Isolation and evaluation of three novel native

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NOTE Isolation and evaluation of three novel native promoters in Brassica napus

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Limin Wu, Aliaa El-Mezawy, and Saleh Shah

Abstract: To provide effective and specific native promoters for canola (Brassica napus L.) genetic modification, three promoters were isolated by genome walking from this species. These three promoters were fused to the uidA reporter gene (GUS) and were independently used to generate populations of transgenic canola plants. Plants transformed with BnPGPro-GUS (B. napus putative germin promoter) exhibited GUS activity in all the tissues tested at a level comparable to those transformed with CaMV35 S promoter. This indicates that BnPGPro may serve as a native constitutive promoter for canola. The other two promoters, BnPro3-GUS and BnPro5-GUS (B. napus, promoter 3 and 5), exhibited GUS activity in various tissues. None of these two promoters expressed in embryo, however. These novel Brassica native promoters can be used to modify canola genes for various purposes. Key words: canola, constitutive, GUS, tissues-specific, promoter. Résumé : Afin de fournir des promoteurs efficaces et spécifiques pour la modification génétique du canola (Brassica napus L.), les auteurs ont isolé trois promoteurs par gene-walking chez cette espèce. Ils ont fusionné ces trois promoteurs au gène rapporteur uidA (GUS) et les ont utilisés indépendamment pour générer des populations de plants de canola transgéniques. Les plantes transformées avec le BnPGPro-GUS (B. napus promoteur putatif de germine) montrent l'activité GUS dans tous les tissus testés, a` un degré comparable a` ceux transformés avec le promoteur CaMV35S. Ceci indique que le BnPGPro peut servir comme promoteur constitutif d'origine pour le canola. Les deux autres promoteurs, BnPro3-GUS and BnPro5-GUS (B. napus Promoteurs 3 et 5) montrent une activité GUS dans divers tissus. Cependant, aucun de ces deux promoteurs ne s'exprime dans l'embryon. On peut utiliser ces nouveaux promoteurs pour modifier des gènes de canola a` diverses fins. [Traduit par la Rédaction] Mots-clés : canola, constitutif, GUS, spécifique au tissu, promoteur.

Introduction Population experts anticipate the global population will grow to 9 billion by roughly 2050. To feed the 9 billion people, the world will need 70%–100% more food, which is a great challenge to the world (Godfray et al. 2010). To cope with the challenge, one of the important strategies suggested by scientists is developing and deploying more genetically modified (GM) crops to increase yield and (or) improve yield stability (Fedoroff et al. 2010). Even though there is an opinion against using agricultural biotechnology, GM crops were grown on 170.3 million hectares in 28 countries in 2012 and benefited 17.3 million farmers, a 100 fold increase in hectares between 1996 and 2012 (James 2012). To develop GM crops, the availability of different types of promoters is important for modifying genes for specific purposes. For example, constitutive CaMV35 S promoter was used to develop Bt (Bacillus thuringiensis) cotton, so that the whole plant gained protection from insects by expression of Bt gene throughout the plant (Bakhsh et al. 2010; Odell et al. 1985). Falco et al. (1995) increased lysine of canola and soybean seeds with seed-specific promoter Pv 5=, because the lysine content of other parts of the plant is not important. Canola is one of the most important oilseed crops. Made in Canada, this crop adds $15.4 billion annually in Canadian economic activity alone (Canola Council of Canada 2011). For increasing canola production to meet global demand in future, new cultivars with improved seed meal and resistance to biotic and abiotic stresses are desirable. To develop such canola cultivars through biotechnology, promoters of different kinds are required to drive the introduced genes in the desired target tissues. The napin promoter from Brassica napus L. was widely used for gene expression in seed embryos (Bagheri et al. 2010; Lock et al. 2009;

Wei et al. 2010). However, very few native and other tissue-specific promoters are available for canola. There is a tremendous need and opportunity for canola seed meal improvement through seed coat modification (Hickling 2007). Therefore, we have searched for seed coat-specific promoters for canola and several such promoters have become available (El-Mezawy et al. 2009; Nesi et al. 2009; Wu et al. 2010, 2011). Surprisingly, some putative seed coat-specific promoters turned out to be very useful constitutive or tissue-specific native and novel promoters for canola. In this study, the discovery of three such promoters from canola is reported so that these promoters can be used by researchers to control constitutive or tissuespecific gene expression for modification of this important crop.

