遮荫与外源脱落酸喷施对青杨(Populus cathayana Rehd.)不同种群抗旱性的影响

杨树具有分布广、适应性强的特征，在生态环境治理和解决木材短缺方面均占有重要位置。青杨(Populus cathayana Rehd.)是青杨派树种的重要成员之一，也是生长较迅速、易繁殖的重要杨树资源。本研究选取了来自不同气候地区的青杨两种群为材料，采用植物生态学、生理学和生物化学的研究方法，系统地研究了青杨对干旱与遮荫、干旱与外源脱落酸(ABA)喷施的生长、形态、生理和生化响应及种群间差异，研究成果可为我国干旱半干旱地区的造林以及生态恢复提供理论依据和科学指导。主要研究结论如下：1.青杨在干旱胁迫下的适应机制为：生长性状及生物量的分配变化：干旱胁迫下虽然植株生长受抑，株高、基茎及各部分生物量都显著减小，但有相对较多的生物量向根部分配，根/冠比以及细/粗根比增加。青杨对干旱胁迫的光合作用表现为：干旱胁迫降低了青杨的净光合速率、蒸腾速率、气孔导度以及光合氮利用效率，提高了瞬时用水效率。干旱还引起了活性氧的产生，使得膜脂过氧化产物丙二醛(MDA)增加，同时也增强了植物抗氧化酶系统(如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性的增加)及非酶系统的能力(如抗坏血酸(AsA)含量的增加)。干旱降低了植物叶片的相对含水量，而促进了渗透调节物质(游离脯氨酸及可溶性糖)的积累，增加了植物的渗调能力。干旱下青杨两种群的内源ABA含量显著增加，碳同位素组分(δ13C)也显著提高。这些结果证明植物遭受干旱胁迫后发生一系列的形态、生理和生化响应，这些变化能提高植物在干旱下的存活和生长能力。2.青杨两种群对干旱胁迫反应的种群差异：与来自湿润地区的汉源种群相比，来自干旱地区的乐都种群在干旱条件下生物量向根系分配的可塑性更强，同时具有更强的抗氧化系统能力，所受到活性氧的伤害也更少，并且累积更多的脯胺酸和ABA，具有更高的δ13C。这些都说明了乐都种群对干旱的适应性比汉源种群更强。两种群对干旱的响应差异应归于它们的用水策略的不同：汉源种群来自湿润地区，采用了耗水型的用水策略，抗旱能力较弱；而乐都种群，来自干旱地区，通常采用节水型的用水策略，有更强的抗旱能力。3.遮荫对青杨两种群抗旱性的影响：遮荫对青杨抗旱性的影响决定于遮荫程度的不同，我们的结果表明中度的遮荫可以有效的提高干旱下植物的生长，对干旱胁迫有明显的缓解作用，具体体现在中度遮荫下受旱植物的叶片相对含水量得到提高，使得植物体内水分状况得到了改善；光合速率并未降低，植物光合氮利用效率增加，说明中度的遮荫并未明显限制植物的碳获得；抗氧化酶活性与膜脂过氧化产物MDA含量的同时降低，说明中度遮荫下所受到的活性氧伤害减少；中度遮荫下的ABA及δ13C的变化也不如在全光下变化明显，这也说明中度遮荫缓解了干旱胁迫。但是重度的遮荫却对干旱胁迫有明显的加剧作用，主要表现在重度遮荫降低了植物的光合速率，严重抑制了植物的生长；同时重度遮荫下脯胺酸含量和抗氧化酶活性的急剧下降，导致了植物渗调能力的下降及膜脂过氧化产物MDA的显著升高；重度遮荫还显著降低了内源ABA的累积和δ13C，降低了植物的抗旱能力。此外，青杨两种群在对干旱和遮荫的响应中，也表现出种群差异。汉源种群，来自湿润且年日照辐射较少的地区，表现出相对更强的耐荫性和需水性。而乐都种群，来自干旱且年日照辐射丰富的地区，表现出相对更强的耐旱性和需光性。这说明了植物对环境胁迫的耐受性是其长期适应原生境的结果，并且来自不同气候地区的两种群在面临环境胁迫时会采取不同的生存策略。4. 外源ABA喷施对青杨两种群抗旱性的影响：外源ABA的喷施可以提高两种群的抗旱性，具体表现为外源ABA喷施促进了青杨根系的生长，显著提高了干旱下植物的根/冠比和细/粗根比，减少了比叶面积；在生理生化方面，外源ABA降低了干旱下植物叶片的气孔导度，降低了蒸腾速率和净光合速率，但提高了瞬时用水效率，提高了叶片的相对含水量，增加了干旱下植物的保水能力。外源ABA进一步增加了干旱下植物内源ABA的积累，促进了植物渗调物质如脯胺酸和可溶性糖的积累，增加了抗氧化酶系统(如SOD、APX、CAT)的活性和非酶系统AsA的含量，降低了活性氧(如超氧阴离子(O2和过氧化氢(H2O2))对植株的伤害。此外，外源ABA还进一步提高了干旱下植物的δ13C，提高了植物的长期用水效率，由此提高了植物的抗旱能力。另一方面，两种群对外源ABA和干旱的响应也有所差别。来自湿润地区的汉源种群，对干旱较为敏感，所受干旱的影响也较大，而外源ABA的喷施对汉源种群抗旱性的提高作用也更为突出。乐都种群，由于其长期适应干旱地区的生长，本身已具有较强的抗旱能力，因此外源ABA喷施对其抗旱性的提高不如对汉源种群的效果明显。由此我们可以得出对于一些抗性弱或干旱敏感的物种或者种群，可以采用外施ABA的方法来提高其抗性。Poplars play an important role in lumber supply, and are important component ofecosystems due to their wide distribution and well adaptation. Populus cathayana Rehd.,which belongs to Populus Sect. Tacamahaca Spach, is one of the most important resources ofpoplars for its fast growth and reproductive. In this study, different populations of P.cathayana were used as experiment material to investigate the adaptability to drought stressand population differences in adaptability, and the effects of shade and exogenous abscisicacid (ABA) application on the drought tolerance. Our results could provide a strongtheoretical evidence and scientific direction for the afforestation, and rehabilitation ofecosystem in the arid and semi-arid area, and provide a strong evidence for adaptivedifferentiation of different populations, and so may be used as criteria for species selectionand tree improvement. The results are as follows:1. A large set of parallel response to drought stress: Drought stress caused pronouncedinhibition of the growth and increased relatively dry matter allocation into the root. For thetwo populations, the shoot height, basal diameter and total biomass were decreased but theroot/shoot ratio and fine root/coarse root ratio were increased under drought conditions；Drought stress caused pronounced inhibition of photosynthesis, decreased the stomatalconductance, transpiration rate, and photosynthetic nitrogen-use efficiency (PNUE) butincreased the instantaneous water use efficiency. Drought significantly improved the levels ofreactive oxygen species and malondialdehyde (MDA) and to induce the entire set ofantioxidative systems including the increase of activities of superoxide dismutase (SOD),ascorbate peroxidase (APX), catalase (CAT) and ascorbate (AsA) content. Drought decreased the leaf relative water content (RWC) but improved the capability of osmotic adjustmentindicated by the higher proline accumulation. Drought also increased the ABA content andcarbon isotope composition (δ13C), which indicating the long period water use efficiency wasimproved under drought. These results demonstrate that there are a large set of