A plant defensin gene from Orychophragmus violaceus can improve Brassica napus ’ resistance to Sclerotinia sclerotiorum

A plant defensin gene, named as Ovd, was cloned from Orychophragmus violaceus (L.) O. E. Schulz and subsequently introduced into Brassica napus genome by Agrobacterium tumefaciens-mediated transformation method in sense and antisense forms under the control of the CaMV 35S promoter. Genomic PCR analysis confirmed the integration of the sense and antisense Ovd into the host genome. Quantitative RT-PCR showed that the expression of Ovd in the sense plant line was stronger than non-transformed plant and antisense plant. The lesion size on detached leaves of the transgenic plants and nontransformed control caused by Sclerotinia sclerotiorum mycelia was examined. Lesion size was reduced in sense transgenic plants compared to nontransformed control (15 20% reduction area), while it was enlarged in antisense transgenic plants compared to nontransformed control (14 20% enlargement). The results showed that the over-expressing transgenic B. napus had higher resistance to S. sclerotiorum.


INTRODUCTION
Plant defensins are a class of antimicrobial cysteine-rich peptides whose structural and functional properties resemble those of insect and mammalian defensins (Broekaert et al., 1995).They also can protect seeds, seedlings and plants from attacking of soil-bore pathogens (Terras et al., 1995).Some studies have demonstrated transgenic expression of plant defensins can result in the enhanced resistance of host plants against pathogen (Terras et al., 1995;Lai et al., 2002;Gao et al., 2000;Koike et al., 2002).Furthermore, inhibition of the growth of a broad range of fungi (pathogen) by plant defensins has no concomitant of toxicity to either mammalian or plant cells (Thomma et al., 2002).Therefore, plant defensins are not only important components of host defense, but also can be used to generate transgenic crops with improved pathogen resistance (Thomma et al., 2002).
Brassica napus is one of the most important oilseed crop in many countries, ranking third only to soybean and palm oil in global production.A great deal of effort has gone into improving the quality and disease resistances of B. napus using both classical breeding and biotechnological techniques.The plant pathogenic fungus Sclerotinia sclerotiorum is an ubiquitous inhabitant of soils in many parts of the world (Boland and Hall, 1994).In China, it is the first pathogen of oilseed.It causes stem rot on oilseed rape (B.napus) and leads serious losses in yield every year in the middle and low drainage areas of Yangtse River (Zhang et al., 2003).So it is urgent to improve resistance of the oilseed.
Orychophragmus violaceus (L.) O. E. Schulz, cultivated as an ornamental plant in China, is a valuable oil-seed resource of Cruciferae with high amounts of oleic (20.32%), linoleic (53.17%) and palmitic (14.31%) acids and low amount of linolenic (4.76%) and erucic (0.94%) acids in its seed oil (Luo et al., 1994).O. violaceus has a strong disease and insect resistance (Luo et al., 1995).In this paper, we describe the molecular cloning of a plant defensin coding sequence from germinating O. violaceus seeds and transformation of the cloned gene into B. napus.The over-expressing transgenic plants showed higher resistance to S. sclerotiorum.

RNA isolation and cloning of the plant defensin gene (Ovd)
O. violaceus seeds were germinated on MS medium.Total RNA was extracted from the germinating seeds at 3 d using the RNA extraction Kit (TIANGEN, P. R. China).The cDNA synthesis was performed with the RevertAid TM First Strand cDNA Synthesis Kit (MBI Fermentas, Canada).The full coding sequence of defensin gene was cloned using the primers P1: 5′-ATGGCTAAGTTT GCTTCC-3′ (sense) and P2: 5′-TTAACATGGGAAATAACAGATAC -3′ (antisense).PCR was performed under the following condition: 94 C for 30 s, 50 C for 40 s and 72 C for 30 s.The PCR product was purified and then cloned into pMD18-T vector (TaKaRa Biotechnology Co. Dalian, P. R. China) for sequencing.The resulted plasmid was named pMD18-T-Ovd.

Comparative and bioinformatic analyses
Comparative and bioinformatic analyses of Ovd were carried out online at the websites (http://www.ncbi.nlm.nih.gov).The ORF sequence and deduced amino acid sequence were analyzed and the sequence comparison was conducted through database search using BLAST program (http://www.ncbi.nlm.nih.gov).The signal peptide was predicted with SignalP3.0(http://www.cbs.dtu.dk/services/SignalP/).The phylogenetic analysis of Ovd and the plant defensins from other species was aligned with MEGA4.1 using default parameters.Phylogenetic tree was constructed using MEGA version 4.1 from CLUSTAL X 2.0 alignments.The neighbor-joining method was used to construct the tree.

