濒危植物大花黄牡丹种子休眠与萌发特性研究

本文以中国极危种大花黄牡丹种子为研究材料，对其种子生物学、休眠与萌发特性进行研究，采用变温层积和激素处理解除种子休眠，并通过生物抑制物测试、胚培养、激素含量动态变化的测定，研究种子休眠的原因，从根本上解决了大花黄牡丹迁地保护过程中种子繁殖的技术难题，并初步探讨了大花黄牡丹致濒因素与种子休眠及萌发特性的关系，结果表明： 1.大花黄牡丹种子饱实度高，活力强，种皮较坚硬，但不存在吸水障碍，干燥条件下易因失水而丧失活力。迁地保护冷室中盆播18个月方可出苗，出苗率约4%;变温层积实验表明该种具有典型的下胚轴休眠和上胚轴生长抑制。 2.胚组织培养表明：带胚乳和种皮的胚不能萌发，剥除种皮和胚乳后，胚根可萌发，胚芽不生长，GA3处理上胚轴可促进生长，说明种皮和胚乳是导致大花黄牡丹种子下胚轴休眠的关键因素，而上胚轴生长抑制与胚本身关系更密切。 3.抑制物质提取实验表明：大花黄牡丹种子胚乳、种皮、胚等各部位的浸提液均存在抑制小白菜种子萌发的物质，且抑制作用依次增强，说明胚乳和胚是其下胚轴休眠的主要原因。经过暖层积（15 ℃/90 d）种子（未解除上胚轴生长抑制）的胚根、子叶、胚轴浸提液对小白菜和已解除休眠的大花黄牡丹种子的萌发均有不同程度的抑制作用，并依次减弱，说明种胚本身的抑制物质是导致生理休眠的主要原因。 4.变温层积处理和外源激素实验表明：新鲜种子采收后15 ℃暖层积3个月生根率可达85%，下胚轴休眠解除对温度要求严格，高于或低于15 ℃及变温条件均不利于下胚轴萌发;暖层积90d、根长大于6 cm种子再经过60～80 d/ 5 ℃冷层积，即可有效解除大花黄牡丹种子上胚轴的生长抑制，出芽率达80%，最终出苗率68%。不同浓度GA3及不同处理时间促进上胚轴伸长实验结果显示，GA3　400 mg/L浸种根长大于1.5 cm的种子2 h，出芽率可达100%，可以完全解除上胚轴的生长抑制作用。 5.休眠萌发过程中种子各部位内源激素含量动态变化分析结果说明，初始状态脱落酸含量高是导致大花黄牡丹种子下胚轴休眠、上胚轴生长抑制的主要原因之一，且上胚轴抑制与子叶、下胚轴和根的脱落酸含量密切相关;同时认为种子各部位初始生长素含量水平低是导致其休眠的另一个主要原因;变温层积过程中胚各部位脱落酸含量的急剧下降和生长素的迅速升高是解除休眠的关键;同时发现赤霉素在解除休眠和促进萌发过程中起着重要的促进作用，外源GA3能够有效地打破上胚轴的深度休眠。玉米素核苷在休眠与萌发进程中变化趋势与生长素和赤霉素相似，说明其对种子胚根和上胚轴的萌发和生长具有一定的促进作用。

Effects of gibberellin A(3) on growth and microcystin production in Microcystis aeruginosa (cyanophyta)

