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菌物学报 jwxt@im.ac.cn 15 January 2015, 34(1): 38‐52 Mycosystema ISSN1672‐6472 CN11‐5180/Q © 2015 IMCAS, all rights reserved. 研究论文Research paper DOI: 10.13346/j.mycosystema.130198 基金项目:国家教育部博士点基金项目《冬虫夏草复合体居群DNA序列生态地理变异式样研究》(20125301110001)、国家火炬计划产业化示范项目《云南云百草牌虫草系列产品产业化》(2013GH021540)和云南省科技计划民营经济发展专项资金项目《云南虫草生物资源可持续产业化开发》(2014XA051) *Corresponding author. E‐mail: hongyu@ynu.edu.cn; herbfish@163.com 收稿日期:2013‐10‐10,接受日期:2013‐12‐27 云南虫生真菌粉棒束孢遗传分化研究代永东虞泓*陈自宏杨俊媛云南大学中草药生物资源研究所云百草实验室 云南 昆明 650091 摘 要:对云南粉棒束孢8个当地居群和蝙蝠蛾拟青霉2个当地居群进行ITS测序和RAPD扩增分析,结合GenBank中相关序列,对粉棒束孢开展遗传多样性、居群遗传结构及其种内分化研究。共获得大范围内地理距离远的6个居群共97条粉棒束孢ITS序列,共有33种单倍型,单倍型多样性Hd=0.546和总核苷酸多样性Pi=0.00276,显示粉棒束孢在物种水平上遗传多样性较低。云南粉棒束孢共37条序列,有14种单倍型(10种为云南特有),具有较高单倍型多样性和核苷酸多样性(Hd=0.659,Pi=0.00274);单倍型聚类和网状分支分析表明云南粉棒束孢单倍型类型丰富,遗传多样性高,暗示云南为粉棒束孢多样性分布中心之一。ITS序列分析表明,云南当地居群间遗传分化系数Fst=51.95%;RAPD分析表明,居群间遗传分化系数Gst=0.5547,基因流Nm=0.4014;说明云南当地居群粉棒束孢分化剧烈。居群遗传距离与地理距离相关性研究表明,粉棒束孢居群遗传距离与地理距离无明显相关。中性检验和失配分析表明粉棒束孢经历过近期居群扩张。结合单倍型聚类和网状分支分析,表明Hap 19为扩张建群单倍型,但原始祖先单倍型(Hap 1)依然是粉棒束孢居群中最优势单倍型(频率为48.45%),表明粉棒束孢并不存在明显的因地理原因造成的生殖隔离。值得重视的是,通过ITS单倍型和RAPD分析,支持将蝙蝠蛾拟青霉作为粉棒束孢异名处理。 关键词:ITS序列分析,RAPD扩增分析,单倍型,居群扩张,相关性 Genetic differentiation of an entomogenous fungus Isaria farinosa in Yunnan, China DAI Yong‐Dong YU Hong* CHEN Zi‐Hong YANG Jun‐Yuan Yunnan Herbal Laboratory, Institute of Herbal Biotic Resources, Yunnan University, Kunming, Yunnan 650091, China Abstract: In order to detect the genetic diversity, population genetic structure and intraspecific differentiation of Isaria farinosa, the ITS sequencing and RAPD amplification were conducted in 8 local populations of Isaria farinosa and 2 local populations of Paecilomyces hepiali in Yunnan. Together with the ITS sequences in GenBank, a total of 97 ITS sequences of I. farinosa were obtained in the 6 populations from widely separated geographical locations. The 33 haplotypes were discovered in the 代永东等/云南虫生真菌粉棒束孢遗传分化研究 菌物学报 39populations sampled of I. farinosa. The haplotype (gene) diversity was Hd=0.546 and nucleotide diversity was Pi=0.00276, showing lower degree of genetic diversity at the species level. A total of 14 haplotypes were found in the local populations of Yunnan, and 10 of them were not found elsewhere. The haplotypes’ clustering and network analyses demonstrated a richer genetic diversity of I. farinosa in Yunnan. The coefficient of genetic differentiation from ITS sequence analysis was Fst=51.95% among the 8 local populations in Yunnan. The gene