建立螺旋藻转基因体系初报

从钝顶螺旋藻单细胞克隆的制备、重组平台的 克隆、同源重组表达质粒的构建、质粒的电激转化和报告基因的表达等方面对螺旋藻转基因 表达系统进行了研究，初步建立起螺旋藻转基因体系。用次氯酸钠溶液处理获得无菌培养系 ；用钠、钙离子混合液处理，获得了可再生的螺旋藻单细胞。用含EDTA的培养基预培养和用 培养基洗涤可分别去除胞外核酸酶活性和抑制胞内核酸酶活性。以氯霉素乙酰转移酶基因替 换大肠杆菌表达质粒pBV220的抗性选择标记，同时插入系列同源重组片段和萤火虫荧光素酶 基因，构建了同源重组表达载体pBVCS01-04L。电激转化螺旋藻S6-4品系单 细胞，获得了具有稳定氯霉素抗性的培养系，并用SDS-聚丙烯酰胺凝胶电泳证实了报告基 因的表达。

A Preliminary Report on Transgenic Expression System for Spirulina platensis

The transgenic expression system for Spirulina plat ensis was conducted, including the preparation of axenic single-cells, the co nstruction and tramsformation of homologous recombinant plasmid and the expressi on of reporter genes. Axenic single-cells with regeneration capacity of the alg a were obtained by treating with 0.4% sodium hypochloride for 5min and an ion mi xture of sodium and calcium at the concentration of 500mM, pH7.5. The external n uclease activity was eliminated by washing with medium and the internal nuclease activity was inhibited by EDTA treatment. 　　The pBV220 was chosen as the starting plasmid and then plasmid pBVC was constr ucted by replacing the Ampr gene on the starting plasmid with the Cat gene on the plasmid pIJ4813. Four random homologous recombinant fragments (Spr01 -04) cloned by PCR were inserted into plasmid pBVC respectively, producing fo ur homologous recombinant plasmids pBVCS01-04. Finally firefly luciferase gene was inserted into pBVCS01-04, producing the homologous recombinant expression plasmids pBVCS01-04L, which have inherited cIts857, PRPL, rrmBT1T2, Cat, Spr and luc+. The cells of S. platensis strain S6-4 were transformed with four homologous recombinant expression plasmid pBVCS01-04L by electroporation and then spread on selective Zarrouk medi um after vigor restoring for 4 days in liquid medium without chloromycetin. Clon es with chlorlmycetin resistance were obtained after cultivation at 25℃ for 30 days. Chloromycetin resistance was performed over 20 following generations. The transformants were verified by SDS-PAGE electrophoresis showing the band of luc iferase.