CN101501196A - Modified dicamba monooxygenase enzyme and methods of its use - Google Patents

Abstract

The present invention provides modified variants of dicamba monooxygenase (DMO). The present invention relates to the unexpected discovery that cells expressing this DMO exhibit high levels of tolerance to the herbicide dicamba. Compositions and methods of use comprising nucleic acids encoding DMOs are provided.

Description

Modified dicamba monooxygenases and methods of use thereof

Background of the invention

This application claims priority to U.S. Provisional Patent Application Serial No. 60/811,152, filed June 6, 2006, and U.S. Patent Application Serial No. 11/758,657, filed June 5, 2007, the disclosures of which are incorporated herein by reference in their entirety .

1. Field of invention

The present invention generally relates to the field of biotechnology. More specifically, the present invention relates to modified dicamba monooxygenases capable of conferring tolerance to the herbicide dicamba in transgenic organisms.

2. Description of related fields

The methods of producing field crops such as corn, soybean, and cotton have changed dramatically over the past few decades due to the introduction of traits such as insect resistance and herbicide tolerance through the use of plant genetic engineering techniques. These changes have resulted in greater productivity per hectare, reduced production costs, greater flexibility and efficiency in production schemes, reduced pesticide use and, in the case of insect resistant cotton, improved farmer health. Genetically modified crops have thus been widely adopted and are now grown on millions of acres around the world. However, for GM crops to remain competitive in the marketplace, traits that add new value are needed.

Although new traits that improve the quantity and quality of agricultural and horticultural crops have emerged and will continue to emerge at an accelerated rate in the next few years, there is still a need for traits that improve methods of production of food, feed, and other products. For example, although genetically modified plants tolerant to the herbicides glyphosate, bromoxynil, sulfonylureas, and other herbicide treatments are currently available, the range of weeds controlled and through the development of additional herbicide-tolerant crops can be addressed. There are gaps in the processing options for . Furthermore, the emergence, although often localized and variable, of weeds tolerant to the above-mentioned herbicides creates the need for additional or alternative weed control measures.

Although transgenic herbicide tolerance has proven valuable in a commercial setting, plants tolerant to other herbicides are therefore needed to avoid over-reliance on any single herbicide and to increase options for managing difficult-to-control weed species. In particular, there is a need to develop herbicide tolerance for herbicides that are environmentally friendly and highly effective in controlling weeds. Dicamba is one such example of an effective and environmentally friendly herbicide that has been used by farmers for over 40 years. Dicamba is especially useful for controlling annual and perennial broadleaf weeds and several narrowleaf weeds in corn, sorghum, millet, pasture, hay, pasture, sugarcane, asparagus, turf, and grassseed crops (Crop Protection Reference, 1995). Unfortunately, dicamba can injure many commercial crops and dicots, such as soybean, cotton, pea, potato, sunflower and canola, which are particularly sensitive to even low levels of the herbicide. Nonetheless, dicamba appears to be effective in controlling weed growth and is thus an important tool in agriculture.

Recently, a gene encoding dicamba monooxygenase (DMO), which confers tolerance to dicamba, was isolated from Pseudomonas maltophilia (US Patent No. 7,022,896). DMO is involved in the conversion of the herbicide dicamba (3,6-dichloro-o-anisic acid) to the non-toxic 3,6-dichlorosalicylic acid. This gene is disclosed in US Patent No. 7,022,896 as conferring tolerance to dicamba in plants expressing the DMO gene. However, the development of variants of this gene would be of great benefit. Such variants may have altered efficiency under specific environmental conditions. In this way, a variant optimized for the specific circumstances in which it will be intended to be used and which may exhibit particularly beneficial kinetic characteristics can be selected. The variant may in particular show maximum efficiency under different temperature or pH conditions and thus be selected for a particular crop species depending on intracellular conditions and/or expected crop growth conditions.

Summary of the invention

In one aspect, the invention provides an isolated nucleic acid sequence selected from the group consisting of: a) a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 1; b) a nucleic acid sequence comprising a sequence of SEQ ID NO: 2; and c) encoding and SEQ ID NO: The polypeptide of NO: 1 has a nucleic acid sequence of a polypeptide having at least 90% sequence identity, wherein the latter polypeptide has dicamba monooxygenase activity and comprises a cysteine at a position corresponding to amino acid 112 of SEQ ID NO: 1 . In other embodiments, DNA vectors are provided comprising a DMO encoding a nucleic acid described herein operably linked to a promoter. The promoter may be functional in plant cells. In some embodiments, a nucleic acid sequence encoding a dicamba monooxygenase can be operably linked to a chloroplast transit peptide.

In another aspect, the present invention provides a polypeptide sequence at least 90% identical to SEQ ID NO: 1, wherein the polypeptide has dicamba monooxygenase activity and comprises at a position corresponding to amino acid 112 of SEQ ID NO: 1 cysteine.

In yet another aspect, the invention provides a host cell or tissue transformed with a nucleic acid encoding a dicamba monooxygenase described herein. In some embodiments, the host cell can be a plant cell. In other embodiments, a plant cell may be defined as a dicot cell or a monocot cell. In specific embodiments, the host cell is a soybean, cotton, corn or canola plant cell. In further embodiments, tissue cultures comprising the transgenic cells described herein are provided.

In yet another aspect, the present invention provides transgenic plants transformed with a nucleic acid encoding a dicamba monooxygenase described herein and progeny thereof. In some embodiments, a plant can be defined as a dicot or a monocot. In particular embodiments, the plant is a soybean, cotton, corn or canola plant.

In yet another aspect, the present invention provides a method of producing a dicamba-tolerant plant comprising introducing into the plant a transformation construct provided herein. In one embodiment of this method, introducing the transformation construct may be performed by stably transforming one or more plant cells and regenerating said one or more cells into dicamba-tolerant plants. In another embodiment, a dicamba-tolerant plant is produced by selfing a parent plant or crossing it with a second plant, wherein the parent plant and/or the second plant comprise the transformation construct and the dicamba-tolerant plant The transformation construct is inherited from a parent plant and/or a second plant.

In a further aspect, the invention provides a method of producing food or feed comprising: a) obtaining a plant or part thereof of the invention as provided herein; and b) preparing food or feed from said plant or part thereof. In one embodiment of the invention said plant part is a seed. In certain other embodiments, the food or feed is oil, meal, protein, grain, starch or protein. In other embodiments, the feed comprises forage or pasture plants, such as hay. The invention also provides methods of producing fibers, pharmaceuticals, neutraceuticals, and industrial chemicals, including biofuels, and any other product from the plants provided herein.

In yet another aspect, the present invention provides a method of controlling weed growth in a crop growing environment comprising a plant of the invention as provided herein or seeds thereof, comprising applying dicamba to the crop growing environment in an amount effective to control weed growth herbicide. In some embodiments of the invention, the dicamba herbicide may be applied to the top of the crop growing environment. In particular embodiments, the amount of dicamba herbicide does not damage the plants of the invention or their seeds and damages plants of the same genotype as plants lacking the DMO-encoding nucleic acids provided by the invention.

In yet another embodiment of the present invention, plants are provided that comprise the DMO-encoding 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 Such coding sequences. In specific embodiments, the plant comprises a transgene that confers one or more additional beneficial traits such as herbicide or pest/insect tolerance. For example, as described below, tolerance to one or more herbicides may be provided in addition to dicamba tolerance, among other beneficial traits. The present invention thus provides in particular plants comprising a DMO-encoding nucleic acid according to the invention "stacked" in any desired combination with additional transgenic traits.

Brief description of the drawings

The following drawings form part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings together with the detailed description of specific embodiments presented herein.

Figure 1. Schematic diagram of the cassette used to genetically engineer the dicamba monooxygenase gene (DMOc) for expression in higher plants using the FLt36 promoter from peanut chlorotic stripe disease virus, tobacco etch virus (TEV leader sequence) translational enhancer sequence, and terminator region from the pea Rubisco small subunit gene. Another genetically engineered form of the DMOc gene was prepared containing the transit peptide coding region from the pea Rubisco small subunit gene for chloroplast localization of the DMO between the TEV translational enhancer region and the coding region of the DMOc.

