Knowledge Resource Center for Ecological Environment in Arid Area
| 石果衣真菌功能基因组初步研究 | |
| 其他题名 | A preliminary research on the functional genomic of lichen-forming fungus Endocarpon pusillum |
| 王延延 | |
| 出版年 | 2014 |
| 学位类型 | 博士 |
| 导师 | 魏江春 |
| 学位授予单位 | 中国科学院大学 |
| 中文摘要 | 地衣是一类典型的互惠共生联合体，地衣化是真菌的一种重要共生方式。地衣化赋予地衣型真菌很多独特的生物学特性，例如极强的抗逆能力。不同于其它被广泛研究的真核生物，在分子生物学水平上对地衣型真菌的了解甚少。造成这种现状的原因很多，例如地衣型真菌缓慢的生长速度及共生菌、藻分离困难等。目前的地衣生物学研究由于缺少遗传操作系统，几乎不能实现其基因功能的体内验证。然而，想要破解地衣的遗传信息首先要从地衣型真菌或其光合共生物的基因组测序工作开始。 \n本研究所采用的地衣型真菌材料是石果衣（Endocarpon pusillum），它分离自干旱荒漠地区的优势地衣，其共生藻为柯氏复球藻（Diplosphaera chodatii）。为了探究地衣化对地衣型真菌的影响及抗旱的分子机制，我们对石果衣真菌的基因组和转录组进行测序。主要研究结果如下： \n（1）石果衣真菌基因组测序拼接成37.5 Mb的基因组，预测出9,285个基因。生物信息分析结果为揭示地衣型真菌的一些生物学特性提供了分子证据，例如：同宗结合繁殖方式和抗旱基因的注释。比较基因组分析指出石果衣真菌中一些蛋白家族的进化揭示了菌、藻之间的互作关系。石果衣共生菌、藻的共培养实验验证一些涉及共生体间碳、氮传递的基因的表达变化，以及三个无信号肽的lectin基因在菌、藻识别过程中的表达变化。同时，确定了共生菌、藻合适的生物量比例及接触时间。 \n（2）比较转录组分析找到1,781个在PEG诱导干旱胁迫条件下差异表达的基因。参照其它抗旱真菌和植物，一些通用的抗逆机制基因在石果衣真菌中也是差异表达的，然而，有关渗透压调节机制的基因的表达变化在石果衣真菌中不同，而且，不同于其它真核生物，一些干旱适应机制的基因在石果衣真菌中为差异表达基因，说明石果衣真菌可能是干旱适应物种。荧光定量实验验证了23个转录组中差异基因在地衣体自然脱水过程中的表达变化，结果说明共生状态和分离培养状态下的石果衣真菌抗旱机制大体一致。 \n（3）为了进一步研究石果衣真菌的抗旱机制，利用双向电泳、质谱技术结合的方法，对PEG诱导干旱胁迫条件下的石果衣真菌进行了差异蛋白质组的比较分析。实验中对蛋白质样品制备方法进行了优化，找到合适的地衣型真菌蛋白质组提取方法。2DE图谱分析结果表明，在干旱胁迫条件下，共检测到58个蛋白丰度差异表达变化在1.5倍以上的蛋白点。其中49个蛋白点通过MALDI-TOF-TOF分析得到有效鉴定。但是，只有22%的基因在蛋白质水平和转录水平有较高的一致性，说明石果衣真菌抗旱相关基因大部分是受复杂的翻译后修饰作用调控的，值得进一步的深入研究。 \n本研究在基因组水平上揭示石果衣真菌的生物学特性提供信息，并对石果衣共生菌、藻之间识别和互作进行初步研究，有利用进一步深入研究地衣型真菌抗旱性分子机制，本研究所取得的结果将为开展地衣功能基因组学奠定基础。 |
| 英文摘要 | Lichen is a classic mutualistic organism and the lichenization is one of most important fungal symbioses. Lichenization makes the lichen-forming fungi have many unique biological characteristics, such as extremely strong resistance and tolerance to harsh environment. Unlike other well-studied eukaryotic systems, the lichen-forming fungus is not well understood molecularly. The lichen-forming fungi has been somewhat neglected for several reasons, such as slowly groth rate and difficult to separate from lichen thalli. There has any genetic manipulation method been established on either the fungus or the alga, it is not possible to analyze the gene function in vivo. However, the lack of genetic information of lichens could be compensated by the sequencing of the genomes of lichen-forming fungi and its symbiotic partner. \nThis work uses the lichen-forming fungus Endocarpon pusillum, which is living in symbiosis with the green alga Diplosphaera chodatii Bialsuknia as a lichen in the arid regions, and the genome and transcriptome were sequenced to explore the impact of licheniztion on lichen-forming fungi and the molecular mechanisms of drought resistance. The main results were obtained as follows: \n(1) A total of 9,285 genes were annotated in the 37.5 Mb genome of E. pusillum. Analyses of the genes provided direct molecular evidence for certain natural characteristics, such as homothallic reproduction and drought-tolerance. Comparative genomics analysis indicated that the expansion and contraction of some protein families in the E. pusillum genome reflect the specific relationship with its photosynthetic partner (D. chodatii). Co-culture experiments using the lichen-forming fungus E. pusillum and its algal partner allowed the functional identification of genes involved in the nitrogen and carbon transfer between both symbionts, and three lectins without signal peptide domains were found to be essential for the symbiotic recognition in the lichen; interestingly, the ratio of the biomass of both lichen-forming fungus and its photosynthetic partner and their contact time were found to be important for the interaction between these two symbionts. \n(2) The comparative transcriptome analysis indicated that a total of 1,781 genes were differentially expressed between samples cultured under normal and PEG-induced drought stress conditions. Similar to those in other drought resistance plants and fungi, the common drought-resistant mechanisms were differentially expressed in E. pusillum. However, the expression change of genes involved in osmotic regulation in E. pusillum is different, which might be the evidence for the feature of drought adaptation. Interestingly, different from other organisms, some genes involved in drought adaption mechanisms showed significantly different expressions between the presence and absence of drought stress in E. pusillum. The expression of 23 candidate stress response genes was further confirmed by quantitative real-time PCR using dehydrated E. pusillum lichen thalli. \n(3) To further understand the drought resistant mechanisms, we conducted the comparative proteome analysis for proteins isolated from E. pusillum cultured under normal and PEG-induced drought stress conditions. The method of protein sample preparation was optimized and the proper mthod was proposed in this study. 58 protein spots, which changed significantly (≥ 1.5 folds), were detected by 2DE. Among these protein spots, 49 differentially expressed proteins were identified by MALDI-TOF-TOF. However, only 22% of these genes have the same change trend between transcription and translation level. This results suggest that most drought resistant genes in E. pusillum were controled by post-translation modification, which deserve further research. \nThe present study lays a genomic analysis of the lichen-forming fungus E. pusillum for demonstrating its general biological features and the traits of the interaction between this fungus and its photosynthetic partner D. chodatii, and will provide a valuable resource for future research on lichen-forming fungi and shall facilitate future functional studies of the specific genes related to drought resistance. |
| 中文关键词 | 共生菌 ; 共生藻 ; 地衣化 ; 共生 ; 共生相关基因干旱适应 ; 干旱抗性 |
| 英文关键词 | Mycobiont Phycobiont Lichenization Symbiosis Symbiosis-related gene Drought adaption Drought resistant |
| 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/2XILL650/287420 |
| 推荐引用方式 GB/T 7714 | 王延延. 石果衣真菌功能基因组初步研究[D]. 中国科学院大学,2014. |
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