红酵母RS1耐酸铝机制的分泌蛋白组学研究

Aluminum (Al) toxicity is a major constraint for growth and production of crops in acidic soil. Currently, the mechanism of Al tolerance in plants has been extensively investigated, but little is known about these in microorganisms. Extracellular proteins are directly involved in response to various stresses, but so far there are no reports related to these under acid and Al stress. Rhodotorula taiwanensis RS1 was isolated form acidic soil with high acid and Al tolerance. It was showed that under different pH and Al concentration, the proteins secreted by R. taiwanensis RS1 were significantly different from these under the normal environment; however, the exact mechanism is still not clear. From the point of extracellular proteins, the different proteins and their dynamic changes will be systematically investigated by secretome under different pH and Al concentration, as well as under acid and Al stress. Combined with the physiological and biochemical characteristics, the function of extracellular proteins in the high acid and Al tolerance mechanism will be demonstrated. Furthermore the key proteins responsible for high acid and Al tolerance will be explored, interaction network of extracellular proteins related to acid and Al tolerance will be built, and then the mechanism of acid and Al tolerance in R. taiwanensis will be explained. The study will provide theoretical basis and gene resource for the soil bioremediation and breeding of the acid and Al tolerant crops, and is of great significance for improving productivity of crops in acidic soil.

铝毒害是酸性土壤作物生长和产量的主要限制因子之一。目前，植物的耐铝机制已有大量研究，但是微生物耐酸铝机制的研究相对较少。微生物分泌蛋白是参与胁迫响应的重要活性分子，但至今还没有酸铝胁迫下分泌蛋白的研究报道。红酵母（Rhodotorula taiwanensis）RS1是本课题组从酸性土壤中分离获得的强耐酸铝微生物。前期实验表明在不同pH和铝浓度下，其分泌蛋白谱带存在显著的差异，但具体的作用机制仍不清楚。本课题将从分泌蛋白的视角，采用分泌蛋白组学技术系统地解析红酵母RS1在不同pH和铝浓度以及酸铝共胁迫下的差异分泌蛋白及其动态变化规律，结合生理生化特征阐明差异分泌蛋白的生物学功能，发掘关键耐酸铝分泌蛋白，构建酸铝胁迫下分泌蛋白的互作网络，阐明红酵母耐酸铝机制。本课题的实施将为酸性土壤修复和耐酸铝作物的遗传改良提供理论基础和基因资源，对提高酸性土壤作物生产力具有实践意义。

酸性土壤占据全世界大约40%-50%可耕地面积。自上世纪80年代以来，我国主要的农田土壤酸度在不断升高，酸性土壤面积仍在不断扩大。酸性土壤中的离子毒害（Al和H等）严重抑制微生物生长，土壤酶活力以及作物生长和产量。提高酸性土壤的生产力对我国的粮食安全和农业可持续发展具有重要的意义。一些土壤微生物表现出极高的酸性和铝毒耐受能力，由此可借助抗酸铝微生物减轻植物的铝毒害。同时，微生物耐酸铝基因的发掘也将为耐酸铝作物的选育提供科学依据。目前，植物的耐铝机制已有大量研究，但是微生物耐酸铝机制的研究相对较少，其具体的作用机制尚不明晰。.本项目以酸性油茶园土壤中自行筛选获得的高耐酸铝红酵母（Rhodotorula taiwanensis）RS1为研究对象。实验发现，红酵母RS1对酸性环境有着极大的偏好性，在酸胁迫下仍能维持偏中性6.0的胞内环境。而这很可能是由于H+-ATPase的大量诱导表达发挥质子泵的作用。此外，本项目从分泌蛋白的视角，系统地剖析了红酵母RS1在不同pH和铝浓度下的差异分泌蛋白。胞内pH的细微变化导致了胞外分泌蛋白种类和含量的极大差异。通过SDS-PAGE和高分辨率质谱鉴定显示分泌型天冬氨酸蛋白酶在酸胁迫下大量诱导表达。进一步，通过生物学验证发现其在酸胁迫下发挥作用。同时，本项目对红酵母RS1进行随机诱变，从其诱变细胞中筛选出铝敏感突变体alsm1，采用转录组测序，在RNA水平来全面分析红酵母RS1在高铝胁迫下的响应，剖析其耐铝机制。鉴于酵母在耐酸铝中表现出的极端优异的性能，本项目通过对酸性土壤来源的红酵母RS1耐酸铝机制的研究，阐明红酵母RS1对酸性高铝环境的响应和适应机制，为改善酸性土壤生态系统和耐酸铝作物的遗传改良提供更多的理论基础和基因资源，对提高酸性土壤作物生产力具有一定的意义。