钝顶螺旋藻和乳酸乳球菌对驼背鲈非特异性免疫力影响及其诱发的免疫器官特异表达基因研究

Hamperback grouper,Cromileptes altivelis, is a rare and precious seawater fish. With high economic and ornamental values, its aquaculture prospect is bright. In recent years, its artificial breeding technique has been broken through and large sum of fry could be obtained in Hainan. Our research group has successfully improved the survival rate and growth rate of hamperback grouper by adding Spirulina platensis and Lactococcus lactis in its feed. However, the effect of Spirulina platensis and Lactococcus lactis on its immunity and its genetic regulation mechanism was still not clear, and the immunology character of hamperback grouper is blank either. In this project, the operation scheme of Spirulina platensis and Lactococcus lactis will be optimized and the effect of these two feed addatives on the nonspecific immunity of hamperback grouper will be studied. Then, the specific expression genes of the main immuno-related organs (headkidney and spleen) transcripts after being induced by Spirulina platensis and Lactococcus lactis will be identified by Solexa high-throughput sequencing technique, Real-Time PCR and bioinformatics analysis. The results of this study will not only explore the function mechanisms of these two immunostimulants’ effect on improving the nonspecific immunity of hamperback grouper from serological and transcriptomic level, but also be helpful for understanding the immunological characteristcs of hamperback grouper and promoting the sustainable development of its culture.

驼背鲈属珍稀名贵海水鱼类，具有重要的经济价值，养殖发展前景广阔。近年来，我国海南等地已突破了驼背鲈的人工繁育技术并实现了其苗种的规模化生产，本项目组也通过在饵料中添加钝顶螺旋藻和乳酸乳球菌等措施突破了驼背鲈养殖中成活率低和生长速度慢等瓶颈。然而，有关钝顶螺旋藻和乳酸乳球菌对驼背鲈免疫功能的影响及其免疫调节机制尚不清楚，对驼背鲈免疫生物学特征的研究迄今仍属空白。为此，本项目将在优化钝顶螺旋藻和乳酸乳球菌使用方案和确定其对驼背鲈非特异性免疫力影响的基础上，采用Solexa高通量测序和实时定量荧光PCR相结合技术，并借助生物信息学分析等手段确定经该2种饵料添加剂诱导后驼背鲈头肾和脾脏等主要免疫器官转录组的特异表达基因，项目成果不仅可从血清学水平和转录组水平阐明该2种饵料添加剂提高驼背鲈非特异性免疫力的作用机制，还对了解驼背鲈的免疫生物学特征和促进驼背鲈人工养殖可持续发展有重要意义。

驼背鲈又名老鼠斑，是珍稀名贵的海水鱼类，具有重要的经济价值，养殖发展前景广阔。为深入了解益生菌和微藻对驼背鲈生长、免疫功能的影响及其免疫调节机制，本项目首先从4种益生菌中筛选得到了一株来源于野生驼背鲈肠道且能够显著提高驼背鲈生长速度及抗病力的益生菌（乳酸乳球菌HNL12），该益生菌能较好地耐受人工胃肠液且具有较高的自凝集活性；在基础饲料中添加三种不同浓度的HNL12连续拌料投喂4周后，添加浓度为1×108 CFU/g HNL12能显著提高驼背鲈的体重、增重率和特定生长率；非特异性免疫指标检测表明，1×108 CFU/g HNL12添加组能显著提高驼背鲈的巨噬细胞呼吸爆发活性、SOD、ACP和LZM活性。以哈维氏弧菌攻毒结果表明，HNL12添加组能显著提高驼背鲈的抗病力，相对保护率最高达到53.14%。比较转录组的结果表明，投喂1×108 CFU/g HNL12的驼背鲈组与对照组相比，共获得差异表达基因307个；GO及KEGG富集分析显示，这些差异表达基因的功能和参与的通路主要和感染性疾病、抗原呈递及其他免疫系统应答相关。qRT-PCR结果显示，驼背鲈的几个免疫相关基因均显著性上调，且趋势与转录组测序结果一致。根据前期研究结果和养殖生产的使用效果，进一步研究益生菌HNL12与裂壶藻粉联用对驼背鲈生长和非特异性免疫功能的影响，结果表明，以1×108 CFU/g HNL12和1%裂壶藻联用投喂驼背鲈，比单独使用益生菌或裂壶藻均能显著促进驼背鲈的生长、提高其非特异性免疫力及抗病力。比较转录组测序结果显示，菌藻联用组比基础饲料组，共获得差异表达基因157个；GO及KEGG富集分析显示，这些差异表达基因的功能和参与的通路主要和感染性疾病、抗原呈递、消化系统及其他免疫系统应答相关。qRT-PCR结果显示，乳酸乳球菌与裂壶藻联用能显著提高驼背鲈免疫相关及脂质代谢相关基因的表达，且显著下调炎症因子的表达。本项目研究结果对了解驼背鲈的免疫生物学特征和促进驼背鲈人工养殖可持续发展有重要意义。