Gene Expression and Regulation of Bioluminescence: Functional Analysis of the Regulatory Gene of the lux Regulon form Vibrio fischeri

• Main Authors: Hui-Yi Chen, 陳惠
• Other Authors: Juey-Wen Lin
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/54920807387796787431

碩士 === 國立中興大學 === 分子生物研究所 === 81 === For the purpose of simplicity, only the regulatory genes, luxR and luxI, were used for studying in this work. A series of gene order rearrangement constructions and in trans comple- mentary tests were designed to investigate the requirement of the sequences of the luxR and luxI genes, and functional analy- sis of the LuxR protein and autoinducer (AI). The luciferase genes, luxA-luxB, from V. harveyi were used as reporter gene to monitor the expression of the R- and L-promoter. The results show that the sequences of the luxR and luxI genes were the ne- gative control elements for the expression of the R- and L-pro- moter. The LuxR-AI complex is the positive control element for both the R- and L-promoter, and directly bound to the R&R se- quence. The sequence of the luxR gene repressed the expression of the L-promoter much stronger than the effect of the sequence of the luxI gene to the R-promoter. Nucleotide sequence analysis showed that an reversed repeat sequence, termed R3- reversed re- peat, was found on the sequence of the luxR gene, and it might be the LuxR motif-binding locus. The site-directed mutagenesis was used to inspect the function of this site. The results of the luxR mutations show that the premature translation of the LuxR protein repressed the expression of the following reporter genes. It suggested that the sequence of the luxR gene might re- gulate the lux regulon by attenuation-like termination. Further modification in this site was be done to define the function of the R3-reversed repeat, but the result can't confirm R3-reversed repeat is LuxR protein motif-binding locus for negative feedback control. The LuxR protein might bind on one specific site of the luxR sequence to enhance the attenuation-like transcriptional termination and negative feedback control the expression of L- operon.