Functional Complementation Of Nitrate Reductase-deficient Mutants In 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. Nitrate reductase(NR) is a key and rate-limiting enzyme in assimilation of nitrate, which has been found to have single copy in a number of higher plant and alga genomes. It has been demonstrated that NR can be induced by nitrate and repressed by ammonium, and NR-deficient mutant can be easily isolated by chlorate-resistance. Hence, transformation systems have been established in several green algae, such as chlymadomonas reinhardtii, chlorella sorokiniana and volvox carteri, based on NR gene functional complementation.Both chlorate and nitrate are substrates for the enzyme, and each is a competitive inhibitor of the reduction of the other. The toxic effect of chlorate on algal growth can therefore reflect the induction and suppression of NR. In our previous studies three NR-deficient mutants were isolated and characterized by the method of chlorate toxicity to wild type D. salina cells. Also, NR cDNA was cloned from wild type D. salina. The purpose of this work was to rescue the mutant phynotype of NR-deficient mutants of D. salina with the wild-type NR gene of this alga by in vivo complementation. In this study, the three NR-deficient mutants were further identified by growth experiments and NR activity assay, and the results showed that the NR-deficient mutants could not grow on solid medium with nitrate as the sole nitrogen resource and their NR activity were not detected. Also, the copy number of the NR gene in D. salina genome was analyzed by Southern blot, and the results revealed that there was a single copy of the NR gene in the genome of D. salina. In addition, the effects of nitrogen resource on regulation of the NR gene were studied by Northern blot and NR activity assay. The Northern blot results revealed that the transcription of the NR gene could be induced by nitrate and repressed by ammonium, as well as NR activity increased and came to peak value at the third day under the condition of nitrate induction.The results from the studies above confirmed the feasibility of the NR gene functional complementation in D. salina. Here, a plasmid pMDDCA-NR harboring duplicated carbonic anhydrase promoter(DCA) and wild-type NR cDNA was constructed and transformed into NR-deficient mutant cells. After screening on solid selective medium, individual transformants were isolated. One of them was identified by NR activity assay, in which NR activity was rescued by 48.1%. A 410bp fragment of plasmid pMDDCA-NR was amplified with the genome DNA of the transformant as template, and Southern blot analysis confirmed the integration of wild-type NR cDNA in the genome of the transformant. Further studies showed that the transcripts of the NR gene in the transformant was enhanced by nearly 3 folds compared to the corresponding NR-deficient mutant.In conclusion, the NR gene functional complementation was accomplished in D. salina, indicating that the initial establishment of selection system with the NR gene as a selective marker has been successfully established. The study may lay a solid foundation for the development of D. salina bioreactor which produces valuable proteins.