Environmental. factors that affect the growth and microcystin production of microcystis have received worldwide attention because of the hazards microcystin poses to environmental safety and public health. Nevertheless, the effects of organic anthropogenic pollution on microcystis are rarely discussed. Gibberellin A(3) (GA(3)) is a vegetable hormone widely used in agriculture and horticulture that can contaminate water as an anthropogenic pollutant. Because of its common occurrence, we studied the effects of GA3 on growth and microcystin production of Microcystis aeruginosa (M. aeruginosa) PCC7806 with different concentrations (0.001-25mg/L) in batch culture. The control was obtained without gibberellin under the same culture conditions. Growth, estimated by dry weight and cell number, increased after the GA3 treatment. GA3 increased the amounts of chlorophyll a, phycocyanin and cellular-soluble protein in the cells of M. aeruginosa PCC7806, but decreased the accumulation of water-soluble carbohydrates. In addition, GA3 was observed to affect nitrogen absorption of the test algae, but to have no effect on the absorption of phosphorus. The amount of microcystin measured by enzyme-Linked immunosorbent assay (ELISA) increased in GA3 treatment groups, but the stimulatory effects were different in different culture phases. It is suggested that GA3 increases M. aeruginosa growth by stimulating its absorbance of nitrogen and increasing its ability to use carbohydrates, accordingly increasing cellular pigments and thus finally inducing accumulation of protein and microcystin. (C) 2007 Elsevier GmbH. All rights reserved.

长余辉发光材料的合成，缺陷及发光性质

近十年间，长余辉发光材料备受人们关注，其研究和开发得到了迅猛的发展。究其原因，长余辉材料是一种新型能量存储与电子俘获材料，它不仅可以应用到紧急照明与显示等传统领域，而且在高能射线探测、光纤温度计、工程陶瓷的无损探测以及超高密度光学存储与显示等高新科技领域具有潜在的应用价值。这一时期，研究兴趣主要集中在稀土离子特别是Eu2+激活的蓝色和绿色长余辉材料如铝酸盐、嫁酸盐、硅酸盐、硅铝酸盐、锗酸盐及氧化物等体系，而严重医乏红色长余辉材料。长余辉发光主要由三个过程，也就是能量吸收、能量存储与能量释放即余辉发光组成。而整个过程主要涉及两个中心，即发光中心和陷阱中心，它们之间的能量传递是产生余辉发光的主要原因。因此，本论文围绕这两个中心，选择以下两个角度为切入点：（a）选择具有较高发光性能的现有荧光体，通过各种辅助激活离子的共掺杂，主动引入陷阱中心；（b）选择具有丰富陷阱中心的现有荧光体，主动引入具有余辉发光性能的主激活离子，致力于新型长余辉特别是红色长余辉发光材料的开发，最终获得了红、绿、蓝三色共十种长余辉体系。其中，新型红色长余辉体系有：β-Zn3(PO4)2:Mn2+、γ-Zn3（PO4）2:Mn2+、Zn3B2O6:Mn2+和Y2O3：Eu3+；新型绿色长余辉体系有：Ca8Mg（SiO4）4Cl2:Eu2+、Zn4B6O13:Mn2+、α-Zn3(PO4)2：Mn2+和Zn2SiO4:Mn2+。我们还对已有红色长余辉体系Y2O2S:Eu3+和caO：Eu3＋进行了余辉性能的改善。在合成上述长余辉材料的基础之上，详细地分析并研究了组成、结构、缺陷和余辉性能之间的关系，得到了影响长余辉发光的一般规律。此外，首次在多晶粉末中报道了β-Zn3(PO4)2:Mn2+，Ga3+和Zn2SiO4:Mn2+，Al3+的红色和绿色光激励长余辉发光现象。

