Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E. coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate
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- Kahrizi, D., Salmanian, A.H., Afshari, A. et al. Plant Cell Rep (2007) 26: 95. doi:10.1007/s00299-006-0208-4
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Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This is a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity for glyphosate, followed by its transfer to plants is one of the most effective approaches for the production of glyphosate-tolerant plants. In this study, we chose to focus on amino acid residues glycine96 and alanine183 of the E. coli (k12) EPSPS enzyme. These two amino acids are important residues for glyphosate binding. We used site directed mutagenesis (SDM) to induce point mutations in the E. coli EPSPS gene, in order to convert glycine96 to alanine (Gly96Ala) and alanine183 to threonine (Ala183Thr). After confirming the mutation by sequencing, the altered EPSPS gene was transferred to rapeseed (Brassica napus L.) via Agrobacterium-mediated transformation. The transformed explants were screened in shoot induction medium containing 25 mg L−1 kanamycin. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there was a significant difference between the transgenic and control plants. It was observed that transgenic plants were resistant to glyphosate at a concentration of 10 mM whereas the non-transformed control plants were unable to survive 1 mM glyphosate. The presence and copy numbers of the transgene were confirmed with PCR and Southern blotting analysis, respectively.
KeywordsEPSPSGlyphosate resistancePlant transformationRapeseed (Brassica napus L.)Site-directed mutagenesis
Oilseed rape (B. napus L.) is one of the most important oil seed crops (ANZFA 1999). It is grown commercially in 50 countries with a combined harvest of over 40 million metric tones. Canola is a genetic variant of B. napus with low levels of glucosinolates and erucic acid. It is cultivated for its seeds which represent a major source of edible vegetable oil. Pressed cake obtained from canola oil extraction is also used in livestock feed. However, one of the major restrictions in canola production is effective weed control. The presence of weeds in rapeseed plantation reduces crop quantity and quality (Kishore et al. 1992; Kuiper et al. 2000). For effective control of weeds the non-selective, broad-spectrum and post-emergence herbicide glyphosate [N-(phosphonomethyl) glycin] is widely used and recommended. Since this herbicide also affects rapeseed, production of glyphosate-tolerant rapeseed is highly essential (Holt et al. 1993; Kuiper et al. 2000).
The primary mode of action in planta for glyphosate is by competitive inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; E.C.18.104.22.168), which catalyses the penultimate and main step of the prechorismate part of the plastid-localized shikimate pathway (Steinruken and Amrhein 1980). This protein is a key enzyme in the shikimic acid pathway for the biosynthesis of aromatic amino acids and other aromatic compounds and is ubiquitous in plants and microorganisms, including bacteria and fungi. EPSPS protein have been isolated and characterized from microorganisms, with some exhibiting tolerance to glyphosate and others sensitive to inhibition by glyphosate (Cajacob et al. 2004). EPSPS converts shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. Of the several known PEP dependent enzymatic reactions, EPSPS is the only enzyme that interacts with PEP as an enzyme–substrate complex (E.S3P) and not as a free enzyme (Padgett et al. 1991). In plants, as much as 20% of all fixed carbon flows through the shikimate pathway leading to the formation of aromatic amino acids such as tyrosine, phenylalanine and tryptophan, as well as tetrahydrofolate, ubiquinone and vitamins K and E (Boocock and Coggins 1983; Sost and Amrhein 1990; Haslam 1993; Franz et al. 1997; Gruys and Sikorski 1999).
Inhibition of EPSPS by glyphosate appears to be competitive with respect to PEP. Glyphosate forms a stable but non-covalent ternary complex with the enzyme and S3P (Ream et al. 1992; Marzabadi et al. 1996; McDowell et al. 1996). The manipulation of the bacterial EPSPS gene to reduce its affinity to glyphosate and transformation of this altered gene to plants is one of the most effective methods for production of glyphosate-tolerant plants (Wang et al. 2003). Glyphosate resistance was first reported in transgenic tobacco expressing the Pro101 to Ser substitution in the mutant version of EPSPS obtained from Salmonella typhimurium (Comai et al. 1985; Stalker et al. 1985). Another report of a mutation in the EPSPS of E. coli conferring glyphosate resistance in transgenic petunia involved the Gly96 to Ala substitution (Kishore et al. 1986; Padgette et al. 1991).
The native and modified CP4-EPSPS and GOX genes from Agrobacterium sp. (strain CP4) and Ochromobactrium anthropii (strain LBAA) respectively were used for glyphosate plant production separately and simultaneously. The modified CP4 gene product has a low affinity with glyphosate and GOX gene encodes the glyphosate oxidoreductase enzyme that degrades this herbicide. Several agronomic crops including corn, cotton, rapeseed, potato, soybean, sugar beet and tomato transformed with both CP4 and GOX genes together (Meilan et al. 2002).
