松乳菇及其分离菌株的RAPD-PCR分析研究
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摘要
论文首次对松乳菇及其分离菌株进行了RAPD-PCR分析研究。采用筛选并改进的适宜于松乳菇及其分离菌株DNA抽提的四种不同方法，提取了来源于醴陵山区的松乳菇子实体及其通过本实验室组织分离并经摇瓶纯培养得到的5株菌株的基因组DNA，摸索出了一套松乳菇及其分离菌株DNA的高效提取方法——综合DNA抽提法。对松乳菇RAPD的反应体系及反应条件等进行了一系列的探索，并在此基础上对松乳菇及其分离菌株的DNA进行了分子水平上的鉴别研究，对分离菌株的稳定性进行了初步分析。实验对100条10bp的3’端随机引物进行了筛选，筛选出了条带清晰、重复性好的25条引物，并用这25条引物对松乳菇及其分离菌株的DNA进行了扩增，共获得185个扩增标记位点，平均每条引物7.4个。通过对松乳菇子实体及其分离培养菌株共6个样品DNA的遗传指纹图谱分析，统计出了它们之间的遗传相似系数，5个分离菌株与松乳菇子实体间的相似性性系数都比较高，在0.8000～0.9784之间。其中2号和3号菌株与1号样品(子实体)之间的DNA相似系数均在0.9351以上，尤其是2号和1号间相似系数高达0.9784。4号菌株是传代二次的松乳菇分离菌株，它与1号样品(子实体)之间的DNA相似系数为0.8541。5号和6号菌株是传代三次的松乳菇分离菌株，它们与子实体之间的DNA相似性系数分别为0.8000和0.8108，相似系数均比较高。相似系数的统计分析显示松乳菇子实体与分离菌株同种。同时以相似系数为依据，进行了UPMGA聚类分析，6株供试样品在0.8左右的相似水平上聚成一大类，聚类分析结果亦显示松乳菇子实体与5株分离菌株同种。从两种分析结果均可以判定5个分离菌株是松乳菇菌种(Lactarius deliciosus)。进一步根据相似系数及UPMGA聚类图分析了分离菌株的遗传稳定性，分析表明遗传变异是绝对的，而稳定性是相对的。本研究摸索出了一套松乳菇及其分离菌株DNA的高效提取方法，为松乳菇分离菌株的鉴别分析找到了一个便捷而又科学的新途径，并为松乳菇在分子生物学方面的进一步研究提供了参考和依据。
It' s the first time for this dissertation to analyze Lactarius deliciosus and isolated strains with RAPD-PCR. Four different DNA extractions which are improved after selection are used in this research. The DNA of Lactarius deliciosus and isolated strains are extracted, Lactarius deliciosus come from LiLing mountainous area of china, and the isolated strains which are cultivated in shaking flask are obtained from tissue culture of Lactarius deliciosus. A highefficiency DNA extraction, synthetical extraction, which can be used for Lactarius deliciosus is obtained. A series of searches are experimented about the conditions of RAPD-PCR , with which the heredity differentia between Lactarius deliciosus and isolated strains are analysed on the molecule level. Moreover, the heredity stability of isolated stains is analysed. Twenty five primers whose DNA fingerprinting patterns are clear and repeated highly are selected from one hundred 10bp random primers. 185 loci are obtained in the amplification for the experi
mental samples with these 25 primers, 7.4 loci per primer averagely. The similarity coefficients are calculated on the base of the six samples' DNA fingerprinting patterns. The similarity coefficients which range from 0.8000 to 0.9784 are high between Lactarius deliciosus and isolated strains. The similarity coefficients between strain 2, strain 3 and sample 1 are all over 0.9351. Especially, the similarity coefficient of sample 1 and sample 2 is higher to 0.9784. Strain 4 is the second generation from Lactarius deliciosus, and the similarity coefficient of sample 1 and sample 4 is 0.8541. The similarity coefficients between Lactarius deliciosus to strain 5 and strain 6, which are isolated and cultivated from generation to generation for third times, are 0.8000 and 0.8108 respectively. These similarity coefficients are high. The conclusion that Lactarius deliciosus and isolated strains are the same species can be drawn. Furthermore, cluster analysis(UPMGA) is applied in the light of the six samples' similari
ty coefficients. The result of the cluster analysis shows that six samples cluster together at similarity coefficient 0.8, more or less. The initial conclusion that Lactarius deliciosus and five isolated strains are the same species can also be obtained. Both in the similarity analysis and the cluster analysis, the conclusion that Lactarius deliciosus and isolated
