溶磷细菌的分子改良及其溶磷作用机制

It's a problem of fertilizer science needed to be solved immediately that how to increase phosphate availability in soil. There has been much interest over the last decade in bio-solubilization of phosphate, maybe because of the potential use of phosphate-solubilizing microorganisms considered as a cost-effective, aesthetically pleasing technology. Therefore, understanding molecular mechanism and genetical modification of phosphate-solubilizing bacteria is essential in exploiting the soil phosphate resources. In this research, based on reverse genetics method, two phosphate-solubilizing bacterium (PSB) such as Bacillus megaterium and phosphate bacteria 9320 were focused and studied in the following four aspects: (1) Three genes including acpA, GDH and pqq were isolated from two phosphate-solubilizing bacterium by RACE method, respectively. (2) The recombinant plasmid pHT-acpA, pEASY-GDH and pEASY-pqq were constructed, respectively, and transformed into E. coli to identify transgenic bacteria with the phosphate dissolving function. (3) The recombinant plasmid pHT-acpA, pEASY-GDH and pEASY-pqq were further transformed into Bacillus megaterium and phosphate bacteria 9320, respectively, to endow transgenic PSB with dissolved both inorganic and organic phosphorus capacity. (4) The phosphate dissolving capacity of transgenic PSB was examined in Lab and field experiments, population and composition of PSB in soil and powdered rock phorphate in the medium with transgenic PSB by scanning transmission electron microscope (SEM), the expression of acpA, GDH and pqq gene by Northern blot analysis. These results from the research of transgenic PSB with recombinant plasmid pHT-acpA, pEASY-GDH and pEASY-pqq would play important roles in gaining better insight into mechanism of phosphate dissolving mechanism, widening our knowledge of bio-solubilization of phosphate, and promoting phosphate-solubilizing bacterial fertilizer from an experimental stage to a large-scale practical application stage.

土壤无效磷素的活化是肥料学科急需解决的科学问题。溶磷细菌以其廉价、高效、环保等优势受到国内外研究关注。研究溶磷细菌溶磷机制、研制高效溶磷菌株意义重大。本研究依据反向遗传学原理，利用基因工程技术，以巨大芽胞杆菌（解有机磷）和磷细菌9320（解无机磷）为研究对象，（1）克隆酸性磷酸酶（acpA）、葡萄糖脱氢酶(GDH)和吡咯喹啉醌合成（pqq）三个溶磷基因；（2）构建表达载体，在大肠杆菌表达三个基因，验证基因溶磷功能；（3）将三个基因转入溶磷细菌，获得分子改良的溶磷工程菌，赋予溶磷菌同时溶解难溶无机磷及有机磷的能力；（4）通过室内溶磷效果分析、田间溶磷效果验证、扫描电镜观测溶磷菌分布及解磷过程、Northern分析溶磷基因表达，从田间、生理、细胞及分子水平深入揭示溶磷机制。本研究旨在解决土壤有效磷含量低的实际问题，分子改良溶磷细菌，深化认识溶磷机制，推动溶磷菌肥从实验室阶段向田间应用阶段迈进。

土壤无效磷素的活化是肥料学科急需解决的科学问题。溶磷菌以其廉价、高效、环保等优势受到国内外研究关注。本项目以巨大芽胞杆菌和柠檬酸杆菌为研究对象，开展以下研究：（1）酸性磷酸酶（acpA）、葡萄糖脱氢酶(GDH)和吡咯喹啉醌合成基因（pqqE）的克隆、表达和功能研究；（2）酸性磷酸酶、葡萄糖脱氢酶溶磷工程菌的获得及功能鉴定；（3）溶磷工程菌的室内及田间溶磷效果验证。取得重要结果如下：（1）巨大芽胞杆菌acpA基因长度为627bp，具有催化磷酸单酯键水解的功能；柠檬酸杆菌gdh3基因长度为2391bp，具有将D-葡萄糖转化成葡萄糖内酯的功能；（2）在酸性磷酸酶工程菌中，acpA基因表达产物为分泌蛋白，酶活力为18.65 U/mg，工程菌对磷酸肌醇、磷脂、核酸三种有机磷源均表现出较好的解磷效果，以葡萄糖为碳源可显著增强工程菌溶解有机磷能力，工程菌内acpA基因在培养14d内保持高水平表达；（3）在葡萄糖脱氢酶工程菌中，gdh3基因表达产物为膜蛋白，酶活力为28.35 U/mg，接种工程菌可使含磷矿粉的液体培养基中速效磷含量增加46.75-242.26µmol/L，透射电镜能谱图中接种工程菌的磷矿石残体中P和Ca元素含量明显减少，工程菌内gdh3基因表达量随培养时间延长而增加，在培养14d时表达量最高；（4）施用ACP和GDH菌剂后，盆栽玉米地上部干重显著增加，地上部磷累积量分别增加35.6%和61.02%，苗期土壤有效磷含量分别增加了11.77%和13.87%，在收获期，工程菌处理下的土壤有效磷含量下降约7%，而未接种菌的土壤有效磷含量下降约40%，工程菌在盆栽土壤中能很好定殖，定殖数量随着定殖时间的延长呈现先增加再下降的趋势，GDH工程菌的溶磷效果优于ACP工程菌，GDH菌剂与磷酸钙配施溶磷效果最好。本项目克隆的acpA和gdh3基因可为培育磷高效农作物新品种以及生物工程菌提供重要基因资源，研发的溶磷工程菌，在活化土壤无效磷素方面具有重大应用价值。本研究成果可为论证溶磷细菌的生物学功能提供新的认识和实验证据，对于指导溶磷菌的合理使用提供理论依据，对于提高土壤磷素利用效率、减少磷肥投入有重大实践意义。