内生菌Pantoea alhagi PS-2胞外多糖在提高水稻苗盐胁迫抗性中的作用模式解析

Endophytes which significantly increase salt tolerance of plants by colonization and symbiotic interaction, has become an environmentally friendly and economical strategy for crops growth in salinization soil. However, their mechanisms for enhancing salt tolerance of hosts are still not entirely clear. In the early of this project, a endophytic Pantoea alhagi PS-2 (PS-2 ) was found that could enhance salt resistance of rice seedling and its extracellular polysaccharide (EPS) played an important role during the process. This project aims to reveal the physiological mechanism of EPS on PS-2 promoting salt tolerance of rice seedling. In this project, the synthesis key gen of EPS will be firstly confirmed depending on the EPS deficient mutant Pantoea alhagi PS-2eps- and be marked with fluorescent tags to study its expression in rice seedling under salt stress. Then, the elicited H2O2 signaling by EPS in rice seedling will be researched through technologies of chemical quantitative, fluorescence staining, non-invasive micro-test and RT-PCR, and the rice genes responding to EPS will also be studied by transcriptome sequencing to build the interaction between H2O2 signal and gene response. At last, the possible recognition protein of EPS in rice will try to find by Full-Down experiment, then we will come up with a hypothesis that EPS recognition→signal eliciting→gene expressing→stress resistance responding.

一些植物内生菌由于能显著提高共生作物的盐胁迫抗性，已成为一种环境友好、经济有效的促进作物在盐渍化土壤中生长的策略。而内生菌提高宿主耐盐性的机制却不完全清楚。本项目前期获得一株能增强水稻苗盐胁迫抗性的内生菌Pantoea alhagi PS-2并发现该效应与其胞外多糖有关。因此，本申请旨在解析PS-2胞外多糖在内生菌增强水稻苗盐胁迫抗性中的生理机制。本申请将借助前期构建的胞外多糖表型缺失突变株获得胞外多糖合成关键基因，利用荧光标签考察该基因在水稻应对盐胁迫时的表达差异；同时通过化学定量、荧光染色、非损伤微测及RT-PCR技术研究PS-2胞外多糖对水稻苗H2O2信号的激发效应，并借助转录组测序发掘水稻苗对胞外多糖的响应基因，构建受胞外多糖激发的信号与基因互作模型；最后利用Pull-Down技术，发掘胞外多糖识别蛋白，建立从多糖识别→信号激发→基因表达→抗逆响应的内生菌胞外多糖作用模式假说。

本项目首先证明P. alhagi NX-11能促进水稻生长，增强水稻抗逆。并进一步确定了胞外多糖对P. alhagi NX-11增强水稻抗逆的作用，同时解析了胞外多糖结构。通过Crispr/Cas9无痕敲除体系成功构建了胞外多糖定向缺失菌株ΔpspD，通过水培模拟盐胁迫明确了胞外多糖在P. alhagi NX-11增强水稻抗盐中的关键作用。此外，分析了P. alhagi NX-11胞外多糖诱导下的P. alhagi NX-11根际特异性定殖的基础。确定了胞外多糖促进P. alhagi NX-11的根系定殖，同时分析了胞外多糖与根系分泌物的交互作用。鉴定了胞外多糖诱导下水稻根系分泌物中两种差异最为明显的根系分泌物组分，棕榈酸和丙酰胺。明确了棕榈酸对P. alhagi NX-11的促进作用及丙酰胺对P. alhagi NX-11的抑制作用。探究了胞外多糖与丙酰胺的相互作用保护P. alhagi NX-11免受丙酰胺抑制的机制。通过构建水稻根系的简化合成菌群，阐述了胞外多糖诱导下水稻根系分泌物的变化对根际菌群的选择性装配机制。借助转录组测序发掘水稻苗对胞外多糖的响应基因, 木菠萝凝集素基因OsJRL经NX-11及其胞外多糖诱导均显著上调，基于凝集素为植物识别多糖及结合多糖的蛋白，初步推测其可能特异性识别NX-11的胞外多糖。对其他细菌多糖的研究具有借鉴意义。

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