单季稻水田生态系统水量平衡及节水研究

本文通过观测确定了水稻生长季蒸发蒸腾量和水面蒸发量的相关关系，并利用这一关系和水量平衡原理，评估了下辽河平原地区单季稻水田生态系统的水量平衡情况；试验研究了保留犁底层、深翻打破犁底层和30cm土底层下铺设塑料地膜与育秧膜四种土壤处理措施的稻田生态系统水量平衡的特征和水分利用效率。研究表明，北方单季稻水田生态系统的蒸发蒸腾量和水面蒸发量呈良好的线性关系；用此关系估算下辽河平原各水稻灌区的蒸发蒸腾量变化规律为：营口>盘锦>辽阳>铁岭>沈阳。下辽河平原地区水稻生长季的总用水量为1773.4～2194.3mm，其中降水占总用水量的27.2%～35.8%，其余水量依靠灌溉供给，平均每亩灌水量在1138.9～1597.6mm之间，南部滨海灌区高于中部地区。除蒸发蒸腾损失外，下辽河平原地区水田总用水量的65%～72.3%由渗漏损失，该区总体的渗漏特征是中部高于南部的北部。在沈阳郊区潮棕壤条件下进行的第三年实验结果表明，与保留犁底层处理相比，深翻处理渗漏水量多18.7%；地膜处理与保留犁底层处理的渗漏水量基本持平；育秧膜阻渗处理的渗漏水量少29.4%。显然，犁底层具有保水作用，深翻打破犁底层加剧稻田水分渗漏；地膜阻渗效果已不明显，而育秧膜仍具有明显的阻渗节水效果。

维生素C二步发酵菌系2980生长代谢及其生产调控

本文研究了维生素C生产菌系2980的生长和产酸代谢规律，主要环境因子的影响及生产调控。研究结果表明：2980混合菌系在种子罐和300M3发酵罐混合培养时，呈现一种协同共生的关系。在流加糖发酵过程中，2-酮基-L-古龙酸的合成有两个高峰期：8-20小时和24-40小时高峰期。L-山梨糖脱氢酶总酶活迅速上升，2-酮基-L-古龙酸还原酶活性始终呈平缓上升趋势。环境因子对棍菌间的协同共生影响其显著。在种子罐培养中，采取适宜的接种量、通气量、p日、温度和营养，可获较佳的菌群状态。在发酵罐内，生长期不同，对’环境的温度、通气量、介质和p日值要求不同。对数生长期和稳定生长期，提高温度、p日值、通气量、接种量和采用较低的初始糖浓以及添加FeCl3,有利于菌系生长和产酸代谢。新的调控工艺，种液可较快调整到最佳接种状态且衰亡较慢，发酵罐内佩狗菌系结构和菌群状态得到改善。发酵平均转化率为89.51%，较之对照提高4.21%,平均周期为51.2小时，较之对照缩短4.8小时。

Vc二步发酵新菌系B529-V6的构建及其分子生物学特性

从芽抱杆菌属、酵母属二十株菌中筛选到一株优良伴生菌B529，与新选育出的产酸菌V6构成一新菌系B529-V6。新菌系B529-V6表现出了较强的高浓度L-山梨糖耐受能力、较高的底物代谢速率和较高的2-KGA转化能力，在8％山梨糖浓度的发酵培养基中培养48h，糖酸转化率较对照菌系提高了5.94％；山梨糖浓度提高至10％，其生长代谢受影响程度较小，且能不同程度地利用葡萄糖和山梨醇为底物，合成维生素C前体-2-酮基-L-古龙酸（2-KGA），其发酵产物2-KGA经反相高效液相色谱分析其质量符合工业化生产要求。对新菌系生长代谢规律及调控进行了研究，4M3罐和300M3罐发酵实验表明：种子培养基的碳源、葡萄糖／山梨糖浓度比、氮源、生长因子、接种种液质量及环境因子，均可影响新菌系的生长代谢。4M3罐发酵，新菌系具有周期短、糖酸转化率高等特点，连续4批发酵平均转化率较对照菌系提高10.18％，周期缩短23.7％。在300M3罐发酵试运行期间，新菌系糖酸转化率达到90.10％，较原生产菌系提高3.95％，发酵周期平均缩短1.3小时，显示出了较高的应用价值，在东北制药总厂进行了推广应用。研究了新产酸菌V6的基本生物学特性，分析了GC moL％含量，165rDNA同源性，鉴定其在系统发育学上应归入Ketoguloigenium 属，暂命名为Ketogulonigenium sp．V6。选用限制性内切酶Hind IH对新产酸菌V6染色体DNA进行了部分消化，应用载体pGEM-3zf（+）构建了v6的基因文库。结合阳性转化子在以L－山梨糖为唯一碳源培养基上的生长特性，利用PCR技术从该基因文库中，筛选到一株含有L-山梨糖还原酶（sR）基因的阳性克隆，并利用pET-32a（+）表达载体，实现了sR酶基因在大肠杆菌AD494（DE3）中的表达。SDS-PAGE电泳分析测定SR融合蛋白分子量大约在65kD左右，除去硫氧化还原蛋白、S-Tag和His-Tag蛋白，可推测出天然sR酶蛋白分子量约53 kD左右，与从SR基因推测出的分子量大小相符。另外，SR酶学特性研究表明，还原型辅酶II（NADPH）是sR酶蛋白的最适电子供体，其最适反应pH为7.0，pH6.5时保持稳定，酶活力较高；最适反应温度为50 ℃，30 ℃时热稳定性较好；lmM的Cu~(2+)，Fe~(3+)和Mn~(2+)对该酶活抑制作用较大。

