Search

The gene fatty acid desaturase 2 (FAD2) exists in multiple copies in the Brassica napus genome and encodes an enzyme that catalyzes the conversion of oleic acid to linoleic acid. In the present study, we characterized the regulatory region controlling the expression of an FAD2 gene located on chromosome C5 of Brassica napus and named it BnFAD2-C5. A long intron was found within the 5′-untranslated region (5′-UTR) of the BnFAD2-C5 gene. This intron, compared with an intron-less control, conferred up to a sixfold increase in green fluorescent protein (GFP) expression in transgenic Arabidopsis, thus suggesting that it makes function through intron-mediated enhancement. The sequence containing the promoter and intron was identified to promote high levels of gene expression in genital organs, particularly in seeds, using qRT-PCR and transgenic Arabidopsis. We identified the different promoter regions responsible for the tissue-specific gene expression through a deletion analysis of the BnFAD2-C5 promoter and a β-glucuronidase and GFP reporter system. The results showed that the −1020 to −319 bp region primarily controls BnFAD2-C5 gene expression in the root, whereas the −1020 to −581 bp region controls expression in the stem, the −581 to −319 bp region controls expression in the leaf, and the −1257 to −1020 bp region probably controls expression in the floral parts. The −319 to −1 bp region is also important, conferring high-level transcription in the seeds. The transcription of BnFAD2-C5 could be induced by salicylic acid and jasmonic acid, and the relative response elements were identified in the −1257 to −1020 bp region and −319 to −1 bp region, respectively.

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.

In the present study, we characterized the transcriptional regulatory region (KF038144) controlling the expression of a constitutive FAD2 in Brassica napus . There are multiple FAD2 gene copies in B. napus genome. The FAD2 gene characterized and analyzed in the study is located on chromosome A5 and was designated as BnFAD 2A5-1. BnFAD 2A5-1 harbors an intron (1192 bp) within its 5′-untranslated region (5′-UTR). This intron demonstrated promoter activity. Deletion analysis of the BnFAD 2A5-1 promoter and intron through the β-glucuronidase (GUS) reporter system revealed that the − 220 to − 1 bp is the minimum promoter region, while − 220 to − 110 bp and + 34 to + 285 bp are two important regions conferring high-levels of transcription. BnFAD 2 transcripts were induced by light, low temperature, and abscisic acid (ABA). These observations demonstrated that not only the promoter but also the intron are involved in controlling the expression of the BnFAD 2A5-1 gene. The intron-mediated regulation is an essential aspect of the gene expression regulation.