寒地根瘤菌III型效应因子对大豆宿主特异性的影响

Type III effectors of rhizobium played an important role for the construction of symbiosis between rhizboium and soybean. Different type III effectors has special functions on the soybean cultivar specificity. In this project, we try to elucidate the effect of type III effectors including NopA/B/C/M/T/P/L and Hrp1/2/3 on the cultivar specificity, which were derived from cold field rhizobium HD001 isolated by ourselves. Firstly, we carried out the identification of nodulation efficiency of HD001 on 205 soybean germplasms to acquire 30 germplasms with significantly difference of nodulation phenotypes. Secondly, we clone the coding sequences of 10 type III effectors and construct various mutations of special type III effectors, including mutations of loss of functions and deletion mutations. Last, by tri-parental mating to construct the rhizobium mutants harbored the mutated type III effectors, to perform the mutant complementation test to further confirm the specificity of type III effectors on 30 soybean germplasms. On the other side, the expression of nodulation-specific genes including ENOD40 and NIN was analyzed, and the tissue-specific localization of type III effector proteins was investigated. These results could supply a good theory for the construction of highly efficient and broad host rhizobium and the functional study of type III effectors.

III型效应因子是共生结瘤关系建立的关键信号分子，直接影响根瘤菌的品种特异性，从而影响结瘤效率。本项目针对自主分离的寒地生态型根瘤菌HD001中10个III型效应因子（NopA/B/C/M/T/P/L和Hrp1//2/3），研究对其宿主特异型的影响。首先，鉴定HD001在205份东北大豆核心种质上的结瘤效率，筛选出结瘤特征存在显著差异的30份大豆种质；然后，克隆10个效应因子基因，对其进行突变（功能失活突变和功能域缺失突变）；最后，利用三亲杂交获得效应因子突变的根瘤菌株，结合突变体回补实验鉴定10个效应因子对30份大豆结瘤效率的影响，明确各效应因子及其功能域对品种特异性的作用。同时，分析效应因子对结瘤特异基因（ENOD40和NIN等）的转录表达影响及效应因子在根瘤中的积累模式。研究结果将为创制对大豆品种具有广谱性且高效结瘤的根瘤菌及研究III型效应因子作用机制奠定基础。

本项目通过对根瘤菌的基因组测序注释，深入细致的获得了根瘤菌基因组中固氮基因和结瘤相关基因的编码序列。在此基础上针对获得的III型效应因子，进行了深入研究，获得了NopAA、NopL、NopP、NopD、NopB等III型效应因子的突变体。并克隆以上5个III型效应因子的编码序列，通过构建到原核表达载体，获得了带有HIS标签的融合表达蛋白。完成了III型效应因子NopL突变体的回补实验。通过结瘤实验获得III型效应因子突变体对结瘤性状的影响，得到了700余份大豆种质资源的结瘤表型分析。针对NopAA进行了蛋白生化活性分析，证明NopAA具有糖基水解酶活性。研究结果为深入细致的研究 III型根瘤菌在共生结瘤建立过程中的作用具有奠定了重要基础，尤其是了解多个III型效应因子在相互作用是否在共生结瘤过程中具有独立或者协同作用。