沈阳城市森林生态系统健康与管理研究

本文系统研究了沈阳城市森林的布局与结构、城市森林功能、城市森林病虫害发生与树木健康状况和城市自然资源与社会经济状况等指标对沈阳城市森林生态系统健康与管理的影响。同时一，采用2种生态系统健康评价方法对沈阳城市森林生态系统健康状况进行了评价，并提出了沈阳城市森林生态系统健康管理的对策。研究结果如下：1、截至2004年末，沈阳城市森林植被覆盖率已经达到35％，城市森林林地分布基本合理，但需要进一步加强道路林地、居住区林地和城郊大面积生态林建设。2、沈阳城市森林以乔木为主，乔灌株数比为1.7：1，乔灌的覆盖度比约为7:1。3、沈阳城市森林不同类型林地中植物组成不同。公园林地中有74个属，137个种（变种）；庭院林地中有53个属，104个种（变种）；居住区林地中有45个属，81个种（变种）；道路林地中有43个属，94个种（变种）；运河风景林地中有75个属，142个种（变种）；棋盘山风景林地中有48个属，118个种（变种）。4、公园林地、庭院林地、居住区林地、道路林地和运河风景林地的Shannon一Wiener多样性指数分别为2.78、3.05、3.15、3.18和3.18，均匀度指数分别为0.56、0.66、0.72、0.70和0.64。除了棋盘山风景林地外，沈阳城市森林中栽植总量超过乔木总量5％的乔木树种有7个属，分别为李、柳树、杨树、桧柏、榆树、槐树和银杏，7种树木总量达到了全部乔木总量的82.09％；栽植总量超过灌木总量5％的灌木树种也有7个属，分别为水腊、丁香、李属，小聚、玫瑰、忍冬和连翘，7个属灌木总量达到了全部灌木总量的87.92％。5、公园林地、庭院林地、道路林地和防护林地中OBH＜20cm、20cm＜DBH＜60cm和DBH＞60cm树木的比例分别为：57.9％、40.0％、2.1％，49.2％、47.8％、3.0％，65.3％、33.1％、1.6％和64.6％、34.9％、0.5％，表明沈阳城市森林树木的规格总体上偏小。6、经样方调查和CITYgreen模型计算，沈阳城市森林的生态效益约2.0亿USD／yr.。公园林地、庭院林地和风景林地的景观指标相对较高；道路林地和居住区林地的景观效果一般；防护林地的景观效果较差。7、目前已经发现的沈阳城市森林病害约600余种，虫害约700余种，其中杨树主要病虫害39种，柳树的主要病虫害有33种，榆树和槐树的主要病虫害均为，1种。杨柳树腐烂病、光肩星天牛、天幕毛虫、桃红颈天牛和美国白蛾等是近10年来沈阳城市森林中普遍发生和造成严重危害的主要病虫害。沈阳城市森林主要树木的平均健康指数为2.68，处于一般健康状态。8、沈阳城市森林的土壤和水资源状况均不利于树木的健康生长，沈阳的社会经济发展也有待于进一步提高。9、经过生物指示物法（光肩星天牛为生物指示物）、专家权重法、公众问卷调查和对比研究，沈阳城市森林生态系统总体上处于亚健康状态。10、通过对沈阳城市森林资源、管理状况的调查研究和健康状况的评价，本文提出了沈阳城市森林生态系统健康管理的对策，包括合理规划沈阳城市森林林地布局，增加道路林地、居住区林地和城郊林地的面积和植被覆盖率；调整树木种类组成，避免单一或少数树种的大量栽植，提高生物多样性水平；保护大树和古树；增加城市森林管理资金的投入；应用先进技术，采取科学的病虫害防治和植物养护方法，促进树木的健康生长等。This project systematically studied the urban forest ecosystem health and management in Shenyang. The study explored factors, such as urban forest structure, distribution, pests, aesthetic value, ecological benefit, natural resources and socieo-economic status, that affecting the urban forest ecosystem health and management. Two methods were used to evaluate the ecosystem health. This project also proposed Shenyang's urban forest ecosystem health management strategies. The research results can be summarized as follows: 1. As of the end of 2004, urban forest coverage in Shenyang is about 35%, and is in relatively even patch distribution pattern. However, the street trees and roadside forest patches, residential block forest patches should be enhanced. 2. Trees are the major component of the Shenyang s urban forest, followed by shrubs. The quantity ratio of tree to shrub is about 1.7:1, and the coverage ratio of trees to shrub is about 7:1. 3. Species composition varies by location. There are 74 genera, 137 species (including varieties) in the public parks; 53 genera, 104 species (and var.) in the green spaces of the institution (including school), factory, and company; 45 genera, 81 species (var.) in residential blocks; 43 genera, 94 species (var.) in streets and roadside forest patches; 75 genera, 142 species (var.) in the Canal landscape forest patches; 48 genera, 118 species (var.) in the Qipan Mountain recreation forest. 