Cloning Of A Putative Low Affinity Sulfate Transporter Gene And Its Correlation With Cd Toxicity In Brassica Napus

Full-length cDNA encoding a putative low-affinity sulfate transporter (LAST), (accession number DQ091257), was isolated by RT-PCR and 5' RACE from Brassica napus 72 h after sulfate deficiency. The cDNA is 2150 bp long with an open reading frame (ORF) of 1959 bp, a 35-nucleotide(nt)-5' untranslated region (UTR) and a 153-nt 3' UTR. The ORF encodes a 653-amino acid protein with a predicted molecular weight of 72 kD and an isoelectric point of 9.5.The deduced protein displays typical 12 membrane-spanning domains and the STAS (after Sulphate Transporter and AntiSigma factor antagonist) domain matching with the general feature of sulfate transporters. The predicted protein was compared with a sulfate transporter from A. thaliana and several other higher plant sulfate transporters. The comparison shows a higher protein sequence similarity to low-affinity sulfate transporters than to high-affinity sulfate transporters. Phylogenetic relationships among the plant sulfate transporters indicate that the predicted protein falls into the group 2 of BjLAST from B. juncea, AtSultr2;1 and AtSultr2;2 from A. thaliana, and SHST3 from Stylosanthes hamata, and has the lowest phylogenetic distance from AtSultr2;2. Therefore, the putative sulfate transporter gene cloned from B. napus should encode a low-affinity sulfate transporter. It has been designated as BnSultr2;2.Examination of steady-state mRNA levels revealed that BnSultr2;2 transcripts were enhanced in leaves of sulfate-deficient plants. The leaf BnSultr2;2 expression was also upregulated by 20-120 μM Cd exposure for 72 h or 40 μM Cd exposure for different time. Under the same condition, the root BnSultr2;2 expression, however, was severely suppressed. To understand correlation between sulfate uptake and Cd stress, we simultaneously isolated another cDNA encoding a known high-affinity sulfate transporter (BnSultr1;1, accession number AJ416460) from roots. RT-PCR analysis demonstrated that BnSultr1;1 expressed only in roots, and its expression was upregulated by both sulfate-deficiency and Cd exposure. To relate the expression of sulfate transporters to sulfur...