一株分离于酸性土壤的红酵母强耐铝机制研究

Alumimum can inhibit the growth of all the organisms including plants, animals, and microbes in acidic soils. There were more studies on the mechanisms of plant aluminum tolerance than those of microorganism aluminum tolerance. Microorganims which are the main components of soil organims play an important role in the soil ecosystem modulation and soil biodiversity maintance. We isolated an organism with high aluminum tolerance from acid soils, and this microorganism can grow at 200 mM of aluminum medium. This microorganism was indentified as red yeast (Rhodotorula). Our primary results showed that the changes of endocelluar organic acids and cell surface components contributed to the high aluminum tolerance of this red yeast. Using high-aluminum-tolerant Rhodotorula, middle-aluminum-tolerant Cryptococcus, aluminum-sensitive Saccharomyces cerevisiae as materials, this project will research the changes of cell membrane lipid, cell wall polysaccharide, endocellular pH and organice acid, plasma membrane H+-ATPase, membrane lipid peroxidation, and cell surface components under aluminum stress concering the aluminum-excluding mechanism and aluminum-tolerant mechanism. Also, the protenomics and gene expression will be done in order to find the genes and proteins related to aluminum tolerance. We will attempt to inoculate this red yeast into acid soils and observe its effects on the pH and aluminum forms of acid soils. All of these results will be taken together to explain the mechanisms responsible for the high aluminum tolerance of this red yeast, which will help improve the production and maintain the sustainable development in acidic soils.

铝抑制了酸性土壤中植物、动物、微生物在内的各种生物的生长和发育。植物耐铝机制以往研究较多，而对于微生物耐铝机制还不清楚。微生物是土壤生物的主体，在土壤微生态系统调节和生物多样性维持等方面具有重要作用。我们从酸性土壤中筛选得到一株强耐铝微生物，经鉴定为红酵母，其可以在高达200mM铝浓度下生长。已获得的结果表明，细胞体内有机酸和细胞表面性质的变化与其强耐铝有关，但是确切机制仍不清楚。本项目将采用该强耐铝红酵母、中等耐铝隐球酵母、不耐铝酿酒酵母为材料，从耐铝的内部忍耐机制和外排排斥机制两个方面出发，研究铝胁迫下细胞膜脂、细胞壁多糖、细胞内pH和有机酸、质膜H离子ATP酶、膜脂过氧化、细胞表面组分的变化，结合蛋白组学和基因表达研究，找出红酵母耐铝相关的蛋白和基因。并将该红酵母接种于酸性土壤中，观察其对土壤性质的影响。依此来阐明红酵母的耐铝机制，为酸性土壤生产力发挥和可持续利用提供一定帮助。

植物一般仅能忍耐微摩尔级浓度的铝，一些微生物却能够在高达上百毫摩尔级浓度铝中生长。目前微生物高耐铝机制还不清楚，研究微生物高耐铝机制有可能发现新的重要耐铝机制。本项目选用一种高耐铝红酵母Rhodotorula taiwanensis （Rt）、一种不耐铝红酵母R. mucilaginosa（Rm）和Rt的铝敏感突变体alsm1（Al sensitive mutant 1）为试验材料，在生理、组学和分子生物学三个层面研究了Rt高耐铝机制，取得了3个主要方面进展。（1）通过比较在不同供铝条件下细胞生长、细胞铝含量和分布、细胞铝Morin染色、细胞壁厚度、细胞壁组分和铝吸附特征、细胞表面电势等生理指标，获得的结果表明Rt耐铝的主要机制是Rt具有较低细胞表面负电性，排斥带正电铝离子，更少的铝接近细胞，可以在更高铝浓度介质中生长，而不耐铝红酵母Rm细胞表面具有较高负电性，吸附了更多带正电铝离子，对铝很敏感。（2）通过蛋白质组学和转录组学相结合的方法，从组学角度构建了红酵母Rt响应铝毒的生物系统网络结构，为全面了解Rt耐铝机制提供了研究视野。（3）采用cDNA文库构建和转化不耐铝酿酒酵母的策略，初步筛选得到多个与Rt高耐铝相关的基因，这为进一步揭示Rt耐铝的分子机制提供了靶标基因。项目研究成果目前已发表SCI收录论文4篇，中文论文1篇（已接受），还有2篇英文论文正在准备投稿中。项目执行期间，获得中国土壤学会科技奖一等奖，本项目的研究成果是该奖项成果的其中一部分。