ES2359749T3 - ENZIMA DICAMBA MONOOXIGENASA MODIFIED AND PROCEDURE OF USE. - Google Patents

Abstract

A nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 1; b) a nucleic acid molecule comprising the sequence of SEQ ID NO: 2; and c) a nucleic acid molecule that encodes a polypeptide with at least 90% sequence identity with the polypeptide of SEQ ID No. 1, wherein the polypeptide has dicamba monooxygenase activity and comprises cysteine at a position corresponding to amino acid 112 of SEQ ID NO. 1.

Description

Background of the invention

1. Field of the invention

The invention relates in general to the field of biotechnology. More specifically, the invention relates to modified dicamba monooxygenase enzymes capable of conferring tolerance to the dicamba herbicide in transgenic organisms.

2. Description of the related technique

Procedures for the production of field crops, such as corn, soybeans and cotton, have changed dramatically over the past decade due to the introduction of traits such as insect resistance and herbicide tolerance through the use of engineering techniques. plant genetics These changes have led to greater productivity per hectare, reduced production costs, greater flexibility and efficiency in production regimes, decreased use of pesticides and, in the case of insect-resistant cotton, improved health in farmers. Transgenic crops have therefore achieved widespread adoption and are now grown on millions of acres worldwide. However, for GM crops to remain competitive in the market, they will now require new value-added features.

Although new features have appeared that improve the quantity and quality of agricultural and horticultural crops and will continue to appear at an increasing rate in the coming years, there is a demand for features that improve the procedures for the production of food, feed and other products. For example, although transgenic plants that are tolerant to treatments with glyphosate, bromoxynil, sulfonylureas and other herbicides herbicides are currently available, there are gaps in the spectrum of controlled weeds and treatment options that can be addressed through the development of tolerant crops. additional herbicides In addition, the appearance of weeds resistant to the herbicides indicated above, although it is generally located and contained in a variable way, imposes the need for supplementary or alternative control measures of weeds.

As it has been shown that transgenic herbicide tolerance is valuable in a commercial environment, plants tolerant to other herbicides are needed to avoid excessive dependence on any individual herbicide and to increase the options for handling difficulties in controlling weed species. Of particular interest is the development of herbicide tolerance for herbicides that are both environmentally friendly and highly effective in controlling weeds. Dicamba is one such example of an effective and environmentally friendly herbicide that farmers have used for more than 40 years. The dicamba is especially useful for the control of annual and perennial broadleaf weeds and various herbaceous weeds in corn, sorghum, small grains, grass, hay, forest pasture, sugarcane, asparagus, grass, and grass seed crops (Crop Protection Reference, 1995). Unfortunately, the dicamba can damage many commercial crops and dicotyledonous plants such as soybeans, cotton, peas, potatoes, sunflowers, and canola, which are particularly sensitive even at low levels of the herbicide. Despite this, the dicamba is highly effective in controlling weed growth and is therefore an important tool in agriculture.

Recently, a gene encoding the dicamba monooxygenase (BMD) of Pseudomonas maltophilia that confers tolerance to dicamba (US Patent No. 7,022,896) was isolated. BMD is involved in the conversion of the herbicide dicamba (3,6-dichloro-o-anisic acid) into a non-toxic 3,6-dichlorosalicylic acid. This gene is described in U.S. Patent No. 7,022,896 that provides tolerance to dicamba in plants expressing the DMO gene. However, the development of variants of this gene would be of great benefit. Such variants could potentially have an altered expression efficiency under specific environmental conditions. In this way, a variant that is optimized for a specific environment in which it is intended to be used could be selected, and can show particularly beneficial kinetic characteristics. The particular variant can show maximum efficiency at different temperatures or pH conditions, and therefore could be selected for a particular crop species depending on the intracellular conditions and / or the expected growth conditions of the crop.

Summary of the invention

In one aspect, the invention provides a nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 1; b) a nucleic acid sequence comprising the sequence of SEQ ID NO: 2; and c) a nucleic acid sequence encoding a polypeptide with a sequence identity of at least 90% with the polypeptide of SEQ ID No. 1, where the polypeptide has dicamba monooxygenase activity and comprises cysteine at a position corresponding to amino acid 112 of SEQ ID NO. 1. In other embodiments, a DNA vector is provided comprising a DMO nucleic acid described herein functionally linked to a promoter. The promoter can be functional in a plant cell. In certain embodiments, the nucleic acid sequence encoding the dicamba monooxygenase may be functionally linked to a transit peptide in chloroplasts.

In another aspect, the invention provides a polypeptide sequence with an identity of at least 90% with SEQ ID No. 1, where the polypeptide has dicamba monooxygenase activity and comprises cysteine at a position corresponding to amino acid 112 of SEQ ID No. 1.

In yet another aspect, the invention provides a host cell or tissue transformed with a nucleic acid encoding dicamba monooxygenase described herein. In certain embodiments, the host cell may be a plant cell. In other embodiments, the plant cell can be defined as a dicot plant cell or a cell of a monocot plant. In specific embodiments, the host cell is a plant cell of soy, cotton, corn or rapeseed. In other embodiments, a tissue culture is provided comprising a transgenic cell described herein.

In yet another aspect, the invention provides a transgenic plant, and the offspring thereof, transformed with a nucleic acid encoding dicamba monooxygenase described herein. In certain embodiments, the plant can be defined as a dicot or monocot plant. In specific embodiments, the plant is a soybean, cotton, corn or rapeseed plant.

In yet another aspect, the invention provides a method for producing a dicamba tolerant plant comprising introducing into the plant a transformation construct provided herein by stably transforming one or more plant cells and regenerating the one or more cells in a plant. Dicamba tolerant. In a preferred embodiment, the method further comprises crossing a parental plant with itself or a second plant, where the parental plant and / or the second plant comprises the transformation construction and the dicamba tolerant plant inherits the transformation construction of the parental plant and / or the second floor.

In yet another aspect, the invention provides a process for producing food or feed comprising: a) obtaining a plant of the invention provided herein or a part thereof; and b) prepare food

or I think from the plant or part of it. In one embodiment of the invention, the part of the plant is a seed. In certain additional embodiments, the food or feed is oil, flour, protein, grain, starch or protein. In other embodiments, the feed comprises a forage or grass plant such as hay. The invention also provides methods for producing fibers, pharmaceutical agents, nutraceutical agents, and industrial chemical compounds, including biofuels, as well as any other product derived from a plant provided herein.

In yet another aspect, the invention provides a method for controlling weed growth in a crop growth environment comprising a plant of the invention provided herein.

or a seed thereof, which comprises applying to the crop growth environment an amount of the effective dicamba herbicide to control the growth of weeds. In certain embodiments of the invention, the dicamba herbicide can be applied over the upper part of the crop growth environment. In specific embodiments, the amount of the dicamba herbicide does not damage the plant of the invention or the seed thereof and damages a plant of the same genotype as the plant that lacks a DMO coding nucleic acid provided by the invention.

In yet another embodiment of the invention, there is provided a plant comprising a DMO coding nucleic acid provided by the invention and at least one other transgenic coding sequence including, for example, at least two, three, four, five or more of said sequences. encoders In particular embodiments, the plants comprise a transgene that confers one or more additional beneficial traits, such as herbicide tolerance.

or pests / insects. For example, tolerance can be provided to one or more herbicides in addition to dicamba, as well as another beneficial trait, as described below in this document. The invention, therefore, provides plants comprising a DMO coding nucleic acid of the present invention "stacked" in any desired combination with additional transgenic features.

Brief description of the figures

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by reference to one or more of these graphs in combination with the detailed description of the specific embodiments presented herein.

FIG. 1. Cassette scheme used for the genetic design of the dicamba monooxygenase (DMOc) gene for expression in higher plants using the FLt36 promoter of the peanut chlorotic striatum virus, the translation enhancer sequence of tobacco engraving virus ( TEV leader), and a terminator region of the small subunit gene of the Pea Rubisco. Another genetically engineered version of the DMOc gene that was prepared contained a region encoding a transit peptide of the small subunit gene of the Pea Rubisco for localization in the chloroplast of the BMD between the translation enhancing region of the TEV and the coding region of DMOc.

FIG. 2. DNA, RNA and protein transfer panels that demonstrate the presence and expression of the genetically modified BMD gene in T1 generation transgenic tobacco plants. Lanes Q to V represent the DNA, mRNA and BMD samples extracted from various tobacco plants of the T1 generation. Extracts from a non-transgenic tobacco plant are represented in the WT lane while the Ox lane shows

5 a restriction digestion product of the cloned DMO gene construct (upper panel) and the MO37 kDa DMO enzyme super-produced in E. coli (lower panel). The large subunit of 55 kDa from Rubisco was detected in protein transfer by adding antibodies against Rubisco to DMO antibodies and Rubisco detection served as an internal standard to compare total protein loads in each lane. Equal amounts of RNA were loaded into each lane that were measured by staining with ethidium bromide of a duplicate gel. The arrows indicate the location of the DNA, mRNA or protein band of BMD.

FIG. 3. Effect of the treatment with dicamba at 2.2 kg / ha in two T1 tobacco plants, one containing the genetically modified DMOc gene that lacks a transit peptide coding sequence in chloroplasts (right) and one that lacks of the DMOc gene (second from the right). The transgenic plant on the right shows little, if any, damage due to dicamba treatment. The two plants on the left were not treated with

15 dicamba and represent a non-transgenic plant (left) and a transgenic plant that contains the DMOc gene (second from the left).