4. The Shannon-Woener indices varies in parks, in institution, factory, and company yards, in streets and roadside forest patches, in residential blocks.there are 2.78, 3.05, 3.18, 3.15, 3.18, respectively; and the evenness indices are 0.56, 0.66, 0.70, 0.72, 0.64, respectively. Besides the Qipan Mountain forest patches, trees of 7 genera, Prunus spp., Salix spp., Populus spp., Sabina spp., Ulmus spp., Robinia spp. and Ginkgo biloba are of more than 5% the total urban trees, respectively. In fact, trees from these 7 genera are about 82% of all trees in Shenyang's urban forests. In terms of shrubs, species of 7 genera, Ligustrum spp., Syringa spp., Prunus spp., Berberis spp., Rosa spp., Lonicera spp., and Forsythia spp. are more than 5% the total urban shrubs, respectively. 88% of all the shrubs in Shenyang s urban forest are from these 7 genera. 5. The diameter class of DBH<20cm, 20cm60cm of trees in parks, in institution, factory, and company yards, in in streets and roadside forests, and in protective forests are 57.9%, 40.0%, 2.1%; 49.2%, 47.8%, 3.0%; 65.3%, 33.1%, 1.6%; and 64.6%, 34.9%, 0.5%, respectively. The result indicated that the DBH is relatively small in Shenyang s urban forests. 6. Using random plots sampling and CITYgreen model calculation, the ecological benefit of Shenyang's urban is about 0.2 billion US dollars per year. The aesthetic value of parks, institution and company yards, and scenary forest patches is relative high; however, the aesthetic value of protective forest patches is low. 7. There are more than 600 types of diseases and more than 700 kinds of insects in Shenyang's urban forest. Among them, Populus spp., Salix spp., Ulmus spp. and Robinia spp., are attached mainly by 39, 33, 11, and 11, difference types of pests respectively. The rot disease of the Populus spp. and Salix spp., and the insects such as, Anoplophora glabripennis, Malacosoma neustria, Aromia bungii, and Hyphantria cunea are major pests in Shenyang s urban forest for the past 10 years. The trees health condition is relatively poor by having the average health index of 2.68 for major species. 8. The urban soil and water resource are not favorable to tree's growth in Shenyang, and so is the socieo-economic status. 9. Through using bio-indicator, professional and public evaluation, and comparision with other urban forests, it can be concluded that the status of Shenyang s urban forest is not very healthy in general. 10. To improve the urban forest ecosystem health in Shenyang, a better urban forest design need to be implemented to enhance the area and vegetation coverage of the street and roadside forests, the residential block forests, and the surburb forest Species biodiversity need to be enhanced by adjust the species composition of urban forest trees to reduce the single or several tree species populations and to plant more varieties. A better fundings for urban forest health management need to be budgeted. Advanced technologies in tree care and scientific measures to control pests need to be adopted to prove better care for plants and to keep trees grow healthfully.

