Cloning and Application of lux Genes from Vibrio orientalis

• Main Authors: Yan-Wei Huang, 黃彥瑋
• Other Authors: Douglas J.H. Shyu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/89604666031997168514

碩士 === 國立屏東科技大學 === 生物科技研究所 === 100 === Communication among bacteria with quorum-sensing system is accomplished through the exchange of signaling molecules called autoinducers. When the density of autoinducers accumulated to a certain concentration, bacteria responded by specialized gene expression such as genes involved in biofilm formation, bioluminescence, virulence and sporulation. Currently it still remains unclear about the light-emitting and regulatory mechanism of the marine luminous bacteria V. orientalis. It is speculated that the expression of bioluminescence genes in V. orientalis is also regulated by the quorum sensing system. In this study, the lux genes of V. orientalis are identified with the same five major structural genes luxCDABE in contrast to other luminous bacteria. The synthesis of luciferase is mediated by luxA and luxB genes, and that of fatty acyl reductase, acyl transferase, and acyl protein synthase, comprising the fatty acyl reductase complex for providing the substrate for luciferase, are mediated by luxC, luxD, and luxE genes, respectively. Moreover genes of lux operon are found varied in different luminous bacteria. Among these, the luxAB genes could be used as reporter genes for monitoring promoter and terminator activities after cloning into the vectors for the construction of reporter vectors. Upstream sequences of V. orientalis lux operon are analyzed to predict the potential promoter region and the regulatory elements involved in the activation and inhibition of gene expression. Serial deleted DNA fragments from upstream sequences are cloned and analyzed by the promoter reporter vector for this purpose. Possible DNA sequences of rho-independent terminators are also cloned and analyzed by the terminator reporter vector. The results of this study would provide both useful information for the investigation of lux genes regulation in V. orientalis and facile tools for the monitoring of potential promoter and terminator activities from genes of various organisms.