FIG. 4. DCSA training versus time by DMOw.

FIG. 5. Determination of the optimum test pH for DMOw.

FIG. 6. Determination of the optimum test temperature for DMOw.

FIG. 7. Determination of the optimum pH for BMDD.

FIG. 8. Determination of the optimum temperature for BMDD.

FIG. 9. Summary of the optimum temperature and pH conditions for DMOc and DMOw.

FIG. 10. Kinetics in steady state for DMOw.

FIG. 11. Kinetics in steady state for DMOc.

25 FIG. 12. Effects of pre-incubation of BMDc for 45 minutes at 30 ° C in 50 mM TRIS pH 7.5 and 100 mM KPi pH 7.0.

FIG. 13. DMOc assays with the enzyme settling for one week and stored at 4 ° C in TRIS buffer (the two tests on the left; tests before and after storage, respectively) and KPi buffer (the two tests on the right; tests before and after of storage, respectively).

FIG. 14. Construction of the genetically engineered dicamba monooxygenase gene for homologous recombination and expression in tobacco chloroplasts.

FIG. 15. Demonstration of the homoplastic state of the chloroplast genomes of transgenic tobacco lines transformed with a DMO gene designed for homologous recombination and expression in tobacco chloroplasts. The panel on the left shows a construction for the integration of BMD into the chloroplast by homologous recombination (as shown in FIG. 14). The bar above the left addressing sequence refers to an amplified DNA fragment for the repair of the hybridization probe labeled with digoxigenin. The panels on the right show DNA transfers: Lane 1 contains size markers. Lane 2 contains DNA from non-transgenic tobacco plants. Lanes 3-11 contain DNA isolated from transgenic plants shortly after the first round of selection and regeneration in the presence of spectinomycin (upper panel) and after several rounds of selection and regeneration when apparent homoplasticity of the chloroplast genome (lower panel) was obtained. . DNA for DNA transfer analysis was isolated from transgenic and non-transgenic plants and was digested with restriction enzymes with BamH I before electrophoretic separation and probing of the transferred DNA with a labeled DNA fragment complementary to the " left addressing sequence "of the chloroplast genome transformation vector (ie, the probe

45 of hybridization labeled with digoxigenin). The 5.6 kb DNA band corresponds to the chloroplast DNA fragment containing the DMO gene and the 3.3 kb band corresponds to the homologous native chloroplast band lacking an inserted DMO gene construct.

FIG. 16. T1 generation homoplastic transgenic tobacco plants that contain a dicamba gene encoded in the chloroplast treated with dicamba at a level of 28 kg / ha (plants 1-2 and plants 3-4 were obtained from two independently transformed R0 plants).

FIG. 17. Expression of BMD and sensitivity and resistance to dicamba treatment in non-transgenic and transgenic tobacco plants that contain the BMD gene in the chloroplast genome. The protein transfer was probed with antibodies against BMD: Lane 1 contains purified BMD. Lane 2 is white and lane 3 contains protein extracts from non-transgenic tobacco plants. Lanes 4 and 8 contain proteins isolated from "false-positive" tobacco plants that exhibit antibiotic resistance during spectinomycin selection, but lack the intact BMD gene. Lanes 5-7 contain extracts of transgenic plants that express BMD encoded by the BMD gene integrated into the chloroplast genome. S = dicamba sensitive plants at 0.56 kg / ha; R = dicamba resistant plants at 5.6 kg / ha. Almost equal amounts of the extracts were loaded in lanes 4-8 measured by the amount of protein from the large subunit of Rubisco detected with anti-Rubisco antibodies although significantly more protein was loaded from non-transgenic plants in lane 3. The arrow indicates the position of the BMD protein.

FIG. 18. Comparison of a part of the wild-type BMD polypeptide sequence with conserved regions of other iron-sulfur oxygenases showing that BMD is unique, with low identity with known enzymes, but W112 (arrow) is conserved in other oxygenases of iron-sulfur and is joined by two conserved domains, Rieske and Fe no heme (SEQ ID NO: 4-23).

Detailed description of the invention

The invention provides variants of dicamba monooxygenase (BMD) comprising a cysteine in a position corresponding to position 112 of the BMD shown in SEQ ID NO: 1, referred to herein as DMOc. It was shown that DMOc produces a high level of tolerance to the herbicide dicamba when expressed in transgenic plants. The results were surprising since the altered position of the amino acid is highly conserved in other iron-sulfur oxygenases. Of 78 iron-sulfur oxygenase sequences analyzed from 45 species, the 52 oxygenase sequences with at least 15% identity had a W corresponding to the position of amino acid 112 of SEQ ID No. 1, despite the total identity higher than only 38%. This position is also linked by two conserved functional domains (FIG. 18). The high level BMD of herbicide tolerance was therefore unexpected.

The analysis of Michaelis-Menten parameters for DMOc in relation to the unaltered sequence (DMOw; US Patent No. 7,022,896) revealed that enzymes were different in terms of catalytic efficiencies: DMOc was five times more effective than DMOw and DMOc seemed to have a higher renewal rate and a stronger bond to the substrate. In addition, DMOc worked better at lower pH conditions and higher temperature relative to the native enzyme. These results indicated the potential to select BMD variants for use in a particular transgenic plant based on expected conditions of use, such as crop growth conditions. One aspect of the invention, therefore, involves identifying a candidate crop growth environment for at least a first crop species, and identifying the most suitable BMD enzyme for that environment based on kinetics, for example DMOc and DMOw . For example, a person skilled in the art can, in particular embodiments, select a DMOc coding sequence for use in plants that have lower pH conditions in plant and / or in the case of culture environments with higher temperatures relative to others. plant species or cultivation environments, respectively. The dicamba can be applied by incorporation into the soil (prior incorporation to the plant); pulverizing the earth (pre-outcrop); and on the upper part of the plants (post-outcrop treatment), although the levels of tolerance to dicamba may differ at various times during plant growth.

As indicated above, tolerance to extremely high levels of the dicamba herbicide was obtained in transgenic plants expressing DMOc. In tobacco, for example, which is normally sensitive even at very low levels of dicamba, transgenic plants that expressed BMDs were created that were tolerant to dicamba treatment at 5.6 kg / ha or more, for example, 10-20 times more than the rates of application in the field normally recommended for the control of broadleaf weeds. When the DMOc gene was inserted into the chloroplast genome of tobacco plants, a tolerance to dicamba was obtained at least 28 kg / ha. Transgenic soybean, tomato and Arabidopsis thaliana plants were also created that harbored a DMOc gene encoded in the nucleus and were found to be tolerant at high levels of dicamba. For example, the insertion of DMOc in the nuclear genome of soybean plants produced tolerance to treatments of 2.8 kg / ha, thus allowing the use of dicamba to control weeds in plant fields expressing DMOc.

Therefore it has been shown that DMOc is effective in conferring tolerance to dicamba without the need for additional coding sequences such as P. maltophilia, strain DI-6, ferredoxin or reductase. The modified BMD gene was stably inherited as a Mendelian gene with no apparent loss of penetrance or expression. Although somewhat stronger expression was obtained with a chloroplast transit peptide, transgenic plants with a DMO transgene lacking the transit peptide coding sequence also showed a high level of tolerance to post-outcrops dicamba.

A. Nucleic acids and recombinant constructs

1. Dicamba monooxygenase (BMD)

In one embodiment of the present invention, DNA constructs comprising a nucleic acid encoding a dicamba monooxygenase polypeptide comprising a cysteine at a position corresponding to position 112 of SEQ ID NO: 1 are provided. Exemplary BMD coding sequence as SEQ ID NO. 2. This sequence, in addition to comprising cysteine at position 112 of SEQ ID NO. 1, included the addition of a GCC codon (alanine) after the ATG start codon to add a site of Nco I restriction in relation to the native coding sequence and to facilitate cloning. The polypeptide of SEQ ID No. 1, therefore, also included an additional Ala residue immediately after the Met encoded by the start codon. The transit peptide sequence was cleaved from the plasmid with Bgl II and EcoR I and then cloned into the BamH I and EcoR I sites of the pBluescript II KS + vector. This construct was used as a template in a PCR reaction with primers that added Nco I restriction sites at each end of the transit peptide coding sequence. Digestion of the PCR product with Nco I allowed the insertion of the coding sequence of the transit peptide into the ATG start codon site of the modified DMO gene.

Thus, in one embodiment of the invention, sequences encoding the polypeptide of SEQ ID No. 1 are provided including, but not limited to, SEQ ID No. 2. As is well known in the art, homologous sequences can be easily prepared and used. and derivatives of these sequences. For example, a nucleic acid encoding a DMO polypeptide having a sequence identity of at least 90% can be used with the DMOc polypeptide of SEQ ID NO: 1, including an identity of at least about 92%, 94%, 95%, 96%, 97%, 98%, 99% or more with said sequences. A nucleic acid that shows a sequence identity of at least 90% can also be used with the nucleic acid sequence provided as SEQ ID NO: 2, including an identity of at least about 92%, 94%, 95%, 96% , 97%, 98%, 99% or more with said sequence and encoding a BMD comprising a cysteine at position 112. In one embodiment, the sequence identity is determined using the Sequence Analysis software package of the GCG Wisconsin Package ( Accelrys, San Diego, CA), MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis. 53715) with default parameters. Said software matches similar sequences by assigning degrees of similarity or identity.

