【作者】 汪曙；

【导师】 林毅；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 霍山石斛与铜皮石斛是安徽省霍山县主要产有的两种石斛，前者为中药石斛中的上品，品质较后者好，但生长量很低，而后者生长量大。为改良霍山石斛种质，前人以霍山石斛为父本，铜皮石斛为母本培育出杂交石斛（F1代）。本文通过生长、生理指标及主要有效成分含量测定对杂交石斛与其亲本进行了全面比较并分析了杂交石斛的超亲优势；同时提取纯化了杂交石斛及其亲本的重要药效成分石斛多糖，并进行了理化性质鉴定，紫外、红外光谱分析，单糖组成气相色谱分析，蛋白质含量及氨基酸组成分析；还对三种石斛多糖的体外抗氧化作用进行了初步研究。结果如下：1.霍山石斛杂交种（杂交石斛）生长量积累表现为近铜皮石斛的特性，有效成分含量积累则表现为近霍山石斛的特性，在生长和有效成分积累方面结合了两个亲本之间的优势，具有很大的开发利用前景。 2.杂交石斛及其亲本新茎的生长高峰期是5-8月，此后生长减缓；8月中旬茎粗最高，后稍有下降。在此期间杂交石斛的茎长、节数、节间长、叶数、叶面积、单株鲜重和干重等生长指标与铜皮石斛相近，明显高于霍山石斛。与一年茎相比，霍山石斛基本不再生长，但铜皮石斛、杂交石斛还有生长。一年与二年生杂交石斛的茎长、节数、单株鲜重和干重均比亲本有明显的优势；三年生杂交石斛除茎粗稍有优势，茎长、节数、节间长、单株鲜重和干重均不及铜皮石斛，但较霍山石斛仍有极大优势，单株鲜重接近其5倍，单株干重是其6.2倍。3.杂交石斛及其亲本新茎生长期中叶片叶绿素含量5月较低，6月明显增高，7月到8月初稍有降低，11月初最高。其间杂交石斛叶绿素含量一直低于霍山石斛，11月初才开始高于霍山石斛；但从8月中旬开始就极显著高于铜皮石斛。一年生杂交石斛叶绿素含量、吸收光谱峰值处的吸光度以及光合速率均极显著高于两亲本。4.多糖含量均为二年茎最高，三年茎下降较大；杂交石斛二年茎与三年茎多糖含量与霍山石斛接近，低于铜皮石斛。生物碱含量在三年中均为逐年增高，杂交石斛二年茎与三年茎生物碱含量与霍山石斛接近，极显著高于铜皮石斛。杂交石斛二年茎可溶性蛋白质含量极显著高于两亲本，三年茎可溶性蛋白质含量接近霍山石斛，极显著高于铜皮石斛。其中霍山石斛三年茎可溶性蛋白质含量增加很多，较二年茎增长2.5倍。5.三种石斛多糖的理化性质及组成分析结果为：三种多糖为白色絮状固体，均溶于水，不溶于高浓度的乙醇、丙酮等有机溶剂；鉴定反应呈现多糖的特征颜色反应；为非淀粉类中性多糖，不含还原糖。紫外光谱分析均未发现260nm和280nm附近核酸和蛋白质的特征吸收峰，红外光谱分析3500~2800cm-1及1400~1200 cm-1的一些吸<WP=4>收峰表明为糖类化合物。霍山石斛、铜皮石斛及杂交石斛多糖的单糖组成及摩尔比分别为L-鼠李糖—L-阿拉伯糖—D-甘露糖—D-葡萄糖=0.087：1：102：79.5，L-鼠李糖—L-阿拉伯糖—D-木糖—D-甘露糖—D-葡萄糖=0.4：1：1.51：63.0：35.4，L-鼠李糖—L-阿拉伯糖—D-甘露糖—D-葡萄糖=0.17：1：81.9：52.1；三种多糖中蛋白质含量低于0.6%，含有16种常见氨基酸，各种氨基酸相对百分含量有所不同。6.体外抗氧化作用初步研究结果为：在实验浓度范围内（分别为0.5~6 mg/ml、0.1~4 mg/ml），三种石斛多糖对·OH及O2˙ˉ均表现出一定的抑制作用，其作用随着浓度的增加而增大。经计算霍山石斛、铜皮石斛及杂交石斛多糖对·OH的半数清除浓度分别为6.79、 6.75和 7.09mg/ml，对O2˙ˉ的半数抑制浓度分别为3.04、 3.44和 3.02mg/ml。1 mg/ml甘露醇对·OH的清除率为7.64%，1 mg/ml维生素C对O2˙ˉ的抑制率为20.87%，均低于同等浓度霍山石斛、铜皮石斛及杂交石斛多糖对·OH的清除率和对O2˙ˉ的抑制率。说明三种多糖均有较强的·OH 和O2˙ˉ清除能力。此外三种石斛多糖对体外温育和Vc-Fe2+系统诱导的肝匀浆的脂质过氧化均有一定的抑制作用，但未表现出剂量效应；并可减轻Vc-Fe2+系统诱导所致小鼠肝线粒体氧化损伤程度，且表现出一定的剂量效应。更多还原

【Abstract】 D. huoshanense and D. moniliforme is the main species of dendrobium producted in Huoshan County, Anhui Province. D. huoshanense is the best among various dendrobium, its character is better, but its growth is greatly lower than D. moniliforme. To improve dendrobium species in Huoshan, researchers breeded hybrid through interspecific hybridization (D. huoshanense as male parent, and D. moniliforme as female parent). This article fully compares hybrid and its parents by growing, physiologic index and content measuring of effective components, and analyses over-parent heterosis of hybrid. Meanwhile purify and analyze their important medicinal components—polysaccharides through ultraviolet and infrared spectrum, physical and chemical properties determining, gas chromatograph analysis of sugar composing, protein content measuring and chromatogram analysis of amino acids. Besides we have an initial research on anti-oxidation actions of three kinds of dendrobium polysaccharides. Results are as follows: 1. The growth of hybrid is close to D. moniliforme, the accumulation of effective components of hybrid is close to