高度、叶面积上表现出累加效应，而在CAT上交互作用显著；但在青杨中干旱则加剧增强UV-B对栅栏组织与海绵组织的影响，在植株高度、叶面积及比叶面积上表现出显著的交互作用。据碳同素分析，在水分充足的条件下，无论是康定杨，还是青杨，增强UV-B均导致其长期用水效率的提高，然而当两种胁迫共同作用时，长期用水效率则表现出差异，在青杨中，长期用水效率得到进一步增高，而康定杨中干旱的效应被增强UV-B所减轻。 3. 田间试验表明，杨树的生长、生理特征都受到养分和增强UV-B的影响。施肥对杨树的影响表现为：提高了叶面积、生物量及SOD的活性，降低了抗坏血酸含量。对于施肥作用，两种杨树的反应也有区别：在康定杨中施肥显著增加了的叶片长度、宽度及光合色素的含量，降低了净光合速率、气孔导度及胞间CO2浓度；在青杨中，则SOD、GPX、APX活性表现增加。从试验看出，施肥对来自于高海拔地区的康定杨(3500 m)的影响较大，对来自低海拔的青杨(1500 m)影响较小，这与它们对原产地的生境适应有一定关系。在康定杨生长的高海拔地区，低温度和湿度不能为地上凋落物或土壤中的根分解提供理想的条件，造成当地土壤的低养分状况，所以当肥料施用以后，效果显著。 经过增强UV-B处理，杨树叶片中UV-B吸收物质含量、GPX的活性得到提高，而脯氨酸、丙二醛、可溶性蛋白质、叶绿素及类胡萝卜素含量没有受到影响。对于增强UV-B两种杨树受到的影响也有所不同：在青杨中增强UV-B导致叶面积缩小，生物量、净光合速率降低，APX的活性及长期用水效率的提高，而对康定杨的这些指标没有产生显著影响，相反抗氧化酶的活性明显高于青杨。这些差异性是由于两种杨树对原产地不同UV-B背景的长期适应结果。康定杨长期生长在较高UV-B环境中，对UV-B有较强的耐性。而青杨适应于较低的UV-B环境，对增强UV-B较为敏感。 试验中施肥也影响了植株对增强UV-B的反应，不过这种影响与杨树的种类及测定指标有一定的相关性。例如，在缺肥的情况下，青杨的长期用水效率和康定杨的叶绿素含量都受到增强UV-B的显著影响，而施肥以后这种影响变得不显著。在缺肥的条件下，GPX、APX在青杨中的活性、GPX在康定杨中的活性对增加UV-B反应不敏感；而施肥以后则变化显著，同样胞间CO2浓度在康定杨也有类似的变化。 For past decades, Ultraviolet radiation, especially UV-B reaching the Earth’s surface increased because of depletion of ozone layer resulted from emission of NxO and CFC’s from human activities. In this experiment, different species of Populus section Tacamahaca Spach from different UV-B background were selected as a model plant to assess the effects of enhanced UV-B radiation. Morphological and physiological traits induced by enhanced UV-B were observed and the different responses between P. kangdingensis and P. cathayana were discussed, furthermore the influences of drought and fertilizer on responses induced by enhanced UV-B were studied. Since poplars play an important role in lumber supply, and are important component of ecosystems due to their fast growth and wide adaptation, the study could provide a strong theoretical evidence and scientific direction for the afforestation, and rehabilitation of ecosystem. The results are as follows: 1. The experiment conducted in a greenhouse indicated that morphological and physiological traits of two poplars were affected by enhanced UV-B radiation. Enhanced UV-B radiation not only reduced biomass, leave area and internode length, but also increased leaf thickness and SOD activity as well as MDA concentration and electrolyte rate. However, no significant changes in leaf numbers, root shoot ratio, and total chlorophyll and chlorophyll component were observed. There were different responses to enhanced UV-B radiation between two species. Compared with P. kangdingensis, cuttings of P. cathayana, exhibited lower height increment and smaller leaf area. In addition, there were significant differences in free proline, soluble protein, and UV-B absorbing compounds, and the activity of SOD and GPX, long-term WUE between them. Differences in plant height, biomass, leaf area, free proline concentration, and long-termed WUE showed that P. cathayana were more affected by enhanced UV-B radiation than P. kangdingensis. In contrast, more increase of specific leaf mass, leaf thickness, and soluble sugar, and UV-B absorbing compounds, and activity of SOD and GPX were observed in P. kangdingensis. According to these results, we suggested that P. kangdingensis from high elevation, which adapted to higher UV-B environments, had more tolerance to enhanced UV-B than P. cathayana from low elevation, which adapted to lower UV-B environment. We believe it was the difference of leaf thickness, specific leaf mass, and UV-B absorbing compounds as well as the activity of SOD and GPX resulted in lower adaptation of P. cathayana to enhanced UV-B radiation. 2. Growth and physiological traits of two poplars were affected by both drought and enhanced UV-B radiation. Moreover, it was observed that when two stresses applied together drought could exacerbate UV-B effects or decrease sensitivity to UV-B. In the experiment, drought significantly decreased plant height, leaf numbers, leaf area, and increased leaf thickness, and ABA, and CAT activity of two poplars. There were significant interspecific differences to drought stress. Exposed to drought, soluble protein and proline concentration were increased in P. cathayana but not in P. kangdingensis. However, more changes in CAT and long-term WUE were observed in kangdingensis. Different change in long-term WUE suggests that two poplars adapted different water-use strategies. P. kangdingensis employ a conservative water-use strategy, whereas P. cathayana employ a prodigal water-use strategy. Based on the differences in leaf area, accumulation of free proline and ABA, CAT activity as well as long-term WUE, we believed that P. kangdingensis from high elevation had a greater tolerance to drought than P. cathayana