虫生真菌粉棒束孢分子系统学研究

1、分类号 Q94 密级 公 开 UDC 编号 硕士研究生学位论文题 目 虫生真菌粉棒束孢分子系统学研究Study on Molecular Systematics of an Entomogenous Fungus Isaria farinosa (Holmskiold) Fries学院（所、中心） 生命科学学院 专业名称 植物学 研究生姓名 代永东 学号 1201000961 导师姓名 虞 泓 职称 教 授 二零一三 年 五 月作者简介：代永东，男，2014级云南大学博士研究生，从事虫草类真菌遗传与系统研究。2006-2010就读于四川农业大学，2010-2013云南大学植物学专业硕士研究生。

2、电话mail： 或 摘 要粉棒束孢Isaria farinosa (Holm.) Fries，又被称为虫花棒束孢，虫草棒束孢。粉棒束孢在生物防治、食品、医药和保健领域表现出巨大的潜在价值。然而，基础研究尚显薄弱，关于粉棒束孢系统学研究，现有资料零散不充分，也不全面，甚至观点相悖，导致这一物种系统分类出现混淆和模糊，开发应用也出现混乱。本研究对粉棒束孢及相关种属进行分子系统学研究，主要获得以下结果：通过对6株粉棒束孢和1株蝙蝠蛾拟青霉菌株培养和显微形态观察，确定粉棒束孢形态结构具多型特征，主要体现为瓶梗

3、形态和着生方式以及孢子连结方式。而粉棒束孢与蝙蝠蛾拟青霉二者形态显微结构存在重叠特征，不具有种特异性。本研究对采自滇中、滇西、滇东等地的粉棒束孢共29菌株进行ITS测序，在全29条，长502bp的ITS序列中，共10个变异位点，相互间没有显著性差异。对GenBank中粉棒束孢ITS序列整理，共得到75条序列，其中6条长度仅300余bp，9条为错误鉴定；而另外60条与测序数据无差异。通过BLAST比对，还获得除上述60条以外与粉棒束孢高度相似36条序列，其中，18条标识为Paecilomyces sp.或Isaria sp.等未给定完整种名的序列，另18条标识为其他非粉棒束孢真菌，分别是3条蝙蝠

4、蛾拟青霉Paecilomyces hepiali、3条球孢白僵菌 Beauveria bassiana、4条蛹虫草Cordyceps militaris、5条枚烟色棒束孢Isaria fumosorosea、2条鲜红棒束孢Isaria amoenerosea和1条新几内亚刺束梗孢Akanthomyces novoguineensis。经过序列特征分析，建议将这些序列全部视为粉棒束孢。对这125条进行聚类分析，获得4个分支，但遗传距离较小，分支间不具有明显的系统发育信息。通过对粉棒束孢及相关种属共计80个物种144条ITS序列进行分析，确定棒束孢属具有3个分支，相互间为多系类群。粉棒束孢与蝙蝠蛾

5、拟青霉聚为一支，与其他棒束孢种被蚧霉菌、白僵菌、蛹虫草等隔开。同时，ITS区的RNA结构模拟表明ITS序列二级结构具保守性。对粉棒束孢等44物种共计63条序列进行3-5基因（18S rDNA、28S rDNA、tef-1、rpb1和rpb2）系统学分析，结果发现棒束孢属物种形成3个分支，各类群的物种组成与ITS结果一致。粉棒束孢与蝙蝠蛾拟青霉无显著差异，聚为紧密一支，表明二者属于同一个系统学种。粉棒束孢与Cordyceps tuberculata (无性型为Akanthomyces aculeatus)和Cordyceps confragosa (无性型为Lecanicillium atten

6、uatum)遗传距离较小，但它们无性型属于3个不同的属，表明无性型形态之间并不具有严格的系统发育关系。另外，多基因系统学支持将细束梗孢Akanthomyces和球束梗孢Gibellula分支纳入到狭义虫草属（Cordyceps s. s.）中，但支持率较低。通过对350余条虫草类真菌18S rDNA分析，确定虫草类真菌18S rDNA I型内含子分布，为18S rRNA序列第560、1164、1210和1425位点。由此设计3对内含子特异性引物，实现虫草类真菌18S rDNA I型内含子定量检测。对5株粉棒束孢18S rDNA I型内含子进行测序，获得粉棒束孢18S rDNA位点1164和14