A polynucleotide molecule expressing a DMO polypeptide can be obtained by techniques well known in the art in view of the present description. Therefore, BMD variants provided in this document can be prepared that have the ability to degrade the dicamba and be tested for activity according to the methodology described in this document. Such sequences can also be identified, for example, from suitable organisms including bacteria that degrade the dicamba (US Patent No. 5,445,962; Krueger et al., 1989; Cork and Krueger, 1991; Cork and Khalil, 1995). One means of isolating a cloned BMD sequence is by nucleic acid hybridization, for example, with a library constructed from the source organism, or by RT-PCR using mRNA from the source organism and primers based on the BMD described. The invention, therefore, encompasses the use of nucleic acids that hybridize under stringent conditions with a BMD coding sequence described herein. One skilled in the art understands that conditions can become less stringent by increasing the salt concentration and lowering the temperature. Therefore, hybridization conditions can be easily manipulated, and therefore will generally be the procedure of choice depending on the desired results. An example of high stringency conditions is 5X SSC, 50% formamide and 42 ° C. By washing under these conditions, for example, for 10 minutes, those sequences that do not hybridize with a particular target sequence under these conditions can be removed. An embodiment of the invention, therefore, comprises the use of a BMD-encoding nucleic acid that is defined as a hybridizer under 5X SSC wash conditions, 50% formamide and 42 ° C for 10 minutes with a nucleic acid according to SEC. ID No. 2.

Variants can also be chemically synthesized using the BMD polynucleotide sequences described herein in accordance with techniques well known in the art. For example, DNA sequences can be synthesized by phosphoramidite chemistry in an automated DNA synthesizer. Chemical synthesis has several advantages. In particular, chemical synthesis is desirable because the codons preferred by the host in which the DNA sequence will be expressed to optimize expression can be used. An example of such a sequence that was optimized for dichotyledonous expression using the codons of Arabidopsis thaliana is the BMD sequence shown in SEQ ID NO. 3. The polypeptide, which has been predicted to have an Ala, Thr, Cys in Positions 2, 3, 112, respectively, are given in SEQ ID No. 1. The remaining Ala in position 2 was added relative to the wild type BMD as a result of the addition of a codon for alanine immediately after ATG start codon to simplify vector construction, as explained below.

Not all codons have to be altered to obtain an improved expression, but preferably at least the codons rarely used in the host are changed to the codons preferred by the host, for example, codons most frequently used in the host and generally translated more easily than rare non-preferred codons. High levels of expression can be obtained by changing more than about 50%, much more preferably at least about 80%, of the codons not delivered by the codons preferred by the host. The codon preferences of many host cells are known (PCT documents WO 97/31115; PCT WO 97/11086; EP 646643; EP 553494; and U.S. Patent Nos. 5,689,052; 5,567,862; 5,567,600; 5,552 .299 and 5,017,692). Codon preferences of other host cells can be deduced by methods known in the art. In addition, using chemical synthesis, the sequence of the DNA molecule or its encoded protein can be easily changed to, for example, optimize its expression (for example, eliminate secondary mRNA structures that interfere with transcription or translation), add sites of unique restriction at appropriate points, and eliminate protease cleavage sites.

Modifications and changes in the polypeptide sequence of a protein such as the BMD sequences provided herein can be made while retaining enzymatic activity. The following is an analysis based on changing the amino acids of a protein to create an equivalent polypeptide, or even a modified, improved and corresponding coding sequences. In particular embodiments of the invention, BMD sequences can be altered in this way and used in the methods of the invention. Amino acid changes can be achieved by changing the codons of the DNA sequence.

It is known, for example, that certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as binding sites in the substrate molecules. As it is the interactive capacity and the nature of a protein that defines the biological functional activity of that protein, certain substitutions can be made in the amino acid sequence of a protein sequence and, of course, the underlying DNA coding sequence, and nevertheless Get a protein with similar properties. Therefore it is contemplated that various changes can be made in the BMD peptide sequences described herein and the corresponding DNA coding sequences without appreciable loss of their usefulness or biological activity.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic index of amino acids to confer interactive biological function in a protein is generally understood in the art (Kyte et al., 1982). It is appreciated that the relative hydropathic character of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. . Each amino acid has a hydropathic index assigned based on its hydrophobicity and charge characteristics (Kyte et al., 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is known in the art that amino acids can be substituted for other amino acids that have a similar hydropathic index or value and still produce a protein with similar biological activity, that is, still obtain a functionally equivalent biological protein. In making such changes, the substitution of amino acids whose hydropathic indices are in ± 2, those in ± 1 are particularly preferred, and those in ± 0.5 are even more particularly preferred. Therefore, the observation that a BMD that had a substitution of a tryptophan at position 112 with cysteine had biological activity and produced tolerant plants at high levels of dicamba, was surprising given the different hydropathic indices between native and altered amino acids and by both those skilled in the art would not use it to create functional variants according to the prior art.

It is also understood in the art that the substitution of similar amino acids can be done efficiently based on hydrophilicity. US Patent 4,554,101 indicates that the higher average local hydrophilicity of a protein, controlled by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in US Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); Alanine (0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another that has a similar hydrophilicity value and still obtain a biologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. Exemplary substitutions that take these characteristics and various previous characteristics into consideration are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Again, the activity of DMOc was surprising given the hydrophilicity values so different between the altered and native amino acids and those skilled in the art would not use this substitution to create functional variants according to the prior art.

The modification of a BMD sequence according to the invention can be guided by the consideration of the domains conserved within the enzyme. For example, it is shown below that the DMO enzyme contains functional domains such as a group of Rieske iron-sulfur and a binding site for free iron (see FIG. 18, for example). This information, combined with knowledge in the art regarding functional domains and protein modification, can generally be used, therefore, to generate modified BMD enzymes while maintaining enzymatic activity within the scope of the invention (see, for example, Mason and Cammack, 1992; Jiang et al., 1996).

2. Transformation constructions

A DMO coding polynucleotide used in accordance with the invention will typically be introduced into a cell as a construct comprising expression control elements necessary for effective expression. Methods for functionally linking expression control elements to coding sequences are well known in the art (Maniatis et al., 1982; Sambrook et al., 1989). Expression control sequences are DNA sequences involved in any way in the control of transcription. Suitable expression control sequences and methods for using them are well known in the art. A particular promoter can be used, with or without enhancer elements, a 5 'untranslated region, transit peptides or signal for the targeting of a protein or RNA product to a plant organelle, particularly to a chloroplast and 3' untranslated regions such as polyadenylation sites. A person skilled in the art will know that various enhancers, promoters, introns, transit peptides, signaling targeting sequences, and 5 'and 3' untranslated regions (UTR) are useful in the design of expression vectors in effective plants, such as those disclosed, for example, in U.S. Patent Application Publication 2003/01403641.

Promoters suitable for the present use and other uses are well known in the art. Examples describing such promoters include U.S. Patent 6,437,217 (RS81 Corn Promoter), U.S. Patent 5,641,876 (Rice Actin Promoter), U.S. Patent 6,426,446 (Promoter RS324 corn), U.S. Patent 6,429,362 (PR-1 Corn Promoter), U.S. Patent

6,232,526 (A3 corn promoter), United States patent 6,177,611 (corn constitutive promoters), United States patents 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), United States Patent 6,433,252 (L3 oleosin promoter of corn), United States Patent 6,429,357 (rice actin 2 promoter as well as rice actin 2 intron), patent United States 5,837,848 (specific root promoter), United States patent 6,294,714 (light inducible promoters), United States patent 6,140,078 (salt inducible promoters), United States patent 6,252 .138 (pathogen-inducible promoters), U.S. 6,175,060 (phosphorus deficiency-inducible), U.S. 6,635,806 (the gamma-coixin promoter), and patent application United States Serial No. 09 / 757,089 (corn chloroplast aldolase promoter). Additional promoters that may find use are a nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried by tumor inducing plasmids of Agrobacterium tumefaciens), promoters of caulimovirus such as the 19S promoter of cauliflower mosaic virus (CaMV) (Lawton et al., 1987), the 35S promoter of CaMV (Odell et al., 1985), the 35S promoter of the fig mosaic virus (Walker and col., 1987), the sucrose synthase promoter (Yang et al., 1990), the R gene complex promoter (Chandler et al., 1989), and the chlorophyll-binding protein gene promoter a / b, etc. The CaMV35S promoter (US Pat. Nos. 5,322,938; 5,352,605; 5,359,142; and 5,530,196), an FMV35S promoter (United States patents 6,051,753; can be particularly beneficial for use with the present invention. 5,378,619), PC1SV (e.g., U.S. Patent 5,850,019, and SEQ ID No. 24), and AGRtu.nos promoters (access to GenBank V00087; Depicker et al., 1982; Bevan et al., 1983 ).

Benefit for the expression of heterologous genes can be obtained by the use of a sequence encoding a transit peptide. Transit peptides generally refer to peptide molecules that when bound to a protein of interest direct the protein to a particular tissue, cell, subcellular location, or cellular organelle. Examples include, but are not limited to, chloroplast transit peptides, nuclear targeting signals, and vacuolar signals. A chloroplast transit peptide is of particular utility in the present invention to direct the expression of a DMO enzyme to chloroplasts. It is expected that the function of BMD will be facilitated by endogenous reductases and ferredoxins found in plant cells to degrade the dicamba. Vegetable chloroplasts are particularly rich in reductases and ferredoxins. Accordingly, in a preferred embodiment for the production of dicamba-tolerant transgenic plants, a sequence encoding a peptide that will direct the degrading oxygenase of dicamba into the chloroplasts can be used. Alternatively or additionally, endogenous reductase and / or ferredoxin can also be expressed in a cell.

