Oilseed rape (Brassica napus L.) is one of the most important crop for the production of vegetable oils in the temperate zones of the world (Downey and Robbelen, 1989). The properties of edible vegetable oils are determined to a large extent by the relative content of triacylglycerol fatty acids (Kinney et al., 2002). Fatty acids also determine many of the nutritional and health properties of edible fats and oils. The Brassica genus is classified into two basic groups: high erucic acid rapeseed (HEAR) and low erucic acid rapeseed (LEAR). Earlier studies had shown that diets rich in erucic acid are associated with fibriotic myocardium and increased blood cholestrol level, and are therefore, undesirable for human consumption (Gopalan et al., 1974). Oils having high levels of erucic acid have found widespread applications for non-edible purposes such as: manufacture of polymers, cosmetics, lubricants, plasticizers, surfactant detergents and pharmaceuticals (Princen and Rothfus, 1984; Murphy and Sonntag, 1991; Ohlrogge, 1994). Regulation during the development of the seed is necessary to implement strategies that change the erucic acid content. In many higher plants, C16:1 and C18:1 fatty acids are the major components of seed storage triacylglycerols (TAG) but in HEAR, the oil is different since erucic acid (C22:1 D13) represents 45 to 60% of the total fatty acids. Erucic acid biosynthesis is catalysed by the membrane-bound oleoyl-CoA elongase complex through four successive reactions (Bernert and Sprecher, 1977; Fehling and Mukherjee, 1991; Domergue, 2000). The elongation mechanism has now been well characterized due to the isolaton of the FAE1 gene (encoded by 3-ketoacyl-CoA synthase, CE). However, organization of the acyl-CoA elongase in the membrane, the developmental regulation of very-long-chain mono-unsaturated fatty acids (VLCMFA) and triacylglycerol biosynthesis remain unclear (Barret et al., 1998).
The pathway, for the biosynthesis of C18 fatty acid, occurs in the plastid, thereafter the C18 fatty acids are exported out into the cytosol for further modification including the synthesis of VLCFA, such as erucic acid (Millar and Kunst, 1997). The elongation of chain lengths greater than C18 are catalyzed by an enzyme complex known as elongase complex (Harwood, 1988). The key enzyme of the elongase complex, b-ketoacyl-CoA synthase (KCS) was shown to be encoded by the FAE1 gene and responsible for erucic acid synthesis (Lassner et al., 1996). The gene encoding the enzyme, 3-ketoacyl-CoA synthase, was characterized in Arabidopsis thaliana (FAE1) and jojoba (James et al., 1995; Lassner et al., 1996). Two homologous sequences have been isolated from embryos of B. napus (Bn-FAE1.1 and Bn-FAE1.2). Bn-FAE1.1 encodes a protein of 506 amino acids and Bn-FAE1.2, a protein of 505 amino acids, both of which share 98.2% identity (Barret et al., 1998). Modification of the fatty acid composition to make rapeseed oil more competitive in various segments of the food and idustrial oil markets has been an important issue for both plant breeding and molecular genetics in recent years (Friedt and Luhs, 1998; Katavic et al., 2000; Jaworski and Cahoon, 2003).
In this paper, we report the isolation and cloning, in sense and antisense orientation, of the seed specific promoter and FAE gene from B. napus. Effects of this constructs on the erucic acid content of transgenic rapeseed plants were studied.
MATERIALS AND METHODS
Plant materials: The LEAR (PF) and HEAR (Maplus) cultivars of Brassica napus L. were used in this study. These cultivars were grown under similar condition in an experimental filed. Genomic DNA was extracted from young leaves by a protocol based on the Cetyl Trimethyl Ammonium Bromide (CTAB) method (Murray and Thampson, 1980).
Plasmids and bacterial strains: The BluescriprtII SK+ plasmid and Escherchia coli DH5a were used for cloning and sequencing. The plasmid pBI121 (Novagen) and A. tumefaciens LBA4404 were used for plant transformation.
