HK1136604B - Chloroplast transit peptides for efficient targeting of dmo and uses thereof - Google Patents

Abstract

The invention provides for identification and use of certain chloroplast transit peptides for efficient processing and localization of dicamba monooxygenase (DMO) enzyme in transgenic plants. Methods for producing dicamba tolerant plants, methods for controlling weed growth, and methods for producing food, feed, and other products are also provided, as well as seed that confers tolerance to dicamba when it is applied pre- or post-emergence.

Description

Chloroplast transit peptides for efficient targeting of DMO and uses thereof

Background

This application claims priority from U.S. provisional patent application 60/891,675 filed on 26/2/2007 and U.S. patent application 11/758,659 filed on 5/6/2007, the disclosures of which are incorporated herein by reference in their entirety.

Technical Field

The invention relates to the technical field of plant biology. More specifically, the invention relates to the identification and use of chloroplast transit peptides that allow efficient processing and localization of dicamba monooxygenase in plants.

Description of the related Art

DMO (dicamba monooxygenase) catalyzes the degradation of the herbicide dicamba (3, 6-dichloro-o-anisic acid) to the non-toxic 3, 6-dichlorosalicylic acid (3, 6-DCSA) in plants, thereby conferring herbicide tolerance. The activity of DMO requires two intermediate proteins for the transfer of electrons from NADH to dicamba: reductase and ferredoxin (U.S. Pat. No.7,022,896; Herman et al, 2005). However, dicamba tolerance in transgenic plants has been demonstrated by transformation with DMO alone, indicating that plant endogenous reductases and ferredoxin can be replaced in electron transfer. Plant ferredoxin involved in electron transfer is located in the plastid. Thus, in order to obtain efficient performance of DMO and thus improved dicamba tolerance, targeting of DMO to chloroplasts is required.

In many cases, this targeting can be achieved by the presence of an N-terminal extension called the Chloroplast Transit Peptide (CTP) or plastid transit peptide. If the expressed polypeptide is to be compartmentalized in the plant plastid (e.g., chloroplast), the chromosomal transgene of bacterial origin must have a sequence encoding a CTP sequence fused to a sequence encoding the expressed polypeptide. Thus, localization of the exogenous polypeptide into the chloroplast is typically accomplished by operably linking a polynucleotide sequence encoding a CTP sequence to the 5' region of the polynucleotide encoding the exogenous polypeptide. CTP is removed during the transfer into the plastid through a processing step. However, the processing efficiency may be affected by the amino acid sequence of CTP and the sequence near the amino terminus of the peptide.

Weeks et al (U.S. Pat. No.7,022,896) describe the potential use of maize cab-m7 signal sequences (see, Becker et al, 1992 and PCT WO 97/41228; GenBank accession number X53398) and pea glutathione reductase signal sequences (Creissen et al, 1992 and PCT WO97/41228) in targeting DMOs to plant plastids, but do not give data on processing or targeting efficiency. Pea Rubisco small subunit (RbcS) CTP, which contains a 27 amino acid sequence that includes a pea Rubisco small subunit coding sequence, has also been used to target DMO to chloroplasts (e.g., U.S. provisional application 60/811,152). However, it has been found during western blot (western blot) analysis that this pea RbcS CTP produces a correctly processed DMO protein band (-38 kDa), but also produces a larger band (-41 kDa) corresponding to 27 amino acids of the DMO and RbcS coding regions. Additional amino acids may adversely affect the activity of DMO. In addition, the additional protein in the transgenic product poses regulatory hurdles due to incomplete processing of the DMO, and additional efforts in product characterization are required for product registration purposes in government agencies, thereby increasing the cost of product registration. Thus, there is a need to identify CTPs that efficiently produce properly processed DMO, thereby providing the advantages of complete DMO activity and ease of product characterization.

Summary of The Invention

One aspect of the invention relates to a recombinant DNA molecule comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a nucleotide sequence encoding dicamba monooxygenase, wherein the nucleotide sequence encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11. In certain embodiments, the recombinant DNA molecule comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 12-22. In certain embodiments, the recombinant DNA molecule comprises a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs: 24. 26, 28, 30, 32, 34, 36, 38, and 40. A DNA construct comprising a DNA molecule operably linked to a promoter functional in a plant cell is also an aspect of the present invention.

In another aspect, the invention includes a plant cell transformed with a DNA molecule comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a nucleotide sequence encoding dicamba monooxygenase, wherein the chloroplast transit peptide sequence is selected from the group consisting of SEQ ID NOs: 1-11. In certain embodiments, the recombinant DNA molecule comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 12-22. In certain embodiments, the DNA molecule comprises a nucleotide sequence encoding a polypeptide selected from the group consisting of SEQ id nos: 24. 26, 28, 30, 32, 34, 36, 38, and 40, wherein the DNA molecule is operably linked to a promoter functional in a plant cell. In a particular embodiment, the DNA molecule comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 23. 25, 27, 29, 31, 33, 35, 37 and 39.

In certain embodiments, the plant cell is a dicot plant cell. In other embodiments, the plant cell is a monocot plant cell. In particular embodiments, the plant cell is a soybean, cotton, maize, or rapeseed plant cell. The invention also relates to plant tissue cultures comprising such cells, to transgenic seeds and transgenic plants comprising such cells. In certain embodiments, the transgenic seed or plant is a dicot seed or plant. In other embodiments, the transgenic seed or plant is a monocot seed or plant. The transgenic seed or plant may be a soybean, cotton, maize or rapeseed seed or plant.

The present invention further relates to a method for producing a dicamba tolerant plant comprising: introducing into a plant cell, and regenerating a plant therefrom, a recombinant DNA molecule comprising a nucleotide sequence encoding a chloroplast transit peptide operably linked to a nucleotide sequence encoding dicamba monooxygenase, wherein the sequence encoding a chloroplast transit peptide is selected from the group consisting of SEQ ID NOs: 12-22. In certain embodiments, the recombinant DNA molecule comprises a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs: 24. 26, 28, 30, 32, 34, 36, 38, and 40. The DNA molecule may be operably linked to a promoter that is functional in a plant cell. The method can further comprise producing a dicamba tolerant plant by crossing the parent plant with itself or a second plant, wherein the parent plant and/or the second plant comprises the DNA construct, and the dicamba tolerant plant inherits the DNA construct from the parent plant and/or the second plant.

A method of expressing dicamba monooxygenase in a plant cell is a further aspect of the invention, the method comprising operably linking a selected CTP to a sequence encoding dicamba monooxygenase.

In another aspect, the invention relates to a method of controlling weed growth in a crop growing environment, comprising: such plants or seeds thereof are planted and a crop growing environment is applied with dicamba herbicide in an amount effective to control weed growth. The dicamba herbicide may be applied to the crop growing environment from above, whereby the amount of dicamba herbicide does not damage the plant or its seed but damages a plant or seed of the same genotype as the plant or its seed but lacking the construct.

A further aspect of the invention relates to a method of producing a food, feed or industrial product comprising:

a) obtaining a plant comprising a nucleotide sequence encoding a promoter functional in a plant cell operably linked in a 5 'to 3' direction to a nucleotide sequence encoding a chloroplast transit peptide and a nucleotide sequence encoding dicamba monooxygenase or a portion thereof; b) preparing food, feed, fiber or industrial products from said plant or part thereof.

In certain embodiments of the method, the food or feed is a grain, meal, oil, starch, flour, or protein. In other embodiments of the method, the industrial product is a biofuel, fiber, industrial chemical, pharmaceutical, or nutraceutical.

Dicamba tolerant seeds comprising DNA encoding a chloroplast transit peptide operably linked to DNA encoding dicamba monooxygenase, which provide protection against pre-emergence (pre-emergence) application of dicamba, are a further aspect of the invention. In certain embodiments, the dicamba tolerant seed comprises a nucleotide sequence encoding a chloroplast transit peptide, e.g., a sequence selected from seq id NOs: 12-22. The dicamba tolerant seed may further comprise a polynucleotide encoding a sequence selected from SEQ ID NOs: 24. 26, 28, 30, 32, 34, 36, 38, and 40.

Another aspect of the invention relates to a method of increasing the standability of a monocot plant comprising: a) obtaining and growing a plant produced by crossing a parent plant with itself or a second plant, wherein the parent plant and/or second plant comprises the DNA construct and the dicamba tolerant plant inherits the DNA construct from the parent plant and/or second plant; b) plants were treated with dicamba. In certain embodiments, the plant is a maize plant. In other embodiments, parameters related to stand ability including the shape, number, length and/or configuration of the strut roots, percent lodging and yield may be measured.

Brief Description of Drawings

FIG. 1: use of a CTP-DMO construct for the correct processing of DMO and for providing dicamba tolerance.

Detailed Description

In accordance with the present invention, compositions and methods are provided for more efficient expression and transport of Dicamba Monooxygenase (DMO) polypeptides to chloroplasts in plant cells. The compositions and methods of the invention are therefore useful in enhancing tolerance of plants and cells to the herbicide dicamba. Increased expression of DMO and tolerance to dicamba can be achieved, inter alia, by targeting DMO to chloroplasts with a Chloroplast Transit Peptide (CTP).

Surprisingly, however, the inventors have found that certain CTPs do not function well in combination with DMO. For example, some CTPs do not result in sufficient protein expression. This includes improper expression of the protein, as well as production of proteins of varying sizes and incomplete in vivo activity. This can lead to incomplete herbicide tolerance and complicate the registration approval. The present invention provides CTPs which, when used in combination with DMO, provide unexpected benefits including, but not limited to: increased levels of transport to chloroplasts, enhanced herbicide tolerance in transgenic plants expressing DMO, desirable levels of correct size protein expression, and appropriate post-translational modifications. One example of a CTP that provides unexpected benefits when combined with DMO is the transit peptide CTP2, including SEQ ID NO: 4 or 5, and comprises a nucleic acid encoding SEQ id nos: 15 and 16. In other embodiments, a pea (Pisum sativum) ribulose bisphosphate carboxylase-oxygenase small subunit CTP coding sequence is used, for example, a sequence represented by SEQ ID NO: 2 or encodes SEQ ID NO: 13. thus, a DNA construct comprising a DMO coding sequence operably linked to CTP2 and/or a pea rubisco small subunit CTP transit peptide coding sequence constitutes an aspect of the present invention, as does the protein encoded thereby.

Dicamba monooxygenase from Pseudomonas maltophilia strain DI6 (Herman et al, 2005; U.S. patent publication 20030115626; GenBank accession AY786443, the sequence encoding DMO of which is incorporated herein by reference) catalyzes the detoxification of the herbicide dicamba. DMO is part of a three-component system for detoxifying dicamba into nontoxic 3, 6-dichlorosalicylic acid (3, 6-DCSA), and requires reductase and ferredoxin functions to transfer electrons as described above. Since endogenous plant ferredoxin involved in electron transfer is localized in the plastid, it is preferred that the DMO is targeted to the plastid (e.g., chloroplast) in order to obtain efficient activity of the DMO and tolerance to dicamba, e.g., in dicotyledonous plants or enhanced tolerance to dicamba, e.g., in monocotyledonous plants.

Chloroplast Transit Peptide (CTP) was tested for efficiency in targeting DMO to plastids and DMO processing. The plastid localization and processing of DMOs associated with these CTPs varies from none, partial, to none. It was found that only some CTPs allowed complete processing of DMO to the correct size. Thus, the ability of any given CTP to provide complete and efficient DMO processing based on its protein or nucleotide sequence is unexpected and unexpected.

Furthermore, it has also been found in arabidopsis that in the absence of a suitable CTP, little or no expression of DMO correlates with little or no dicamba tolerance. This suggests that chloroplast targeting is important for dicamba detoxification and thus for tolerance. CTPs that allow efficient processing of DMO are useful in targeting DMO to the plastids, e.g., chloroplasts, of crop plants, thus providing the following advantages: full DMO activity and higher tolerance to dicamba, as well as ease of product characterization and reduced registration costs.

Chimeric DNA molecules comprising DNA encoding a chloroplast transit peptide operably linked to DNA encoding dicamba monooxygenase can be prepared by molecular biology methods well known to those skilled in the art (e.g., Sambrook et al, 1989). The present invention provides CTPs operably linked to known DNA molecules encoding DMOs, including those listed in table 1, for use in increasing expression of DMO in plants.

