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| 侧耳属分子系统学及分子鉴定的研究 | |
| 其他题名 | Molecular phylogeny of Pleurotus with reference to molecular identification of species |
| 杨志辉 | |
| 出版年 | 2008 |
| 学位类型 | 博士 |
| 导师 | 姚一建 |
| 学位授予单位 | 中国科学院微生物研究所 |
| 中文摘要 | 侧耳[Pleurotus (Fr.) P. Kumm.]一类非常重要的栽培食用菌，其产量占世界栽培蘑菇总产量的25%；侧耳也是一种环保菌，可以用环境废物的处理；侧耳一些种类具有捕食线虫的能力，可以作为生防菌用于农业上线虫病害的防治。\n 本研究在侧耳属形态学研究的基础上，利用mt-SSU、rDNA ITS和IGS等3个片段的序列对糙皮侧耳复合群、刺芹侧耳复合群及相关类群（不产生孢梗束的单系菌丝的侧耳种类）进行了分子系统学和物种形成的研究，发现了糙皮侧耳和肺形侧耳杂交对佛州侧耳形成具有重要作用，序列分析表明佛州侧耳对糙皮侧耳复合群、刺芹侧耳复合群及相关类群侧耳种类系统发育关系推断有很强的干扰作用。杂交种的发现使得最近分化的不产生孢梗束的单系菌丝侧耳种类分子系统学得到很好的解决。发现了中国的阿魏侧耳不同与欧洲的刺芹侧耳复合群，可能具有独立的起源。同时结合寄主、营养类型、温度和地理分布等综合信息探讨最近分化的单系菌丝系统的侧耳种类的物种分化原因和演化方向，发现了生态因素是这些复合群种类快速分化的主要原因，它们朝着木本植物向多年生草本植物，森林生态系统向干旱荒漠的草原生态系统，腐生向弱寄生，喜高温向低温的方向演化。\n 在糙皮侧耳复合群、刺芹侧耳复合群及相关类群研究的分子系统学的研究基础上，利用mt-SSU、LSU rDNA和ITS rDNA序列重建了侧耳属种类的系统发育关系。在这3个基因中，mt-SSU提供的信息位点数最少，单个基因不能重建侧耳属的系统学关系；LSU rDNA变异较大，双系菌丝类型种类以及产生孢梗束的单系菌丝的种类在系统发育树的末端形成稳定的分支；ITS序列变异太大，整个侧耳属种类的序列比对困难。系统发育分析表明菌丝系统和孢梗束的有无在侧耳属的演化中有重要作用。在分子系统学研究的基础上，结合传统形态学的属下分组系统，重新建立了侧耳属的属下分组系统，并与形态学分组进行了比较。\n 本研究以侧耳属12个种类为例，设计了一种基于ITS片段序列分析的PCR-RFLP方法用于菌物种类的快速分子鉴定的方法。 基于侧耳ITS序列系统发育分析的结果，利用本研究开发的内切酶自动筛选的软件PRE Auto，筛选限制性内切酶，以区分不同侧耳种类的多个序列。将ITS序列导入PRE Auto软件，根据系统发育种的界定进行分组后，分析限制性内切酶的识别位点、计算序列酶切后的片段长度，通过序列组间和组内平均系数对每个限制性内切酶进行打分，分值用于指导选择限制性内切酶的酶切图谱，以便于快速确定用于区分不同侧耳种类的候选酶。总共85个代表151条侧耳种类的ITS序列用于分析，筛选了2992种限制性内切酶，最终确定了利用3种内切酶、两步法鉴定12个侧耳种类。该方法用12个侧耳种的28个样品进行了验证，除了4个错误鉴定样品以外，限制性内切酶酶切结果与PRE Auto程序预测的带型相同。测定了14个样品（包括4个起始鉴定错误的样品）的ITS序列，系统发育分析证实了PCR-RFLP的分子鉴定结果，该方法也可用于其它生物类群的分子鉴定。此外，还设计了贝盖侧耳特异性引物，利用nest-PCR技术对其进行快速分子鉴定。也利用AFLP指纹技术对糙皮侧耳复合群和刺芹侧耳复合群的种类进行了聚类分析和种类鉴定。 |
| 英文摘要 | Species of Pleurotus, commercially called oyster mushrooms, are a group of the most important edible fungi, accounting for 25% of the total world production of commercially cultivated mushrooms. Pleurotus species are also used as a tool for environmental control in the biotransformation and biodegradation of industrial effluents owing to their unique ligninolytic system. Further, some species of Pleurotus are known as predaceous fungi and have the potential for controlling nematode diseases of agricultural crops. \n Based on morphological research of Pleurotus, phylogenetic relationships and speciation among the P. ostreatus-complex, the P. eryngii-complex and the allied taxa were investigated with the sequences analysis of multiple loci of mt-SSU, rDNA ITS and IGS. The results revealed that species hybidization between P. ostreatus and P. pulmonarius might played important role in the speciation of P. floridanus. The phylogenetic relationship among the P. ostreatus-complex, the P. eryngii-complex and the allied taxa were clarified based on the finding of the hybrid species, P. floridanus. Further, P. ferulae from China was found to be phylogenetically different from the P. erygnii-complex from Europe in the sequence analysis of a single gene and multi-genes, when P. floridanus was excluded. The combinated analyses of molecular data with the information of host, ecology and biothophy indicated that ecological factors such as temperature and hosts evidently affected species divergence and evolutionary directions among the P. ostreatus-complex, the P. eryngii-complex and their allied taxa, which evoluved from woods to herbs, from forest to dried grassland, from saprotroph to weak parasite and from high temperature to low temperature.\n Sequences of a single mt-SSU locus are inadequate for the phylogenetic inference of the genus Pleurotus due to its low variation. Each species of dimitic hyphal system and monomitci hyphal system with synnematoid fructications in the genus Pleurotus forms a well-supported terminal branche in phylogenetic tree of rDNA LSU. It is difficult to align ITS sequences within the genus Pleurotus because of its high variation; however, ITS sequences were very useful for the phylogenetic analysis of species of monomitic hyphal system without synnematoid fructications. The results of phylogentic analysis indicated hyphal system and synnematoid fructifications might be the important evolutionary characters in Pleurotus. Intrageneric sections were proposed and estimated in the genus Pleurotus, based on the results of phylogenetic analysis combined with the traditionally morphologhical intrageneric sections and subgenus.\n A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of ITS sequences was designed to rapidly identify fungal species, with twelve members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms. In addition, P. calyptratus was rapidly identified with the designed species-specific primers and nest-PCR technique. Several species of P. ostreatus-complex and the P. eryngii-complex were also clustered and dertermined by AFLP fingerprints. |
| 中文关键词 | 侧耳属 ; 糙皮侧耳复合群 ; 刺芹侧耳复合群 ; 分子系统学 ; 物种形成 ; 分子鉴定 ; PRE Auto程序 ; PCR-RFLP技术 ; AFLP分子标记 |
| 英文关键词 | Pleurotus P. ostreatus complex P. eryngii complex Molecular phylogeny Speciation Molecular identification PRE Auto program PCR-RFLP technique AFLP marker |
| 语种 | 中文 |
| 国家 | 中国 |
| 来源学科分类 | 微生物学 |
| 来源机构 | 中国科学院微生物研究所 |
| 资源类型 | 学位论文 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/286674 |
| 推荐引用方式 GB/T 7714 | 杨志辉. 侧耳属分子系统学及分子鉴定的研究[D]. 中国科学院微生物研究所,2008. |
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