Creation of glyphosate-resistant Brassica napus L . plants expressing DesC desaturase of cyanobacterium Synechococcus vulcanus

Aim. Creation of glyphosate-resistant canola plants expressing bifunctional hybrid desC::licBM3 gene. In the hybrid gene the sequence of DesC desaturase of cyanobacterium S. vulcanus without plastid targeting was fused with the sequence of thermostable lichenase reporter LicBM3 gene. Methods. Agrobacterium tumefaciensmediated transformation, PCR, quantitative and qualitative determination of lichenase activity, genetic analysis. Results. Transgenic canola plants, carring the enolpyruvat shikimat phosphate syntase gene (epsps), conferring on plants resistance to phosphonomethyl glycine herbicides (Roundup), as well as the desC::licBM3 gene, were selected. The presence of transgenes was confimed by multiplex PCR. The epsps gene expression in canola was shown at the transcription level, during in vitro growth and after greenhouse herbicide treatment. Activity of the licBM3 gene product as a part of hybrid protein allowed quantitative and qualiative estimation of the desaturase gene expression. Inheritance of heterologous genes and their expression in the first generation were investigated. Conclusions. Transgenic canola plants were obtained, the presence of trangenes in plant genome was proved and expression of the target genes was detected.

Introduction.Due to climate changes, the plant resistance to stress factors of various origin, including low temperatures and phytopathogens, becomes of great importance.
One of the plant adaptive mechanisms to cold is an increase in the unsaturation of fatty acid residues in cellular membranes, sustaining the required membrane fluidity at low temperatures [1].An important role in this process is attributed to fatty acid desaturases, catalyzing the transformation of a single bond between carbon atoms in acyl chains (C-C) into the double bond (C=C).According to the current data, the synthesis of D9-mono-unsaturated fatty acids in higher plants occurs in plastids, while the formation of additional double bonds may occur in both plastids and endoplasmatic reticulum.However, some reports demonstrate the cloning of genes, encoding D9-acyl-lipid desaturases, which supposedly function outside plastids [2].
The application of biotechnological approaches allows the obtaining of plants with the increased content of mono-and polyunsaturated fatty acids in the membrane lipids.The Agrobacterium tumefaciensmediated transformation resulted in the introduction of the gene coding for D9-acyl-lipid desaturase of cyanobacterium Anacystic nidulans into the Nicotiana tabacum L. genome.This enzyme catalyzes the formation of cys-double bond in position D9 in both 16-and 18-carbon saturated fatty acids.Due to this fact the created plants have considerably increased number of unsaturated fatty acids in the majority of membrane lipids which leads to significant enhancement of cold tolerance [2].Similar changes in cold tolerance were observed in tobacco plants, obtained via direct (using polyethylene glycol) transformation of protoplasts using vectors, containing either cDNA of D9-desaturases of cyanobacterium A. nidulans or cDNA of D9-desaturase of cold-resistant potato strain Solanum commersonii [3].The introduction of heterologous desaturases of various origin results in comparable increase of cold tolerance.The expression of D9-desaturase of cyanobacterium S. vulcanus [4] and FAD7 desaturase of Arabidopsis thaliana L. [5] in tobacco leaves also allows the transformants to endure considerable cold stress.The potato plants of Solanum tuberosum L. become more resistant to low temperatures after the introduction of D12-desaturase gene of Synechocystis sp.PCC 6803 [6], as well as the gene of D9-desaturase of wild potato S. commersonii [7].
The data on an impact of stress factors on avocado fruit demonstrate that the increase in the activity of D9desaturase (AvFAD9) resulted in higher resistance to the pathogen fungus Colletotrichum gloeosporioides [8].
Brassica napus L. is the third among the most important oil-bearing crops in the world (after palm and soya, faostat.fao.org/site/567/default.aspx#ancor)by the amount of yielded oil.The selection of Brassica napus L. is aimed first of all at the increase in the yield, oil-bearing, and the improvement of oil quality.This may be achieved also by obtaining plants, resistant to phytopathogenic fungi and with increased cold tolerance.
This work was aimed at canola plants, resistant to Roundup herbicide, which express the desaturase DesC of cyanobacterium S. vulcanus (without the signal of transporting into plastids) as a part of the bifunctional reporter gene for further testing of transgene lines for resistance to stress factors of different origin.