from low elevation，which is the result of adaptation to local environment. In high elevation area, trees are prone to suffer from physiological drought because of un-movable water caused by frost. Besides lower temperature enable the plants had greater adaptability to frost as a results the requirement of water is reduced Enhanced UV-B radiation decreased shoots height, leaf area, and increased specific leaf mass and thickness of palisade and sponge layer as well as APX activity and UV-B absorbing compounds in both species. Whereas, leaf numbers, ABA content, soluble protein and CAT activity showed no differences to enhanced UV-B radiation. Interspecific differences were also observed. Compared with P. kangdingensis, P. cathayana showed lower shoot height and smaller leaf area, higher SOD activity. Besides, variation in UV-B absorbing compounds was found. These differences suggested that P. kangdingensis from high elevation (3500 m) was more tolerant to enhanced UV-B radiation than P. cathayana from low elevation (1500 m). Compared with morphological and physiological changes induced by enhanced UV-B radiation under well-watered conditions, drought exacerbated or decreased these changes. However, these effects vary with parameters measured. When two stresses applied together, shoot height and leaf area further decreased while leaf thickness further increased. Under well-watered conditions, enhanced UV-B radiation significantly increased proline content, but such effect was not observed under drought conditions. The effect of drought on enhanced UV-B radiation was related to species. For example, drought reduced the effects of enhanced UV-B radiation on palisade parenchyma and sponge mesophyll in P. kangdingensis, and additive effects in shoot height and leaf area and interactive effect CAT activity were observed. In contrast, for P. cathayana drought significantly exacerbated the effects of enhanced UV-B radiation on palisade parenchyma and sponge mesophyll; there were noticeable interaction in shoot height, leaf area and specific leaf mass. As far as long-term WUE is concerned, it was increased by enhanced UV-B radiation under well-watered conditions in both species. While different effect was observed between two species in combination of two stresses. Long-term water use efficiency was further increased in P. cathayana whereas the effect was less significant in P. kangdingensis. 3. The field experiment showed that growth and physiological traits of poplars were affected by nutrition and enhanced UV-B radiation. Fertilization significantly increased leaf area, biomass and SOD activity, reduced Ascorbic acid concentration. There was interspecific difference in response to fertilization. For P. kangdingensis, fertilization significantly increased leaf width, leaf length and photosynthetic pigments content while net photosynthetic rate and stomatal conductance, intercellular CO2 concentration were significantly decreased. However, for P. cathayana, these parameters were unaffected except the increase of SOD, GPX and APX activity. From above, it could concluded that P. kangdingensis from high elevation was more affected by fertilization than P. cathayana, This difference was due to adaptation to local environment., The low temperature and moisture where P. kangdingensis was collected can not provided optimum to decompose roots and litter fall as a result the nutrition in soil was poor. Exposed to enhanced UV-B radiation, for both species UV-B absorbing compounds and GPX activity were significantly increased while proline, MDA, soluble protein, chlorophyll, carotenoids were not affected. Different responses were also observed between the two species. Enhanced UV-B radiation caused significant decreases in leaf area, biomass, net photosynthetic rate and increase in APX activity and long-term WUE in P. cathayana but not in P. kangdingensis. In addition, activity in antioxidant enzymes was much higher in P. kangdingensis than in P. cathayana. In the experiment fertilization affected responses of cuttings to enhanced UV-B radiation, but it concern species and parameters measured. Long-term WUE in P. cathayana and chlorophyll in P. kangdingensis were significantly increased by enhanced UV-B radiation under non-fertilization treatments while the increase was not found under fertilization treatment. In contrast, under no fertilization treatment enhanced UV-B radiation did not affected GPX and APX activity in P. cathayana and GPX in P. kangdingensis while significant increase appeared after application of fertilization. Similar effect of enhanced UV-B radiation on intercellular CO2 concentration in P. kangdingensis was observed.

