Isolation And Expression Of The Bioluminescence Genes Of Vibrio Qinghaiensis

The luminescent bacteria are those bacteria that contain lux genes and could emitblue-green light in the process of their normal metabolism. The majority species ofluminescent bacteria are halophilic and found in saltwater environment, except the terrestrialPhotorhabdus species and Vibrio qinghaiensis sp.-Q67,Vibrio cholerae biovar albensis whichsomewhat unique in their ability to also flourish in freshwater environments. Being proved tobe less toxic and sensitive, lux genes have been used as reporters of many biological functions,particularly for long-term studies of living cells. Compared with the reporter genes, such aslacZ and xylE, luxCDABE has certain advantages. It is real-time and additional substrateswere unnecessary for signal generation. However, characteristics of low temperature tolerance,saltwater environment requirement and narrow pH adaptability, restrict the further applicationof luminescence. Luciferase with better performance is expected and perhaps Q67is one ofthe candidates.The objective of the study is isolation of luxAB and luxCDABE genes, and cloned theminto suitable prokaryotic expression vectors to produce the desired protein in E. coli and it canbe used as a reporter gene for further biotechnology applications. The second goal is todevelop a better understanding of the basic biochemical properties of Q67and luciferase. Themain conclusions are as following.（1）16S rDNA of Q67was obtained, and the phylogenetic tree shows that theluminescent bacteria can be divided into four genera, including Vibrio, Photobacterium,Shewanella and Photorhabdus (Xenorhabdus).16S rDNA sequence of Q67shows99%identity to that of Vibrio anguillarum (NCBI access NO. KC404866.1) and Vibrio ordalii(NCBI access NO. AY530930.1).（2）luxAB and luxCDABE fragments were obtained through Polymerase ChainReaction (PCR), and ORF of luxC, luxD, luxA, luxB, luxE were found and1446bp、954bp、 1143bp、975bp and1155bp were contained respectively.（3）Primary structure, secondary structure and tertiary structure of proteins coded byluxCDABE were predicted. The prediction of chemical and physical properties shows that αsubunit of luciferase was stable and all proteins are hydrophilic. Phylogenetic tree shows thatsequence of Q67luciferase to be highly homologous to the proteins from Vibrio choleraestrain Albensis.(4) The recombinant plasmids PluxAB and PluxCDABE were successfully transformedinto Top10and created T-PluxAB、T-PluxCDABE and they expressed successfully inprokaryotic cells. The maximum light intensity of T-PluxCDABE was13without decanaladded. When induced by IPTG, resuspended with new medium, and decanal added beforelight measurement, light intensity can be increased largely.