Agrobacterium tumefaciens strain, plasmid and culture
The Ovd ORF fragment was amplified from pMD18-T-Ovd using the above primers with Pfu DNA Polymerase (TaKaRa Biotechnology Co. Dalian，P.R. China) and phosphorylated.Plasmid pBI121 (Chen et al., 2003) was digested with SmaI and Ecl136II to remove the β-glucuronidase gene and was ligated with the phosphorylated Ovd ORF fragment.The constructed vectors were designated as sense and antisense respectively (Figure 1).Then the recombinant plasmids were introduced into Agrobacterium tumefaciens (strain EHA105).
A single colony of A. tumefaciens was inoculated into 50 ml of liquid LB medium containing 20 mg/l streptomycin, 50 mg/l kanamycin and 40 mg/l rifampicin in an Erlenmeyer flask and shaken at 220 rpm overnight in the dark at 28°C. 10 ml of bacterial suspension were pelleted and resuspended in 250 ml liquid LB medium containing 20 mg/l streptomycin, 50 mg/l kanamycin and 40 mg/l rifampicin, cultured at 28°C at 250 rpm in the dark until the OD600 reached 0.6 -0.8.For transformation, the culture was centrifuged at 5000 rpm for 5 min and the pellets were resuspended in 250 ml MS liquid medium (pH 5.6) with 19.62 mg/l acetosyringone (Sigma, USA).The bacteria suspension was cultured in the darkness at 120 rpm at 28°C for an hour before infection of plant cells.

Plant material and culture condition
Seeds of B. napus line 84100-18 presented by Professor Mao-lin Wang which had low resistance to S. sclerotiorum were rinsed in 70% (v/v) ethanol for 1 min, then surface-sterilized for 12 min in 0.1% (w/v) mercuric chloride (HgCl2) solution.After that, the seeds were rinsed four times in ddH2O and germinated on MS medium (Murashige and Skoog, 1962) in the darkness for 2 d followed by a light intensity (1600 lux) for an additional 3 -4 d at 25 ± 2°C and a 16-h day photoperiod.Hypocotyls were excised and used as explants.
All media were supplemented with 30 g/l sucrose and readjusted to pH 5.8 with 1 M NaOH before autoclaving.6-benzylaminopurine (BA), 2, 4-D, NAA (a-naphthaleneacetic acid) were added just before autoclaving.Silver nitrate (AgNO3), acetosyringone and antibiotics were filter sterilized with a 0.2 m membrane and added into the autoclaved media.0.8% (w/v) agar was added to solidify the media.The media for plants were summarized in Table 1.

Optimization the concentration of kanamycin
Sensitivity of the hypocotyl explants to kanamycin was determined
by culture the explants on the media with different concentrations of kanamycin.Hypocotyls from the sterile seedlings were cut into about 1 cm segments and pre-cultured for 2 d on medium II, then transferred to fresh regeneration media K0-K6 at 10-day intervals (Table 1).The experiment was repeated three times with 50 hypocotyl segments per treatment.

Transformation, selection and plant regeneration
The A. tumefaciens-mediated transformation procedure in B. napus was referred to the published results (Cardoza and Stewart, 2003;De Block et al., 1989;Fry et al., 1987;Moloney et al., 1989).Five to six days after sowing, hypocotyls from the sterile seedlings were cut into about 1 cm segments and pre-cultured for 2 d on medium II.
Then the explant segments were immersed in the activated A. tumefaciens suspension (OD600 = 0.6 -0.8) for 1 min with slightly shaking.The immersed hypocotyls were patted dry on sterile filter paper, then co-cultured for 2 days on medium II without kanamycin in the dark.
After 2 days of cocultivation, the explants were transferred to medium III containing 10 mg/l kanamycin and 500 mg/l carbenicillin.The explants were subcultured at 10-day intervals to fresh medium of the same composition.The small shoots were formed after 2 -4 weeks.The healthy shoots (2 cm or longer) were removed from the hypocotyl explants and transferred directly to rooting medium IV.Rooted shoots were propagated with either the top shoot or stem pieces with an axial knob on medium IV or transferred directly to the greenhouse.