Chloroplast transit peptides from any gene encoded in the nucleus and whose product targets the polypeptide to the chloroplast can be tested for efficient expression of DMO. Chloroplast transit peptide sequences can be isolated or synthesized. The nucleotide sequence encoding the CTP may be optimized for expression in dicots, monocots, or both. By operably linking each to a DMO coding sequence, the following transit peptides were tested: PsRbcS-derived CTPs (SEQ ID NOS: 1 and 2: pea ribulose bisphosphate carboxylase-oxygenase small subunit CTP; Coruzzi et al, 1984); AtRbcS CTP (SEQ ID NO: 3: Arabidopsis thaliana rubisco small subunit 1A CTP; CTP 1; U.S. Pat. No. 5,728,925); AtShkG CTP (SEQ ID NO: 4: Arabidopsis thaliana 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS); CTP 2; Klee et al, 1987); AtShkGZm CTP (SEQ ID NO: 5: CTP2 synthetic; codon optimized for monocot expression; SEQ ID NO: 14 of WO 04009761); PhShkG CTP (SEQ ID NO: 6: Petunia hybrida EPSPS; CTP 4; codon optimization for monocot expression; Gasser et al, 1988); TaWaxy CTP (SEQ ID NO: 7: wheat (Triticum aestivum) granule-bound starch synthase CTP synthesized for codon optimization for maize expression: Clark et al, 1991): OsWaxy CTP (SEQ ID NO: 8: Rice (Oryza sativa) starch synthase CTP; Okagaki, 1992); NtRBCS CTP (SEQ ID NO: 9: Nicotiana tabacum) ribulose 1, 5-bisphosphate carboxylase small subunit chloroplast transit peptide; Mazur, et al, 1985); ZmAS CTP (SEQ ID NO: 10: maize (Zea mays) anthranilate synthase α 2 subunit gene CTP; Gardiner et al, 2004); and RgASCTP (SEQ ID NO: 11: Ruta graveolens (Ruta graveolens) anthranilate synthase CTP; Bohlmann, et al, 1995). Encoding the amino acid sequence of SEQ ID NO: 1-SEQ ID NO: 11 are as set forth in SEQ ID NOs: 12-SEQ ID NO: 22.

Other transport peptides that may be useful include the maize cab-m7 signal sequence (Becker et al, 1992; PCT WO97/41228) and the pea (Pisum sativum) glutathione reductase signal sequence (Creissen et al, 1995; PCT WO 97/41228). CTPs with additional amino acids from the coding region of the gene that are part of or fused to the coding region of the gene, such as the AtRbcS CTP (which comprises the transit peptide, the 24 amino acids of the mature rubisco protein, followed by a repeat of the last 6 amino acids of the transit peptide), can be used to produce DMO. ZmAS CTP7 also contained an additional 18 amino acids from the coding region of the gene. Other CTPs may also be used to produce DMO, which have additional amino acids (e.g., 27 amino acids) from the coding region of the gene that are part of the coding region of the gene, such as PsRbcS CTP, followed by amino acids (e.g., 3 amino acids) introduced by cloning methods. CTPs with fewer amino acids (e.g., 21 amino acids) encoding a full-length CTP, such as RgAs CTP, can also be used to produce DMO. Preferably, a nucleotide sequence encoding the full length CTP is used. One or more nucleotide additions or deletions may be included to facilitate cloning of CTPs. These additions or deletions may precede or follow other expression elements and coding regions, resulting in one or more modifications of the encoded amino acid, e.g., at or near the restriction enzyme recognition site.

In one embodiment, the invention relates to a nucleic acid sequence encoding a chloroplast transit peptide that hybridizes to any one of SEQ ID NOs: 1-11, including at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity to such sequences, including 100% identity. In particular embodiments, the nucleic acid sequence encodes a polypeptide that is identical to SEQ ID NOs: 1-11. In another embodiment, the nucleic acid sequence encoding CTP is identical to SEQ ID NOs: 12-22, including at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity, including 100% identity, to one or more of these sequences. Polypeptide or polynucleotide comparisons of these sequences and any other sequences described herein and determination of identity can be performed as is well known in the art, for example, using MEGAlign (DNAStar, inc., 1228s.park St., Madison, WI), with default parameters. Such software matches similar sequences by determining the degree of similarity or identity.

DMO can be targeted to other organelles, such as mitochondria, by using a pro sequence to take advantage of the ferredoxin redox system present in the organelle. Alternatively, DMO can be targeted to both chloroplasts and mitochondria by a dual targeting peptide to take advantage of the two ferredoxin redox systems for more efficient work. Such elements are known to those skilled in the art. For example, mitochondrial prosequences are described in Silva Filho et al, (1996). The nucleic acid sequence encoding the dual targeting peptide sequence can be identified from the following nucleotide sequences encoding proteins known to be targeted to both chloroplasts and mitochondria: Zn-MP (Moberg et al, 2003), glutathione reductase (Rudhe et al, 2002; Creissen et al, 1995) and histidyl-tRNA synthetase (Akashi et al, 1998). For example, as shown in table 1, in the case of encoding SEQ id nos: 24. examples of DMO coding sequences that may be used in this regard are found in the sequences of 26, 28, 30, 32, 34, 36, 38, 40 polypeptides.

Table 1 DMO and DMO variants used.

DMO and/or variants

Protein SEQ ID

DNA SEQ ID

Length of protein

Amino acid predicted at position 2

Predicted amino acid at position 3

Predicted amino acid at position 112

Codon usage

DMO-Cat(A)

24

23

340

Ala

Thr

Cys

Dicotyledonous plant

DMO-Cat(L)

26

25

340

Leu

Thr

Cys

Dicotyledonous plant

DMO-Wat(L)

28

27

340

Leu

Thr

Trp

Dicotyledonous plant

DMO-Cnat(A)

30

29

340

Ala

Thr

Cys

Bacteria

DMO-Wat(A)

32

31

340

Ala

Thr

Trp

Dicotyledonous plant

DMO-Wnat(T)

34

33

339

Thr

Phe

Trp (at 111)

Bacteria

DMO-Cnat(L)

36

35

340

Leu

Thr

Cys

Bacteria

DMO-Wmc(L)

38

37

340

Leu

Thr

Trp

Monocotyledon

DMO-Wmc(A)

40

39

340

Ala

Thr

Trp

Monocotyledon

In certain embodiments, the nucleic acid encoding dicamba monooxygenase hybridizes with a nucleic acid encoding SEQ id nos: 24. 26, 28, 30, 32, 34, 36, 38, and 40, including at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, and higher sequence identity to these sequences. In certain embodiments, the nucleic acid is identical to SEQ ID NOs: 23. 25, 27, 29, 31, 33, 35, 37, or 39, including at least about 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, and greater sequence identity to one of these sequences. In further embodiments, dicamba monooxygenase can be a variant of any such sequence and/or can be an engineered synthetic DMO molecule, for example, as described in U.S. provisional application 60/884,854 entitled "DMO Methods and molecules," filed on 12.1.2007, the entire disclosure of which is specifically incorporated herein by reference.

Variants of DMO having the ability to degrade auxin-like herbicides, as well as glyphosate or other herbicide tolerance genes, can be readily prepared according to standard methods and assayed for activity. These sequences can also be identified by techniques known in the art, for example, from suitable organisms including bacteria capable of degrading biotin-like herbicides such as dicamba or other herbicides (U.S. Pat. No. 5,445,962; Cork and Krueger, 1991; Cork and Khalil, 1995). One method of isolating DMO or other sequences is by, for example, nucleic acid hybridization to a library constructed from the source organism, or RT-PCR using mRNA from the source organism and primers based on the disclosed desaturases. The invention thus encompasses the use of nucleic acids that hybridize under stringent conditions to the DMO coding sequences described herein. Those skilled in the art understand that less stringent conditions can be provided by increasing the salt concentration and decreasing the temperature. Thus, hybridization conditions can be easily controlled, and are generally methods selected according to the intended results. An example of high stringency conditions is 5 XSSC, 50% formamide and 42 ℃. By washing under such conditions, for example, for 10 minutes, those sequences that do not hybridize to the particular target sequence under these conditions can be removed.

Variants can also be synthesized chemically, for example, using known DMO polynucleotide sequences according to techniques well known in the art. For example, the DNA sequence can be synthesized on an automatic DNA synthesizer by phosphoramidite chemistry (phosphoroamidite). Chemical synthesis has many advantages. In particular, chemical synthesis is desirable because codons preferred by the host in which the DNA sequence is to be expressed can be used to optimize expression. Not all codons need to be changed to obtain improved expression, but preferably at least those codons that are rarely used in the host are changed to host-preferred codons. High levels of expression can be obtained by altering more than about 50%, most preferably at least about 80% of the codons to host-preferred codons. The codon bias of many host cells is known (e.g., PCT WO 97/31115; PCT WO 97/11086; EP 646643; EP 553494; and U.S. Pat. Nos.: 5,689,052; 5,567,862; 5,567,600; 5,552,299 and 5,017,692). Codon usage for other host cells can be deduced by methods known in the art. In addition, the sequence of a DNA molecule or its encoded protein can be readily altered using chemical synthesis, for example to optimize expression (e.g., to eliminate mRNA secondary structures that interfere with transcription or translation), to add unique restriction sites at convenient sites, and to delete protease cleavage sites.

Modifications and changes can be made to the polypeptide sequence of a protein, such as the DMO sequence provided herein, while preserving or altering enzyme activity as desired. An exemplary method of generating DMO sequences is provided in U.S. provisional application 60/884,854 filed on 12.1.2007. The following is a discussion of the generation of comparable or even improved, modified polypeptides and corresponding coding sequences based on altering the amino acids of the protein. It is known, for example, that certain amino acids may be replaced by other amino acids in a protein structure without significant loss of ability to bind to structures on the substrate molecule such as binding sites. Because the interactive capacity and nature of a protein defines the biological functional activity of the protein, certain amino acid sequence substitutions may be made in the protein sequence, and of course in its DNA coding sequence, while still obtaining a protein with similar properties. Thus, it is envisaged: various changes can be made to the DMO peptide sequences or other herbicide tolerance polypeptides and corresponding DNA coding sequences described herein without significantly diminishing their biological utility or activity.

In making such changes, the hydropathic index of amino acids is considered. The importance of the hydrophilic amino acid index in conferring interactive biological functions on proteins is generally understood in the art (Kyte et al, 1982). It is generally accepted that the relative hydrophilic character of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Based on their hydrophobic and charge properties, each amino acid has been assigned a hydropathic index (Kyte et al, 1982), which 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); glutamic acid (-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 replaced by other amino acids having similar hydrophilicity indices or scores while still producing proteins having similar biological activities, i.e., still obtaining biologically functionally equivalent proteins. When such a conversion is performed, amino acid substitutions having a hydrophilicity index within. + -.2 are preferred, amino acid substitutions within. + -.1 are particularly preferred, and amino acid substitutions within. + -. 0.5 are even more preferred.

It is also understood in the art that substitution of like amino acids can be made efficiently on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 suggests that the maximum local average hydrophilicity of a protein, controlled by the hydrophilicity of adjacent amino acids, is related to the biological properties of the protein. As described in U.S. patent No. 4,554,101, the following hydrophilicity values were assigned to the amino acid residues: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic 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 should be understood that: an amino acid may be replaced by another amino acid with a similar hydrophilicity value, while still obtaining a biologically equivalent protein. Among such changes, amino acid substitutions having a hydrophilicity value within. + -.2 are preferred, amino acid substitutions within. + -.1 are particularly preferred, and amino acid substitutions within. + -. 0.5 are even more preferred. Exemplary permutations that take these and various of the above features into account 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, and valine, leucine and isoleucine.

