论文标题:南瓜种质资源遗传多样性与多糖多样性的研究
Studies on Genetic and Polysaccharide Diversity in Cucurbita Germplasm Resources
论文作者
论文导师 向长萍,论文学位 博士,论文专业 蔬菜学
论文单位 华中农业大学,点击次数 100,论文页数 139页File Size8111K
2006-09-01论文网 http://www.lw23.com/lunwen_7580067/
Cucurbita;; germplasm genetic diversity;; morphology;; RAPD;; polysaccharide;; extraction;; purification;; identification;; anti-oxidative
南瓜(Cucurbita)是十分重要的瓜类蔬菜作物，因其栽培简单，产量高，易于贮运，而且营养丰富，深受人们的喜爱。本研究以本实验室近年来收集的70份南瓜资源为研究对象，从形态性状与分子水平上分析了这些资源的遗传多样性，有利于对这些种质资源的收集、保存、鉴定、创新和有效利用。同时以功能型南瓜品种的选育为最终目的，首次对不同品种南瓜种质资源从提取、分离纯化、生物活性的研究等多方面展开对南瓜重要的生物活性物质——多糖的探讨性研究，以探明高活性南瓜多糖的资源条件，为保健品质育种材料的选择、选配以及功能性南瓜的选育提供一定的理论依据。实验内容主要如下： 1．对70份南瓜种质45个植物学性状进行鉴定和分析，平均变异系数为32.16％，平均多样性信息指数为1.25。基于形态数据的聚类分析，70份南瓜种质被分为三大类群——中国南瓜、美洲南瓜、印度南瓜。主成分分析表明，前三个主成分的累计方差贡献率为67.31％，利用形态性状对南瓜分类时，首先应考虑的是种子、瓜梗和花的特征，其次是根据植株的生长习性分类，然后是根据瓜的形状进行分类。 2．从100个引物中筛选出21个随机引物，对70份南瓜种质进行了RAPD分析，共扩增出168条带，其中156条表现多态性，占总数的92.9％。平均每个引物产生7.43条多态带。基于RAPD标记的聚类分析，将70份南瓜种质划分为了三大类。同时在三大类群内，又将种质进行了细分，第一类分为了三组，第二类分为了六组，第三类分为了五组，其结果与地域来源和形态特征有一定的相关性。主坐标分析与系统聚类分析结果基本一致。 3．对植物学性状和RAPD两种标记间的相似系数矩阵的相关性进行Mantel检验，得出形态标记与分子标记的相关系数为0.6306；将植物学性状和RAPD标记的聚类结果，按CONSEN程序中进行作图，获得两种聚类结果的一致树图。表明形态标记与分子标记聚类结果存在着一定程度的一致性，也存在差异，应结合使用。 4．研究了南瓜多糖的热水提取工艺和微波辅助提取工艺，对其工艺参数进行了优化，得出水提法提取南瓜多糖的过程中，料液比是影响多糖提取率的最关键因素，南瓜多糖提取的最佳工艺条件是以1:40的料液比，在70℃水浴条件下，提取3次，每次提4h。而在微波提取工艺中，影响多糖提取率各因素的主次关系为：微波强度＞微波时间＞料液比，微波提取法最适工艺条件为：微波强度60％，微波时间20min，料液比1:30。通过体外抗氧化实验证明，不同方法提取的南瓜多糖均具有一定的抗氧化活性，微波工艺对南瓜多糖的体外抗氧化活性在超氧阴离子自由基(O_2~-)清除能力上有微弱的下降趋势。微波工艺在南瓜多糖提取上的有一定的应用前景。 5．研究了南瓜多糖体外清除自由基以及抗氧化的作用。结果表明南瓜多糖可以清除·OH、O_2~-，抑制·OH所致丙二醛的产生，减少·OH所致红细胞溶血，减轻·OH所致肝线粒体肿胀程度。 6．本文首次对56个不同品种南瓜多糖的提取率、含量，多糖中中性糖、糖醛酸含量及其体外抗氧化活性进行了分析，不同南瓜品种的多糖的各个指标含量的变幅和变异系数较大，表明南瓜多糖特性和生物活性存在品种的多样性。主成分分析将56份南瓜种质按多糖含糖量和清除自由基能力的强弱分为了四组，第四组的三份种质46(02-4)、38(H34)、5(H13-3)多糖含量及多糖清除自由基的能力均较强，在保健品质育种上比较有应用价值。 7．采用DEAE-52纤维素柱层析和Sephadex G-200凝胶柱层析相结合的方法对两个品种南瓜多糖进行分级，获得六个精制多糖组分ZPP1a，ZPP2，ZPP3和YPP1，YPP2，YPP3a。紫外光谱分析表明六个多糖不含有核酸和蛋白质等杂质，是均一多糖。对六个多糖的基本特性和理化性质进行了研究，并用红外光谱对其结构进行了初步表征。表明中国南瓜02-4和印度南瓜H34提取的多糖各级分在基本特性、生物活性及结构上存在着差异。综合评价各个指标表明中国南瓜02-4优于印度南瓜H34，因而具有更好的开发利用前景。
Pumpkin (Cucurbita) is one of the major vegetable crops in Cucurbit family. Due to its easy cultivation, high yield, long shelf life and abundant nutriments, pumpkin is very popular in China. In this paper, 70 accessions in cucurbita from different countries or regions were analysised by using morphological and RAPD markers. The study on the genetic diversity in cucurbita can benefit the conservation, classification, identification, genetic enhancement and effective utilization of cucurbita germplasm resource. In this paper, aiming to select Pumpkin which has high content of polysaccharide, we also discussed the diversity of the different pumpkin polysaccharide from several aspects including the extraction, purification, identification and physiological activity. The polysaccharide analysis of 56 accessions provided some useful information and facilitated the selection of pumpkin breed possessed biological activity. The main research contents and results were as follows: 1. In 45 morphological assessments, the average coefficient of variation was 32.16%. The average Shannon"s Information index among the assessments was 1.25. The 70 accessions were clustered into 3 groups based on the morphological data: Cururbita moschata, C. pepo, C. maxima. The results of principal components analysis showed that the accumulative variance contribution of the first three principal components was 67.31%. The characteristics of fruit stem, seed and flower, fruit shape and growth habit are the primary factors affecting cucurbita germplasm classification according to morphological characters. 