›› 2018, Vol. 30 ›› Issue (10): 1705-1714.DOI: 10.3969/j.issn.1004-1524.2018.10.14

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Application of bacterial-one-hybrid (B1H) system in screening factors interacting with methanol dehydrogenase gene promoter in a methylotrophic bacteria strain

ZOU Qiqi, QI Shanshan, XIE Luhan, XIN Qi, LI Junyue, ZHANG Mengning, GE Xin*   

  1. College of Life Science, Hebei University, Baoding 071002, China
  • Received:2018-01-15 Online:2018-10-25 Published:2018-11-02

Abstract: To construct the transcription factor sublibrary of methylotrophic bacteria and screen the proteins interacting with the methanol dehydrogenase promoter, we found many candidate genes by analyzing the genome and transcriptome databases of methylotrophic bacteria (MP688) in the use of some key words searching the genome annotation and transcriptome result, then amplified the target genes by PCR (polymerase chain reaction) and construct a series of transcription factor sublibrary. Finally B1H and EMSA (electrophoretic mobility shift assay) methods were employed to screen and verify the candidate factors. We had constructed a total of 32 transcriptional factor clones, and two transcriptional factors which have strong interaction with methanol dehydrogenase promoter were screened out. For bacteria having detailed genomic sequence information, the construction of the transcriptional factor sublibrary is better than the construction of the genomic library or cDNA library. B1H system can be successfully applied in screening the DNA binding proteins in methylotrophic bacteria. And the establishment of this method laid a solid foundation to clarify the regulatory mechanism of methanol dehydrogenase gene in the process of development and metabolic product in the methylotrophic bacteria.

Key words: transcriptional factor, gene library construction, bacterial-one-hybrid system

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