Introduction

Plant lipids are altered to improve oil quantity and quality for using in food, bioenergy, and industrial bioproducts (Suh et al. 2015). Canola oils containing high oleic acid and low linoleic acid have attracted researchers’ attention, owing to their attributes associated with oleic acid, including high oxidation stability, extended shelf life, and limited unpleasant odor (Maher et al. 2007). Some genes involved in the biosynthesis and accumulation of oleic acid have been target genes in studies seeking to improve the quality of rapeseed oils; however, little is known about the regulatory mechanisms of these genes. Studies on the regulation of these genes would provide a theoretical foundation for improving the quality of vegetable oils.

The oleic acid desaturase 2 (FAD2) gene encodes an enzyme that catalyzes the conversion of oleic acid to linoleic acid (Baud and Lepiniec 2010). Higher plants generally contain one or more FAD2 gene copies, such as Arabidopsis, which has one copy of FAD2 (Beisson et al. 2003); cotton, which has 3 copies (Pirtle et al. 2001); sesame, which has one copy (Jin et al. 2001); soybeans, which have 2 copies (Bilyeu et al. 2003); flax, which has 2 copies (Chen et al. 2015); and Brassica napus, which has 4 copies (Lee et al. 2013). Different FAD2 gene copies have different expression patterns. For example, in normal-type sunflower, the FAD2-1 gene is highly expressed, especially in developing embryos, but the FAD2-2 and FAD2-3 genes are weakly expressed (Martínez-Rivas et al. 2001). In sesame, the FAD2 gene is expressed with a seed-specific pattern (Kim et al. 2006).

Yang et al. (2011) have detected two major quantitative trait loci (QTLs) for oleic acid content in the linkage groups (LGs) of chromosomes A5 and C5, which explain 60–70 % of the variance in oleic acid content and are located in the FAD2 gene. Moreover, cloning and aligning the coding sequences of the FAD2 genes in high- and low- oleic acid rapeseeds indicate that the high oleic acid content might be explained by the expression of the FAD2 gene. The expression of FAD2 gene in high oleic acid rapeseed is lower than that in the standard rapeseed (Guan et al. 2012), and the oleic acid content can be significantly improved by knocking down FAD2 expression using RNAi in Brassica napus and Brassica campestris (Jung et al. 2011; Peng et al. 2010). All the above findings suggest that the functions of the FAD2 genes depend on their ability to be expressed efficiently. To improve oleic acid content in rapeseed, studying the expression and regulation patterns of both the FAD2 genes is necessary. Xiao et al. (2014) have studied the transcription regulation of the FAD2 gene on chromosome A5, which has a constitutive expression pattern and is regulated by positive and negative cis-elements. To explain the regulatory functions of both FAD2 genes in fatty acid synthesis in Brassica napus, a study of the FAD2 gene on chromosome C5 should be conducted to determine how FAD2 is expressed and what causes it to be expressed, which would allow plants with the desired oil characteristics to be engineered.

Introns not only maintain the specific structure and function of chromosomes but also determine a gene’s expression level and its spatial and temporal expression (Deyholos and Sieburth 2000). Introns are induced by internal and external signals and regulate spatial and temporal gene expression during seed development (Bolle et al. 1996; Kim et al. 2006; Xiao et al. 2014). The first intron found to stimulate gene expression was in an animal system, and the observation was extended to plants when Callis et al. (1987) demonstrated that the first intron of Adh1 in maize increases the expression of several genes. Introns with an enhancement function are usually located in the 5′-untranslated region (UTR) (Kim et al. 2006). Intron-mediated enhancement (IME) in dicots is usually in the range of approximately 2- to 10-fold and is much lower than that in monocots, which can reach more than 100-fold (Clancy and Hannah 2002). Typically, introns that are located nearer to the 5′end of a gene have a greater enhancement of expression than those at the 3′end (Parra et al. 2011). The 5′UTR of the FAD2 gene on chromosome A5 in Brassica napus contains an intron that is responsible for IME (Xiao et al. 2014). Given the homology between chromosomes A5 and C5, the question arises as to whether the BnFAD2-C5 intron might also be involved in IME and whether it can induce BnFAD2-C5 gene expression spatially and temporally.

