Isolation and characterization of nitrate reductase-deficient mutants of Dunaliella salina

摘要

A method is presented to isolate mutants of Dunaliella salina with defects in NO_3~-metabolism. Wild-type D.salina cells treated with Ethynetrosourea (ENU) were recovered in three kinds of medium which uses NO_2, urea, NH_4~+ as N-source. The mutants library were obtained after more than ten times. Chlorate-resistant selection both in liquid and solid media. The Final selection was performed using 96 well-plate for single cell colonies. The nitrate reductase (NR) activity of all the single cell colonies was examined by sulfanilamide colormetry and three of them were zero. DNA sequencing and amino acid homology studies indicated that the three strains of D.salina contained the same mutations in NR: 19 point mutations in all, 16 of them were base changes and 3 new bases were presented in three different sites which resulting in frame-shift mutation and the change of amino acids. The function of every mutation remains to be studied, but the mutants can be established now. The NR-deficient mutants will be promising tools used as a specific selectable marker in study of transgenic Dunaliella salina and for investigations of the nitrate assimilation pathway on the molecular level.