Directed Evolution of Dunaliella salina Ds-26-16 and Salt-Tolerant Response in Escherichia coli
نویسندگان
چکیده
Identification and evolution of salt tolerant genes are crucial steps in developing salt tolerant crops or microorganisms using biotechnology. Ds-26-16, a salt tolerant gene that was isolated from Dunaliella salina, encodes a transcription factor that can confer salt tolerance to a number of organisms including Escherichia coli (E. coli), Haematococcus pluvialis and tobacco. To further improve its salt tolerance, a random mutagenesis library was constructed using deoxyinosine triphosphate-mediated error-prone PCR technology, and then screened using an E. coli expression system that is based on its broad-spectrum salt tolerance. Seven variants with enhanced salt tolerance were obtained. Variant EP-5 that contained mutation S32P showed the most improvement with the E. coli transformant enduring salt concentrations up to 1.54 M, in comparison with 1.03 M for the wild type gene. Besides, Ds-26-16 and EP-5 also conferred E. coli transformant tolerance to freezing, cold, heat, Cu2+ and alkaline. Homology modeling revealed that mutation S32P in EP-5 caused the conformational change of N- and C-terminal α-helixes. Expression of Ds-26-16 and EP-5 maintained normal cellular morphology, increased the intracellular antioxidant enzymatic activity, reduced malondialdehyde content, and stimulated Nitric Oxide synthesis, thus enhancing salt tolerance to E. coli transformants.
منابع مشابه
Proteomic Analyses Reveal the Mechanism of Dunaliella salina Ds-26-16 Gene Enhancing Salt Tolerance in Escherichia coli
We previously screened the novel gene Ds-26-16 from a 4 M salt-stressed Dunaliella salina cDNA library and discovered that this gene conferred salt tolerance to broad-spectrum organisms, including E. coli (Escherichia coli), Haematococcus pluvialis and tobacco. To determine the mechanism of this gene conferring salt tolerance, we studied the proteome of E. coli overexpressing the full-length cD...
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