设施菜田盐渍化土壤解磷菌溶磷关键基因挖掘及其促进磷素利用的分子机制

The excessive use of fertilizers in facilities vegetable soils not only significantly increased the salinization, but also caused the accumulation and immobilization of phosphorus element. The lack of available soluble phosphorus in soil has become an important limiting factor for agricultural production. Phosphate-solubilizing bacteria in soils play an important role in phosphorus cycling as well as transformation. Therefore, it is valuable to study the distribution characteristics and the soil enzymes subjected to bacteria community structure shift as well as identification of major genes involved in releasing-phosphorus, and giving proper regulations will help to improve the phosphorus utilizing efficiency in the soils due to its solid form. Nevertheless, neither details of the phosphate-solubilizing bacteria responded to the slat stress, nor the main genes involved in this process have been reported. The purpose of this study will be going to use the isolated phosphate-solubilizing bacteria to observe the dynamic process of solubilizing strains colonized in salinized soil in vegetable crop roots and explore its influencing factors by using fluorescence in situ hybridization and scanning electron microscopy. The key genes will be identified from phosphorus solubilizing bacteria and phosphorus deficiencies mutants will be constructed. Meanwhile the Southern Blot, PCR-DGGE, and qPCR techniques will be used to explore the dynamic process and changes in bacteria community structures in greenhouse salinized soil, with the main aims to identify the molecular mechanism of phosphate solubilizing groups to express effective genes on activation of solid phosphorus, and to improve the abundance of soil enzyme activities which are related to the pathway of phosphorus transformation. Moreover, cloned phosphorus-solubilizing genes and plasmids will be transformed into Escherichia coli and Pichia pastoris to verify their functions. The interpretation of this mechanism will be helpful to solve the issues about the improvement of salinized soil and rational fertilization in facilities vegetable soils, and to provide the theoretical basis for reducing the usage of chemical fertilizer.

设施菜田化肥过量使用导致土壤盐渍化程度显著升高及磷元素的大量积累固定。土壤缺少可利用的磷成为农业增产的重要限制性因素。解磷菌在固态磷的转化和循环利用中起着重要作用，对解磷菌溶磷关键基因的挖掘并加以调控将有助于提高磷的利用率。本研究拟采用分离获得的多株解磷菌，通过筛选和鉴定解磷菌溶磷的关键基因并构建溶磷缺陷株，运用荧光原位杂交和电镜扫描技术观察解磷菌定殖于盐渍化土壤蔬菜作物根部的动态过程并探明其影响因素。利用Southern Blot、PCR-DGGE和qPCR技术分析调控解磷菌溶磷关键基因表达的因素，同时克隆溶磷基因并构建质粒转化至大肠杆菌和毕赤酵母进行溶磷功能验证。目的为阐述解磷菌溶磷关键基因对固态磷活化利用的分子机制，揭示溶磷关键基因对提高盐渍化土壤酶活并促进磷素转化的途径。对该机理的探索有助于改善设施土壤的磷素利用，为合理施肥和降低化肥的使用量提供科学的理论依据。

设施蔬菜种植过量使用化肥导致土壤盐渍化程度显著升高，且伴随着磷素的大量积累固定。设施土壤缺少可利用的磷素成为农业增产的重要限制性因素。解磷菌是存活于土壤、植株根部的一类重要微生物群体，并且在土壤固态磷的转化和循环中起着重要角色，探索解磷菌溶磷关键基因并加以调控将有助于提高磷的利用率。本项目通过鉴定解磷菌溶磷的关键基因，观察解磷菌定殖于盐渍化土壤蔬菜作物根部的动态过程并探明其影响因素。分析调控解磷菌溶磷关键基因表达的因素，同时对关键的溶磷基因进行溶磷功能验证。目的为阐述解磷菌溶磷关键基因对固态磷活化利用的分子机制。研究结果表明设施蔬菜设施土壤中无论是细菌还是真菌，其种类都在发生显著改变，且结构更加趋向于单一化。解磷菌株在不同磷素水平下，调控溶磷的基因数目显著不同。经过比对分析，发现上调基因有531个，下调基因有514个。通过GO和KEGG分析，解磷菌可通过调控细胞壁的信号通路、ABC信号通路以及甘油脂代谢途径调控磷素的转化。针对解磷菌选取了3个上调基因和3个下调基因进行qRT-PCR验证，调控的表达量与转录测序结果一致。对其中一个关键基因的序列克隆分析鉴定和功能验证，初步结果显示溶磷活性有所提升，但活性相对原始解磷菌株并非完全相同。在设施瓜菜土壤中接种解磷菌(N3和M01)，可提高土壤α-葡萄苷酶、β-纤维二糖苷酶、几丁质酶的酶活，并且显著提高土壤中的速效磷和速效钾的含量，说明土壤酶活的提高可促进磷素的转化。而缺陷菌株对溶磷作用显著降低，对单个溶磷基因的克隆分析结果显示单一基因溶解磷素的效果和水平有限，说明解磷菌的溶磷是一个综合调控的过程。转录组测序结果表明，解磷菌可诱导植株(辣椒和甜瓜)表达相关基因并调控不同的代谢途径，可显著促进甜瓜和辣椒幼苗对磷素的吸收。对解磷菌溶磷机理的解析有助于改善设施土壤的磷素利用，为降低化肥的使用量提供科学的理论依据。