The DNA encoding a chloroplast targeting sequence can preferably be placed upstream (5 ') of a BMD coding sequence, but can also be placed downstream (3') of the coding sequence, or both upstream and downstream of the sequence. coding sequence A particular chloroplast transit peptide (CTP) can be designed to fuse to the N-terminus of proteins that have to be directed into the plant chloroplast. Many proteins located in the chloroplasts are expressed from nuclear genes in the form of precursors and are directed to the chloroplast by a CTP that is removed during the import stages. Examples of chloroplastic proteins include the small subunit (RbcS2) of ribulose-1.5, carboxylase bisphosphate, ferredoxin, ferredoxin oxidoreductase, protein I and protein II of the light uptake complex, and thioredoxin F. demonstrated in vivo and in vitro that non-chloroplast proteins can be directed to the chloroplast through the use of protein fusions with a CTP and that a CTP is sufficient to direct a protein to the chloroplast. For example, it has been shown that the incorporation of a suitable chloroplast transit peptide such as the EPSPS CTP from Arabidopsis thaliana (Klee et al., 1987), and the EPSPS CTP from Petunia hybrida (della-Cioppa et al. , 1986) directs heterologous sequences of the EPSPS protein to chloroplasts in transgenic plants. Other exemplary chloroplast targeting sequences include the signal sequence of cab-m7 corn (Becker et al., 1992; PCT document WO 97/41228) and the signal sequence of glutathione reductase of the pea (Creissen et al., 1991; document PCT WO 97/41228). In the present invention, AtRbcS4 (CTP1; U.S. Patent 5,728,925), AtShkG (CTP2; Klee et al., 1987), AtShkGZm (synthetic CTP2; see SEQ ID No. 14 of WO04009761), and PsRbcS (Coruzzi et al., 1984) may be of particular benefit, for example, with respect to the expression of a DMO polypeptide.

A 5 'UTR that functions as a translational leader sequence is a genetic element of DNA located between the promoter sequence of a gene and the coding sequence. The leading translation sequence is present in the fully processed mRNA upstream of the translation initiation sequence. The leading translation sequence may affect the processing of the primary transcript in mRNA, mRNA stability or translation efficiency. Examples of translation leading sequences include corn and petunia heat shock protein leaders (U.S. Patent No. 5,362,865), plant virus cover protein leaders, plant rubisco leaders among others (Turner and Foster, 1995). In the present invention, the 5 'UTRs that may, in particular, find benefit, are GmHsp (U.S. Patent 5,659,122), PhDnaK (U.S. Patent 5,362,865), AtAnt1, TEV (Carrington and Freed, 1990 ), and AGRtunos (access to GenBank V00087; Bevan et al., 1983).

The 3 'untranslated sequence, the 3' transcription termination region, or the polyadenylation region indicates a DNA molecule attached to and located downstream of the coding region of a gene and includes polynucleotides that provide the polyadenylation signal and others. regulatory signals capable of affecting transcription, mRNA processing or gene expression. The polyadenylation signal works in plants causing the addition of polyadenylate nucleotides at the 3 'end of the mRNA precursor. The polyadenylation sequence can be obtained from the natural gene, from a variety of plant genes, or from DNA-T genes. An example of a 3 'transcription termination region is the 3' region of the nopaline synthase (nos 3 '; Fraley et al., 1983). The use of different 3 'untranslated regions has been described (Ingelbrecht et al., 1989). Polyadenylation molecules of a RbcS2 gene from Pisum sativum (Ps.RbcS2-E9; Coruzzi et al., 1984) and AGRtu.nos (Rojiyaa et al., 1987, access to Genbank E01312) in particular may be beneficial for use with the invention

An expression unit of the BMD-encoding polynucleotide molecule can be attached to a second polynucleotide molecule in an expression unit that contains genetic elements for a selectable / titratable marker or for a gene that confers a desired trait. Genes commonly used to explore supposedly transformed cells include -glucuronidase (GUS), -galactosidase, luciferase, and chloramphenicol acetyltransferase (Jefferson, 1987; Teeri et al., 1989; Koncz et al., 1987; De Block et al., 1984), the green fluorescent protein (GFP) (Chalfie et al., 1994; Haseloff et al., 1995; and PCT application WO 97/41228).

The second polynucleotide molecule can include, but is not limited to, a gene that acts as a selection marker. An additional second gene or gene may provide a desirable characteristic associated with plant morphology, physiology, growth and development, production, nutritional enhancement, resistance to diseases or pests, or tolerance to chemical environments or compounds and may include genetic elements comprising resistance to herbicides (U.S. Patents 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; 5,463,175), production Increased (U.S. Patent RE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; 5,716,837) , insect control (U.S. Patents 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,468,523; 6,326,351; 6,313 .378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 5,959,091; 5,942,664 ; 5,942,658, 5,880,275; 5,763 .245; 5,763,241), resistance to fungal diseases (US Patents 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; 6,506,962), virus resistance (U.S. Patents 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; 5,304,730), nematode resistance (U.S. Patent 6,228,992) , resistance to bacterial diseases (U.S. Patent 5,516,671), plant growth and development (U.S. Patent 6,723,897; 6,518,488), starch production (U.S. Patent 6,538,181; 6,538,179 ; 6,538,178; 5,750,876; 6,476,295), modified oil production (United States patents 6,444,876; 6,426,447; 6,380,462), high oil production (United States patents 6,495,739; 5,608 .149; 6,483,008; 6,476,295), modified fatty acid content (US Patents 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461; 6,459,018), high protein production (U.S. Patent 6,380,466), fruit ripening (U.S. Patent 5,512,466), animal nutrition and Enhanced Human (US Patents 6,723,837; 6,653,530; 6,541,259; 5,985,605; 6,171,640), biopolymers (U.S. Patent RE37,543; 6,228,623; 5,958,745 and U.S. Patent Publication No. US20030028917), environmental stress resistance (U.S. Patent 6,072,103), pharmaceutical peptides and secretable peptides (U.S. Patents 6,812,379; 6,774,283; 6,140,075; 6,080,560), improved processing features (U.S. Patent 6,476,295), improved digestibility (U.S. Patent 6,531,648), low raffinose content (United States patent 6,166,292), production of industrial enzymes (United States patent 5,543,576), improved aroma (United States patent 6,011,199), nitrogen fixation (United States patent 5,229. 114), hybrid seed production (U.S. Patent 5,689,041), fiber production (U.S. Patents 6,576,818; 6,271,443; 5,981,834; 5,869,720) and biofuel production (U.S. patent 5,998,700). Any of these or other genetic elements, methods, and transgenes can be used with the invention as those skilled in the art will appreciate in view of the present disclosure.

An expression unit can be provided in the form of T-DNA between the regions of the right end (RB) and the left end (LB) of a first plasmid together with a second plasmid that carries the transfer and integration functions of the T-DNA in Agrobacterium. The constructs may also contain plasmid structural DNA segments that provide the replication function and antibiotic selection in bacterial cells, for example, an Escherichia coli replication origin such as ori322, a wide host range replication origin such as oriV or oriRi, and a coding region for a selection marker such as Spec / Strp encoding the aminoglycoside adenyltransferase (aadA) of Tn7 that confers resistance to spectinomycin or streptomycin, or a selection marker gene for gentamicin (Gm, Gent). For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, or LBA4404. However, other strains known to those skilled in the art of plant transformation may function in the present invention.

3. Preparation of transgenic cells

The transformation of plant cells can be achieved by any of the techniques known in the art for the introduction of transgenes into cells (see, for example, Miki et al., 1993). Examples of such procedures are believed to include almost any procedure by which DNA can be introduced into a cell. The procedures that have been described include electroporation as illustrated in US Patent No. 5,384,253; bombardment with microprojectiles as illustrated in U.S. Patent Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; Agrobacterium-mediated transformation as illustrated in U.S. Patent Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301; and transformation by protoplasts as illustrated in U.S. Patent No. 5,508,184. Through the application of techniques such as these, the cells of almost any plant species can be stably transformed and selected according to the invention and these cells developed in transgenic plants.

The most widely used procedure to introduce an expression vector into plants is based on the Agrobacterium natural transformation system (for example, Horsch et al., 1985). A. tumefaciens and A. rhizogenes are pathogenic earth bacteria for plants that genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for the genetic transformation of the plant (eg, Kado, 1991). Descriptions of Agrobacterium vector systems and procedures for Agrobacterium-mediated gene transfer are provided in numerous references, including Miki et al., Supra, Moloney et al., 1989, and U.S. Patent Nos. 4,940,838 and

5,464,763. Other bacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium that interact with plants naturally can be modified to mediate gene transfer to several different plants. These symbiotic bacteria associated with plants can become competent for gene transfer by the acquisition of both a deactivated Ti plasmid and a suitable binary vector (Brothers et al., 2005).

B. Tissue cultures and plant regeneration

The regeneration of a plant cell transformed into a fertile plant can be achieved by first cultivating an explant in a budding medium and then in a rooting medium. Sometimes, an explant can be grown in a callus medium before transferring to a budding medium. A variety of means and transfer needs can be applied and optimized for each plant system for plant transformation and recovery of transgenic plants. Accordingly, said culture media and conditions can be modified or substituted with nutritionally equivalent components, or similar procedures for the selection and recovery of transgenic cases.