Figure 2. DNA, RNA and Western blots showing the presence and expression of the genetically engineered DMO gene in _T1 transgenic tobacco plants. Lanes Q to V depict DNA, mRNA and DMO species extracted from various Ti _generation transgenic tobacco plants. Extracts from non-transgenic tobacco plants are depicted in lane WT, while lane Ox shows restriction enzyme digestion products of the cloned DMO gene construct (upper panel) and the approximately 37 kDa DMO overproduced in Escherichia coli (E. coli) Enzymes (below). The approximately 55 kDa large subunit of Rubisco was detected in Western blots by detection with Rubisco antibody added to DMO antiserum, and Rubisco was used as an internal standard to compare the total protein load in each lane. Equal amounts of RNA were loaded in each lane as judged by ethidium bromide staining of duplicate gels. Arrows indicate the positions of DMODNA, mRNA or protein bands.

Figure 3. Effect of dicamba treatment at 2.2 kg/ha on two _T1 tobacco plants, one containing a genetically engineered DMOc gene lacking the chloroplast transit peptide coding sequence (right), one lacking the DMOc gene (right the second). The transgenic plants on the right panel show little, if any, damage from dicamba treatment. The two plants on the left were not treated with dicamba and represent transgenic plants (left) and transgenic plants containing the DMOc gene (second from left).

Figure 4. DMOw induced DCSA formation over time.

Figure 5. Determination of optimal assay pH for DMOw.

Figure 6. Determination of the optimum assay temperature for DMOw.

Figure 7. Determination of the optimum pH of DMOc.

Figure 8. Determination of the temperature optimum for DMOc.

Figure 9. Overview of temperature and pH optima for DMOc and DMOw.

Figure 10. Steady state kinetics of DMOw.

Figure 11. Steady state kinetics of DMOc.

Figure 12. Effect of pre-incubation of DMOc for 45 min at 30°C in 50 mM TRIS pH 7.5 and 100 mM KPi pH 7.0.

Figure 13. TRIS buffer (two assays on the left; assays before and after storage, respectively) and KPi buffer (two assays on the right; DMOc assays in pre- and post-storage assays, respectively).

Figure 14. Dicamba monooxygenase constructs genetically engineered for homologous recombination and expression in tobacco chloroplasts.

Figure 15. Elucidation of the homoplastid state of the chloroplast genomes of transgenic tobacco lines transformed with DMO genes designed for homologous recombination and expression in tobacco chloroplasts. The left panel shows the construct used to integrate DMO into the chloroplast by homologous recombination (as shown in Figure 14). The bar above the guide sequence on the left indicates the DNA fragment that was amplified to prepare the digoxigenin-labeled hybridization probe. Southern blots are shown on the right: lane 1 contains size markers. Lane 2 contains DNA from non-transgenic tobacco plants. Lanes 3-11 contain chloroplasts isolated from transgenic plants shortly after the first round of selection and regeneration in the presence of spectinomycin (upper panel) and when the apparent homoplast of the chloroplast genome was obtained after several rounds of selection and regeneration (lower panel). DNA. DNA for Southern blot analysis was isolated from transgenic and non-transgenic plants and subjected to restriction enzyme digestion with BamH I, followed by electrophoretic separation and use of a tagged DNA fragment complementary to the "left guide sequence" of the chloroplast genome transformation vector (i.e., foreign Dixigenin-labeled hybridization probe) to probe the blotted DNA. 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 the inserted DMO gene construct.

Figure 16. _T1 homoplasmic transgenic tobacco plants containing a chloroplast-encoded dicamba monooxygenase gene treated with dicamba at a level of 28 kg/ha (plants 1-2 and plants 3-4 from two independently transformed R ₀ plants).

Figure 17. DMO expression and sensitivity and resistance to dicamba treatment in non-transgenic tobacco plants and transgenic tobacco plants containing the DMO gene in the chloroplast genome. Western blot probed with DMO antibody: Lane 1 contains purified DMO. Lane 2 is blank and lane 3 contains protein extracts from non-transgenic tobacco plants. Lanes 4 and 8 contain proteins isolated from "false positive" tobacco plants that displayed antibiotic resistance during selection with spectinomycin but lacked the intact DMO gene. Lanes 5-7 contain extracts from transgenic plants expressing DMO encoded by the DMO gene integrated into the chloroplast genome. S = plants sensitive to 0.56 kg/ha of dicamba; R = plants resistant to 5.6 kg/ha of dicamba. Almost equal amounts of extracts were loaded into lanes 4-8 as judged by the amount of Rubisco large subunit protein detected with anti-Rubisco antibody, whereas significantly more protein from non-transgenic plants was loaded into lane 3 . Arrows indicate the location of DMO proteins.

Figure 18. Comparison of parts of the wild-type DMO polypeptide sequence with conserved regions of other iron-sulfur oxygenases, showing that DMO is unique and has low identity to known enzymes, but W112 (arrow) in other iron-sulfur oxygenases It is conserved among enzymes and is surrounded by two conserved domains, Rieske and Non-Haem Fe (SEQ ID NOS: 4-23).

Detailed description of the invention

The present invention provides a dicamba monooxygenase (DMO) variant, referred to herein as DMOc, comprising a cysteine at a position corresponding to position 112 of the DMO shown in SEQ ID NO: 1. DMOc has been shown to confer high levels of tolerance to the herbicide dicamba when expressed in transgenic plants. The results were surprising because the altered amino acid positions are highly conserved in other iron-sulfur oxygenases. Of the 78 iron-sulfur oxygenase sequences analyzed from 45 species, all 52 with at least 15% identity had a W at the position corresponding to amino acid 112 of SEQ ID NO: 1, although the highest The overall identity is only 38%. This position is also surrounded by two conserved functional domains (Figure 18). The resulting high level of herbicide tolerant DMOc was unexpected.

Mie-Mann parameter analysis of DMOc relative to the unchanged sequence (DMOw; U.S. Patent No. 7,022,896) revealed that the enzymes differ in catalytic efficiency: DMOc is 5-fold more efficient than DMOw and DMOc appears to have a higher number of renewals and is more stringent substrate binding. In addition, DMOc functions better at lower pH conditions and higher temperatures relative to native enzymes. These results indicate the possibility of selecting DMO variants for specific transgenic plants based on desired use conditions, such as crop growth conditions. One aspect of the invention thus relates to identifying a candidate crop growth environment for at least one crop species and identifying a DMO enzyme suitable for that environment based on the kinetics of eg DMOc and DMOw. For example, those skilled in the art can, in particular embodiments, select the DMOc coding sequence for having lower pH conditions in the plant (inplanta) relative to other plant species or growth environments and/or to have higher pH conditions under the conditions of the growth environment. in high temperature plants. Dicamba is applied by incorporating the soil (pre-plant incorporation); spraying the soil (pre-emergence); and spraying the tops of the plants (post-emergence treatment), while tolerance levels to dicamba can vary at different times during plant growth rather different.

As noted above, tolerance to very high levels of the herbicide dicamba was obtained in transgenic plants expressing DMOc. In e.g. tobacco, which is usually sensitive to even very low levels of dicamba, transgenic plants expressing DMOc were generated which tolerated dicamba treatments of 5.6 kg/ha or more, e.g. The application rate is 10-20 times higher. When the DMOc gene is inserted into the chloroplast genome of tobacco plants, dicamba tolerance to at least 28 kg/ha is obtained. Transgenic soybean, tomato and Arabidopsis thaliana plants carrying the nuclear-encoded DMOc gene were also generated and found to be tolerant to high levels of dicamba. For example, insertion of DMOc into the nuclear genome of soybean plants confers tolerance to the 2.8 kg/ha treatment, allowing the use of dicamba to control weeds in fields of DMOc-expressing plants.

Thus it was shown that DMOc is effective in conferring tolerance to dicamba without the need for additional coding sequences such as Pseudomonas maltophilia strain DI-6, ferredoxin or reductase. The modified DMO gene is genetically stable as a Mendelian gene without significant loss of penetrance or expression. Transgenic plants with the DMO transgene lacking the transit peptide coding sequence also showed high levels of post-emergence dicamba tolerance, although somewhat stronger expression was obtained with the chloroplast transit peptide.

A. Nucleic Acids and Recombinant Constructs

1. Dicamba monooxygenase (DMO)

In one embodiment of the present invention, there is provided a DNA construct comprising a nucleic acid encoding a dicamba monooxygenase polypeptide comprising, at a position corresponding to position 112 of SEQ ID NO: 1 cysteine. An exemplary DMO coding sequence is provided herein as SEQ ID NO:2. In addition to the cysteine at position 112 of SEQ ID NO: 1, the sequence also includes the addition of a GCC codon (alanine) to the Nco I restriction enzyme site relative to the native coding sequence after the ATG start codon and easy cloning. Thus, the polypeptide in SEQ ID NO: 1 also includes an additional Ala residue immediately after Met encoded by the start codon. The transit peptide sequence was excised from the plasmid with Bgl II and EcoR I and 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 using primers that added Nco I restriction sites at either end of the transit peptide coding sequence. Digestion of the PCR product with Nco I allowed insertion of a transit peptide coding sequence in the site of the ATG start codon of the modified DMO gene.