parallelchanges in the morphological, physiological and biochemical responses when plants areexposed to drought stress; these changes may enhance the capability of plants to survive andgrow during drought periods.2. Difference in adaptation to drought stress between contrasting populations of P.cathayana: Compared with the Hanyuan population (wet climate), the Ledu population (dryclimate) showed higher root/shoot ratio and water use efficiency, exhibited higherantioxidative systems capability thus resulting in less oxidative damage, accumulated moreABA and free proline content under drought conditions. The results suggested that there weredifferent water-use strategies between the two populations. The Ledu population, whichcomes from dry climate region, with higher drought tolerance, may employ a conservativewater-use strategy, whereas the Hanyuan population, which comes from wet climate, withlower drought tolerance, may employ a prodigal water-use strategy. These variations indrought responses may be used as criteria for species selection and tree improvement.3. The effects of shade on the drought tolerance: The reduction in the availability of lightand water affected the morphological and physiological responses of the two P. cathayanapopulations. In addition, the light environment modified the growth responses of P.cathayana seedlings to varying water environments in different ways depending upon theintensity of the light levels considered. There is an apparent alleviation to drought effects bymoderate shade in P. cathayana seedlings, as indicated by the higher leaf RWC, and unchanged net photosynthesis and PNUE, as well as by the lower antioxditative enzymeactivity, MDA, ABA and δ13C levels, which implied moderate shade did not significantlylimited the carbon acquisition or inhibited the plant growth, but ameliorated the detrimentaleffects of drought. On the other hand, an apparent aggravation to drought effects by severeshade was also observed, as indicated by the pronounced decrease of plant growth and net photosynthesis, the lower total biomass, ABA level, δ13C, free proline content andantioxditative enzyme activity and higher MDA accumulation. By contrast, the twopopulations showed different responses to shade and drought. The Hanyuan population,which comes from a riparian basin having a relatively wet climate and less annual solarradiation, is more sensitive to drought but more tolerant to shade. The Ledu population, whichcomes from a mountainous plateau with less rainfall and with more annual solar radiation, ismore tolerant to drought but more sensitive to shade. The results demonstrated that theendurance of plants to stress is a result of long-term evolution and adaptation to theenvironment, as suggested by the different strategies employed by the P. cathayanapopulations originating from contrasting habitats when they were exposed to drought andshade.4. The effects of exogenous ABA application on the drought tolerance: For bothpopulations under drought conditions tested, exogenous ABA application significantlyimproved the root/shoot ratio, fine root/coarse root ratio, and decreased the specifical leaf area.On the physiological and biochemical traits, exogenous ABA application significantlydecreased stomatal conductance, transpiration rate and net photosythesis but increased theinstance water use efficiency and leaf RWC. On the other hand, exogenous ABA applicationsignificantly increased endogenous ABA, proline, solube sugar and AsA content, as well asSOD, APX and CAT activities, thus reduced the damage of reactive oxygen species. Moreover,the long period water use efficiency as indicated by δ13C was also improved by exogenousABA application. In additionally, there was different responsive between the two populationsto drought and exogenous ABA application. The Hanyuan population, which comes from wetclimate region, is more sensitive to drought, and the effect of exogenous ABA is moreobviously than that in the Ledu population, which comes from dry climate region and is moredrought-responsive. Therefore, we can use exogenous ABA application to improve theresistance of plants, especially for the drought- sensitive species or populations.