Nutrient media are prepared in the form of a liquid, but these can be solidified by adding the liquid to materials capable of providing a solid support. Agar is the most commonly used for this purpose. Bactoagar, Hazelton agar, Gelrite, and Gelgro are specific types of solid support that are suitable for plant cell growth in tissue cultures. Some cell types will grow and divide into liquid suspension or solid media or both media.

Receiving cell targets include, but are not limited to, meristematic cells, corns, immature embryos, and gamma cells such as sperm from pollen microspores and ovules. Any cell from which a fertile transgenic plant can be regenerated can be used in certain embodiments. For example, immature embryos can be transformed followed by callus selection and onset and subsequent regeneration of fertile transgenic plants. Direct transformation of immature embryos obviates the need for long-term development of recipient cell cultures. Meristematic cells can also be used (i.e., plant cells with a capacity for continuous cell division and characterized by an undifferentiated cytological aspect, normally joined at plant growth points or tissues such as root tips, stem apices, lateral shoots, etc.) as a recipient plant cell. Because of its undifferentiated growth and its capacity for organ differentiation and totipotence, a complete transformed plant could be recovered from a single transformed meristematic cell.

Somatic cells are of various types. Embryogenic cells are an example of somatic cells that can be induced to regenerate a plant through embryo formation. Non-embryogenic cells are those that typically do not respond in that way.

Certain techniques that enrich the recipient cells within a cell population can be used. For example, the development of type II corns, followed by manual selection and culture of the disintegrable embryogenic tissue generally causes enrichment of the recipient cells for use in, for example, microprojectile transformation.

In certain embodiments, the recipient cells are selected after their growth in culture. Cultured cells can be grown on solid supports or in the form of liquid suspensions. In any case, nutrients can be provided to the cells in the form of medium, and environmental conditions can be controlled. There are many types of tissue culture media composed of amino acids, salts, sugars, growth regulators and vitamins. Most of the media employed in the practice of the invention will have some similar components, although the media may differ in the composition and proportions of the ingredients according to known tissue culture practices. For example, various cell types grow in more than one type of medium, but will show different growth rates and different morphologies, depending on the culture medium. In some media, cells survive but do not divide. The medium composition is also frequently optimized based on the species or cell type selected.

Various types of media suitable for plant cell culture have been previously described. Examples of these means include, but are not limited to, the N6 medium described by Chu et al. (1975) and the MS medium (Murashige and Skoog, 1962). In some embodiments, it may be preferable to use a medium with a somewhat lower ammonia / nitrate ratio, such as N6, to promote the generation of recipient cells by keeping the cells in a pro-embryonic state capable of supporting the divisions. Means for woody plants (WPM) can also be used (Lloyd and McCown, 1981).

The maintenance procedure of cell cultures can contribute to their usefulness as sources of recipient cells for their transformation. Manual selection of cells for transfer to fresh culture medium, the frequency of transfer to fresh culture medium, the composition of the culture medium, and environmental factors including, but not limited to, the quality and quantity of light and temperature they are all factors for the maintenance of calluses and / or suspension cultures that are useful as sources of recipient cells. Alternating the callus between different culture conditions can be beneficial to enrich the recipient cells in a culture. For example, cells can be grown in suspension culture, but transferred to solid medium at regular intervals. After a period of growth in solid medium, the cells can be manually selected to return them to liquid culture medium. Repeating this sequence of transfers to fresh culture medium can be used to enrich the recipient cells. Passing cell cultures through a 1.9 mm sieve can also be useful to maintain the friability of a callus or suspension culture and to enrich transformable cells when using such cell types.

C. Transgenic plants

Once a transgenic cell has been selected, the cell can be regenerated in a fertile transgenic plant using techniques well known in the art. The transformed plants can be subsequently analyzed to determine the presence or absence of a particular nucleic acid of interest in a DNA construct. Molecular analyzes may include, but are not limited to, Southern blots (Southern, 1975) or PCR analysis, immunodiagnostic approaches. Field evaluations can also be used. These and other well known procedures can be performed to confirm the stability of the transformed plants produced by the described procedures. These procedures are well known to those skilled in the art (Sambrook et al., 1989).

Thus, transgenic plants comprising a sequence encoding BMD provided herein can be produced. In particular, economically important plants, including crops, trees, and other plants with DNA constructs of the present invention can be transformed so that they are tolerant to dicamba or have increased tolerance. Plants that are currently considered tolerant to auxin-type herbicides, therefore, can be transformed to increase their tolerance to the herbicide. Some non-limiting examples of plants that may find use with the invention include alfalfa, barley, beans, beets, broccoli, cabbage, carrots, canola, cauliflower, celery, Chinese cabbage, corn , cotton, cucumber, eggplant, leek, lettuce, watermelon, oatmeal, onion, pea, pepper, peanut, potato, pumpkin, radish, rice, sweet corn, sorghum, soy, spinach, zucchini, sugar beet, sunflower, tomato, melon, and wheat.

Once a transgenic plant containing a transgene has been prepared, that transgene can be introduced into any plant that is sexually compatible with the first plant by crossing, without the need to even directly transform the second plant. Therefore, as used herein, the term "offspring" refers to the offspring of any generation of a parental plant prepared in accordance with the present invention, where the offspring comprises a selected DNA construct prepared in accordance with the invention. . A "transgenic plant" can therefore be of any generation. "Crossing" a plant to provide a plant line that has one or more transgenes or alleles added relative to an initial plant line, as described in this document, is defined as the techniques that cause a particular sequence to be introduced into a plant line crossing an initial line with a donor plant line comprising a transgene or allele of the invention. To achieve this, for example, the following steps could be carried out: (a) planting seeds of the first (initial line) and the second (donor plant line comprising a desired transgene or allele) parental plants; (b) grow the seeds of the first and second parental plants in plants that house flowers; (c) pollinate a flower of the first parental plant with pollen of the second parental plant; and (d) collect the seeds produced in the first plant that houses the fertilized flower.

The invention, therefore, provides transgenic plant tissues comprising a DMO coding nucleic acid provided herein. The tissues may have been directly transformed with a DMO coding nucleic acid or inherited the nucleic acid from a progenitor cell. The tissues provided by the invention specifically include, but are not limited to, cells, embryos, immature embryos, meristematic cells, immature panicles, microspores, pollen, leaves, anthers, roots, root tips, flowers and seeds. Any such fabric, including any part of the plant, comprising a nucleic acid described herein, is thus provided by the invention. The seeds, in particular, will find a particular benefit for their use, both for commercial use and for feeding in the form of grain, as well as for planting them to grow additional crops.

EXAMPLES

The following examples are included to illustrate embodiments of the invention. Those skilled in the art should appreciate that the techniques described in the following examples represent techniques discovered by the inventor that work well in the practice of the invention.

EXAMPLE 1

Vector construction to design the DMO gene by generic engineering

The coding sequence of the DMOc variant was initially generated by PCR amplification from a DMOw template. In this amplification, the DMOw coding region was amplified from plasmid pPLH1, which contained the DMOw gene in the form of a 3.5 kbp Xho I / Sst I DNA fragment of P. maltophilia, strain DI-6. For DNA amplification, a 5 'primer was used that inserted an Nco I restriction site near the 5' end of the PCR product and a codon for alanine immediately after the ATG start codon and a 3 'primer that created a site Xba I restriction at the 3 'end of the PCR product (details of the procedure are provided below). Change 112W to 112C was subsequently identified by nucleic acid sequencing. For the creation of the plant transformation vector, the DMOc gene was inserted using the Nco I and Xba I sites added to the 5 'and 3' ends, respectively, of the coding region in the vector pRTL2 (Carrington and Freed, 1990) thereby fusing the coding region to the translation enhancer element of the tobacco engraving virus (TEV leader) of the vector. The 5 'Nco I site was introduced along with the addition of a GCC codon (alanine) after the ATG start codon and an Xba I restriction site was created at the 3' end of the codon region using specifically designed PCR primers . To allow the delivery of DMOc to the chloroplast, the coding region of the chloroplast transit peptide of the pea Rubisco subunit gene (Coruzzi et al., 1983) was placed upstream of the DMO coding region to allow its addressing to chloroplast. The transient peptide coding sequence carried on a Bgl II and EcoR I fragment was cloned into the BamH I and EcoR I sites of the pBluescript II KS + vector. This construct was used as a template in a PCR reaction that inserted an Nco 1 site at both the 3 'end and the 5' end of the transit peptide sequence. The amplified product was cloned into the Nco I site of the pRLT2 vector so that the transit peptide sequence was directly upstream and in phase with the coding region of the DMO gene. A cassette consisting of the TEV leader, the transit peptide region and the BMD DNA coding sequences of the pRTL2 vector with Xho I and Xba I was cleaved and cloned into the vector pKLP36 (US 5,850,019; FIG. 5) using the same restriction sites to bind the cassette to a PClSV promoter and the poly A sequence of PsRbcS2-E9. The new vector was called pKLP36-TEV-TP-DMOc (also called pKLP36-DMOc), and was deposited in the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA on February 2, 2006, and the ATCC access number PTA-7357 was assigned.