Internal and external signals are involved in the regulation of spatially and temporally expressed genes during plant growth. Abscisic acid (ABA) appears to play a key role in plant maturation and desiccation tolerance (Finkelstein et al. 2002). Moreover, the germination of seedlings is sensitive to ABA (Bäumlein et al. 1994). In addition, the repeated copies of ABREs can confer ABA responsiveness to minimal promoters, whereas a single copy of ABRE is not responsive to ABA (Narusaka et al. 2003). The plant hormones salicylic acid (SA) and jasmonic acid (JA) play key roles in the regulation of the defense-signaling network, which is activated in response to invaders (Pieterse et al. 2012). In general, pathogens with a biotrophic lifestyle are more sensitive to SA-induced defenses, whereas necrotrophic pathogens and herbivorous insects are resistant, owing to JA-mediated defenses (Glazebrook 2005; Howe and Jander 2008).

Here, we isolated the full-length BnFAD2-C5 gene from the 5′UTR to the 3′UTR, including a long intron within the 5′UTR. The qRT-PCR results and the analysis of transgenic Arabidopsis with a full-length promoter and intron construct showed that the BnFAD2-C5 promoter promotes high levels of gene expression in the genital organs, especially in seeds. We found that the BnFAD2-C5 intron indeed has an IME function. The regions or cis-elements in the BnFAD2-C5 promoter required for expression in different tissues were assessed in transgenic Arabidopsis using GUS/GFP (β-glucuronidase and green fluorescent protein) as reporter genes and were detected through GUS histochemical analysis and western blotting. Both SA and JA increased BnFAD2-C5 promoter activity, and the relative response elements were also analyzed in the BnFAD2-C5 promoter.

Materials and methods

Plant materials

Fifteen Brassica napus cv. Xiangyou plants were planted in the field, and Arabidopsis thaliana (ecotype Columbia), preserved by our laboratory, was cultivated in an incubator (BINDER, Germany) under a 16/8-h [light (24 °C)/dark (22 °C)] photoperiod.

Gene cloning

Total DNA was isolated with a DNA extraction kit (TaKaRa, Japan) from 15 Xiangyou leaves. The conserved primer pairs P1/P2 from Xiao et al. (2008) were used to amplify the BnFAD2-C5 gene coding sequence. The PCR products were purified using the DNA gel-extraction kit (TaKaRa, Japan) and then ligated with pMD18-T vector (TaKaRa, Japan) at 4 °C for overnight. Then, the ligation products were transformed into E. coli DH5α. Selected ten positive colonies on ampicillin agar plate were sequenced in BoShang Corporation.

To identify the transcription initiation site of the BnFAD2-C5 gene, the full length of BnFAD2-C5 cDNA was isolated. Total RNA was extracted with the CTAB method (Chang et al. 1993) from 15 Xiangyou leaves and was then digested with RNase-free DNase (TaKaRa, Japan) at 37 °C for 30 min to remove genomic DNA. The digested total RNA was used to synthesize cDNA using a SMARTerTM cDNA Amplification Kit (TaKaRa, Japan) according to the manufacturer’s protocol.

Quantitative RT-PCR to characterize BnFAD2-C5 gene expression

Total RNA was extracted from 15 Xiangyou roots, leaves, flowers, developing seeds, and the silique coat 20–30 days after flowering (DAF), using the CTAB method. Genomic DNA was digested from the total RNA with an RNase-free DNase (TaKaRa, Japan) at 37 °C for 30 min. cDNA was synthesized using a cDNA synthesis kit (TaKaRa, Japan) according to the manufacturer’s protocol. Quantitative RT-PCR was conducted using YG-C5-FAD2-F/YG-C5-FAD2-R (Table 1) primers and 2ACTIN as an internal reference gene with the above cDNA as templates. The PCR was performed with a predenaturation at 95 °C for 30 s and 40 cycles of 95 °C for 5 s, and 60 °C for 30 s.