不同抗旱性青稞LEA2/LEA3蛋白基因的克隆与表达

作物的抗旱性是一个多基因控制的、极为复杂的数量性状，植物对干旱在分子水平上的差异反应通过植物组织生理和细胞生物学水平，最终表现为植物抗旱性的不同。在我国，旱地农业超过耕地面积的50%，但水资源短缺，因此培育和选育抗旱高产作物是发展节水型农业最有效的途径。 青藏高原气候恶劣、年均降雨量少，也是世界大麦初生起源中心，因而蕴藏了十分丰富的与抗逆相关的种质资源材料，从这些特殊的资源材料克隆抗旱基因，不仅对培育抗旱、优质、高产大麦新品种具有重要理论意义和经济价值，而且对整个作物抗旱基础和育种应用研究都具重大促进作用。 为了筛选青稞（裸大麦，Hordeum vulgare ssp. vulgare）抗旱性材料，本研究选用来自青藏高原不同地区的84份青稞为材料，在叶片失水率(water loss rate, WLR)检测分析的基础上，选择失水率值差异显著的12个品种，通过相对含水量（relative water content, RWC）和反复干旱法评价其抗旱性，并通过植株对干旱胁迫下的丙二醛（MDA）含量和游离脯氨酸（free-proline）含量变化，了解不同抗旱性材料的生理反应特性。选择抗旱性强弱不同的品种各两份进行LEA2蛋白基因(Dhn6基因)、LEA3蛋白基因(HVA1基因)的克隆，比较LEA蛋白结构差异与作物抗旱性之间的关系。同时，对抗旱性不同的青稞品种受到干旱时间不同的失水变化率（dynamics water loss rate, DWLR）进行了检测；对抗旱性不同的青稞对照材料进行2 h、4 h、8 h和12 h的快速干旱处理，通过SYBR Green实时荧光定量RT-PCR技术对Dhn6基因、Dhn11基因、Dhn13基因和HVA1基因在不同抗旱性材料受到不同干旱时间处理后的相对表达水平进行了检测。本研究对LEA蛋白基因在抗旱性不同的青稞材料中的干旱胁迫分子水平上的差异反应进行了研究，也对植物的抗旱机理进行了初步探讨。主要研究结果如下： 1. 青稞苗期进行离体叶片失水率测定结果表明，来自青藏高原的84份青稞材料的WLR在0.086~0.205gh-1g-1DW之间。选择WLR低于0.1gh-1g-1DW和WLR高于0.18gh-1g-1DW的品种各6份，并对苗期分别进行未干旱及干旱12小时的处理。相对含水量检测结果表明，低失水率青稞材料干旱后的具有更高的相对含水量，盆栽缺水试验也显示叶片失水率低的材料耐旱能力强于失水率高的材料。通过水合茚三酮法测定离体叶片游离脯氨酸的含量，结果表明，所有品种未干旱处理时，游离脯氨酸含量差异不大（17.10~25.74 µgg-1FW）；干旱12小时后，低失水率的品种游离脯氨酸含量明显增高（32.99~53.45µgg-1FW），高失水率品种的游离脯氨酸含量与干旱前变化不明显(P<0.05)。硫代巴比妥酸法测定离体叶片丙二醛(MDA)含量，结果显示，12份所选对照品种中，丙二醛的含量在0.97~2.74nmolg-1FW，干旱12小时后丙二醛的含量显著上升（1.46~4.74nmolg-1FW），高失水率的6个品种的丙二醛含量在未干旱和干旱处理时都明显高于低WLR品种。本研究结果表明青稞的低失水率、低丙二醛含量、高相对含水量和高脯氨酸含量具相关性（P<0.05）。综上研究，我们认为作物失水率的测定可以作为快速检测作物抗旱性的指标之一，因此，强抗旱品种喜玛拉10号(TR1)、品比14号(TR2)和弱抗旱品种冬青8号(TS1)、QB24 (TS2)被选作抗旱基因克隆和表达分析的研究材料。 2. 高等植物胚胎发育晚期丰富蛋白(late embryogenesis abundant proteins, LEA proteins)与植物耐脱水性密切相关，为了探讨青稞LEA蛋白结构差异性与植物抗旱性的关系，本研究以强抗旱品种（喜玛拉10号、品比14号）和弱抗旱品种（冬青8号、QB24）为材料，利用同源克隆法，通过RT-PCR，分别克隆了与抗旱性密切相关的Dhn6基因和HVA1基因。Dhn6基因序列分析结果表明，强抗旱品种品比14号和弱抗旱品种冬青8号Dhn6基因所克隆到的序列为1026bp，它们之间只有5个碱基的差异；喜玛拉10号和QB24克隆到的序列长963bp。在强弱不同的抗旱品种中有22个核苷酸易突变位点，相应的脱水素氨基酸序列推导结果表明，22个核苷酸突变位点中，仅有8个位点导致相应的氨基酸残基的改变，其余的位点系同义突变，另外，21个富含甘氨酸序列的缺失并没有联系作物抗旱性特征。推测这些同义突变位点的氨基酸残基对维持青稞DHN6蛋白的正常结构和功能起着非常重要的作用，也可能DHN6蛋白对青稞长期适应逆境胁迫和遗传进化的结果。对HVA1基因的序列分析结果表明，冬青8号、QB24、品比14号和喜玛拉10号的目的基因核苷酸序列全长分别为661bp、697bp、694bp和691bp，它们都包含1个完整的开放阅读框。相应的LEA3蛋白氨基酸序列结果表明，11个高度保守的氨基酸残基组成基元重复序列的拷贝数与青稞抗旱性之间没有必然关系，在强抗旱品种（喜玛拉10号、品比14号）中三个共同的氨基酸突变位点Gln32、Arg33和Ala195可能对抗旱蛋白的结构和功能有影响；另外，强抗旱青稞品种LEA3蛋白质中11-氨基酸保守基元序列拷贝数和极性氨基酸占蛋白的比例更高，推测LEA3蛋白中基元序列拷贝数和极性氨基酸占蛋白的比例对该蛋白的结构和功能影响更大。 3. LEA蛋白基因的表达水平的上调与植物的耐脱水性密切相关，我们对强抗旱性材料（喜玛拉10号、品比14号）和弱抗旱材料（冬青8号、QB24）进行干旱处理2 h、4 h、6 h、8 h和10 h的失水变化率进行测定，结果表明弱抗旱品种在2~4小时之间失水率变化最明显，而四个对照品种的失水率在8小时后和24小时的失水率值变化不大。进一步提取青稞苗期进行2 h、4 h、8 h和12 h的干旱处理后的总RNA，通过SYBR Green实时荧光定量RT-PCR技术对青稞脱水素基因(Dhn6、Dhn11和Dhn13)和LEA3蛋白基因(HVA1)的相对表达水平受干旱时间和作物抗旱性的影响进行了检测。研究发现，抗旱性不同的青稞品种随干旱处理的时间延长，Dhn6、Dhn11、Dhn13和HVA1基因的相对表达水平不同。 Dhn6基因的相对表达水平在强抗旱青稞品种干旱8小时后快速上升，但在弱抗旱青稞品种干旱处理12小时后检测到更高表达量；Dhn11基因在对照青稞抗旱品种的表达累积水平随干旱时间的延长持续下降；整个干旱过程中，Dhn13基因的相对表达水平在弱抗旱品种持续上升，在强抗旱品种中干旱处理8小时快速上升并达到最高，干旱12小时后降低。与脱水素基因相比较，强抗旱青稞品种在干旱2小时后HVA1基因的相对表达水平显著升高，相对表达量随干旱处理的时间持续上升，在干旱12小时后达到最高；与之相比较，在整个干旱过程中，弱抗旱品种的相对表达水平显著低于强抗旱品种，在干旱8小时之前弱抗旱品种的相对表达水平变化不明显；在干旱8~12小时后却显著上升。上述结果表明，不同的LEA蛋白在植物耐脱水过程中的干旱表达累积水平不同；干旱不是诱导高等植物Dhn11基因表达的主要因素；植物的抗旱性不同，不同LEA蛋白基因对干旱的反应有差异。推测某些LEA蛋白基因的干旱胁迫早期表达累积程度与植物的抗旱性直接相关；其中，Dhn11基因和Dhn12基因不同的表达模式可能与干旱调控表达顺式作用成分(dehydration responsive element, DRE)的有无或结构上的差异有关。 本研究结果认为，(1)失水率和相对含水量可作为植物抗旱性检测的指标之一；(2) DHN6同义突变位点的氨基酸残基对维持该蛋白的正常结构和功能起着重要作用；(3) 11-氨基酸保守基元序列拷贝数和极性氨基酸的比例对LEA3蛋白结构和功能有重要影响；(4)LEA蛋白表达随着干旱胁迫程度而增加，但Dhn11基因并不受干旱诱导表达；(5)作物的抗旱性不同，LEA蛋白对干旱的累积反应并不相同，干旱早期LEA蛋白的累积程度可能会影响植物的抗旱性。 