A DNA construct comprising a CTP sequence operably linked to a DMO sequence may be expressed in a test system such as protoplasts, transiently or stably transformed plant cells, etc., by being operably linked to a promoter that is functional in plants. Examples of promoters that describe this include U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter; OsActl), U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611 (constitutive maize promoter), U.S. Pat. No. 5,322,938, U.S. Pat. No. 5,352,605, U.S. Pat. No. 5,359,142 and 5,530,196(35S promoter), U.S. Pat. No. 6,433,252 (maize L3 protein promoter), U.S. Pat. No.7 (rice actin 2 promoter and rice actin 2 intron), U.S. Pat. No. 5,837,848 (root-specific promoter), U.S. Pat. No. 6,294,714 (light inducible promoter), U.S. Pat., PClSV promoter), SEQ ID NO: 41, us patent 6,635,806 (gamma-coixin promoter) and us patent 7,151,204 (maize chloroplast aldolase promoter). Other promoters which may be used are the nopaline synthase (NOS) promoter (Ebert et al, 1987), the octopine synthase (OCS) promoter, which is carried on a tumor-inducible plasmid of Agrobacterium tumefaciens (Agrobacterium tumefaciens), cauliflower mosaic virus promoters such as the cauliflower mosaic virus (CaMV)19S promoter (Lawton et al, 1987), the CaMV35S promoter (Odell et al, 1985), the figwort mosaic virus 35S-promoter (Walker et al, 1987), the sucrose synthase promoter (Yang et al, 1990), the R gene complex promoter (Chandler et al, 1989), and the chlorophyll a/b binding protein gene promoter, among others. In the present invention, promoters of CaMV35S (e 35S; U.S. Pat. No. 5,322,938; 5,352,605; 5,359,142 and 5,530,196), FMV35S (U.S. Pat. No. 6,051,753; 5,378,619), peanut chlorotic stripe cauliflower mosaic virus (PClSV; U.S. Pat. No. 5,850,019), At.Act 7 (accession # U27811), At.ANT1 (U.S. Pat. No. 20060236420), FMV.35S-EF1a (U.S. Pat. No. 20050022261), eIF4A10 (accession # X79008) and AGRtu. nos (GenBank accession number V00087; Depicker et al, 1982; Bevan et al, 1983), triose phosphate isomerase of rice (OsTPI; U.S. Pat. No.7,132,528) and rice actin gene (OsAct 15; U.S. Pat. application 0162010) may be particularly useful.

The 5' UTR, which functions as a translation leader, is a DNA genetic element located between the promoter sequence and the coding sequence of a gene, and may be included between the promoter and the CTP-DMO sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation initiation sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability, or translation efficiency. Examples of translation leader sequences include maize and petunia heat shock protein leaders (U.S. Pat. No. 5,362,865), plant viral coat protein leaders, plant rubisco leaders, GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and free, 1990), and AGRtunos (GenBank accession No. V00087; Bevan et al, 1983), among others (Tumer and Foster, 1995). In the present invention, 5' UTRs which may be particularly beneficial are GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No. 5,362,865), AtAnt1, TEV (Carrington and free, 1990), OsAct1 (U.S. Pat. No. 5641876), OsTPI (U.S. Pat. No.7,132,528), OsAct15 (U.S. publication No. 20060162010), and AGRtunos (GenBank accession No. V00087; Bevan et al, 1983).

By 3 'untranslated sequences, 3' transcription termination region or polyadenylation region is meant a DNA molecule linked to and located downstream of a structural polynucleotide molecule and includes polynucleotides that provide polyadenylation signals and other regulatory signals capable of affecting transcription, mRNA processing or gene expression. Polyadenylation signals function in plants to cause the addition of polyadenylic acid to the 3' end of the mRNA precursor. The polyadenylation sequence may be derived from the natural gene, from a variety of plant genes, or from a T-DNA gene. These sequences may be included downstream of the CTP-DMO sequence. An example of a3 ' transcription termination region is the nopaline synthase 3 ' region (nos 3 '; Fraley et al, 1983). The use of different 3' untranslated regions is illustrated (Ingelrecht et al, 1989). Polyadenylated molecules from the pea RbcS2 gene (Ps. RbcS 2-E9; Coruzzi et al, 1984), AGRtu. nos (Genbank accession number E01312), E6 (accession number # U30508) and TaHsp17 (wheat low molecular weight calorimetric shock protein gene; accession number # X13431) are particularly advantageous for use in the present invention.

In addition to the expression elements described above, an intron may be required between the promoter and the 3' UTR in order to express the coding region, particularly in monocotyledonous plants. "Intron" refers to a polynucleotide molecule that can be isolated or identified from intervening sequences in genomic copies of a gene, and can generally be defined as a region that is spliced out during pre-translational mRNA processing. Alternatively, the intron can be synthetically produced. The intron itself may contain sub-elements such as cis-elements or enhancer regions that affect transcription of an operably linked gene. A "plant intron" is a natural or non-natural intron that functions in a plant cell. Plant introns may be used as regulatory elements to regulate the expression of one or more genes operably linked. The polynucleotide molecule sequence in the transformation construct may comprise an intron. Introns may be heterologous with respect to the polynucleotide sequence that may be transcribed. Examples of introns include the maize actin intron (U.S. Pat. No. 5641876), the maize HSP70 intron (ZmHSP 70; U.S. Pat. No. 5,859,347; U.S. Pat. No. 5,424,412) and the rice TPI intron (OsTPI; U.S. Pat. No.7,132,528), and are useful in the practice of the present invention.

The CTP-DMO construct may be tested for providing the correct processing of DMO in a test system such as protoplasts or transiently or stably transformed monocot or dicot plant cells by methods known to those skilled in the art of plant tissue culture and transformation. According to the present invention, any technique known in the art for introducing transgenic constructs into plants can be used (see, e.g., Miki et al, 1993). It is believed that suitable methods for plant transformation include virtually any method by which DNA can be introduced into cells, such as electroporation as described in U.S. Pat. No. 5,384,253; the particle bombardment methods described in U.S. Pat. nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861 and 6,403,865; agrobacterium-mediated transformation methods as described in U.S. Pat. nos. 5,635,055, 5,824,877, 5,591,616, 5,981,840, and 6,384,301; and the protoplast transformation method described in U.S. Pat. No. 5,508,184. By applying techniques such as these, cells of virtually any plant species can be stably transformed and these cells can be developed into transgenic plants. U.S. Pat. nos. 5,846,797, 5,159,135, 5,004,863, and 6,624,344 disclose techniques that are particularly useful in cotton conversion. For example, techniques for transforming Brassica (Brassica) plants are specifically disclosed in us patent 5,750,871; techniques for transforming soybeans are disclosed, for example, in Zhang et al, 1999, U.S. Pat. No. 6,384,301, and US 7,002,058. Techniques for transforming maize are disclosed in WO 9506722. Some non-limiting examples of plants that may be used in the present invention include alfalfa, barley, beans, sugar beet, broccoli, cabbage, carrot, canola, cauliflower, celery, chinese cabbage, corn, cotton, cucumber, dried beans, eggplant, fennel, green sword bean, gourd, leek, lettuce, melon, oat, okra, onion, pea, pepper, pumpkin, peanut, potato, pumpkin, radish, rice, sorghum, soybean, spinach, squash, sweet corn, sugar beet, sunflower, tomato, watermelon, and wheat.

The next step in achieving the introduction of foreign DNA into recipient cells to produce transgenic plants generally involves identifying transformed cells for further culture and plant regeneration. In order to improve the ability to identify transformants, it may be necessary to use a selectable or screenable marker gene together with the transformation vector prepared according to the present invention. In this case, the population of cells that may be transformed is typically subsequently tested by exposing the cells to one or more selective agents, or the cells are screened for a trait of a desired marker gene.

Cells that survive exposure to the selective agent, or cells that score positive in the screening assay, may continue to be cultured on media that supports plant regeneration. Any suitable plant tissue culture medium, for example, MS or N6 medium (Murashige and Skoog, 1962; Chu et al, 1975), may be altered by inclusion of other substances such as growth regulators. The tissue may be maintained on a basal medium containing growth regulators until sufficient tissue is available to begin plant regeneration work, or repeated rounds of manual selection are performed until the morphology of the tissue is suitable for regeneration, typically at least two weeks, and then transferred to a medium that facilitates seedling formation. The cultures were transferred periodically until sufficient seedling formation occurred. Once seedlings are formed, they are transferred to a medium that facilitates root formation. Once enough roots are formed, the plants can be transferred to soil for further growth and maturation.

To confirm the presence of the foreign DNA or "transgene" in the regenerated plant, various tests may be performed. Such assays include, for example, "molecular biology" assays, such as southern and northern blotting and PCR^TM(ii) a "biochemical" assays, such as detecting the presence of protein products, for example, by immunological methods (ELISA and western blotting) or by enzymatic function; plant part tests, such as leaf or root tests; and, by analyzing the phenotype of the entire regenerated plant.

Once the transgene is introduced into a plant, the gene can be introduced into any plant compatible with the first plant by crossing, without the need to directly transform a second plant at all. Thus, the term "progeny" as used herein refers to any generation of progeny of a parent plant made according to the present invention, wherein the progeny comprises a selected DNA construct made according to the present invention. Thus, a "transgenic plant" can be any generation. As disclosed herein, "crossing" a plant to provide a plant line having one or more added transgenes or alleles relative to the original plant line is defined as a technique by which the original line is crossed with a donor plant line comprising a transgene or allele of the present invention, resulting in the introduction of a particular sequence into the plant line. To achieve this, for example, the following steps may be performed: (a) planting seeds of a first (initial line) and a second (donor plant line containing the desired transgene or allele) parent plant; (b) growing seeds of the first and second parent plants into flowering plants; (c) pollinating the flower of the first parent plant with pollen of the second parent plant; and (d) harvesting seed produced on the parent plant having the fertilized flower.

Stably transformed plant tissues and plants can be tested for dicamba tolerance by proper processing of DMO proteins. Providing dicamba tolerance in crop plants can be used to design a method of controlling weed growth in a growing environment, comprising: applying to the crop growing environment an amount of dicamba herbicide effective to control the growth of weeds. Dicamba herbicide is applied to the crop growth environment from above in an amount that does not damage crop plants or seeds transformed with the CTP-DMO construct, but does damage crop plants having the same genotype but lacking the CTP-DMO construct.

The preparation of the herbicidal compositions for use in the present invention will be apparent to those skilled in the art based on the disclosure. Such commercially available compositions typically contain components such as surfactants, solid or liquid carriers, solvents, and binders in addition to the active ingredient. Examples of surfactants that may be used for application to plants include the alkali metal, alkaline earth metal or ammonium salts of: aromatic sulfonic acids, for example lignosulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid, and fatty acids, arylsulfonates, alkyl ethers, lauryl ethers, fatty alcohol sulfates and fatty alcohol glycol ether sulfates, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or naphthalenesulfonic acid with phenol and formaldehyde, condensates of phenol or phenolsulfonic acid with formaldehyde, condensates of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl-or nonylphenol, tributylphenyl polyglycol ether, alkylaryl polyether alcohols, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenol, salts of phosphorylated triarylphenol ethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin-sulfite waste liquors or methylcellulose, or mixtures thereof. A common example of the use of surfactants is about 0.25 wt% to 1.0 wt%, more commonly about 0.25 wt% to 0.5 wt%.

The composition applied to the plants may be solid or liquid. Where solid compositions are used, it may be desirable to include one or more carrier materials and active compounds. Examples of carriers include mineral earths such as silica, silica gel, silicates, talc, kaolin, magnesia clay (attaclay), limestone (limestone), chalk (chalk), loess (loess), clay, dolomite (dolimite), diatomaceous earth (diatomous earth), calcium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal grits, bark grits, wood flour and nut shell grits, cellulose flour, attapulgite (attapulgites), montmorillonite (montmorillonites), mica (mica), vermiculite (vermiculites), synthetic silica and synthetic calcium silicate, or mixtures thereof.

For the liquid solution, a water-soluble compound or salt such as sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium bisulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium bicarbonate, ammonium thiosulfate, ammonium dihydrogenphosphate, sodium hydrogenphosphate, ammonium thiocyanate, ammonium sulfamate, or ammonium carbamate may be contained.

Other exemplary ingredients in the herbicidal composition include: binders such as polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethyl cellulose, starch, vinylpyrrolidone/vinyl acetate copolymer and polyvinyl acetate or mixtures thereof; lubricants, such as magnesium stearate, sodium stearate, talc or polyethylene glycol or mixtures thereof; antifoaming agents, such as silicone emulsions, long-chain alcohols, phosphate esters, acetylene glycols, fatty acids or organofluorine compounds; and chelating agents, such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of nitrilotriacetic acid or salts of polyphosphoric acid or mixtures thereof.

Dicamba from about 2.5g/ha to about 10,080g/ha may be used, including from about 2.5g/ha to about 5,040g/ha, from about 5g/ha to about 2,020g/ha, from about 10g/a to about 820g/h and from about 50g/ha to about 1,000g/ha, from about 100g/ha to about 800g/ha and from about 250g/ha to about 800 g/ha.

The CTP-DMO construct may be linked to one or more polynucleotide molecules comprising genetic elements for a screenable/scorable/selectable marker and/or to confer another desirable trait. Genes commonly used to screen presumably transformed cells include beta-Glucuronidase (GUS), beta-galactosidase, luciferase and chloramphenicol acetyltransferase (Jefferson, 1987; Tereri et al, 1989; Koncz et al 1987; De Block et al, 1984), Green Fluorescent Protein (GFP) (Chalfie et al, 1994; Haseloff and Amos, 1995; and PCT application WO 97/41228). Non-limiting examples of selectable marker genes are described, for example, in Miki and McHugh, 2004.