Materials and methods Plant material As was reported previously (El-Mezawy et al. 2009), a double haploid canola line DH12075 (B. napus) was used for transformation in this study. Plant growth conditions, seed sterilization, and germination for in vitro culture were performed as described previously (Wu et al. 2010). Dimensional electrophoresis (DE) Total proteins were isolated from seedling, root, stem-peel, leaf, flower, pod, embryo, and seed coat according to the method described by Hajduch et al. (2005). The proteins from 0.2 g tissue samples were suspended in 500 ␮L rehydration buffer and 5 ␮L tributylphosphine that were purchased from BioRad (Hercules, California, USA). Then 100 ␮L of this 505 ␮L mixture was cup loaded on IPG strip (pH 3–10) for first dimension (isoelectric focus-

Received 2 October 2012. Accepted 26 February 2013. L. Wu, A. El-Mezawy, and S. Shah. Alberta Innovates-Technology Futures, P.O. Bag 4000, Hwy 16A & 75 Street, Vegreville, AB T9C 1T4, Canada. Corresponding author: Limin Wu (e-mail: [email protected]).

Botany 91: 414–419 (2013)

Published at on 4 March 2013.

Wu et al.


Table 1. Primers used in this study. Gene specific primers for genome walking


BnPGProP1 BnPGProP2 BnPro3P1 BnPro3P2 BnPro5P1 BnPro5P2


Primers for gene expression analysis


BnG3 F BnG3 R BnG5 F BnG5 R BnActinF BnActinR


Promoter used in construct

Primers for amplification of promoters


BnPGPro (385 bp)

BnPGProF BnPGProR BnPro3 F BnPro3 R BnPro5 F BnPro5 R


Promoter BnPGPro BnPro3

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Gene BnG3 BnG5 BnActin

BnPro3 (2045 bp) BnPro5 (1527 bp)

ing), followed by SDS-PAGE, as described by Hajduch et al. (2005). The separated proteins were visualized by staining with silver nitrate. The putative seed coat-specific proteins were then cut out from a separate Colloidal Blue (Invitrogen, Carlsbad, California, USA) stained gel (the proteins from seed coat were rerun and stained with Colloidal Blue), digested with trypsin and analysed by automated matrix-assisted laser desorption/ionization time-offlight mass spectrometric (MALDI-TOF MS) peptide mapping followed by extensive database searches (Henzel et al. 1993). Molecular techniques Genomic DNA was prepared from young leaves of B. napus plants using DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA). The primers GUSF (5=-CATCGCAGCGTAATGCTCTA-3=) and GUSR (5=-AATCACCACGATGCCATGTT-3=) were used for amplification of part of the uidA reporter gene (GUS) in transgenic plants, which produced a 537 base pair (bp) fragment. Total RNA was prepared from eight tissues (seedling, root, leaf, stem-peel, flower, pod, embryo, and seed coat) using RNeasy Plant Mini Kit (QIAGEN) and was treated with DNase to remove DNA using DNA-freeTM Kit (Ambion, Austin, Texas, USA). OneStep RT-PCR Kit (QIAGEN) was used for gene expression analysis. The program was reverse transcription for 30 min at 50 °C; initial PCR activation for 15 min at 95 °C; 3-step cycling that includes denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C, for 30 cycles; followed by a final extension for 10 min at 72 °C. Genome walking for promoters was performed for B. napus putative germin gene (BnPGG), B. napus putative seed coat-specific genes 3 and 5 (BnPSCSG3, 5; short named BnG3 and 5) as described in the protocol of GenomeWalkerTM Universal kit (Clontech, Mountain View, California, USA). The gene specific primers used for genome walking, primers for gene expression analysis and primers for amplification of promoters used for preparing constructs are given in Table 1. All the three pair primers used for building constructs had engineered BamHI (GGATCC) and PstI (CTGCAG) sites (Table 1).