Materials and Methods.Plant material.Aseptically cultivated spring canola plants, Obreey cultivar, were used as material for transformation.The seeds were kindly provided by N. V. Slisarchuk (National Scientific Center "Institute for Soil Science and Agrochemical Research", UAAS).
Genetic transformation was performed using leaf explants in accordance to the method, previously suggested by us [9].The transformation was performed simultaneously with two vectors, each of them was cloned in A. tumefaciens, GV3101 strain.Vector pBISN-desC::licBM3 contains genes desÑ (D9-desaturase) of cyanobacterium S. vulcanus and licBM3 (thermostable lichenase) of Clostridium thermocellum [10], fused in one reading frame under the control of 35S promoter of cauliflower mosaic virus, and the selective gene of neomycinphosphotransferase II (nptII) under the control of nos promoter.Vector ðÑÂ133 carries genes epsps (target) under the control of 35S promoter of cauliflower mosaic virus and bar (selective) under the control of nos promoter.The regenerants were selected on the media with phosphinotricin (PPT, 5 mg/l).
PCR-analysis.The total DNA was isolated from the leaf tissue of the transformed plants using the method [11].The reaction was performed with 40 ng of plant DNA as well as the corresponding primers in the concentration of 0.5 µM and nucleoside triphosphates -500 µM, 1 unit of Taq DNA-polymerase, the reaction buffer contained 50 mM ÊÑl, 10 mM tris-HCl (pH 9, 25°Ñ), 0.1% triton X-100 and 2 mM MgCl 2 .The total volume of the mixture was 20 µl.The gene epsps was identified using the primers, amplifying the fragment of 498 b.p. [12].During multiplex PCR the genes desC and licBM3 were determined using the primers, amplifying fragments of 949 and 642 b.p. respectively [13].DNA, isolated from the non-transformed plants (negative control) and 1 ng of plasmid vector (positive control) were amplified with the same primers and in the same conditions using the Mastercycler personal thermocycler (Eppendorf, Germany).The parameters of amplification reaction corresponded to [12,13].The PCR products were analyzed by electrophoresis in 1 % agarose gel in tris-acetate buffer.
The isolation of the total RNA and RT-PCR was performed according to [14].
Testing for resistance to glyphosate.The sterile solution of N-phosphonomethylglycin (2.5 mg/l) was added to the hormone-free nutrient medium MS [15] after autoclaving to test the resistance in vitro.The root formation and general state of plants were estimated three weeks later.Three-week-old adapted plants were sprayed with Uragan Forter 500 SL herbicide under cover following the manufacturer's recommendations (Syngenta, Switzerland).The working solution contained 2.5 mg/l of glyphosate.The impact of the preparation was estimated 7 days later.
The qualitative evaluation of the thermostable lichenase activity was performed according to [13].
The quantitative evaluation of the thermostable lichenase activity was performed according to the modified method [16], estimating the concentration of free reducing sugars after the reaction of total protein extracts of the leaf tissue with lichenan.The leaves were ground in a singlefold volume of 100 mM tris-HCl buffer (pH 8.0), containing 0.1 M NaCl, 5 mM Na 2 -EDTA and 10 mM mercaptoethanol, and centrifuged at 13,000 g (4°Ñ) for 5 min.The supernatant (20 µl) was added to 50 µl of 0.5 % aqueous solution of lichenan, diluted with water to 500 µl and incubated at the temperature of 65-70 °Ñ for 95 min.Then we introduced 500 µl of DNS-reagent (1 % dinitrosalicylic acid and 0.05 % sodium sulfite in 1 % sodium hydroxide solution), 165 µl of 40 % K-Na-tartrate and kept the mixture in the water bath at 95-100 °Ñ for 10 min, cooled till 4 °Ñ, and kept for 15-20 min at room temperature.
The concentration of reducing sugars was evaluated using the calibration chart for glucose.The activity of the enzyme, forming 1 µmol of reducing sugars per 1 s, was accepted as a unit of activity.The specific activity was evaluated per protein amount.
The determination of total soluble protein was performed using Bradford's method [17].
Results and Discussion.Three lines of canola plants on the basis of Obreey cultivar with selective genes nptII and bar, and target genes epsps and desC::licBM3, were obtained by simultaneous cocultivation of canola explants with two agrobacterial vectors (pCB133 and pBISNdesC::licBM3).
