海洋苔藓动物共附生微生物杀虫活性先导化合物的研究

As human health was seriously jeopardized by the traditional chemical pesticide, the process of discovering novel biorational pesticide was more and more urgent. The applicant found the crude extracts of marine bryozoans Bugula neritina and Cryptosula pallasiana exhibited significant cytotoxicities when using MTT method to screen active constituents from 12 marine bryozoans. Besides, a lot of bromized alkaloids showing significant cytotoxicties were also isolated. The analysis of references and primary chemical ecology indicted that these secondary metabolites were produced by symbiotic and epiphyte microorganisms of marine bryozoans. The symbiotic and epiphyte microorganisms were survived by their host and can produce definite novel chemical constituents to resist enemy's invasion..Based on the above research, we will use symbiotic and epiphyte microorganisms of marine bryozoans B. neritina and C. pallasiana as our study object, use biological test by Artemia Salina and Plutella xylostella to screen insecticidal active strain, apply host characteristic spectroscopy to isolate insecticidal active constituents quickly from active strains of marine bryozoans, deploy modern spectroscopy to determine the definite structures of the isolated compounds, combine the results of insecticidal activities both by in vivo and in vitro to discuss their structure-function relationship, and finally found 1 or 2 potential lead compounds as the friendly environmental microorganic pesticides to establish a home base for the development of novel biorational pesticides.

传统化学农药杀虫剂严重危害人类健康，研发新型无公害绿色农药迫在眉睫。申请人采用MTT法对我国海域的12种苔藓虫进行活性筛选，发现苔藓虫Bugula neritina和Cryptosula pallasiana的总提物具有显著的细胞毒活性，并从中获得多个含溴生物碱、甾体等活性物质。文献及生态学分析结果表明这些物质可能源于苔藓虫共附生微生物。而海洋共附生微生物一方面依赖于寄主生存又产生一些结构新颖的特殊化学物质抵御外敌入侵。.基于此，本课题以苔藓虫B. neritina和C. pallasiana共附生微生物为研究对象，以卤虫和小菜蛾为指示害虫筛选杀虫活性菌株，运用寄主特征谱学导向快速追踪分离活性菌株中具有杀虫活性的微量成分，运用多种波谱学方法确定化合物的结构，结合体内外杀虫活性探讨其构效关系，发现1-2个高效低毒的环境友好型微生物源杀虫活性先导化合物，为开发新型无公害绿色农药杀虫剂奠定基础。

传统化学农药在杀虫剂市场中占主导地位，但是其造成的环境污染、损害人类健康、破坏生态环境等缺点越来越受到人们的关注，研发新型安全无公害绿色农药杀虫剂已迫在眉睫。海洋苔藓动物及其共附生微生物中富含的潜在杀虫活性物质将为解决这一难题提供突破口。.在国家自然科学基金委的支持下，本项目得以顺利开展和实施。课题已取得了多个创新性结论：首次对海洋苔藓动物总合草苔虫Bugula neritina的共附生微生物进行了杀虫活性物质研究，明确总合草苔虫共附生微生物分布以细菌为主，其次为真菌和放线菌，所占比例分别为67%、28%和5%，确定其共附生微生物杀虫活性菌株主要集中在真菌中的青霉菌属和曲霉菌属，并对总合草苔虫共附生真菌的多样性进行了探讨；通过形态观察与分子鉴定技术鉴定了2株活性菌株的种属分别为Aspergillus versicolor BZ-31（GenBank序列登记号：KF848935）和Penicillium polonicum EZ-17（GenBank序列登记号：KF848936）；从总合草苔虫共附生杀虫活性真菌中分离鉴定出了27个化合物，其中包括4个新化合物和1个新天然产物；化合物versicolorin C (7) 在72h、1000ppm浓度下，对3龄粘虫幼虫的校正死亡率为88.5%，12h时的拒食活性为100%，化合物2(Z) -烯庚醇(12)在24h对孑孓的LC50为3.03 mg/L，麦角甾醇 (13) 为多株真菌的共同杀虫活性成分，在24h对3龄粘虫幼虫的LC50为346.78 mg/L；拒食中浓AFC50为143.36 mg/L；另外，还从南海总合草苔虫与细薄星芒海绵Stelletta tenuis中分离鉴定出了36个化合物，其中包括10个新化合物和3个新天然产物，且部分化合物显示出显著的细胞毒活性。.以上研究结果表明，总合草苔虫共附生真菌次生代谢产物versicolorin C (7)、2(Z)-烯庚醇(12)和麦角甾醇(13)可以作为杀虫活性先导化合物加以开发和利用。这项研究工作一方面阐明了总合草苔虫共附生真菌A. versicolor BZ-31和P. polonicum EZ-17的杀虫活性物质基础，也为将这两株海洋真菌开发为生物源杀虫剂奠定了理论基础，具有潜在的巨大社会效益和经济效益。