Research Of Nitrate Reductase Gene Expression Regulatory Elements And Establishment Of Inducible Expression System For Phaeodactylum Tricornutum

Genetic transformation is generally required for basic research and applied biotechnology. However, it is usually quite difficult due to technical limitations of existing methods. Here we report an inducible expression system for optimization of microalgal transformation vector. Initially, nitrate reductase gene was cloned from eukaryotic microalga Phaeodactylum tricornutum, a widely used forage species and a promising candidate as bioreactor for the large-scale production of value-added proteins.Phylogenetic analysis of the nitrate reductase indicated that it was clustered into diatoms, separate from other green and red algae, green plants and fungi. Bioinformatic analysis on the cloned upstream sequence of the nitrate reductase gene indicated that it contained the core promoter elements and other regulatory elements. A transformation construct was made containing reporter gene CAT cassette (encoding chloramphenicol acetyltransferase conferring chloramphenicol resistance) driven by the cloned putative nitrate reductase promoter and terminator.The construct was transferred into P. tricornutum and Chlorella vulgaris by electroporation. Expression of CAT in transformed microalgae conferred resistance to the antibiotic chloramphenicol and enabled growth in selective medium. The overall transformation efficiency of both microalgae is relatively high compared with that so far reported. The expression of CAT was induced in the presence of nitrate and repressed in the presence of ammonium as sole nitrogen source. Our results identified an inducible expression system from P. tricornutum and its function in recombinant gene expression, also providing more gene regulation elements with important potential for biotechnological industry.