Functional Analysis of the Regulation and Enhancement of the luxU-luxV genes from Photobacterium leiognathi

• Main Authors: Wang Pei-Ju, 王蓓茹
• Other Authors: Juey-Wen Lin
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/90582081764852323003

碩士 === 國立中興大學 === 生物化學研究所 === 91 === Photobacterium leiognathi ATCC 25521 genome library was constructed to select the specific regulatory gene(s) by bioassays in E. coli. The luxU and luxV gene were cloned. Regulations of the luxU and luxV genes are independent. The luxU gene contains a weak cAMP-CRP binding site and half repressed gene expression in presence of glucose. In contrast, the luxV gene was obviously not enhanced by glucose directly. The luxU and luxV genes transcription are independent can be proved by RT-PCR. Three transcriptional initiation sites of the luxU gene were identified by primer extension. The P(III)-promoter is identified as a Class I cAMP-CRP -dependent promoter which transcription from +1(1*). Bioassay demonstrated that of the luxU gene’s expression was reduced when luxV gene coupled with the luxU gene. DNＡ binding assay showed both the LuxV and LuxU can bind [lux]R&R(PR) DNA, it confirms that the LuxV and LuxU were coupled for the luxU down-regulation. The LuxU protein apparently enables to enhance bioluminesence of PL741 lux operon in E. coli, but no function for ATCC 25521 lux operon. DNA binding assay proved that the LuxU binds PL741 [lux]R&R(PR), but not binds ATCC 25521 [lux]R&R. The specific genes were selected from ATCC 25521 genome library that enhanced by the LuxU. ufoII and motX genes apparently enhanced by the LuxU. Sequence analysis showed that the LuxU binding sequence is TGT-N8-12-ACA. The similar sequence also resided in of PL741 [lux]R&R(PR), but not in ATCC 25521 [lux]R&R. It suggests that the conserved sequence could be recognized by the LuxU to enhance the gene expression.