2. Twenty one random primers which used in the experiment produced 168 consistent and differential amplification products. Among them, 157 bands (93.5%) were polymorphic. Cluster analysis revealed three clusters: Cururbita moschata, C. pepo, C.maxima. This conclusion is completely consistent with the traditional cultivar classification. And within C.moschata, the dendrogram shows the existence of three sub clusters. Within C. pepo, the dendrogram shows the existence of six sub clusters. Within C.maxima, the dendrogram shows the existence of five sub clusters. This conclusion is correlative with their origins and morphotypes. Principal coordinate analysis also revealed three clusters. The results from these two methods were similar. 3. Comparison of the data obtained by morphological markers and molecular makers was made by the method of Mantel test. The r correlation value scope was 0.6306 between the morphological markers and molecular markers; A consensus tree based on morphological marker and molecular markers was produced by CONSEN module in NTSYS. The result shows that there are some difference and also some comparability between the genetic relationship among cucurbita accessions revealed by RAPD and that by morphological markers. 4. In order to study the hot water extraction and microwave extraction technology of Pumpkin polysaccharide, we used the methods of single factor test and orthogonal design test of L9 (3~4) and their antioxidant activities were studied simultaneously. The result showed that the highest extraction rate of Pumpkin polysaccharide by hot water extraction could be obtained with a ratio of solid to liquid with 1:40, 70℃for 4h and 3 times extraction. In microwave extraction technology, the optimal conditions were as follows: the radio of solid to liquid with 1:30 and the extraction time of 20min under the microwave intension of 60%. In contrast to the hot water extraction method, the extraction rate of the microwave technique was higher and the extraction time was shorter. Meanwhile, anti-oxidative results showed Pumpkin polysaccharide extracted by the two methods was effective to anti-oxidation. 5. The scavenging effect of Pumpkin polysaccharide on·OH and O_2~-were studied. The result showed that Pumpkin polysaccharide had a significant effect on scavenging·OH and O_2~-. It could inhibit the generation of MDA in liver homogenate, the swelling of mitochondria and hemolysis of RBC induced by·OH. The conclusion was that Pumpkin polysaccharide had the effect of anti-oxidation induced by OH. 6. Polysaccharide was extracted from 56 cultivars of pumpkin, and their composition and activity were analyzed. The results showed that there were obvious differences not only in extracting rate, but also in content of neutral sugar, uronic acid of PP, which led to different inhibiting effects on hydroxyl radical (·OH) and superoxide anion radical(O_2~-).In conclusion, diversity existed in characteristics and bioactivities of PP among pumpkin cultivars. The 56 accessions were clustered into 4 groups based on the composition and activity data of PP by the method of principal coordinates analysis. In the fourth group, 3 accessions content of PP and inhibiting effects on hydroxyl radical (OH) and superoxide anion radical(O_2~-) are also better. 7. Two PP from two different pumpkin varieties were graded by DEAE-52 cellulose column and Sephadex G-200 column. Six components, ZPPla, ZPP2, ZPP3, YPP1, YPP2, YPP3a were obtained. By UV spectrum analysis, six components were shown to be homogeneous. Their physical, chemical characters, composition and activity were studied, and their structures were analyzed by IR (Infra-Red) spectra. The results showed that there were typical characteristic absorption peaks of polysaccharide in their spectrograms. It testified that the six components were polysaccharide. The results also showed that diversity existed in characteristics and bioactivities of six polysaccharide components between H34 and 02-4. In conclusion, 02-4 is prior to H34 and could be a useful breeding germplasm.
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