Search

Based on the sequence of a novel expressed sequence tag (EST), the full-length cDNA of 1 017 nucleotides was cloned from Brassica napus cv. Xiangyou 15 through rapid amplification of cDNA ends (RACE). The gene was designated as Bnhol34 (HQ585980), encoding a protein of 338 amino acids. BLAST analysis showed no high degree of sequence identity to any known gene. The calculated molecular weight of the Bnhol34 protein was 36.23 kDa, and the theoretical isoelectric point was 8.74. The Bnhol34 was also cloned from a high oleic acid mutant 854-1 through homologous cloning. There was no difference between the two Bnhol34 genes. Bnhol34 was localized in a tissue-specific manner in B. napus, and its expression level was about eight-fold greater in Xiangyou 15 seeds than in 854-1. The promoter region sequences of Bnhol34 were then isolated from Xiangyou 15 and 854-1, and a 93-bp deletion was found to occur in the Bnhol34 promoter region of 854-1. Three abscisic acid-responsive cis-elements (ABRE) were identified in the promoter region of Xiangyou 15. Real-time PCR analyses revealed that exogenous abscisic acid increased Bnhol34 expression by about four-fold in Xiangyou 15 seeds, yet did not change Bnhol34 expression in 854-1. It appeared that Bnhol34 might be abscisic acid insensitive in 854-1.

FAD2 gene is essential for converting oleic acid to linoleic acid in plant seeds. From Xiangyou 15, a cultivar of Brassica napus, 56 FAD2 DNA clones and 47 FAD2 cDNA clones were obtained including 6 new copies of FAD2, which were designated FAD2P1 to FAD2P6. These new FAD2 copies ranged from 1141 bp to 1157 bp in length and contained no introns in their open reading frames. They had 96.1% homology in nucleotide sequence and shared more than 87.0% of nucleotides identity to the published FAD2 gene (AY577313). The deduced amino acid sequences of the 6 FAD2 copies presented 1–12 stop codons within the coding regions, which prevented from coding functional proteins. These copies were transferred into Saccharomyces cerevisiae through vector pYES2.0, and the expression products showed no functions to synthesize 16:2 and 18:2 fatty acids. These results indicate that they are pseudogenes of FAD2.