大豆根际溶磷菌WJ1和WY1溶磷特性及溶磷机制的研究

Phosphate-solubilizing bacteria（PSB） can change insoluble phosphate into plant available state. But the study in-depth of biological regulatory pathways and application effect for PSB were greatly limited by the different of phosphate solubilization conditions, research means limited and understanding of the phosphate- solubilizing mechanism in the bacteria-specific not clear. PSBs (Enterobacter sp.WJ1 and Pseudomonas sp.WY1) of the production of acid with significant differences isolated in preliminary work from the northeast soybean rhizosphere soil were studied in this subject by the use of HPLC and Real-time PCR. It was quantitative assaied that solubilization capacity, the quantity and type of organic acids, the growth rate, enzyme activity and gene expression of PSBs under different culture conditions. The optimum culture conditions of phosphate solubilization and the relationship among of phosphate solubilization ability, characteristics of acid production and gene expression of PSBs in different conditions were clear. The role of organic acids in the strains WJ1 and WY1 phosphate solubilization process was analyzed and the regulation law of gene expression under external factors was found. The phosphate solubilization mechanism of WJ1, and WY1 form the specific habitat conditions was revealed and it will provide the theoretical basis for a comprehensive understanding of the regulation mechanism of phosphate- solubilizing microorganisms in the process of phosphate dissolved.

溶磷菌能够将难溶性磷酸盐溶解、转化为植物可利用状态，但由于溶磷作用条件不同、研究手段所限，对溶磷菌特异性溶磷机理的认识并不清楚，大大限制了溶磷菌生物调控途径研究的深入与应用效果。本课题以前期从东北大豆根际土壤中分离到两株溶磷能力强、但产酸性能具有明显差异的菌株Enterobacter sp.WJ1和Pseudomonas sp.WY1为研究对象，利用HPLC和Real-time PCR，通过对溶磷菌在不同培养条件下的溶磷量、产有机酸种类和数量、菌株生长速率以及菌体内溶磷基因表达量、酶活进行定量分析测定。明确菌株最佳溶磷培养条件以及不同培养条件下菌株溶磷能力、产酸特性、溶磷基因表达之间的关系。探明有机酸在菌株WJ1和WY1溶磷过程中的作用与菌株溶磷过程中外界因素对基因表达调节的作用规律。揭示从特定生境条件下获得的WJ1和WY1菌株特有的溶磷机制，为全面理解溶磷微生物的溶磷调控机理提供依据。

针对大部分农田土壤由于磷肥过度施用而造成富磷状态，土壤溶磷微生物可有效提高土壤中植物可利用磷含量，可以减少化肥使用的情况，课题组以大豆根际土壤中分离到的无机磷溶磷菌Pseudomonas sp.WY1和Enterobacter sp.WJ1两菌株为研究材料，从菌株溶磷量、有机酸分泌及酶学与分子角度几个方面，研究两菌株的溶磷特性及溶磷机制。结果表明，两菌株最高溶磷量分别为558µg•mL-1和478µg•mL-1，两菌株具有较好的溶磷能力，利用这两株菌开发生物菌肥具有一定的可行性；甲基红试验和培养物pH值测定试验结果表明：2菌株在溶磷过程中可使培养物的pH值下降，pH 5时，生长受到轻微的影响，当pH小于4时，会明显阻碍菌株的生长。GC-MS对菌株在溶磷过程中产生的有机酸进行分析表明，两菌株均分泌多种有机酸，α-酮戊二酸在溶磷过程中表达量丰富，但菌株间分泌有机酸的种类不完全相同。溶磷过程中分泌有机酸造成培养基pH值降低，影响菌体生长，而菌体数量决定各菌株的溶磷量；成功的从两菌株克隆到溶磷直接氧化途径中的关键酶基因—葡萄糖脱氢酶（GDH）基因，片段大小分别为2007 bp和2066 bp；两菌株在不同磷源、不同pH值培养基中GDH活性及基因表达量不同，菌株WY1在高可溶性磷条件下基因表达量最高，磷胁迫条件下基因表达量较低，而WJ1在不同磷源条件下GDH基因表达量都较低。GDH基因表达量及酶活的变化与WJ1菌株溶磷量没有直接的关系，两菌株溶磷存在不同的机制，依赖于GDH的直接氧化途径并非是WJ1菌株溶磷的主要途径，菌株WY1的溶磷特点基本符合直接氧化途径的特点。本项目获得两株溶磷能力较好的细菌菌株，并在菌株溶磷的分子机制方面取得一些重要进展。这不仅为生物菌肥的开发增添了新的菌源，为溶磷微生物的应用提供科学依据，同时为揭示溶磷菌溶磷的动态变化机制提供理论基础。