百脉根侵染线形成必需的基因ITD6在共生互作中分子机理的研究

During symbiotic nitrogen fixation between legumes and rhizobia, a key step for a effective nodule formation is the host root hair cell form a tunnel-like infection thread (ITs), a structure in which bacteria entry into plant root cortex from epidermis cell. But the mechanisms and plant genes that contribute to the initiation and developments of ITs are still not fully understood. In order to identify genes required for IT formation, we screened mutants from Louts japonicus EMS mutagenesis pool for defects infection thread formation. One of these mutants called itd6 (infection-thread deficient 6), which only can entrap bacteria in the infection pocket and occasional form few abnormal infection thread, but it can not form effective nodule to fix nitrogen. ITD6 encode a function unknown protein which only have long coiled-coil domain by map-based cloning, sequencing and function complementation assay. ITD6 was induced by rhizobia infection and it likely a new target gene of NIN, which is a specific transcriptor gene for symbiotic nitrogen fixation. This project will further explore ITD6 biological function to reveal the molecular mechanisms of infection threat formation through genetic, cellular and biochemical methods. This research will provide scientific basis for engineering non-legume plants with symbiotic nitrogen fixation ability.

在豆科植物与根瘤菌共生固氮体系中，有效根瘤形成的关键是宿主植物根毛内形成侵染线，根瘤菌沿着侵染线从表皮细胞向皮层细胞延伸。因此侵染线的正确起始与延伸是有效根瘤形成的首要保障。但侵染线形成的分子机制尚不清楚。申请人在前期工作中，在百脉根中获得一个侵染线缺陷突变体itd6（infection-thread deficient 6），该突变体只能形成侵染点和极少数异常侵染线，但不形成有效固氮根瘤。经过图位克隆，并结合基因测序、互补实验等研究表明，ITD6编码一个含Long coiled-coil结构域的功能未知蛋白。对ITD6的表达调控研究表明ITD6受结瘤因子诱导，很可能是共生固氮特异的转录因子NIN的靶基因。本项目拟在已有的研究基础上，通过遗传、细胞、生化等方法，进一步探索ITD6的生物学功能，揭示共生固氮过程中侵染线形成的分子机制，为探索非豆科植物共生固氮提供科学依据。

核孔是核内外物质运输的主要通道，在植物免疫和共生过程中，核孔蛋白起着至关重要的作用。在本研究中，我们发现一个编码coiled-coid结构域的蛋白是侵染线形成必需的。受根瘤菌ITD6特异表达，而且结瘤特异的转录因子NIN能够结合到ITD6的启动子上激活它的表达。另外，ITD6能够与核孔蛋白NUP和RNA-binging protein1（RBP1）相互作用，并且与nup85一样，itd6和nup突变体中mRNA在细胞核中的量积累，这说明ITD6的突变减弱了细胞核中mRNA 的出核转运过程。