顶头孢霉pcbAB-pcbC双向启动子区域的克隆与应用

Cloning of Bidirectional pcbAB-pcbC Promoter Region from Cephalosporium acremonium and Its Application

用PCR方法从丝状真菌顶头孢霉中克隆出全长 1 3kb的pcbAB_pcbC双向启动子DNA片段 ,通过转化子对博莱霉素的抗性证明了该启动子在顶头孢霉中的双向启动功能。另外 ,利用所克隆的pcbAB_pcbC双向启动子构建了一个用于顶头孢霉转化的质粒pYG13,并成功地将该质粒转化入顶头孢霉。pYG13含有博莱霉素抗性基因和透明颤菌血红蛋白基因 (vgb) ,Southern杂交和CO结合实验分析显示vgb整合到顶头孢霉的基因组DNA中并表达了有活性的透明颤菌血红蛋白。

kb DNA fragment containing the full_length bidirectional pcbAB_pcbC promoter region was cloned from filamentous fungus Cephalosporium acremonium by PCR amplification, and the function of the promoter region was confirmed with the expression of bleomycin resistant gene. Using the bleomycin resistance as dominant selective marker, plasmid pYG13 which contains Vitreoscilla hemoglobin gene(vgb) was constructed with the bidirectional promoter, and successfully transformed into Cephalosporium acremonium. Southern blotting and Carbon monoxide_binding analysis reveal that vgb gene is integrated into the genomic DNA of Cephalosporium acremonium and functional vgb gene was expressed in Cephalosporium acremonium.