Thus, in one embodiment of the present invention, there is provided a sequence encoding a polypeptide of SEQ ID NO: 1, including but not limited to SEQ ID NO: 2. Homologous sequences and derivatives of these sequences can be readily prepared and used as known in the art. For example, a polypeptide encoding a DMOc having at least 90% sequence identity to SEQ ID NO: 1, including at least about 92%, 94%, 95%, 96%, 97%, 98%, 99% or more to this sequence, can be used Nucleic acids of identity to DMO polypeptides. Also useful are nucleic acids having at least 90% sequence identity to the nucleic acid provided by SEQ ID NO: 2, including at least about 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to this sequence. and encoding a DMO containing a cysteine at position 112. In one embodiment, 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 . The software matches similar sequences by assigning degrees of similarity or identity.

According to the present disclosure, polynucleotide molecules expressing DMO polypeptides can be obtained by techniques well known in the art. Variants of the DMOs provided herein that are capable of degrading dicamba can thus be prepared and assayed for activity according to the methods disclosed herein. Such sequences can also be identified, for example, from suitable organisms, including dicamba-degrading bacteria (US Patent No. 5,445,962; Krueger et al., 1989; Cork and Krueger, 1991; Cork and Khalil, 1995). One method of isolating cloned DMO sequences is by, for example, suitable hybridization to a library constructed from the source organism, or RT-PCR using mRNA from the source organism and primers designed based on the disclosed DMO. The invention thus includes the use of nucleic acids that hybridize under stringent conditions to the DMO coding sequences described herein. Those skilled in the art understand that the stringency of the conditions can be made less stringent by increasing the salt concentration and lowering the temperature. Thus, hybridization conditions can be readily manipulated and will generally be the method of choice depending on the desired outcome. An example of highly stringent conditions is 5X SSC, 50% formamide and 42°C. By washing under such conditions, such as 10 minutes, those sequences which do not hybridize to the specific target sequence under these conditions will be removed. One embodiment of the invention thus includes the use of DMO to encode nucleic acids defined as nucleic acids that hybridize to the nucleic acid of SEQ ID NO: 2 under wash conditions of 5X SSC, 50% formamide, and 42° C. for 10 minutes.

Variants can also be chemically synthesized using the DMO polynucleotide sequences described herein according to techniques well known in the art. For example, DNA sequences can be synthesized by phosphoramidite chemistry in an automated DNA synthesizer. Chemical synthesis has many advantages. In particular, chemical synthesis is desirable because codons preferred by the host (in which the DNA sequence will be expressed) can be used to optimize expression. An example of such a sequence optimized for expression in dicots using Arabidopsis codon usage is the DMO sequence shown in SEQ ID NO:3. The polypeptides predicted to have Ala, Thr, Cys at positions 2, 3, and 112, respectively, are given in SEQ ID NO: 1. An Ala residue was added at position 2 relative to wild-type DMO due to the addition of an alanine codon immediately after the ATG start codon to simplify vector construction (as described below).

It is not necessary that all codons be changed to obtain improved expression, but preferably at least codons that are rarely used in the host are changed to codons that are preferred by the host, e.g., more frequently used in the host and often than rare non- Preferred codons are codons that are more easily translated. High level expression can be obtained by changing greater than about 50%, most preferably at least about 80%, of the non-preferred codons to host-preferred codons. The codon preferences of many host cells are known (PCT 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 for other host cells can be deduced by methods known in the art. Furthermore, using chemical synthesis, the sequence of a DNA molecule or the protein it encodes can be readily altered, e.g., to optimize expression (e.g., to eliminate mRNA secondary structures that interfere with transcription or translation), to add unique restriction sites at convenient locations, and removal of protease cleavage sites.

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

It is known, for example, that certain amino acids can substitute for other amino acids in protein structures without appreciable loss of ability to interact with structures, such as binding sites on substrate molecules. Since it is the interacting capabilities and properties of a protein that define the biological functional activity of that protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, in the underlying DNA coding sequence and still result in a protein with similar properties. It is thus contemplated that various changes may be made in the DMO polypeptide sequences and corresponding DNA coding sequences described herein without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interacting biological functions on proteins is well known in the art (Kyte et al., 1982). It is recognized that the relatively hydrophilic character of amino acids contributes to the resulting protein's secondary structure, which in turn determines the protein's interaction with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydrophobicity index based on their 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); -3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is known in the art that amino acids can be substituted by other amino acids with a similar hydropathic index or score and still result in a protein with similar biological activity, ie still result in a biologically functionally equivalent protein. When making such changes, amino acid substitutions with a hydropathic index within ±2 are preferred, within ±1 are especially preferred, and within ±0.5 are even more preferred. Here, tryptophan at position 112 was replaced with cysteine, taking into account the different hydropathic indices between natural and altered amino acids and would not be used by those skilled in the art to generate functional variants according to the prior art The observation that DMO is biologically active and results in plants tolerant to high levels of dicamba is surprising.

It is also understood in the art that similar amino acid substitutions can be made efficiently based on hydrophilicity. US Patent 4,554,101 discloses that the maximum local average hydrophilicity of a protein (as controlled by the hydrophilicity of its neighboring amino acids) is related to the biological properties of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamine Acid (+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 amino acid having a similar hydrophilicity value and still result in a biologically equivalent protein. In such alterations, amino acid substitutions with hydrophilicity values within ±2 are preferred, amino acid substitutions within ±1 are especially preferred, and amino acid substitutions within ±0.5 are even more especially preferred. Exemplary substitutions that take into account these and many of the foregoing features are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine amides; and valine, leucine, and isoleucine. Again, the activity of DMOc is surprising considering the very different hydrophilicity values between the altered and natural amino acids and this substitution would not be used by those skilled in the art to generate functional variants according to the prior art.

The modification of the DMO sequence according to the invention can be guided by taking into account conserved domains within the enzyme. For example, it is illustrated below that DMO enzymes contain functional domains such as Rieske iron-sulfur clusters and binding sites for free iron (eg, see Figure 18). This information, combined with knowledge in the art about functional domains and modifications of proteins, can thus generally be used to generate modified DMO enzymes while retaining enzymatic activity within the scope of the invention (see, e.g., Mason and Cammack, 1992; Jiang et al. ., 1996).

2. Transformation Constructs

The DMO-encoding polynucleotides used in the present invention will generally be introduced into cells as constructs containing the expression control elements necessary for efficient expression. Methods for operably 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 that refer in any way to the control of transcription. Suitable expression control sequences and methods of using them are well known in the art. In particular, promoters, with or without enhancers, 5' untranslated regions, transit peptides or signal peptides for targeting protein or RNA products to plant organelles, especially chloroplasts, and 3' untranslated regions, such as polyadenylation, can be used. acidification site. Those skilled in the art will know that a variety of enhancers, promoters, introns, transit peptides, targeting signal sequences and 5' and 3' untranslated regions (UTRs) can be used to design efficient plant expression vectors, as disclosed in the U.S. Patent Application Those described in 2003/01403641.

Promoters suitable for current and other uses are well known in the art. Examples describing such promoters include US Patent 6,437,217 (maize RS81 promoter), US Patent 5,641,876 (rice actin promoter), US Patent 6,426,446 (maize RS324 promoter), US Patent 6,429,362 (maize PR-1 ), U.S. Patent 6,232,526 (maize A3 promoter), U.S. Patent 6,177,611 (constitutive maize promoter), U.S. Patents 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Patent 6,433,252 (maize L3 oleosin promoter) , U.S. Patent 6,429,357 (Rice Actin 2 Promoter and Rice Actin 2 Intron), U.S. Patent 5,837,848 (Root-Specific Promoter), U.S. Patent 6,294,714 (Light-Inducible Promoter), U.S. Patent 6,140,078 (Salt inducible promoter), U.S. Patent 6,252,138 (pathogen-inducible promoter), U.S. Patent 6,175,060 (phosphorus deficiency-inducible promoter), U.S. Patent 6,635,806 (γ-coixin chloroplast aldolase promoter). Additional promoters that can be used are the nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried on the tumor-inducing plasmid of Agrobacterium tumefaciens), cauliflower Mosaic virus promoters, such as cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987), CaMV 35S promoter (Odell et al., 1985), Scrophulariaceae mosaic virus 35S-promoter (Walker et al., 1987), sucrose synthase promoter (Yang et al., 1990), R gene composite promoter (Chandler et al., 1989), and chlorophyll a/b binding protein gene promoter and the like. Of particular interest for use in the present invention may be CaMV35S (U.S. Patent Nos. 5,322,938; 5,352,605; 5,359,142; and 5,530,196), FMV35S (U.S. Patent Nos. 6,051,753; 5,378,619), the PC1SV promoter (e.g. U.S. Patent No. 5,850,019, and SEQ ID NO: 24) , and AGRtu.nos (GenBank accession number V00087; Depicker et al, 1982; Bevan et al., 1983) promoter.

