青藏高原梭罗草对低温适生芽孢杆菌促生作用的应答分子机制探究

Kengyilia thoroldiana is a kind of important grass breed and pasture resource applied to prevent meadow degradation and vegetation recovering in Qing-Tibet Plateau. Based on the ecological idea of “what is taken from the plateau is used in the interests of the plateau”, Bacillus isolated from Qing-Tibet Plateau were used to promote growth and improve output of Kengyilia thoroldiana in our previous research. However, the mechanisms of Kengyilia thoroldiana response to growth promotion effect of low-temperature adapting Bacillus was rarely reported. The project were focused on the interaction between low-temperature adapting Bacillus isolated from Qing-Tibet Plateau with Kengyilia thoroldiana. On the base of understanding growth-promoting rule of Bacillus RYS9 on grass, RNA-Seq technique would be performed to explore differently gene expression (DGE) in Kengyilia thoroldiana response to Bacillus strain RYS9. For the purpose to interpret growth-promoting molecular mechanism of Kengyilia thoroldiana response to Bacillus RYS9, the technology of bioinformatics analysis, plant transgene, transient expression and protein interaction were used to further explore expression pattern, function, subcellular location and regulation pathway of key growth-promoting genes in Kengyilia thoroldiana. The research could provide theoretical basis of utilizing PGPR Bacillus to improve growth-promoting of pasture, also provide new idea and target genes for improving growth and biomass of Kengyilia thoroldiana, possess important practical significance for ecological vegetation recovering and ecological husbandry development in Qing-Tibet Plateau.

梭罗草是青藏高原防止草甸退化、恢复生态植被的重要牧草资源。基于“取之于高原，用之于高原”的生态理念，我们前期利用分离自青藏高原的根围促生芽孢杆菌来处理梭罗草，促生效果显著，但有关梭罗草应答低温适生芽孢杆菌促生作用的机制尚无研究。本项目拟以低温适生根围芽孢杆菌RYS9对梭罗草的促生互作为研究对象，在明确RYS9对梭罗草促生规律的基础之上，借助转录组测序（RNA-Seq）技术挖掘梭罗草应答RYS9促生作用的关键差异表达基因；采用生物信息学、植物转基因、瞬时表达及蛋白互作等方法深入研究关键基因的功能、亚细胞定位及调控通路，阐释梭罗草应答芽孢杆菌RYS9促生作用的分子机制。本研究不仅为利用芽孢杆菌促进梭罗草生长提供理论依据，也为促进梭罗草快速生长、提高生物量提供新的靶标基因，对于青藏高原草地生态植被恢复及畜牧业具有重要现实意义。

青藏高原高寒牧草生长缓慢、生物量低，加之过度放牧等人为因素导致高寒草甸植被退化严重。本项目以高原低温适生根围促生芽孢杆菌对高原牧草梭罗草的促生互作为研究对象，在明确芽孢杆菌对梭罗草促生规律基础上，借助转录组测序（RNA-Seq）及全基因组测序技术挖掘梭罗草应答芽孢杆菌促生作用的实时差异表达基因及关键通路，阐释梭罗草应答芽孢杆菌促生作用的分子机制。.在青海极端生境中分离筛选到2株促生效果良好的生防菌株KKLW、KKD1，分别能够在NaCl浓度为11%、13%的生长环境及10℃低温条件下正常生长。提取菌株产生的次级代谢产物，经过质谱分析及HPLC纯化，菌株KKLW中含有表面活性素、伊枯草菌素、泛革素等次级代谢产物。通过转录组测序（RNA-Seq）分析，菌株KKLW与梭罗草分别互作2 h、4 h、8 h、12 h后，KKLW能够介导梭罗草体内植物激素信号转导、植物抗病反应等多条信号传导途径及相关基因的调控表达，进而整体促进植株生长发育并诱导植物免疫防御能力。.对菌株KKLW进行全基因组测序注释，KKLW体内含有锌离子转运蛋白znuABC、锰离子转运蛋白ppaC和gene2479、镁离子转运蛋白corA和moxR、葡萄糖脱氢酶gdh、生物素合成酶bioB及合成吲哚乙酸的关键基因trpC等多个促生长相关基因。其中，gene0211和gene2479在芽孢杆菌模式菌株中尚未发现，根据数据库注释该基因编码铁离子转运蛋白。预测菌株KKLW体内含有次级代谢产物基因簇，共发现13个基因簇，分别编码形成表面活性素、泛革素、枯草杆菌素等能抑制真菌及细菌病害的物质。其中，有4个未知基因簇尚未在数据库中报道，推测其形成新的次级代谢产物。.该项目研究结果不仅为利用根围促生芽孢杆菌促进高原牧草生长提供理论依据，也为促进高原牧草快速生长、提高生物量提供新策略和靶标基因，对于青藏高原草地生态植被恢复及生态畜牧业发展具有重要意义。