Isolation And Characterization Of Nitrate Reductase-deficient Mutants Of Dunaliella Salina

In study of transgenic Dunaliella salina (D. salina), it is critical to establish a specific selectable marker to test whether a target gene is expressed, especially stably expressed. Antibiotics and herbicides resistance have been used as selectable markers for nuclear transformation of D. salina. However, Selectable marker genes conferring antibiotic or herbicide resistance have three major problems which limit their usefulness for application: (1) the selective agents have negative effects on proliferation and differentiation of plant cells; (2) there is uncertainty regarding the environmental impact of these selectable marker genes; (3) their gene products of the transgenic cells may cause human to be insensitive to or tolerant of the selective agent.In assimilation of nitrate, a main nitrogen source of organisms, NR is considered as the key and rate-limiting enzyme. Transformation systems based on complementing nitrate reductase-deficient mutants have been developed in a number of filamentous fungi and algae. The nitrate assimilation system offers several inherent advantages for transformation, such as high frequencies, ease of recipient isolation through resistance to chlorate, and low background growth after transformation.Chlorate, a nitrate analogue and substrate for both prokaryotic and eukaryotic NRs, has been widely and successfully used to select NR-deficient mutants in fungi, algae and higher plants, which have been shown to be resistant to chlorate toxicity in mutants but not in wild types of theirs. This fact is generally explained by assuming that chlorate itself is...