钝顶螺旋藻表达载体p215 t-spp的构建及转化  

Construction and Transformation of Expression Vector p215t-spp of Spirulina platensis

作　　者：王芬[1] 叶霁[1] 曹媛媛[1] 张海生[1] 甘旭华[1] 唐欣昀[1] 

机构地区：[1]安徽农业大学生命科学学院,安徽合肥230036

出　　处：《激光生物学报》2009年第1期12-16,共5页Acta Laser Biology Sinica

基　　金：安徽省教育厅自然基金项目(2006KJ173B;2007jq1052)

摘　　要：用合适的限制性内切酶消化载体p215 t,胶回收获得含有gfp基因及amp基因大片段。设计引物,采用PCR扩增无菌钝顶螺旋藻A9藻株的强启动子片段;在T4连接酶的作用下进行体外连接重组,构建了带有螺旋藻启动子和gfp报告基因的新型表达载体p215 t-spp。将该载体转入钝顶螺旋藻,利用报告基因的表达,在荧光显微镜下观察并记录转化藻细胞,p215 t-spp质粒显著提高转化率。研究了不同PEG浓度、转化时间及冰浴处理对转化率的影响。采用1%PEG能有效促进细胞吸收DNA,获得10.5‰的转化率。实验初步证明,带有螺旋藻启动子的报告基因gfp能够在钝顶螺旋藻中顺利表达。The plasmid p215t was digested by the restriction enzymes and the fragment with gfp and amp gene was recovered. The promoter fragment of Spirulina A9 strain was obtained by PCR. Then the fragments above were recombined by T4 DNA ligase, and the new plasmid p215t-spp with gfp and amp gene was constructed. The new plasmid was transformed into Spirulina platensis. The evidences of fluorescence microscopy observation and electrophoretogram proved that the plasmid had been constructed successfully. Plasmid p215t-spp could increase the transformation rate remarkably. The effects of PEG concentration and time on transformation were studied. Treatment of 1% PEG could help cells to absorb DNA fragments and 10.5 %, transformation rate was obtained. This experiment proved that report gene gfp with the promoter of S. platensis could be expressed successfully in S. Platensis and E. coll.

关 键 词：钝顶螺旋藻 启动子 GFP基因 PEG转化 转化率 

分 类 号：Q943.1[生物学—植物学]