Advantages may also be gained by expressing heterologous genes using sequences encoding transit peptides. A transit peptide generally refers to a peptide molecule that, when attached to a protein of interest, directs that protein to a specific tissue, cell, subcellular location, or organelle. Examples include, but are not limited to, chloroplast transit peptides, nuclear targeting signals, and vacuolar signals. Chloroplast transit peptides are particularly useful in the present invention for directing the expression of the DMO enzyme to the chloroplast. Degradation of dicamba by endogenous reductases and ferredoxins found in plant cells is expected to promote DMO function. Plant chloroplasts are especially rich in reductases and ferredoxins. Thus, in a preferred embodiment for the production of transgenic dicamba-tolerant plants, sequences encoding peptides which direct dicamba-degrading oxygenases to chloroplasts may be used. Alternatively or additionally, a heterologous reductase and/or ferredoxin may also be expressed in the cell.

The DNA encoding the chloroplast targeting sequence can preferably be placed upstream (5') of the sequence encoding the DMO, but can also be placed downstream (3') of the coding sequence, or both upstream and downstream of the coding sequence. In particular, chloroplast transit peptides (CTPs) can be engineered to be fused to the N-terminus of proteins to be directed to the plant chloroplast. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and directed to chloroplasts by CTPs, which are removed in the import step. Examples of chloroplast proteins include ribulose-1,5,-bisphosphate carboxylase small subunit (RbcS2), ferredoxin, ferredoxin oxidoreductase, light-harvesting complex protein I and protein II, and thioredoxin F. It has been demonstrated in vivo and in vitro that non-chloroplast proteins can be targeted to chloroplasts by using protein fusions to CTP and that CTP is sufficient to target proteins to chloroplasts. For example, it has been shown that in transgenic plants, incorporation of appropriate chloroplast transit peptides, such as the Arabidopsis thaliana EPSPS CTP (Klee et al., 1987) and Petuniahybrida EPSPS CTP (della-Cioppa et al., 1986) will The heterologous EPSPS protein sequence is targeted to the chloroplast. Other exemplary chloroplast targeting sequences include the maize cab-m7 signal sequence (Becker et al., 1992; PCT WO 97/41228) and the pea glutathione reductase signal sequence (Creissen et al., 1991; PCT WO 97/41228) . In the present invention, AtRbcS4 (CTP1; U.S. Patent 5,728,925), AtShkG (CTP2; Klee et al., 1987), AtShkGZm (CTP2 synthetic; see SEQ ID NO of WO04009761; 14), and PsRbcS (Coruzzi et al., 1984) for example It is especially beneficial in the expression of DMO polypeptides.

The 5'UTR, which functions as a translation leader sequence, is a genetic element of DNA located between the promoter sequence and the coding sequence of a gene. The translation leader sequence is present in the fully processed mRNA upstream of the translation initiation sequence. The translation leader sequence can affect the processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences include maize and morning glory heat shock protein leaders (US Pat. No. 5,362,865), plant viral envelope protein leaders, plant rubisco leaders, and the like (Turner and Foster, 1995). In the present invention, 5'UTRs of particular interest are found to be GmHsp (US Patent 5,659,122), PhDnaK (US Patent 5,362,865), AtAnt1, TEV (Carrington and Freed, 1990), and AGRtunos (GenBank Accession No. V00087; Bevan et al. , 1983).

3' untranslated sequence, 3' transcription termination region, or polyadenylation region refers to a DNA molecule that is linked to and located downstream of the coding region of a gene and that includes polyadenylation signals that provide and are capable of affecting transcription, mRNA processing or other regulatory signals of gene expression. The polyadenylation signal functions in plants to cause the addition of poly(A) nucleotides at the 3' end of the pre-mRNA. The polyadenylation sequence can be from a native gene, from various plant genes, or from a T-DNA gene. An example of a 3' transcription termination region is the nopaline synthase 3' region (nos3'; Fraley et al., 1983). The use of different 3' untranslated regions has been described (Ingelbrecht et al., 1989). Polyadenylation molecules from the pea (Pisum sativum) RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al., 1984) and AGRtu.nos (Rojiyaa et al., 1987, Genbank Accession No. E01312) are especially useful for the present invention. benefit.

A DMO-encoding polynucleotide molecule expression unit can be linked to a second polynucleotide molecule in the expression unit containing a screenable/scorable marker or genetic element of a gene conferring a desired trait. Commonly used genes for screening putatively transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase, and chloramphenicol acetyltransferase (Jefferson, 1987; Teeri et al. , 1989; Koncz et al., 1987; De Block et al., 1984), green fluorescent protein (GFP) (Chalfie et al., 1994; Haseloff et al., 1995; and PCT application WO97/41228).

The second polynucleotide molecule may include, but is not limited to, a gene as a selectable marker. The second or another gene may provide desirable characteristics associated with plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance and may include genetic elements, so述遗传元件包含除草剂抗性(美国专利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)，增加的产量(美国专利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)，昆虫控制(美国专利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)，真菌疾病抗性(美国专利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)，病毒Resistance (US Patent 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; 5,304,730), nematode resistance (US Patent 6,228,992), bacterial disease resistance (US Patent 5,516,671), plant growth and development (US Patent 6,723,897; 6,518), production (US Patents 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), altered oil production (US Patents 6,444,876; 6,426,447; 6,380,462), high oil yield (US Patents 6,495,739; 5,608,149; 6,483,079 modified fatty acid content)国专利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)，高蛋白质产量(美国专利6,380,466)，果实成熟(美国专利5,512,466)，增强的动物和人类营养(美国专利6,723,837 6,653,530; 6,5412,59; 5,985,605; 6,171,640), biopolymers (US Pat. RE37,543; 6,228,623; 5,958,745 and US Patent Publication No. US20030028917), environmental stress resistance (US Pat. Secreted peptides (US Patents 6,812,379; 6,774,283; 6,140,075; 6,080,560), improved processing traits (US Patent 6,476,295), increased digestibility (US Patent 6,531,648), low raffinose (US Patent 6,166,292), industrial enzyme production ( 5,543,576), improved flavor (US 6,011,199), nitrogen fixation (US 5,229,114), hybrid seed production (US 5,689,041), fiber production (US 6,576,818; 6,271,443; 5,981,834; 5,869,720) and biofuel production (US Patent 5,998,700). Any of these or other genetic elements, methods and transgenes may be used in the present invention, as will be understood by those of skill in the art with reference to this disclosure.

The expression unit can be provided in Agrobacterium as a T-DNA between the right border (RB) and left border (LB) regions of the first plasmid together with a second plasmid carrying the T-DNA transfer and integration functions . The construct may also contain a plasmid backbone DNA segment that provides replication function and antibiotic selection in bacterial cells, such as an E. coli origin of replication, such as ori322, a wide host range origin of replication, such as oriV or oriRi, and the coding region for a selectable marker, Such as Spec/Strp encoding Tn7 aminoglycosyladenylate transferase (aadA), which confers resistance to spectinomycin or streptomycin, or the gentamicin (Gm, Gent) selectable marker gene. For plant transformation, the host bacterial strain is usually 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

Transformation of plant cells can be accomplished by any of the techniques known in the art for introducing transgenes into cells (see, e.g., Miki et al., 1993). Examples of such methods are considered to include almost any method of introducing DNA into cells. Methods that have been described include electroporation as set forth in US Patent No. 5,384,253; US Patent Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; Agrobacterium-mediated transformation as described in 6,384,301; and protoplast transformation as described in US Patent No. 5,508,184. By applying techniques such as these, cells of almost any plant species can be stably transformed and selected according to the present invention and these cells developed into transgenic plants.