Table 1 List of primers used in experiments
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Isolation of the BnFAD2-C5 promoter and intron

On the basis of the homology between Brassica napus and Brassica oleracea, the specific primers C5 (pro)-F/C5-FAD2-R were used to amplify the 5′flanking region, including the 1257 bp promoter and the 1123 bp intron, from the genomic DNA of 20 DAF rapeseeds by PCR using HS polymerase (TaKaRa, Japan). The PCR program was 1 cycle of 95 °C for 5 min, 95 °C for 50 s, 56 °C for 50 s, and 72 °C for 3 min; 30 cycles of 95 °C for 50 s, 56 °C for 50 s, and 72 °C for 3 min; and 1 cycle at 72 °C for 10 min. The PCR products were analyzed on 1.5 % agarose gels, and fragments were recovered and purified with a DNA gel-extraction kit (TaKaRa, Japan) and then ligated with pMD18-T vector at 4 °C for overnight. Then, the ligation products were transformed into E. coli DH5α. The selected positive colonies were sequenced by the BoShang Corporation.

Construction of the promoter-GUS/GFP fusion vectors and transformation of Arabidopsis

To clone the 5′-deletion series of the BnFAD2-C5 promoter into the binary vector pCAMBIA1303, the 5′flanking region of BnFAD2-C5 was amplified using the gene-specific primers PBn-R as the reverse primer and PBn-F1, PBn-F2, PBn-F3, PBn-F4, and PBn-F5 as the forward primers. The PCR products were cloned into between the HindIII and NcoI sites of the pCAMBIA1303 vector after purification from the agarose gels and were named constructs PBn-1, PBn-2, PBn-3, PBn-4, and PBn-5.

The 5′flanking region, including the promoter and intron, was amplified using the primers PBn-F1/IBn-r, and the products were inserted between the HindIII and NcoI sites of the pCAMBIA1303 vector to generate the Bn-1 construct. The pCAMBIA1303 vector was digested with HindIII and SalI to remove the CAMV35S promoter and create the negative control, and the pCAMBIA1303 vector with the CAMV35S promoter was the positive control. The full length of the BnFAD2-C5 intron was inserted into the negative control and positive control to generate the Bn-2 and CAMV35S + intron constructs, respectively.

All the constructs were introduced into agrobacterium tumefaciens strain GV3101 via the freeze–thaw method (An 1987). Arabidopsis was transformed by the floral dip method (Clough and Bent 1998). A large number of the collected seeds were sterilized in 75 % ethanol for 5 min and then in 26 % sodium hypochlorite for 10 min, and the seeds were then rinsed with sterile water five times. Finally, seeds were germinated on selective medium (4.45 g/l MS, 3 % sucrose, 2.5 g/l phytagar, and pH 6.0) containing 50 mg/l hygromycinb. The plants with well-developed roots were transferred to the soil to obtain the T2 generation.

Exogenous salicylic acid and jasmonic acid application

The T2 generation seeds of BnP-1, BnP-2, and BnP-4 were selected with MS medium with hygromycinb. After 7 days, the positive seedlings of BnP-1 and BnP-2 were treated with salicylic acid, and the positive seedlings of BnP-4 were treated with jasmonic acid. All the seedlings were treated with various hormone concentrations for 24 h under a 16/8-h photoperiod [light (24 °C)/dark (22 °C)].

Histochemical analysis of GUS and western blotting

The roots, stems, leaves, flowers, and developing seeds were cut and incubated immediately in GUS staining buffer [1 M NaH2PO4, 0.5 M Na2HPO4, 0.1 M K3[Fe(CN)6], 0.1 M K4[Fe(CN)6]·3H2O, 0.5 M Na2EDTA(pH 8.0), 5 % methanol, 0.12 % TritonX-100, and 0.1 mM X-Gluc] for 16 h at 37 °C. The stained tissues were then rinsed with 70 % ethanol until the chlorophyll had been cleared completely, and the images were photographed using an SZX2-ILLB stereomicroscope (Olympus, Japan).

