花柳珊瑚中Avermectins类化合物的放线菌来源及其在基因和蛋白水平防御海洋污损生物附着机制研究

The avermectins, produced by Streptomyces avermitilis, are one of the most effective antibiotic pesticides for animals and plants. Gorgonian corals of the genus Anthogorgia (family Acanthogorgiidae) are prolific in Beibu Gulf. Our investigation on the gorgonian coral A. caerulea collected from Beibu Gulf led to the isolation and characterization of four avermectin homologs, including two new avermectin derivatives, avermectin B1c (1) and avermectin B1e (2), as well as two known compounds, avermectin B2a (3) and ivermectin A1a (4). Compounds 1-4 were evaluated for antilarval settlement activity, the results displayed that the EC50 values of compounds 1-4 were 15.81, 6.25, 4.81, and 7.78 mg/ml, respectively, and the LD50 values of compounds 1-4 were >200 mg/ml. We isolated 23 Streptomyces strains from A. caerulea on media (2216E, NA and ISP2 ) supplement with seawater. Three of them belong to S. malachitospinus, S. violaceorubidus, and S. rubrogriseus, which is similar to S. avermitilis..In order to obtained highly efficient and non-toxic antifouling compounds for developing marine new antifouling agents with originality and independent intellectual property, the project plants to further investigate avermectin derivatives from the marine gorgonian-associated Streptomyces sp..In this study, we aim at 1) obtained gorgonian-associated Streptomyces sp. from A. caerulea; 2) isolating avermectin derivatives from the EtOAc layer of Streptomyces sp. by 1H NMR and HPLC; 3) examining the antibacterial activity of avermectin derivatives against four larval settlement-inducing bacteria L. hongkongensis, M. luteus, Rhodovulum sp., and Ruegeria sp., which were isolated from marine natural biofilms; 4) investigating the antilarval effect of avermectin derivatives against larval settlement of Balanus amphitrite and Hydroides elegans; 5) studying the mode-of-action at gene and protein level of lead avermectin derivative.

Avermectins类化合物当今生物农药市场中最受欢迎产品，具有广谱杀虫、杀螨、杀线虫活性。Anthogorgia caerulea为中国北部湾海域优势珊瑚种。我们在前期研究中从花柳珊瑚A. caerulea中获得了4个具有防御纹藤壶幼虫附着活性的Avermectins类化合物，推测该类化合物是由其共附生放线菌产生。项目组还初步对A. caerulea中相关可培养放线菌进行了研究，发现了3株链霉菌属菌株。本申请项目拟在前期研究基础上，寻找能产生Avermectins类化合物的共附生海洋放线菌，获得目标菌株。优化发酵和培养条件，运用多种现代高效分离和分析手段，快速分离鉴定Avermectins类化合物，测试其防御海洋污损生物附着能力，在基因和蛋白水平研究显效活性的Avermectin化合物对华美盘管虫幼虫的影响机制，为研制具有原创性和自主知识产权的海洋新型无毒绿色防污剂奠定基础。

全世界记录的海洋污损生物有4000余种。海洋大型植物和海洋固着动物的体表就如同所有人工设施的表面一样，是海洋污损生物潜在的附着位点。生长在海洋的婀娜多姿柳珊瑚却没有海洋污损生物附着。从柳珊瑚 A. caerulea 中获得了72株不同种属共附生放线菌，发现目标菌株S. avermectinius。通过发掘优化目标菌株S. avermectinius发酵条件，获得最优发酵条件和培养基。分离鉴定了11个Avermectins 类化合物，发现了显效活性化合物Avermectins Blc。探讨了显效活性Avermectins Blc在基因和蛋白水平防御海洋污损生物附着的作用机制。野外挂板实验，发现含Avermectins Blc的防污漆具有良好的抗海洋污损生物附着活性。本项目共发表论文7篇，其中SCI收录论文5篇，申请国家发明专利2项。项目组1人获聘四级研究员，1人晋升副高级职称，1人晋升中级职称。培养博士研究生1名，硕士研究生2名。