PCR analysis of the transgenic plants
DNA was isolated from leaves according to CTAB method (Doyle and Doyle, 1990).PCR analysis of putative transgenic shoots was performed to verify the plant defensin gene transformed into B. napus.Primer sets used were: CaMV 35S promoter specific sense primer (5′-GACTAGTGCAAGACCCTTCCTC-3′) coupled with Ovd sense primer P1 or antisense primer P2.PCR was performed with 30 cycles at 94°C for 40 s, 50°C for 45 s and 72°C for 30 s.All reactions were preceded by a primary denaturation step at 94°C for 5 min.Amplified DNA was separated on 1.5% (w/v) agarose gel.Non-transgenic plant was used as the negative control and A. tumefaciens with vector pBI121 harboring sense and antisense Ovd with CaMV 35S promoter fragment were used as positive control.

Expression analysis of Ovd by quantitative RT-PCR
Total RNA was isolated from collected leaves of the sense plant lines (s6, s8 and s12), non-transformed plant and antisense plant line (a3, a4 and a7) selected randomly, using the RNA extraction Kit (TIANGEN, P. R. China).The cDNA synthesis was performed with the RevertAid TM First Strand cDNA Synthesis Kit (MBI Fermentas, Canada).Real-time PCR was performed using the Light-Cycler Quick System 350S (Roche Diagnostics K.K.) with SYBR Premix Ex Taq (Takara, China).Each PCR reaction contained 1 SYBR Premix Ex Taq, 0.2 μM of each primer and 2 μl of a 1:5 dilution of the cDNA in a final volume of 20 μl.The following PCR program was used: initial denaturation, 95°C, 60 s; PCR, 40 cycles of 95°C, 10 s, 57°C, 15 s, 72°C, 15 s.In melting curve analysis, PCR reactions were denatured at 95°C, reannealed at 55°C, then a monitored release of intercalator from PCR products or primer dimmers by an increase to 95°C with a temperature transition rate of 0.1°C s -1 .To create a standard curve, homologous standards for each gene were used as external standards in all experiments.cDNA quantities were calculated by the second derivative maximum methods of Light-Cycler Software Ver.3.5 (Roche Diagnostics) and all quantifications were normalized using β-actin mRNA as an internal control.The primers were used as follows: Ovd specific (forward 5'-TTTCTGCTTTCGAGGCACCAAC-3', reverse 5'-TGATACAGAA GGGACGAGTGTTCAC-3'), actin specific (forward 5'-GTGGGGAT GGAAGCTCCTG-3', reverse 5'-GTG ATCTCTTTGCTCATACGGTC -3').

Fungal resistance bioassays
Ten leaves excised from each plant line were placed into a box bedded with wet-paper and subsequently inoculated with the mycelial agar plug in a diameter of 5-mm cultured from this fungus separately.Plugs were placed on the adaxial surface, near the midvein.The boxes were covered with plastic film and then kept at 20°C in dark.The lesion diameter was measured after inoculation at 48, 72 and 96 h to evaluate the level of resistance.The results were analyzed with SPSS 13.0 statistic analysis software (SPSS Inc., USA).

Clone, comparative and bioinformatic analyses of a plant defensin gene from O. violaceus
A pair of primers was designed according to the plant defensin gene sequence of B. napus (GeneBank U59459) and used to amplify its homologous gene in cDNA of O. violaceus' seeds.The cloned gene is named as Ovd.The obtained sequence was 243 bp in length and encoded a putative pre-protein of 80 amino acids (Figure 2).Its forecasted molecular weight is 9 kDa.The richest amino acid residue was Ala (13.8%), followed by Cys (10%).The first 87 bp DNA sequence encodes 29 residues signal peptide analyzed with SignalP3.0(http://www.cbs.dtu.dk/services/SignalP/).The 29-amino acid signal peptide with the signal peptide cleavage site between A 29 and Q 30 was identified from the Ovd full-length cDNA sequence, which was consistent with the signal peptide cleavage site of Rs-AFP1 (Raphanus sativus antifungal protein 1) and Rs-AFP2 (R. sativus antifungal protein 2) (Terras et al., 1995).The mature protein was 51 aa in length with a molecular weight of 5.8 kDa.It included 8 Cys that may form four structure-stabilizing disulfide bridges as reported before which were strictly conserved in plant defensins (Broekaert et al., 1995;Lay and Anderson, 2005).A glycine (position 13, according to the mature protein), a serine (position 8), an aromatic residue (position 11) and a glutamic acid (position 29) were also conserved as reported by Lay and Anderson (2005).In comparison of the cDNA sequence of Ovd with the sequences of other AFP genes in the NCBI database using BLAST search program，it was found that it has 93% identity with Rs-AFP1，90% with Rs-AFP2 and Sa-AFP (Sinapis alba antifungal protein gene), and 89% with Bn-AFP (B.napus antifungal protein gene).The amino acid sequence alignment showed the high identity of Ovd with AFPs in the NCBI database, for example 96% identity with Rs-AFP1, 91% with Rs-AFP2, 91% with Brassica oleracea defensin, 88% with Sa-AFP, and 87% with Arabidopsis thaliana putative plant defensin PDF1.1 (data not shown).Sequence alignment using DNAMAN also showed the mature protein of Ovd had highly similar with other plant defensins in Brassicaceae (Figure 3).The cDNA sequence has been submitted to GeneBank (GeneBank FJ489240).