Amplification and cloning of the FAE gene and its promoter: The FAE promoter and gene were amplified from genomic DNA by PCR method using appropriate primers (primers number 1 and 2 for promoter and primers number 3 and 4 for FAE1 gene) which were designed on the basis of published data (Accession no. AF275254 for FAE1 promoter and AF274750 for FAE1 gene). The PCR reaction for both amplifications was performed in a total 50 µl final volume, using 2.5 mM of each dNTPs, 10 pmol of each primer, 2.5 mM Mg2+ and 2.5 units of pfu DNA polymerase. Thermocycler was programmed for one cycle at 94°C for 5 min, followed by thirty cycles at 95°C for 1 min; 63.5°C for 1 min; 72°C for 1.5 min and one cycle at 72°C for 10 min, as a final extention. This programme was used for both amplifications. The PCR products were electrophoresed on 1% (w/v) agarose gel and visualised by ethidium bromide (Eth-Br) staining and UV transilluminator. The resulted bands (promoter and gene) were purified using the PCR clean-up gel extraction protocol (MN company-Germany). The purified FAE gene was double digested with Cfr9I and SacI enzymes and cloned into the pSK+ vector which was digested with the same enzymes. The promoter region was double digested with HindIII and Cfr9I enzymes and ligated into the same position in the pSK+ vector. The ligation mixture were used for transformation of E. coli competent cells. The presence of inserts in the transformed colonies were screened by selection on MacConkey agar medium containing 100 mgl-1 ampicillin and colony PCR with specific primers. The recombinant plasmids were further analysed by sequencing (in both direction with T3 and T7 standard primers). The sequenceing results were compared with other sequences deposited in the Gene-bank using the BLAST software (Altschul et al., 1990) and ClustalW program (Thompson et al., 1994).
Sense and antisense construction of FAE1 genes: In the pBI121 binary vector, the CaMV35S promoter was replaced by the FAE1 promoter through digestion with HindIII and Cfr9I restriction endonucleases and ligation procedure. The modified pBI121 vector was digested with Cfr9I and SacI restriction enzymes to eleminate the b-glucuronidase (GUS) gene. The FAE1 gene which was amplified with primers 2 and 3 (Table 1) was digested with the same enzymes. The sense costruct was prepared via ligation of these two components. For antisense construction, the FAE1 gene was replaced with the same gene which was amplified with primer number 5 as the forward and number 6 as the reverse primer (Table 1). Therefore this PCR fragment has SacI site at the begining and Cfr91 at the end of the gene. The presence and orientation of both costructs in recombinant pBI121 were analysed by PCR and restriction enzyme digestion.
Plant tissue culture, transformation and regeneration: Seeds of B. napus were surface sterilized with 1.5% (v/v) sodium hypochlorite and 0.01% (v/v) Triton X-100 by vigorous shaking for 10 min. The seeds were washed 3 times in sterile distilled water and were germinated aseptically on MS medium (Murashige and Skoog, 1962) in glass bottles (15-20 seeds per bottle) at 25°C in a 16 h light/8 h dark photoperiod. Plant transformation and regeneration were performed by procedure which described by Moloney and colleagues (1989). In brief, the 5 days old cotyledons were excised in such a way that they included approximately near 2 mm of petiole at the base. Care was taken to eliminate the apical meristem which sometimes adheres to the petioles. The excised cotyledons were placed on MS medium containing 3% (w/v) sucrose and 0.7% (w/v) agar enriched with 4.5 mgl-1 benzyladenine (BAP) as a cytokinine. Single colonies of the A. tumefaciens strain LBA4404 containing the modified binary plasmid pBI121 (sense and antisense constructs) were grown overnight at 28°C in LB medium supplemented with 50 mgl-1 kanamycin. Explants were then inoculated with A. tumefaciens for 20-30 seconds and the cultivation was continued on the same medium which solidified with agar (8 gl-1) at 25°C in the dark. After 2 days of co-cultivation, explants were transferred to the same medium containing 15 mgl-1 kanamycin (for selection of transgenic plant cells) and 200 mgl-1 cefotaxime (for elemination of Agrobacteria). Subculturing was carried out at 10 days intervals. Transgenic plants were selected on the basis of kanamycin resistance, mature plants were regenerated and cultured in perlite, and were then transferred to soil and grown to maturity.