青藏高原东缘川西云杉林皆伐后灌木生长、繁殖与更新的研究

植物群落及其环境在干扰后的演替格局和过程的研究，是群落和生态系统动态研究的一个热点。选取青藏高原东缘山原区川西云杉林皆伐后，从草地过渡到灌丛的关键阶段的4 个皆伐迹地（恢复时间为8 a、10 a、16 a 和21 a），研究皆伐及自然恢复过程对林下典型灌木银露梅（Potentilla glabra）和唐古特忍冬（Lonicera tangutica）的生长与繁殖能力的影响以及灌木植物在迹地上的更新情况，分析灌木在不同生境中的适应对策和适应能力的差异,为揭示青藏高原东缘山原区迹地植被从草甸到灌丛演替的过程和特点及促进迹地演替与植被恢复进程提供理论依据和技术支撑。研究主要结论如下：1）皆伐后银露梅生长和繁殖能力显著提高，但对唐古特忍冬的影响不明显。皆伐后银露梅丛基径、高度和各部分生物量都显著增加（P < 0.05），但唐古特忍冬只有叶生物量和地下生物量增加，总生物量和其余构件生物量无显著变化。皆伐后，银露梅的结实数量、结实株数、不结实株数和结实株/不结实株比例显著增加（P < 0.05）。自然恢复过程中，银露梅和唐古特忍冬生长能力以及银露梅的结实量都表现出降低的趋势。随着迹地自然恢复时间的增加，银露梅和唐古特忍冬的基径、高度、丛叶片数和各部分生物量有减少的趋势。银露梅的结实株数增加, 但结实数量减少。2）皆伐对银露梅和唐古特忍冬生物量分配模式影响不一致。原始林和迹地中（除CT85）银露梅的生物量大小关系皆为：地下>茎>侧枝>叶。唐古特忍冬在原始林中的生物量大小关系为：茎＞地下部分＞侧枝＞叶,而皆伐后生物量的分配情况改变，生物量大小关系变为：地下部分＞茎＞侧枝＞叶。随着自然恢复时间的增加，银露梅减少了地下生物量的分配，而唐古特忍冬增加了地下生物量的分配。3）皆伐和恢复时间的增加改变了迹地物种组成，促使阳性乔、灌木在迹地上定居。4 个迹地上共出现了灌木15 种，乔木3 种，没有出现天然云杉和冷杉幼苗。随着恢复时间的增加，迹地上的灌木物种由原始林下的耐阴物种逐步发展为以针刺悬钩子（Rubus pungens）为主的阳性灌木。4）影响灌木幼苗密度和幼树密度的因子不一致。灌木幼苗密度与灌木层盖度显著负相关，与苔藓层盖度显著正相关。幼树密度与草本层盖度正相关，与苔藓层盖度、灌木层盖度和高度负相关。5）研究发现在青藏高原东缘山原区皆伐15～20 a 后，迹地仍以草本植物为主，推测皆伐后至少20 a 以上迹地才可能向灌丛阶段过渡，比高山峡谷地区的演替进程至少推迟了20 a。银露梅和唐古特忍冬在皆伐后自然恢复过程中表现出不同的生长与繁殖策略是由两个物种的生物学特性的差异引起的。银露梅比唐古特忍冬更适应迹地退化环境。促进青藏高原东缘山原林区迹地森林恢复一方面是尽量减少人为活动的破坏，另一方面，可以通过在迹地中播种适当的乡土乔、灌木种子（如白桦、银露梅）等人工措施，以加快演替进程。The succession pattern and process of plant community and their environments is a hot spotin community and ecosystem dynamic study. Four clearcuts were chosen in Rangtang(recovery time of 8 a、10 a、16 a and 21 a), which represented the key stage of thecommunity evolved from grass stage to shrub stage in the eastern margin of theQinghai-Tibetan Plateau. The growth and reproduction of the Potentilla glabra andLonicera tangutica and the natural regeneration of shrub plants in the primary Piceabalfouriana forest and 4 clearcuts were studied to explore how clear cutting andnatural recovery process affected the understory shrub species during the 21 years inthe eastern margin of the Qinghai-Tibetan Plateau. The main results were below.1) The growth and reproduction of P. glabra significantly increased after forestclear cutting.. But it was not so significant as to the L. tangutica. The organismbiomass and total biomass of P. glabra were increased obviously after clear cutting（P< 0.05）. But only leaves and underground biomass of L. tangutica increasedsignificantly after clear cutting（P < 0.05）. The number of fruit and growth of P. glabraincreased significantly after clear cutting too（P < 0.05）. The ramet height, basaldiameter , organism biomass and friut number of P. glabra and L. tangutica reducedas the increase of recovery time.2) The biomass allocation patterns varied between P. glabra and L. tangutica inthe primary forest and clearcuts. The biomass allocation of P. glabra both in primary forest and clearcuts was followed as: underground part > stem > branch > leaves.However, the biomass allocation of L. tangutica had changed after the clear cutting.The biomass allocation of L. tangutica in the primary forest was followed as: stem >underground part > branch > leaves and it was underground part > stem > branch >leave in clearcuts. The biomass allocation of P. glabra and L. tangutica varied amongclearcuts. Aboveground biomass was increased while underground biomass decreasedfor P. glabra with the increase of recovery period. However, the L. tangutica showedthe reverse changing pattern.3) Clear cutting and recovery time had changed the species composition of theclearcuts. There were 15.shruby species and 3 tree species in the four clearcuts. Nospruce and fir seedlings were found. In the early stage after clear cutting, there wereonly understory shrub species from the primary Picea balfouriana forest. The sunnyspecies, especieally Rubus pungens invaded intensly as the increase of recovery time.4) There was a significant negative relationship between density of seedlingswith shrub layer coverage and positive correlation with moss coverage. The saplingshad significantly positive correlation with herb layer coverage and negativecorrelation with moss coverage, shrub layer coverage and height.5）Comparing to studies in Miyalou, a nearby high mountain and canyon area,the secondary sucession in this subalpine plateau areas lagged at least 20 years.P. glabra and L. tangutica showed different growth and reproduction strategies toclear cutting and natural recovery , which may associated with the difference of theirbiological characters. P. glabra was more adaptive to the clear cutting than the L.tangutica. Two suggestions were probably recommended to promote the recoveryprogress in the subalpine plateau areas based on the results of this study. Limitanthropogenic disturbance, and meanwhile sow native tree and shrub seeds inclearcuts.

