高效电穿孔转化淡水藻类——微拟球藻。
High efficiency transformation by electroporation of the freshwater alga Nannochloropsis limnetica.
机构信息
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
出版信息
World J Microbiol Biotechnol. 2019 Jul 22;35(8):119. doi: 10.1007/s11274-019-2695-9.
The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 μg mL of zeocin or 5 μg mL of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11 × 10 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well. High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.
微藻属的拟球藻由于其脂质含量高，被认为是生产生物燃料最有前途的生物之一。近十年来，已建立了海洋拟球藻物种的转化系统，然而，该属中唯一已知的淡水物种——网纹藻的遗传操作尚不可用。基于已建立的海洋拟球藻物种电转化方案，在网纹藻中建立了核遗传转化，同时确定了电穿孔的适当抗生素选择浓度和电场强度。对于网纹藻平板转化子的选择，证明 0.07 μg mL 的 zeocin 或 5 μg mL 的 Hygromycin B 就足够了，使用 2-mm 电穿孔管，单个脉冲的电场强度为 2200 至 2600 V 时，转化效率<2×10-1。电穿孔前用 10 mM 醋酸锂和 3 mM 二硫苏糖醇预处理网纹藻可使转化效率提高数百倍，在 12,000 V/cm 的电场强度下获得了最高的 10-11×10-1 的转化效率。我们的结果有助于扩大该淡水物种的生物技术应用，并为其他微藻的成功电转化提供了手段。高效转化淡水拟球藻用 10 mM 醋酸锂和 3 mM 二硫苏糖醇预处理网纹藻可使转化效率提高数百倍。
Zotero 插件