花生慢生根瘤菌中III型分泌系统基因的分布和结构特征及其在菌植互作中的功能探究

Type III secretion system (T3SS) is a very important protein translocation system for interactions and signaling exchange between gram-negative bacteria and their eukaryotic host. Many rhizobia belonged to different genus or species have been found with functional T3SS which had different effects on the nodulation in symbiosis by comparative genomics during recent several years. The researches for T3SS in rhizobia reported at present were all focused on the rhizobia symbiotic with soybean, clover, ect., which entered into the legumes through infection thread. But it is unknown that the T3SS genes structure and their functions for nodulation by “crack entry” invasion. Therefore, we will use the bradyrhizobia isolated from peanuts as study material firstly and test the T3SS conserved genes characterization of the distribution in these Bradyrhizobium strains, which had few detailed research, after finishing the phylogenetic analysis of these bradyrhizobial strains. And the T3SS genes structure will also be characterized by comparative genomics. We will deeply study the tts and nop genes functions in T3SS during the rhizobia interacting with its host by using the technique of homologous double exchange to obtain the different mutants in T3SS. The results of our research can show the roles and mechanisms of T3SS in the “crack entry” invasion, which afford the rational guidance for the screening high effective peanut bradyrhizobial strains. It is also very helpful for comprehensive understanding the evolutionary adaption of rhizobia when they build the symbiotic relation with leguminous host.

III型分泌系统是革兰氏阴性细菌与其真核宿主之间相互作用、信号交流十分重要的蛋白运输系统。近年来，通过比较基因组学研究，在不同种属的根瘤菌中发现了对共生结瘤有影响的III型分泌系统。目前已报道的根瘤菌III型分泌系统的研究都集中在与大豆、苜蓿等共生的根瘤菌中，它们都以“侵入线”方式侵染宿主；而在以“裂隙”方式侵染的根瘤菌中该系统的基因组成和功能还是未知。因此，本项目率先以“裂隙”方式侵染且缺乏系统研究的花生慢生根瘤菌为研究材料，在解析其系统发育关系的基础上，考察III型分泌系统在其中的分布，并运用比较基因组学分析该系统的基因组成。同时利用同源双交换技术获得该系统中不同基因的突变体后，探究它们在菌植互作过程中的功能。本项目的研究能够明确地揭示III分泌系统在“裂隙”侵染过程中的作用机理，从而为花生高效共生菌株的筛选提供理论依据；也有助于全面理解在与宿主建立共生关系过程中根瘤菌的适应性进化。

III型分泌系统是革兰氏阴性细菌与其真核宿主之间相互作用、信号交流十分重要的蛋白运输系统。近年来，通过比较基因组学研究，在不同种属的根瘤菌中发现了对共生结瘤有影响的III型分泌系统。目前已报道的根瘤菌III型分泌系统的研究都集中在与大豆、苜蓿等共生的根瘤菌中，它们都以“侵入线”方式侵染宿主；而在以“裂隙”方式侵染的根瘤菌中该系统的基因组成和功能还是未知。因此，本项目率先以“裂隙”方式侵染且缺乏系统研究的花生根瘤菌为研究材料，在广东省主要花生种植区收集花生根瘤菌菌株216株，采用多位点基因序列分析技术解析其系统发育关系，发现了14个未定种的新遗传种群。通过代表菌株与不同花生品种的结瘤实验及PCR检测T3SS在花生根瘤菌的分布，进一步获得12株花生根瘤菌菌株。经基因组测序并运用比较基因组学分析，获得12株菌株中T3SS的基因簇组成。同时利用基因同源重组技术，获得代表菌株HY4、MM6和MZ5的T3SS的不同基因突变体，通过结瘤试验和石蜡切片分析，探究了T3SS在菌植互作过程中的功能。本项目的研究明确地揭示III分泌系统在“裂隙”侵染过程中发挥重要作用，从而为花生高效共生菌株的筛选提供理论依据；也为全面理解在与宿主建立共生关系过程中根瘤菌的适应性进化奠定基础.