The pKLP36-DMOc vector was used to transform tobacco, Arabidopsis and tomato plants. For soybean transformation, the DMOc cassette was cut from pKLP36-TEV-TP-DMOc in the form of an EcoR I / Acc I segment and cloned into pPZP101 digested with EcoR I / Acc I (Hajdukiewicz et al., 1994) to get the right and left limits. This vector (pPZP101 + DMOc cassette) was then cut with ScaI and the DMOc cassette was cloned into the binary vector pPTN200 (see below), a derivative of pPZP201 (Hajdukiewicz et al., 1994), which contains a bar cassette flanked by T-DNA boundaries on the left and right and allows the selection of transformants in regeneration in the presence of the Basta herbicide. The new binary vector of two T-DNAs was named pPTN348 and was used for soybean transformation. The pPTN200 vector was prepared by first cloning nos-element bar promoter from pGPTV-bar (Becker et al., 1992) in the form of a PstI / BamHI segment in pPZP201 (see Hajdukiewicz et al., 1994) and the resulting plasmid was called pPTN193. The nos terminator of pE7113-GUS (see Mitsuhara et al., 1996) was cloned into pPTN 193 downstream of the nos-element bar promoter to obtain the bar cassette.

Restriction enzymes and other enzymes were obtained from Fermentas or Invitrogen. DIG-11-dUTP (alkali labeled), CSPD (ready to use), DIG III molecular weight markers, anti-digoxigenin-AP (Fab fragments) and blocking reagent were obtained from Roche. The prehybridization solution, ULTRAhyb, was obtained from Ambion. The molecular weight marker DIG-RNA I was obtained from Roche. The (donkey) rabbit anti-IgG antibody, bound to peroxidase and the Hybond ECL (nitrocellulose) membrane were obtained from Amersham Biosciences. DNA, RNA and protein transfers, recombinant DNA techniques, and other molecular biology procedures were performed using conventional techniques (Ausubel et al., 1995).

EXAMPLE 2

Production and analysis of transgenic plants

Tobacco, tomato, soybeans and Arabidopsis were used for the transgenic expression of the genetically modified DMOc gene and the confirmation of dicamba tolerance in plants expressing the gene. The DMOc coding sequence in the binary vector pKLP36 was introduced into A. tumefaciens strain C58C1 containing the deactivated Ti plasmid pMP90 (Koncz and Schell, 1986) by triparental mating (Ditta 1980). The resulting transconjugants were used for the transformation of tobacco (cv Xanthi) and tomato (cv Rutgers) using the leaf disc protocol described by Horsch et al. (Horsch 1985). Arabidopsis thaliana was transformed by the floral immersion technique (Clough and Bent, 1998). The transformation of the soybean varieties Thome and NE-3001 was carried out by the Agrobacterium-mediated transformation system of cotyledonal nodule (Zhang et al., 1999).

Agrobacterium-mediated gene transfer from the DMOc gene to the nuclear genome of tobacco plants produces several T1 generation plants independently obtained. Plants were tested for the presence and expression of the DMOc gene using DNA, RNA and protein transfer analysis. FIG. 2 illustrates that, although all the transgenic plants (lanes 1-6) in this analysis contained the same DNA fragments after restriction enzyme digestion as the cloned DMO gene (lane 8), the level of mRNA and protein transcripts BMD varied significantly between transformants. For example, the plant whose extracts are represented in lane 5 shows relatively high levels of BMD mRNA but very low levels of the enzyme. In contrast, almost equal levels of BMD mRNA were coupled in the extracts shown in lane 3 with a high level of BMD expression. However, it was shown that events with strong expression could be obtained routinely by this procedure.

The plants in the greenhouse were sprayed with commercial grade solvent and dicamba (Clarity; BASF) using a motor-driven rail sprayer of compressed air with a 8002E flat fan nozzle traveling at 0.83 m / s (1 , 87 mph). The additives included: 28% urea, 1.25% v / v ammonium nitrate and 1.0% v / v non-ionic surfactant. The solution containing dicamba at various concentrations was applied at 182 l / ha (40 gallons per acre). Soybean field plantations were sprayed with Clarity herbicide at 2.8 kg / ha (2.5 lb / ac).

Tobacco plants, like most dicotyledonous plants, are quite sensitive to dicamba treatment. This was illustrated by comparison of untreated or treated non-transgenic tobacco plants with increasing amounts of dicamba. Symptoms of herbicide damage were easily detected after spraying dicamba at a level of 0.017 kg / ha. The symptoms are quite severe at 0.28 kg / ha and 0.56 kg / ha, the levels normally used for weed control in agricultural applications.

Post-outcrop treatment of transgenic tobacco plants containing BMDD with 5.6 kg / ha (10 to 20 times higher than normal application rates) caused few, if any, symptoms while a non-transgenic plant suffered serious damage. . The damage to the lower leaves of the transgenic plants could be doubled by spraying the plants with the solvent solution containing surfactant used as a vehicle for the application of dicamba. The leaves produced after the treatment of transgenic plants with dicamba showed no visible signs of damage. Transgenic tomato plants that carried the genetically modified DMOc gene also showed no damage when sprayed with high levels of dicamba, in this particular case, first with 0.56 kg / ha and then with 5.6 kg / he has. Arabidopsis thaliana expressing the DMOc gene also showed a strong tolerance to dicamba treatment. In this study, the concentration of dicamba used provided a dose of 1.12 kg / ha. An unexpected finding was the observation that tobacco plants transformed with a DMOc gene that lacked the coding region of the transit peptide were also tolerant of post-outcropping treatments with dicamba at concentrations only slightly below the average of the plants they housed DMOc genes with coding regions of the transit peptide. In this study, the treatments were compared using 2.2 kg / ha of dicamba in two T1 tobacco plants, one that carried BMD that lacked a transit peptide of chloroplasts and the other that completely lacked the DMOc gene due to segregation. genetics. The last plant was completely susceptible to damage caused by dicamba treatment and succumbed to treatment (FIG. 3). The transgenic plant that carried the DMOc gene that lacked the transit peptide was completely tolerant of dicamba treatment at 2.2, kg / ha. Generic studies of the inheritance of the DMOc gene in transgenic tobacco plants also demonstrated that the trait was inherited in most plants in a normal Mendelian manner and that it maintained the original levels of expression with respect to herbicide tolerance.

In soybeans, more than 50 R0 transgenic soybean events were produced and seeds of generation T1, T2, and T3 were collected. As a binary vector system of Agrobacterium tumefaciens was used, both transgenic plants that harbored a marker gene and transgenic plants without a marker containing the DMOc gene were recovered. In any case, most transgenic soybean lines showed significant tolerance to dicamba treatment at 2.8 kg / ha and 5.6 kg / ha under greenhouse conditions and strong tolerance to dicamba at 2.8 kg / ha ( the highest level tested) in two years of field trials. These results suggest a wide safety margin for transgenic soybeans and other crops that carry the DMOc gene together with a highly effective control of a wide range of broadleaf weeds.

The high levels of resistance to dicamba in GM soybean plants that house the DMO gene indicate the ability to apply dicamba in soybean fields to greatly suppress the competition of broadleaf weeds without damage to the crop. In addition, dicamba resistant crops can be an important complement to current weed control options using transgenic herbicide tolerant crops. That is, they can be a valuable strength in the strategies to control weeds resistant to existing herbicides and to suppress the appearance of additional weeds resistant to herbicides that could ultimately threaten the long-term use and value of current herbicides. and herbicide tolerant crops.

EXAMPLE 3

Overexpression, purification and comparison of the enzymatic properties of DMOw and DMOc

A. Cloning and overexpression

The wild-type (DMOw) and variant (DMOc) BMD coding sequences were cloned from plasmids pMON95900DMO (DMOw) and pMON58499DMO (DMOc) into vector pET28b (Novagen, San Diego, CA) and introduced by transformation into BL21 cells from Escherichia coli (Novagen, San Diego, CA). The cells were grown in 1 liter of Luria-Bertani broth at 37 ° C at an absorbance at 600 nm of 0.4 to 0.6. Protein expression was induced by adding 50 FeM Fe (NH4), 100 NaM Na2S, and 1mM isopropyl beta-thiogalactopyranoside (IPTG) and the cells were then changed at 15 ° C. After 48-72 hours at 15 ° C, the cells were collected by centrifugation at 10,000 xg for 20 minutes. For later use, the cells were stored at -20 ° C.

The yield of protein expression in E. coli for DMOw and DMOc was different. Although the production of DMOw was about 100 to 150 mg of pure protein per liter of LB medium, the production of DMOc was 10 times lower, or about 10 to 15 mg of pure protein per liter. This was not anticipated since E. coli does not have rare codons for cysteine and there is only one codon for tryptophan, but in both cases the ability to produce heterologous proteins in E. coli was shown regardless of yield. The amount of protein in the inclusion bodies was low in both cases, suggesting that the protein mainly remains in the soluble fraction.

His-labeled recombinant DMOw protein was purified from Pseudomonas maltophilia, strain DI-6 and His-labeled recombinant DMOc protein expressed in E. coli strain BL21, until homogeneous by Ni-NTA column chromatography. The cells were suspended in lysis buffer (100 mM NaPi pH 8.0, 300 mM NaCl, and 10 mM imidazole) and altered by sonication. The cell lysate was centrifuged at 55000xg for 1 hour. The supernatant was loaded on a Ni-NTA column, which was washed with wash buffer (100 mM NaPi pH 8.0, 300 mM NaCl, and 20 mM imidazole) to remove proteins that do not specifically bind to the resin. His-labeled protein was eluted with elution buffer (100 mM NaPi pH 8.0, 300 mM NaCl, and 250 mM imidazole). For the purification of DMOw, a step gradient was sufficient to obtain the 95% pure enzyme, while for DMOc, a linear gradient of 20 to 250 mM of imidazole concentration was needed to achieve the same level of purity. The enzyme eluted from the column was pure at about 95% according to the estimate by protein transfers (western blots) of the enzyme after size fractionation in electrophoresis in that of SDS-polyacrylamide. A single main band at approximately 40 kDa (37.3 kDa DMO enzyme plus 3 kDA for the His brand) indicated that the correct protein had been super-produced.