Analysis of transgenic plants: PCR and digestion analysis of integrated constructs- Total DNA was extracted from leaves of putative transformants according to the Murray and Thampson (1980) protocol. Total DNA from transformed and control plants were analysed for the presence of the sense, antisense constructs and the nptII gene by PCR. The primer pairs used for DNA amplification were: nptF and nptR primers for the nptII gene, senF and senR primers for the sense construct and antF and antR primers for the antisense construct (Tabel 1). For further confirmation, each of two PCR products were digested at the SacI site.
Southern blot analysis- Southern hybridization was conducted according to Sambrook and Russell (2001) procedure with some modification. For each plant, 15 mg of genomic DNA was digested with restriction endonuclease EcoRV and separated by electrophoresis on a 0.8 % (w/v) agarose gel. The DNA fragments in the gel were then transferred to a nylon membrane. Prehybridization and hybridization were performed using a standard method essentially as described by the protocol. Two probes were prepared by the PCR DIG Probe Synthesis Kit (Roche, Germany). The first probe was obtained from recombinant pBI121 vector with the primers (Table 1) senF and senR for detection of sense construct and another was prepared from the same template with the primers antF and senR for detection of the antisense construct. After hybridization with DIG labeled probes (65°C, overnight), the membranes were washed twice with washing buffer (maleic acid 0.1 M, NaCl 0.15 M, 3%(v/v) tween 20, pH 7.5) and detected by the AP- conjugated anti-digoxigenin antibody and NBT/BCIP color substrate solution by the procedure described by the manufacturer.
Fatty acid analysis- Total fatty acids from the putative transgenic and control plants were converted into methyl esters by incubation at 70°C for 30 min in the presence of 1 ml of absolute methanol containing 10 % (w/v) of boron trifluoride. After addition of 1.5 ml of 2.5% (v/v) aqueous NaCl, the methyl esters were extracted with hexane and analysed by GC.
PCR amplification of genomic DNA from the HEAR variety of B. napus with specific primers (1,2 and 3,4 in Table 1) generated two PCR products; these two fragments, The FAE gene and its promoter, have sizes of approximately 1500 bp and 1400 bp respectively. The FAE gene and its promoter were isolated and further analysed by restriction enzyme digestion (Fig. 1a,b,c), and clonesd into the pSK+ plasmid separately. The clones were confirmed by PCR, restriction enzyme analysis and sequencing. The authentic PCR fragments were subcloned into a plant binary vector (pBI121) and the resulting clones and orientation of constructs were confirmed by PCR and restriction enzyme digestion (Fig. 2a,b). These constructs were transferred to A.tumefaciens LBA4404 by the freeze-thaw standard method (Höfgen and Willmitzer, 1988). The Agrobacterium strains were then used to transform B. napus using an Agrobacterium-mediated petiole cotyledonary transformation. The best morphogenic response was shown by 5 days old explants. According to our results the optimum medium for shoot regeneration was the MS medium contain 4.5 mgl-1 of BAP, leadind to 85% shoot regeneration after 20 days of culture.
Explants from the two varieties of B. napus (PF and Maplus) were co-cultivated with the Agrobacterium strain carrying the recombinant binary vector. Transformed shoots were first transferred to shoot elongation medium (MS medium without any hormones) (Fig. 3a,b) and then in MS medium containing with 2 mgl-1 of indol-butyric acid (IBA) and 25 mgl-1 kanamycin (a lethal concentration of antibiotic for nontransformed shoots). The transgenic plants had a regeneration frequency of approximately about 29% in the medium containing 25 mgl-1 of kanamycin. After acclimatization of rooted plantlets to in vivo conditions, they were allowed to flower and set seed (data not shown).
