cloning and functional analysis of the specific genes that enhance bioluminescence of the lux operon from photobacterium leiognathi

• Main Authors: chia-hsiu huang, 黃嘉秀
• Other Authors: juey-wen lin
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/81252731537230360596

碩士 === 國立中興大學 === 生物化學研究所 === 90 === Abstract The genes concerned with bioluminescence of Photobacterium leiognathi are formed the lux operon. The lux operon of P. leiognathi ATCC 25521 cloned in E. coli showed dim; it suggests that the specific regulatory gene(s) is required for the lux operon. Genome library was constructed to select the specific regulatory gene(s) of P. leiognathi lux operon by in trans complementation bioluminoassays in vivo. Two clones, which can enhance the luminance of P. leiognathi lux operon in E. coli, were selected. It included the luxR[T] and crp genes; LuxR[T] and CRP were identified by DNA sequence analysis. LuxR[T] is the response regulator protein of two-component regulatory system. The crp gene encodes cyclic AMP receptor protein CRP. The regulatory region R&R of the luxR[T] gene includes Promoter[H], Promoter[F], Promoter[D] conserved sequences that could be recognized by σ32-RNA polymerase, σ54-RNA polymerase and σ70-RNA polymerase. It implies that the luxR[T] gene could be activated in various situations. Only Promoter[H] conserved sequence for σ32-RNA polymerase was found in the regulatory region R&R of the crp gene. Sequence analysis suggests that LuxR[T] could recognized the conserved sequence 5’-TGT-N8-12-ACA-3’ resided 357th to 366th bp in the regulatory region R&R. Doubtlessly, CRP could formed cAMP-CRP dimer to bind the 5’-TAGTGTGACCA-3’ sequence resided upstream -62 of the regulatory region R&R. The result demonstrates that the P. leiognathi lux operon is regulated by the cAMP-CRP and two component regulatory system of the luxR[T] gene.