日本、华北落叶松幼——成龄相关选择模型及早期选择

为了加速日本、华北落叶松的遗传改良进程，缩短育种周期，本文建立了用于早期选择的年遗传增益最大模型 (MAGG) 和最大未来净值模型 (MFNW)。遗传力平方根比率(g)、幼-成龄遗传相关系统(r) 和遗传预测系数等参数可用8种随时间函数来逼近，根据残差平方和与非线性回归相关指数，以二次抛物线式拟合的精度最高。不同模型利用的评价标准及参数逼近的函数不同，估算的最佳选择年龄 (tmax) 也不同。对于 MFNW模型，tmax的确定受生物学因子和经济学因子的共同影响。估算的结果表明，日本、华北落叶松的早期选择都是有效的。如果轮伐期（T）为20年，考虑改良的性状为材积，对于日本落叶松可用早期的树高作为选择指标，初选在2龄进行，第二次选择在7－8龄，早期选择的相对效率 (RSE) 分别为 14.2037 和 2.5017－1.9378；对于华北落叶松，当用树高作为早期选择指标时，第一次选择在3龄 (RSE=3.1711)，第二次选择在 9－10 (RSE=1.5492－1.4561)，当用树高年生长最作为早期选择指标时，第一次选择在4龄 (RSE=1.1276)，第二次选择在 7－8 (RSE=1.3343－1.3436)。本文对不同轮伐期的tmax和其他性状的早期选择也作了探讨。

酸雨对重庆地区不同植被下土壤的影响

在地处亚热带，酸雨污染严重的重庆地区土壤上，近一年的定位研究表明：1）酸雨促进了马尾松针叶林林内降水的酸化和元素的淋失。在重污染的城区针叶林，其冠流和干流pH分别为3.71和2.75，离子含量分别是郊区冠流、干流的1.2-3.2倍和1.1-2.7倍。2）森林死地被物层对酸性降水有中和、缓冲作用，其中阔叶林死地被物层作用大于针叶林。3）酸雨有助于马尾松针叶林黄壤的酸化和土壤溶液中离子含量的增加。重污染的城区与轻污染的郊区比较，土壤溶液pH低0.2-0.8，而离子含量却普遍增高。4）在重庆地区的土壤溶液中，SO~(2-)_4是优势阴离子，占所测阴离子总量的60-90％，但土壤溶液中Al~(3+)含量却很低。目前土壤溶液中Al~(3+)不可能对树木产生毒害作用。