The most widely utilized method for introducing expression vectors into plants is based on the natural transformation system of Agrobacterium (e.g., Horsch et al., 1985). A. tumefaciens and A. rhizogenes are phytopathogenic soil bacteria that genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of plants (eg, Kado, 1991). Descriptions of Agrobacterium vector systems and Agrobacterium-mediated gene transfer methods are provided by 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 that interact naturally with plants, such as Sinorhizobium, Rhizobium, and Mesorhizobium, can be modified to mediate gene transfer into many different plants. These plant-associated commensal bacteria can be rendered competent for gene transfer by obtaining disarmed Ti plasmids and appropriate binary vectors (Brothers et al, 2005).

B. Tissue Culture and Plant Regeneration

Regeneration of transformed plant cells into fertile plants can be achieved by culturing the explants first on germination medium and subsequently on rooting medium. Sometimes, explants can be grown on callus medium and then transferred to germination medium. Various media and transfer requirements for plant transformation and recovery of transgenic plants can be realized and optimized for each plant system. Accordingly, such media and culture conditions may be modified or replaced with nutritionally equivalent components or similar methods for selection and recovery of transgenic events.

The nutrient medium can be prepared as a liquid, but this can be solidified by adding to the liquid a substance capable of providing solid support. Agar is most commonly used for this purpose. Bactoagar, Hazelton agar, Gelrite and Gelgro are specific types of solid supports suitable for the growth of plant cells in tissue culture. Some cell types will grow and divide in liquid suspension or solid media, or both.

Recipient cell targets include, but are not limited to, meristematic cells, callus tissue, immature embryos, and gametic cells, such as microspore pollen, sperm cells, and egg cells. Any cell that can be used to regenerate fertile transgenic plants can be used in some embodiments. For example, immature embryos can be transformed, followed by selection and priming of calli and subsequent regeneration of fertile transgenic plants. Direct transformation of immature embryos avoids the need for long-term development of recipient cell cultures. Meristematic cells (i.e., plant cells capable of sustained cell division and characterized by an undifferentiated cytological appearance (commonly found in growing points or tissues of plants, such as root tips, shoot tips, lateral buds, etc.) can also be used as recipients. somatic plant cells. Because of their undifferentiated growth and capacity for organ differentiation and totipotency, whole transformed plants can be recovered from a single transformed meristematic cell.

Somatic cells are of many types. An embryogenic cell is an example of a somatic cell that can be induced to regenerate a plant through embryogenesis. Non-embryogenic cells are those cells that do not normally respond in this way.

Certain techniques for enriching recipient cells within a population of cells can be used. For example, Type II callus development, followed by artificial selection and culture of fragile embryogenic tissue, usually results in enrichment of recipient cells for, eg, microparticle transformation.

In some embodiments, recipient cells are selected after growth in culture. Cultured cells can be grown on solid supports or in liquid suspension. In either case, nutrients can be provided to the cells in the form of culture medium, and the environmental conditions controlled. There are many types of tissue culture media, which contain amino acids, salts, sugars, growth regulators and vitamins. Most media used in the practice of the present invention will have some similar components, while media may vary in composition and proportions of ingredients in accordance with known tissue culture practices. For example, multiple cell types are often grown in more than one medium, but will exhibit different growth rates and different morphologies depending on the growth medium. In some media, cells survive but do not divide. Media composition is also often optimized based on the species or cell type chosen.

Various types of media suitable for plant cell culture have been described previously. Examples of such media include, but are not limited to, N6 medium described by Chu et al. (1975) and MS medium (Murashige & Skoog, 1962). In some embodiments, it is preferred to use a medium with a somewhat lower ammonia/nitrate ratio, such as N6, to maintain the cells in a proembryonic state capable of continued division to facilitate production of recipient cells. Woody Plant Medium (WPM) can also be used (Lloyd and McCown, 1981).

Methods of maintaining cell cultures can facilitate their use as a source of recipient cells for transformation. Manual selection of cells for transfer to fresh medium, frequency of transfer to fresh medium, composition of the medium, and environmental factors including, but not limited to, light quality and quantity, and temperature are maintained for use as recipient cell sources factors in callus and/or suspension cultures. Alternating callus between different culture conditions is beneficial for enriching recipient cells in culture. For example, cells can be grown in suspension culture, but transferred to solid medium at regular intervals. After a period of growth on solid media, cells can be manually selected for return to liquid media. Repeating this sequence of transfers to fresh medium can be used to enrich recipient cells. Passing the cell culture medium through a 1.9 mm sieve can also be used to maintain the fragility of callus or suspension cultures and enrich for transformable cells when using such cell types.

C. Transgenic plants

Once a transgenic cell has been selected, the cell can be regenerated into a fertile transgenic plant using techniques well known in the art. Transformed plants are then analyzed to determine the presence or absence of the particular nucleic acid of interest in the DNA construct. Molecular analysis may include, but is not limited to, Southern blot (Southern, 1975) or PCR analysis, immunodiagnostic methods. Field assessments can also be used. These and other well-known methods can be performed to demonstrate the stability of transformed plants produced by the disclosed methods. These methods are well known to those skilled in the art (Sambrook et al., 1989).

Transgenic plants comprising the DMO coding sequences provided herein can thus be produced. In particular, economically important plants, including crops, trees, and other plants can be transformed with the DNA constructs of the invention such that they are dicamba-tolerant or have increased tolerance. Plants currently thought to be tolerant to an auxin-like herbicide can thus be transformed to increase their tolerance to that herbicide. Some non-limiting examples of plants that may be used in the present invention include alfalfa, barley, bean, sugar beet, broccoli, cabbage, carrot, brassica, cauliflower, celery, Chinese cabbage, corn, cotton, cucumber, eggplant, leek, Lettuce, melon, oats, onions, peas, peppers, peanuts, potatoes, zucchini, radishes, rice, sweet corn, sorghum, soybeans, spinach, squash, sugar beets, sunflowers, tomatoes, watermelons, and wheat.

Once a transgenic plant containing the transgene has been prepared, the transgene can be introduced by crossing into any plant that is vegetatively compatible with the first without directly transforming the second plant. Thus, as used herein, the term "progeny" refers to a descendant of any generation of a parent plant prepared according to the present invention, wherein the progeny comprise a selected DNA construct prepared according to the present invention. A "transgenic plant" can be a transgenic plant of any generation. As disclosed herein, "crossing" with a plant to provide a plant line with one or more added transgenes or alleles relative to the starting plant line is defined as the technique by combining the starting line with the Crossing of a transgenic or allelic donor plant line results in specific sequences being introduced into the plant line. To achieve this, the following steps can be carried out, for example: (a) planting the seeds of the first (starter line) and second (donor plant line comprising the desired transgene or allele) parent plants; ( b) growing the seeds of the second and second parent plants into flowering plants; (c) pollinating the flowers from the first parent plant with pollen from the second parent plant; and (d ) to harvest the seeds produced on the first plants with fertilized flowers.

The present invention thus provides transgenic plant tissues comprising the DMO-encoding nucleic acids provided herein. The tissue may have been directly transformed with a DMO-encoding nucleic acid or may have inherited nucleic acid from a progenitor cell. Tissues provided by the invention specifically include, but are not limited to, cells, embryos, immature embryos, meristematic cells, immature tassels, microspores, pollen, leaves, anthers, roots, root tips, flowers and seeds. The invention thus provides any such tissue, including any plant part, comprising a nucleic acid described herein. Seeds are especially beneficial in the form of grains for commercial or food applications, and for planting to grow additional crops.

Example

The following examples are included to illustrate embodiments of the invention. Those of skill in the art will appreciate that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain substances of chemical or physiological relative may be substituted for those described herein to achieve the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Example 1

Vector Construction of DMO Gene for Genetic Modification

DMOc variant coding sequences were initially generated by PCR amplification from the DMOw template. In this amplification, the coding region of DMOw was amplified from plasmid pPLH1 containing the DMOw gene as a 3.5 kbp Xho I/Sst I fragment of DNA from Pseudomonas maltophilia strain DI-6. For DNA amplification, use the 5' primer and the 3' primer, the 5' primer inserts a Nco I restriction site near the 5' end of the PCR product and the codon for alanine immediately after the ATG start codon, the 3' primer An Xba I restriction site was created at the 3' end of the PCR product (details of the procedure are provided below). The 112W to 112C change was subsequently identified by nucleic acid sequencing.

