华癸中慢生根瘤菌typA基因在根瘤菌类菌体分化中的作用机制研究

After invading the IRLC legumes, rhizobia will differentiate into bacteroid in nodule with nitrogen fixing activity. It was reported that bacteroid terminal differentiation was controlled by bacA from rhizobia and nodule-specific cysteine-rich peptides (NCRs) from host plant. However, the signal responsing and molecular interactive mechanism during bacteroid differentiation have not yet been expounded, more research and experiment evidences are needed. Recently, our laboratory has identified a TypA protein from Mesorhizobium huakuii which can interact with the BacA protein by bacterial two hybrid. The phenotypes of the typA mutant during bacteroid differentiation was highly similar to those in bacA mutant. TypA protein belongs to a type of ribosome-binding GTPase. It was hypothesized that TypA plays an essential role in bacteroid differentiation by interacting with BacA. The aims of this proposal is to answer following questions. 1) Does and how TypA interacts with BacA under symbiotic conditions and determines the bacteroids differentiation? Meanwhile, 2) By analyzing the global proteome of typA mutant, the responding signalling pathway for the bacteroid differentiation mediated by the typA will be discovered, and a few key functional peoteins will also be identified. By this project, the anticipated results will expound the functional mechanism of the typA gene to mediate bacteroid differentiation in M. huakuii, and as well as , it will provide advanced research progresses and novel insights into the molecular mechanism to control bacteroid differentiation in indeterminate-type nodules.

根瘤菌侵染IRLC类豆科植物后，将在根瘤中分化为具有固氮能力的终极形式类菌体。类菌体分化受根瘤菌bacA基因和宿主植物的根瘤特异富含半胱氨酸（NCR）多肽调控，但类菌体分化中的信号响应和分子互作机制仍需进一步揭示和阐明。本项目的前期工作中，从华癸中慢生根瘤菌中筛选得到与BacA相互作用的TypA蛋白，它编码一种核糖体结合型GTPase酶，并发现typA突变体在类菌体分化中的表型与bacA高度相似。本申请拟开展研究，旨在获得直接证据，一方面阐明TypA是否以及如何与BacA相互作用，从而调控根瘤菌类菌体分化发育；同时，基于对TypA突变体根瘤的全局蛋白组测定，结合转录组数据，分析以typA介导的类菌体分化信号途径，查明其中潜在的关键功能蛋白。从而揭示typA在介导华癸根瘤菌类菌体分化中的作用机制，为不定型根瘤类菌体分化的分子机理提供创新的研究进展。

一、项目背景：根瘤菌侵染豆科植物后，在根瘤中分化为有固氮能力的类菌体（bacteroid）。类菌体分化受根瘤菌bacA基因和宿主植物的根瘤特异富含半胱氨酸（NCR）多肽调控。前期从华癸中慢生根瘤菌中筛选得到与BacA相互作用的TypA，它编码一种GTPase酶，并发现typA突变体在类菌体分化中的表型与bacA高度相似。.二、研究内容：（1）考察根瘤菌ΔtypA突变株在与植物共生时的各项表型；（2）验证TypA是否以及如何与BacA相互作用；（3）对TypA突变根瘤做蛋白组和转录组分析，建立以typA介导的类菌体分化信号途径，查明其中的关键功能蛋白。.三、主要结果和关键数据：.1、typA突变根瘤菌与盆栽紫云英的共生表型显示：（1）typA突变导致宿主植物出现缺氮表型，根瘤发育异常。（2）突变体根瘤中大部分植物细胞未被根瘤菌侵染，少量细胞中类菌体分化异常，且出现早衰降解。（3）类菌体Live/dead染色观察，发现从ΔtypA类菌体大部分是未分化的根瘤菌，少量分化的类菌体已经提前衰老死亡，流式细胞仪发现ΔtypA类菌体DNA含量没有明显的分化加倍特征。（4）RT-qPCR和启动子表达定位显示：typA从侵染线阶段就开始表达，并且在根瘤发育的不同时期都有表达，在侵染早期和未成熟根瘤中的表达量相对较高。.2、酵母双杂表明TypA蛋白和BacA的特定结构域有相互作用，进一步工作还在进行中。 .3、蛋白组和转录组分析表明：ΔtypA根瘤中大量根瘤菌蛋白（基因）和植物蛋白（基因）差异表达，其中固氮相关蛋白（基因）显著下调表达。防御相关蛋白显著上调表达。.四、科学意义：现有结果表明typA在根瘤菌侵染早期发挥重要功能，并且在类菌体分化和根瘤发育中发挥着全局表达调控作用。本研究通过揭示typA在介导华癸根瘤菌类菌体分化中的作用机制，为不定型根瘤类菌体分化的分子机理提供创新的研究进展。