五种毛壳霉属和曲霉属真菌代谢产物及细胞毒活性

毛壳霉属(Chaetomium)和曲霉属(Aspergillus)真菌产生多种具有生物活性的化合物。为系统阐明两属微生物的次生代谢物，对三种毛壳霉、两种曲霉真菌分别进行固态发酵，以色谱和波谱技术研究发酵物中的成分，分离鉴定了51个化合物，其中23个为新化合物，测试了部分化合物对肿瘤细胞的活性。 1、从螺卷毛壳霉（C. cochloides）固态发酵物中分离鉴定了11个化合物，3个新化合物为螺卷毛壳霉素A~C（1~3）。化合物1、3及dethio-tetra (methylthio) chetomin（4）对Bre-04、Lu-04和N-04细胞株生长抑制的GI50值为0.05~7.0 μg/mL。 2、从印度毛壳霉（C. indicum）固态发酵物中鉴定的三个异喹啉生物碱印度毛壳霉素A~C（12~14）代表两类骨架新颖的异喹啉生物碱。 3、从巴西毛壳霉（C. brasiliense）固态发酵物中鉴定了11个化合物，其中Mollicellins I~J（15~16）、2-Hydroxymethyl-6-methylmethyleugenin（19）为新化合物。化合物16和Mollicellin H（18）对Bre-04、Lu-04、N-04细胞株生长抑制的GI50值在2.5~8.6 μg/mL。 4、从土曲霉（A. terreus）固态发酵物中鉴定了18个化合物。5个为新化合物为Terretonin A~D（24~27）和Asterrelenin（28），24~27为二倍半萜化合物，28为吲哚生物碱。 5、从杂色曲霉(A. versicolor）固态发酵物中鉴定了16个化合物。9个新的化合物Brevianamides K~N (40~43)、Averins A~C (44~46)和Glyanphenines A~B (47~48)代表三种类型的生物碱。 6、综述了1997－2007年间新的二倍半萜的研究进展。 The fungi of the genera Chaetomium and Aspergillus produce various secondary metabolites with biological activities. In order to systematically study the secondary metabolites, the solid-state fermented rice culture of three species of Chaetomium and two of Aspergillus were chemically studied. By the means of chromatograhy and spectroscopy, 55 compounds were isolated and identified, among of them 23 were new ones. The biological activities of some compounds were investigated. 1. From the fungus C. cochliodes, three new epipolythiodioxopiperazines, chaetocochins A-C (1-3) were isolated, together with 8 known ones (4-11). Compounds 1, 3 and 4 showed growth inhibitory effects against cancer cell lines Bre-04, Lu-04 and N-04 with GI50 values from 0.05 to 7.0 μg/mL. 2. Three novel isoquinolines Chaetoindicins A-C (12-14) were isolated and identified from the fungus C. indicum. Chaetoindicin A, Chaetoindicins B-C represented two classes of novel carbon skeletons. 3. Three new compounds, Mollicellins I-J (15-16), and 2-hydroxymethyl-6-methylmethyleugenin (19), were isolated from C. brasiliense. Compound 16 and Mollicellin H (18) showed growth inhibitory effects against cancer cell lines Bre-04, Lu-04 and N-04 with GI50 values from 2.5 to 8.6 μg/mL. 4. Eighteen compounds were isolated from the fungus A. terreus. Terretonin A-D（24 - 27）and Asterrelenin（28） are new compounds belonging to sesterterpoids and indole-ralated alkaloid, respectively. 5. From the fungus A. versicolor, sixteen secondary metabolites, including nine new ones, Brevianamides K-N (40-43), Averins A-C (44-46), and Glyanphenines A-B (47-48), were isolated and identified. Brevianamides K-N (40-43), Averins A-C (44-46), and Glyanphenines A-B (47-48) represented three classes of alkaloids. 6. New sesterterpenes and their bioactivities reported from 1997 to 2007 were summarized.