Nucleotide molecules that confer another desirable trait include, but are not limited to: genes that provide desirable traits related to plant morphology, physiology, growth and development, yield, nutrient enrichment, disease or insect resistance, or environmental or chemical tolerance may also include genetic elements including: herbicide resistance (U.S. Pat. No. 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), increased yield (U.S. Pat. No. 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), pest control (U.S. Pat. No. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,110,464; 3667172; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 36942, 365,880,275; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 6,537,756; 36, Altered oil production (U.S. Pat. No. 6,444,876; 6,426,447; 6,380,462), high oil production (U.S. Pat. No. 6,495,739; 5,608,149; 6,483,008; 6,476,295), altered fatty acid content (U.S. Pat. No. 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. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhanced animal and human nutrition (U.S. Pat. No. 6,723,837; 6,653,530; 6,541,259; 5,985,605; 6,171,640), biopolymers (U.S. Pat. RE37,543; 6,228,623; 5,958,745 and U.S. patent publication US 200300917), environmental stress tolerance (U.S. Pat. 6,072,103), pharmaceutical and secretable peptides (U.S. Pat. No. 6,812,379; 6,774,283; 6,140,075; 6,080,560), improved processing traits (U.S. Pat. 6,476,295), improved digestibility (U.S. Pat. 6,531,648), low levels of raffinose (U.S. Pat. 6,166,292), industrial enzyme production (U.S. Pat. 6,166,292), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seed production (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. No. 6,576,818; 6,271,443; 5,981,834; 5,869,720), and biofuel production (U.S. Pat. No. 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 based on the present disclosure.

In addition, one or more polynucleotide molecules linked to the CTP-DMO construct may achieve the above-mentioned plant characteristics or phenotypes by encoding RNA molecules that result in targeted suppression of endogenous gene expression (e.g., via antisense, inhibitory RNA (rnai), or co-suppression mediated mechanisms). The RNA may also be a catalytic RNA molecule (i.e., a ribozyme) designed to cleave a desired endogenous mRNA product. Thus, any polynucleotide molecule that encodes a transcribed RNA molecule that affects a phenotypic or morphological change of interest can be used in the practice of the present invention.

Also disclosed are methods of producing a food, feed, or industrial product comprising a plant comprising a CTP-DMO construct or a part of such a plant, and preparing a food, feed, fiber, or industrial product from the plant or part thereof, wherein the food or feed is a grain, meal, oil, starch, flour, or protein, and the industrial product is a biofuel, fiber, industrial chemical, pharmaceutical, or nutraceutical.

Another aspect of the present invention relates to a method of improving the standability of a monocot plant comprising: a) obtaining and planting a plant produced by crossing a parent plant with itself or a second plant, wherein the parent plant and/or the second plant comprises the DNA construct and the dicamba tolerant plant inherits the DNA construct from the parent plant and/or the second plant; and b) treating the plant with dicamba. Parameters related to stand ability including number, shape, length or structure of strut roots, percent lodging and yield can be measured. In certain embodiments, the plant is a maize plant.

Examples

The following examples are presented to illustrate embodiments of the invention. Those skilled in the art will understand that: the techniques disclosed in the following examples 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 agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would 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.

Example 1

Preparation of CTP-DMO construct for transformation

The DNA constructs shown in table 2, comprising a CTP operably linked to a DMO gene or variant thereof between a plant promoter and a polyadenylation signal sequence, were prepared according to standard methods (e.g., Sambrook et al, 1989). These constructs were tested in the maize protoplast system or in stably transformed Arabidopsis or soybean plants as described below.

Example 2

Analysis of CTP-DMO constructs in maize protoplasts

Maize leaf pulp protoplasts were prepared from 12-day yellow seedlings (from LH200x LH5 cross). The middle part of the second leaf (approximately 6cm long) was cut into 0.5mM strips with a razor blade and after 30 minutes of vacuum filtration, in a flask containing 2% (w/v) cellulase RS, 0.3% (w/v) eductase R10 (all from Karlan Research Products Corp, Santa Rosa, Calif.), 0.6M mannitol, 10mM MES (pH 5.7) and 1mM CaCl₂The enzyme solution of (a) is digested at 23 ℃ for not more than 2 hours. Protoplast from filtered and digested leaf tissueThe plastids were released by shaking the flask by hand for 5 minutes and isolated by filtration through a 60- μm nylon mesh. Protoplasts were collected by centrifugation at 150g for 2 minutes, washed once with 0.6M cold mannitol solution, centrifuged at 2X 10⁶The suspension was resuspended in cold 0.6M mannitol. Protoplasts were then transformed with 12.5. mu.g DNA using polyethylene glycol (PEG) and incubated for 16-20 hours at room temperature.

Protoplasts were stored at-80 ℃ until analyzed by western blotting (western blot). Protoplasts were thawed on ice and 1-3 volumes of 2 xlaemli sample buffer/dye containing 5.0% β -ME (BioRad) were added to the protoplasts. Aliquots of the protoplast protein samples were then heated to approximately 100 ℃ for 5 minutes and loaded onto preformed Tris-HCL 10% polyacrylamide gels. Electrophoresis was carried out at a constant current of about 80-100Amps for about 35 minutes. The protein was then electrotransferred from the gel to a 0.2 micron nitrocellulose membrane at 100V constant voltage for 1-3 hours. The membranes were blocked with 5% (w/v) milk powder in TBST for 1 hour at room temperature or overnight at 4 ℃. The membrane was probed with goat anti-DMO antibody diluted 1: 200 in TBST for 1 hour. TBS was used to remove excess antibody by washing 3 times for 5 minutes. Membranes were probed with peroxidase-conjugated rabbit anti-goat IgG (Sigma, St. Louis, Mo.) diluted 1: 7,500 in TBST containing 0.5% (w/v) milk powder for 1 hour. Excess peroxidase conjugate was removed by washing 3 times with TBST for 5 minutes each. All procedures, including blocking and all other incubations, were performed at room temperature, except those specifically noted. Bands showing immunological activity using the ECL detection System (Amersham Biosciences, Piscataway, NJ) and were visualized in Kodak BioMax^TMExposure on MS film. The presence of an immunologically active band of appropriate size indicates the correct processing and localization of DMO (table 1). Thus, for example, CTP4, using a protein operably linked to DMO and transformed into corn protoplasts, produced an immunologically active band of 38kDa after western blot analysis.

Example 3

Testing of various CTP-DMO constructs in Arabidopsis

Arabidopsis thaliana Columbia ecotype plants were transformed according to the method developed by Clough and Bent (1998). The seeds obtained by this method were inoculated onto plant selection media containing dicamba at various concentrations of 0.5, 1.0 to 2.0 or 4.0 mg/l. The plates were incubated at 4 ℃ for 48 hours and then transferred to a Percival incubator set at 23.5 ℃ with a 16 hour photoperiod. Seeds transformed with the CTP-DMO construct were grown into plants on dicamba containing medium and primary and secondary leaves were grown, while untransformed seeds and negative segregants (segregants) died or did not grow primary and secondary leaves. By passingTransgenic plants that were detected as 3' UTR positive by PCR assay were used for further analysis.

3-5 leaf perforations from transgenic Arabidopsis plants were used for Western blot analysis. Protein extraction was performed in a Harbil paint shaker with 500-. The samples were spun at 3000rpm for 3 minutes at 4 ℃. To an aliquot of the supernatant was added an equal volume of 2x Laemmli sample buffer/dye containing 5.0% β -ME (cat. No.161-0737 BioRad). The remaining steps of the western blot analysis were the same as in example 2. The presence of an immunologically active band of the correct size indicates the correct processing and localization of DMO (table 2). For example, as shown in table 2, in a comparison of the bands seen after transformation of arabidopsis with pMON73749 or pMON73725, using an RbcSnoc-CTP lacking the 27 amino acid coding sequence from pea rubisco produced a properly processed DMO localized to the chloroplast, while using an RbcS CTP containing the 27 amino acid coding sequence produced 2 immunologically active bands.

Example 4

Testing of CTP-DMO constructs in Soybean

Transgenic soybean (e.g., cvs. thorne, NE3001 and a3525) plants were obtained by agrobacterium-mediated transformation of soybean (e.g., Zhang et al, 1999; US 7,002,058) using standard procedures. 3-5 leaf perforations from transgenic soybean plants were used for western blot analysis. Protein extraction was performed in a Harbil paint shaker with 500-. The samples were spun at 3000rpm for 3 minutes at 4 ℃. To an aliquot of the supernatant was added an equal volume of 2x Laemmli sample buffer/dye (BioRad)/5.0% β -ME. The remaining steps of the western blot analysis were the same as in example 2. The presence of an immunologically active band of the correct size indicates the correct processing and localization of DMO (table 2).

Soybean plants transformed with the construct encoding DMO linked to a pea rubisco transit peptide of the other 24 amino acids attached to the rubisco coding region and 3 amino acids added due to the introduction of restriction enzyme recognition sites showed a 17-20% injury rate when treated with 0.5lb of dicamba at the pre-emergence stage followed by 2lb of dicamba at the post-emergence (V6) stage. This was compared to soybean plants transformed with a construct encoding DMO linked only to the pea rubisco transit peptide, which showed a damage rate of approximately 12%. These results indicate that the use of a transit peptide without additional amino acids results in the production of a single DMO activity (rather than multiple partially or differently processed polypeptides) and higher tolerance to dicamba. The production of a single form of enzyme also results in ease of product characterization and reduced registration costs.

Example 5

Efficient production of DMO and higher dicamba tolerance requires CTP

Several constructs as described in example 3 (FIG. 1) were used to transform Arabidopsis thaliana Columbia ecotype plants. Transformed seeds were selected on plant tissue culture media containing dicamba at various concentrations from 0.5, 1.0 to 2.0 mg/l. Seeds transformed with the CTP-DMO construct were grown into plants on media containing dicamba and primary and secondary leaves were grown, while untransformed seeds and negative segregants died or did not grow primary and secondary leaves. Transgenic plants grown and tested positive for DMO genes were used for further analysis.

As shown in figure 1, plants transformed with the CTP-free construct showed little or no tolerance to dicamba. When treated with 0.5lb/a dicamba at the pre-emergence stage followed by 2lb/a dicamba at the post-emergence (V6) stage, soybean plants transformed with a DNA construct encoding a DMO not linked to a CTP showed no pre-emergence tolerance, while plants transformed with a construct in which DMO is linked to a CTP showed pre-and post-emergence tolerance to dicamba.

Example 6

Production of dicamba-tolerant transgenic maize plants

To test the use of DMO genes to provide dicamba tolerance to monocots, transgenic corn plants comprising DMO genes (e.g., SEQ ID NOS: 29, 33, 35, 37, 39), with or without transit peptides (e.g., tawax, CTP1, CTP2 synthesis, CTP4) were generated under the control of plant gene expression elements such as promoters (e.g., PClSV, e35S, OsAct1, ospti, OsAct15) and introns (e.g., OsAct1, OsAct15, ospti, ZmHSP 70). The expression element comprises the first intron and flanking UTR exon sequences from the rice actin 1 gene, further comprising 12nt of exon 1 at the 5 ' end and 7nt of exon 2 at the 3 ' end, and also a3 ' UTR (e.g., TaHsp 17).

Transgenic maize plants are essentially produced by the method described in U.S. patent application 20040244075. Dicamba tolerance of transgenic corn events with a single copy was evaluated in single-site replicate experiments. 6 events from each of the 6 constructs were used. The experimental design was as follows: columns/items: 1; and (3) treatment: 0.5lb/a dicamba at stage V3, followed by 1lb/a dicamba at stage V8 (BASF, Raleigh, NC); repeating:2; line spacing: 30 inches; land length: a minimum of 20 feet; plant density: about 30 plants/17.5 feet; channel (alley): 2.5 feet. The entire plot was evenly fertilized to obtain agronomically acceptable crops. To control corn rootworm, a soil insecticide is applied at a period of cultivation of 1000 feet per 5 ounces of soil, e.g. in rows3G (Syngenta Crop Protection, Greensboro, NC, USA). If an infestation of black cutworm (black cutworm) is observed, the application is carried out in an amount of 4-8 ounces per acre3.2EC (FMC Corporation, Philadelphia, Pa.). In addition, spray insecticide programs are used to control lepidopteran pests on all terrain, including european corn borer, corn earworm, and fall armyworm. Applied in an amount of 4-8 ounces per acre every 3 weeks3.2EC to control lepidopteran pests; approximately 4 administrations. Applied before emergence asXtra 5.6L (Monsanto, St. Louis, Mo.) and Degree(Monsanto, St. Louis, Mo.) to keep plots free of weeds. Throughout the experiment, if weed development was observed in the untreated check, PERMIT (Monsanto, St. Louis, MO) or PERMIT (R) was applied by hand weeding or after emergence of the soil(Bayer, Research Triangle Park, NC) controls weeds.