Drought resistance was a complex trait which involved multiple physiological and biochemical mechanisms and regulation of numerous genes. Because its complex traits, it is difficult to understand the mechanisms of drought resistance in plants. Plants respond to water stress through multiple physiological mechanisms at the cellular, tissue, and whole-plant levels. Tibetan hulless barley, a pure line, is a selfing annual plant that has predominantly penetrated into the Qinghai-Tibetan Plateau and remains stable populations there. The wide ecological range of Tibetan hulless barley differs in water availability, temperature, soil type and vegetation, which makes it possess a high potential of adaptive diversity to abiotic stresses. This adaptive genetic diversity indicates that the potential of Tibetan hulless barley serves as a good source for drought resistance alleles for breeding purposes. 12 contrasting drought-tolerant genotypes were selected to measure relative water content (RWC), maldondialdehyde (MDA) and proline content, based on values of water loss rate (WLR) and repeated drought methods from Tibetan populations of cultivated hulless barley. As a result of the screening, sensitive and tolerant genotypes were identified to clarify relationships between characteristics of LEA2/LEA3 genes sequences and expression and drought-tolerant genotypes, associated with resistance to water deficit. In addition, dynamics water loss rate (DWLR) was measured to observe the changes on diffrential drought-tolerant genotypes. Real-time quantitative RT-PCR was applied to detect relative expression levels of Dhn6, Dhn11, Dhn13 and HVA1 genes in sensitive and tolerant genotypes with 2 h, 4 h, 8h and 12 h of dehydration. In the present study, differential sequences and expression of LEA2/LEA3 genes were explored in Tibetan hulless barley, associated with phenotypically diverse drought-tolerant genotypes. 1. The assessments of WLR and RWC were considered as an alternative measure of plant water statues reflecting the metabolic activity in plants, and the parameters of MDA and proline contents were usually consistent with the resistance to water stress. The values of detached leaf WLR of the tested genotypes were highly variable among 84 genotypes, ranging from 0.086 to 0.205 g/h.g DW. The 12 most contrasting genotypes (6 genotypes with the lowest values of WLR and 6 genotypes with the highest values of WLR) were further validated by measuring RWC, MDA and free-proline contents, which were well watered and dehydrated for 12 h. Results of RWC indicated that the values of 12 contrasting genotypes RWC ranged from 89.94% to 93.38% under condition of well water, without significant differences, but 6 genotypes with lower WLR had higher RWC suffered from 12 h dehydration. The results indicated that lower MDA contents, lower scores of WLR and higher proline contents were associated with drought-tolerant genotypes in hulless barley. Remarkably, proline amounts were increased more notable in 6 tolerant genotypes than 6 sensitive genotypes after excised leaves were dehydrated for 12 h, with control to slight changes under condition of well water. Results of MDA contents showed that six 6 tolerant genotypes had lower MDA contents than the 6 sensitive genotypes under both stressed and non-stressed conditions. As a result of that screening, drought- resistant genotypes (Ximala 10 and Pinbi 14) and drought-sensitive genotypes (Dongqing 8 and QB 24) were chosen for comparing the differential characteristics of LEA2/LEA3 genes and their expression analysis. It was conclusion that measurements of WLR could be considered an alternative index as screening of drought-tolerant genotypes in crops. 