Screening, Sequence Analysis And Characterization Of Novel Plasmids From The Bacteria Isolated From The Arctic Sea-ice And Jiaozhou Bay Sediments

A plasmid is an extra chromosomal DNA molecule separate from the chromosomal DNA which is circular and double-stranded and capable of replicating independently. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., in Saccharomyces cerevisiae). Plasmids are one of the greatly important tools in the field of genetic modification and engineering and hold large potential in the establishment of novel expression systems of which some (e.g., cold-adapted expression systems) have been widely utilized. In addition, plasmids may carry genes that provide the bacteria resistance against naturally occurring antibiotics or proteins produced acting as toxins or pathogen. Research on these novel plasmids from marine environment is beneficial for elucidating the gene pool and the reshuffling, acquisition and exchange of genetic material.In this study, seven novel plasmids were obtained through screening the bacterial strains from the Arctic sea-ice and the coastal sediment of Jiaozhou Bay. In the first part of this thesis, two cryptic plasmids designated pSM327 and pSM429 were obtained from the bacterial strains Pseudoalteromonas sp. BSi327 and Pseudoalteromonas sp. BSi429 from the Arctic sea-ice. Through random restriction enzyme digestion and gene walking, the whole sequences of these two plasmids were obtained. Characterization of pSM327 and pSM429 was performed from the respects of plasmid sequences analysis and transcriptional detection of open reading frames. The plasmid pSM327 consisted of 6,114 bp with a GC content of 37%, which did not have obvious homology to other plasmids. Three putative open reading frames were predicted on the sense strand pSM327 and they all had transcriptional activities according to the results of RT-PCR. This plasmid replicated by the rolling-circle mechanism. The plasmid pSM429 consisted of 3,827 bp with a GC content of 28% and it has four putative ORFs on the sense strand in which three ORFs had transcriptional activities. This plasmid replicated by the theta mechanism. All of the experiments above laid a solid foundation for the ensuing application on the molecular level. In the second part of this thesis, five novel small plasmids were obtained by screening 120 bacterial strains from the marine sediment of Jiaozhou Bay. Plasmid biodiversity was investigated based on the results of BLAST and RT-PCR. From the analysis of plasmid sequence, ORFs on plasmids pA5-5 and pB2-12 showed some similarity to other nucleic acid sequences. But the other three plasmids displayed very little or even no homology to other sequences. From the results of RT-PCR, orf4 and orf7 on pZY5 had transcriptional activities but orf3 had no transcriptional activity; both orf1 and orf4 on pB2-12 had transcriptional activities. Besides, no plasmid was obtained by screening the bacterial strains from the sediments from the South China Sea.