植物激素对土壤污染早期诊断研究

通过菲污染诱导动态响应预实验确定了小麦为供试植物，赤霉素(GA3)、细胞分裂素的玉米素和玉米素核苷总含量(Z&ZR)以及脱落酸(ABA)为植物激素供试指标，并且确定试验终点为小麦生长14d。在预实验的基础上，以草甸棕壤为供试土壤，采用室内培养箱盆栽方法，进行多环芳烃菲、芘、荧蒽和苯并(a)芘单一，两-两复合以及三-三复合与3种植物激素的剂量-效应关系研究；3种植物激素对土壤重金属镉、铅、铜和锌单一污染诱导的响应关系研究。并以多环芳烃类的菲和重金属类的镉为代表污染物，进行小麦芽期生长指标及植物激素测定，研究植物激素与生长指标对污染物的响应及敏感性比较。 多环芳烃单一及复合污染诱导实验结果表明：ABA对菲、芘、荧蒽、苯并(a)芘单一及复合污染诱导的响应趋势一致，即随着污染诱导浓度增加，ABA含量显著增加；GA3与四种多环芳烃单一及复合污染诱导存在一定的响应关系，但响应趋势呈多样性变化；对于多环芳烃单一及复合污染诱导，随着污染物浓度增加，Z&ZR含量出现了先增加后减少的趋势，表现为显著的污染诱导抑制效应。ABA和Z&ZR对多环芳烃污染诱导响应敏感，这为其成为土壤污染诊断指标提供了可能性。 在不同的镉、铅、铜、锌污染处理中，小麦体内ABA含量均出现明显增加趋势，与多环芳烃污染胁迫响应一致；GA3含量对镉、铅、铜、锌四种重金属污染胁迫响应趋势相同，均为先增加后减少。对于土壤镉和铅污染胁迫Z&ZR表现为先增加后减少的响应趋势，而对于铜和锌污染胁迫，Z&ZR含量变化与镉、铅胁迫时不同。 植物激素与生长指标对污染胁迫的响应及敏感性比较研究表明，对多环芳烃菲污染诱导，植物激素含量同生长指标相比具有显著的敏感性。对重金属镉污染诱导，内源激素含量和生长指标敏感性都显著，小麦根和芽中ABA含量显著增加，进一步证明了其在植物污染抗逆中的作用机理。

土壤镉污染的遗传及生态毒性研究

针对蚕豆根尖微核试验方法现存的问题并结合土壤自身的特性，进行了蚕豆微核试验方法改进的试验研究。在此基础上，本文选择蚕豆幼苗次生根根尖为供试材料，以细胞有丝分裂指数（MI）、染色体畸变率（CAF）和微核率（MNF）为试验指标，开展了我国典型土壤（红壤、棕壤和黑土）中低剂量（0~10 mg•kg-1）镉污染遗传毒性剂量-效应关系研究。研究针对蚕豆根尖微核遗传毒性评价指标“污染指数”的缺陷，首次提出“毒性指数”。“毒性指数”将有丝分裂指数、染色体畸变率和微核率指标所得数据结合，评价土壤镉遗传毒性水平。与此同时，研究选择蚕豆幼苗叶片为供试材料，进行了以抗氧化酶活性（SOD、POD和CAT）和植物激素含量（ABA、GA3和Z&ZR）为指标的镉污染胁迫生态毒理试验研究，并比较了上述三类指标对土壤镉污染的毒性响应及敏感性。 蚕豆根尖微核方法改进试验结果表明，在三种备选试验材料中，蚕豆初生根、次生根根尖细胞和芽尖细胞均对镉胁迫响应显著。与芽尖细胞相比，根尖细胞MI、CAF和MNF之间具有较好的相关性；而次生根尖细胞比与初生根尖细胞对镉响应更敏感，遗传毒性效应显著相关。在上述方法学研究基础上，将蚕豆幼苗次生根根尖微核试验应用到典型土壤镉污染的研究中，结果表明，同一剂量下，红壤中镉的遗传毒性效应最强，棕壤次之，黑土最不显著。土壤pH值与有机质含量对镉的遗传毒性影响较大。 抗氧化防御酶活性试验结果表明，POD和SOD在0~10 mg•kg-1镉浓度范围内先升高后降低，对镉毒性的指示效果最明显；CAT反应与SOD、POD相反，其作用机理有待进一步探讨。植物激素试验结果表明，ABA和Z&ZR对镉胁迫响应敏感，其含量与镉浓度存在良好的剂量-效应关系；GA3随着镉浓度的增加表现出无规律性，表明它对污染物的响应具有选择性。 试验数据显示，不同指标对镉胁迫均具有响应，但响应的域值及敏感度不同。比较而言，蚕豆根尖微核遗传毒性指标指示效应更明显。将各指标联合使用可使土壤镉污染的遗传和生态毒性诊断更为全面、有效。本研究为土壤镉污染早期诊断敏感指标筛选及指标组合提供了实验依据，为土壤重金属污染早期诊断奠定试验和理论基础。