The Roundup Ready canola was produced by transforming the CP4 and GOX genes together. The glyphosate tolerance of Roundup Ready canola has been demonstrated in field tests starting in 1992 and in additional field tests conducted throughout growing regions in United States, Canada, Europe and Australia. Roundup Ready canola was first planted commercially in 1996 in Canada. In the 2000 growing season, approximately 2.2 million hectares of Roundup Ready canola were planted in Canada and USA (James 2003).
In the present study, we mutated Gly96 to Ala and Ala183 to Thr in the Escherichia coli (k12) EPSPS gene using site directed mutagenesis (SDM), in order to investigate the effect of the simultaneous substitutions of the two amino acids on enzyme affinity towards glyphosate in transgenic rapeseed plants (B. napus L.).
Materials and methods
Enzymes and chemicals
All chemicals, culture media, plant growth regulators and antibiotics were purchased from Merck (Germany) at the highest purity available, unless stated otherwise. Restriction enzymes and other DNA-modifying enzymes were obtained from Roche Biochemical and Fermentas. The blunt end PCR cloning kit was from Roche Biochemical (Germany). Glyphosate (Roundup®) was supplied from Razor® Pro Herbicide 4467.
Bacterial strains, plasmids and plant materials
E. coli DH5α was used in all molecular biological experiments and Agrobacterium tumefaciens LBA4404 was used for plant transformation. Bacteria were grown in LB medium at appropriate temperatures (37°C for E. coli and 28°C for Agrobacterium tumefaciens) with shaking (200 rpm). Plasmid pUC18 (MBI, Fermentas) was used for routine cloning and sequencing and plasmid pBI121 (Novagen) was used as a binary plant expression vector. B. napus L. cultivar PF7045/91 was used as experimental plant material.
Recombinant DNA techniques
Unless otherwise stated, standard DNA methodologies were used (Sambrook and Russel 2001). Oligonucleotide synthesis and DNA sequencing reactions were carried out at MWG-Biotech AG (Ebersberg, Germany).
EPSPS gene amplification and cloning
Isolation of genomic DNA from E. coli (K12) was carried out according to Sambrook and Russel (2001). The EPSPS gene was amplified by PCR using specific primers P1 forward: 5′-CGGGATCCATGGAATCCCTGACGTTACAA-3′ and P2 reverse: 5′-GCGGATCCTCAGGCTGCCTGGCTAATC-3′ with a BamHI site at the 5′ end of each primer (underlined). Restriction enzyme analysis was carried out using BglII, HincII and TaqI. The authentic PCR product (1.3 kb) was cloned into the pUC18 plasmid and sequenced from both directions with M13 standard primers, using the dideoxy chain termination method.
Fragments amplified by internal primers were purified by agarose gel electrophoresis and using a DNA purification kit (Roche Diagnostic). Each pair of purified fragments was attached to each other in order to assemble the mutated gene. The attachment was carried out by the PCR based primer extension. For introducing both mutations into the EPSPS gene, the same strategy was applied. The EPSPS gene with the Gly96 to Ala mutation was used as a template and primers P5 and P6 were used to introduce a second mutation. With this strategy, the fifth intermediate fragment (500 bp with two mutations) was produced which was attached to fragment D, resulting in an EPSPS gene with two mutations.
Cloning and sequencing of the mutated genes
Mutated genes were cloned into the BamHI site of the vector pUC18. The presence of insert in recombinant plasmids was confirmed by restriction enzyme digestion and PCR. The plasmids with desired inserts were sequenced by the dideoxy chain termination method using standard M13 forward and reverse primers. Gene sequences were compared with other sequences stored in gene bank and aligned using Blast (www.ncbi.nlm.nih.gov) to confirm the fidelity of mutations as well as the absence of any undesired mutation.
Mutated genes were recovered as XbaI/SacI fragments from pUC18 derivatives and cloned into the XbaI/SacI site of binary plant transformation vector pBI121 downstream of cauliflower mosaic virus 35S promoter. The binary plasmids were individually transferred to A. tumefaciens strain LBA4404 using the freeze and thaw method (Höfgen and Willmitzer 1988).
Plant transformation and selection procedures
Seeds of B. napus cultivar PF 7045/91 were surface sterilized according to Moloney et al. (1989). Single colonies of Agrobacterium tumefaciens harboring the modified binary vector were grown overnight at 28°C in LB medium supplemented with 50 mg L−1 kanamycin.
Plant transformation was carried out as described by Moloney et al. (1989). After root formation on selection medium, the plantlets were transferred to potting mix supplemented with liquid fertilizer. The plants were grown in a misting chamber (80% relative humidity) for 2–3 weeks at 25°C, with a 16 h light and 8 h dark photoperiod and light intensity of 60–80 μEm−2 s−1. After 3 weeks, plants were transferred to the greenhouse and allowed to flower and set seed.