Dicamba tolerance of maize inbred lines transformed with DNA constructs comprising DMO transgenes was tested by measuring strut root damage when treated with 0.5lb/a dicamba at stage V3 followed by 1lb/a dicamba at stage V8. The strut root damage was visually assessed by counting the number of plants in a row that showed a "atypical" morphology of strut root fusion compared to typical morphologies of "finger-like" structures. As shown in table 4, maize plants transformed with DNA constructs encoding DMO not linked to CTPs (pMON73699, pMON73704) showed higher levels of strut root damage, i.e., low levels of protection after dicamba treatment. DNA constructs encoding DMO linked to CTP (pMON73716, pMON73700, pMON73715, pMON73703) showed lower levels of strut root damage, i.e., higher levels of protection after dicamba treatment.

Table 4. percentage of strut root damage exhibited by transgenic maize transformed with DNA constructs carrying DMO when tested for dicamba tolerance.

Inbred/construct	Details of	Injury of strut root
			01CSI6	Dicamba-sensitive inbred lines	95.4
LH244	Dicamba-tolerant inbred lines	93.8
			pMON73699	PC1SV/I-OsAct1/DMO-Wmc/TaHsp17	93.2
pMON73704	e35S/I-OsAct1/DMO-Wmc/TaHsp17	91.3
			pMON73716	PC1SV/I-OsAct1/TaWaxy/DMO-Wmc/TaHsp17	78.8
pMON73700	PC1SV/I-OsAct1/CTP1/DMO-Wmc/TaHsp17	74.4
			pMON73715	PC1SV/I-OsAct1/CTP2syn/DMO-Wmc/TaHsp17	68.2
pMON73703	e35S/I-OsAct1/CTP1/DMO-Wmc/TaHsp17	68.8

From these studies performed in different plant species (further, for example, examples 3,4 and 8), it can be seen that chloroplast transit peptides can be used for efficient targeting of DMO and complete production of DMO activity, resulting in higher tolerance to dicamba. Furthermore, CTP-DMO expression in maize provides pre-emergence tolerance to dicamba.

Example 7

Construction of efficient DMO expression units

Several genetic elements may influence the efficient expression of a gene, such as a promoter, chloroplast transit peptide sequence, intron, 5 'UTR, coding region of a gene, 3' UTR. However, it is unclear which combinations work best. There is a need for efficient DMO expression units or constructs to produce improved products such as dicamba tolerant seeds and plants. Several DMO expression units are constructed by operably linking each of various promoters, CTPs, DMO variants, and 3' UTRs to obtain efficient DMO expression units for product development. These constructs are transformed into soybean by methods known in the art (e.g., U.S.6,384,301, U.S.7,002,058 or Zhang et al, 1999). Transgenic seeds were obtained and tested for tolerance to dicamba herbicide before and after emergence. Table 5 shows the percentage of damage caused by dicamba when seeds and plants were treated with 0.5 lb/acre of dicamba before emergence followed by 2 lb/acre of dicamba at the V6 stage after emergence (lower damage means higher tolerance). Seeds transformed with the CTP-free DNA constructs pMON68939 and pMON73723 were not tolerant to pre-emergence applications of dicamba, indicating that to obtain pre-emergence tolerance to dicamba, DMO needs to be targeted to the chloroplast. Plants transformed with pMON68939 and pMON73723 (without CTP) showed injury rates similar to 55% and 57%, respectively, of wild type soybean (60%) after V3 post-treatment with dicamba in an amount of 1lb/a, while plants transformed with pMON68938 (with CTP) showed very low injury rates. These results indicate that CTP is required to achieve pre-and post-emergence tolerance to dicamba in soybean.

Table 5. percentage of damage exhibited by soybean plants transformed with specific DMO expression units and treated with dicamba before and after emergence.

Expression unit	pMON name	% damage
			PC1SV/CTP2syn/DMO-Wat(A)/nos	73724	9
e35S/CTP1/DMO-Wat(L)/nos	68938	12
			PC1SV/RbcSnoc/DMO-Wat(A)/nos	73725	12
PC1SV/RbcSnoc/DMO-Wat(L)/nos	73728	12
			PCSV/CTP1/DMO-Wat(A)/nos	73729	13
PC1SV/CTP2syn/DMO-Wat(L)/nos	73727	13
			ANT1/CTP1/DMO-Wat(L)/nos	68945	14
PC1SV/RbcSnoc/DMO-Wat(A)/nos	73730	15
			PC1SV/RbcS-CTP/DMO-Cnat(A)/nos	68934	17

[0097]

Act7/CTP1/DMO-Wat(L)/nos	68942	17
			FMV.35S-EF1a/CTP1/DMO-Wat(L)/nos	68940	17
PClSV/RbcS-CTP/DMO-Cnat(A)/E9	84254	20
			FMV/CTP1/DMO-Wat(L)/nos	68941	29
eIF4A10/CTP1/DMO-Wat(L)/nos	68943	60
			e35S/CTP1/DMO-Cat(A)/nos	68937	62
e35S/CTP1/DMO-Cnat(L)/nos	68946	73
			e35S/DMO-Wat(A)/nos	68939	100 (front)
PC1SV/DMO-Wat(A)/nos	73723	100 (front)

Example 8

Production of dicamba-tolerant transgenic cotton plants

To test the use of the DMO gene in providing dicamba tolerance to cotton, transgenic cotton plants were generated. Several DNA constructs were generated which harbored a DMO coding region (e.g., SEQ ID NO: 23, 25, 27, 29, 31, 35) with a transit peptide (e.g., PsRbcS CTP, CTP1, CTP2) under the control of a plant gene expression element such as a promoter (e.g., PClSV, FMV or E35S) and a 3' UTR (e.g., E6; accession # U30508), and transformed into cotton (Gossypium hirsutum) as described below. The media used are listed in table 6.

Cotton cv Coker130 seedlings were grown ex vivo and sections of the embryonic axis excised and inoculated into liquid suspensions of Agrobacterium tumefaciens harboring DNA constructs, blotted dry, and co-cultured for 2 days. The inoculated hypocotyl explants were then transferred to glucose selection medium for 4 weeks, sucrose selection medium for 1 week, and glucose selection medium for an additional 4 weeks to induce callus. Cultures were incubated at 16/8 (light/dark) cycles and 28 ℃. The kanamycin-resistant calli were then transferred to UMO medium and cultured at 28-30 ℃ for 16-24 weeks in the dark to induce embryogenic calli. Embryogenic callus was then harvested from these calli and maintained on TRP + medium at 28-30 ℃ under dark conditions for up to 4-16 weeks. Small embryos from embryogenic callus were harvested and germinated on SHSU medium at 28-30 ℃ under 6/8 (light/dark) cycling conditions. The normally-appearing plantlets are then transferred to soil to obtain mature cotton plants. The transgenic nature of the transformants was confirmed by DNA detection.

TABLE 6 composition of various media used for cotton transformation.

Dicamba (0.5lb/a) in an amount of 561g ae/ha in the growth phase V4-5BASF, Raleigh, NC) transformed cotton plants containing DNA constructs each containing a different combination of DMO coding region with transit peptide, promoter and 3' UTR were found to be tolerant as a post-emergence treatment, whereas untransformed cotton plants showed a damage rate of 79% -86%. Transgenic plants showing more than 95% tolerance (equal to less than 5% damage) were selected for further study. Transgenic plants are also tolerant to subsequent post-emergence treatment with dicamba. For example, plants treated with 0.5 lb/acre of dicamba at stage V3-4 followed by 1 or 2 lb/acre of dicamba at stage V5 or later were still tolerant to dicamba. This example shows that the DMO gene can provide dicamba tolerance to cotton at various growth stages,dicamba can thus be applied at various stages to obtain effective weed control.

Example 9

Method for improving corn uprightness

Certain monocotyledonous plants such as corn produce rootstocks that grow from nodes above the surface of the earth and help support the plant and absorb water and nutrients from the upper layers of the soil during the reproductive stage. A healthy strut root system becomes important if the plant encounters high winds or the underground root system becomes weaker due to root nematode infestation or lack of ground water. To control broadleaf weeds, synthetic herbicides such as dicamba and 2,4-D are allowed to be used on monocotyledons such as corn. Dicamba is the fifth most widely applied herbicide for post-emergence weed control in corn. While the optimum amount for controlling broadleaf weeds is 280-560 grams per hectare (g/h) or 0.25-0.5 lb/acre, the average usage in corn is 168g/h or 0.15 lb/acre, as dicamba can damage corn at higher application rates and under certain environmental conditions such as hot days. In addition, several maize hybrids, such as DKC61-42, DKC64-77, DKC63-46, DKC66-21, and DKC61-44, and inbreds, such as 01CSI6, 16IUL2, 70LDL5, and 90LCL6, were sensitive to dicamba administration. Sensitivity is manifested in a number of ways, such as the occurrence of onion peeling (onion leaf), ear malformations, reduced plant height, or abnormal formation of pillar roots, for example, fused or twisted roots. The stub roots become more numerous nodules, tend to grow together, and do not grow into the soil to support the plant. This can result in poor corn crop stand ability, higher lodging susceptibility and ultimately yield loss. Some herbicidal products containing dicamba, e.g.BANVEL, MARKSMAN, DISTINCT, norhstar, and CELEBRITY PLUS, can cause these effects. Increasing the tolerance of corn to dicamba is also useful in protecting corn fields grown closer to dicamba tolerant crop species (e.g., soybean, cotton), and among other things allows forHigh application rates of dicamba.

This example provides a method of improving the stand ability of maize and other monocots comprising introducing a DMO gene in maize and treating the maize with dicamba. In one embodiment, the DMO gene is expressed under the control of a constitutive promoter that is also capable of expressing DMO in the node region and/or in the strut root. In another embodiment, the DMO gene is expressed under the control of a chimeric constitutive and node/strut root specific promoter. In another embodiment, the DMO gene is expressed under the control of a root-specific promoter such as RCc3 or a variant thereof (e.g., SEQ ID NOs: 1-6 found in US 20060101541). Expression of DMO in the corbel roots results in no or less damage to the corbel roots, resulting in higher uprightness, less lodging and thus higher yield in maize.

R1 or F1 seeds from 3 single copy events of maize plants transformed with various DMO constructs (listed in table 7) germinated in 4.0 "trays. Healthy plants were transplanted into approximately 10 "pots. Germination and growth media include Redi-earth (Scotts-Sierra Horticultural Products Co., Marysville, Ohio). The pots were placed on capillary pads in 35 inch x 60 inch fiberglass watering trays for sub-watering during the test to maintain optimal soil moisture for plant growth. Pots were fertilized with Osmocote (14-14-14 slow release; Scotts-Sierra Horticultural Products Co., Marysville, Ohio) at an amount of 100gm/cu.ft. to maintain plant growth during the greenhouse trial. Plants were grown in a greenhouse at a day/night temperature of 29 ℃/21 ℃ and a relative humidity of 25% to 75% to simulate the growth conditions in the warm season of late spring. Supplemental light of approximately 600 muE is provided as needed for a minimum 14 hour light cycle.

Dicamba was applied with an orbital sprayer using a Teejet 9501E flat fan nozzle (Spraying Systems Co, Wheaton, IL) with an atmospheric pressure set to a minimum of 24psi (165 kpa). The nozzle was held approximately 16 inches above the plant tip for spraying. The spray volume was 10 gallons per acre or 93 liters per hectare. Application was started when the plants reached the V4-5 leaf stage.

Plants of maize inbred lines transformed with a DNA construct comprising DMO expression units were tested for prop-root damage and lodging by treatment with 2 lb/acre or 4 lb/acre dicamba at stage V4-5 and evaluation of the plants for prop-root damage (0%; no visible damage, to 100%, complete death of the plant) and lodging (degree of tilt) 24 days after treatment.