The PCR products of BnPGPro (385 bp, see Supplementary Fig. 11) and pGreen binary vector (Hellens et al. 2000) were both double digested with BamHI and PstI, and ligated to make the binary vector BnPGPro:uidA/pGreen-NPTII (Fig. 1). The PCR products of BnPro3 (2045 bp, see Supplementary Fig. 2) were directly used for sub-cloning using pCR®8/GW/TOPO® TA Cloning kit (Invitrogen, Carlsbad, California, USA). After sequence confirmation for identity and orientation, the promoter from the correct plasmid was recombined with pMDC162 binary vectors (Curtis and Grossniklaus 2003) to construct BnPro3:uidA/pMDC162-HYG (Fig. 1). The PCR products of BnPro5 (1527 bp, see Supplementary Fig. 3) and binary vector pCambia2381Z ( daisy/cambia/585.html; modified from pCambia1381Z by replacing the hygromycin resistance gene with a kanamycin resistance gene for plant selection; GenBank accession nos. AF234290-AF234316) were double digested with BamHI and PstI, and ligated to make BnPro5:uidA/pCambia2381Z-NPTII (Fig. 1). Plasmids and Agrobacterium strains All three binary vectors (Fig. 1) were introduced into Agrobacterium tumefaciens strain GV3101 by freeze and thaw method (Chen et al. 1994) and were used for canola transformation. The vectors contain the neomycin phosphotransferase II (NPTII) gene (Kanr), except BnPro3:uidA/pMDC162-HYG that contains hygromycin B phosphotransferase (HPT) gene (Hygr), for plant selection. The A. tumefaciens strain GV3101 carrying the vector was grown on Luria Broth (LB, GIBCOBRL, Burlington, Ontario, Canada) supplemented with 50 mg·L−1 rifampicin, 20 mg·L−1 gentamycin, and 50 mg·L−1 kanamycin. Agrobacterium-mediated canola transformation and plant regeneration Canola cotyledon explants were transformed with A. tumefaciens containing one of the three constructs according to Moloney et al. (1989) with some modifications as described in Wu et al. (2010).

data are available with the article through the journal Web site (

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Botany Vol. 91, 2013

Fig. 1. Binary vectors constructed for testing the activation of three Brassica native promoters. BnPGPro, B. napus putative germin promoter; BnPro3, 5, B. napus promoter 3 and 5, respectively. BamHI RB














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BnPro3:uidA/pMDC162-HYG PstI RB

NOS poly-A


BamHI BnPro5



35S poly-A



The selection of transformed shoots was performed as described previously (Wu et al. 2011). Canola embryo assay and GUS assays Canola embryo assay for the expression of the selection marker gene, which confirms if the transgenic plants are true or false and also indicates the number of transgenic loci in each transgenic event (by showing segregation ratio of young embryos for resistance and susceptibility when cultured on antibiotic selection plates), and histochemical assay for GUS activity were done as described in Wu et al. (2010). Fluorogenic assay for GUS activity was performed according to Jefferson et al. (1987). The tissues used for fluorogenic assay were root, leaf, stem, flower, pod (without seeds inside), seed coat, and embryo. For promoter strength determination, one homozygous BnPGPro transgenic plant (BnPGPro-9-3, second generation) and one homozygous 35 S-PBI121 transgenic plant, both with single copy transgenic loci were used.

Fig. 2. Expression patterns of two Brassica napus candidate genes. BnG3, 5, B. napus gene 3, 5; BnActin, B. napus Actin gene that is a housekeeping gene. Se, seedling; R, root; St-p, stem-peel; L, leaf; F, flower; P, pod; Em, embryo; SC, seed coat; N, negative control (no template). Stem-peels, leaves, seed pods, seed coats, and embryos were collected at 25 DAP (days after pollination), the flowers were collected after opened, roots and seedlings were collected at cotyledon stage (1 week after seeding in the soil at greenhouse). The RT-PCR results showed that the two putative seed coat-specific genes also expressed in other tissues.

Results and discussion Isolation and characterization of putative seed coat-specific proteins by 2-DE It was expected that the genes coding for seed coat-specific proteins should have seed coat-specific promoters. Therefore, proteins from seven canola tissues (seedling, root, stem-peel, leaf, flower, pod, and embryo) were compared with those of seed coat in 2-DE gels. Two unique seed coat proteins named BnPSCSP3 and 5 (B. napus putative seed coat-specific protein 3, 5; short named BnP3 and 5) were identified. These two proteins were detected only in the seed coat, not in any other tissues (see Supplementary Fig. 4). They were subjected to MS analysis. The peptide mass fingerprint analysis resulted in two candidate cDNAs (one B. napus cDNA: gi|32520798 for BnP3; and one B. rapa cDNA: gi|50885557 for BnP5), which code for the two proteins (see Supplementary Table 1). BnP3 is the homolog of BnD22 that is a drought-induced protein (Downing et al. 1992). The closest homolog of BnP5 is Arabidopsis lyrata hypothetical protein (gi|297834517), about which no detail is available. Expression patterns of putative seed coat-specific genes It was hypothesised that the tissue-specific promoter should direct the corresponding gene expressed in specific tissue(s). Therefore, before promoter hunting, expression patterns of the two candidate genes from protein analysis were determined by RT-PCR in eight tissues that were used for 2-DE analysis. Our results showed that these genes expressed in several other tissues in addition to the expected seed coat tissue (Fig. 2). BnG3 expressed in stem-peel, flower, pod and seed coat, and BnG5 expressed in all the tissues, except embryo (Fig. 2).