Bioassay with glyphosate challenging under greenhouse conditions
Putative transgenic plantlets were clonally propagated (via auxiliary buds culture) for glyphosate treatment replication and accuracy. In this experiment, we used 10 independent transgenic rapeseed lines. Fifteen clones from each line were used for five glyphosate doses in three replications. Then we used 150 transgenic plantlets for the glyphosate challenging experiment. These plantlets were transferred to soil under greenhouse conditions and tested for glyphosate tolerance by spraying them with the five doses (1, 2.5, 5, 7.5 and 10 mM) of herbicide Roundup® (active ingredient isopropylamine salt of glyphosate, 41%). Non-transformed plants were kept as control. One week after the first application, the second round of glyphosate spraying was carried out on surviving plants. For statistical analysis, t-test (p<0.01) was used for comparison of glyphosate resistance in the control and transgenic plants.
PCR screening of transformants
Genomic DNA was extracted from young leaves of green putative transgenic and non-transgenic (control) plants, by using the cetyl-trimethyl ammonium bromide (CTAB) method (Murray and Thompson 1980). Integration of the desired gene into the plant genome was confirmed by PCR of CaMV 35S Promoter/EPSPS and EPSPS/Nos terminator regions. Two pairs of specific primers were designed, the first pair contained a forward primer from the CaMV 35S promoter region (35SF: 5′-GGCGAACAGTTCATACAGAGTCT-3′) and a reverse primer from the mutated EPSPS region (ER: 5′-TCGCGTTGCGGCGTTACCGAGGA-3′), resulting in 800 bp amplification products. The second pair consisted of a forward primer from the transferred EPSPS region (P3: see above) and a reverse primer from the nopaline synthase (Nos) terminator region (NR: 5′-CGCGCGATAATTTATCCTAGT-3′) that amplifies a 1030 bp fragment. Each of the above PCR products was digested with BamHI for further confirmation. Digestion of the first PCR product produced 300 and 500 bp fragments and the second PCR product produced 1030 and 200 bp fragments.
Southern blot analysis
For Southern blot analysis, total genomic DNA was isolated from leaves of T0 glyphosate resistant and non-transformed control plants. Genomic DNA (15 μg) was digested with HindIII and separated on 0.7% (w/v) agarose gels, then transferred to nylon membranes. Prehybridization and hybridization were performed using a standard method (Sambrook and Russel 2001). A partial fragment (800 bp in size) was obtained from PCR amplification of the EPSPS gene using primers from the CaMV 35S promoter and the EPSPS gene and subjected to DIG DNA labeling (Roche Applied Science Gmbh, Germany) and used as a probe in hybridization experiments. It is worthy of mention that we analyzed six independent transgenic lines in three separate Southern blot experiments (in each experiment we tested only two transgenic lines and a control plant) but the results of one experiment has been shown.
To confirm the transcription of the mutated EPSPS gene, RT-PCR was performed according to the manufacturer's instructions (MBI Fermentas). Total RNA was extracted from leaves of glyphosate resistant and control rapeseed plants using an RNA isolation kit (MBI, Fermentas). First strand cDNA was generated using the E. coli EPSPS specific primer (5′-GCGGATCCTCAGGCTGCCTGGCTAATC-3′). PCR amplification of the 1300 bp fragment of the above gene was achieved by using the first strand synthesis as template with primers 5′-CGGGATCCATGGAATCCCTGACGTTACAA-3′ and 5′-GCGGATCCTCAGGCTGCCTGGCTAATC-3′ under the following conditions: 30 cycles of 94°C for 1 min, 63°C for 1 min and 72°C for 1 min, and a final extension at 72°C for 10 min.
Selection and assay for glyphosate tolerance
The survival frequencies of transgenic plants in 1, 2.5, 5, 7.5 and 10 mM glyphosate were 95, 93, 72, 64, and 51%, respectively.
Stable integration of the transgene
The above two PCR products from putative transgenic plants were further analyzed with restriction enzyme digestion. BamHI digestion of the 800 and 1030 bp fragments resulted in the formation of 500, 300 bp and 800, 230 bp fragments, respectively (Fig. 6B).
Southern blot analysis of transformants
The last 50 years have witnessed a major shift with in medicine and agriculture towards almost total dependence on toxic chemicals designed to control unwanted organisms (Malik et al. 1989). Glyphosate is a post-emergence, non-selective herbicide used in weed control programs around the world since its commercialization in 1974. Despite its widespread and long-term use, weeds have evolved limited resistance to glyphosate (Malik et al. 1989; Baerson et al. 2002).
