【摘要】 将钝顶螺旋藻(Spirulina platensis)A9藻株在24℃培养,经2 mmol/L的EDTA预处理24 h;采用功率300 W的超声波处理70 s获得单细胞样品,以本实验室构建的携带gfp基因的质粒p215t转化A9藻株单细胞藻液,利用Amp作为选择标记,使单细胞在平板上再生长出单藻落,获得17株具有Amp抗性的转化藻株,转化率3.73‰。在390 nm紫光激发下,生长30天的转化藻丝体发出稳定绿色荧光;培养45天后具有绿色荧光的藻丝出现断裂、具有荧光藻丝长度缩短的现象。实验结果初步表明:报告基因gfp在螺旋藻中得到稳定有效的表达,可以采用单细胞再生形成单藻落技术进行螺旋藻的基因克隆。更多还原

【Abstract】 A9 strain of Spirulina platensis was cultured at 24 ℃ and treated with 2 mmol/L EDTA for 24 hours.Single cell system was obtained by ultrasonic treatment with power of 300 W for 70 s.The sample of single cell system was transformed with plasmid p215t with gfp gene,and amp was used as selective marker.The single cell sample was successfully regenerated as single colonies on plates with Amp and 17 transformants were obtained with transformation rate being about 3.73 ‰.Observed by fluorescence microscope with 390 nm as stimulation light,parts of the transformants filaments presented stable green fluorescence when grew for 30 days.After 45 days part of the green fluorescent transformants trichomes began to break and the length of the trichomes with green fluorescentce were shortened.The results showed that techniques of the single cell regeneration could be applied for gene cloning in Spirulina,and the foreign gfp gene has been effectively expressed in S.platensis.更多还原