摘要
海洋球石藻Emiliania huxleyi是一种全球广泛分布的真核浮游植物,该种不仅是海洋碳、硫循环和全球气候变化的重要指示物种,而且能够产生丰富的次级代谢生物活性物质,在生物技术领域也具有很好的应用前景。本文通过分析氨苄青霉素、卡那霉素、G418、氯霉素、链霉素、新生霉素及嘌呤霉素等7种常用抗生素对海洋球石藻生长的影响,确定G418可作为该藻阳性转化藻株的抗性筛选试剂,其对应的抗性基因neo则作为该藻表达载体构建中的抗性筛选标记。在此基础上克隆了绿色荧光蛋白基因gfp、抗性标记基因neo及E.huxleyi BOF92内源性岩藻黄素-叶绿素a/c结合蛋白基因的启动子fcp,以pUC18为基础载体,构建了pUC18-fcp-gfp和pUC18-fcp-neo两个重组表达载体,以电转化方法共转化球石藻细胞并结合选择性固体培养基筛选,成功获得了被转化的球石藻细胞。海洋球石藻遗传转化系统的建立为进一步开展该种相关的基础生物学研究及其在生物技术领域的应用奠定了基础。
The marine coccolithophore Emiliania huxleyi is a eukaryotic microalga species crucial to the study of global biogeochemical cycles and climate modeling and also much of interest to those in biotechnology due to the capable of abundant bioactive metabolites production.Here,seven different kinds of antibiotics including ampicillin,kanamycin,G418,chloramphenicol,streptomycin,novobiocin and puromycin were used for the screening of antibiotic resistance.G418 was chosen most suitable selective antibiotics and the corresponding resistance gene＂neo＂as the marker for E.huxleyi genetic system.The promoter of the endogenic fucoxanthin chlorophyll a/c-binding protei n gene＂fcp＂was cloned fromE.huxleyi BOF92 strain.A construct was made containing the green fluorescent protein reporter gene＂gfp＂and screened G418 resistance gene＂neo＂.The resultant recombinant transformation vectors pUC18-fcp-gfp and pUC18-fcp-neo were co-transferred into E.huxleyi by electroporation.Transformants were obtained upon G418 selection.The results presented the new genetic transformation system for E.huxleyi,providing additional genetic resource with potential for exploring basic biological questions and biotechnological applications.
出处
《海洋学报》
CAS
CSCD
北大核心
2016年第8期103-114,共12页
基金
国家自然科学基金(41576166)
福建省科技重点项目(2015Y0039)
厦门市南方海洋研究中心项目(14GZP71NF35)
