微拟球藻抗生素抗性分析和选择标记筛选

Antibiotic resistance of Nannochloropsis oculata and determination of selection markers for its genetic transformation

为了筛选到微拟球藻Nannochloropsis oculata的筛选标记,研究了微拟球藻对新霉素(Neomycin)、链霉素(Streptomycin)、潮霉素(Hygromycin)、壮观霉素(Spectinomycin)和博来霉素(Zeocin)的敏感性。每种抗生素都设置液体培养和固体培养实验并且浓度梯度相同。新霉素和链霉素的浓度梯度为:0、100、200、400、600、800μg/ml。潮霉素的浓度梯度为0、25、50、100、125、150μg/ml。壮观霉素浓度梯度为0、40、80、100、150、200μg/ml。博来霉素浓度梯度则为0、0.1、0.2、0.4、0.5、1μg/ml。结果表明,微拟球藻对新霉素、链霉素、潮霉素和壮观霉素不敏感,而对博来霉素高度敏感。在固体培养时,0.5μg/ml博来霉素即可完全抑制微拟球藻生长;在液体培养时,1μg/ml博来霉素即可完全抑制其生长。博来霉素可作为微拟球藻遗传转化的选择抗生素。ble基因为其阳性筛选的选择标记基因。

To determine the selection marker of N.oculata,the sensitivity to 5 antibiotics（Neomycin,Streptomycin,Hygromycin,Spectinomycin,and Zeocin） was determined in this study.For each antibiotic,liquid medium and solid medium were both adopted with the same concentration gradient.The concentration gradient of Neomycin and Streptomycin was 0,100,200,400,600,and 800μg/ml,while that of Hygromycin was 0,25,50,100,125,and 150μg/ml,and that of Spectinomycin was 0,40,80,100,150,and 200μg/ml.The concentrations of Zeocin were much lower,which were 0,0.1,0.2,0.4,0.5,and 1μg/ml.It was found that N.oculata is not sensitive to Neomycin,Streptomycin,Hygromycin or Spectinomycin,but highly sensitive to Zeocin.Zeocin can completely inhibit the growth of N.oculata when its concentration is 0.5μg/ml in solid medium or 1μg/ml in liquid medium.Zeocin could serve as a selection reagent in the genetic transformation of N.oculata,and the ble gene could be the selection marker.