Construction Of Ectoine Biosynthetic Gene Integrated Expression Engineering Strain And Its Salt Tolerance Determination

Objective Microbial community and diversity of bacteria and archaea in the water and sediment mixture samples were investigated by using Illumina high-throughput sequencing platform.The microbial resources in Salt Lake were further explored.The prokaryotic heterologous expression technology was used to clone and to construct the p ET-28a(+)-promoter+ect ABC and p Cold?DNA-promoter+ect ABC,and finally detected the salt tolerance growth ability of the recombinant expression strain.Methods Using DNA extraction kit for Chaka Salt Lake water and sediment mixture samples whole genome DNA extraction.Sequencing of the reference sample quality requiremented on the appropriate proportion of mixture,entrusting by the high-throughput sequencing.Promoter+ect ABC gene cluster sequences were obtained by PCR amplification of model strains Halomonas sp.QHL1 genome-wide.The target genes were successfully connected to the cloning expressing recombinant plasmid.After that,induced protein expressed purpose.SDS-PAGE detected the targe protein.To test the salt tolerance of target protein,it provided certain basis for later experiment.Results The species annotation OTU(Operational taxonomic unit)numbers of bacteria and archaea were 421 and 317,respectively,belonging to definite taxonomic bacteria of 14 phylum 28 classes 170 genera and classified archaea of 5 phylum 4 classes 34 genera.The bacterial dominant populations were Bacillus.Furthermore,dominant archaea populations were Halonotius.A 3335 bp sequence including ect ABC and its promoter 420 bp upstream regulation sequence was obtained.SDS-PAGE showed: ect A,ect B and ect C three genes that encode proteins of 192,422 and 129 amino acids respectively,consistenting with the prediction results.Salt gradient experiments showed E.coli BL21 compared with recombinant strains resistant to salinity increased by 50%.Conclusion Preliminary revealed the Chaka Salt Lake in bacteria and archaea community structure and species diversity,for the development of halophilic bacteria and subsequent microbial resources mining provides theory basis;Cloned promoter+ect ABC building the recombinant expression strains.For promoter+ect ABC gene cluster in strain industry played an important role in the quantitative production to provide basic experiment basis.