岷江干旱河谷三种蔷薇植物繁殖更新研究

繁殖更新是植物生活史的重要阶段，在退化生态系统中，植物繁殖更新能力往往较差，是植被恢复的限制环节，因而也成为恢复研究重点和核心。本研究选择岷江干旱河谷广泛分布的三种蔷薇：多苞蔷薇（R. multibracteata）、黄蔷薇（R. hugonis）和川滇蔷薇（R. soulieana）为研究对象，通过野外调查，在查明其生长、繁殖更新状况的基础上，采用控制和模拟实验，对种子和幼苗阶段进行了深入研究，综合分析更新潜力，并提出相对应的促进更新和植被恢复措施。主要结论如下： 1）三种蔷薇在岷江干旱河谷广泛分布，生长和繁殖状况良好，结实量大。各生长指标：株高、基径和冠幅，繁殖指标：结实数量、重量和单果重量都具有显著的空间差异性。基径对多苞蔷薇结实量影响最大；而冠幅对黄蔷薇结实量影响最大。海拔和纬度是对蔷薇生长和繁殖影响最大的环境因素，随着海拔和纬度的升高，植株生长更高大，结实量增加；坡度和坡向对其生长和繁殖也有一定影响，随着坡度 和坡向增加，蔷薇生长和结实受到抑制。 2）三种蔷薇在岷江干旱河谷更新现状不佳, 但更新潜力大。活力种子比率低，动物取食以及两年生幼苗的大量死亡是蔷薇更新的主要限制因素。多苞蔷薇和黄蔷薇的结实率低，川滇蔷薇较高。三种蔷薇种子产量大，但种子质量较差，更新具有充足的种源。三种蔷薇都能形成持久种子库，种子库中种子总量大，但有效种子少，黄蔷薇被动物啃食的比例很高，多苞蔷薇和川滇蔷薇也有一部分种子受到动物破坏。三种蔷薇幼苗库组成特征表现为，当年生幼苗所占比例很高，年龄较大幼苗所占比例小。 3）三种蔷薇都具有不同程度休眠，未经处理种子的发芽率极低。黄蔷薇休眠程度最深，为深度生理休眠；多苞蔷薇为中度生理休眠；川滇蔷薇为非深度生理休眠。三种蔷薇种子在形态上发育成熟，种皮具有透水性。蔷薇果果肉和瘦果中含有抑制物质，其浸泡液抑制了油菜种子萌发，果肉抑制作用更强，果肉和瘦果浸泡液的抑制程度分别为：川滇蔷薇>黄蔷薇>多苞蔷薇。切割和硫酸腐蚀提高了川滇蔷薇种子的发芽能力，而对多苞蔷薇和黄蔷薇没有影响。完全去除瘦果果皮和种皮提高了多苞蔷薇种子发芽率，但对黄蔷薇没有影响。赤霉素和烟水对蔷薇种子萌发没有促进作用。三种蔷薇打破休眠所需低温层积时间分别为：黄蔷薇>多苞蔷薇>川滇蔷薇。对于多苞蔷薇和川滇蔷薇，层积前对种子进行硫酸腐蚀或暖温层积能缩短低温层积时间，提高发芽率。对于多苞蔷薇，变温层积中暖温层积和低温层积具有一定的负补性，即延长暖温层积可以缩短种子萌发对低温层积的需要。 4）多苞蔷薇种子形态特征和种子休眠与萌发在不同海拔梯度间存在较大差异。种子采集时间、采集季节和干藏影响多苞蔷薇和川滇蔷薇的种子休眠。多苞蔷薇果实大小、种子大小和千粒重、种皮厚度随海拔升高而增加，而种子饱满率和活力随海拔升高而降低，种子休眠程度也随海拔升高而增加。种皮厚度与种子大小、千粒重成正相关关系，硫酸腐蚀后的种子经过不同时间的低温层积后，种子发芽率与种皮厚度、种子大小、千粒重、海拔成正相关关系。2006 年采集川滇蔷薇和多苞蔷薇种子休眠程度较2005 年低。种子休眠随种子年龄增加而减弱。高温和干旱能减轻多苞蔷薇和川滇蔷薇种子休眠。 5）三种蔷薇的生长和生物量积累在干旱胁迫条件下受到抑制，而生物量分配、叶片形态特征和水分利用特征等都发生了变化。三种蔷薇的根、茎、叶各器官生物量以及总生物量等在干旱胁迫下明显减小，叶片脱落数量增加。在干旱胁迫条件下，较多的生物量分配到地下部分，从而这使R/S 明显增加。比叶面积（SLA）和冠层面积比（LAR）对干旱胁迫的反应不敏感，仅有部分物种在干旱胁迫条件下发生了变化，并且其变化特点在不同年龄幼苗之间有一定差异。干旱胁迫对WUE 的影响在不同物种间存在差异。多苞蔷薇和黄蔷薇的WUE 随着干旱胁迫的增加而增大， 而川滇蔷薇的WUE 则随干旱胁迫增加而减小。在干旱胁迫条件下，多苞蔷薇和黄蔷薇叶片脱落量和生物量减小幅度较川滇蔷薇大，表明其抗旱能力较强。在干旱胁迫条件，三种蔷薇两年生幼苗的生物量减小幅度较当年生幼苗小，表明两年生幼苗的抗旱能力更强。 6）两种植被恢复措施中，幼苗移栽比播种具有更好的植被恢复效果。播种后，蔷薇种子的发芽率较高，但出苗率都很低，即使出苗，幼苗也几乎在一月内全部死亡。 三种微生境条件下（灌木、半灌木和裸地），种子出苗和幼苗成活没有差异。移栽幼苗总体死亡率都比较低，小于20％。特别是两年生幼苗死亡率更低，小于2％。移栽后的幼苗生长状况良好，在整个生长季中，各生长指标不断增加。生境对幼苗的存活率没有显著影响，但对于幼苗的生长和生物量积累有一定影响，裸地更有利于幼苗生长和生物量积累。与当年生幼苗相比，两年生幼苗具有更高的成活率。总之，三种蔷薇在干旱河谷分布广泛、生长繁殖状况良好，结实量大，具有丰富种源，繁殖更新潜力大，但繁殖更新状况不佳；种子散布后动物对种子的取食、种子的深度休眠过程、种子出苗以及当年生幼苗的存活和定居是更新的主要限制环节。水分是影响结实、种子休眠解除和萌发，幼苗存活和定居的最主要的限制因素。在植被恢复中，应在种子成熟季节大量采集种子，在室内打破休眠后进行人工播种，培育两年生幼苗，通过幼苗移栽方式进行植被恢复。川滇蔷薇应栽种在相对湿润的过渡区，而多苞蔷薇和黄蔷薇可以应用于核心区植被恢复。 Regeneration is an important phase in plant life cycle. It has been a key component of ecological restoration in degradation ecosystem in which plants commonly has poor regeneration. In this paper, we investigated the natural growth, propagation and regeneration status of native three rose species, Rosa multibracteata, R. hugonis and R. soulieana, and analysis the limitation in seed germination and seedling establishment stages. Advice on facilitating the use of these plants in restoration based on the results has been proposed. The results were as follows: 1) Three rose plants widely distributed in the dry valley of the Minjiang River, and made a good performance in growth and propagation. There were significant spatial differences in each growth parameter, such