丰度加权法分析冬虫夏草RAPD多态性高度差异及动态变化

北京大学学报（医学版） ›› 2014, Vol. 46 ›› Issue (4): 618-628.

丰度加权法分析冬虫夏草RAPD多态性高度差异及动态变化

姚艺桑1，高凌1，李玉玲2，马少丽2，吴子媺1，谈宁芝1，吴建勇3，倪陆群4，朱佳石3,5△

（1.美国如新华茂公司北京临床药理研究中心，北京100088； 2.青海省畜牧兽医科学院草原研究所，西宁810016； 3.香港理工大学应用生物学和化学科技系，香港； 4. Department of Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, CA 92093, USA； 5. NS Center for AntiAging Research, Provo, UT 84601, USA）

出版日期:2014-08-18 发布日期:2014-08-18

Amplicon density-weighted algorithms for analyzing dissimilarity and dynamic alterations of RAPD polymorphisms of Cordyceps sinensis

YAO Yi-sang1, GAO Ling1, LI Yu-ling2, MA Shao-li2, WU Zi-mei1, TAN Ning-zhi1, WU Jian-yong3, NI Lu-qun4, ZHU Jia-shi3,5△

(1. Pharmanex Beijing Clinical Pharmacology Center, Beijing 100088, China; 2.Institute of Grassland Research, Qinghai Academy of Animal Husbandry and Veterinary Science, Xining 100088, China; 3. Department of Applied Biology and Chemistry Technology, Hong Kong Polytechnic University, Hong Kong; 4. Department of Mechanical and Aerospace Engineering,University of California San Diego, La Jolla, CA 92093, USA; 5. NS Center for AntiAging Research, Provo, UT 84601, USA)

Online:2014-08-18 Published:2014-08-18

摘要/Abstract

摘要： 目的：检测在冬虫夏草僵虫体、子座和子囊果部位多菌共存和差异表达导致的随机扩增多态性DNA（random amplified polymorphic DNA，RAPD）分子标记多态性及其在冬虫夏草成熟过程中的动态变化。方法：采用模糊、整体性RAPD分子标记多态性研究法，选用20条随机引物，应用ZUNIX丰度加权相似度公式和丰度加权聚类法，比对冬虫夏草3个成熟期僵虫体、子座和子囊果部位RAPD分子标记多态性，以及与中国被毛孢的差异。结果：丰度非加权算法忽略了RAPD共有扩增子的丰度差异，而丰度加权ZUNIX相似度算法和聚类法，将扩增子的丰度差异及其在冬虫夏草成熟过程中的动态变化所蕴含的全部菌物分子信息汇入分析，准确获取样本间相似度和构建聚类图。冬虫夏草全部样本的总相似度为0.42，小于总相异度0.58。3个成熟期子座样本的相似度为0.57，僵虫体为0.50。同株冬虫夏草的子座和僵虫体样本的相似度随着成熟过程出现低→高→低的变化。子囊果部位与成熟子座的相似度最高（0.87），汇入一个聚类分枝；未成熟子座和僵虫体聚为另一分枝；这两个分枝聚为一群。而成熟中子座和僵虫体聚为一个分枝，与成熟僵虫体聚为另一群。中国被毛孢与冬虫夏草样本的相似度为0.55~0.69，与冬虫夏草聚类群之间被外群对照蛹虫草拟青霉分隔。结论：冬虫夏草各部位的RAPD分子标记多态性丰富，随着冬虫夏草的成熟而变化；冬虫夏草与中国被毛孢的RAPD多态性存在巨大差异，支持冬虫夏草是多菌和多种突变基因型菌共生的统一微生态系统的学说，不支持“冬虫夏草是一种真菌”和“中国被毛孢是冬虫夏草的无性世代”的学说。

关键词: 冬虫夏草, 随机扩增多态DNA技术, 丰度加权算法, 生物学标记

Abstract: Objective：To examine the dynamic maturational alterations of random amplified polymorphic DNA (RAPD) molecular marker polymorphism resulted from differential expressions of multiple fungi in the caterpillar body, stroma and ascocarp portion of Cordyceps sinensis (Cs). Methods: Used the fuzzy, integral RAPD molecular marker polymorphism method with 20 random primers; used density-weighted cluster algorithms and ZUNIX similarity equations; compared RAPD polymorphisms of the caterpillar body, stroma and ascocarp of Cs during maturation; and compared RAPD polymorphisms of Cs and Hirsutella sinensis (Hs). Results: Density-unweighted algorithms neglected the differences in density of the DNA amplicons. Use of the density-weighted ZUNIX similarity equations and the clustering method integrated components of the amplicon density differences in similarity computations and clustering construction and prevented from the loss of the information of fungal genomes. An overall similarity 0.42 (< the overall dissimilarity 0.58) was observed for all compartments of Cs at different maturation stages. The similarities for the stromata or caterpillar bodies of Cs at 3 maturational stages were 0.57 or 0.50, respectively. During Cs maturation, there were dynamic Low→High→Low alterations of the RAPD polymorphisms between stromata and caterpillar bodies dissected from the same pieces of Cs. The polymorphic similarity was the highest (0.87) between the ascocarp and mature stroma, forming a clustering clade, while the premature stroma and caterpillar body formed another clade. These 2 clades merged into one cluster. Another clade containing the maturing stroma and caterpillar body merged with mature caterpillar body, forming another cluster. The RAPD polymorphic similarities between Hs and Cs samples were 0.55-0.69. Hs were separated from Cs clusters by the outgroup control Paecilomyces militaris. Conclusion: The wealthy RAPD polymorphisms change dynamically in the Cs compartments with maturation. The different RAPD polymorphism for Hs from those for Cs supports the hypothesis of integrated micro-ecosystem Cs with multiple fungi, but does not support the “single fungal species” hypothesis for Cs and the anamorph-teleomorph connection between Hs and Cs.

Key words: Cordyceps sinensis, Random amplified polymorphic DNA technique, Densityweighted algorithms, Biological markers

姚艺桑, 高凌, 李玉玲, 马少丽, 吴子媺, 谈宁芝, 吴建勇, 倪陆群, 朱佳石. 丰度加权法分析冬虫夏草RAPD多态性高度差异及动态变化[J]. 北京大学学报（医学版）, 2014, 46(4): 618-628.

YAO Yi-Sang, GAO Ling, LI Yu-Ling, MA Shao-Li, WU Zi-Mei, TAN Ning-Zhi, WU Jian-Yong, NI Lu-Qun, ZHU Jia-Shi. Amplicon density-weighted algorithms for analyzing dissimilarity and dynamic alterations of RAPD polymorphisms of Cordyceps sinensis[J]. Journal of Peking University(Health Sciences), 2014, 46(4): 618-628.

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