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以p ET-28a-RFP质粒为模板,PCR扩增红色荧光蛋白(red fluorescent protein,RFP)基因,并克隆到真核表达载体p TE11上,置于RP27启动子调控之下,成功构建表达载体p TE11-RFP。采用PEG介导转化法,将p TE11-RFP转入银耳芽孢中。提取转化子基因组DNA,RFP基因特异性引物扩增获得与目的基因大小一致的特异条带;日光下肉眼观察转化子略显红色,荧光显微镜观察有明显的红色荧光。以上结果证明RFP基因已成功转入银耳芽孢并进行表达,RP27启动子可以调控外源基因RFP在银耳芽孢中的正确表达,为进一步研究外源基因在银耳芽孢生物反应器中的高效表达奠定了一定的基础。
The red fluorescent protein (RFP) gene was amplified by PCR using p ET-28a-RFP plasmid as a template and cloned into the eukaryotic expression vector p TE11. The RFP gene was placed under the control of RP27 promoter and the expression vector p TE11-RFP. Using PEG-mediated transformation, p TE11-RFP was transferred into Tremella fuciformis. The genomic DNA of the transformant was extracted and specific bands of RFP gene were amplified by PCR to obtain the specific bands with the same size as the target gene. The naked eye showed that the transformant was slightly red when observed under fluorescent microscope and obvious red fluorescence was observed under fluorescence microscope. The above results prove that the RFP gene has been successfully transformed into the spore of T. treponema and expressed. The RP27 promoter can regulate the expression of the foreign gene RFP in the spore of T. gonorrhoeae, and lay the foundation for further study on the efficient expression of the foreign gene in the spore bioreactor A certain basis.