B. Test for BMDD and BMD and steady state kinetics

Protein concentrations were determined by Bradford assay with rabbit IgG as a standard. Proteins were separated by SDS-PAGE and stained with Coomassie blue. DMO activity was measured after DCSA formation that was separated by HPLC (Waters Corporation, Milford, MA) using a Discovery C18 column (Supelco, Sigma-Aldrich, St. Louis, MO). The retention time for DCSA was 8 minutes and for dicamba it was 9.5 minutes. For kinetic studies, DCSA was detected and quantified by fluorescence emission at 420 nm (excitation wavelength of 310 nm) after separation in the HPLC column of the reaction mixture. Established concentrations of DCSA (12 and 24 M) were used as quantification standards.

Dicamba stock solution (100, 200, 400, 800, 1000, 2000, 5000, and 10000 M), 0.1 M KPi pH 7.2, 0.1 M FeSO4, 0.1 M NADH, and 1M MgCl2. The tests were performed at 30 ° C for 20 minutes and the reaction was stopped by the addition of 40 µL of H2SO4. For activity measurements, BMD was coupled with an excess of purified ferredoxin and reductase from P. maltophilia strain DI-6.

Since the test for BMD activity was a discontinuous test, it was important to establish the time during which the test has to proceed to obtain significant kinetic parameters. Therefore the test must proceed under initial conditions since the amount of DCSA produced is linear during the time the test is proceeding (FIG. 4). The results suggested that the test could proceed between 20 to 30 minutes and still maintain linearity. FIG. 5 shows that the optimum pH for the test performed in the presence of 0.1 M Kpi buffer was 7.2 and the optimum temperature was approximately 37 ° C (FIG. 6).

C. Analysis of kinetic data

The Michaelis-Menten parameters were determined by adjusting the data to a non-linear steady state equation (Equation 1). Data were analyzed using Sigma plot 8.0 (Jandel Scientific).

V0 = Vmax * [S] / (Km + [S]) Equation 1

5 The optimum pH and temperature for DMOw and DMOc were also determined. The optimum pH was measured at 30 ° C for 20 minutes and the optimum temperature determination was also measured for 20 minutes at pH 7.2 for both forms of the enzyme. The results are summarized in FIG. 7-9 and are analyzed below.

Studies show that DMOw and DMOc differ in kinetic properties. For example, the Michaelis-Menten parameters calculated for DMOw and DMOc are: for DMOw, Km = 49 ± 7 M and Vmax = 633 ± 24

10 nmoles / min / mg, and for BMDc, Km = 20.5 ± 5 M and Vmax = 676 ± 37 nmoles / min / mg. These results are shown in FIG. 10 and 11 and are summarized in the following Table 1. In addition, two additional analyzes performed for DMOw and DMOc produced similar results (Table 2 and 3).

As can be seen, in terms of catalytic efficacy, the DMOw and DMOc enzymes have different properties: DMOc is an enzyme five times better than DMOw by this analysis. The pH profile for BMDD is

15 different from DMOw. First, it seems that DMOc is sensitive to the buffer system used (TRIS vs. KPi) by comparison with DMOw (FIG. 9, 12, and 13). Second, DMOc shows stationary activity over a wide pH range when tested in KPi buffer by comparison with TRIS when DMOc activity decreases with increases in pH units. The temperature profiles for BMD incubated in KPi or TRIS buffer are similar.

20 Looking at the temperature profiles between these two forms of the enzyme, DMOw worked better at 37 ° C while DMOc worked better at somewhat lower temperatures (FIG. 9). FIG. 9 indicates a lower optimum temperature for BMDD, which may be useful in transgenic plants soon in the growing season.

Table 1. The steady state kinetic parameters for DMOw and DMOc.

Enzyme

Km (M) Vmax (U / mg) kcat (s-1) Kcat / Km (M-1s -1)

Dmow

49 ± 7 X 10-6 633 ± 24 X 10-3 36.63  7.47 X 105

BMDD

20 ± 5 X 10-6 676 ± 37 X 10-3 70.41  35.21 X 105

Table 2. Summary of the Michaelis-Menten parameters for DMOw.

Study No.

Rsqr Vmax (nmoles / min / mg) Km (M)

one.

0.983 633 ± 24 49 ± 7

2.

0.988 583 ± 18 46 ± 5

3.

0.987 590 ± 19 46 ± 5.5

Table 3. Summary of the Michaelis-Menten parameters for DMOc.

Study No.

Rsqr Vmax (nmoles / min / mg) Km (M)

one.

0.933 713 ± 43 21 ± 6

2.

0.948 676 ± 37 20 ± 5

EXAMPLE 4

30 Bioinformatic analysis of conserved regions of BMD

A bioinformatic analysis was performed to compare the BMD polypeptide sequence with other iron-sulfur oxygenases and to identify conserved regions. Initially, 78 sequences were selected for analysis based on the value cut-off point and Ie-08 and a 70% coverage of the BMD sequence over the sequence alignment. Further analysis of these 78 sequences revealed the presence of two domains that had been identified in other studies, including the Rieske and nonheme Fe domains (Herman et al., 2005). Of these 78 sequences, 68 contained both domains, while 10 had only one of the domains. The 68 molecules with the two domains were used for further motive analysis.

The alignment of the 68 molecules with both domains at different levels of identity revealed a new WXWX motif. Although some sequences did not contain the motif, the phylogenetic analysis indicated that the molecules without the motive were in certain clades of the phylogenetic tree that do not pretend to the same group as the molecules with the motive. Those molecules without the motif, therefore, were removed from the original database, leaving 52 remaining sequences that were re-aligned for further analysis.

The 52 re-aligned sequences showed conservation around two W residues that contained the following format: WX1WX2G (W is Trp, G is the Gly residue, X1 is a non-polar residue, and X2 is any amino acid). The second W in this case corresponds to position 112 of SEQ ID No. 1. The WXG motif of WX1WX2G has recently been presented and proteins with the WXG motif are related to the cellular secretion system (Desvaux et al., 2005).

Tryptophan (W) and cysteine (C) are remnants with remarkably different sizes. W is a large remainder, while C is a relatively small one. Since both W and C are polar amino acids, they share some common characters, such as proton donation. Since the rest of W is encoded by TGG and Cys by TGC and TGT, certain conversions in the third code (G-> C or G-> T) can produce a nonsense mutation of W to C or C to W Such conversions have been identified in nature and the bio-functions and activities were changed by these mutations (see, for example, the BRCA1 gene in hereditary breast and ovarian cancer (Xiaoman and Jinghe, 1999); deficiency of the factor of coagulation XII (Wada et al., 2003), and the mutation of lipoprotein lipase in hyperlipoproteinemia type I (Hoffmann et al., 2000)).

The previous results, therefore, indicated that, although BMD is unique and has low identity with known enzymes, W112 is conserved in other related iron-sulfur oxygenases. In addition, position 112 is linked by two conserved functional domains (FIG. 18). In addition, conversions from W to C typically affect bioactivity. Therefore, the finding that DMOc produced a functional enzyme with higher kinetic parameters than those of the wild-type DMOw enzyme and produced a high tolerance to dicamba when expressed in transgenic plants was particularly surprising.

EXAMPLE 5

The BMD encoded in chloroplast produced a high level of resistance to dicamba

To determine whether BMD could function exclusively inside the chloroplasts and to explore the possibility of limiting the "propagated gene" through the entrained pollen, constructions based on the vector pFMDV1 were created (for example, Svab et al., 1990 ) to allow the integration of the DMOc gene into the genome of tobacco chloroplast by homologous recombination and isolation of the transformants using selection for antibiotic resistance (FIG. 14). In this construction, the coding region of the DMOc gene is directed by the chloroplast psbA gene promoter that contains the complete 5'UTR sequence of psbA. Initial DNA transfer analyzes of antibiotic-resistant transgenic plants (FIG. 15A) demonstrated the presence in the chloroplastic genomes of both the DMOc transgene (5.6 kb band) and the native gene region (3.3 band kb) replaced by homologous integration of the DMOc gene (i.e., the chloroplasts were heteroplastic for the native gene and the DMOc transgene). Regeneration and repeated selection of transgenic plants in medium containing antibiotic produced seemingly homoplastic chloroplasts that harbored the DMOc gene fragment but not the replaced native gene region (FIG. 15B).

The T1, T2 and T3 generations of the offspring of two independently obtained chloroplast transformants were tested for tolerance to dicamba treatment at various doses. All showed high tolerance levels. In fact, the transformants of the chloroplastic genome showed no apparent damage (other than "solvent-only damage" in the lower leaves) when sprayed at a rate of 28 kg / ha (25 1b / ac) (FIG. 16) and Only transient damage was observed when the plants were treated with extremely high applications of 112 and 224 kg / ha dicamba. At these extremely high levels, initial damage was caused primarily by the surfactants and other components of the solvent in which the dicamba was supplied; the tissues that grow from the damaged apex presented almost normal to normal phenotypes, which shows the absence of a decrease in growth rates after the initial atrophy and retained the ability to produce habitual quantities of seeds and in their usual quality.