The green fluorescent protein (GFP) in the pCAMBIA1303 vector after GUS was quantitatively detected by western blotting. Total protein was extracted from the T2 generation transgenic Arabidopsis thaliana roots, stems, leaves, flowers, and seeds according to Noir et al. (2005). An 8 µl protein sample was mixed with 2 µl of 5 × SDS-PAGE sample loading buffer (Beyotime, China) and was preheated for 10 min at 65 °C to denature the protein completely, and samples were loaded onto SDS-PAGE gels. The SDS-PAGE gels were composed of 10 ml of 12 % separating gel and 5 ml of 3 % stacking gel, and the electrophoresis procedure was 180 V for 1.5 h and then 80 V for 1.5 h using a Bio-Rad electrophoresis apparatus. Afterward, the protein was transferred into a 0.45 µm PVDF membrane by trans-membrane electrophoresis. Then, the membrane with the protein of interest was incubated with blocking buffer (5 % skim milk powder solution) for 1 h, washed, incubated for 1 h with primary antibody GFP (Beyotime, China), and washed 5 times, incubated with a secondary antibody (HRP-labeled goat anti-mouse IgG) for 1 h, and subjected to a final wash step. The processes of blocking primary and secondary antibody incubation were all performed on a shaking Table (54 rpm). The last step was the detection of the protein. ECL luminescence solution was added onto the PVDF membrane, which was then placed under an X-ray film for 5 min. The X-ray film was then developed for 2 min and then fixed for 2 min. After drying of the X-ray film, it was scanned with a GS-800 Calibrated Densitometer, and the GFP content was calculated with the Quality One software.

Results

Expression analysis of the BnFAD2-C5 gene

At the beginning of our study, the sequencing of the Brassica napus genome had not been completed. Homologous cloning and cRACE technology were used to clone the coding sequence (CDS) and the 5′ and 3′ untranslated regions (UTR) of the BnFAD2-C5 gene, to characterize its expression and regulation patterns. As shown in Fig. 1, the BnFAD2-C5 gene comprises a 1155 bp open reading frame (ORF) that encodes 384 amino acids, a 1150 bp intron from position +146 to position +1269 within the 5′UTR, and a 351 bp 3′UTR downstream of the CDS.

Fig. 1
figure 1

Structure of BnFAD2-C5 gene in rapeseed genome

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The expression pattern of the BnFAD2-C5 gene was obtained by quantitative RT-PCR with a pair of specific primers, which were designed on the basis of the sequences of the 3′UTR region. The BnFAD2-C5 gene was primarily expressed in the genital organs of Brassica napus, including the flower and seed (Fig. 2a). Then, we constructed a vector with a full-length promoter and a full-length intron inserted in front of GUS/GFP and transformed Arabidopsis plants to analyze the expression pattern of the BnFAD2-C5 gene in the T2 transgenic Arabidopsis generation. As shown in Fig. 2b, the expression of the reporter gene GFP in seeds was much higher than that in other organs, including the root, stem, and leaf. This result was consistent with the results of the histochemical staining. The seeds with the target gene were stained more deeply, and the staining of the root, stem, leaf, and flower was similar. On the basis of these results, we concluded that BnFAD2-C5 promoter promotes high levels of expression in the genital organs, especially in the seeds.

Fig. 2
figure 2

Assay of expression pattern of BnFAD2-C5 gene. Quantitative of BnFAD2-C5 gene (a) and GFP expression in transgenic Arabidopsis with construct Bn-1 which involves the full length of promoter and intron (b). Western blot was used for the quantitative analysis of GFP protein. In this progress, ten independent transgenic lines (T2) were examined. The highest and lowest ones were excluded in our data analyses. The values are average ± error

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Sequence analysis of the promoter region of the BnFAD2-C5 gene

Transcriptional regulation is key to gene expression, and the promoter is a crucial factor in transcriptional regulation. A 1257 bp fragment of the promoter-like region of BnFAD2-C5 was amplified. Subsequently, the promoter sequence of BnFAD2-C5 was analyzed for known cis-acting elements through a web search of publicly available databases (http://www.dna.affrc.go.jp/htdocs/PLACE and http://www.intra.psb.ugent.be:8080/PlantCARE). Putative TATA-box and CAAT-box elements were found to be located at −139 and −61 bp, respectively.

