PCR-IDENTIFICATION OF A NICOTIANA-PLUMBAGINIFOLIA CDNA HOMOLOGOUS TO THE HIGH-AFFINITY NITRATE TRANSPORTERS OF THE CRNA FAMILY

A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homol ogues of this family have been cloned from a higher plant (barley). Ba sed on six of the peptide sequences most strongly conserved between th e barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from o ther plant species. The utility of these primers was demonstrated by R T-PCR with cDNA made from poly(A)(+) RNA from barley, C. reinhardtii a nd Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbag inifolia was used as probe to isolate a full-length cDNA clone which e ncodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumb aginifolia. Northern blotting showed that the Nrt2 transcripts are mos t strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reduc tase genes: their expression was strongly induced by nitrate but was r epressed when reduced forms of N were supplied to the roots.