西藏胡黄连和鸡矢藤的化学成分及黄芪多糖的提取工艺研究

本论文由四部分组成，前三部分为实验论文，第四部分为文献综述。第一、二部分分别报道了中药西藏胡黄连和鸡矢藤的化学成分研究结果。从两种药用植物中共分离和鉴定了32个化学成分，其中3个为新化合物。第三部分为黄芪多糖的提取工艺研究。第四部分概述了近年来植物多糖的研究进展。 第一章为西藏胡黄连化学成分研究。通过正、反相硅胶柱层析等分离方法从药用植物西藏胡黄连（Picrorhiza scrophulariiflora Pennell）的根茎中共分离纯化出7个化合物。运用MS、1H-NMR、13C-NMR、DEPT、HSQC和HMBC等现代谱学方法，结合理化分析对这些化合物的结构进行了分析鉴定。7个化合物中有两个是酚性的葡萄糖苷类成分：西藏胡黄连酚苷D (1)、4-O-β-D-(6-O-vanilloyl glucopyranosyl) vanillic acid (6)；四个苯乙基苷类化合物：plantamajoside (2)、plantainoside D (3)、西藏胡黄连苷A (4) 和西藏胡黄连苷F (5)；一个苯基小分子化合物：香豆酸甲酯 (7)。其中化合物1和5未见文献报道，确定为新化合物；化合物3为首次从该种植物中分到。 第二章为鸡矢藤化学成分研究。从鸡矢藤（Paederia scandense (Lour) Merrill）全草中分离出25个化合物，通过理化常数和波谱数据鉴定了它们的结构。25个化合物中包括一个蒽醌类成分：茜根定-1-甲醚 (1)；两个香豆素：异东莨菪香豆素 (2)和5-羟基-8-甲氧基吡喃香豆素 (3)；两个香豆素-木脂素化合物：臭矢菜素 B (4)和臭矢菜素 D (5)；一个木脂素：异落叶松树脂醇 (6)；两个黄酮：diadzein (7)和蒙花苷 (8)；三个三萜类化合物：齐墩果酸 (9)、乌苏酸 (10)和 3-O-β-D-吡喃葡萄糖基乌苏烷 (11)；三个甾体及其糖苷：b-谷甾醇 (12)、胡萝卜苷 (13)和(24R)-豆甾-4-烯-3-酮 (14)；六个小分子化合物：对羟基苯甲酸 (15)，咖啡酸 (16)，香豆酸 (17)，丁烯二酸 (18)，3,5-二甲氧基-4-羟基苯甲酸(19)，咖啡酸-4-O-β-D-吡喃葡萄糖苷(20)；五个环烯醚萜类化合物：鸡矢藤苷 (21)，鸡矢藤酸 (22)，鸡矢藤酸甲酯 (23)，saprosmoside E (24)和paederoside B (25)。其中化合物25未见文献报道，为新化合物。化合物1～8、11、14、15～20为首次从该化合物中分离得到。同时对鸡矢藤中环烯醚萜类化合物做了高效液相-串联质谱(HPLC-MSn)分析，探讨了这类化合物的质谱裂解规律。 第三章为黄芪多糖的提取工艺研究。首先确定了黄芪多糖含量的测定方法，并进行了方法学验证；其次探讨了黄芪中黄芪多糖的提取工艺，确定以酶法-Sevag法联用来去除黄芪多糖中的蛋白质，可使其提取物中黄芪多糖总含量达到70％以上。 第四章为近年来植物多糖的研究进展。主要包括植物多糖的提取纯化、多糖的定性定量检测方法、多糖的结构分析和多糖的药理活性。 This dissertation consists of four parts. The first and second parts reports the studies on the chemical constituents of medicinal plants of Picrorhiza Scrophulariiflora and Paederia scandens. The third part is about the extract technique of Astragalan Polysaccharide (APS). The last part reviews the progress of the studies on plant polysaccharides. 　 The first chapter is about the chemical constituents of P. Scrophulariiflora which is widely used as an important medicine to treat various immune-related diseases. A new phenyl glycoside, scrophenoside D (1) and a new phenylethyl glycoside, scroside F (5), together with five known compounds, plantamajoside (2), plantainoside D (3), scroside A (4), 4-O-β-D-(6-O-vanilloylglucopyranosyl) vanillic acid (6); and methyl-p-coumarate (7) were isolated from the stems of P. scrophulariiflora. Their structures were elucidated by spectroscopic and chemical methods. The second chapter is about the chemical constituents of medicinal herb of P. scandens. Twenty-five compounds were isolated and purified by normal and reversed phase silica gel column chromatography. By physicochemical properties and spectral analysis, their structures were identified as rubiadin-1-methylether (1), isoscopoletin (2), 5-hydroxyl-8-methoxyl-coumarin (3), cleomiscosin B (4), cleomiscosin D (5), isolariciresinol (6), diadzein (7), linarin (8), oleanolic acid (9), ursolic acid (10), 3-O-β-D-glucopyranosyloxyl-ursane (11), b-sitosterol (12), b-daucosterol (13), (24R)-stigmast-4-ene-3-one (14), p-hydroxyl-benzoic acid (15), caffic acid (16), coumaric acid (17), trans-butenedioic acid (18), 3,5-dimethoxyl-4-hydroxylbenzoic acid (19), caffeic acid 4-O-β-D-glucopyranoside (20), paederoside (21), paederosidic acid (22), paederosidic acid methyl ester (23), saprosmoside E (24), paederoside B (25). Among them, compound 25 is a new compound. Compounds 1～8、11、14、15～20 were isolated from this plant for the first time. Futhermore, we studied the HPLC-MSn analysis and investigation of fragmentation behavior of the sulfur-containing iridoid glucosides. The third chapter is about the extracting process of Astragalan Polysaccharide (APS). The method of the content determination is built. The optimum condition of extraction of polysaccharides from Radix Astragali is defined and the more effective way to remove protein is combined enzyme method with Sevag method, by which the content of polysaccharides extract can be up to 70%. The last part is a review of the research progress of the plant polysaccharides, which includes its extraction, isolation, purification, determination, structure analysis, and pharmacology.