as ramet height, basal diameter, crown diameter and propagation parameters including hip number of a clump, hip mass of a clump and a hip mass. Basa diameter was the most important growth parameter influencing fruit number for R. multibracteata and crown diameter was for R. hugoni. Altitude and latitude had the greatest effect on the growth and propagation of rose plants among environmental conditions. Each parameter of growth and propagation increased with the increase of altitude and latitude. In addition, the increase of slope and aspect limited the growth and propagation. 2) Three rose plants had poor natural regeneration, but great regeneration potential. Low seed viability, predation and higher mortality of current year old seedlings were the limitation in regeneration. R. multibracteata and R. hugonis had higher fruiting rates than R. souliean. All three plants produced a great number of seeds, while their viability was poor. Three rose plants had persistent seed banks, with high total seed number but very low viable seed density. Predation was most severe in R. hugonis, and it also existed to some degree in R. multibracteata and R. soulieana. The seedling age-structure was characteristic of current-year seedlings predominating and few older seedlings were observed. 3) Three rose seeds were dormant and untreated seeds germinated with very low germination percentages. The rose seeds had morphological mature embryos, and achenes were permeable. Some inhabit substances existed in hips and achenes for the extracts of hips and achenes inhibited germination of Brassica campestris. The inhibition effect of the extracts of three rose hip and achenes was R. soulieana>R. hugonis>R. multibracteata. Mechanical and H2SO4 scarification increased R. soulieana germination but had no effect on germination of R. hugonis and R. multibracteata seeds. Full removal of pericarp and testa improved the germination of R. multibracteata but did not affect R. hugonis germination. GA3 and smoke water had no positive effect on rose seed germination. The periods of cold stratification required to released seed dormancy was R. hugonis > R. soulieana >R. multibracteata. H2SO4 scarification and warm stratification shortened cold stratification to release dormancy for R. soulieana and R. multibracteata. Warm stratification had complementary effect for cold stratification, i.e. the longer warm stratification seeds received, the shorter cold stratification were required to obtain the same germination percentage. Three rose seeds had different kinds of dormancy; R. hugonis has deep physiological dormancy, R. multibracteata with intermediate physiological dormancy and R. souliean non-deep physiological dormancy. 