7、25的 I型内含子，内含子序列分析显示在种间存在明显差异，但种内高度保守。因此，可以作为物种鉴定的辅助指标。利用8条RAPD引物对26株粉棒束孢和3株蝙蝠蛾拟青霉进行扩增，获得条带清晰的电泳谱带。谱带统计和聚类分析发现，在UPGMA聚类中距离为0.75处，供试菌株可分为5个分支，表现出丰富的遗传信息；同时，蝙蝠蛾拟青霉分散在粉棒束孢分支中，不具有独特的分枝地位。RAPD分析支持将蝙蝠蛾拟青霉归并至粉棒束孢。综上所述，本文利用多基因测序和RAPD分析等方法，进行粉棒束孢分子系统学研究，解析艳虫草菌科种属系统发育关系。同时，构建出粉棒束孢与近缘种属系统发育树，确定棒束孢属为多系类群，粉棒束孢为独立

8、分支，澄清了蝙蝠蛾拟青霉为粉棒束孢同物异名，并建议将蝙蝠蛾拟青霉作为异名。根据粉棒束孢产孢结构多型性和基因组遗传异质性，暗示粉棒束孢正处于种内分化之中。关键词：粉棒束孢； ITS； 多基因片段； 系统发育； 多系群IVAbstractIsaria farinosa (Holmskiold) Fries，is also called Cordyceps links to Isaria fungus. It shows great potential value in the field of biological control, food, medicine and health care.

9、However, the basic research on the species level has been still significant weak, the systematic information of Isaria farinosa has also been neither sufficient nor comprehensive, and even contradictory, resulting confusion and fuzz in classification, phylogenetic systematics and application of this

10、 fungus.In the present study, the molecular systematics of Isaria farinosa and its related species were investigated, the results were as follows.By morphology and microscopy observation of 6 strains of Isaria farinosa and 1 strain of Paecilomyces hepiali, it was comfirmed that Isaria farinosa was w

11、ith multi-type feature which mainly reflected in phialide forms and the location, along with the pattern of conidia connection. It was evident that the morphology features of these 2 species were overlapped, and there were no species-specificity between them. Through sequencing 29 strains of Isaria

12、farinosa collected from the northern, eastern and central regions of Yunnan, China. The 10 variable sites were found in all sequences of lengh 502bp, with no significant difference each other. By collecting, analyzing and packing ITS of Isaria farinosa in the GenBank database, the 75 sequences were

13、obtained. Six of them were no more than 300 bp because of length deficiency, and nine were confirmed to be misidentified in classification. Another 36 sequences were obtained by BLAST online, with being highly similar to Isaria farinosa. There were 18 sequences marked as Paecilomyces sp., Isaria sp.

14、 or Fungal sp. etc. And the rest 18 sequences were tagged as the others fungi, i.e. Akanthomyces novoguineensis, Beauveria bassiana, Cordyceps militaris, Isaria amoenerosea and Paecilomyces hepiali. It was suggested that all of these 36 sequences be considered as Isaria farinosa according to pairwis

15、e distance analysis. Four clades were divided based on the clustering analysis, but the genetic distance was so little that they didnt reveal any significant phylogenetic information.The 144 ITS sequences from 80 species were analyzed and determined in the genus Isaria. The three clades were classif

16、ied in a polyphyletic group of the genus Isaria. Isaria farinosa and Paecilomyces hepiali were clustering into the same clade. They were separated from the others Isaria species. Meanwhile, it was shown that the secondary structure were conservative according to the RNA secondary structure simulatio

17、ns of ITS1 and ITS2 regions. The 63 multi-genes contained 3-5 genes (18S rDNA、28S rDNA、tef-1、rpb1 and rpb2) were analyzed in 40 species. It was indicated that three clades were divided in the polyphyletic tree of the genus Isaria, with being the same topological structures as the ITS phylogenetic tr