flow from RAPD analysis was Nm=0.4014. It was shown that a great genetic differentiation was presented in the local populations of I. farinosa in Yunnan. The results of mantel tests revealed that there were no significant correlations between the average genetic distance and the geographic distance among geographically distant or local populations. Neutrality test and mismatch analysis revealed that the species went through a recent population expansion. By combining the results from haplotypes clustering and network analysis, it was inferred that the haplotype (Hap 19) might be the founder. However, the original ancestor haplotype (Hap 1) also had absolute predominance. These results indicated that I. farinosa did not experience an obvious reproductive isolation. In addition, P. hepiali could be considered the synonym of I. farinosa based on ITS haplotypes and RPAD analyses. Key words: ITS sequence, RAPD amplification, haplotype, population expansion, correlation 粉棒束孢Isaria farinosa (Holmsk.) Fr.,异名粉拟青霉 Paecilomyces farinosus (Holmsk. ex Gray) A.H.S.Br & G.Sm.,又称虫花棒束孢,虫草棒束孢,为丝状真菌棒束孢属Isaria Fries模式种,系统分类置于艳虫草菌科Cordycipitaceae Kreisel ex G.H. Sung, J.M. Sung, Hywel‐Jones & Spatafora。粉棒束孢为主要昆虫病原真菌,在森林和农田生态系统较湿润且草木茂盛的生境中,均为虫生真菌优势种(王四宝2003;陈名君2008)。粉棒束孢寄主范围广泛,在自然界中能够侵染多种害虫,是抑制害虫居群数量的重要因素,在生物防治上具有重要地位(蒲蛰龙和李增智1996)。 20世纪80年代以来,注意到粉棒束孢有性型可能是麦角菌科虫草Cordyceps Fr.或虫壳菌Torubiella Boud.时,开始对其活性成分进行研究。粉棒束孢含有多糖、虫草酸等功效成分已有大量研究(江艳华 2005;杨涛和董彩虹 2011)。目前已经从粉棒束孢培养菌丝体中分离20余种新化合物(Cheng et al. 2004,2006;胡丰林和李增智 2007;Zhang et al. 2009;车永胜 2011)。研究发现粉棒束孢具有广泛的药理活性,如降血脂、止咳祛痰、镇静、护肝利胆、抗肿瘤、解痉等(口如琴 1995;王学新 2011;张琛等2012)。从天然冬虫夏草Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel‐Jones & Spatafora分离到此真菌(Zhang et al. 2009),在生物医药领域内表现出较高的药用价值。鲁增辉等(2013)对粉棒束孢活性成分和药效进行了综述,表明该种具有广阔的开发应用前景。 关于粉棒束孢遗传特征已有报道。Chew et al.(1998)使用RAPD电泳图谱分析技术对加拿大东部20株粉棒束孢进行扩增分析,结果发现这一分布广、寄主多、形态变异大的物种遗产差异与地理环境无明显相关。黄勃等(2001)使用RAPD对不同来源不同寄主的13株粉棒束孢及另外3种拟青霉和白僵菌进行研究,聚类显示粉棒束孢能同其他物种明显分开,粉棒束孢菌株间差异表现为地理的相关,和寄主不相关。黄勃等(2003)通过对20株粉棒束孢ITS序列分析,菌株间有一定多态性,但与寄主和地理的相关性均无明显表现。孙绍红等(2011)利用ISSR研究安徽省粉棒束孢居群遗传异质性特征,结果发现居群遗传异质性较高,居群聚类结果与地理分布相符。 从已有资料来看,关于粉棒束孢遗传结构研究大多局限于当地较小地理范围,技术方法比较单 ISSN1672‐6472 CN11‐5180/Q Mycosystema January 15, 2015 Vol. 34 No. 1 ‐myco.im.ac.cn 40 一,结果也不统一,使得对于粉棒束孢的遗传特征认识仍然比较模糊。课题组近几年调查中国西南地区,特别是云南虫生真菌时,获得昆明、楚雄、德钦等地大量粉棒束孢。研究发现,关于该地区粉棒束孢研究非常缺乏(黄勃等 2001,2003;韩燕峰 2007)。本文拟应用ITS测序和RAPD电泳图谱分析对云南粉棒束孢居群遗传结构进行研究。为综合比较云南粉棒束孢遗传特点,对GenBank中有确切地理来源粉棒束孢ITS序列一并分
本文标题:云南虫生真菌粉棒束孢遗传分化研究
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