Through this database analysis, many cis-elements were identified, including a Dof core, an E-box, a salicylic acid-responsive element (TCA motif), a methyl jasmonate-responsive element (CGTCA motif), abscisic acid-responsive elements (ABRE, MYB, and MYC), root hair specific elements (ARS element and RHE), an anoxic-responsive element (GC motif), and light regulation elements (Box I, Box II, Box III, Box IV, GAG, G-box, and GT-1 consensus). Furthermore, AGAAA was involved in pollen-specific expression, GATA was required for high-level, light dependent, and tissue-specific expression, and ACGT was required for the endosperm-specific expression and the 5′UTR Py-rich stretch that conferred high transcription levels (Fig. 3).

Fig. 3
figure 3

Nucleotide sequences of the 5′ flanking region and intron of BnFAD2-C5. A black box represents the transcription initiation site identified by 5′-RACE and is designated as the +1 position; the numbering on the left refers to nucleotide sequences; the sequences of the BnFAD2-C5 intron are shown in lowercase letters; several potential cis-elements are underlined and designated with the names of each of the motifs

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In many plants, such as cotton (Pirtle et al. 2001), soybeans (Heppard et al. 1996), sesame (Kim et al. 2006), oilseed (Xiao et al. 2014), and rice (http://www.cdna01.dna.aVrc.go.jp/cDNA), the FAD2 genes have a large intron within the 5′-UTR. We also found an 1123 bp-length fragment of the intron in the BnFAD2-C5 gene. This intron had 43.3 % T content, 25.9 % A content, and GT-AG dinucleotides at both ends, which are well-known characteristics of higher plant introns. The intron sequence was also evaluated for known cis-acting elements through a web search of PLACE and PlantCARE. The promoter-like cis-elements, the TATA-box element, and CAAT-box element were at positions +1241 and +1040, respectively. Several potential cis-elements, including ACGT, MYB, MYC, Dof, WRKY, HSE, G-box, E-box, GARE, CAT box, TC-rich, and the GT-1 consensus, were predicted to be located within that region (Fig. 3). Although the promoter and the intron shared a number of sequence elements, the overall sequence identity was only 37.5 %, thus revealing that the promoter and intron did not arise from tandem replication.

The BnFAD2-C5 intron is involved in intron-mediated enhancement (IME)

Kim et al. (2006) and Xiao et al. (2014) have reported that, in sesame and oilseed, respectively, the single intron sequence slightly activates the activity of the promoter. Thus, to characterize the BnFAD2-C5 intron, we cloned the intron sequence, including the short 5′UTR (137 bp), inserted it into a pCAMBIA1303 vector to construct Bn-3, and deleted CAMV35S from the pCAMBIA1303 vector to construct the negative control. The Bn-3 construct and the negative control construct were transformed into Arabidopsis and were detected in various tissues in the T2 generation, including the root, stem, leaf, flower, and seed, through histochemical staining. However, the results of the staining showed no differences between the Bn-3 construct and the negative control construct (Fig. 4a), thus indicating that the BnFAD2-C5 intron does not serve as a promoter, even though it has putative promoter-like elements as determined through bioinformatics.

Fig. 4
figure 4

Function analysis of BnFAD2-C5 intron. a GUS histochemical analysis of Bn-3 construct and negative control construct; b Scheme figure of Bn-3 construct

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When the intron was inserted after the BnFAD2-C5 promoter, the GFP expression was approximately fourfold higher than that of the construct with only the promoter sequence, thus suggesting that the intron improved the expression level of the BnFAD2-C5 gene (Fig. 5a). For the construct with the intron inserted downstream of the CAMV35S promoter, as compared with the construct with only the promoter CAMV35S, the expression levels in the root, stem, leaf, and even the flower decreased, but the GFP expression was approximately 1.6-fold higher in the seed (Fig. 5b).

Fig. 5
figure 5

BnFAD2-C5 intron analyses in transgenic Arabidopsis. a Comparative analyses of GFP in various tissues between the construct with BnFAD2-C5 intron downstream after itself promoter or not using histochemical and quantitative (western blot) methods. b Comparative analyses of GFP in various tissues between the construct with BnFAD2-C5 intron downstream after CAMV35 promoter or not using histochemical and quantitative (western blot) methods. The values are average ± standard error

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All the results revealed that the BnFAD2-C5 intron is involved in intron-mediated enhancement (IME). Moreover, the BnFAD2-C5 intron served as its own promoter in various transgenic Arabidopsis tissues but promoted the exogenous CAMV35S promoter only in the seeds of transgenic Arabidopsis.