川西亚高山针叶林主要针叶树种年轮生态学研究

米亚罗地区是四川西部较为典型的亚高山针叶林区域之一。为建立该地区主要针叶树种岷江冷杉、云杉、紫果云杉和红杉的年轮宽度年表资料，了解不同海拔高度岷江冷杉原始林和不同恢复过程的人工针叶林及次生混交林树木径向生长规律，结合样地调查，用生长锥钻取了树木芯样做年轮生态学分析。芯样经过标准化程序固定和打磨抛光后，用WinDENDRO图像分析系统测量年轮宽度序列，用COFECHA程序交叉定年和控制测量数据质量，用ARSTAN程序建立了4个主要针叶树种的地区年表和不同海拔高度岷江冷杉林及人工针叶林和次生混交林针叶树的样地年表。 4个主要针叶树种年轮宽度年表的平均敏感度低于0.2，而其晚材宽度年表都具相对较高的平均敏感度。早材宽度与年轮总宽度标准化年表间的相关系数均在0.9以上；晚材宽度与年轮总宽度标准化年表间的相关系数则种间差异较大，红杉的最高，岷江冷杉的最低。岷江冷杉晚材宽度与年轮总宽度的相关性从1970年以后明显下降，而其他种的相关系数则随时间变化较小。树种之间标准化年表显著正相关，而云杉与紫果云杉和红杉与岷江冷杉之间相关系数明显较高。年表序列的第1主分量表达了4个树种树木共同径向生长变化格局；第2至第4主分量分别表达了云杉属和冷杉属、常绿针叶树种和落叶针叶树种以及云杉和紫果云杉树木径向生长变化差异。 不同海拔高度的8个岷江冷杉样地年轮宽度年表序列敏感度大体上随海拔高度升高而降低。各样地早材宽度与年轮总宽度年表之间的相关系数均在0.9以上，且随海拔高度变化不大；晚材宽度与年轮总宽度之间的相关系数随海拔高度的变化较大，并有随海拔升高而降低的趋势。样地年表序列之间相关系数差异很大，高海拔样地年表间多为显著正相关；低海拔样地年表间的相关系数变化不一；高海拔和低海拔样地年表之间相关性较差，且多不显著。样地年表的第1主分量能解释年表序列总方差的37.5%，反映了不同海拔高度岷江冷杉林木共同的径向生长变化格局；第2和第3主分量分别解释总方差的24.5%和18.2%，表现出明显的高海拔和低海拔样地树木间不同的径向生长变化，除一些样地例外，它们一般与低海拔样地年表有正相关，与高海拔样地年表有负相关。在那些另外的样地，海拔以外的其他因素可能也影响了树木径向生长变化。不同海拔高度样地林木的生长抑制和生长释放频率在不同时期表现出较大的差异，表明了不同的干扰历史和林木补充时间。 人工针叶林和次生混交林各样地林木早材宽度与其年轮总宽度年表之间相关系数均高达0.9以上；晚材宽度与年轮总宽度年表之间也都显著正相关，但人工针叶林样地的明显较高。样地年表序列之间的相关关系表现为，林分起源和经营管理相似的样地年表之间的相关系数明显较高，如人工针叶林与人工针叶林尽管树种不同，但样地年表之间显著正相关，而与次生混交林样地年表间关系不显著；反之亦然。综合比较各项生长参数及不同时期的树木径向生长速率，人工针叶林树木的胸径增长至少在40年以内是优于次生混交林的同种（或不同种）针叶树的。不同样地林木生长释放和生长抑制及人工针叶林树木胸高断面积增长分析表明，除严重的人为干扰外，林分郁闭后林木密度过大是造成高频率生长抑制的主要原因，在林分发育的适当时期通过抚育间伐等措施调控林分密度，是保证林木胸高断面积在一定时期内保持较高的连年增长的关键。日本落叶松作为引进的树种，在海拔3100 m左右种植表现良好，近30年来各项生长指标均高于林龄相近的云杉人工林，因此，适当用其作为川西亚高山针叶林采伐迹地快速恢复是合理的。 Miyaluo area is one of the typical regions covered by subalpine coniferous forests in western Sichuan province of southwestern China. To develop the regional tree-ring width chronology series for the dominant conifers such as Abies faxoniana, Picea asperata, P. purpurea and Larix potaninii, and to understand the radial growth patterns of conifers in Abies faxoniana natural forest stands at different altitudes, and in coniferous plantations and natural regenerated mixed stands in their different restoring processes as well, increment cores were sampled in the field together with conventional plots investigations for dendroecological analyses. After the increment cores being prepared according to standard procedures, the ring widths (total-ring and intra-ring widths) were measured with a WinDENDRO image-analysis system, and the measured tree-ring sequences were crossdated and quality-controlled with the software COFECHA. Using the software ARSTAN, we developed tree-ring width based chronology series of the four dominant conifers, eight site-specific Abies faxoniana chronologies, and seven site-specific chronologies of conifers in coniferous plantations and natural regenerated mixed stands. Mean sensitivities for total ring width chronologies of the four sampled dominant conifers were all below 0.2, while those for the latewood width chronologies of the same species were relatively much higher. Correlation coefficients between standard earlywood and total ring width chronologies of the four conifers were all above 0.9, but those between standard latewood and total ring width chronologies exhibited differences among species, with the coefficient of Larix potaninii the highest and that of Abies faxoniana the lowest. Correlation coefficients between latewood and total ring width of A. faxoniana obviously decreased from 1920-1970 for successive 50-year segments with 10-years lag analyses, though the same for the other three species changed unnoticeably with time. Tree-ring standard chronologies among species showed significant positive correlations, with the correlation coefficients between chronologies of Picea asperata and P. purpurea, and of Larix potaninii and Abies faxoniana relatively much higher. The first principal component of tree-ring chronologies represented the common radial growth patterns of the four conifers in Miyaluo area. The second, third and fourth PCs expressed the differences in radial growth responses for the genus Picea and Abies, for the evergreen and deciduous confers, and for the two species of the genus Picea, respectively. In