Isolation of promoters through genome walking Although the promoter of B gene, which is one of the members of barley germin gene family, was seed coat-specific in barley (Wu et al. 2000), the B. napus homologs of barley B gene were identified by BLAST with available ESTs ( The homologous germin EST (EE468156 that includes Bna.14750 unigene, GenBank unigene database, see Supplementary Fig. 5) was used for genome walking to isolate its promoter, B. napus putative germin promoter (BnPGPro). Although the data of expression patterns (not seed coat-specific) of the putative candidates did not match with 2-DE results (seed coat-specific) for the two proteins, we continued to isolate the promoters of the two candidates by considering that the expression pattern of RNA is not always consistent with the expression pattern of protein (Fagard and Vaucheret 2000; Kato et al. 2005). Following the protocol of GenomeWalkerTM Universal kit, the longest PCR product of each promoter was sequenced. After sequence analysis, the BnPGPro was found to be 385 bp (see Supplementary Fig. 1), BnPro3 was 2045 bp (see Supplementary Fig. 2), and BnPro5 was 1527 bp (see Supplementary Fig. 3). The translational start site (ATG) was used as point of reference for these three promoters (GenBank accession nos. HQ916359, HQ916361 and HQ916362; see Supplementary File). Transgenic canola The three B. napus putative seed coat promoters were fused to the reporter gene uidA for testing their expression pattern in canola. A total of 500 canola DH12075 cotyledons were cocultivated with A. tumefaciens carrying each of the three binary Published by NRC Research Press

Wu et al.


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Fig. 3. Genomic PCR of transgenic canola lines. M, 1 kb plus DNA marker; 1–10, BnPGPro-1 to BnPGPro-10; 11–20, BnPro3-1 to BnPro3-10; 21–30, BnPro5-1 to BnPro5-10; N, negative control, nontransgenic DH12075. A 537 base pair fragment of GUS gene was amplified in all transgenic plants, except BnPGPro-3, -4, -6, which confirmed that 27 of the 30 canola plants analyzed were true transgenic plants.

vectors. Twenty-six and 33 kanamycin-resistant primary transformants (T0) were recovered for BnPGPro and BnPro5, respectively. Fifteen hygromycin-resistant primary transformants (T0) were recovered for BnPro3. Presence of the transgene was verified by genomic PCR and “embryo assay” of immature T1 seeds for the first 10 transgenic events of each constructs. The genomic PCR confirmed the presence of transgene in 27 of the 30 lines tested and the embryo assay test also showed expression of the selection marker genes in those same 27 lines (Fig. 3, Table 2). The segregation ratio obtained from the embryo assay test suggested that the transgenic lines had one or two transgenic loci (Table 2). GUS expression patterns directed by the three promoters differ in B. napus Ten T0 plants from each promoter constructs were analysed for their expression pattern in different canola tissues by GUS assay. The results showed that GUS was expressed in all tissues for BnPGPro, which means that the B. napus putative germin promoter is not seed coat-specific, but a constitutive promoter for canola (Fig. 4). For BnPro3, GUS was expressed in stem, flower, pod, and seed coat; and BnPro5 was expressed in all the tissues except embryo (Fig. 4). These results showed that these two putative seed coat-specific promoters corresponding to two apparently unique seed coat proteins are in fact not seed coat-specific, but tissue-specific promoters. Although these two promoters are not seed coat-specific, BnG3 and BnG5 mRNA expression patterns were consistent with their GUS expression. This indicates that these two promoters are useful as tissues-specific promoters: BnPro3 can be used when the transgene expression is unwanted in root, leaf, and embryo; whereas BnPro5 can be used to drive a transgene in canola tissues except embryo. Although the BnPGPro was expected to be seed coat-specific, our results showed that in fact it is a constitutive promoter. Because the B. napus germin gene family includes many members (20 B. napus germin or germin-like unigenes recorded in GenBank, BnPGPro corresponds to Bna. 14750, see Supplementary Fig. 5), the Bna.14750 probably is not the seed coat-specific member of B. napus germin gene family, though it is the closest homolog of barley B gene that is seed coat-specific. We also found that the expression of promoters from two putative seed coat-specific proteins were not seed coat-specific. This might be because the resolution of 2-DE gels was not sensitive enough to detect the week protein signals in other tissues. Expression strength analysis for BnPGPro Our results showed that BnPGPro is a constitutive promoter in B. napus and has the potential to be used for genetically engineering canola. Considering CaMV 35S is a constitutive and widely used promoter in commercial canola, the strength of BnPGPro was compared with that of CaMV 35S by fluorogenic assay. In our preliminary test, GUS activity directed by BnPGPro was no less than that of CaMV 35S promoter in all seven tissues (Fig. 5), at least in the plant we used for comparison. Therefore, the BnPGPro is an excellent candidate that can be potentially used in canola genetic engineering.

