Gene Cloning Of Bop Gene From Halophilic Bacteria Seperated From Eejnoor In Inner Mongolia

Some organisms can grow in extreme environments. The extreme environments including heat, cold, high-pressure, high-salt, alkali, acid, high radiation environment and so on. The microorganisms that exist in such environment called extremophiles. Due to its special resilience, the archaea can grow in high salinity environments. This makes it a good material of biological adaptation of the environment and physiological and biochemical characteristics research. In addition, it has a good application potential on the food industry, pharmaceutical industry, environmental remediation and other chemical industry as its special enzymes and other macromolecules production.The purple membrane protein of archaea is similar with visual rhodopsin, called bacteriorhodopsin. Bacterial rhodopsin (bacteriorhodopsin, BR) is the only protein in the purple membrane of archaea, it is encoded by bop gene. BR is a light-driven proton pump, protons can be transfered from inner membrane to outer membrane in the case of lighting conditions, therefor transmembrane proton gradient is generated, the original light energy was conversed into electrochemical potential energy which can be used in many in life activities. The bacteriorhodopsin has many application potentials. The BR portein has good stability, it is also a kind of green biological functional materials which has no pollution to the environment, so it has very good application prospect.In this study a rhodopsin producing extreme halophilic archaea was isolated from Eejnoor in Inner Mongolia. It was preliminary identified as an archaea belonging to the genus Natrinema using sequence analysis of its16S rDNA and BLAST in NCBI data base.The bop gene fragment of this halophilic archaea strain has been amplified by PCR using the genomic DNA as a template, primers which designed according to the bop gene. Sequence analysis showed that the fragments length is385bp. The bop gene fragment has been cloned in the vector pET-28a, the recombinant expression vector has been named pET-28a-Bop. This sequence of the bop gene has been registered in Genbank, registration No. is KF863690. The results show that the bop gene has an open reading frame with a303bp, encoding101amino acids. Blastp result shows that this protein belongs to bacteriorhodopsin super family and has the typical structure domain of bacteriorhodopsin. Similarity analysis using the BLAST shows that the bop gene encode an amino acid sequence which has a similarity of above98%with the rhodopsin encoding genes AAS87571.1, WP-007110705and AFB77278.1in Genbank. This result indicates that rhodopsin proteins in bacteria and archaea are highly conserved in their structure, and this is closely related with their proton pump function.The growth condition of the strain has been investigated and the results showed that the suitable condition for the growth of the strain in the separation medium is1.03.0mol/L NaCl, pH6.5～8.5, magnesium ion concentration0.4～0.8mol/L. The optimum NaCl concentration is1.5mol/L, the optimum pH value is7.5, and the optimum magnesium ion concentration is0.4mol/L for its growth. This experimental results laid a foundation for the further study of physiological and biochemical characteristics and genetics of the rhodopsin produced by extremely halophilic archaea.