Capturing the target genes of BldD in Saccharopolyspora erythraea using improved genomic SELEX method
作者单位:Institute of Health Sciences School of Life Sciences Anhui University School of Life Sciences Hefei Normal University CAS Key Laboratory of Pathogenic Microbiology & Immunology Institute of Microbiology Chinese Academy of Sciences
会议名称:《中国生物工程学会2014年学术年会暨全国生物技术大会》
会议日期:2014年
学科分类:0710[理学-生物学] 07[理学] 071007[理学-遗传学]
摘 要:BldD(SACE077), a key developmental regulator in actinomycetes, is the first identified transcriptional factor in Saccharopolyspora erythraea positively regulating erythromycin production and morphological differentiation. Although the Bld D of S. erythraea binds to the promoters of erythromycin biosynthetic genes, the interaction affinities are relatively low, implying the existence of its other target genes in S. erythraea. Through the genomic SELEX method that we herein improved, four DNA sequences of S. erythraea A226, corresponding to the promoter regions of SACE306(beta-galactosidase), SACE811(50 S ribosomal protein L25), SACE410(fumarylacetoacetate hydrolase) and SACE014(aldehyde dehydrogenase), were captured with all three BldD concentrations of 0.5μM, 1μM and 2μM, while the previously identified intergenic regions of ery BIV-ery AI and ermE-eryCI plus the promoter region of SACE115, the amfC homolog for aerial mycelium formation, could be captured only when the BldD’s concentration reached 2μM. EMSA analysis indicated that BldD specifically bound to above seven DNA sequences, and qRT-PCR assay showed that the transcriptional levels of above-mentioned target genes decreased when bldD was disrupted in A226. Furthermore, SACE115 and SACE306 in A226 were individually inactivated, showing that SACE115 was predominantly involved in aerial mycelium formation, while SACE306 mainly controlled erythromycin production. This study provides valuable information for better understanding of the pleiotropic regulator BldD in ***, and the improved method may be useful for uncovering regulatory networks of other transcription factors.