To generate the plant transformation vector, the DMOc gene was inserted into the pRTL2 vector (Carrington and Freed, 1990) with NcoI and XbaI sites added to the 5' and 3' ends of the coding region, respectively, thereby fusing the coding region to the tobacco Etch virus (TEV leader) translational enhancer element. Specially designed PCR primers were used to introduce a 5' Nco I site and add a GCC codon (alanine) after the ATG start codon and create an Xba I restriction site at the 3' end of the coding region. To allow delivery of DMOc to chloroplasts, the chloroplast transit peptide coding region from the pea Rubisco subunit gene (Coruzzi et al., 1983) was placed upstream of the DMOC coding region to allow targeting to the chloroplast. The transit peptide coding sequences carried on the Bgl II and EcoR I fragments were 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 where an Nco I site was inserted 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 such that the transit peptide sequence was located directly upstream of the coding region of the DMO gene and in frame with it. The cassette consisting of the TEV leader sequence, transit peptide region, and DMODNA coding sequence was excised from the pRTL2 vector with Xho I and Xba I and cloned into the pKLP36 vector (U.S. 5,850,019; Figure 5), using the same restriction enzyme sites used for This cassette was linked to the PC1SV promoter and the PsRbcS2-E9 polyA sequence. The new vector was labeled pKLP36-TEV-TP-DMOc (also referred to as pKLP36-DMOc) and was deposited with the American Type Culture Collection (ATCC) on February 2, 2006, 10801 University Boulevard, Manassas, Va. 20110-2209 USA, and assigned ATCC deposit number PTA-7357.

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

Restriction and other enzymes were obtained from Fermentas or Invitrogen. DIG-11-dUTP (base-labeled), CSPD (ready to use), DIG III molecular weight marker, anti-digoxigenin-AP (Fab fragment) and blocking reagent were obtained from Roche. The prehybridization solution ULTRAhyb was obtained from Ambion. DIG-RNA molecular weight marker I was obtained from Roche. Anti-rabbit IgG, peroxidase-linked antibody (donkey) and Hybond ECL (nitrocellulose) membranes were obtained from Amersham Biosciences. DNA, RNA, Western blotting, recombinant DNA techniques, and other molecular biology methods were performed using standard techniques (Ausubel et al., 1995).

Example 2

Generation and analysis of transgenic plants

Tobacco, tomato, soybean and Arabidopsis were used to transgenicly express the genetically engineered DMOc gene and to demonstrate dicamba tolerance in plants expressing the gene. The DMOc coding sequence in the binary vector pKLP36 was introduced by triparental mating (Ditta 1980) into Agrobacterium strain C58C1 containing the disarmed Ti plasmid pMP90 (Koncz and Schell, 1986). The resulting transconjugates were used for tobacco (cv Xanthi) and tomato (cv Rutgers) transformation using the leaf disk protocol described by Horsch et al. (Horsch 1985). Arabidopsis were transformed by the floral dip technique (Clough and Bent, 1998). Transformation of soybean cultivars Thorne and NE-3001 was performed by the Agrobacterium cotyledonium-mediated transformation system (Zhang et al., 1999).

Agrobacterium-mediated gene transfer of the DMOc gene to the tobacco plant genome produced _T1 plants of several independent origins. Plants were tested for the presence and expression of the DMOc gene by DNA, RNA and Western blot analysis. Figure 2 illustrates that although all transgenic plants in this analysis (lanes 1-6) contained the same DNA fragment as the cloned DMO gene (lane 8) after restriction enzyme digestion, mRNA transcript and DMO protein levels varied between transformants Significantly different. For example, plants whose extracts are depicted in lane 5 showed relatively high levels of DMO mRNA but very low levels of the enzyme. In contrast, almost the same level of DMO mRNA in the extract shown in lane 3 was coupled with the high expression level of DMO. However, it was shown that events with strong expression could always be obtained by this method.

Plants in the greenhouse were sprayed with solvent and commercial grade dicamba (Clarity; BASF) using a compressed air, motor driven orbital sprayer with a flat fan 8002E nozzle at 1.87 mph. Additives included: 1.25% v/v 28% ammonium urea nitrate and 1.0% v/v nonionic surfactant. Solutions containing dicamba at various concentrations were applied at 182 L/ha (40 gallons per acre). Soybean field plants were sprayed with Clarity herbicide at 2.8 kg/ha (2.5 lb/ac).

Tobacco plants, like most dicots, are quite sensitive to dicamba treatment. This is illustrated by comparing untreated and non-transgenic tobacco plants treated with incremental dicamba. Symptoms of herbicide damage were easily detected after spraying dicamba at a level of 0.017 kg/ha. Symptoms are quite severe at levels commonly used for weed control in agricultural applications: 0.28 kg/ha and 0.56 kg/ha.

Post-emergence treatment of transgenic tobacco plants containing DMOc with 5.6 kg/ha (10 to 20 times higher than normal application rates) caused few, if any, symptoms, while non-transgenic plants suffered severe damage. Injury to the lower leaves of transgenic plants can be replicated by spraying the plants with a surfactant-containing solvent solution (used as a vehicle for dicamba application). The leaves produced by the transgenic plants treated with dicamba showed no visible signs of damage. Transgenic tomato plants carrying the genetically engineered DMOc gene also showed no damage when sprayed with high levels of dicamba, in this particular case first 0.56 kg/ha followed by 5.6 kg/ha. Arabidopsis expressing the DMOc gene also displayed 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 the DMOc gene lacking the transit peptide coding region also tolerated post-emergence treatments at average concentrations of dicamba that were only slightly less tolerant than those with the transit peptide coding region. Plant tolerance of the DMOc gene. In this study, two _T1 tobacco plants were compared treated with 2.2 kg/ha of dicamba, one carrying DMOc lacking the chloroplast transit peptide and the other completely lacking the DMOc gene due to genetic segregation. The latter plants were completely susceptible to the injury caused by the dicamba treatment and died of the treatment (Figure 3). Transgenic plants carrying the DMOc gene lacking the transit peptide were completely tolerant to the 2.2. kg/ha treatment of dicamba. Genetic studies of DMOc gene inheritance in transgenic tobacco plants also showed that this trait is inherited in a normal Mendelian manner in most plants and maintains the original expression level with respect to herbicide tolerance.

In soybean, more than 50 R ₀ transgenic soybean events were generated and T ₁ , T ₂ and T ₃ generation seeds were collected. Since the Agrobacterium tumefaciens binary vector system was used, transgenic plants containing the DMOc gene with and without the marker gene were recovered. In either case, the majority of transgenic soybean lines showed strong tolerance to 2.8 kg/ha and 5.6 kg/ha dicamba treatments under greenhouse conditions and to 2.8 kg/ha (max. Test level) Strong tolerance of dicamba. These results suggest a broad safety margin for transgenic soybeans and other crops carrying the DMOc gene associated with highly effective control of a broad range of broadleaf weeds.

The high level of dicamba tolerance in transgenic soybean plants bearing the DMO gene demonstrates that dicamba can be applied in soybean fields to strongly inhibit competition with broadleaf weeds without crop damage. In addition, dicamba-tolerant crops could be an important addition to current weed control options using genetically modified herbicide-tolerant crops. That is, they are valuable assets in controlling existing herbicide-tolerant weeds and inhibiting the emergence of additional herbicide-tolerant weeds that could ultimately threaten current weed control long-term use and value of herbicide- and herbicide-tolerant crops.

Example 3

Comparison of overexpression, purification and enzymatic properties of DMOw and DMOc

A. Cloning and overexpression

The wild-type (DMOw) and variant (DMOc) DMO coding sequences were cloned from the plasmids pMON95900DMO (DMOw) and pMON58499DMO (DMOc) into the vector pET28b (Novagen,. San Diego, CA) and transformed into Escherichia coli BL21 cells (Novagen, San Diego, CA). Cells were grown in 1 liter of LB medium at 37°C to an absorbance at 600 nm of 0.4 to 0.6. Protein expression was induced by adding 50 μM Fe(NH ₄ )SO ₄ , 100 μM Na ₂ S, and 1 mM isopropyl-β-thiogalactopyranoside (IPTG) and switching cells to 15°C. After 48-72 hours at 15°C, cells were harvested by centrifugation at 10,000 xg for 20 minutes. Cells were stored at -20°C for later use.

The protein expression yields of DMOw and DMOc in E. coli are different. While DMOw yields about 100 to 150 mg of pure protein per liter of LB medium, DMOc yields are 10-fold lower, or about 10 to 15 mg of pure protein per liter. This was unexpected since E. coli has no rare codons for cysteine and only one codon for tryptophan, but in both cases demonstrates that proteins can be produced heterologously in E. coli regardless of yield. The amount of protein in inclusion bodies was low in both cases, suggesting that the protein was mainly retained in the soluble fraction.