Table 2. Segregation of kanamycin/hygromycin resistant and sensitive embryos in the selfed T1 progeny of canola transformed with each of the three constructs. Seedling type Transformation No. of Estimated event germinated ␹2 transgenic designation seeds Resistant Sensitive value loci* BnPGPro-1 BnPGPro-2 BnPGPro-3 BnPGPro-4 BnPGPro-5 BnPGPro-6 BnPGro-7 BnPGPro-8 BnPGPro-9 BnPGPro-10 BnPro3-1 BnPro3-2 BnPro3-3 BnPro3-4 BnPro3-5 BnPro3-6 BnPro3-7 BnPro3-8 BnPro3-9 BnPro3-10 BnPro5-1 BnPro5-2 BnPro5-3 BnPro5-4 BnPro5-5 BnPro5-6 BnPro5-7 BnPro5-8 BnPro5-9 BnPro5-10

32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32 32

28 26 0 0 27 0 29 24 22 25 24 28 28 27 23 28 26 25 29 27 25 22 23 24 29 24 27 28 24 22

4 6 32 32 5 32 3 8 10 7 8 4 4 5 9 4 6 7 3 5 7 10 9 8 3 8 5 4 8 10

2.13 0.67 N/A N/A 1.50 N/A 0.53 0.00 0.67 0.17 0.00 2.13 2.13 1.50 0.17 2.13 0.67 0.17 0.53 1.50 0.67 0.17 0.00 0.00 0.53 0.00 1.50 2.13 0.00 0.67

2 1 0 0 1 0 2 1 1 1 1 2 2 1 1 2 1 1 2 1 1 1 1 1 2 1 1 2 1 1

*In accordance with the expected Mendelian ratio 3:1 or 15:1 at P = 0.05.

The other two promoters, BnPro3 and BnPro5, can be used as tissues-specific promoters for genetic modification of canola. For example, BnPro5 can be used to express Bt gene in all the tissues, except embryo that produces edible oil. This may increase the public acceptance of GM canola for oil production. Cisgenesis and intragenesis are new technologies to genetically modify a recipient organism with all native DNA sequences (promoter, gene, terminator, etc.) from the same or a sexually compatible species as the donor (Andersson et al. 2012; Conner et al. 2007). Because no foreign DNA sequences are introduced into the recipient, the hazards arising from cisgenic plants are similar to those from conventional plant breeding and the perceived hazards arising from intragenic plants are lower than those from transgenic plants (Andersson et al. 2012). Given public concerns over the deployment of GM crops in agriculture, especially for Published by NRC Research Press


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Fig. 4. Activity pattern of three Brassica napus novel native promoters in canola DH12075 transgenic plants. BnPGPro, B. napus putative germin promoter; BnPro3, 5, B. napus promoter 3, 5; a, root; b, leaf; c, stem; d, flower; e, pod; f, seed coat; g, embryo. The pods were collected at 25 DAP (days after pollination) and stained after removing the seeds; and the seeds were then used to separate seed coats and embryos for staining. GUS staining was seen in all the tissues for BnPGPro; in stem, flower, pod and seed coat for BnPro3; and in all the tissues, except embryo for BnPro5.

Fig. 5. Strength determination for BnPGPro and 35 S by fluorogenic assay. BnPGPro, B. napus putative germin promoter; 35 S, CaMV 35 S promoter. The tissues were from one homozygous BnPGPro transgenic plant and one homozygous 35 S-PBI121 transgenic plant (both second generation, GUS staining stable between two generations and DH12075 genotype background). These two plants both had one copy of transgenic loci as indicated by embryo assay test (data not shown). The preliminary fluorogenic assay showed that GUS activity of tissues from this BnPGPro transgenic plant was no less than that of same tissues from the 35 S transgenic plant used in this study. Error bars indicate SD (standard deviation), n = 4. Four samples were harvested and tested for each tissue.

AtGILTpro, Wu et al. 2010, 2011), will give research scientists a relatively broad choice to use them for modification of canola genes to produce better GM canola crops, including cisgenic and intragenic cultivars.

Acknowledgements This research was funded by Genome Canada in the project “Designing oilseeds for Tomorrow's Markets”. We wish to thank (late) Gerhard Rakow, Agriculture and Agri-Food Canada, Canada, for providing DH12075 canola seeds, Paul Semchuk of the University of Alberta for MS analysis, and CAMBIA-Australia for providing binary vectors.


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