Gene nptII, conferring the resistance to kanamycin, was selective in the construction pBISN-desC::licBM3.It is known that a number of difficulties arise at the regeneration stage in the process of using kanamycin with Cruciferae plants.Low doses of this antibiotic promote the occurrence of false transformants, which perish under selective pressure during the subsequent cultivation.High amounts of kanamycin hinder the very process of regeneration [18].The solution may be found in temporary removal of selective pressure which prolongs the process of obtaining transformants.The aim of experiments with transgene lines with two target genes was to obtain the plants with desC gene as a part of the hybrid gene and simultaneous avoiding the negative impact of kanamycin.
It was previously demonstrated that the PPT presence increases the number of canola regenerants during the direct transformation of protoplasts using polyethylene glycol [19].The selection on the media with PPT was successfully performed by us previously for obtaining transgene canola plants with the promoter-free bar gene [9], the gene of animal cytochrome [20], and the gene of human interferon alpha 2b [21].Therefore, in these experiments we also used the vector with T-DNA containing bar gene for the purpose of selecting transformed plants on the media with PPT. 17 canola lines were selected after the regeneration in selective conditions.The data of PCR-analysis demonstrated the presence of introduced target genes (epsps, desC::licBM3) in the nuclear genome of three of them -Bn18a, Bn18b, Bn18c (Fig. 1, a, c).The remaining lines are characterized by the presence of two heterologous genes from pCB133 vector -epsps and bar.The combined integration of two T-DNA was observed with the frequency of 17.6 %.The simultaneous introduction of two and three T-DNA (with the frequency of 30 and 9.5 %, respectively) was observed in the experiments with A. thaliana with simultaneous transformation of plants using three vectors with different genetic constructions [22].The plants with two target genes, introduced by different vectors, were obtained in the work with canola [23].The experiments on introducing different genes in one or several constructions are performed to study and change metabolic pathways, to obtain composite proteins or protein complexes, and to investigate genetic control and regulation [24].We used this approach in our work on the creation of transgene plants with the planned target genes for selection using the most suitable for canola selective agent phosphinotricin.
Further molecular and biological, biochemical and genetic investigations we performed, analyzing the plant lines with four introduced genes.
The expression of epsps gene in canola plants was demonstrated at the level of transcription (Fig. 1, c).In addition, it was tested in vitro while cultivating on the medium with N-phosphonomethyl glycine (2.5 mg/l) and during herbicide treatment in the greenhouse.
The plants of all three lines grew normally in vitro, remaining green and capable of root formation without any additional stimulation on a selective medium with glyphosate (Fig. 2, a).The control non-transformed plants were getting yellow and formed neither new leaves nor roots.
In the greenhouse conditions the transformants withstood spraying with the working solution of glyphosate while control plants withered and were not capable of further growth (Fig. 2, b).
The lichenase plate test (Fig. 3) was positive for all the three lines which proves the expression of the hybrid desC::licBM3 gene.
The initial transformants (Bn18a, Bn18b) were planted in the greenhouse, where they adapted easily, flowered and gave viable seeds by self-pollination.The obtained seeds were cultivated in aseptic conditions on the media with PPT (10 mg/l).No segregation by [the] resistance to phosphinotricin was observed which indicates the integration of more than one copy of bar transgene.The selected PPT-resistant seedlings were passaged on the medium with kanamycin.The seedlings, resistant to both kanamycin and PPT, were tested for the lichenase activity (Fig. 3).
The quantitative evaluation of the enzyme activity demonstrated its absence in the control plants, while its level varied among the transgene lines (Table ).Line 18b was remarkable for the highest lichenase activity.Lines 18a and 18b/25 were characterized by comparable, but considerably lower activity levels, compared to line 18b (by ~40 % ).The lichenase activity for line 18a/2 appeared to be lower than the detection level.The initial transformants (18b and 18a) had higher lichenase activity compared to the first generation plants, obtained by self-pollination of the initial lines (18b/25, 18a/2, 18a/b).
A diverse level of the lichenase activity in the transgene canola lines indicates analogous differences in the expression of the target gene of D9-desaturase as a part of the hybrid gene.The level of gene expression may depend on the integration locus of the foreign DNA and the number of integrated transgene copies.The similar results, reflecting evaluation of the target gene expression by determination of the lichenase