MOLECULAR-CLONING OF HIGHER-PLANT HOMOLOGS OF THE HIGH-AFFINITY NITRATE TRANSPORTERS OF CHLAMYDOMONAS-REINHARDTII AND ASPERGILLUS-NIDULANS

The crnA nitrate transporter from Aspergillus nidulans was identified as belonging to the major facilitator superfamily (MFS) of membrane tr ansporters. Degenerate oligonucleotides corresponding to the crnA sequ ences at the locations of two conserved sequence motifs were designed and used in the polymerase chain reaction (PCR) to amplify related seq uences from barley root poly(A)(+) RNA. A 130 bp cDNA fragment with se quence similarities to crnA was amplified and used as a probe to scree n a barley root cDNA library. Two full-length clones (pBCH1 and pBCH2) were isolated. The nt sequences of pBCH1 and pBCH2 are closely relate d (80% identical) and potentially encode hydrophobic polypeptides of 5 4.7 and 55.0 kDa respectively, with twelve predicted transmembrane dom ains. The encoded polypeptides are 41-43% identical to the A. nidulans CRNA protein and 56-57% identical to NAR-3, a high-affinity nitrate t ransporter from the eukaryotic alga Chlamydomonas reinhardtii. Phyloge netic analysis indicated that crnA, nar-3 and the barley homologues be long to a new family within the MFS, a family that also includes narK, the gene for a nitrite efflux pump in Escherichia coli. In northern b lots, BCH1 hybridised to a mRNA species of 1.9 kb which is rapidly ind uced in barley roots by NO3-, but not by NH4+, and genomic Southern bl ots indicated that there may be seven to ten BCH1-related genes in the barley genome.