Chlamydomonas reinhardtii strains expressing nitrate reductase under control of the cabII-1 promoter: isolation of chlorate resistant mutants and identification of new loci for nitrate assimilation

摘要

The Chlamydomonas reinhardtii strain Tx11-8 is a transgenic alga that bears the nitrate reductase gene (Nia1) under control of the CabII-1 gene promoter (CabII-1-Nia1). Approximately nine copies of the chimeric CabII-1-Nia1 gene were found to be integrated in this strain and to confer a phenotype of chlorate sensitivity in the presence of ammonium. We have used this strain for the isolation of spontaneous chlorate resistant mutants in the presence of ammonium that were found to be defective at loci involved in MoCo metabolism and light-dependent growth in nitrate media. Of a total of 45 mutant strains analyzed first, 44 were affected in the MoCo activity ( 16 Nit), unable to grow in nitrate, and 28 Nit+, able to grow in nitrate). All the Nit) strains lacked MoCo activity. Diploid complementation of Nit), MoCo) strains with C. reinhardtii MoCo mutants and genetic analysis indicated that some strains were defective at known loci for MoCo biosynthesis, while three strains were defective at two new loci, hereafter named Nit10 and Nit11. The other 28 Nit+ strains showed almost undetectable MoCo activity or activity was below 20% of the parental strain. Second, only one strain ( named 23c+) showed MoCo and NR activities comparable to those in the parental strain. Strain 23c+ seems to be affected in a locus, Nit12, required for growth in nitrate under continuous light. It is proposed that this locus is required for nitrate/chlorate transport activity. In this work, mechanisms of chlorate toxicity are reviewed in the light of our results.