The BnFAD2-C5 gene is expressed in various tissues

To define the regulatory sequences that control the expression of the BnFAD2-C5 gene, a series of promoter deletions (BnP-1, BnP-2, BnP-3, BnP-4, and BnP-5) fused to a GUS/GFP reporter gene were constructed on the basis of the bioinformatics analysis (Fig. 3). The deletion constructs included the short 5′UTR (137 bp) and were transformed into the wild Arabidopsis. Up to ten independent primary transformants were regenerated for each construct until the T2 generation. None of the tissues of the transgenic Arabidopsis plants with the BnP-5 promoter fragment could be stained, thus suggesting that this region does not play the role of the promoter and that the BnP-4 is the minimal promoter (Fig. 6a).

Fig. 6
figure 6

BnFAD2-C5 promoter analyses in transgenic Arabidopsis. Histochemical (a, b) of GUS and quantitative (c, d) of GFP expression in transgenic Arabidopsis. In histochemical staining and GFP quantity analyses measurements, ten independent transgenic lines (T2) were examined for each construct. The values are average ± standard error. The seed was transgenic Arabidopsis developing seeds between 7 and 10 DAF. a Model diagram of deleted promoter fragments with different hormone responsive elements

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The GUS histochemical staining showed that the transgenic Arabidopsis root with the BnP-2 promoter fragment exhibited the strongest staining (Fig. 6b), and the data analysis indicated that the highest level in the transgenic Arabidopsis root was with the BnP-2 promoter fragment (Fig. 6d). Furthermore, when the −1020 to −581 and −581 to −319 bp regions were deleted, the GFP activity of the root decreased. It is possible that the region of the −1020 to −319 bp promoter fragment is the main control reporter gene expressed in the root. It is also possible that this promoter fragment primarily controls the BnFAD2-C5 gene expression in the root. As shown in Fig. 6d, when the −1020 to −581 bp region was deleted, the relative content of GFP substantially decreased in the transgenic plants’ stem, thus suggesting that this region maintains the gene expression in the stem, although the histochemical staining was not obvious. In the transgenic Arabidopsis leaves with the BnP-3 promoter fragment, the relative content of GFP was the highest (Fig. 6d), and this corresponded to the staining results (Fig. 6b); however, this content decreased when the −581 to −319 bp region was deleted, thus suggesting that the −581 to −319 bp region primarily controls the expression of the BnFAD2-C5 gene in the leaf. When the −1257 to −1020 bp region was deleted to create construct BnP-2, the relative content of GFP decreased substantially in the flowers of the transgenic Arabidopsis, thus suggesting that the −1257 to −1020 bp region probably controls the expression of the gene in floral organization. However, the relative content of GFP in the transgenic Arabidopsis flower with the BnP-3 and BnP-4 promoter fragments increased, possibly because of the deletion of the negative cis-elements in the −1020 to −581 bp region, which might have inhibited BnFAD2-C5 gene expression in the flower.

Notably, the expression of the BnFAD2-C5 promoter was active in the root, stem, leaf, flower, and especially in the seeds of the transgenic Arabidopsis with a series of deleted promoter fragments. As shown in Fig. 6b, the GUS histochemical staining decreased in the transgenic plants’ seeds from the BnP-1 to the BnP-3 construct, but this effect depended on the BnP-4 construct. This result was consistent with the analysis of the GFP content. The deletion from −1257 to −581 bp decreased the GFP content, thus suggesting that the −1257 to −581 bp region contains some cis-elements, which improves gene expression. However, when the −581 to −319 bp region was deleted, the expression level of GFP increased markedly (Fig. 6c). This result suggested that some negative cis elements exist in the −581 to −319 bp region. As shown in Fig. 6c, the GFP expression in the transgenic Arabidopsis seeds with the −319 to −1 bp promoter fragment was higher than that with the full promoter, thus suggesting that −319 to −1 bp region is the main region driving expression.

