【作者】 彭杰丽；

【导师】 李友国；

【作者基本信息】 华中农业大学 ， 微生物学， 2014， 博士

【摘要】 华癸中慢生根瘤菌7653R (Mesorhizobium huakuii7653R)可以与宿主豆科植物紫云英(Astragalus sinicus)形成生物固氮共生体,也可以在土壤中自由生长。在华癸中慢生根瘤菌7653R的全基因组测序完成之后,为了更好地了解7653R在类菌体分化过程中所发生的生理生化变化,我们采用RNA-Seq和Microarrays技术相结合的方法对7653R在两种代谢条件下(共生和自生)的转录组进行了研究。1.对类菌体的分离方法进行了比较分析,结果表明：如果需要获得完整的类菌体用于后续形态观察,可以使用物理分离的方法；如果需要获得高质量类菌体RNA用于RNA-Seq或Microarrays实验,可以采用Ambion MICROB Enrich试剂盒直接提取和纯化类菌体RNA的方法。应用上述所建立的技术方法,本文成功制备了华癸中慢生根瘤菌7653R在共生和自生条件下的RNA,进行了RNA-Seq和Microarrays实验。2.通过同源比对的生物信息学方法,构建了华癸中慢生根瘤菌7653R全局蛋白质互作网络(protein-protein interaction network, PPI)。3.对华癸中慢生根瘤菌7653R的转录组进行了分析。结果表明：(1)RNA-Seq和Microarrays均鉴定出7653R在两种生长状态下近三千条差异表达的基因。对这些基因进行了共线性分析,发现两种方法检测到的基因及表达特征相似度高,共线性好。(2)采用整合数据的方式(两种方法检测到的差异表达基因的合集),对这些差异表达的基因进行了COG功能分类,代谢途径分析,KEGG富集分析以及GSEA基因集富集分析。结果显示7653R在自生条件下主要是维持基础代谢,而类菌体的功能主要是进行生物固氮。显著上调的基因位于共生质粒上。在类菌体条件下,能量代谢相关基因的表达水平上调明显,而脂肪酸代谢相关基因表达水平变化很小,大多数与细胞周期相关的基因均呈下调表达。(3)利用Cytoscape将基因的差异表达与7653R的全局蛋白质互作网络进行整合分析,解析获得一个与共生固氮密切相关的亚网络。该亚网络包含多个hub基因以及若干未曾报导与固氮过程相关的基因亚网络。(4)鉴于CtrA在细胞分裂和类菌体分化中具有重要调控作用,以CtrA为中心,本文解析获得一个与其直接互作的基因亚网络。这些研究结果对深刻揭示类菌体分化、固氮机理提供了新材料及新视野。4.选择共生固氮子网中的hub基因MCHK-0866及其相邻的MCHK-0867’进行突变体的构建。构建了单基因突变和双基因突变的置换载体,采用三亲本结合转移的方法筛选获得双基因突变的稳定突变株,基于植物盆栽的共生表型检测为结瘤不固氮。5.改造了用于构建细菌单杂交随机文库的转录因子表达载体,建立并优化了构建随机文库的方法。结果显示此方法转化效率高,可获得大量转化子。为构建细菌单杂交随机文库,研究转录组中的重要转录因子及调控打下了工作基础。更多还原

【Abstract】 Mesorhizobium huakuii7653R occurs either in nitrogen-fixing symbiosis with its host plant, Astragalus sinicus, or free-living in the soil. The M. huakuii7653R genome has recently been sequenced. To better understand the complex biochemical and developmental changes that occur in7653R during bacteroid differentiation, RNA-Seq and Microarrays were used to investigate the differential transcriptomes of7653R bacteroids and free-living cells.1. Comparative analysis of the methods of isolating bacteroids. The results showed that use physical method to get complete form of bacteroids for morphological observation or use Ambion MICROBEnrich kit to get high-quality bacteroids RNA. Using above methods, total RNA of bacteroids and free-living cells was extracted for RNA-Seq and Microarrays analysis.2. We constructed a protein-protein interaction (PPI) network for7653R using the method of orthologous.3. RNA-Seq and Microarrays were used to investigate the differential transcriptomes of7653R bacteroids and free-living cells. The results are as follows.(1) Both the two approaches identified about three thousand differentially expressed genes. Correlation between the two methods was high.(2) We have analyzed COG functional categories, biological metabolic pathways, enriched KEGG pathways and Gene set enrichment analysis using the both sources of data following an integrative strategy. These results all showed that free-living cells have a primary role in maintaining basal metabolism, whereas bacteroids have a primary role in nitrogen fixation. The most prominent up-regulation occurred in the symbiosis plasmids. The results suggested that the main energy metabolism is active while fatty acid metabolism is inactive in bacteroid and that most of genes relevant to cell cycle are down-regulated accordingly.(3) Integrated gene expression data into the7653R protein-protein interaction (PPI) network using Cytoscape. A highly inter-connected subnetwork, with function enrichment for nitrogen fixation, was found, and a set of hubs and previously uncharacterized genes participating in nitrogen fixation were identified.(4) In order to identify the subnetwork mediated by CtrA connected with the cell cycle and bacteroid differentiation, we dissected the7653R PPI network by using CtrA as a specific target. Then we got the CtrA subnetwork. The results described here provide a broader biological landscape and novel insights that elucidate rhizobial bacteroid differentiation, nitrogen fixation and related novel gene functions.4. Construction of mutants of MCHK-0866, one of the hub gene in nitrogen fixation subnetwork, and its adjacent gene MCHK-0867. Construction of single-gene mutation (MCHK-0866-or MCHK-0866-) and double-gene mutation vectors, using three parental hybridization to screen. Finally, obtain stable double-gene mutant and plant symbiotic phenotype detection was Fix-.5. Modification of transcription factor expression vector used to build the Bacterial one-hybrid random library. Pre-construction of random library of Bacterial one-hybrid with high transformation efficiency. Lay the foundation for constructing the Bacterial one-hybrid random library and analyzing the expression and regulation of important transcription factors in transcriptome results.更多还原