The shikimate pathway, which occurs in plants and microorganisms, couples with the specificity of glyphosate as an inhibitor of EPSPS, and contributes in a large part to glyphosate's lack of toxicity to animals (Baerson et al. 2002). For the first time, Padgette and his coworkers (1991) reported the isolation of an E. coli B variant, containing a highly glyphosate-tolerant EPSPS. The further analysis of glyphosate- tolerant EPSPS revealed that the altered affinity of the EPSPS gene for glyphosate was the result of a single amino acid substitution of alanine for glycine at residue 96. Among conserved amino acids of this region, a very strong hydrogen bond formed between the nitrogen of glycine at residue 96 and the oxygen from the phosphate group of glyphosate (Schonbrunn 2001). The resistance to glyphosate in a glyphosate-tolerant strain of S. typhimurium has been reported to be the result of a single amino acid substitution of serine for glycine at position 101 (Stalker et al. 1985; Comai et al. 1985).
Alignments of the amino acid sequences of EPSPS from different prokaryotes and plants show that these two amino acids are located in a highly conserved region, but are absent from the active site of the enzyme.
Among conserved amino acids of this region, alanine at position 183 is an important residue for EPSPS–glyphosate interaction. Therefore, substitution for this amino acid can decrease the affinity of the enzyme for glyphosate. Based on what is known about the interaction between glyphosate and its target enzyme, many research groups are currently trying to develop glyphosate resistant transgenic plants.
There are several distinct strategies for engineering herbicide resistance. The most efficient method uses gene encoding naturally or artificially mutated protein. The logic behind this approach is to find a modified target protein that acts as a substitute for the native protein and is resistant to inhibition by the herbicide (Stalker et al. 1985).
Eichholtz and his coworkers (2001) have described a method for creating a modified gene encoding glyphosate-tolerant EPSPS. By using the M13 mutagenesis method they introduced mutations (Gly96Ala and Ala183Thr) into a 660 bp fragment, which replaced the wild type segment. These changes in the EPSPS increased its tolerance to the glyphosate herbicide, and it also exhibited lower Km values for phosphoenolpyruvate than other variant EPSPS enzymes.
In comparison to other previous techniques, the PCR based SDM is more simple, reliable and reproducible. In this technique, two PCR fragments are joined together by overlap extension to produce mutated genes.
Studies have also shown that it is possible to reduce the affinity of the enzyme for glyphosate by introducing changes in other amino acids as well (Baerson et al. 2002).
The active sites of EPSPS have been largely studied and identified. The data indicate that modifications in Lys22 and Lys340 result in enzyme inactivation (Padgette 1988). Thus, due to the importance of these residues in enzyme activity, it is impossible to change them. But in the present work two residues in a conserved motif of the EPSPS gene (Gly96 and Ala183) were chosen to be modified. Although this modification decreases the affinity of the enzyme for glyphosate, it does not lead to any changes in enzyme activity. The mutated EPSPS gene was used to transform Brassica napus. Transformation was mediated by Agrobacterium and the cut surfaces of cotyledoneary petioles containing the target cells. Results showed that this target is a vigorous source of new shoot material leading to very rapid shoot development. The origin of these shoots has been shown by Sharma (1987) to be cells located around the cut end of the petioles. This cut surface is an ideal target for Agrobacterium-mediated transformation as the cells undergoing organogenesis are those most readily accessed.
The value and success of Agrobacterium-mediated plant transformation is measured by the number of independent transformed plants expressing the gene of interest, per explants used. This variable can be a function of the genotype of the species to be transformed, the strain (virulence) of Agrobacterium, the selectable marker, regeneration capacity of the target cells and the accessibility of the bacterium to the regenerable cells. An additional less-frequently quantified variable is the amount of labor required to maintain cultures until transformed shoots are obtained.
We examined the expression of mutant E. coli EPSPS gene in transgenic rapeseed. Furthermore, we used the CaMV 35S promoter to ensure high levels of gene expression in all tissues.
Genome analysis (PCR and digestion) confirmed integration of the desired gene into rapeseed genome. Southern blot analysis of six transgenic lines provided additional evidence for T-DNA integration. Furthermore, it was shown that the tested lines carried a single copy of mutated bacterial EPSPS gene. Studies have shown that it is desirable to have single gene insertion in plants as multiple copies of T-DNA influence the expression of the introduced gene (Stam and Kooper 1997). Moreover, multiple copies of transferred gene are often associated with a reduction of transgene activity, affecting by gene position or suppressing gene expression (Bhalla and Smith 1998). Our finding (about single copy insertion into plant genome) is in agreement with the results of Wang et al. (2003), but is in disagreement with Moloney et al. (1989) that reported multiple copy insertion into rapeseed genome.
Special thanks to Dr. P. Shariati and Dr. Christopher Preston for critical reading of this manuscript. The National Institute for Genetic Engineering and Biotechnology (NIGEB), Iran, has financially supported this research project no. 155.