Exogenous salicylic acid and jasmonic acid application changes BnFAD2-C5 promoter activity

The bioinformatics analysis results showed that some hormone responsive cis-elements were present in the BnFAD2-C5 promoter region. To verify these results and to determine whether hormonal control affects the expression of the BnFAD2-C5 promoter, the T2 generation seedlings, with the relevant promoter fragments, were incubated with various concentrations of salicylic acid (SA) and jasmonic acid (JA), which were predicted to have response elements in the promoter. As shown in Fig. 7, when the seedlings with the BnP-1 promoter fragment containing the TCA element, which responded to SA, were treated with various SA concentrations, the relative content of GFP detected by western blotting increased. However, when the TCA element was deleted (BnP-2, the relative content of GFP did not show obvious changes). Thus, the −1257 to −1020 bp region harbors the SA-response element. Moreover, the seedlings with the BnP-4 promoter fragment containing the CGTCA motif, which responded to JA, were treated with various JA concentrations, and western blot analysis revealed that an exogenous JA application resulted in an approximately 3—5-fold increase in the expression of the reporter gene. These results indicated that the −319 to −1 bp region harbors the JA-response element. All these results enabled us to delineate the region harboring an SA-response element and the region harboring a JA-response element. Furthermore, we predict that these two elements exist at −1150 to −1141 and −39 to −35 bp, respectively.

Fig. 7
figure 7

Quantitative analyses of GFP in transgenic Arabidopsis seedlings after the application of SA (a) and JA (b). a Developing transgenic Arabidopsis seedlings was incubated with 0, 50, 75, and 100 µM for 48 h, then subjected to the western blot analysis of GFP gene. b Dealt with a serious concentration of JA (0, 50, 75, and 100 µM) for 48 h and detected as (a). c Model diagram of different deleted promoter fragments

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Discussion

The functions of the FAD2 copies in Brassica napus

Four FAD2 copies have been identified and located in an N1 linkage group (BnFAD2-A1 and BnFAD2-C1) and an N5 linkage group (BnFAD2-A5 and BnFAD2-C5) in Brassica napus (Scheffler et al. 1997; Smooker et al. 2011; Yang et al. 2012; Lee et al. 2013). Each FAD2 copy is an independent gene, and the cooperation among copies maintains normal development. Thus, studying the function of each FAD2 copy is important.

Of the four copies, only BnFAD2-C1, BnFAD2-A5, and BnFAD2-C5 play a role in fatty acid desaturation and function to different degrees. The BnFAD2-A5 and BnFAD2-C5 genes contribute much more than BnFAD2-C1 (Lee et al. 2013). BnFAD2-C5 is highly expressed in the flower especially in the seed (Fig. 2), whereas BnFAD2-C5 primarily regulates and controls expression in genital organs.

An intron within the 5′UTR is involved in IME function

The previous analysis showed that the single BnFAD2-C5 intron did not have a promoter function, even though it harbored promoter-like elements (Figs. 3, 4). However, when the intron was inserted downstream of the BnFAD2-C5 promoter and the CAMV35S promoter, it improved the gene expression level significantly (Fig. 5). Namely, the BnFAD2-C5 intron improved expression from not only the respective promoter but also the exogenous promoter CAMV35S. An intron with an enhanced effect that is widely distributed in the 5′UTR sequence has been identified in maize (Maas et al. 1991), Arabidopsis and sesame (Kim et al. 2006). The IME function may be a result of the intron splicing effects (Mascarenhas et al. 1990), the functions of specific elements in specific regions of the intron interacting with the promoter regions (Kim et al. 2006) or the length of the intron serving as a spacer (Chung et al. 2006). The phenomenon in which the BnFAD2-C5 intron enhances the CAMV35S promoter activity may be a result of the above three reasons and requires further study.

