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Cloning Of Ectoine Biosynthesis Genes Of A Moderately Halophilic Bacterium Halomonas Sp. Nj223 From Antarctic And Characterization Of Its Function

Posted on:2008-07-15Degree:MasterType:Thesis
Country:ChinaCandidate:Z L ChenFull Text:PDF
GTID:2120360242478809Subject:Biochemistry and Molecular Biology
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Due to its specific geographical environment and seafloor topography features, Prydz Bay in Antarctica maintains a high-salt, low-temperature and high-density characteristics. The specific ecological environment offers rich microbial resources and most of them are still unknown. They often have specific physiology to adapt to the specific circumstances. Thus, They are good materials for the study of adaptation mechanism in moderate halophile.The moderate halophile strain Nj223 was selected from Prydz Bay deep-sea sediment and identified by 16S rDNA as Halomonas sp. Nj223. The types of compatible solutes accumulated by this strain were analyzed and identified. Data shows that it accumulates ectoine and glutamate as the main compatible solutes. To clone the genes encoding enzymes for the biosynthesis of ectoine, a number of primers were designed according to the conservative amino acid sequences. Thus, partial sequences of ectABC were cloned by PCR using the genomic DNA of this strain. After many times of PCR and inverse PCR amplification, we eventually got a 2826bp DNA fragment. Software analysis shows that this fragment contains three genes encoding enzymes for ectoine biosynthesis, ectA, ectB, ectC and their upstream and downstream sequences. To elucidate the function of the gene cluster, recombinant plasmid pET-28a(+)-ectABC was constructed and transformed in E.coli BL21(DE3). The recombinant plasmid was identified by enzyme digestion and sequencing, which shows that it indeed contains ectABC coding genes. Thus, a recombinant pKS002 was constructed. We observed that the recombinant improved salt tolerance in basic medium M63. To verify that the improved salt tolerance was caused by synthesis of ecoine in pKS002, we detected the compatible solutes in the cell by HPLC and 13C NMR. The observation of ectoine peak in 13C NMR shows that this strain can synthesize ectoine to raise its salt tolerance.The three encoding genes, ectA, ectB and ectC, were cloned and expressed in E.coli BL21(DE3). The enzymatic activities of the purified recombinant proteins, EctA, EctB and EctC, were detected by adding their specific substrates. By this means, we can verify the reaction equation and elucidate the biosynthetic pathway for ectoine in vitro. Data shows that the three recombinant proteins have similar enzymatic property.Ectoine is one of the most effective compatible solutes in protection of biological macromolecules, such as DNA or proteins. Therefore, we study the effect of ectoine secreted by Nj223 strain on the protection of lactate dehydrogenase against heating, guanidine hydrochloride, freeze drying and freeze thawing. The data show that ectoine can stabilize lactate dehydrogenase during the treatment. It is also found that ectoine maintain the effective transform of competent cells after repeated freezing thawing.
Keywords/Search Tags:Antarctic moderate halophile, compatible solutes, ectoine, osmotic adaptation mechanism
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