See His-tagged recombinant DMOw protein from Pseudomonas maltophilia strain DI-6 and His-tagged recombinant DMOc expressed in E. coli strain BL21 were purified to homogeneity by Ni-NTA column chromatography. Cells were suspended in lysis buffer (100 mM NaPi pH 8.0, 300 mM NaCl, and 10 mM imidazole) and disrupted by sonication. Cell lysates were centrifuged at 55000 xg for 1 hour. The supernatant was loaded onto a Ni-NTA column, and the column was washed with wash buffer (100 mM NaPi pH 8.0, 300 mM NaCl, and 20 mM imidazole) to remove proteins non-specifically attached to the resin. His-tagged proteins were eluted with elution buffer (100 mM NaPipH8.0, 300 mM NaCl, and 250 mM imidazole). For DMOw purification, a step gradient was sufficient to obtain 95% pure enzyme, while for DMOc a linear gradient of imidazole concentrations from 20 to 250 mM was required to achieve the same level of purity. The enzyme eluted from the column was approximately 95% pure as estimated from Western blot of the enzyme after size fractionation on SDS-polyacrylamide gel electrophoresis. A single major band migrating to ~40kDa (37.3kDa DMOase plus 3kDAHis tag) indicates that the correct protein has been overproduced.

B. Assays and Steady State Kinetics of DMOc and DMOw

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

Stock solutions of dicamba (100, 200, 400, 800, 1000, 2000, 5000, and 10000 [mu]M), 0.1M KPi pH 7.2, 0.1M _FeSO4 , 0.1M NADH, and 1M _MgCl2 were used. The assay was performed at 30 °C _for 20 min and the reaction was quenched by adding 40 μl _H2SO4 . To measure activity, DMO was coupled to excess purified ferredoxin and reductase from Pseudomonas maltophilia strain DI-6.

Because the assay of DMO activity is a discontinuous assay, it is important to establish the time that the assay must be run to obtain meaningful kinetic parameters. Thus, the assay had to be run under initial conditions since the amount of DCSA produced was linear with time for the assay being run (Figure 4). The results suggest that the assay can be run for 20 to 30 minutes and still be linear. Figure 5 shows that the assay performed in the presence of 0.1M Kpi buffer had an optimum pH of 7.2 and a temperature optimum was found to be about 37°C (Figure 6).

C. Kinetic Data Analysis

The Mie-Man parameters (Equation 1) were determined by substituting the data into the nonlinear steady-state equation (Equation 1). Data were analyzed with Sigma plot 8.0 (Jandel Scientific).

V _o ＝Vmax*[S]/(Km+[S]) Equation 1

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

The results indicated that the kinetic properties of DMOw and DMOc were different. For example, the Mie-Mann parameters calculated for DMOw and DMOc are: Km = 49 ± 7 μM and Vmax = 633 ± 24 nmol/min/mg for DMOw and Km = 20.5 ± 5 μM and Vmax = 676 ± 37 nm for DMOc mol/min/mg. These results are shown in Figures 10 and 11 and summarized in Table 1 below. Furthermore, two additional analyzes performed for DMOw and DMOc yielded similar results (Tables 2 and 3).

It can be seen that DMOw and DMOc enzymes have different properties in terms of catalytic efficiency: DMOc is a five-fold better enzyme than DMOw by this analysis. The pH profile of DMOc is different from that of DMOw. First, DMOc appears to be sensitive to the buffer system used (TRIS vs. KPi) compared to DMOw (Figures 9, 12 and 13). Second, DMOc showed stable activity over a wide range of pH when assayed in KPi buffer compared to TRIS (where DMOc activity decreased with increasing pH units). The temperature profiles of DMOc incubated in KPi or TRIS buffer were similar.

Looking at the temperature profile between the two forms of the enzyme, DMOw functions better at 37°C, while DMOc functions better at somewhat lower temperatures (Figure 9). Figure 9 shows a lower temperature optimum for DMOc, which can be used in transgenic plants early in the growth phase.

Table 1. Steady-state kinetic parameters of DMOw and DMOc

enzyme Km(M) Vmax(U/mg) kcat(s ^-1 ) Kcat/Km(M ^-1 s ^-1 ) wxya 49±7×10 ^-6 633±24×10 ^-3 36.63 7.47×10 ⁵ DMOc 20±5×10 ^-6 676±37×10 ^-3 70.41 35.21×10 ⁵

Table 2. Overview of Mie-Mann parameters for DMOw

Research number Rsqr Vmax (nanomol/min/mg) Km (μM) 1. 0.983 633±24 49±7 2. 0.988 583±18 46±5 3. 0.987 590±19 46±5.5

Table 3. Overview of Mie-Mann parameters for DMOc

Research number Rsqr Vmax (nanomol/min/mg) Km (μM) 1. 0.933 713±43 21±6 2. 0.948 676±37 20±5

Example 4

Bioinformatic analysis of conserved regions of DMO

Bioinformatic analysis was performed to compare the polypeptide sequences of DMO and other iron-sulfur oxygenases and to identify conserved regions. Initially, 78 sequences were selected for analysis based on an e-value cutoff of 1e-08 and a DMO sequence coverage of 70% on 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 non-haem Fc domains (Herman et al., 2005). Of these 78 sequences, 68 contained two domains, while 10 had only one domain. 68 molecules with these two domains were used for further motif analysis.

Alignment of these 68 molecules with two domains at different levels of identity revealed a novel WXWX motif. Although some sequences do not contain the motif, phylogenetic analysis shows that molecules without the motif fall into certain clades in the tree that do not belong to the same group as molecules with the motif. Those sequences without this motif were therefore removed from the original data set, leaving 52 remaining sequences that were re-aligned for further analysis.

The 52 re-aligned sequences showed conservation around two W residues of the form: WX ₁ WX ₂ G (W is Trp, G is a Gly residue, X ₁ is a nonpolar residue, X ₂ is any amino acid). The second W in this case corresponds to position 112 of SEQ ID NO:1. WXG of the WX ₁ WX ₂ G motif has recently been reported, and proteins with the WXG motif are associated with the cellular secretion system (Desvaux et al., 2005).

Tryptophan (W) and cysteine (C) are residues of significantly different sizes. W is a large residue and C is a relatively small residue. Because both W and C are polar amino acids, they share some common features such as proton donating. Since the W residue is encoded by TGG and Cys by TGC and TGT, certain transitions (G->C or G->T) in the third codon can generate errors from W to C or from C to W Sense mutation. Such switches have been identified in nature and biological function and activity are altered by those mutations (see, e.g., the BRCA1 gene in hereditary breast and ovarian cancers (Xiaoman and Jinghe, 1999); coagulation factor XII deficiency (Wada et al., 2003), and lipoprotein lipase mutations in type I hyperlipoproteinemia (Hoffmann et al., 2000)).

Thus, the previous results indicate that although DMO is unique and has low identity to known enzymes, W112 is conserved among other related iron-sulfur oxygenases. In addition, position 112 is surrounded by two conserved functional domains (Figure 18). Furthermore, W to C conversion often affects biological activity. It was especially surprising to find that DMOc produces a functional enzyme with superior kinetic parameters than the wild-type DMOw enzyme and when expressed in transgenic plants provides high levels of tolerance to dicamba.

Example 5

Chloroplast-encoded DMOs confer high levels of dicamba tolerance

To determine whether DMO can function only within chloroplasts and to investigate the possibility of limiting "gene dispersal" through pollen floating, constructs were generated based on the pFMDV1 vector (e.g., Svab et al., 1990) to allow the DMOc gene to pass through homologous recombination Integrated into the chloroplast genome of tobacco and used antibiotic resistance selection to isolate transformants (Figure 14). In this construct, the DMOc gene coding region is driven by the psbA chloroplast gene promoter containing the complete psbA 5'UTR sequence. Preliminary Southern blot analysis of antibiotic-resistant transgenic plants (Figure 15A) indicated the presence of the DMOc transgene (5.6 kb band) and the native gene region (3.3 kb band) replaced by homologous integration of the DMOc gene in the chloroplast genome (i.e., Chloroplasts are heterogeneous for native genes and DMOc transgenes). Repeated regeneration and selection of transgenic plants on antibiotic-containing media resulted in apparently homogeneous chloroplasts bearing the DMOc gene segment but without the replaced native gene region (Fig. 15B).