The intron with an IME typically contains a U-rich sequence, and our analysis indicated a 38.7 % U sequence in this region as well as an ABRE/MYB/MYC, E-box, Wbox, and GARE, which promote high-level expression via the corresponding promoter in rapeseed and Arabidopsis (Stålberg et al. 1996; Eulgem et al. 2000). An E-box usually appears with some hormone responsive elements, such as ABRE/MYB/MYC (Liu et al. 2015; Lenka et al. 2015), thus providing a site for bHLH proteins to bind and consequently regulate gene expression. The WRKY/W box is usually stimulated by a pathogen or salicylic acid (SA), and it plays a defense function in plant defense-related genes (Dong et al. 2003). Overall, the elements in this intron are mostly hormone and defense responsive elements, which are crucial factors affecting plant physiology. Thus, at specific stages of growth, the internal hormones produced by plants might stimulate high expression of the BnFAD2-C5 gene. However, this possibility remains to be verified by additional experiments.

Different regions are responsible for the BnFAD2-C5 gene expression in various tissues

Although most oil existed in rape seeds used for oil manufacture, it also distributes in other organizations, such as root, stem, leaf, and flower. We detected BnFAD2-C5 expression not only in the seeds but also in other organs. Transcriptional regulation plays an important role in gene expression, and the promoter is crucial in this process. Here, we demonstrated that the −1020 to −319 bp region harbors ARS and RHE elements, which regulate root-specific gene expression. These elements are located in the −1020 to −581 and −581 to −319 bp regions, respectively. This finding may explain why the expression in the root decreased from BnP-2 to BnP-3, and even to BnP-4. Several CACT elements, which are key elements of the mesophyll expression module (Gowik et al. 2004), are distributed along the full length of the BnFAD2-C5 promoter. The decreased expression of GFP in the leaf with BnP-4 may have been caused by the deletion of most of the CACT in the −1257 to −319 bp region. Another important element regulating gene expression in the flower is the pollen element (Dzelzkalns et al. 1993). Two pollen elements exist in the −1257 to −1020 bp region, and another two are in the BnP-4 construct. Thus, the expression of the reporter gene was high in the flower with the BnP-1 construct, but it decreased approximately fourfold after the deletion of the −1257 to −1020 bp region in the BnP-2 construct. However, the expression in the flower with the BnP-4 construct was as high as that with BnP-1, probably because some negative cis-elements that inhibit the function of pollen element in BnP-4 were deleted.

Although the BnFAD2-C5 promoter is not the seed-specific promoter, it contains some elements that can activate the promoter in seeds. In the −319 to −1 bp region, there are 4 Dof, GAG, GC-motif, G-box, CGTCA motif, 3GT-1 consensus, GATA, TATA-box, and CAAT-box cis-elements. The GT-1 consensus has been identified to promote SCaM-4 gene expression in plants infected by pathogens or treated with NaCl (Park et al. 2004). The Dof and GAG are induced by light, and when they are stimulated by light, the cyPPDK and PEPC genes have high transcription level in maize (Yanagisawa 2000). When the jasmonic acid content increases in the barley plant, the CGTCA motif associates with it and induces high expression of the lipoxygenase1 gene in the barley grain (Rouster et al. 1997). The GATA element strongly improves the transcriptional level of the cab promoter (Lam and Chua 1989), and the CAAT-box mediates the gene expression level by increasing the frequency of transcription initiation (Kusnetsov et al. 1999). Overall, these cis-elements may improve gene expression after stimulation with the relevant factor.

SA, ABA, and JA induce the expression of BnFAD2-C5

The Brassica napus genome originated from Brassica oleracea (CC) and Brassica rapeseed (AA), and high homology exists between B. oleracea and B. rapeseed. The alignment of the 5′flanking sequences between BnFAD2-A5 and BnFAD2-C5 in Brassica napus showed that the highly homologous regions of the promoter were mainly in the 600 bp next to the transcription initiation site (data not shown). We have identified the existence of the ABRE in the BnFAD2-A5 promoter region, which is homologous to the BnFAD2-C5 promoter, as published previously (Xiao et al. 2014). The BnFAD2-C5 promoter also functions with the ABA for its ABRE. We found that a TCA element and the CGTCA motif are responsible for SA and JA responses. These findings may provide some clues as to how to increase the content of oleic acid to improve the quality of oil.