2. Late embryogenesis abundant (LEA) proteins were thought to protect against water stress in plants. To explore the relationships between configuration of LEA proteins and phenotypically diverse drought-tolerant genotypes, sequences of LEA genes and their deduced proteins were compared in Tibetan hulless barley. Results of comparing Dhn6 gene in Ximala 10 and QB24 indicated that absence of 63bp was found, except that only 5 mutant nucleotides were found. While 22 mutant sites were taken place in Dhn6 gene between sensitive and tolerant lines, 14 synonymous mutation sites appeared in the contrasting genotypes. The additional/absent polypeptide of 21 polar amino acid residues was not consistent with phenotypically drought-tolerant genotypes in hulless barley. It was deduced that synonymous mutation sites would play important roles in holding out right configurations and functions on DHN6 protein. The sequencing analysis results indicated that each cloned HVA1 gene from four selected genotypes contained an entire open reading frame. The whole sequence of HVA1 gene from Dongqing 8, QB24, Pinbi 14 and Ximala 10 was respectively 661bp, 697bp, 694bp and 691bp. Results of DNA sequence analyses showed that the differences in nucleotides of HVA1 gene in sensitive genotypes were not consistent with that of tolerant genotypes, except for absence of 33 nucleotides from +154 to +186 (numbering from ATG) in QB24. Database searches using deduced amino acid sequences showed a high homology in LEA3 proteins in the selected genotypes. Multiple sequence alignments revealed that LEA3 protein from Dongqing 8 was composed of 8 repeats of an 11 amino acid motif, less the fourth motif than Pinbi 14, Ximala 10 and QB24. Consistent mutant amino acid residues appeared in contrasting genotypes by aligning and comparing the coding sequence region, including Gln32, Arg33 and Ala195 in tolerant genotypes as compared to Asp32, Glu33 and Thr195 (Thr184 in Dongqing 8) in sensitive lines. It was concluded that consistent appearance of Gln32, Arg33 and Ala195 would contributed to functions of LEA3 protein in crops, as well as higher proportion of 11-amino-repeating motifs and polar amino acid residues. 3. Most of the LEA genes are up-regulated by dehydration, salinity, or low temperature, are also induced by application of exogenous ABA, which increases in concentration in plants under various stress conditions and acts as a mobile stress signal. Higher levels of proteins of LEA group 3 accumulated was correlated well with high level of desiccation tolerance in severely dehydrated plant seedlings. Dehydrins (DHNs), members of LEA2 protein, are an immunologically distinct protein family, and Dhn genes expression is associated with plant response to dehydration. Dynamic water loss rate was measured between sensitive genotypes and tolerant genotypes after they were dehydrated for 2 h, 4 h, 6h and 8 h. Detailed measurements