滇池水华中控藻菌的杀藻相关基因及其功能研究

Algicidal bacteria are commonly considered to be effective in controlling algal blooms, their algicidal compounds are also crucial in both revealing algicidal mechanism and actual application of bloom control. But the purification of algicidal remains inefficient and tough. In our study, Aeromonas sp. were found to be the stable bloom controlling species in Dianchi Lake. The isolate Aeromonas sp.DBL3 shows efficient algicidal function, therefore, DBL3 is a perfect candidate to resolve bloom problem in Dianchi Lake. This study focuses on the algicidal mechanism of DBL3 and aims to find its algicidal genes and reveal the algicidal mechanism. Transposon insertion mutant library of DBL3 will be constructed and the mutants with algicidal function changed significantly are screened based on M. aeruginosa co-culture experiments. Then the potential algicidal genes are verified by Crispr-Cas9 technique. The genomics and algicidal gene of DBL3 were both considered to locate the metabolic pathway associated with algicidal function and find the candidate substances of bloom control. Finally, their algicidal functions are verified by M. aeruginosa co-culture process. Our mode of “functional bacteria-functional genes-function compounds” will be effective to further our understanding of algicidal mechanism, and guide the full excavation and utilization of algicidal compounds. Also it contributes greatly to the biological control of bloom in Dianchi lake and will provide new ideas to the promotion of bacterial control process.

细菌控藻是当前水华生物防治的重要方向。申请人前期研究发现，Aeromonas sp. DBL3是滇池水华过程中稳定存在且具有高效杀藻效果的控藻菌，是开展水华生物防治的理想杀藻菌。但对其杀藻机理还不清楚。为研究杀藻相关的关键基因和分子机制，为此本项目拟通过转座子随机突变技术构建高效控藻细菌DBL3的随机突变库，结合转座子插入位置和突变菌株抑藻功能变化定位控藻基因；利用Crispr-Cas9技术对候选控藻基因进行功能研究；结合细菌基因组数据分析控藻基因所在代谢通路，挖掘潜在的杀藻物质；结合杀藻实验验证候选杀藻物质的生物学功能。这种“功能细菌-功能基因-功能物质”的研究模式将有助于我们更加深入的了解“以菌治藻”中杀藻菌与水华藻的相互作用机理，指导杀藻化合物的开发和利用。这也将有助于滇池水华的生物学防治，并为水华的细菌防治推广提供新的思路。

本项目对滇池的水华期间及不同季节的蓝藻和细菌群落进行了生态研究。结果显示，铜绿微囊藻水华过程中，PAB和FLB在多样性、结构和微生物连接网络方面存在显著差异。PAB群落结构更相似、网络连接更紧密，水华藻和Alphaproteobacteria（尤其是固氮菌(Pleomorphomonas)和潜在溶藻菌(Brevundimonas sp.)）。FLB群落环境因子更敏感，另外细菌多样性高、连通性差、网络规模大、水华藻在互作网络中边缘化。在这两个网络中，细菌类群之间主要是正向互作(>88%的正相关)，而与氮的生物地球化学循环有关的细菌占据网络模块的中心，如Meganema, Pleomorphomonas和Nitrospira等。季节性研究发现，滇池的水体环境特点、藻类群落结构、细菌群落结构有明显的季节性，其中藻类和细菌群落结构以及PAB互作网络与水华严重程度密切相关。水华在夏季和秋季较严重。在不同的季节，滇池的藻类均以蓝藻为主。PAB在不同的季节，群落结构、多样性差异显著，其中多样性在不同季节差异较大，其季节波动与水华严重程度有关。FLB在不同的季节，多样性差异较小，群落结构差异显著。水华的加剧，会显著降低藻类、蓝藻以及PAB细菌多样性，显著改变PAB和FLB的群落结构；简化PAB互作网络并使细菌间互作更紧密，并升高铜绿微囊藻的网络地位；扩大FLB互作网络。PAB对水华严重程度更敏感，而FLB对环境因子更敏感。.通过本项目的开展，从滇池中分离获得30株纯藻和152株细菌，其中铜绿微囊藻有12株，能高效杀灭铜绿微囊藻的细菌有47株。其中Bacillus siamensis Sp37通过分泌吲哚乳酸和tryptoline快速杀灭滇池铜绿微囊藻DCM4，且该菌对微囊藻有特异性杀灭作用，如水华微囊藻、惠氏微囊藻等；吲哚乳酸能和Tryptoline协同溶藻，并主要通过影响藻细胞光合作用过程，促进活性氧产生，引起细胞膜发生脂质过氧化，DNA损伤等细胞伤害，导致藻细胞死亡。这个过程也会有效降低藻细胞中微囊藻毒性含量，并提高蛋白质和糖类的含量；目前已获得该菌的5个与控藻功能相关的基因，与琥珀酸脱氢酶和glucose-1-phosphate cytidylyltransferase等有关。.本项目进一步解析了水华过程中微生物生态过程以及溶藻菌的溶藻机制，对水华的微生物防治具有指导意义。