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High-oleic acid rapeseed is a new and promising rapeseed variety because it produces oil with high-quality health and nutrient properties. To identify differentially expressed proteins in a high-oleic acid rapeseed line, self-bred seeds (20 to 35 days after pollination) of a high- and a low-oleic acid rapeseed near-isogenic line (oleic acid contents of 81.4 and 56.2%, respectively) were used as raw materials for iTRAQ (isobaric tags for relative and absolute quantitation) analysis. In total, 24,755 peptides, 14,455 unique peptides, and 4726 proteins were identified. Among them, 137 differentially expressed proteins were assigned pathway annotations, accounting for 3.84% of the total proteins (3568). Three differentially expressed proteins, PSaB (gi|383930478), Lhcb6 (gi|50313237), and ALD (gi|15227981) were subjected to western blotting analyses. The results were consistent with the iTRAQ data, demonstrating the satisfactory quality of the iTRAQ experimental procedures and data. The transcript levels of 19 genes involved in fatty acid metabolism and photosynthesis were analyzed by qPCR. The transcript levels were consistent with the protein levels for 10 of these genes, but inconsistent for the other nine genes. Among the differentially expressed proteins were key enzymes in fatty acid biosynthesis and polyunsaturated fatty acid biosynthesis (acyl-CoA thioesterase, 3-ketoacyl-CoA reductase, omega 6 reductase, and acyl-CoA dehydrogenase), oleic acid biosynthesis (acyl-CoA thioesterase and omega 6 reductase), and seed lipid content (long-chain acyl-CoA synthetase). These results provide fundamental functional proteomics information for further breeding work on high-oleic acid rapeseed. This information will also be useful for research on the molecular mechanism of oleic acid synthesis.

A high oleic acid rapeseed material M15 (derived from Xiangyou 15 variety) has been received more attention for its significant effect for human health. And it has almost the same physiological characteristic with Xiangyou 15 variety. To find out the difference between high oleic acid rapeseed material and Xiangyou 15 seedling, a comparative proteomic approach based on 2-DE and mass spectrometry was adopted. A total of 277 protein spots showed a significant change in intensity by more than 2.0-fold from M15 compared with Xiangyou 15 variety. Among them, 48 spots that changed at least 3.0-fold were excised from gels and successfully identified by MALDI-TOF/TOF MS. The identified proteins involved in metabolism of carbohydrate and energy (75%), stress and defense (8.3%), photosynthesis (6.3%), protein metabolism (2.1%) and other functions (8.3%). Then real-time quantitative PCR (qPCR) analysis was used to verify the expression levels of differentially expressed proteins, but the results did well agree with the proteomic results. In this work, most of the proteins involved in metabolism of carbohydrate and energy have higher expression in M15, which may reveal M15 has higher metabolism ability. These results provided much information to understand the differences between high oleic acid rapeseed material and Xiangyou 15 variety, which will be useful to screen high oleic rapeseed materials in seedling period.

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.

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.