of WLR at the early stage of dehydration (2, 4, 6, and 8 h) showed that WLR was stabilizing after 8 h, and there were no significant changes between these values and WLR after 24 h. Drought stress was applied to 10-day-old seedlings by draining the solution from the container for defined dehydration periods. Leaf tissues of the selected genotypes were harvested from control plants (time 0); and after 2, 4, 8, and 12 h of dehydration. Differential expression trends of Dhn6, Dhn11, Dhn13 and HVA1 genes were detected in phenotypically diverse drought-tolerant hulless barleys, related to different time of dehydration. Results of quantitative real-time PCR indicated that relative level of HVA1 expression was always higher in tolerant genotypes, rapidly increasing at the earlier stages (after 2-4 h of dehydration). However, HVA1 expressions of sensitive genotypes had a fast increase from 8 h to 12 h of stress. Significant differences in expression trends of dehydrin genes between tolerant genotypes and sensitive lines were detected, mainly in Dhn6 and Dhn13 gene, depending on the duration of the dehydration stress. The relative expression levels of Dhn6 gene were significantly higher in tolerant genotypes after 8 h dehydration, by control with notable higher expression levels after 12 h water stress in sensitive ones. The relative expression levels of Dhn13 gene tended to ascend during exposure to dehydration in drought-sensitive genotypes. However, fluctuate trends of Dhn13 expression level were detected in drought-resistant lines, including in lower expression levels of 12 h dehydration as compared to 8 h water stress. It was conclusion that (1) diverse LEA proteins would play variable roles in resisting water stress in plants; (2) expression of Dhn11 gene was not induced by dehydrated signals because of the trends of expression descended in contrasting genotypes suffered from water deficit and (3) variable accumulations on LEA proteins would be appear in diverse drought-tolerant genotypes during dehydrations. It is deduced that higher accumulations of Dhn6 and Dhn13 expression in 8 h dehydration are related to diverse drought-tolerant lines in crops. The present results indicated that different dehydrin genes would play variable functional roles in resisting water stress when plants were suffered from water deficit. The authors suggest physiologically different reactions between resistant and sensitive genotypes may be the results of differential expression of drought-resistant genes and related signal genes in plants. In addition, contrarily induced expression of Dhn11 and Dhn12 was related to dehydration responsive element (DRE) in barleys. The present study indicated that (1) measurements of WLR and RWC could be considered as one index of drought-tolerant screenings; (2) synonymous mutation sites would play important roles in holding out right configurations and functions on DHN6 protein, (3) higher proportion of 11-amino-repeating motifs and polar amino acid residues would contribute to functions on LEA3 protein, (4) the longer drought, the more accumulation on LEA proteins, except for Dhn11 gene in crops and (5) differential responses on expression of LEA protein genes would result in physiological traits of drought tolerance in plants.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