4）The seeds traits and dormancy of R. multibracteata showed significant difference across altitudes. Year and season of seed collection had significant effect on seed dormancy for both R. souliean and R. multibracteata. Hip size, seed size, seed weight, seed coat thickness and seed dormancy level increased with the increase of the altitude. There were positive relations between seed coat thickness with seed size and seed weight. Germination percentage of seeds treated with H2SO4 scarification following different periods of cold stratification showed positive relation with seed coat thickness, seed size, seed weight and altitude. Seeds of R. souliean and R. multibracteata collected in 2006 had low dormancy level than those collected in 2005. Seed dormancy decreased with increasing seeds age. High temperature and drought were associated with low dormancy level. 5) Seedling growth, the total dry mass and their components of seedlings were reduced, while leaf senescence accelerated under drought stress. More biomass allocation to root system resulted in higher R/S ratio under drought. Water-use efficiency (WUE) of R. multibracteata and R. hugonis increased, while it declined for R. soulieana under drought stress. R. soulieana seedlings had poor drought-resistance capacity it had more senescent leaves, and its reduction of biomass was stronger than two other rose plants under drought. The reduction degree of one year old seedlings under drought stress was slighter than that of current year seedlings. Therefore, one year old seedling was more drought-resistent compared to current year seedlings. 6）Planting seedlings may have better effect in comparison with direct seeding. Most seeds germinated after seeding, but seedling emergence was very low. More than 80 % seedlings from direct seeding died within a months after emergence. Seedling emergence and survival rate did not show difference among microhabitats. Mortality rates of seedlings artificially planted in microhabitats were general lower than 20 %, and the mortality rate of one year old seedlings was lower than 2 %. Each grow parameter including plant height, leaf number and branch number continually increased after planting. Microhabitat type had effect on the growth parameter and biomass production, but it did not influence the seedling survival. Bare land tended to facilitate seedling growth. One year old seedlings had higher survival rate than current year seedlings. In conculsion, the three rose had wide distribution in the dry valley of the Minjiang River. They produced many seeds and had tolerance to drought stress to some degree. But they had poor regeneration in habitats may be caused by predation, seed dormancy，and high mortality in current year seedlings. We recommend that rose plants should be utilized in restoration by planting two-year old seedlings in spring. A large quantity of seeds should be collected artificially in autumn, release seed dormancy in room, and then cultivate two-year old seedlings by seeding in particular container. R. soulieana seedling probably be planted in transition area, and R. multibracteata and R. hugonis can be used in core area of the dry valley of the Minjiang River.