圈养大熊猫遗传结构研究

大熊猫(Ailuropoda melanoleuca)是我国特有的濒危野生动物之一，迁地保护已经成为大熊猫物种保护的一个重要方面。当前大熊猫圈养种群数量增长很快，但是其“多雄配一雌”的交配（配种方式），以及生产过程中记录遗失等原因，造成圈养种群普遍存在亲子关系不清、谱系混乱等问题。为了加强遗传管理，有必要进行亲子关系鉴定、完善谱系；还需要检测种群的基因多样性水平，并在此基础上提出相应的遗传管理建议。 本研究应用9个具有高度多态性的大熊猫微卫星标记，对来自成都大熊猫繁育研究基地2006和2007年度出生的17只大熊猫幼崽及其全部候选父母共37个样品做了基因型分析；然后应用最大似然法，判断幼崽的父－子关系。同时，还对来自卧龙大熊猫保护研究中心的31只大熊猫个体也做了基因分型。将两个种群的数据进行比较：1）等位基因多样性和杂合度水平；2）通过F统计法，分析两个种群的遗传分化水平；3）通过遗传距离法，对所有个体进行聚类分析。 研究结果表明： 1）在母子关系不清的情况下，9个微卫星标记联合的父亲鉴定排除概率E为0.940090；而在母子关系确实的条件下，E= 0.993933。由于本研究中所有后代的母亲都是清楚的，因此这9个微卫星位点能够有效用于圈养大熊猫的亲子鉴定。似然法分析也表明，本研究所获得的亲子鉴定结果置信度在95％以上。 2）2005年种源交换后，成都大熊猫的等位基因多样性和杂合度水平都略高于卧龙种群（但没有达到显著水平），两个种群间的遗传分化水平也有所降低。但是，与卧龙相比，成都种群面临较大的近交压力。 基于以上研究结果，我们建议：进一步加强种源交换和基因交流，把两个种群当作一个遗传单元(MU)来进行管理。 Giant panda (Ailuropoda melanoleuca) is one of the endangerd wildlife endemic to China, and the ex-situ breeding become more and more important for the conservation of this speices. Although the captive population is expanding rapidly, the uncertainty occurs because the paternities of cubs are not clear due to the breeding pattern of “multiple male to single female,”as well as the records lost, resulting in errors in the studbook. For this reason, the paternity of the cubs and the genetic diversity of the captive giant pandas should be tested carefully to get information for the genetic management in the future. 9 polymorphism microsatellite markers were used to do paternity assignment for the 17 cubs born in 2006 and 2007 from Chengdu Research Base for Giant Panda Breeding (CGB) based on the maximum-likelihood methods. A total of 37 individuals were sampled, including all the candidate dams and sires. These samples were also used for comparing with 31 individuals sampling from Wolong China Research and Conservation Center for the Giant Panda (WCG). The comparing indexes were: 1) Allelic diversity and heterozygosity; 2) Genetic differentiation based on F-statistic; 3) Cluster analysis based on genetic distance. The results show that: 1) If the mother is unkown, the combined exclusion probability using these 9 loci is 0.940090. If the mother is known then the exclusion probability is 0.993933. Since the dam-offspring relationship is known in captive populations, the results could resolve unknown paternities in the study. And the confidence level of the results is 95% based on the likelihood methods. 2) The allelic diversity and the heterozygosity of CGB were higher than WCG (n ot significant), and the genetic differentiation was reduced a little since the genetic exchange between two populations in 2005. However, the population of CGB will be threatening by inbreeding seriously than that of WCG. From above, we suggest to reiforce the genetic exchange and geneflow between CGB and WCG, and these two populations should be regarded as one genetic management unit (MU).

