利用饵料微藻表达抗菌肽及其初步应用

为了解抗菌肽在饵料微藻中表达后的抗菌特性，构建海洋微拟球藻(Nannochloropsis oceanica)、湖泊微拟球藻(N.limnetica)和三角褐指藻(Phaeodactylum tricornutum)的抗菌肽(源自虹鳟，Cath-1a)表达质粒，分别转化相应的微藻，检测转化子中抗菌肽的表达量和体外抑菌效果，将藻株作为鱼饲料添加剂喂食斑马鱼，初步分析了抗菌肽及藻体自身的岩藻黄素和多不饱和脂肪酸对鱼免疫系统的影响。结果表明，外源抗菌肽在3种微藻中均可以成功表达，体外抑菌试验表明，仅三角褐指藻对水产领域常见致病菌爱德华氏菌(Edwardsiella tarda)有一定的抑菌效果，然而抗菌肽的表达并未使3种藻株的体外抑菌性增加。添加藻粉对斑马鱼的生长无明显影响，通过检测鱼体肝脏中与抗氧化和免疫相关基因的表达水平及丙二醛的含量,表明添加藻粉可增强斑马鱼的抗氧化和抗炎症能力，表达抗菌肽(PtC组)能进一步提高斑马鱼的免疫力。另外，添加Pt6(富含岩藻黄素)藻粉组比添加PtC的抗炎效果更显著，表明三角褐指藻中的岩藻黄素和二十碳五烯酸对增强鱼的抗病能力具有潜在作用。

Expression of Antimicrobial Pepetides in Microalgae for Aquaculture and Its Preliminary Application

To understand the roles of antimicrobial peptides expressed in microalgae for aquaculture, expression vectors containing Cath-1a (from rainbow trout) gene were constructed and introduced to Nannochloropsis oceanica, N. limnetica and Phaeodactylum tricornutum, respectively. Randomly selected transformants were confirmed by PCR and Western Blot. Positive clones were used for the in vitro bacteriostatic experiments, and some lines were used as additives to feed zebrafish in order to test the effect of different nutrients on the immune system of fish. The results showed that all the three algal strains had successfully expressed exogenous antimicrobial peptides. Only P. tricornutum showed a bacteriostatic effect on Edwardsiella tarda, a common pathogenic bacterium in aquaculture, and there was no significant difference in the bacteriostatic effect between the wild-type and transformants of the three species of microalgae. The growth of zebrafish fed with algae as additives had no significant difference compared with the control (without additives). The expression levels of antioxidant and immune related genes together with the content of malondialdehyde (MDA) in the liver of zebrafish indicated that zebrafish fed with additives showed stronger antioxidant and anti-inflammatory ability than the control, and expression of antimicrobial peptides (PtC, a wild-type P. tricornutum strain Pt1 transformant expressing Cath-1a gene) could further enhance the immunity of zebrafish. In addition, the anti-inflammatory capacity of the group with Pt6 (a wild-type P. tricornutum strain with high fucoxanthin content) additives is more significant than that of the group with PtC additives, which indicated that fucoxanthin and eicosapentaenoic acid (EPA) in P. tricornutum might enhance disease resistance of zebrafish.