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.

RNAi沉默烟草GA 20-氧化酶基因研究

赤霉素是一种高效能的广谱植物生长调节剂，为五大植物激素之一，具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶，它位于GA合成途径的中心位置。本研究根据烟草（Nicotiana tabacum）GA 20-氧化酶基因序列，设计2对分别含有特定酶切位点的特异引物，以烟草基因组DNA为模板，扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧，再经BamH I和Sac I双酶切回收约700 bp的目的片段，插入到双元载体质粒p2355中，成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌（Agrobacterium tumefaciens）EHA105中并转化烟草叶片细胞，经卡那霉素选择培养，PCR及GUS组织染色鉴定，获得转基因烟草植株。以EHA105-p2355转化的烟草，获得41株转基因植株，均没有矮化表型；而以EHA105-p23700转化的烟草，获得转基因植株14株，其中具有矮化表型的烟草10株，表明反向重复序列转录产物能形成发夹RNA(hpRNA)，产生小分子干扰RNA（small interferring RNA，简称siRNA），干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明，矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制，表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶（Ent-kaurene synthase，KS）基因，下游的GA-3β羟化酶基因进行了RT-PCR分析，结果显示上游基因的表达没有规律性变化，而下游基因表达量亦降低。上述结果表明，GA 20-氧化酶基因的表达被有效地干扰了，表达受到抑制，从而影响植株体内GA3的合成，影响植株的生长发育，导致植株矮化。并推测，GA 20-氧化酶基因受到抑制，可能影响下游基因的表达。并且通过干旱胁迫测试，发现矮化植株相对于野生型植株及不含干扰片段的转基因植株，对干旱的耐受力有了很大的提高，具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.

外源激素对马铃薯试管苗耐盐性的影响

以马铃薯栽培品种甘农薯2号试管苗为试材,研究了在0.4%、0.6%NaCl胁迫下外源激素GA3、6-BA、IAA对试管苗耐盐性的影响。结果表明:3种激素均对马铃薯试管苗受到的盐胁迫具有一定缓解作用,使盐胁迫下的试管苗叶绿素含量提高、脯氨酸和丙二醛含量降低,其中GA3和IAA提高叶绿素含量的效果显著,GA3降低脯氨酸和丙二醛含量的效果比6-BA和IAA明显。

内源激素含量的变化与白桦成花关系的研究

采用酶联免疫吸附法(ELISA)测定有花史白桦大树与无花史白桦幼树的4种内源激素脱落酸(ABA)、细胞分裂素(iPA)、生长素(IAA)、赤霉素(GA3)含量的动态变化,研究其与白桦成花的关系。研究结果表明:白桦有花史大树与无花史幼树的4种内源激素含量动态变化明显不同。在雌、雄花芽的生理发端期,有花史大树的IAA含量保持相对稳定,而高含量的ABA和iPA含量的逐渐降低以及GA3含量的逐渐升高可能有利于白桦成花。

