基于RNA-seq的丛枝菌根真菌Gigaspora rosea基因构成及其共生发育相关基因的分析

发布时间：2018-08-07 11:25

【摘要】：丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)可与地球上大多数植物根系形成一种独特的互惠共生体——丛枝菌根(Arbuscular mycorrhiza,AM)。AM可以显著改善宿主植物的营养、水分代谢及其抗逆性,因而对现代可持续农业具有重要意义。AMF属于专性活体营养型(Obligate biotrophy)生物,其生物学及遗传学复杂性极大的限制了人们对其发育的分子生物学机制研究。尽管模式种Rhizophagus irregularis组学数据的发布为我们了解AMF遗传组成的全貌打开了第一扇门,然而AMF作为一支独立而古老的真菌进化分支(Glomeromycota,球囊菌门),其中包含了丰富的遗传多样性和生理生态多样性,因而R.irregularis组学数据所呈现出来的特点能否代表整个AMF类群的普遍特征目前还不得而知。为了更全面地了解AMF遗传构成的特点和探索其共生发育的分子生物学机制,本研究选取一个在遗传和生理生态特征上与模式种R.irregularis(Glomerales)有着显著差异的菌根真菌物种Gigaspora rosea(Diversisporales)作为研究对象,采用二代核酸测序技术(RNA-sequencing)及相关生物信息学分析技术,对其转录组结构和功能进行了全面的研究,主要结果如下:1.针对G.rosea的生理特征,本研究优化了相关实验方法并运用这些方法成功制备了高质量、能代表各个典型发育阶段的高通量测序核酸(RNA)样本,克服了以往AMF分子生物学研究尤其是大规模组学研究中一直存在的AMF核酸含量不足和质量不高的问题。2.运用CLC genomics workbench软件对高通量测序所得的读段序列(reads)进行分类和从头拼接(de novo assembly),得到86332个非冗余G.rosea虚拟转录本序列,平均长度643bp。通过与Refseq蛋白质数据库比对,只有15346个转录本可以被注释成数据库中已知基因,表明AMF中存在着大量的家系特异的孤儿基因(Lineage-specific orphan genes)。蛋白质功能注释(GO和KOG)及与其他不同类群的真菌比较显示AMF中显著富集了大量的信号转导相关基因,这表明其与宿主植物互作过程中可能涉及密集的信号交换过程。全局性代谢通路(KEGG)分析结果表明AMF仍保留了绝大多数的初级代谢基因,而缺失的少数代谢基因可能造成了AMF的专性活体营养特性。AMF中重要生理特性相关基因的深度注释表明AMF的基因构成具有一些不同于其他类群真菌的鲜明特点,这在很大程度上反映了它们特殊的生理生态学特征和进化优势。基于多基因比对的系统发育关系研究(phylogenomics)清晰地表明了AMF与毛霉亚门(Mucoromycotina)真菌关系更近,而非之前所认为的双核亚界真菌。3.通过比较根内共生发育阶段和根外菌丝阶段的转录组发现G.rosea在根内发育过程中有上千个转录本出现显著的差异表达。其中上调表达的基因主要涉及氧化还原过程(oxidation-reduction process)、蛋白质水解(proteolysis)、跨膜转运(transmembrane transport)等过程。通过比较进化距离遥远的两种宿主植物共生条件下G.rosea转录组的异同发现,G.rosea基因在不同宿主中的表达调控表现出异乎寻常的一致性:有将近一半的基因出现了相似的表达模式。这种一致性说明G.rosea很可能采用了一种非常保守的基因表达调控策略来实现其对所有宿主植物的侵染和定殖。此外,通过与亲缘关系较远的模式种R.irregularis的转录组数据比较分析本文还发现了四十多个表达模式高度保守的AMF基因:这些基因在不同AMF种类、不同宿主植物种类中都呈现明显上调。这些表现出保守表达模式的G.rosea核心基因的注释表明分泌性蛋白(Secreted protein)介导的信号转导、转运蛋白(Transporter)介导的营养交换、脂肪酸链延长及修饰相关酶类介导的脂类合成等过程在AM共生的建立过程可能发挥着非常核心的作用。在AMF组学数据仍然十分有限的情形下,这些研究结果不仅为将来的AMF基因功能研究提供了海量的基因数据资源,也为Glomeromycota类群内部及其与其他真菌的比较基因组或转录组学研究提供了可能。
[Abstract]:Arbuscular mycorrhizal fungi (AMF) can form a unique mutualistic symbiotic with most of the roots of the earth - Arbuscular mycorrhiza (AM).AM can significantly improve the nutrition, water metabolism and resistance of the host plant. Therefore, it is of great significance to modern sustainable agriculture. The biological and genetic complexity of the specific living body (Obligate biotrophy) organisms greatly restricts the study of molecular biological mechanisms for its development. Although the publication of the model Rhizophagus irregularis data has opened the first door to our understanding of the complete picture of the genetic composition of AMF, AMF is an independent one. The ancient fungal evolutionary branch (Glomeromycota), which contains rich genetic diversity and ecophysiological diversity, is not yet known whether the characteristics of the R.irregularis group data represent the general characteristics of the whole AMF group. In order to understand the characteristics of the AMF genetic composition and explore it more comprehensively The molecular biological mechanism of symbiotic development, this study selects a mycorrhizal fungal species, Gigaspora rosea (Diversisporales), which has significant differences in genetic and physiological characteristics with the pattern species R.irregularis (Glomerales) as the research object, using two generation nucleic acid sequencing technology (RNA-sequencing) and related bioinformatics analysis techniques. The main results are as follows: 1. in view of the physiological characteristics of G.rosea, this study optimizes the relevant experimental methods and uses these methods to successfully prepare high quality and high throughput sequencing nuclear acid (RNA) samples representing