D. huoshanense. The hybrid integrates superiority of parents on growth and accumulation of effective components，having vast vistas for development and utilization.2. The growth peek of new stem of hybrid and its parents is from May to August, after that their growth slows down. The stem perimeter reaches the highest in the middle ten days of August, and then it drops a little. In the period, the stem length, the stem paragraphs, the length of stem paragraph, the leaf number, the leaf area, the fresh and net weight of each plant all are close to D. moniliforme, and apparently higher than D. huoshanense. Compared to one-year-stem, D. huoshanense almost stops growing, but D. moniliforme and hybrid still grows. As to one-year-hybrid and two-year-hybrid, the stem length, the stem paragraphs, the fresh and net weight of each plant all have an apparent advantage over its parents. But as to three-year-hybrid, except for the stem perimeter, the stem length, the stem paragraphs, the length of each stem paragraph, and the fresh and net weight of each plant all are inferior to D. moniliform, but they still have extreme advantage over D. huoshanens, the fresh weight of each plant is about five times, and the net weight of each plant is 6.2 times.3. As to the chlorophyll content of leave during the growth of new stem, it is lower in May, increases in June, decreases a little in summer, and reaches the highest in November. In the <WP=6>period, the chlorophyll content of hybrid is lower than D. huoshanense until November, but apparently higher than D. moniliform from the middle ten days of August. As to one-year-hybrid, the chlorophyll content, A value at the peak of its absorption spectrum and the photosynthesis rate all are markly higher than its parents. 4. As to the content of polysaccharides, the two-year-stem all reach the highest, and the content all decrease much the third year. The content in two-year-stem and three-year-stem of hybrid is close to D. huoshanense, lower than D. moniliforme. As to the content of alkaloids, they all increase year by year in three years. The content in two-year-stem and three-year-stem of hybrid is close to D. huoshanense, and markly higher than D. moniliforme. And as to soluble protein content, the two-year-stem of hybrid is higher than its parents, the three-year-stem of hybrid is close to D. huoshanense, markly higher than D. moniliforme. The content in three-year-stem of D. huoshanense increases a lot, reaching 2.5 times as much as two-year-stem. 5. The analysis results of three kinds of dendrobium polysaccharides are as follows: They are white solid, soluble in water, not soluble in organic solvent such as ethanol, et al. And they all have characteristic colour reactions of polysaccharides. They are neutral polysaccharides, containing no starch, no reductive sugar. In ultraviolet spectrum there isn’t absorption peak of nucleic acid and protein, and the abs更多还原