大赖草总DNA转化小麦引起的HMW-GS基因变化及大赖草HMW-GS基因的克隆

应用花粉管通道技术将新疆大赖草总DNA导入小麦，用高重序列分析方法，已为大赖草总DNA转入小麦提供了初步的分子证据。在转 化后代中选育出稳定遗传的大穗变异株系，分析表明，这些转化株中蛋白质含量明显增高(13%-17%)。对基因供体新疆大赖草、受 体春麦761、转化株的高分子量谷蛋白亚基(HMW-GS)进行了SDS-PAGE分析，发现这些转化株中HMW-GS发生了很大变化，并在此基础 上，用来自小麦基因组的四对特异引物，以PCR方法扩增供体、受体以及转化株的1Ax、1Bx、1Dx及1Ay、1By、1Dy型HMW-GS全基因 ，比较他们扩增产物的差异，结果表明，受体与转化株在HMW-GS基因1Ax、1Bx位点上的扩增产物差异不大，在HMW-GS基因位点1Dx 和y型基因上的扩增产物有较大差异，说明了受体在基因位点1Dx、1Ay、1By和1Dy上可能发生了多位点插入，可能由于这些基因位 点上的插入引起了转化株的高分子量谷蛋白亚基(HMW-GS)的变化，这就再一次为大赖草总DNA导入提供了直接的分子证据。虽然大 赖草总DNA导入提高了小麦蛋白质的含量，改变了HMW-GS的组成，部分改变了品质评分，但我们感到这些转化株在品质改良方面仍 有很大余地，如何更好地利用新疆大赖草蛋白质的优良特性及避免总DNA导入给转化株带来的不良性状，一个大赖草HMW-GS基因正 被分离及克隆，并准备将其利用于未来的品质育种当中。Total DNA of Leymus racemosus had been transformed into wheat through pollen tube pathway. Analysis of the repeated gene sequence had provided an elementary proof. Some variant cultivars with big ear were screened from their offsprings, and their protein content increased greatly from 13% (receptor)to 17%(transformed). The result from SDS-PAGE analysis of high-molecular-weight glutenin subunits(HMW- GS) respectively in donor(Xinjiang Leymus racemosus), receptor(spring wheat cultivar 761)and transformed wheats, showed the HMW-GS composition changed in the transformed plants. On the basis of the research, Four special pairs primers from wheat(T.aestivum L.) genome were used to amplify complete coding regions of HMW-GS genes on 1Ax、1Bx 、1Dx and 1Ay、1By、1Dy loci of donor、receptor and the big ear transformed cultivars. By comparing amplified PCR products. Faint differences were found among receptor and transformed cultivar's 1Ax、1Bx PCR amplifed products and apparent differences on those of 1Dx、y-typePCR product. We gueseed that there may be some DNA inserts in 1Dx 、1By、1Dy loci resulted in the changes of the HMW-GS among transformed cultivars. This provides second direct molecular witness to the exogenous DNA introduction. Even though the transformed plants have higher protein content, changed HMW-GS composition, partially improved process quality, there still leave much work to improve quality. In order to make full use of the excellent property of Leymus racemosus protein and avoid the disadvantages introducced by total DNA transformation, a HMW-GS gene of Leymus racemosus was being isolated and cloned.