various typical developmental stages and overcome the previous AMF molecular biology research. In particular, the problem of insufficient AMF nucleic acid content and poor quality that existed in large-scale omics research,.2. uses CLC genomics workbench software to classify read segment sequence (reads) and splice (de novo assembly) from high throughput sequencing (de novo assembly), and obtains 86332 non redundant G.rosea virtual transcriptional sequences, the average length 643bp. passes through. Compared with the Refseq protein database, only 15346 transcripts can be annotated into a known gene in the database, indicating a large number of family specific orphan genes in AMF (Lineage-specific orphan genes). Protein functional annotations (GO and KOG) and comparison with other different groups of fungi show that a large number of letters have been enriched in AMF. Signal transduction related genes, which suggest that their interaction with host plants may involve intensive signal exchange processes. The results of the global metabolic pathway (KEGG) analysis show that AMF still retain most of the primary metabolic genes, and the missing few metabolic genes may cause important physiological characteristics in the specific active nutrition of AMF.AMF. The deep annotation of related genes indicated that the gene composition of AMF had some distinct characteristics different from other groups of group fungi, which largely reflected their special ecophysiological characteristics and evolutionary advantages. The phylogenetic relationship based on multiple gene alignment (phylogenomics) clearly indicated that AMF and Mucor (Mucoromycot) INA) the fungal relationship was closer, but not the previously considered biuclear subboundary fungus.3. found that there were significant differences in the expression of G.rosea during the development of the root, including the redox process (oxidation-reductio), which was mainly involved in the redox process (oxidation-reductio). N process), protein hydrolysis (proteolysis), transmembrane transport (transmembrane transport) and other processes. By comparing the similarities and differences of the G.rosea transcriptome in the two species of host plants that are far distant, the expression of the G.rosea gene in different hosts shows uncommon consistency: nearly half of the genes appear. Similar expression patterns. This consistency indicates that G.rosea is likely to use a very conservative gene expression regulation strategy to achieve its infection and colonization of all host plants. In addition, more than 40 expression patterns are also found by comparison of the transcriptional data of R.irregularis, a pattern species that is far closer to the relationship. The conserved AMF gene: these genes are obviously up-regulated in different species of AMF and in different species of host plants. These show that the annotation of G.rosea core genes in the conservative expression pattern indicates that secretory protein (Secreted protein) mediated signal transduction, transport protein (Transporter) mediated nutrition exchange, fatty acid chain extension and repair are shown. The process of lipid synthesis mediated by related enzymes may play a very important role in the establishment of AM symbiosis. Under the condition that the data of AMF are still very limited, these results not only provide a large amount of genetic data for the future research of AMF gene function, but also for the internal and other Glomeromycota groups. Comparative genomics or transcriptome studies of fungi provide the possibility.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93

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