【作者】 钟凤林；

【导师】 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2006， 硕士

【摘要】 龙眼是福建重要的亚热带特色水果。然而，人们对龙眼的遗传背景了解甚少，生产上存在的品种老化、良莠不一等突出问题，严重限制了龙眼的资源开发利用和生产的发展。前人应用同工酶、RAPD分析等标记对小样本的龙眼遗传资源进行了初步研究，而在整个有代表性的遗传资源的分析却未见报道。因此有必要应用RAPD分子标记技术结合POD同工酶分析技术对龙眼的遗传资源进行较为全面的遗传资源分析。本研究优化了龙眼POD同工酶提取和电泳体系；摸索出适合龙眼叶片基因组DNA的提取方法；建立和优化了龙眼RAPD反应体系；采用RAPD分子标记技术，辅以POD同工酶分析，结合聚类分析，对以福建龙眼遗传资源为主的95份国内外龙眼遗传资源，进行了遗传资源的遗传多样性和亲缘关系分析。主要研究结果如下： 一、龙眼POD同工酶提取和电泳体系的优化。针对POD同工酶酶的活性和酶带数受到材料新鲜度、提取液的成分、电泳和染色方法的影响，经过反复试验，改进确立了龙眼POD同工酶提取和电泳的优化体系，首次确定供试材料以叶龄为3～6月龄的龙眼叶片为佳，以低浓度的磷酸缓冲液(0.05 mol／L pH7.5的PBS加0.1％的Triton)为宜。同工酶电泳采取聚丙烯酰胺凝胶电泳法，染色方法采用醋酸-联苯胺染色法。改进优化的龙眼POD同工酶提取和电泳的体系，使以往只能获得的POD同工酶酶谱从8条增加到17条。 二、龙眼遗传资源亲缘关系的POD同工酶分析。整个酶谱共产生17条酶带划分为4大酶区。其中第Ⅰ酶区和第Ⅲ酶区是龙眼POD的活性区，在这两个区域的酶带比较固定，且活性大。第Ⅱ酶区和第Ⅳ酶区是龙眼POD的弱性区，所产生的酶带活性较弱。第二条、第三条酶带(迁移率分别为0.220、0.253)是龙眼样品的特征谱带。POD同工酶聚类分析结果把95份龙眼遗传资源分为2个品种类群并细分为7组。 三、龙眼叶片DNA提取方法的建立。针对龙眼叶片富含单宁和多糖的特性，采用两种提取DNA的方法：一种把Tris替代Tris-HCl的改良CTAB法；另一种为加溶解液先除掉可溶性多糖再加入裂解液的改良CTAB法。两种方法都能有效地克服多糖等物质的干扰，更加简便地从龙眼叶片提取较高质量和产量的DNA。 四、龙眼RAPD-PCR反应体系的优化。在参考一般RAPD分析的反应程序的基础上，经过反复试验，确立适宜的PCR扩增程序：94℃预变性5min，94℃1min，38℃40s，72℃2min，共45个循环，最后置4℃保温。优化的龙眼RAPD-PCR反应体系为：25μL反应总体积更多还原

【Abstract】 Longan(Dimocarpus longana Lour.) is one of the most important subtropical fruit trees in Fujian province. But the development of Longan resources are restricted severity for knowing little about the genetic background. The genetic diversity and the genetic relationship among cultivars of longan were studied by using the random amplified polymorphic DNA technique (RAPD)and the analysis of POD sioenzyme in this paper. The major results obtained from the study are summarized as follows:1. The method of POD isoenzyme extract the system of electrophoresis were optimized. According to the factors which effect the activity of POD isoenzyme and number of the bands in the gel such as the fresh and tender of the material, the component of the extracting buffer, the methods of electrophoresis and dyeing and so on, the method fit for extracting the POD isoenzyme and its system of electrophoresis were established after iterative experiments. The longan leaves age from 3 to 6 months were the best material for POD isoenzyme. The 0.05mol/L PBS (pH7.5) adding 0.1%Triton was the adaptive extracting buffer. PAGE was chosen for electrophoresis, methods of acetic acid and benzidine for dyeing.2. An analysis of the genetic relationship of longan resources using the analysis POD isoenzyme. The whole zymogramic includes 17 bands and was divided into 4 regions. RegionⅠ and Ⅲ are active regions, the bands of the enzymes in the two regions were more fixed and active. The activity of the enzymes in Region Ⅰ and Ⅲ were weaker. The second and third bands (whose RF were respectively 0.220 and 0.253) were the fundamental bands of longan. Using clustering analysis by POD isoenzyme, the 95 genetic resources of longan were divided into 2 types and 7 subtypes.3. Establish the method of longan genomic DNA extraction from leaves. Some secondary metabolites such as polyphenols, polysaccharides and pigments were rich in the leaves which are easy to form complex with DNA. The complex was insoluble and could inhibit theactivity of Taq更多还原