Study on QSPR of alcohols with a novel edge connectivity index F-m

A novel edge degree f(i) for heteroatom and multiple bonds in molecular graph is derived on the basis of the edge degree delta(e(r)). A novel edge connectivity index F-m is introduced. The multiple linear regression by using the edge connectivity index F-m and alcohol-type parameter delta, alcohol-distance parameter L can provide high-quality QSPR models for the normal boiling points (BPs), molar volumes (MVs), molar refraction (MRs), water solubility(log(1/S)) and octanol/water partition (logP) of alcohols with up to 17 non-hydrogen atoms. The results imply that these physical properties may be expressed as a liner combination of the edge connectivity index and alcohol-type parameter, 6, alcohol-distance parameter, L. For the models of the five properties, the correlation coefficient r and the standard errors are 0.9969,3.022; 0.9993, 1.504; 0.9992, 0.446; 0.9924,0.129 and 0.9973,0.123 for BPs, MVs, MRs, log(1/S) and logP, respectively. The cross-validation by using the leave-one-out method demonstrates the models to be highly reliable from the point of view of statistics.

预应力结构抗滑桩设计计算方法及其在滑坡治理中的应用研究

Landslide is a kind of serious geological hazards and its damage is very great. In recent years, landslides become more and more frequent along with increase of scale of engineering constructions and cause greater loss. Consequently, how to protect landslides has become important research subject in the engineering field. This paper improves the method how to compute landslide thrust and solves the irrational problem in the design of piles because of the irrational landslide thrust according to the theory and technology of existed anti-slide piles and pre-stressed cable anti-slide piles. Modern pre-stressing technology has been introduced and load balancing method has been used to improve the stressing behavior of anti-slide piles. Anchor cables, anti-slide piles and modern pre-stressing technology have been used to prevention complicated landslide. It is an important base to select values for the landslide thrust. An improved method to calculate design thrust of anti-slide piles has been presented in this paper on the base of residual thrust method by comparing existing methods to select values of landslide thrust in the design of anti-slide piles. In the method, residual landslide thrust behind the anti-slide piles and residual skid resistance before the piles has been analyzed, equitable distribution of residual landslide thrust behind the piles has been realized, and the method to select value of design thrust becomes more reasonable. The pre-stressed cable anti-slide piles are developed from the common anti-slide piles and are common method to prevent landslide. Their principle is that internal force of anti-slide piles is adjusted and size of section is diminished by changing constraint conditions of anti-slide piles. For landslides with deep slip surface and large scale of slopes, limitation of the method appears. Such landslides are in need of long piles and anchor cables which are not only non-economic but also can generate larger deformation and leave potential danger after prevention. For solving the problem, a new kind of anti-slide piles, inner pre-stressing force anti-slide piles, is presented in this paper, and its principle is that an additional force, which is generated in the inner anti-slide piles by arranging pre-stressed reinforcement or tight wire in a certain form in interior of anti-slide piles and stretching the steel reinforcement or tight wire, may balance out the internal force induced by landslide thrust whole or partly (load balancing method). The method will change bending moment which anti-slide piles are not good at bearing into compressive stress which piles are good at bearing, improve stressing performance of anti-slide piles greatly, diminish size of section, and make anti-slide piles not fissured in the natural service or postpone appearance of the fissures, and improve viability of anti-slide piles. Pre-stressed cable anti-slide piles and inner pre-stressing force anti-slide piles go by the general name of pre-stressed structure anti-slide piles in the paper, and their design and calculation method is also analyzed. A new calculation method is provided in the paper for design of anti-slide piles. For pre-stressed structure anti-slide piles, a new computation mode is firstly presented in the paper on the foundation of cantilever piles. In the mode, constraint form of load-bearing section of the anti-slide piles should be confirmed according to reservoir conditions in order to figure out amount of pre-stress of the anchor cables, and internal force should be analyzed for the load-bearing section of pre-stressed structure anti-slide piles so as to confirm anchorage section of anti-slide piles. Pre-stressed cables of the pre-stressed cable anti-slide piles can be arranged as required. This paper analyzes the load-bearing section of single-row and double-row pre-stressed cable anti-slide piles and provides a calculation method for design of the pre-stressed cable anti-slide piles. Inner pre-stressing force anti-slide piles are a new kind of structural style. Their load-bearing section is divided into four computation modes according to whether pre-stressed cables are applied for exterior of the anti-slide piles, and whether single-row or double-row exterior pre-stressed cables are applied. The load balancing method is used to analyze the computation modes for providing a method to design the inner pre-stressing force anti-slide piles rationally. Pre-stressed cable anti-slide piles and inner pre-stressing force anti-slide piles are applied to research on Mahe landfall in Yalong Lenggu hydropower station by the improved method to select value of design thrust of anti-slide piles. A good effect is obtained in the analysis.