Molecular analysis of transgenic plants: Genomic DNA of putative transgenic and non-transgenic plants were analysed for persence of the nptII gene by PCR using the nptF and nptR primers. PCR amplification produced a fragment of 1340 bp in the transgenic plants, but no amplification was observed in the control plants (Fig. 4). PCR analysis for sense and antisense constructs with specific primers (described in materials and methods) generated amplified fragments of 996 bp and 521bp, respectively (Fig. 5a,b). These two PCR products from the putative transgenic plants were further analysed with restriction enzyme digestion. Digestion of the 996 bp PCR product with the SacI enzyme produced 238 bp and 758 bp fragments, while that of the and 521 bp PCR product produced 238 bp and 283 bp fragments (Fig. 6a,b).
Southern blot analysis of transformants: Four antisense and four sense (data not shown) transgenic and one non-transgenic lines were analysed by Southern blotting. Extracted genomic DNA samples were digested with EcoRV and were hybridized with a Dig labeled 521 bp long probe that consisted of the FAE gene and nopalyne synthase (Nos) terminator sequences (Fig. 7a). As there is an EcoRV site in the recombinant T-DNA construct, the number of hybridization bands indicated the number of integration copies (Fig. 7b). The results of the Southern blotting analysis showed that some of the transformed plants had only a single gene insertion.
Fatty acid analysis: Seeds of 30 self pollinated kanamycin resistant T0 plants carrying the FAE gene (15 plants contain the antisense construct and 15 plants containing the sense construct) were harvested. Bulk samples of 100 seeds per plant were analysed for fatty acid composition by GC (Fig. 8). Five plants bearing the antisense construct had erucic acid levels of approximately 33% (comparing to the HEAR cultivar as control contained 48% erucic acid )(Fig. 8a). Four transformed T0 plants containing the sense construct showed an increases of over 10% in erucic acid levels (the LEAR cultivar as control contained 1.2% erucic acid) (Fig 8b), while two lines of the LEAR transgenic plants showed a decrease of approximately 0.2% in erucic acid level.
The FAE1 gene has previosly been isolated from A. thaliana (James et al., 1995), B. napus (Han et al., 2001), B. campestris (Das et al., 2002) and B. oleracea (Barret et al., 1998). The evidence that the FAE1 gene is responsible for erucic acid synthesis was provided when a b-ketoacyl synthase (coded by FAE1 gene) isolated from Simmondsia chinensis was able to complement the mutation in fatty acid elongation and thus restore the erucic acid level in zero-erucic acid lines of B. napus (Lassner et al., 1996). It has been suggested that the variation in erucic acid levels is due to the differences in the FAE gene sequences belonging to the low and high erucic acid genotypes (Das et al. 2002). Sequence analysis revealed that the FAE gene was a 1521 bp fragment that starts with an ATG initiation codon and after alignment has shown a significant sequence similarity to the other b-ketoacyl CoA synthase gene belonging to B. napus (AF274750, U 50771), B. juncea (Y 11007), S. chinensis (U 37088) and A. thaliana (U 29142). Analysis of the genomic DNA of B. napus FAE clones from HEAR cultivars revealed that they have continuous coding regions which are devoid of any interruption by introns. Similar results were also reported earlier (James et al., 1995; Barret et al., 1998; Fourmann et al., 1998; Venkateswari et al., 1999; Han et al., 2001; Das et al., 2002). The promoter sequence contains several A/T-rich elements also present in other seed-specific promoters which have been shown to enhance gene expression (Thomas, 1993; Sandhu et al., 1998). In addition, the analysis of transgenic plants containing the GUS gene under the control of the seed-specific promoter from the B. napus Askari cultivar revealed that GUS activity was only detected in developing seeds of transgenic rapeseed plants, however there was no color development in leaves or other organs such as roots, stems or buds (Han et al., 2001). It is clear that, reduction in erucic acid content of the seeds of oil producing plants has positive nutritional effects, but this reduction can also change fatty acid composition of cell membrane or has other diverse effects on plant growth and development. Therefore, it is nacessary and very important to control the erucic acid level only in the seeds in transgenic canola plant.
