微孢子虫(microsporidia)长期专性细胞内寄生,其基因组表现出显著的减缩性。利用家蚕微孢子虫(Nosema bomby-cis)基因组数据库探究在其显著减缩的基因组中是否有编码序列发挥调控序列的作用,结果发现在DNA转录调控中发挥重要作用的细胞分裂周期蛋白(cell division cycle protein)的N端编码序列具有典型的真核生物启动子序列特征。设计引物扩增该基因N端590 bp包含启动子基序的序列(C启动子序列),将该启动子序列克隆到载体启动子(MET25-P)被终止序列(CYC1-T)替代的pUG35载体上,构建由该启动子序列启动下游绿色荧光蛋白(GFP)表达的克隆载体ΔpUG35-CYC1-T-C-yEGFP-CYC1-T,并将其电击转化酿酒酵母CEN.PK2菌株,经筛选及诱导培养后,在荧光显微镜下观察到酵母中有GFP表达,证实了家蚕微孢子虫细胞分裂周期蛋白的N端编码序列包含具有启动子活性的序列,表明家蚕微孢子虫基因组减缩后,部分编码序列可能扮演了调控序列的角色。研究结果也为微孢子虫启动子的活性验证提供了一个简便的途径。
As to long term intracellular parasitism,the genome reduction of microsporidia was significant.We surveyed the genome data of Nosema bombycis to identify whether there is any coding sequence acting as the promoter sequence in such significantly reduced genome.We found that there are typical characteristics of the eukaryotic promoter in the N-terminal coding sequence of cell division cycle protein(CDCP) which plays an important role in regulation of DNA transcription.We designed specific primers to amplify this gene's N-terminal 590 bp which contained the promoter motif sequence(C promoter sequence).Subsequently,we cloned it into the pUG35 vector of which the promoter(MET25-P)was replaced with the termination sequence CYC1-T to construct the vector ΔpUG35-CYC1-T-C-yEGFP-CYC1-T that had the 590 bp promoter sequence to activate expression of its downstream green fluorescent protein(GFP).We transformed this vector into the Saccharomyces cerevisiae CEN.PK2 strain by electroporation,and obtained the positive clone.After screening and induction culture,fluorescence microscopic observation showed signals of GFP expression,verifying that the N-terminal coding sequence of cell division cycle protein of N.bombycis has promoter activity.This phenomenon indicates that some coding sequences could act as regulatory elements in the reduced genome of N.bombycis.Our work also provides a simple method to test the promoter activity of microsporidia.