The results were consistent with the possibility that reduced ferredoxin in tobacco chloroplasts could be the donor to the BMD of the electrons necessary for oxidation of dicamba in DCSA. As a direct test of this possibility, the ability of spinach purified ferredoxin to withstand the conversion of dicamba into DCSA in the presence and absence of purified BMD from P. maltophilia, strain DI-6, or super-produced and purified from E. coli (Table 4). The results demonstrated that reduced spinach ferredoxin or Clostridium was fully capable of donating electrons to BMD in vitro as measured by degradation of dicamba or appearance of DCSA.

Tables 4A-B. Purified dicamba monooxygenase can use reduced chloroplast ferredoxin or Clostridium reduced ferredoxin as an electron source to catalyze the in vitro conversion of dicamba into 3,6-dichlorosalicylic acid.

Table 4A-Dicamba degradation

Type of reaction

Dicamba degradation (%)

(Ferr + Red) DI-6 + NADH

0

(Oxi + Ferr + Red) DI-6 + NADH

86

(Oxi) DI-6 + (Ferr) Spinach + (Ferr: Oxidored) Spinach + NADPH

83

(Oxi) DI-6 + (Ferr: Oxidored) Spinach + NADPH

ND

(Oxi) DI-6 + (Ferr) Spinach + (Ferr: Oxidored) Spinach + No NADPH

ND

(Oxi) DI-6 + (Ferr) clostridium + (Ferr: Oxidored) Spinach + NADPH

82

(Ferr) clostridium + (Ferr: Oxidored) spinach + NADPH

ND

Table 4B-DCSA Training

Type of reaction

DCSA training (%)

(Ferr + Red) DI-6 + NADH

ND

(Oxi + Ferr + Red) DI-6 + NADH

100

(Oxi) DI-6 + (Ferr) Spinach + (Ferr: Oxidored) Spinach + NADPH

95

(Oxi) DI-6 + (Ferr: Oxidored) Spinach + NADPH

2.5

(Oxi) DI-6 + (Ferr) Spinach + (Ferr: Oxidored) Spinach + No NADPH

1.2

(Oxi) DI-6 + (Ferr) clostridium + (Ferr: Oxidored) Spinach + NADPH

90

(Ferr) clostridium + (Ferr: Oxidored) spinach + NADPH

1.5

ND, Not determined

Although the results of FIG. 2 showed that the levels of BMD produced were variable and sometimes the

BMD levels did not correlate very much with dicamba tolerance levels, the results demonstrated 10 the ability to obtain a high dicamba tolerance on a regular basis. The production of

DMOc from both a DMOc gene located in the nucleus and a DMOc gene located in the chloroplast in

transformants In nuclear transformants none constituted an exceptionally high level of total DMOc with

ratio to total proteins and the amount of DMOc in the chloroplast transformants was not very different and at

times lower than nuclear transformants. Estimates of relative enzyme activity in extracts without 15 cells from foliar tissue samples indicated that a higher percentage of the BMDD produced in the cells is active.

Chloroplasts that BMD synthesized in the cytoplasm and supposedly transferred to the chloroplasts.

In all the plants analyzed, tolerance to dicamba was achieved without cotransformation with genes from

ferredoxin or reductase. The results showed that the plants contained one or more molecules that could

transfer necessary electrons to the BMD to allow the conversion of dicamba into 3,6-dichlorosalicylic acid (DCSA). The initial addressing of the BMD to the chloroplasts using a transit peptide sequence is

potentially addressed using reduced ferredoxin abundantly available in chloroplasts. To this

In this regard, it is of interest to note that the transformation of tobacco plants with a gene construction of DMOc

lacking a coding sequence of the chloroplast peptide unexpectedly produced plants that were

tolerant to post-outcrop treatment with dicamba. The results of limited trials with a small amount of T1 generation plants, however, indicated that the tolerance level obtained with these plants

GM was slightly lower on average than that obtained with tobacco plants that produced BMD that

It contained a transit peptide. These observations raise interesting questions regarding the molecules in

GM plants that can productively donate electrons to BMD. The fact that chloroplasts

homoplastics that produce BMD internally from a DMOc gene integrated in the chloroplast genome show resistance to extremely high levels of dicamba (FIG. 16) and the fact that purified BMD can function in vitro with reduced chloroplastic ferredoxin of the Spinach (Table 4) suggests that chloroplastic ferredoxin can interact productively with BMD to allow electron transfer. However, the source of electrons for BMD produced from nuclear genes that lack a chloroplast transit peptide coding sequence remains unknown. Assuming that ferredoxins do not reside outside the plant's chloroplasts, the possibility that an unknown cytoplasmic protein can provide DMO with a continuous supply of electrons should be considered. Alternatively, the BMD itself may contain a free chloroplast transit peptide that allows sufficient BMD to enter the chloroplasts to provide protection against the dicamba that moves into the cell after the dicamba treatment.

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<110> CLEMENTE, THOMAS E.

DUMITRU, RAZVAN FENG, PAUL C. C. FLASINSKI, STAN WEEKS, DONALD P.

<120> MODIFIED DMO ENZYME AND USE PROCEDURES

<130> MONS: 093WO

<140> PCTUS0770514

<141> 06-06-2007

<150> UNKNOWN

<151> 06-06-2007

<150> 11 / 758,657

<151> 06-05-2007

<150> 60 / 811.152

<151> 06-06-2006

<160> 24

<170> PatentIn Ver. 2.1

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<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 10

image 1

<210> 11

<211> 57

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 11

image 1

<210> 12

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 12

image 1

<210> 13

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 13

image 1

<210> 14 <211> 60

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 14

image 1

<210> 15

<211> 57

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 15

image 1

<210> 16

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 16

image 1

<210> 17

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 17

image 1

<210> 18

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 18

image 1

<210> 19

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 19

image 1

<210> 20

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 20

image 1

<210> 21

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide <400> 21

image 1

<210> 22

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 22

image 1

<210> 23

<211> 58

<212> PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 23

image 1

<210> 24

<211> 433

<212> DNA <213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic primer

<400> 24

image3

Claims (7)

 1.- A nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule encoding the polypeptide of SEQ ID NO: 1; b) a nucleic acid molecule comprising the sequence of SEQ ID NO: 2; Y c) a nucleic acid molecule encoding a polypeptide with at least 90% identity of sequence with the polypeptide of SEQ ID No. 1, in which the polypeptide has dicamba monooxygenase activity and it comprises cysteine in a position corresponding to amino acid 112 of SEQ ID NO. 1. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes the dicamba monooxygenase encoded by plasmid pKLP36-TEV-TP-DMOc (ATCC accession number PTA-7357). 3. A DNA construct comprising the nucleic acid molecule of claim 1 attached in a manner functional to a promoter. 4. The construction of claim 3, wherein (i) the promoter is works in a plant cell; or

(ii)

 The nucleic acid molecule is functionally linked to a chloroplast transit peptide. 5.- A polypeptide sequence comprising an amino acid sequence with at least 90% identity

with SEQ ID No. 1, in which the polypeptide has dicamba monooxygenase activity and comprises cysteine in a position corresponding to amino acid 112 of SEQ ID No. 1. 6. A plant cell transformed with the nucleic acid molecule of claim 1. 7. The plant cell of claim 6, wherein

(i)

 the plant cell is a dicot plant cell, preferably the plant cell dicot is a plant cell of soy, cotton, corn or rapeseed; or

(ii)

 The plant cell is a monocot plant cell. 8.- A plant tissue culture comprising the plant cell of claim 6. 9.- A transgenic plant transformed with the nucleic acid molecule of claim 1. 10.- The transgenic plant of claim 9, wherein

(i)

 the plant is a dicot plant, preferably the plant is a soybean, cotton, corn or rapeseed plant; or

(ii) the plant is a monocot plant. 11. A process for producing a dicamba tolerant plant comprising introducing the construction of claim 3 into the plant stably transforming an initial plant cell and regenerating the cell in said dicamba tolerant plant.  12. The method of claim 11, wherein

(i)

 the dicamba tolerant plant is produced by crossing a parental plant with itself, wherein the parental plant comprises the transformation construction of claim 3 and the dicamba tolerant plant inherits the transformation construction of claim 3 from said parental plant or

(ii)

 the dicamba tolerant plant is produced by crossing a parental plant with a second plant, in which the parental plant and / or the second plant comprises the transformation construction of claim 3 and the dicamba tolerant plant inherits the transformation construction of the claim 3 of said parental plant and / or the second plant.

13. A method for controlling weed growth in a crop growth environment comprising a transgenic plant of claim 9 or a seed thereof comprising the nucleic acid molecule of claim 1, wherein said The method comprises applying to the crop growth environment an amount of the effective dicamba herbicide to control the growth of weeds.  14. The method of claim 13, wherein (i) the dicamba herbicide is applied to the upper part of the crop growth environment; or (ii) the amount of the dicamba herbicide does not damage said transgenic plant of claim 9 or seed thereof comprising the nucleic acid molecule of claim 1, and damages a plant of the same genotype as the transgenic plant of claim 9 which It lacks the nucleic acid of claim 1. 15. A process for producing a food, feed or industrial product comprising: a) obtaining the transgenic plant of claim 9 or a part thereof comprising the nucleic acid molecule of claim 1; and b) prepare the food, feed or industrial product from the plant or part thereof. 16. The method of claim 15, wherein (i) the food or feed is oil, flour, grain, starch, flour flower, or protein; or 10 (ii) the industrial product is biofuel, fiber, industrial chemical compounds, a pharmaceutical agent or nutraceutical.