高效溶藻菌B1分泌的含氮化合物对有毒赤潮异湾藻杀藻机制和产毒影响

According to the reports and the applicant’s previous researchs, it was found that secreted nitrogen compounds which were isolated from algicidal bacteria and identified before can significantly kill toxic algae. Such secreted nitrogen compounds are expected to be effective algicidal substances for toxic red tides treatment. However, the algicidal mechanism and generation mechanism of the pure nitrogen compounds isolated from the sterile culture filtrate of the algae are still unclear. The project intends to select harmful akashiwo which has broken out frequently around the country in recent years as target algae and choose nitrogen algicidal compounds of structurally similar secreted by the efficient algicidal strain B1 as object and verify such five containing compounds’ algaecidal effect and synergistic algicidal effect on the target algae. Besides, the project intends to make relevant biological toxicity assessment and study the production regularity of the target algae’s hemolytic toxin under the effect of nitrogenous compounds. In combination with multiplex fluorescence staining, RT-PRC,TUNEL and other molecular biology detection technology, the researchers plan to use modern instruments like flow cytometry analyzer, SEM, TEM, phytoplankton fluorescence instrument to track , observe and study algal cells’ physiological activity, changes in morphology and structure, interaction process and apoptosis of algae. After a series of relevant experiment, make the research of functional gene annotation and metabolic pathway of the obtained strain B1 gene information. It can help provide scientific basis for developing a new efficient algaecide and the construction of the engineering strains of algae lysis. Besides, it can also help provide scientific basis for biological treatment of toxic red tide.

文献报道和申请人前期研究发现，已经分离和鉴定溶藻菌分泌的含氮化合物能显著地杀死有毒赤潮藻，有望成为赤潮防治新物质。但从杀藻菌发酵液中分离提取的含氮杀藻化合物，其胁迫下对有毒赤潮藻溶藻机制和藻毒素产生规律不清楚。项目拟选取国内频发和危害严重的有毒赤潮异湾藻为目标藻种，以分离鉴定出的高效溶藻菌株B1菌分泌的结构相似含氮溶藻化合物为研究对象，验证探明该物质对目标藻的杀藻效果和协同杀藻作用，并进行生物毒性评价。研究含氮化合物胁迫下对目标藻产溶血毒素的影响。采用流式细胞仪、SEM 、TEM、浮游植物荧光仪等现代仪器、结合多重荧光染色、RT-PRC、TUNEL等分子生物学检测技术，对含氮化合物胁迫下藻细胞生理活性、形态和结构变化和藻的凋亡现象进行追踪、观察和研究；对获得B1菌全基因信息进行功能基因注释和代谢通路的研究,为构建溶藻工程菌株、生物杀藻剂和有毒赤潮生物治理提供科学依据。

项目选取在国内频发的赤潮异湾藻为研究对象，探究揭示B1菌分泌的5种含氮化合物对目标藻的溶藻机理和杀藻过程产毒及毒素合成机制、脂肪酸的代谢等。 . 研究结果表明，5种含氮物质的溶藻效率与加药浓度和时间呈正相关，在96小时内对藻的抑制率都可以达到80%以上。藻细胞的抑制是通过影响细胞抗氧化系统、光合系统，通过减少线粒体膜电位增高，使细胞膜的通透性增加。使细胞膜完整性和细胞核结构受到破坏，导致细胞内ROS含量增加，从而诱发藻细胞发生程序性死亡。 . 投加溶藻物质48小时后，应用代谢组学方法对藻细胞的代谢物和代谢途径进行了研究。共鉴定出了28种差异代谢物，涉及了光合作用、抗氧化酶合成、细胞膜的渗透性和信号转导及氨基酸转运等生物过程。卟啉和叶绿素合成的抑制表明光合素色合成的抑制，导致叶绿素a、叶绿素c和类胡萝卜素等细胞色素的减少；L-谷氨酸、甘氨酸的增加标志了促进谷胱甘肽代谢，即谷胱甘肽抗氧化物酶合成增加，以应对逐渐升高的 ROS；甘油、甘露醇和肌醇等醇类的降低表明糖类合成降低，这影响到了后续细胞的碳水化合物代谢以及细胞膜的渗透压的失衡。. 研究证实赤潮异湾藻溶血毒素具有明显的染色体损伤遗传毒性。溶藻物质次黄嘌呤和 Nω-乙酰组胺能够降低藻毒素的染色体损伤遗传毒性。. 产毒及毒素合成机制初探表明,藻细胞毒素合成和释放是细胞应对外界压力释放的一种自身的防御机制。藻细胞在100mg/mL浓度的次黄嘌呤胁迫下在第五天时，对加药第五天和未加药藻体中的脂肪酸变化进行归一化法分析推断可知,C18：4可能就是藻毒素的合成物质。 对脂肪酸代谢通路的研究发现，与投药相比未投药的脂肪酸氧化相关酶均下调，说明投药后细胞体内脂肪酸氧化基因上调，进一步说明细胞投药后主要以消耗脂肪酸来提供细胞所需能量。. 该研究结果有望为构建溶藻工程菌株、开发生物杀藻剂和为有毒赤潮生物治理提供科学依据。

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