Progeny of _T1 , _T2 and _T3 generations of chloroplast transformants from two independent sources were tested for tolerance to treatment with different doses of dicamba. All showed high levels of tolerance. Indeed, when sprayed with dicamba at a rate of 28 kg/ha (25 lb/ac), chloroplast genome transformants showed no apparent damage (as opposed to "solvent only damage" to lower leaves) (Figure 16) and when sprayed with 112 and 224 kg/ha of extremely high dicamba applications treated plants, only temporary damage was observed. At these extremely high levels, the initial damage is mainly caused by surfactants and other components in the solvents used to deliver dicamba; tissue growing from the damaged apex shows a near-normal to normal phenotype, After the initial growth retardation showed no decrease in growth rate and maintained the ability to produce the usual number and quality of seeds.

The results are consistent with the possibility that reduced ferredoxin in tobacco chloroplasts may be the DMO electron donor required for the oxidation of dicamba to DCSA. As a direct test of this possibility, the support of dicamba by purified spinach ferredoxin was examined in the presence and absence of DMO purified from Pseudomonas maltophilia DI-6 strain or overproduced and purified from E. coli. Ability to convert to DCSA (Table 4). The results indicate that reduced ferredoxin from spinach or Clostridium is fully capable of donating electrons to DMO in vitro, as measured by dicamba degradation or DCSA appearance.

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

Table 4A - Degradation of Dicamba

response type Degradation of dicamba (%) (Ferr+Red) _DI-6 +NADH 0 (Oxy+Ferr+Red) _DI-6 +NADH 86 (Oxy) _DI-6 + (Ferr) _spinach + (Ferr:Oxidored) _spinach + NADPH 83 (Oxy) _DI-6 + (Ferr:Oxidored) _spinach + NADPH ND (Oxy) _DI-6 + (Ferr) _Clostridium + (Ferr:Oxidored) _Spinach + No NADPH ND (Oxy) _DI-6 + (Ferr) _Clostridium + (Ferr:Oxidored) _Spinach + NADPH 82 (Ferr) _Clostridium + (Ferr:Oxidored) _Spinach + NADPH ND

Table 4B - Formation of DCSA

response type Formation of DCSA (%) (Ferr+Red) _DI-6 +NADH ND (Oxy+Ferr+Red) _DI-6 +NADH 100 (Oxy) _DI-6 + (Ferr) _spinach + (Ferr:Oxidored) _spinach + NADPH 95 (Oxy) _DI-6 + (Ferr:Oxidored) _spinach + NADPH 2.5 (Oxy) _DI-6 +(Ferr) _Spinach +(Ferr:Oxidored) _Spinach +No NADPH 1.2 (Oxy) _DI-6 + (Ferr) _Clostridium + (Ferr:Oxidored) _Spinach + NADPH 90 (Ferr) _Clostridium + (Ferr:Oxidored) _Spinach + NADPH 1.5

ND, not determined

Although the results in Figure 2 indicate that DMO levels produced are variable and that sometimes DMOc levels do not correlate strongly with dicamba tolerance levels, the results indicate that high levels of tolerance to dicamba can be consistently obtained. Production of DMOc from the nuclear-localized DMOc gene and from the chloroplast-localized DMOc gene in transformants is shown. In nuclear transformants, an additional high level of total DMOc was constituted relative to total protein, and the amount of DMOc in chloroplast transformants was not significantly different and sometimes lower than in nuclear transformants. Estimation of relative enzyme activities in cell-free extracts of leaf tissue samples indicated that a higher percentage of DMOc produced in chloroplasts was more active than DMOc synthesized in the cytoplasm and thought to be translocated into chloroplasts.

Dicamba tolerance was achieved without co-transformation with the ferredoxin or reductase genes in all plants analyzed. The results indicated that the plants contained one or more molecules that could transfer the required electrons to DMO to allow the conversion of dicamba to 3,6-dichlorosalicylic acid (DCSA). Using the transit peptide sequence, the initial targeting of DMO to the chloroplast targets the potential utilization of reduced ferredoxin that is fully available in the chloroplast. In this regard, it is important to note that transformation of tobacco plants with the DMOc gene construct lacking the chloroplast peptide coding sequence unexpectedly resulted in plants tolerant to the post-emergence treatment of dicamba. However, results from limited trials with a small number of _T1 generation plants indicated that the level of tolerance obtained with these transgenic plants was on average slightly lower than that obtained with tobacco plants producing DMOc containing the transit peptide. These observations raise interesting questions about molecules in transgenic plants that could productively supply electrons to DMO. The fact that homogeneous chloroplasts producing DMO endogenously from the DMOc gene integrated into the chloroplast genome showed resistance to extremely high levels of dicamba (Fig. Both the fact of function (Table 4) suggest that chloroplast ferredoxin can productively interact with DMO to allow electron transfer. However, the electron source of DMO produced from nuclear genes lacking the chloroplast transit peptide coding sequence remains unknown. Given that ferredoxin is not located outside the chloroplast of plants, the possibility must be considered that an unknown cytoplasmic protein might provide DMO with a stable electron supply. Alternatively, the DMO itself may contain available chloroplast transit peptides that allow sufficient DMO to enter the chloroplast to provide protection against dicamba entry into the cell following dicamba treatment.

* * * * * *

All of the compositions and/or methods disclosed and claimed herein can be made and practiced in light of the present disclosure without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and/or methods and steps or sequence of steps of the methods disclosed herein without departing from the concept, spirit and scope of the present invention. More particularly, it will be apparent that certain substances of chemical and physiological relevance may be substituted for those described herein and the same or similar results will be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

references

The references listed below are incorporated herein by reference to the extent that they supplement, explain, provide a background, or teach the methods, techniques and/or compositions used herein.

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sequence listing

<110>CLEMENTE. THOMAS E.

DUMITRU, RAZVAN

  FENG, PAUL C.C.

 FLASINSKI, STAN

WEEKS, DONALD P.

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Claims (24)

1. An isolated nucleic acid molecule selected from the group consisting of: A) a nucleic acid molecule encoding a 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 encoding a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 1, wherein the latter polypeptide has dicamba monooxygenase activity and at amino acid 112 corresponding to SEQ ID NO: 1 Cysteine is included in the position. 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule encodes dicamba monooxygenase encoded by plasmid pKLP36-TEV-TP-DMOc (ATCC Accession No. PTA-7357). 3. A DNA construct comprising the nucleic acid molecule of claim 1 operably linked to a promoter. 4. The construct of claim 3, wherein the promoter is functional in plant cells. 5. The construct of claim 3, wherein said isolated nucleic acid molecule is operably linked to a chloroplast transit peptide. 6. A polypeptide sequence comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1, wherein the polypeptide has dicamba monooxygenase activity and comprises at a position corresponding to amino acid 112 of SEQ ID NO: 1 cysteine. 7. A plant cell transformed with the nucleic acid molecule of claim 1. 8. The cell of claim 7, wherein said plant cell is a dicot plant cell. 9. The cell of claim 7, wherein said plant cell is a monocotyledonous plant cell. 10. The cell of claim 7, wherein said dicot plant cell is a soybean, cotton, corn or rapeseed plant cell. 11. A plant tissue culture comprising the cells of claim 7. 12. A transgenic plant transformed with the nucleic acid molecule of claim 1. 13. The transgenic plant of claim 12, wherein said plant is a dicot. 14. The transgenic plant of claim 12, wherein said plant is a monocot. 15. The transgenic plant of claim 12, wherein said plant is a soybean, cotton, corn or rapeseed plant. 16. A method of producing a dicamba-tolerant plant comprising introducing the construct of claim 3 into a plant. 17. The method of claim 16, comprising introducing the construct of claim 3 into a plant by stably transforming a starting plant cell and regenerating said cell into a dicamba-tolerant plant. 18. The method of claim 16, wherein a dicamba-tolerant plant is produced by selfing a parent plant or crossing a second plant, wherein the parent plant and/or the second plant comprise a transformation construct and the A dicamba-tolerant plant inherits the transformation construct from said parent plant and/or a second plant. 19. A method of controlling the growth of weeds in a crop growing environment comprising the plant of claim 12 or its seeds, comprising applying to said crop growing environment an amount of dicamba herbicide effective to control weed growth. 20. The method of claim 19, wherein the dicamba herbicide is applied on top of said crop growing environment. 21. The method of claim 19, wherein the amount of the dicamba herbicide does not damage the plant of claim 12 or its seeds and damages a plant having the same genotype as the plant of claim 12 but lacking the nucleic acid of claim 1 . 22. A method of producing food, feed or industrial product comprising: a) obtaining the plant or part thereof of claim 12; and b) preparing food, feed or industrial products from said plants or parts thereof. 23. The method of claim 22, wherein the food or feed is oil, meal, grain, starch, flour or protein. 24. The method of claim 22, wherein said industrial product is a biofuel, fiber, industrial chemical, pharmaceutical or nutritional product.

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