【作者】 秦素玲；

【导师】 刘震；

【作者基本信息】 河南农业大学 ， 森林培育， 2006， 硕士

【摘要】 悬铃木(Platanus acerifolia)为世界著名的行道树和庭荫树，素有“行道树之王”的美称。但每年冬芽萌发，老果脱落，新花、新果生长之际，老果脱落产生的绒毛，新生的雄花序散落的大量花粉都从树上飘落下来，漫天飞舞，不仅污染环境，而且会刺激呼吸道，造成呼吸道障碍，皮肤瘙痒、迷眼、过敏等危害，严重影响人们的生活及身体健康。因此，如何解决悬铃木的果毛污染问题具有重要的现实意义。长期以来，众多研究者在此方面做了不同的探索和尝试，如利用物理、化学因素进行人工诱变，采用树冠嫁接，修剪控果等方法，也采用过AC合剂等化学药剂的处理，并取得了一定的进展，但尚没有从根本上解决问题。 我们从6年前开始，以郑州为中心，调查附近各大城市与乡村，通过自然选种途径，对形态上自然变异产生的无球少球悬铃木单株进行跟踪调查，并且采用过氧化物酶、苹果酸脱氢酶、乙醇脱氢酶、酯酶四种同工酶进行分析鉴定和聚类分析，结合形态标记和同工酶标记初步选育出了无球悬铃木类型和少球悬铃木类型。本文在以上研究的基础上，对形态标记为无球少球悬铃木类型中生长状况良好的6株单株进行AFLP分子标记，通过进行悬铃木叶片DNA提取、酶切连接、预扩增、选择性扩增、银染等实验过程，得到了清晰的悬铃木无球少球品种的AFLP指纹图谱。并且与同工酶标记相比较，从而从分子水平上鉴定无球少球悬铃木品种的遗传变异和遗传稳定性，结论总结如下： 1)在悬铃木叶片DNA提取过程中，采用CTAB法和酚-氯仿法两种方法，提取到了更理想的适于AFLP实验的悬铃木叶片的组DNA。试验中通过采用饱和酚-氯仿相结合来变性蛋白质，变性效果比单一使用氯仿彻底；为了防止酚类氧化造成DNA降解，提高了巯基乙醇的浓度，并加入了PVP；纯化时采用DNA干燥后复溶重复抽提的办法，解决了由于悬铃木叶片中含有较多酚类物质及其他次生物质而较难提取，组DNA较难被限制性内切酶消化等问题，为悬铃木的分子系统学研究奠定了基础。 2)优化了AFLP分子标记反应体系，找出最适合酶切连接的DNA浓度，完成了悬铃木叶片DNA提取、酶切连接、预扩增、选择性扩增、银染等试验过程。通过试验筛选出6对适合悬铃木AFLP分子标记的引物，得到清晰的少球无球悬铃木品种的指纹图谱。 3)利用AFLP技术确定出无球少球悬铃木与有球悬铃木的遗传差异与多态性。筛选出的6对引物对所得到的条带数在21-49之间，共得到217条带，平均每个引物对得到36条带：多态性条带数在15-43之间，平均28条；多态性比率为65.2％-87.7％，平均78.6％。AFLP分子标记技术作为检测种内植物遗传变异是十分有效的工具，6对引物共检测到特异性条带55条，隐性特异性条带48条。其中在引物对M74P50的电泳图谱中，分子量约为428bp处，无球品种均有显著的条带而少球无球品种均无条带，究竟此DNA片段是不是控制悬铃木结果的基因，还有待于进一步的研究证实。 4)通过AFLP分子标记遗传图谱和聚类分析可以看出，1、2、3号为无球单株，4、5号为少球单株。从形态特征来看，虽然6号品种形态表现为无球性状(6号形态特征枝条通直；树皮青灰色，粗糙；叶形掌状，叶缘有少量锯齿，叶基平截，叶先端尾尖，叶裂三裂，三出脉；无球)，但在遗传水平上却同7号(形态特征枝条通直；树皮青灰色，粗糙；叶形掌状，叶缘有锯齿，叶基近平截，叶先端渐尖，叶浅裂三更多还原

【Abstract】 none-or-little cone varieties of Platanus acerifolia were identified by AFLP techniques in this paper. The main results were as follows:1. During DNA extraction of Platanus acerifolia, leaves obtained by water cultivate are supervisor than those obtained directly in fiedle. The quantity of DNA in CTAB method is worse than in hydroxybenzene-chloroform method. Through the new method in DNA extraction of remelting after dryness two times, high quality and pure DNA suitable to AFLP is received. So the problem that DNA of Platanus acerifolia difficult to extract by means of so many hydroxybenzene substances is solved. That would exert important effects on molecular systematics.2. By finding suitable DNA concentration in restriction-ligase reaction and using 6 primers, a better silver-staining AFLP system for Platanus acerifolia was developed.Choosing primer combinations 3+3 and 3+2 obtained high stability and polymorphism while the bands of primer conbinations 2+2 were too denseness to stat.3. Identified genetic variation and polymorphism of Platanus acerifolia by AFLP techniques. The bands available from each of 6 primers were between 21 and 49 , the total were 217 and the average was 28. The polymorphic bands were between 15 and 43, and the average was 28. As a tool of identification of genetic variation , AFLP is very effective. The total of the specific bands were 55(48). In the silver-stained results of primer combinations M74P50, there were clear bands at 428bp of all the none cones Platanus acerifolia. Whether it is the gene controlled blooming and fruit or not, more experiments are expected.4. Through genetic map and clustering analysis, it was regarded the tree of number 1、 2and 3 as none cone variety of Platanus acerifolia and the tree of number 4、5、6 as little cones variety. From morphological characteristics, number 6 variety is none cone variety, but has genetically near with number 7 which is cone variety.5. compared results from AFLP techniques with peroxidase isozyme and by finding the same更多还原