Molecular evolution analysis
Ovd was the first defensin gene cloned from O. violaceus plant.And it had highly similar with other plant defensins in Brassicaceae.Therefore it would be interesting to investigate its evolutionary position among the phylogenetic tree of various plant defensins.Using MEGA version 4.1 from CLUSTAL X 2.0 alignments, a phylogenetic tree of plant defensins in Brassicaceae was constructed.According to the phylogenetic tree, Ovd had higher identity with Rs-AFP1, Rs-AFP2, B. oleracea defensin and Brassica juncea defensin (Figure 4), which was closely related to these defensins.Apparently, all the plant defensins in Brassicaceae are derived from a common ancestor in evolution, suggesting that they share a common evolutionary origin.All the analysis results strongly suggest that Ovd is a plant defensin.

Transgenic plants obtained by the optimized transformation and regeneration procedure
To study the function of Ovd, the sense and antisense Ovd ORF fragments were ligated into pBI121 vector to make the over expression and inhibiting expression vector, respectively.The pBI121-Ovd sense and pBI121-Ovd antisense vector were transformed into A. tumefaciens (strain EHA105).Since the hypocotyls explants of B. napus line 84100-18 were sensitive to kanamycin and 10 mg/l kanamycin in the media was enough to inhibit normal green shoot differentiation, this concentration was set as selection for transformation.
The hypocotyl explants of seedlings of 5-6d were pre-cultured for 2 d on the medium applied with 1 mg/l 2, 4-D, 2.5 mg/l AgNO 3 and 19.62 mg/l acetosyringone (Table 1).After 2 d pre-cultivation, the explants had no overgrowth of the callus and cells were in the meristematic state which was helpful to transformation and regeneration.Then hypocotyls were dipped into bacterium solution and cultured on the same medium as the pre-cultivation medium.After two days co-cultivation they were transferred to medium III with 2 mg/l 6-BA, 2.5 mg/l AgNO 3 , 500 mg/l carbenicillin and 10 mg/l kanamycin for organogenesis and selection.About 2 -4 weeks, green shoot grew out from the ends of hypocotyl segments.These shoots rooted on medium IV after 2 -4 weeks.The successful incorporation of the transgene was verified by the genomic PCR (Figure 5).No positive result occurred with DNA isolated from control plants (non-transformed).Of random selected 13 independent sense kanamycin   resistant plants and 8 independent antisense kanamycin resistant plants, 10 and 3 plants had positive band respectively.Analysis of the expression of Ovd was performed by quantitative RT-PCR.The expression values of the individual genes were normalized using the expression level of β-actin as an internal standard.Mean expression values and SE values were calculated from the results of three independent experiments.The result (Figure 6) showed that Ovd mRNA was more abundant in sense plant lines than other lines.The expression level of Ovd was the highest in the sense transgenic plant line, while the expression level in the antisense plant line was the lowest.On the other hand this also verified Ovd has been trans-formed and expressed in B. napus.

