【作者】 徐柯；

【导师】 乔代蓉；

【作者基本信息】 四川大学 ， 微生物学， 2003， 硕士

【摘要】 本论文以四川，湖北，浙江，上海16个麦冬（Ophiopogon japonicus （L.f.）Ker-Gawl.）自然居群及2个同属野生亲缘种沿阶草为试材，利用RAPD技术研究了麦冬的遗传多样性，同时探讨了实验条件的优化问题。针对RAPD标记影响因素众多、结果重复性低的特点，对RAPD分析中PCR扩增的各种条件进行了梯度测试，包括反应缓冲液中Mg²⁺浓度、Taq酶量、引物浓度、模板浓度、PCR循环数等。得到了适于麦冬RAPD分析的主要实验条件。在RAPD分析中使用了40个随机引物对18个样本进行分析，经反复实验，筛选出8个有效引物，产生了504条多态性带，得到了18个样品的RAPD指纹图谱。应用最短距离法获得了18份材料的遗传距离矩阵及聚类分析树系图。分析发现：沿阶草与麦冬亲缘关系较远；四种绵阳麦冬在带型上无任何分别，聚类上归为一类，说明绵阳麦冬具有很强的道地性；而川大大叶型麦冬与其他居群的亲缘关系最远；不同地域及叶型的麦冬居群间遗传差异较大。 为了从麦冬干燥块根中制备DNA用于RAPD分析，以便于建立一种快速高效实用的分子鉴定方法，又从绵阳营城匍匐型麦冬干燥块根（成药）中直接提取总DNA，用上述40个10碱基随机引物对其进行RAPD扩增，筛选出11个有效引物，同时从鲜叶及硅胶干燥保存的叶片提取DNA进行对比实验。结果从电泳结果看，三者中制备的DNA在用于RAPD扩增时并无明显差异。说明从麦冬中药成品中制备的DNA具有很好的完整性和足够的浓度，完全可以用于RAPD分析。 本论文还对将RAPD标记转化为更为稳定单一的SCAR标记进行了一足。二研究。挑选了两条特异片段进行克隆、测序，再设计特异引物进行扩增验证二虽然由于时间关系验证工作还未完成，但为进一步的工作积累了经验。更多还原

【Abstract】 The use of the RAPD technique was investigated on 16 natural populations of O.japonicus (L.f.) Ker-Gawl. in Sichuan, Hubei, Zhejiang, Shanghai, and two wild relational species, O. bodinieri Levl. and O.bockianus Diels, which belong to the same genus with O.japonicus (L.f) Ker-Gawl., after optimizing PCR conditions. Due to high sensitivity of RAPD analysis to reaction conditions, main factors affecting the results including concentrations of Mg2+, Taq DNA ploymerase, primers and templates, and number of PCR cycle etc., were examined. Conditions applicable to RAPD analysis of O.japonicus were determined. The screening of 40 decamer oligonucleotides allowed the selection of 8 primers used for the analysis. A total of 504 RAPD markers were produced from the 8 selected primers. Fingerprints of 18 samples had been constituted. Relationships among the 18 populations were clustered using the least distance method analysis in a dendrogram. The largest distance was found between O.bockianus Diels, O. bodinieri Levl. and O.japonicus (L.f.) Ker-Gawl.. There had no difference in four samples of Mianyang City during dusting. It investigated their special characters were related to geographic regions. The largest distance was found between big leaf type population in SichuanUniversity and the other populations. Large genetic variation occurred among the different clime and leaf-type populations.To establish a kind of rapid and efficacious molecular method to identify the Ophiopogon japonicus(L.f.)Ker-Gawl. by officinal directly, we prepare DNA from its dried root for RAPD. Extracted total DNA from dried root of the sprawled sample of Mianyang Yingcheng. Same 40 random decarner primers were screened for RAPD fragments.il identifiable primers were selected. Extracted DNA from leaves dried through silica gel and fresh leaves for contrastive experiment in the same time. In result, there were no any obvious different RAPD results in the three kinds of template DNA from electrophoresis. It was concluded that the quality and quantity of the template DNA prepared from officinal was enough for RAPD.Research of transferring RAPD marker to SCAR marker also had been done. Select two specific fragments to clone and sequence. Then design specific primers to amplify the fragments as verification. Though the verification had not been completed, experience for advancing works had been accumulated.更多还原