As shown in table 7, maize plants transformed with the DNA construct containing the DMO expression unit showed little or no damage to the rootlet and lodging compared to untransformed control inbred lines and plants transformed with the selectable marker expression unit alone (pMON 73746). This example shows that plants comprising DMO can be used to provide improved stand-up ability when treated with dicamba.

Table 7. maize plants transformed with various DMO constructs showed little or no branch root damage and lodging when treated with dicamba.

Reference to the literature

The following listed documents are incorporated herein by reference to supplement, explain, provide a background or teach methods, techniques and/or compositions employed herein.

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Sequence listing

<110> Monsanto technology Co

Feng，Paul C.C.

Malven，Marianne

Stanislaw Flasinski

<120> chloroplast transit peptides for efficient targeting of DMO and uses thereof

<130>MONS：107WO

<140> unknown

<141>2007-06-06

<150>11/758,659

<151>2007-06-05

<150>60/891,675

<151>2007-02-26

<160>41

<170>3.3

<210>1

<211>84

<212>PRT

<213> pea

<400>1

Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala

1 5 10 15

Ser Arg Gly Gln Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys Ser

20 25 30

Met Thr Gly Phe Pro Val Arg Lys Val Asn Thr Asp Ile Thr Ser Ile

35 40 45

Thr Ser Asn Gly Gly Arg Val Lys Cys Met Gln Val Trp Pro Pro Ile

50 55 60

Gly Lys Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Thr Arg

65 70 75 80

Asp Ser Arg Ala

<210>2

<211>57

<212>PRT

<213> pea

<400>2

Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala

1 5 10 15

Ser Arg Gly Gln Ser Ala Ala Met Ala Pro Phe Gly Gly Leu Lys Ser

20 25 30

Met Thr Gly Phe Pro Val Arg Lys Val Asn Thr Asp Ile Thr Ser Ile

35 40 45

Thr Ser Asn Gly Gly Arg Val Lys Cys

50 55

<210>3

<211>85

<212>PRT

<213> Arabidopsis thaliana

<400>3

Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala

1 5 10 15

Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala

20 25 30

Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser

35 40 45

Asn Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro Ile Glu Lys

50 55 60

Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly

65 70 75 80

Gly Arg Val Asn Cys

85

<210>4

<211>76

<212>PRT

<213> Arabidopsis thaliana

<400>4

Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu

1 5 10 15

Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val

20 25 30

Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser

35 40 45

Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg

50 55 60

Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys

65 70 75

<210>5

<211>76

<212>PRT

<213> Arabidopsis thaliana

<400>5

Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu

1 5 10 15

Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val

20 25 30

Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser

35 40 45

Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg

50 55 60

Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys

65 70 75

<210>6

<211>72

<212>PRT

<213> petunia

<400>6

Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro

1 5 10 15

Asn Ser Asn Phe His Lys Pro Gln Val Pro Lys Ser Ser Ser Phe Leu

20 25 30

Val Phe Gly Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser Met Leu Val

35 40 45

Leu Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile

50 55 60

Ser Ala Ser Val Ala Thr Ala Cys

65 70

<210>7

<211>69

<212>PRT

<213> wheat

<400>7

Met Ala Ala Leu Val Thr Ser Gln Leu Ala Thr Ser Gly Thr Val Leu

1 5 10 15

Ser Val Thr Asp Arg Phe Arg Arg Pro Gly Phe Gln Gly Leu Arg Pro

20 25 30

Arg Asn Pro Ala Asp Ala Ala Leu Gly Met Arg Thr Val Gly Ala Ser

35 40 45

Ala Ala Pro Lys Gln Ser Arg Lys Pro His Arg Phe Asp Arg Arg Cys

50 55 60

Leu Ser Met Val Val

65

<210>8

<211>77

<212>PRT

<213> Rice

<400>8

Met Ala Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe

1 5 10 15

Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe

20 25 30

Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu

35 40 45

Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val

50 55 60

Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Cys

65 70 75

<210>9

<211>57

<212>PRT

<213> tobacco

<400>9

Met Ala Ser Ser Val Leu Ser Ser Ala Ala Val Ala Thr Arg Ser Asn

1 5 10 15

Val Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Ala

20 25 30

Ala Ser Phe Pro Val Ser Arg Lys Gln Asn Leu Asp Ile Thr Ser Ile

35 40 45

Ala Ser Asn Gly Gly Arg Val Gln Cys

50 55

<210>10

<211>65

<212>PRT

<213> corn

<400>10

Met Glu Ser Leu Ala Ala Thr Ser Val Phe Ala Pro Ser Arg Val Ala

1 5 10 15

Val Pro Ala Ala Arg Ala Leu Val Arg Ala Gly Thr Val Val Pro Thr

20 25 30

Arg Arg Thr Ser Ser Arg Ser Gly Thr Ser Gly Val Lys Cys Ser Ala

35 40 45

Ala Val Thr Pro Gln Ala Ser Pro Val Ile Ser Arg Ser Ala Ala Ala

50 55 60

Ala

65

<210>11

<211>72

<212>PRT

<213> Ruta graveolens

<400>11

Met Gly Ala Ala Ala Thr Ser Met Gln Ser Leu Lys Phe Ser Asn Arg

1 5 10 15

Leu Val Pro Pro Ser Arg Arg Leu Ser Pro Val Pro Asn Asn Val Thr

20 25 30

Cys Asn Asn Leu Pro Lys Ser Ala Ala Pro Val Arg Thr Val Lys Cys

35 40 45

Cys Ala Ser Ser Trp Asn Ser Thr Ile Asn Gly Ala Ala Ala Thr Thr

50 55 60

Asn Gly Ala Ser Ala Ala Ser Ser

65 70

<210>12

<211>252

<212>DNA

<213> pea

<400>12

atggcttcta tgatatcctc ttccgctgtg acaacagtca gccgtgcctc tagggggcaa 60

tccgccgcaa tggctccatt cggcggcctc aaatccatga ctggattccc agtgaggaag 120

gtcaacactg acattacttc cattacaagc aatggtggaa gagtaaagtg catgcaggtg 180

tggcctccaa ttggaaagaa gaagtttgag actctttcct atttgccacc attgacgaga 240

gattcccggg cc 252

<210>13

<211>171

<212>DNA

<213> pea

<400>13

atggcttcta tgatatcctc ttccgctgtg acaacagtca gccgtgcctc tagggggcaa 60

tccgccgcaa tggctccatt cggcggcctc aaatccatga ctggattccc agtgaggaag 120

gtcaacactg acattacttc cattacaagc aatggtggaa gagtaaagtg c 171

<210>14

<211>255

<212>DNA

<213> Arabidopsis thaliana

<400>14

atggcttcct ctatgctctc ttccgctact atggttgcct ctccggctca ggccactatg 60

gtcgctcctt tcaacggact taagtcctcc gctgccttcc cagccacccg caaggctaac 120

aacgacatta cttccatcac aagcaacggc ggaagagtta actgtatgca ggtgtggcct 180

ccgattgaaa agaagaagtt tgagactctc tcttaccttc ctgaccttac cgattccggt 240

ggtcgcgtca actgc 255

<210>15

<211>228

<212>DNA

<213> Arabidopsis thaliana

<400>15

atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60

tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca 120

cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180

tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc 228

<210>16

<211>228

<212>DNA

<213> Artificial sequence

<400>16

atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60

tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca 120

cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180

tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc 228

<210>17

<211>216

<212>DNA

<213> Artificial sequence

<400>17

atggcccaga tcaacaacat ggcccagggc atccagaccc tgaaccctaa ctctaacttc 60

cacaagccgc aagtgcccaa gtctagctcc ttcctcgtgt tcggctccaa gaagctcaag 120

aatagcgcca attccatgct ggtcctgaag aaagactcga tcttcatgca gaagttctgc 180

tcctttcgca tcagtgcttc ggttgcgact gcctgc 216

<210>18

<211>207

<212>DNA

<213> Artificial sequence

<400>18

atggcggcac tggtgacctc ccagctcgcg acaagcggca ccgtcctgtc ggtgacggac 60

cgcttccggc gtcccggctt ccagggactg aggccacgga acccagccga tgccgctctc 120

gggatgagga cggtgggcgc gtccgcggct cccaagcaga gcaggaagcc acaccgtttc 180

gaccgccggt gcttgagcat ggtcgtc 207

<210>19

<211>231

<212>DNA

<213> Rice

<400>19

atggcggctc tcaccacgtc ccagctcgcc acctcggcca ccggcttcgg catcgccgac 60

aggtcggcgc cgtcgtcgct gctccgccac gggttccagg gcctcaagcc ccgcagcccc 120

gccggcggcg acgcgacgtc gctcagcgtg acgaccagcg cgcgcgcgac gcccaagcag 180

cagcggtcgg tgcagcgtgg cagccggagg ttcccctccg tcgtcgtgtg c 231

<210>20

<211>171

<212>DNA

<213> tobacco

<400>20

atggcttcct cagttctttc ctctgcagca gttgccaccc gcagcaatgt tgctcaagct 60

aacatggttg cacctttcac tggccttaag tcagctgcct cattccctgt ttcaaggaag 120

caaaaccttg acatcacttc cattgccagc aacggcggaa gagtgcaatg c 171

<210>21

<211>195

<212>DNA

<213> corn

<400>21

atggaatccc tagccgccac ctccgtgttc gcgccctccc gcgtcgccgt cccggcggcg 60

cgggccctgg ttagggcggg gacggtggta ccaaccaggc ggacgagcag ccggagcgga 120

accagcgggg tgaaatgctc tgctgccgtg acgccgcagg cgagcccagt gattagcagg 180

agcgctgcgg cggcc 195

<210>22

<211>216

<212>DNA

<213> Ruta graveolens

<400>22

atgggtgcag cggcaacgtc gatgcaatcc cttaaattct ccaaccgtct ggtcccaccc 60

agtcgccgtc tgtctccggt tccgaacaat gtcacctgca ataacctccc caagtctgca 120

gctcccgtcc ggacagtcaa atgctgcgct tcttcctgga acagtaccat caacggcgcg 180

gccgccacga ccaacggtgc gtccgccgcc agtagc 216

<210>23

<211>1023

<212>DNA

<213> Artificial sequence

<400>23

atggccactt tcgttagaaa cgcttggtac gttgctgcac ttcctgagga gttgagcgag 60

aagcctctag gaagaactat cctcgatact ccactagctc tctatcgtca acctgacgga 120

gttgtcgctg ccctgcttga tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180

ctagtcaacg gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag 240

tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt gcgatcattc 300

cctgtcgtgg aaagagacgc attgatctgg atctgccctg gagatccagc actcgcagat 360

cccggtgcta tccctgactt tgggtgtcgt gttgatccag cttaccgtac tgtcggaggt 420

tacggtcacg tggactgcaa ctacaagctc cttgtggata acctcatgga tcttggacac 480

gctcagtacg tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag 540

gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg cacaccctca 600

gttctcatgg ctaagttctt gcgtggtgct aacacaccag ttgacgcctg gaacgacatc 660

cggtggaata aggtgtcggc tatgctgaac ttcatcgcgg tcgcgccgga agggacgccg 720

aaggagcagt caatccactc ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780

tgccattact tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt 840

gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt cgttgaagct 900

atcgaaaggc ggagggctta cgtcgaagcg aacgggatca gacccgccat gttgtcctgc 960

gacgaggcag ccgtcagggt atccagggag attgagaagc tcgaacaact agaagcggcg 1020

tga 1023

<210>24

<211>340

<212>PRT

<213> Artificial sequence

<400>24

Met Ala Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu

1 5 10 15

Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu

20 25 30

Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile

35 40 45

Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly

50 55 60

His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln

65 70 75 80

Cys Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn

85 90 95

Val Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Cys

100 105 110

Pro Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly

115 120 125

Cys Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val

130 135 140

Asp Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His

145 150 155 160

Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg

165 170 175

Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met

180 185 190

Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg

195 200 205

Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys

210 215 220

Val Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro

225 230 235 240

Lys Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu

245 250 255

Thr Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly

260 265 270

Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln

275 280 285

Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg

290 295 300

Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys

305 310 315 320

Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln

325 330 335

Leu Glu Ala Ala

340

<210>25

<211>1023

<212>DNA

<213> Artificial sequence

<220>

<400>25

atgctcactt tcgttagaaa cgcttggtac gttgctgcac ttcctgagga gttgagcgag 60

aagcctctag gaagaactat cctcgatact ccactagctc tctatcgtca acctgacgga 120

gttgtcgctg ccctgcttga tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180

ctagtcaacg gacatctccs gtgtccatat cacggtctgg aatttgacgg aggtggccag 240

tgtgtccaca acccgGacgg caacggagcc cgccctgctt ctctgaacgt gcgatcattc 300

cctgtcgtgg aaagagacgc attgatctgg atctgccctg gagatccagc actcgcagat 360

cccggtgcta tccctgactt tgggtgtcgt gttgatccag cttaccgtac tgtcggaggt 420

tacggtcacg tggactgcaa ctacaagctc cttgtggata acctcatgga tcttggacac 480

gctcagtacg tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag 540

gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg cacaccctca 600

gttctcatgg ctaagttctt gcgtggtgct aacacaccag ttgacgcctg gaacgacatc 660

cggtggaata aggtgtcggc tatgctgaac ttcatcgcgg tcgcgccgga agggacgccg 720

aaggagcagt caatccactc ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780

tgccattact tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt 840

gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt cgttgaagct 900

atcgaaaggc ggagggctta cgtcgaagcg aacgggatca gacccgccat gttgtcctgc 960

gacgaggcag ccgtcagggt atccagggag attgagaagc tcgaacaact agaagcggcg 1020

tga 1023

<210>26

<211>340

<212>PRT

<213> Artificial sequence

<220>

<400>26

Met Leu Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu

1 5 10 15

Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu

20 25 30

Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile

35 40 45

Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly

50 55 60

His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln

65 70 75 80

Cys Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn

85 90 95

Val Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Cys

100 105 110

Pro Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly

115 120 125

Cys Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val

130 135 140

Asp Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His

145 150 155 160

Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg

165 170 175

Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met

180 185 190

Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg

195 200 205

Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys

210 215 220

Val Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro

225 230 235 240

Lys Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu

245 250 255

Thr Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly

260 265 270

Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln

275 280 285

Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg

290 295 300

Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys

305 310 315 320

Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln

325 330 335

Leu Glu Ala Ala

340

<210>27

<211>1023

<212>DNA

<213> Artificial sequence

<220>

<400>27

atgctcactt tcgttagaaa cgcttggtac gttgctgcac ttcctgagga gttgagcgag 60

aagcctctag gaagaactat cctcgatact ccactagctc tctatcgtca acctgacgga 120

gttgtcgctg ccctgcttga tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180

ctagtcaacg gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag 240

tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt gcgatcattc 300

cctgtcgtgg aaagagacgc attgatctgg atctggcctg gagatccagc actcgcagat 360

cccggtgcta tccctgactt tgggtgtcgt gttgatccag cttaccgtac tgtcggaggt 420

tacggtcacg tggactgcaa ctacaagctc cttgtggata acctcatgga tcttggacac 480

gctcagtacg tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag 540

gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg cacaccctca 600

gttctcatgg ctaagttctt gcgtggtgct aacacaccag ttgacgcctg gaacgacatc 660

cggtggaata aggtgtcggc tatgctgaac ttcatcgcgg tcgcgccgga agggacgccg 720

aaggagcagt caatccactc ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780

tgccattact tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt 840

gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt cgttgaagct 900

atcgaaaggc ggagggctta cgtcgaagcg aacgggatca gacccgccat gttgtcctgc 960

gacgaggcag ccgtcagggt atccagggag attgagaagc tcgaacaact agaagcggcg 1020

tga 1023

<210>28

<211>340

<212>PRT

<213> Artificial sequence

<220>

<400>28

Met Leu Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu

1 5 10 15

Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu

20 25 30

Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile

35 40 45

Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly

50 55 60

His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln

65 70 75 80

Cys Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn

85 90 95

Val Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp

100 105 110

Pro Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly

115 120 125

Cys Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val

130 135 140

Asp Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His

145 150 155 160

Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg

165 170 175

Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met

180 185 190

Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg

195 200 205

Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys

210 215 220

Val Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro

225 230 235 240

Lys Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu

245 250 255

Thr Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly

260 265 270

Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln

275 280 285

Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg

290 295 300

Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys

305 310 315 320

Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln

325 330 335

Leu Glu Ala Ala

340

<210>29

<211>1023

<212>DNA

<213> Artificial sequence

<220>

<400>29

atggccacct tcgtccgcaa tgcctggtat gtggcggcgc tgcccgagga actgtccgaa 60

aagccgctcg gccggacgat tctcgacaca ccgctcgcgc tctaccgcca gcccgacggt 120

gtggtcgcgg cgctgctcga catctgtccg caccgcttcg cgccgctgag cgacggcatc 180

ctcgtcaacg gccatctcca atgcccctat cacgggctgg aattcgatgg cggcgggcag 240

tgcgtccata acccgcacgg caatggcgcc cgcccggctt cgctcaacgt ccgctccttc 300

ccggtggtgg agcgcgacgc gctgatctgg atctgtcccg gcgatccggc gctggccgat 360

cctggggcga tccccgactt cggctgccgc gtcgatcccg cctatcggac cgtcggcggc 420

tatgggcatg tcgactgcaa ctacaagctg ctggtcgaca acctgatgga cctcggccac 480

gcccaatatg tccatcgcgc caacgcccag accgacgcct tcgaccggct ggagcgcgag 540

gtgatcgtcg gcgacggtga gatacaggcg ctgatgaaga ttcccggcgg cacgccgagc 600

gtgctgatgg ccaagttcct gcgcggcgcc aatacccccg tcgacgcttg gaacgacatc 660

cgctggaaca aggtgagcgc gatgctcaac ttcatcgcgg tggcgccgga aggcaccccg 720

aaggagcaga gcatccactc gcgcggtacc catatcctga cccccgagac ggaggcgagc 780

tgccattatt tcttcggctc ctcgcgcaat ttcggcatcg acgatccgga gatggacggc 840

gtgctgcgca gctggcaggc tcaggcgctg gtcaaggagg acaaggtcgt cgtcgaggcg 900

atcgagcgcc gccgcgccta tgtcgaggcg aatggcatcc gcccggcgat gctgtcgtgc 960

gacgaagccg cagtccgtgt cagccgcgag atcgagaagc ttgagcagct cgaagccgcc 1020

tga 1023

<210>30

<211>340

<212>PRT

<213> Artificial sequence

<220>

<400>30

Met Ala Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu

1 5 10 15

Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu

20 25 30

Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile

35 40 45

Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly

50 55 60

His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln

65 70 75 80

Cys Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn

85 90 95

Val Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Cys

100 105 110

Pro Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly

115 120 125

Cys Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val

130 135 140

Asp Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His

145 150 155 160

Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg

165 170 175

Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met

180 185 190

Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg

195 200 205

Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys

210 215 220

Val Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro

225 230 235 240

Lys Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu

245 250 255

Thr Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly

260 265 270

Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln

275 280 285

Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg

290 295 300

Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys

305 310 315 320

Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln

325 330 335

Leu Glu Ala Ala

340

<210>31

<211>1023

<212>DNA

<213> Artificial sequence

<220>

<400>31

atggccactt tcgttagaaa cgcttggtac gttgctgcac ttcctgagga gttgagcgag 60

aagcctctag gaagaactat cctcgatact ccactagctc tctatcgtca acctgacgga 120

gttgtcgctg ccctgcttga tatttgtccg catcgcttcg ctccgttgag tgacggtatt 180

ctagtcaacg gacatctcca gtgtccatat cacggtctgg aatttgacgg aggtggccag 240

tgtgtccaca acccgcacgg caacggagcc cgccctgctt ctctgaacgt gcgatcattc 300

cctgtcgtgg aaagagacgc attgatctgg atctggcctg gagatccagc actcgcagat 360

cccggtgcta tccctgactt tgggtgtcgt gttgatccag cttaccgtac tgtcggaggt 420

tacggtcacg tggactgcaa ctacaagctc cttgtggata acctcatgga tcttggacac 480

gctcagtacg tgcaccgcgc taacgcccaa acagacgcct tcgatagact tgagcgtgag 540

gtgatcgttg gcgacggcga gatccaggcg ctcatgaaga tccctggtgg cacaccctca 600

gttctcatgg ctaagttctt gcgtggtgct aacacaccag ttgacgcctg gaacgacatc 660

cggtggaata aggtgtcggc tatgctgaac ttcatcgcgg tcgcgccgga agggacgccg 720

aaggagcagt caatccactc ccgaggaacc catatcctta ctcctgagac cgaggcaagc 780

tgccattact tcttcggtag ttcccgcaac ttcggtatag acgatccaga gatggacggt 840

gttctcagga gctggcaagc tcaagccctg gtgaaggagg acaaagtggt cgttgaagct 900

atcgaaaggc ggagggctta cgtcgaagcg aacgggatca gacccgccat gttgtcctgc 960

gacgaggcag ccgtcagggt atccagggag attgagaagc tcgaacaact agaagcggcg 1020

tga 1023

<210>32

<211>340

<212>PRT

<213> Artificial sequence

<220>

<400>32

Met Ala Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu

1 5 10 15

Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu

20 25 30

Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile

35 40 45

Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly

50 55 60

His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln

65 70 75 80

Cys Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn

85 90 95

Val Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp

100 105 110

Pro Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile pro Asp Phe Gly

115 120 125

Cys Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val

130 135 140

Asp Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His

145 150 155 160

Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg

165 170 175

Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met

180 185 190

Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg

195 200 205

Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys

210 215 220

Val Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro

225 230 235 240

Lys Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu

245 250 255

Thr Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly

260 265 270

Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln

275 280 285

Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg

290 295 300

Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys

305 310 315 320

Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln

325 330 335

Leu Glu Ala Ala

340

<210>33

<211>1020

<212>DNA

<213> Pseudomonas maltophilia

<400>33

atgaccttcg tccgcaatgc ctggtatgtg gcggcgctgc ccgaggaact gtccgaaaag 60

ccgctcggcc ggacgattct cgacacaccg ctcgcgctct accgccagcc cgacggtgtg 120

gtcgcggcgc tgctcgacat ctgtccgcac cgcttcgcgc cgctgagcga cggcatcctc 180

gtcaacggcc atctccaatg cccctatcac gggctggaat tcgatggcgg cgggcagtgc 240

gtccataacc cgcacggcaa tggcgcccgc ccggcttcgc tcaacgtccg ctccttcccg 300

gtggtggagc gcgacgcgct gatctggatc tggcccggcg atccggcgct ggccgatcct 360

ggggcgatcc ccgacttcgg ctgccgcgtc gatcccgcct atcggaccgt cggcggctat 420

gggcatgtcg actgcaacta caagctgctg gtcgacaacc tgatggacct cggccacgcc 480

caatatgtcc atcgcgccaa cgcccagacc gacgccttcg accggctgga gcgcgaggtg 540

atcgtcggcg acggtgagat acaggcgctg atgaagattc ccggcggcac gccgagcgtg 600

ctgatggcca agttcctgcg cggcgccaat acccccgtcg acgcttggaa cgacatccgc 660

tggaacaagg tgagcgcgat gctcaacttc atcgcggtgg cgccggaagg caccccgaag 720

gagcagagca tccactcgcg cggtacccat atcctgaccc ccgagacgga ggcgagctgc 780

cattatttct tcggctcctc gcgcaatttc ggcatcgacg atccggagat ggacggcgtg 840

ctgcgcagct ggcaggctca ggcgctggtc aaggaggaca aggtcgtcgt cgaggcgatc 900

gagcgccgcc gcgcctatgt cgaggcgaat ggcatccgcc cggcgatgct gtcgtgcgac 960

gaagccgcag tccgtgtcag ccgcgagatc gagaagcttg agcagctcga agccgcctga 1020

<210>34

<211>339

<212>PRT

<213> Pseudomonas maltophilia

<400>34

Met Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu Glu

1 5 10 15

Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu Ala

20 25 30

Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile Cys

35 40 45

Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly His

