The Reaserch Of Promoters And NADH Reductase II In G. Oxydans

Gluconobacter oxydans is unsurpassed by other organisms in its ability to incompletely oxidize a great variety of carbohydrates,alcohols and related compounds. Hence, the organism is used for several important biotechnological processes. Since the genome sequence of Gluconobacter oxydans had been published, the molecular biology of the organism has grown rapidly. Through modern biotechnology to improve its genetic characteristics is an effective way to further study and clarify its role in biological catalysis.In this research, we constructed a promoter-pribe vector pBBRlMCS5-TFP by using a promoterless TFP gene and a wide-host plasmid pBBRlMCS-5, in order to clone a number of promoter fragments from G oxydans M5 genome. Then we detected the fluorescence intensity of different clonies by fluorescence microplate reader and select the promoter with the highest fluorescence intensity, which was named Lhp. This selected fragment has typical zones of-35 and-10 by sequence analysis.A pair of primer was designed to obtain the full length of NADH:Q reductase respectively. Then it was inserted into the recombinant vector pBBR1MCS5-LhpTFP, under control of the Lhp promoter. The enzyme activity was verified by NADH oxidation activity, indicating that the expression of NADH:Q reductaseⅡgene was increased in Gluconobacter oxydans. This result implied that the Lhp promoter fragment we screened could start gene transcription effectively. At the same time, the NADH:Q reductase gene was overexpressed in Gluconobacter oxydans.In the respiratory chain of Gluconobacter oxydans, there is only one type of NADH:Q reductase, NDHⅡ. It has been a promising target of new antibiotics.Hence, in order to explore the relationship of structure-function of the protein, an efficient approach for expression of NADH:Q reductaseⅡgene recombinant in E.coli was proposed. The NADH:Q reductaseⅡgene was amplified by means of PCR and the expression vector (pET28-ndh) was constructed. The effects of different expression conditions were investigated systematically. Under the optimized culture conditions (25℃,0.4 mM IPTG,10 h), the high purity and activity protein was obtained and purified by Ni-affinity chromatography. This could lay the foundation for the futher study of respiratory chain in Gluconobacter oxydans...