交易型领导的结构和作用机制研究

The Multifactor Leadership theory developed by Bass (1985) has become the new paradigm of leadership research. The empirical results of the effectiveness of transformational and transactional leadership in the literature, however, are not consentient. Researchers in China found the different structure of transformational leadership, but have not developed the transactional leadership. This study attempts to investigate three key questions in the unique Chinese socio-economic context: 1) what is the structure of transactional leadership in China? 2) What are the differences between western countries and China? And 3) what is the relationship between the transformational and transactional leadership mechanism? This study examines data collected from 3,500 participants, using Explored Factor Analysis (EFA), Confirmed Factor Analysis (CFA), Hierarchical Regression Analyses, partial correlations and other statistics methods. The major finings are listed as follows： Firstly, inductive methods was used to explore the structure of transactional leadership and the result show that transactional leadership is a four dimensions structure which includes contingent reward, contingent punishment , process control and anticipated investment. Reliability analysis, item analysis, EFA and CFA show the reliability and validity of the transactional leadership questionnaire we designed is good enough, the design of the item is effectively and properly. Contrast to other researches, anticipated investment emphasis on the leader’s recessive investment for subordinate, and this kind of transaction is quite special under the Chinese culture. While the content of the contingent reward with the contingent punishment is wider than the contingent reward in the western country, and the process control is wider than the management by exception and including goal setting and the management during the process. Secondly, hierarchical regression analyses showed that transformational and transactional leadership were significant positively related with in-role performance, extra-role performance, satisfaction and leadership effectiveness while negatively related to intention to leave. The effects of transactional and transformational leadership are different. Transactional leadership could significantly predict intention to leave controlling for transformational leadership, while transformational leadership could significantly predict in-role performance, extra-role performance, satisfaction and leadership effectiveness controlling for transactional leadership. Thirdly, the income level and the rank of subordinates are the moderators between the transformational, transactional leadership and leadership effectiveness. The leadership effectiveness of transactional leadership would decrease as the rank of subordinates increased, while the leadership effectiveness of transformational leadership would increase as the rank of subordinates increased. Transactional leadership is positively related to the effectiveness when the level of the subordinate income is low, but negatively related to the effectiveness when the level of the subordinate income is high. However the income level of the subordinate could not influence the leadership effectiveness of transformational leadership.

Developing a toolkit for assessing the impact of health library services on patient care

Urquhart,C., Spink, S., Thomas, R. & Weightman, A. (2007). Developing a toolkit for assessing the impact of health library services on patient care. Report to LKDN (Libraries and Knowledge Development Network). Aberystwyth: Department of Information Studies, Aberystwyth University. Sponsorship: Libraries and Knowledge Development Network/ NHS

O comportamento do consumidor de software de gestão e contabilidade das PMEs em Portugal, França e Espanha

Dissertação de Mestrado apresentada à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Empresariais.

Object Matching in Distributed Video Surveillance Systems by LDA-Based Appearance Descriptors

Establishing correspondences among object instances is still challenging in multi-camera surveillance systems, especially when the cameras’ fields of view are non-overlapping. Spatiotemporal constraints can help in solving the correspondence problem but still leave a wide margin of uncertainty. One way to reduce this uncertainty is to use appearance information about the moving objects in the site. In this paper we present the preliminary results of a new method that can capture salient appearance characteristics at each camera node in the network. A Latent Dirichlet Allocation (LDA) model is created and maintained at each node in the camera network. Each object is encoded in terms of the LDA bag-of-words model for appearance. The encoded appearance is then used to establish probable matching across cameras. Preliminary experiments are conducted on a dataset of 20 individuals and comparison against Madden’s I-MCHR is reported.

Job retention and turnover: a study of child protection and welfare social workers in Ireland

Retaining social workers in child protection and welfare organisations has been identified as a problem in Ireland (McGrath, 2001; Ombudsman for Children, 2006; Houses of the Oireachtas, 2008) and internationally (Ellet et al., 2006; Mor Barak et al., 2006; Tham, 2006). While low levels of retention have been identified, there is no research that examines the factors in Ireland that influence the retention of social workers. In this thesis, data is analysed from qualitative interviews with 45 social workers in the Health Service Executive South about what influences their decisions to stay in or leave child protection and welfare social work. These social workers’ views are examined in relation to quantitative research on the levels of turnover and employment mobility of child protection and welfare social workers employed in the same organisation. Contrary to expectations, the study found that the retention rate of social workers during the period of data collection (March 2005 to December 2006) was high and that the majority of social workers remained positive about this work and their retention. The quality of social workers’ supervision, social supports from colleagues, high levels of autonomy, a commitment to child protection and welfare work, good variety in the work, and a perception that they were making a difference, emerged as important factors in social workers’ decisions to stay. Perceptions of being unsupported by the organisation, which was usually described in terms of high caseloads and demanding workloads, a lack of resources, work with involuntary clients and not being able to make a difference, were the most significant factors in social workers’ decisions to leave and/or to want to leave. Social workers felt particularly professionally unsupported when they received low quality and/or infrequent professional supervision. This thesis critiques the theories of perceived organisational support theory, social exchange theory and job characteristics theory, and uses the concept of ‘professional career’, to help analyse the retention of social workers in child protection and welfare.