18、ee. It was important that Isaria farinosa and Paecilomyces hepiali were clustered into the same clade with no significant difference, suggesting they should be the same phylogenic species. There were much smaller genetic distances among Isaria farinosa, Cordyceps tuberculata and Cordyceps confragosa

19、, but their anamorphs were classified into different genara Isaria, Akanthomyces and Lecanicillium, respectively. It was suggested that asexual forms couldnt be inferred as strict phylogenetic relationships. Moreover, Akanthomyces and Gibellula clades were supported to classify into the genus Cordyc

20、eps sensu suto according to the multi-genes systematic analyses, but a lower support values.Based on analysis of more than 350 sequences of 18S rDNA in Cordyceps fungi，the distributions of the group I introns were determined in 18S rDNA. They were located in 560, 1164, 1210 and 1425 site in 18S rRNA

21、. The three pairs of intron-specific primers were designed, and it was possible to detect quantitatively the group I intron of 18S. The 18S rDNA of five strains of Isaria farinosa were amplified and sequenced. The group I introns were located in 1164 and 1425 site. Compared with others related speci

22、es, it was showed that the group I intron of 18S was significantly interspecific difference but highly conserved within species. So it was considered that the group I intron in 18S rDNA could be an auxiliary marker for species identification.The 29 samples of Isaria farinosa were amplified by 8 pair

23、s of RAPD primers, and detected according to clear electrophoretic bands. It was statistically analyzed that the 29 samples were divided into five groups at the distance 0.75 in the UPGMA tree. Abundant genetic variation was discovered in the 29 samples of Isaria farinosa. It was also found that Pae

24、cilomyces hepiali dispersed in these branches, supporting that Paecilomyces hepiali should be incorporated into Isaria farinosa. It was appeared that a certain degree of genetic heterogeneity within Isaria farinosa. In summary, the multi-gene sequencing and RAPD analysis were used to research the mo

25、lecular systematics of Isaria farinosa. The phylogeny of Cordycipitaceae was explained. A comprehensive classification and identification system was constructed. And the taxonomic status of Isaria farinosa was examed among polyphylic relationship with other Isaria spp. It was suggested that Paecilom

26、yces hepiali should be as the synonym of Isaria farinosa. Sporulation structure polymorphism and genetic heterogeneity were found in Isaria farinosa. All results indicated that Isaria farinosa was in the progress of variation and active phase of differentiation. Keywords: Isaria farinosa； ITS； multi

27、ple gene sequencing； phylogeny； polyphyly目 录目录摘 要IAbstractIII缩略词表iii第一章 粉棒束孢及近缘种属系统学研究概况11.1 虫生真菌棒束孢属系统分类研究进展11.2 虫草类虫生真菌分子系统学研究进展21.2.1 广义虫草属系统分类研究概况31.2.2 虫草真菌主要无性型拟青霉属系统分类概况51.3 粉棒束孢分类及资源利用研究现状81.3.1 分类研究现状81.3.2 生防价值利用现状91.3.3 保健食品、医药产业价值91.4 本研究目的意义10参考文献12第二章 粉棒束孢和蝙蝠蛾拟青霉形态及显微形态研究172.1 试验材料172.

28、2 试验方法172.2.1 菌种分离纯化172.2.2 菌株形态观察182.3 试验结果与分析182.3.1 6株粉棒束孢及1株蝙蝠蛾拟青霉形态描述182.3.2 试验菌株间的相互比较202.4 讨论22参考文献24附图26第三章 粉棒束孢种内ITS rDNA序列分析273.1试验材料283.2 试验方法303.2.1 粉棒束孢样品ITS扩增及测序303.2.2 测序数据处理及分析303.2.3 GenBank中相关数据的获取及处理303.3 试验结果313.3.1 GenBank中粉棒束孢ITS序列完整性和正确性校对313.3.2 粉棒束孢ITS序列种内变异特征323.3.3 聚类树特征分析333.3.4 粉棒束孢各类群间的遗传距离分析34 3.4 讨论35参考文献38第四章 粉棒束孢与近缘种属ITS rDNA系统发育分析414.1 试验材料424.2 试验方法454.2.1 试验样品ITS