Finding appropriate plant cells with high capacity for accepting the foreign gene is an important criteria in plant transformation by Agrobacterium. In B. napus L, different parts of the plant have been used for A. tumefaciens transformation but in these experiments the cut surfaces of cotyledonery petioles were used as the target cells. Results showed that this target is a vigorous source of new shoot material and that shoot development occurs very rapidly. The origin of these shoots has been shown by Sharma (1987) to be cells located in the vincinty of the cut end of the petioles. The value of Agrobacterium-mediated plant transformation is measured by the number of independent transformed plants expressing the gene of interest, per explant used. This can be the effect of the genotype of the species to be transformed, the virulence of Agrobacterium used for transformation, the antibiotic used as selectable marker, regeneration capacity of the target cells and the accessibility of the bacterium to the regenerable cells. By this method both lines of B. napus (PF and Maplus) were successfully transformed and regenerated. In our hands, this transformation and regeneration method can be act genotype-independently. In the control experiments using non-selective regeneration medium, high shoot formation was observed in all explants. In the medium containing kanamycin (25 mgl-1), regeneration of control explants were almost completely inhibited. In transformation experiments, shoot regeneration on non-selective medium were reduced, presumably due to inoculation with Agrobacterium. However on selective medium, many white, non-transformed escape shoots were formed in addition to the green putative transgenic shoots. Putative transformants that rooted on kanamycin-containing medium were confirmed as transgenic by PCR analysis and Southern blotting. Most of the transgenic plants carried one copy of FAE gene and only a few carried two or three copies. Two bands (about 2000 and 3500 bp) were observed on all lanes. Presence of these two bands were due to high sequence similarity (approximately 63%) between the probe and endogenous FAE gene.
Furthermore, the Southern blotting analyses on transgenic rapeseed plant showed that the level of gene expression is correlate with the number and site of integrations of the expression cassette and this is in agreement with some previously reported results (Kahrizi et al., 2007 and Wang et al., 2003). Similar multiple inserts in the case of Brassicaceae may be an intrinsic feature and it may be dependent on the selection schemes and the levels or types of antibiotics used (Moloney et al., 1989). Our results from GC analysis have shown that introduction of the antisense construct appears to have partially silenced the activity of endogenous FAE gene in B. napus. Therefore, the use of antisense technology enabled the reduction of the erucic acid level in HEAR cultivars by approximately15%. In a LEAR variety, the overexpression of the FAE gene led to an increase in erucic acid level by approximately 8%. Comparison of these results have shown that in this expriment the ability to suppress the gene activity via the antisense construct of the FAE gene was stronger than gene silencing mechanisms by its overexpression. The change in erucic acid level in the seeds of transgenic conola, make changes in the level of other fatty acids as well. In transgenic rapeseed (HEAR cultivar, Maplus) with antisense construct of the FAE gene, decrease in the level of erucic acid (C22:1) cause accumulation of upstream fatty acids such as oleic (C18:1) and linoleic (C18:2) (Fig. 8a). Fatty acid composition in transgenic rapeseed plant (LEAR cultivar, PF) with sense construct of FAE gene is different. The GC choromatogram (Fig. 8b) shows that, increase in erucic acid level can cause changes in the level of other fatty acids (C18:1, C18:2, C18:3 and C20:1) as well. These finding can also show the more general effects of FAE gene in erucic acid synthesis and other fatty acid composition in seeds of oil producing plants. Moreover, the transgenic rapeseed plants (LEAR and HEAR) could provide new insight into the complex mechanisms of oil accumulation. Our data show that the sense and antisense technology can be effectivly used to modify the biosynthetic profile of fatty acids, specially that of erucic acid, through targeting the FAE gene into seed of oily plants.
This study was supported by the National Institute of Genetic Engineering and Biotechnology (NIGEB) and Tarbiat Modares University. We would like to thank the LEMBKE company, Germany, for kindly providing us with the Maplus cultivar of B. napus seeds. Our special thanks to Dr. P. Shariati for critical reading of this manuscript.