Synthesis and properties of A-type zeolite membranes by secondary growth method with vacuum seeding

A-type zeolite membranes were successfully synthesized on tubular alpha-Al2O3 supports by secondary growth method with vacuum seeding In the seeding process, a thin, uniform and continuous seeding layer was closely attached to the support surface by the pressure difference between the two sides of the support wall. The effects of seed particle size, suspension concentration, coating pressure difference and coating time on the membrane and its pervaporation properties were investigated. The as-synthesized membranes were characterized by XRD and SEM. The quality of the membranes was evaluated by the pervaporation dehydration of 95 wt. % isopropanol/water mixture at 343 K. High quality A-type zeolite membranes can be reproducibly prepared by the secondary growth method with vacuum seeding under the conditions: seed particle size of 500-1200 nm, suspension concentration of 4-8 g/l, coating pressure difference of 0.0100-0.0250 MPa and coating time of 45-180 s. (C) 2004 Elsevier B.V. All rights reserved.

氮磷营养对小麦水分关系的影响

比较研究了氮磷营养对春小麦水分关系影响的差异。结果表明 ,土壤干旱情况下 ,氮磷营养虽然皆增强了春小麦的渗透调节能力 ,但由于氮磷营养对作物地上地下部生长的不同进促作用而对作物的水分状况产生了完全相反的影响。氮营养增强了作物对干旱的敏感性 ,使其水势和相对含水量大幅度下降 ,蒸腾失水减少 ,自由水含量增加而束缚水含量减少 ,并使膜稳定性降低 ;而磷营养则明显改善了植株的水分状况 ,增大了气孔导度 ,降低了其对干旱的敏感性 ,增加了束缚水含量 ,并使膜稳定性增强

土壤干湿交替对玉米生长速度及其耗水量的影响

在盆栽条件下研究了干湿交替对玉米生长速率、叶片水势、渗透势、气孔导度、相对生长速率和耗水量的影响。结果表明玉米在 3～ 7叶期经历土壤水分缓慢亏缺 ,再进行复水的干湿交替后玉米叶片渗透调节能力明显增加 ,叶片生长表现出补偿效应 ,每次干湿交替后生长速率迅速下降的叶水势趋于下降 ,气孔导度对土壤水分变化非常敏感 ,并在干旱—复水过程中具有后效作用 ,蒸腾耗水量随干—湿交替而具有下降趋势 ,初步证明可在节水灌溉条件下人为控制不同生育时期的供水时间形成干湿交替 ,促进渗透调节能力增强和补偿生长来实现作物高产、高效、优质的目的

小麦抗旱性生理生化研究进展

根据近年有关文献资料 ,从叶水势、渗透调节、光合作用、干旱诱导蛋白、激素调节、膜抗氧化酶等方面 ,对小麦抗旱性研究在生理生化方面所取得的进展作一综述。目前认为 ,作物抗旱性研究的前沿是从分子水平阐明作物由干旱胁迫引起生理生化变化的本质原因 ,并通过基因工程的手段进行抗旱基因的重组 ,从而创造新的抗旱品种 ,将是一个前景诱人的目标。

抗氧化剂对渗透胁迫下沙棘叶片膜脂过氧化的保护作用

以沙棘叶片为材料 ,研究了外源抗氧化物质 Vc、VE、β-胡萝卜素等对 PEG渗透胁迫下细胞膜的保护作用。结果表明 :加入外源 Vc、β-胡萝卜素能有效保护膜系统 ,使膜透性和丙二醛在胁迫条件下增加小于对照 ,尤其在胁迫前期作用明显 ,而对叶绿素的保护作用在后期表现更突出 ,使叶绿素含量维持在较高水平 ;外源 VE 对防止膜透性增加作用不大 ,对减低丙二醛含量与保护叶绿素方面有所贡献。同时也证明沙棘叶片内 Vc在渗透胁迫下含量下降 ,而 Pro含量显著升高。证明这些物质是构成沙棘抗旱性的重要基础。

Synthesis of isomeric bis(amine anhydride)s for novel poly(amine imide)s by Pd-catalyzed amination of 4-chlorophthalic anhydride

Two novel bis(amine anhydride) monomers, N,N'-bis(3,4-dicarboxyphenyl)-1,4-phenylenediamine dianhydride I and N,/N'-bis(3,4-dicarboxyphenyl)-1,3-phenylenediamine dianhydride 11, were prepared via palladium-catalyzed amination reaction of 4-chloro-N-methylphthaliniide with 1,4-phenylenediamine or 1,3-phenylenediamine, followed by alkaline hydrolysis of the intermediate bis(amine imide)s and subsequent dehydration of the resulting tetraacids. A series of new poly(amine imide)s were prepared from the synthesized dianhydride monomers with various diamines in NMP via conventional two-step method.

Nanoporous In2O3 nanocrystal clusters: One-step synthesis, thermal stability and optical property

Nanoporous In2O3 nanocrystal clusters with high surface areas have been synthesized by a one-step solvent-thermal method at a relatively low temperature. On the basis of our experimental data and nanomaterial growth mechanism, a template-assistant dehydration accompanied by aggregation mechanism was proposed to explain their formation. Besides, the influence of the high-temperature treatment on their porous structure and optical properties were studied and compared by various technologies.