strains are the same species can be indicated. What' s more, the heredity differentia and heredity stability of the isolated strains are analysed according to the similarity confident and cluster analysis, the analysis indicate that heredity stability of the isolated strains are relative. A high efficiency DNA extradition for mycorrhizal fungi is obtained. In addition, a new, fast, convenient and scientific approach which is used in the discriminate for the isolated strains of Lactarius deliciosus comes into being. The very convincing references are provided in this dissertation to study the molecule biology of Lactarius deliciosus further. This paper lays a foundation for the research and exploiture on Lactarius deliciosus.
It' s the first time for this dissertation to analyze Lactarius deliciosus and isolated strains with RAPD-PCR. Four different DNA extractions which are improved after selection are used in this research. The DNA of Lactarius deliciosus and isolated strains are extracted, Lactarius deliciosus come from LiLing mountainous area of china, and the isolated strains which are cultivated in shaking flask are obtained from tissue culture of Lactarius deliciosus. A highefficiency DNA extraction, synthetical extraction, which can be used for Lactarius deliciosus is obtained. A series of searches are experimented about the conditions of RAPD-PCR , with which the heredity differentia between Lactarius deliciosus and isolated strains are analysed on the molecule level. Moreover, the heredity stability of isolated stains is analysed. Twenty five primers whose DNA fingerprinting patterns are clear and repeated highly are selected from one hundred 10bp random primers. 185 loci are obtained in the amplification for the experi
mental samples with these 25 primers, 7.4 loci per primer averagely. The similarity coefficients are calculated on the base of the six samples' DNA fingerprinting patterns. The similarity coefficients which range from 0.8000 to 0.9784 are high between Lactarius deliciosus and isolated strains. The similarity coefficients between strain 2, strain 3 and sample 1 are all over 0.9351. Especially, the similarity coefficient of sample 1 and sample 2 is higher to 0.9784. Strain 4 is the second generation from Lactarius deliciosus, and the similarity coefficient of sample 1 and sample 4 is 0.8541. The similarity coefficients between Lactarius deliciosus to strain 5 and strain 6, which are isolated and cultivated from generation to generation for third times, are 0.8000 and 0.8108 respectively. These similarity coefficients are high. The conclusion that Lactarius deliciosus and isolated strains are the same species can be drawn. Furthermore, cluster analysis(UPMGA) is applied in the light of the six samples' similari
ty coefficients. The result of the cluster analysis shows that six samples cluster together at similarity coefficient 0.8, more or less. The initial conclusion that Lactarius deliciosus and five isolated strains are the same species can also be obtained. Both in the similarity analysis and the cluster analysis, the conclusion that Lactarius deliciosus and isolated
strains are the same species can be indicated. What' s more, the heredity differentia and heredity stability of the isolated strains are analysed according to the similarity confident and cluster analysis, the analysis indicate that heredity stability of the isolated strains are relative. A high efficiency DNA extradition for mycorrhizal fungi is obtained. In addition, a new, fast, convenient and scientific approach which is used in the discriminate for the isolated strains of Lactarius deliciosus comes into being. The very convincing references are provided in this dissertation to study the molecule biology of Lactarius deliciosus further. This paper lays a foundation for the research and exploiture on Lactarius deliciosus.
引文
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