Resistance of transgenic plants to S. sclerotiorum
To test the resistance to S. sclerotiorum, sense plants (s6, s8, s12) and antisense plant (a3, a4, a7) were selected randomly.Fungal resistance assays were performed using detached leaves inoculated with an agar plug of S. sclerotiorum mycelia from the actively growing edge of a fungal culture.The lesion diameter was measured after inoculation at 48, 72 and 96 h to evaluate the level of their resistance (Table 2).Comparing the lesion diameters, those of sense transgenic plants were the shortest and lesion diameters of antisense transgenic plants were the longest.Comparing with the non-transformed plant, the difference of the lesion diameters of transformed plants reached significance level (p 0.05 or 0.01).Lesion size was reduced in sense transgenic plants compared to nontransformed control (15 -20% reduction), while it was enlarged in antisense transgenic plants compared to nontransformed control (14 -20% enlargement) (Figure 7).In general, the hierarchy of lesion size was plants with sense Ovd control plants plants with antisense Ovd.This was consistent with the expression level of Ovd performed by quantitative RT-PCR.This result showed that to a certain extent, plant defensin Ovd can confer enhanced resistance to S. sclerotiorum.The line of S. sclerotiorum used in our study was highly pathogenic in Chengdu, so it was possible to affect the resistance of transgenic plants.
B. napus is one of the most important oilseed crops in many countries and S. sclerotiorum is one of the most serious pathogeny of oilseed.For these reason, a great deal of effort has gone into improving the quality and disease resistances of B. napus.Chemical methods have been used to control this disease.However, due to negative environmental effects, they are not a good choice.And there were no rapeseed cultivars found to be immune to Sclerotinia (Zhao and Meng, 2003).So it is urgent to improve resistance of the oilseed.The genetic engineering can target to specific characteristics and is thought to be most practical if efficient, genotype-independent and reproducible transformation and regeneration system were available.Now several plants have been transformed with plant defensin genes.Lay and Anderson (2005) have reviewed these reports.For example, constitutive expression of the radish defensin (Rs-AFP2) enhanced resistance of tobacco plants to the fungal leaf pathogen Alternaria longipes and similarly in tomato to A. solani.Canola (Brassica napus) constitutively expressing a pea defensin had slightly enhanced resistance against blackleg (Leptosphaeria maculans) disease.Expression of the alfalfa defensin (alfAFP) in potatoes enhanced their resistance to the fungal pathogen V. dahliae.These transgenic plants usually showed the resistance to one pathogen, seldom to several pathogens.
In this paper, we cloned a plant defensin gene (Ovd) from O. violaceus and transformed B. napus with Ovd by the A. tumefaciens mediated method.The transformed plants with Ovd sense showed a higher resistance to S. sclerotiorum than non-transgenic plants and the transformed plants with Ovd antisense.To our knowledge, this is the first report of improved higher resistance to S. sclerotiorum by transformation of B. napus with the plant defensin gene (Ovd) derived from O. violaceus.The obtaining of transgenic B. napus with higher resistance to pathogen will improve the crop quality, especially the wider adaptation; consequently the increased output is also expectable.Further studies are on going in our laboratory.

Figure 1 .
Figure 1.Schematic presentation of cloning of pBI121-Ovd recombinant expression plasmid.Plasmid pBI121 was digested with SmaI and Ecl136II to remove the β-glucuronidase gene and was ligated with the phosphorylated Ovd ORF fragment.For the flat-end ligation, two insert directions of (sense and antisense) Ovd fragment were produced.RB and LB were the T-DNA borders.Ori V was the replicating origin.

Figure 2 .
Figure 2. Nucleotide acid sequence and deduced amino acid sequence of Ovd.Eight cysteines are underlined.

Figure 4 .
Figure 4. Phylogenetic analysis of plant defensins from O. violaceus and other plants in Brassicaceae by MEGA version 4.1 from CLUSTAL X 2.0 alignments.The neighbor-joining method was used to construct the tree.The resources of data were the same with those of Figure 3 (Figure 3).

Figure 5 .Figure 6 .
Figure 5. Genomic PCR of transgenic plants.(A) M, DNA molecular weight marker DL2000; C-, Negative control with DNA of the non-transformed plant; C , Positive control with Agrobacterium tumefaciens with pBI121-Ovd sense; Lanes 1-13, PCR of different transgenic plants with primers CaMV 35S and Ovd antisense.(B) M, DNA molecular weight marker DL2000; C-, Negative control with DNA of the non-transformed plant; C , control with A. tumefaciens with pBI121-Ovd antisense; Lanes 1-8: PCR of different transgenic plants with primers CaMV 35S and Ovd sense.

Figure 7 .
Figure 7. Lesion size (in cm 2 ) on detached leaves in response to inoculation with S. sclerotiorum after 48 h (A), 72 h (B) and 96 h (C).Means and SE are shown for three replicates.

Table 1 .
Media for plants in the experiments.

Table 2 .
Comparative effect of S. sclerotiorum attack on transgenic and wild-type B. napus leaves.Fungal resistance assays were performed using detached leaves inoculated with an agar plug of S. sclerotiorum mycelia from the actively growing edge of a fungal culture.The lesion diameter was measured after inoculation at 48, 72 and 96 h to evaluate the level of resistance.Plants of s6, s8 and s12 were transformed plants with Ovd sense.Plants of a3, a4 and a7 were the transformed plants with Ovd antisense.The control was a non-transgenic plant.The average value and P value were obtained by computing data from ten leaves in each plant with SPSS 13.0.Comparing the lesion diameters, those of sense transgenic plants were the shortest and lesion diameters of antisense transgenic plants were the longest.Comparing with the non-transformed plant, the difference of the lesion diameters of transformed plants reached significance level (p 0.05 or 0.01).Means and SE are shown for three replicates.