Experiences of the foster care process in the Republic of Ireland: retrospective accounts from biological children of foster carers

This thesis examines the experiences of the biological children of foster carers. In particular it explores their experiences in relation to inclusion, consultation and decision-making. The study also examines the support and training needs of birth children in foster families. Using a qualitative methodology in-depth, semi-structured interviews were conducted with fifteen birth children of foster carers aged between 18 and 30 years. The research findings show that for the majority of birth children, fostering was overall a positive experience which helped them develop into individuals who were caring and nonjudgemental. However, from the data collected in this study, it is clear that fostering also brings a range of challenges for birth children in foster families, such as managing feelings of loss, grief, jealousy and guilt when foster children leave. Birth children are reluctant to discuss these issues with their parents and often did not approach fostering social workers as they did not have a meaningful relationship in order to discuss their concerns. The findings also demonstrate that birth children undertake a lot of emotional work in supporting their parents, birth siblings and foster siblings. Despite the important role played by birth children in the fostering process, this contribution often goes unrecognised and unacknowledged by fostering professionals and agencies with birth children not included or consulted about foster care decisions that affect them. It is argued here that birth children are viewed by foster care professionals and agencies from a deficit based perspective. However, this study contends that it is not just foster parents who are involved in the foster care process, but the entire foster family. The findings of this study show that birth children are competent social actors capable of making valuable contributions to foster care decisions that affect their lives and that of their family.

Non-product related environmental process and production methods (NPR-PPMs): a view from international trade law and international environmental governance

The primary aim of this thesis is to analyse legal and governance issues in the use of Environmental NPR-PPMs, particularly those aiming to promote sustainable practices or to protect natural resources. NPR-PPMs have traditionally been thought of as being incompatible with the rules of the World Trade Organization (WTO). However, the issue remains untouched by WTO adjudicatory bodies. One can suggest that WTO adjudicatory bodies may want to leave this issue to the Members, but the analysis of the case law also seems to indicate that the question of legality of NPR-PPMs has not been brought ‘as such’ in dispute settlement. This thesis advances the argument that despite the fact that the legal status of NPR-PPMs remains unsettled, during the last decades adjudicatory bodies have been scrutinising environmental measures based on NPR-PPMs just as another expression of the regulatory autonomy of the Members. Though NPR-PPMs are regulatory choices associated with a wide range of environmental concerns, trade disputes giving rise to questions related to the legality of process-based measures have been mainly associated with the protection of marine wildlife (i.e., fishing techniques threatening or affecting animal species). This thesis argues that environmental objectives articulated as NPR-PPMs can indeed qualify as legitimate objectives both under the GATT and the TBT Agreement. However, an important challenge for the their compatibility with WTO law relate to aspects associated with arbitrary or unjustifiable discrimination. In the assessment of discrimination procedural issues play an important role. This thesis also elucidates other important dimensions to the issue from the perspective of global governance. One of the arguments advanced in this thesis is that a comprehensive analysis of environmental NPR-PPMs should consider not only their role in what is regarded as trade barriers (governmental and market-driven), but also their significance in global objectives such as the transition towards a green economy and sustainable patterns of consumption and production.

Typical delay determines waiting time on periodic-food schedules: Static and dynamic tests.

Pigeons and other animals soon learn to wait (pause) after food delivery on periodic-food schedules before resuming the food-rewarded response. Under most conditions the steady-state duration of the average waiting time, t, is a linear function of the typical interfood interval. We describe three experiments designed to explore the limits of this process. In all experiments, t was associated with one key color and the subsequent food delay, T, with another. In the first experiment, we compared the relation between t (waiting time) and T (food delay) under two conditions: when T was held constant, and when T was an inverse function of t. The pigeons could maximize the rate of food delivery under the first condition by setting t to a consistently short value; optimal behavior under the second condition required a linear relation with unit slope between t and T. Despite this difference in optimal policy, the pigeons in both cases showed the same linear relation, with slope less than one, between t and T. This result was confirmed in a second parametric experiment that added a third condition, in which T + t was held constant. Linear waiting appears to be an obligatory rule for pigeons. In a third experiment we arranged for a multiplicative relation between t and T (positive feedback), and produced either very short or very long waiting times as predicted by a quasi-dynamic model in which waiting time is strongly determined by the just-preceding food delay.