50 55 60

Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln Cys

65 70 75 80

Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn Val

85 90 95

Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp Pro

100 105 110

Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly Cys

115 120 125

Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val Asp

130 135 140

Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His Ala

145 150 155 160

Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg Leu

165 170 175

Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met Lys

180 185 190

Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg Gly

195 200 205

Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys Val

210 215 220

Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro Lys

225 230 235 240

Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu Thr

245 250 255

Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly Ile

260 265 270

Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln Ala

275 280 285

Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg Arg

290 295 300

Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys Asp

305 310 315 320

Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln Leu

325 330 335

Glu Ala Ala

<210>35

<211>1023

<212>DNA

<213> Artificial sequence

<400>35

atgctcacct tcgtccgcaa tgcctggtat gtggcggcgc tgcccgagga actgtccgaa 60

aagccgctcg gccggacgat tctcgacaca ccgctcgcgc tctaccgcca gcccgacggt 120

gtggtcgcgg cgctgctcga catctgtccg caccgcttcg cgccgctgag cgacggcatc 180

ctcgtcaacg gccatctcca atgcccctat cacgggctgg aattcgatgg cggcgggcag 240

tgcgtccata acccgcacgg caatggcgcc cgcccggctt cgctcaacgt ccgctccttc 300

ccggtggtgg agcgcgacgc gctgatctgg atctgtcccg gcgatccggc gctggccgat 360

cctggggcga tccccgactt cggctgccgc gtcgatcccg cctatcggac cgtcggcggc 420

tatgggcatg tcgactgcaa ctacaagctg ctggtcgaca acctgatgga cctcggccac 480

gcccaatatg tccatcgcgc caacgcccag accgacgcct tcgaccggct ggagcgcgag 540

gtgatcgtcg gcgacggtga gatacaggcg ctgatgaaga ttcccggcgg cacgccgagc 600

gtgctgatgg ccaagttcct gcgcggcgcc aatacccccg tcgacgcttg gaacgacatc 660

cgctggaaca aggtgagcgc gatgctcaac ttcatcgcgg tggcgccgga aggcaccccg 720

aaggagcaga gcatccactc gcgcggtacc catatcctga cccccgagac ggaggcgagc 780

tgccattatt tcttcggctc ctcgcgcaat ttcggcatcg acgatccgga gatggacggc 840

gtgctgcgca gctggcaggc tcaggcgctg gtcaaggagg acaaggtcgt cgtcgaggcg 900

atcgagcgcc gccgcgccta tgtcgaggcg aatggcatcc gcccggcgat gctgtcgtgc 960

gacgaagccg cagtccgtgt cagccgcgag atcgagaagc ttgagcagct cgaagccgcc 1020

tga 1023

<210>36

<211>340

<212>PRT

<213> Artificial sequence

<400>36

Met Leu Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu

1 5 10 15

Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu

20 25 30

Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile

35 40 45

Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly

50 55 60

His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln

65 70 75 80

Cys Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn

85 90 95

Val Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Cys

100 105 110

Pro Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly

115 120 125

Cys Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val

130 135 140

Asp Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His

145 150 155 160

Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg

165 170 175

Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met

180 185 190

Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg

195 200 205

Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys

210 215 220

Val Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro

225 230 235 240

Lys Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu

245 250 255

Thr Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly

260 265 270

Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln

275 280 285

Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg

290 295 300

Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys

305 310 315 320

Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln

325 330 335

Leu Glu Ala Ala

340

<210>37

<211>1023

<212>DNA

<213> Artificial sequence

<400>37

atgctcacct tcgttaggaa cgcctggtac gtcgccgctc tccctgagga gctgagcgag 60

aagcccttgg gtcgcaccat cctagacact ccgttagccc tttaccgcca gcctgacggc 120

gtagtggcgg ccctgcttga catctgcccg cataggttcg ctccgctcag cgacggcatc 180

ctcgtcaacg ggcatcttca gtgcccgtac cacgggctgg aatttgacgg cggtgggcag 240

tgtgtccaca acccgcacgg caacggcgca cggccagctt ccctcaacgt taggtcgttc 300

cctgttgtcg agcgcgacgc actgatctgg atctggcctg gcgacccagc tctggccgat 360

ccaggagcca ttcccgactt cggttgccgc gtggacccag cctatcggac ggtcggcggt 420

tacgggcacg tcgattgtaa ctataagctc cttgtggaca accttatgga tttgggccac 480

gctcagtacg tgcaccgggc taacgctcag actgacgcct ttgaccgtct cgaaagggag 540

gtcatcgtcg gcgacggaga gattcaggcg ctgatgaaga tccctggagg cacgccctct 600

gtgctcatgg cgaagtttct cagaggcgcg aacacgcccg tggacgcctg gaacgacatc 660

cgctggaata aggtctccgc gatgctgaac ttcatcgccg ttgcgcccga gggcacaccc 720

aaagagcagt caatccacag cagagggacc catattctta caccggaaac cgaggctagt 780

tgccactact tcttcggctc gtcacggaat ttcgggatag acgatccgga gatggacggt 840

gttcttcgat cttggcaagc gcaagctctc gtcaaggaag ataaggtggt cgtggaggct 900

atcgagcgta ggcgcgccta cgttgaggcg aacggtatta ggcccgcgat gctgtcctgc 960

gacgaggccg cagttagagt gtcgcgcgag atagaaaagc tggagcagct agaggccgcc 1020

tga 1023

<210>38

<211>340

<212>PRT

<213> Artificial sequence

<400>38

Met Leu Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu

1 5 10 15

Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu

20 25 30

Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile

35 40 45

Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly

50 55 60

His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln

65 70 75 80

Cys Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn

85 90 95

Val Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp

100 105 110

Pro Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly

115 120 125

Cys Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val

130 135 140

Asp Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His

145 150 155 160

Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg

165 170 175

Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met

180 185 190

Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg

195 200 205

Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys

210 215 220

Val Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro

225 230 235 240

Lys Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu

245 250 255

Thr Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly

260 265 270

Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln

275 280 285

Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg

290 295 300

Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys

305 310 315 320

Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln

325 330 335

Leu Glu Ala Ala

340

<210>39

<211>1023

<212>DNA

<213> Artificial sequence

<400>39

atggccacct tcgttaggaa cgcctggtac gtcgccgctc tccctgagga gctgagcgag 60

aagcccttgg gtcgcaccat cctagacact ccgttagccc tttaccgcca gcctgacggc 120

gtagtggcgg ccctgcttga catctgcccg cataggttcg ctccgctcag cgacggcatc 180

ctcgtcaacg ggcatcttca gtgcccgtac cacgggctgg aatttgacgg cggtgggcag 240

tgtgtccaca acccgcacgg caacggcgca cggccagctt ccctcaacgt taggtcgttc 300

cctgttgtcg agcgcgacgc actgatctgg atctggcctg gcgacccagc tctggccgat 360

ccaggagcca ttcccgactt cggttgccgc gtggacccag cctatcggac ggtcggcggt 420

tacgggcacg tcgattgtaa ctataagctc cttgtggaca accttatgga tttgggccac 480

gctcagtacg tgcaccgggc taacgctcag actgacgcct ttgaccgtct cgaaagggag 540

gtcatcgtcg gcgacggaga gattcaggcg ctgatgaaga tccctggagg cacgccctct 600

gtgctcatgg cgaagtttct cagaggcgcg aacacgcccg tggacgcctg gaacgacatc 660

cgctggaata aggtctccgc gatgctgaac ttcatcgccg ttgcgcccga gggcacaccc 720

aaagagcagt caatccacag cagagggacc catattctta caccggaaac cgaggctagt 780

tgccactact tcttcggctc gtcacggaat ttcgggatag acgatccgga gatggacggt 840

gttcttcgat cttggcaagc gcaagctctc gtcaaggaag ataaggtggt cgtggaggct 900

atcgagcgta ggcgcgccta cgttgaggcg aacggtatta ggcccgcgat gctgtcctgc 960

gacgaggccg cagttagagt gtcgcgcgag atagaaaagc tggagcagct agaggccgcc 1020

tga 1023

<210>40

<211>340

<212>PRT

<213> Artificial sequence

<400>40

Met Ala Thr Phe Val Arg Asn Ala Trp Tyr Val Ala Ala Leu Pro Glu

1 5 10 15

Glu Leu Ser Glu Lys Pro Leu Gly Arg Thr Ile Leu Asp Thr Pro Leu

20 25 30

Ala Leu Tyr Arg Gln Pro Asp Gly Val Val Ala Ala Leu Leu Asp Ile

35 40 45

Cys Pro His Arg Phe Ala Pro Leu Ser Asp Gly Ile Leu Val Asn Gly

50 55 60

His Leu Gln Cys Pro Tyr His Gly Leu Glu Phe Asp Gly Gly Gly Gln

65 70 75 80

Cys Val His Asn Pro His Gly Asn Gly Ala Arg Pro Ala Ser Leu Asn

85 90 95

Val Arg Ser Phe Pro Val Val Glu Arg Asp Ala Leu Ile Trp Ile Trp

100 105 110

Pro Gly Asp Pro Ala Leu Ala Asp Pro Gly Ala Ile Pro Asp Phe Gly

115 120125

Cys Arg Val Asp Pro Ala Tyr Arg Thr Val Gly Gly Tyr Gly His Val

130 135 140

Asp Cys Asn Tyr Lys Leu Leu Val Asp Asn Leu Met Asp Leu Gly His

145 150 155 160

Ala Gln Tyr Val His Arg Ala Asn Ala Gln Thr Asp Ala Phe Asp Arg

165 170 175

Leu Glu Arg Glu Val Ile Val Gly Asp Gly Glu Ile Gln Ala Leu Met

180 185 190

Lys Ile Pro Gly Gly Thr Pro Ser Val Leu Met Ala Lys Phe Leu Arg

195 200 205

Gly Ala Asn Thr Pro Val Asp Ala Trp Asn Asp Ile Arg Trp Asn Lys

210 215 220

Val Ser Ala Met Leu Asn Phe Ile Ala Val Ala Pro Glu Gly Thr Pro

225 230 235 240

Lys Glu Gln Ser Ile His Ser Arg Gly Thr His Ile Leu Thr Pro Glu

245 250 255

Thr Glu Ala Ser Cys His Tyr Phe Phe Gly Ser Ser Arg Asn Phe Gly

260 265 270

Ile Asp Asp Pro Glu Met Asp Gly Val Leu Arg Ser Trp Gln Ala Gln

275 280 285

Ala Leu Val Lys Glu Asp Lys Val Val Val Glu Ala Ile Glu Arg Arg

290 295 300

Arg Ala Tyr Val Glu Ala Asn Gly Ile Arg Pro Ala Met Leu Ser Cys

305 310 315 320

Asp Glu Ala Ala Val Arg Val Ser Arg Glu Ile Glu Lys Leu Glu Gln

325 330 335

Leu Glu Ala Ala

340

<210>41

<211>433

<212>DNA

<213> Artificial sequence

<220>

<400>41

agatcttgag ccaatcaaag aggagtgatg tagacctaaa gcaataatgg agccatgacg 60

taagggctta cgcccatacg aaataattaa aggctgatgt gacctgtcgg tctctcagaa 120

cctttacttt ttatgtttgg cgtgtatttt taaatttcca cggcaatgac gatgtgaccc 180

aacgagatct tgagccaatc aaagaggagt gatgtagacc taaagcaata atggagccat 240

gacgtaaggg cttacgccca tacgaaataa ttaaaggctg atgtgacctg tcggtctctc 300

agaaccttta ctttttatat ttggcgtgta tttttaaatt tccacggcaa tgacgatgtg 360

acctgtgcat ccgctttgcc tataaataag ttttagtttg tattgatcga cacggtcgag 420

aagacacggc cat 433

Claims (8)

1. A recombinant DNA molecule comprising a DNA sequence encoding a chloroplast transit peptide operably linked to a DNA sequence encoding dicamba monooxygenase, wherein the DNA sequence encoding a chloroplast transit peptide encodes the amino acid sequence of SEQ ID NO: 1, and wherein the DNA sequence encoding dicamba monooxygenase encodes the polypeptide of SEQ ID NO: 30.

2. The recombinant DNA molecule of claim 1, wherein said DNA sequence encoding a chloroplast transit peptide is as set forth in SEQ ID NO: 12.

3. The recombinant DNA molecule of claim 1, wherein the DNA sequence encoding dicamba monooxygenase is as set forth in SEQ ID NO: 29, or a sequence shown in fig. 29.

4. The recombinant DNA molecule of claim 1, further comprising a promoter.

5. A method of producing a dicamba tolerant plant comprising: introducing into a plant cell a recombinant DNA molecule comprising a DNA sequence encoding a chloroplast transit peptide operably linked to a DNA sequence encoding dicamba monooxygenase, wherein the DNA sequence encoding a chloroplast transit peptide encodes SEQ ID NO: 1; and regenerating a plant comprising the recombinant DNA molecule from the plant cell.

6. The method of claim 5, wherein the plant is a dicot.

7. The method of claim 5, wherein the plant is a monocot.

8. A method of controlling weeds in a field comprising: cultivating a transgenic plant comprising a recombinant DNA molecule comprising a DNA sequence encoding a chloroplast transit peptide operably linked to a DNA sequence encoding dicamba monooxygenase, wherein the DNA sequence encoding a chloroplast transit peptide encodes SEQ ID NO: 1; and applying dicamba herbicide to the field in an amount effective to control weed growth without damaging the transgenic plants.