general, mean sensitivities of the eight Abies faxoniana site-specific tree-ring width chronologies decreased with increasing altitude. The correlation coefficients between earlywood and total ring width chronologies for all sites reached 0.9, which did not change much with altitude; but those between latewood and total ring width chronologies diversified, with a decreasing tendency from lower altitudinal sites to higher altitudinal sites. Correlation coefficients among site chronologies varied considerably, with significant positive correlations among higher site chronologies, mixed results among lower site chronologies, and poor and insignificant correlations between chronologies of higher site and lower site. The first PC, which represents 37.5% of the total variance, reflected a common radial growth response at sites of different altitudes, and it showed a tendency of explaining more variance with increasing altitude. The second and the third PCs contributed to 24.5% and 18.2% of the total variance, respectively, exhibiting distinctive differences in radial growth responses between low- and high-altitudinal sites. With some exceptions, the radial growth represented by the second and third PCs had a positive correlation with that at the low-altitudinal sites and a negative correlation with that at the high-altitudinal sites. For those exceptional sites, factors other than altitude might also play a role in affecting tree-ring growth variations. Trees in stands of different altitudes showed great differences in frequencies of growth suppressions and releases through times, suggesting different disturbance histories and periods when trees recruiting to the canopy. Correlation coefficients between earlywood and total ring width chronologies for all sites of coniferous plantations and natural regenerated mixed stands were also above 0.9; and the same between latewood width and total ring width chronologies all positively correlated, too, with the coefficients of the coniferous plantations obviously much higher. Correlations among site chronologies showed that the coefficients among sites with similar stand origin and management regimes were much higher than those among sites with different stand origin and management regimes. For example, significant positive correlations were found for chronologies among different coniferous plantations, irrespective of species differences; while insignificant correlations between chronologies of the same conifer from a coniferous plantation and a natural regenerated mixed stand, and vise versa. Integrative comparisons of different tree growth parameters and radial growth rates at different stages indicated that the diameter at breast height (DBH) increments for trees in coniferous plantations were faster than those for trees of the same (or different) species in the natural regenerated mixed stands, at least within their first 40 years of stand development. Analyses of growth releases and suppressions, and basal area increments of trees in different stands demonstrated that over-dense individuals after canopy closure was the main factor resulting in high frequencies of radial growth suppressions, with some exceptions of severe man-made disturbances. Therefore, to ensure a continuous basal area current annual increment in certain periods, tree density controlling through thinning in due time during the stand development process are necessary. It should be mentioned that, as an introduced conifer to Miyaluo area, Larix kaempferi grew quite well at altitude of ca. 3100 m after planting in 1970s. In their near 30 years of stand development, Larix kaempferi trees exhibited faster growth in various parameters than Picea asperata trees of the similar stand age did. Thus we think it reasonable to use Larix kaempferi as a fast restoring species at appropriate sites of cutting blanks of subalpine coniferous forests in western Sichuan.

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.

青藏高原栽培青稞遗传多样性研究

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