[Gene cloning and expression analysis of a male sterility related gene BoDHAR from broccoli]

A differentially expressed cDNA fragment obtained from a cDNA-AFLP analysis, which performed on floral buds of male sterile and fertile lines of cabbage, was used as a querying probe to blast the Genbank and Arabidopsis databases. Based on the assembled homologous cDNA sequences, a full-length cDNA of 633 bp for BoDHAR was cloned by RT-PCR. Furthermore, we have experimentally cloned and sequenced the 5' flanking sequence of gene BoDHAR by genomic walking method based on ligation-mediated PCR. The full length DNA sequence with 1486bp, containing two introns, was achieved. Homologous analysis shows that gene has 82.3% identity at nucleotide level, and 79.6% identity at amino acid level with Arabidopsis dehydroascorbate reductase (DHAR) gene AT1 G19570.1. Structurally, BoDHAR encodes a polypeptide of 210 amino acids, which contains a GST-c-DHAR domain highly conserved among other members of the DHAR superfamily and has multiple phosphorylation sites. Promoter predictions software indicated that the 5' upstream region contained putative transcription signals and conserved sequences, one CAAT-box, one G-box and four TGAC-like motifs. To advance our understanding of gene BoDHAR, tissue expression pattern were analyzed by semi-quantitative RT-PCR. The results indicate that expression level of gene BoDHAR is higher in fertile buds than that in sterile buds, and expressed intensively in the anther.