Single-walled carbon nanotubes binding to human telomeric i-motif DNA under molecular-crowding conditions: More water molecules released

The natural occurrence of the human telomeric G-quadruplex or i-motif in vivo has not been demonstrated and the biological effects of the induction of these structures need to be clarified. Intracellular environments are highly crowded with various biomolecules and in vitro studies under molecular-crowding conditions will provide important information on how biomolecules behave in cells. Here we report that cell-mimic crowding can increase i-motif stability at acid pH and cause dehydration.

Synthesis of Titanate-Based Nanotubes for One-Dimensionally Confined Electrical Properties

Structural tailoring for dimensionally confined electrical properties is fundamentally important for nanodevices and the relevant technologies. Titanate-based nanotubes were taken as a prototype one-dimensional material to study. First, Na0.96H1.04Ti3O7 center dot 3.42H(2)O nanotubes were prepared by a simple hydrothermal condition, which converted into Na0.036H1.964Ti3O7 center dot 3.52H(2)O nanotubes by a subsequent acidic rinsing. Systematic sample characterization using combined techniques of X-ray diffraction, field emission scanning electron microscopy, high resolution transmission electron microscopy, electron paramagnetic resonance, Fourier transform infrared spectroscopy, elemental analyses, and alternative current impedance indicated that both nanotubes possessed a scrolled trititanate-type structure with the (200) crystal face predominant on the tube surface. With increasing temperature, both nanotubes underwent a continuous dehydration process, which however imposed different impacts oil the structures and electrical properties, depending on the types of the nanotubes

Synthesis of bis(amine anhydride)s for novel high T(g)s and organosoluble poly(amine imide)s by palladium-catalyzed amination of 4-chlorophthalide anhydride

Two novel bis(amine anhydride)s, NN-bis(3,4-dicarboxyphenyl)aniline dianhydride (I) and N,N-bis(3,4-dicarboxyphenyl)-p-tert-butylaniline (II), were synthesized from the palladium-catalyzed amination reaction of N-methyl-protected 4-chlorophthalic anhydride with arylamines, followed by alkaline hydrolysis of the intermediate bis(amine-phthalimide)s and subsequent dehydration of the resulting tetraacids. The X-ray structures of anhydride I and II were determined. The obtained dianhydride monomers were reacted with various aromatic diamines to produce a series of novel polyimides. Because of the incorporation of bulky, propeller-shaped triphenylamine units along the polymer backbone, all polyimides exhibited good solubility in many aprotic solvents while maintaining their high thermal properties. These polymers had glass transition temperatures in the range of 298-408 degrees C. Thermogravimetric analysis showed that all polymers were stable, with 10% weight loss recorded above 525 degrees C in nitrogen.The tough polymer films, obtained by casting from solution, had tensile strength, elongation at break, and tensile modulus values in the range of 95-164 MPa, 8.8-15.7%, and 1.3-2.2 GPa, respectively.

Self-assembly of a water chain with tetrameric and decameric clusters in the channel of a mixed-valence (CuCuII)-Cu-I complex

The reaction of Cu(BF4)(2) with pyridine-2,6-dicarboxylic acid (H(2)pydc) and trans-1,2-bis(4-pyridyl)ethylene (bpe) under hydrothermal conditions afforded a porous mixed-valence (CuCuII)-Cu-I coordination polymer. Coexistence of tetrameric and decameric water clusters within the channels of the complex leads to a novel water chain. The metal-organic framework provides both hydrophilic and hydrophobic environments for stabilizing the clusters and retains its integrity upon dehydration and rehydration.

Robust core-shell supramolecular assemblies based on cationic vesicles and ring-shaped {Mo-154} polyoxomolybdate nanoclusters: Template-directed synthesis and characterizations

Substantial progress has been made recently in extending the supramolecular assembly of biomimetic structures to vesicle-based sophisticated nanocomposites and mesostructures. We report herein the successful preparation of unilamellar surfactant vesicles coated with a monolayer of ring-shaped {Mo-154} polyoxometalate (POM) nanoclusters, (NH4)(28)[Mo-154 (NO)(14)O(448)Hi(4)(H2O)(70)].approximate to 350H(2)O, by coulomb attractions using preformed didodecyldimethylammonium bromide (DDAB) surfactant vesicles as templates. The resultant vesicle-templated supramolecular assemblies are robust (they do not disintegrate upon dehydration) both at room-temperature ambient and vacuum conditions, as characterized by conventional transmission electron microscopy (TEM) and atomic force microscopy (AFM). The flexibility of the complex soft assemblies was also revealed by AFM measurements. The effect of POM-vesicle coulomb attractions on the dimensions of the templating vesicles was also investigated by using dynamic light scattering (DLS).Although origins of the structure stability of the as-prepared supramolecular assemblies are not clear yet, the nanometer scale cavities and the related properties of macroions of the POM clusters may play an important role in it.