平阴玫瑰花芽分化期叶片内源激素的变化

对平阴玫瑰花芽分化期叶片甲醇提取物进行IAA、ZR、GA3、ABA的分离、纯化和测定。结果发现,所测的几种激素均表现出明显的变化规律,其中IAA和GA3在花芽分化期含量逐渐下降,且在分化临界期出现一低峰,而ZR和ABA则完全相反。同时经比较分析得出ABA/GA3,ABA/IAA,ZR/GA3,ZR/IAA也表现明显的变化规律,即比值总体趋势是逐渐提高,且均在分化临界期含量出现一飞跃,显然ABA/GA3,ABA/IAA,ZR/GA3,ZR/IAA在平阴玫瑰的花芽分化过程中起着重要的调控作用,由此推测,增加植物体内的ABA、ZR的含量或降低IAA、GA3的含量,都可以促进玫瑰的花芽分化;反之则抑制其花芽分化。

赤霉素、硫脲处理对黄精根状茎和须根发生的影响

为诱导无芽黄精根状茎产生不定芽,选择不同的化学物质进行诱导处理。结果表明,GA3处理效果明显,选用0.4 mg/L的GA3浸泡25 min对黄精根状茎不定芽的产生和须根生长有明显促进作用,高浓度抑制其生长;1%硫脲浸泡处理对黄精无顶芽根状茎萌发新芽和须根有明显的抑制作用。

生长调节剂处理对银杏结实的影响

盛花期对银杏雌株叶面喷施多效唑、B9、GA3、GA3+6- BA。结果表明 ,多效唑和 B9能够明显提高坐果率 ,有效增加来年成花数量。GA3和 GA3+6- BA对提高坐果无影响 ,但能增加种子重量 ,促进纵、横径增加 ,GA3还能够促进翌年成花数量。多效唑处理提高产量的原因 ,一是抑制树体营养生长 ,二是减少落花落果 ,增加植株翌年成花量

土壤镉污染毒性效应的多指标综合评价

以草甸棕壤为供试土壤,以蚕豆幼苗根尖有丝分裂指数、染色体畸变率以及微核率,幼苗叶片内抗氧化酶活性、植物内源激素含量为指标,采用盆栽方法研究了0~10 mg.kg-1镉胁迫对植物细胞的遗传和生态毒性效应.结果表明,在此浓度范围内,蚕豆根尖细胞有丝分裂指数、染色体畸变率以及微核率均随镉浓度增加呈显著的剂量-效应正相关关系,其中微核率变化最为明显,处理组微核率分别为对照组的1.43~3.22倍;蚕豆幼苗叶片内的SOD和POD活性变化呈先升高后下降的趋势;而CAT活性随镉浓度增加其变化规律与SOD、POD相反.此外,镉胁迫下植物激素脱落酸(ABA)、赤霉素(GA3)与细胞分裂素类的玉米素和玉米素核苷总含量(Z&ZR)均表现出低浓度下诱导和较高浓度下诱导率降低的趋势,在镉浓度为2.5 mg.kg-1时3种植物激素含量均达到最高值,分别比对照组增加了6.6%、4.0%和12.6%.研究表明,各指标对污染物的毒性具有响应且响应的域值及其敏感度不同,将各指标综合使用可使土壤镉污染的遗传和生态毒性诊断更为有效.

Synthesis, structural characterization and electrical property of new oxide ion conductors: La3MMo2O12 (M = In, Ga and Al)

New series of oxides, La3MMo2O12 (M = In, Ga and Al), have been prepared by the solid-state reaction. The composition and elemental distribution were analyzed by the energy-dispersive X-ray (EDX) analysis. As determined by the X-ray diffraction (XRD), these compounds have similar crystal structures that can be indexed on a monoclinic cell at room temperature. AC impedance spectra and the DC electrical conductivity measurements in various atmospheres indicate that they are oxide ion conductors with ionic conductivities between 10(-2) and 10(-3) S/cm at 800 degrees C. The conductivity decreases in the order of La3GaMo2O12 > La3AlMo2O12 > La3InMo2O12, implying that the effect of cell volume and polarization associated with In3+, Ga3+ and Al3+ play an important role in the anion transport of these materials. The reversible phase transition was observed in all these compounds as confirmed by the differential thermal analysis (DTA) and dilatometric measurements.