BRPI0312771B1 - METHODS FOR REDUCING TRANSGEN SILENCE AND DETECTING POLYNUCLEOTIDE IN DNA PLANTS, MOLECULE AND CONSTRUCTION AS WELL AS DNA DETECTION KIT - Google Patents

Description

(54) Title: METHODS TO REDUCE TRANSGENE SILENCE AND TO DETECT

POLYNUCLEOTIDE IN PLANTS, MOLECULE AND DNA CONSTRUCT, AS WELL AS DNA DETECTION KIT (73) Holder: MONSANTO TECHNOLOGY LLC, North American Society. Address: 800 North Lindbergh Boulevard, St. Louis, MO 63167, UNITED STATES OF AMERICA (US) (72) Inventor: STANISLAW FLASINSKI

Control Code: BB86314C31EB417B C5DCAD1E72219BF9

Validity Term: 10 (ten) years from 10/02/2018, subject to legal conditions

Issued on: 10/02/2018

Digitally signed by:

Liane Elizabeth Caldeira Lage

Director of Patents, Computer Programs and Topographies of Integrated Circuits

Invention Patent Descriptive Report for METHODS

TO REDUCE TRANSGENE SILENCE AND TO DETECT

POLYNUCLEOTIDE IN PLANTS, MOLECULE AND CONSTRUCTION OF

DNA, AS WELL AS DNA DETECTION KIT.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the genetic engineering of plants. More particularly, a method for constructing an artificial polynucleotide and methods of use for reducing the silencing of transgene in plants. The invention also relates to plant cells containing the artificial polynucleotide in which a plant cell is transformed to express the artificial polynucleotide and the plant regenerated therefrom.

Description of the Related Art

Heterologous genes can be isolated from a source other than the plant into which they will be transformed, or they can be modified or designed to have different or improved qualities. Particular traits or desired qualities of interest to the genetic breeding of plants may include, but are not limited to resistance to insects, fungal diseases and other pests and disease-causing agents, tolerances to herbicides, enhanced stability or shelf life, yield, tolerances to environmental stress and nutritional enhancements.

Traditional molecular biological methods for generating new genes and proteins in general have involved random or targeted mutagenesis. An example of random mutagenesis is a recombination technique known as DNA shuffling as disclosed in US Patents 5,605,793, 5,811,238, 5,830,721, 5,837,458 and International Orders WO 98/31837, WO 99/65927, all all of which are incorporated by reference. An alternative method of molecular evolution involves a stepped extension process (StEP) for mutagenesis and in vitro recombination of nucleic acid molecule sequences, as disclosed in US Patent 5,965,408, incorporated herein by reference. a

l example of targeted mutagenesis is the introduction of a point mutation at a specific site in a polypeptide.

An alternative method, useful when the heterologous gene is from a non-vegetable source, is to design an artificial insecticidal gene that uses the codon most frequently used in the codon use table in the corn plant (Koziel et al., 1993, Biotechnology 11 , 194 to 200). Fischhoff and Perlak (US Patent No. ⁹ 5,500,365, incorporated herein by reference) report the highest expression of Bacillus thuringiensis (Bt) insecticidal protein compared to crop plants when the polynucleotide sequence was modified to reduce the occurrence of destabilizing sequences . It was necessary to modify the wild-type Bt polynucleotide sequence because the naturally-occurring wild-type Bt polynucleotide did not express sufficient levels of insecticidal protein in plants to be agronomically useful.

The heterologous genes are cloned into vectors suitable for plant transformation. Transformation and regeneration techniques useful for incorporating heterologous genes into a plant genome are well known in the art. The gene can then be expressed in the plant cell to display the added trait or trait. However, heterologous genes that normally express well as transgenes can experience gene silencing when more than one copy of the same genes is expressed on the same plant. This can occur when a first heterologous gene is very similar to an endogenous gene DNA sequence in the plant. Other examples include when a transgenic plant is subsequently crossed with other transgenic plants having the same or similar transgenes or when the transgenic plant is re-transformed with a plant expression cassette containing the same or similar gene. Similarly, gene silencing can occur if the implantation of traits uses the same genetic elements used to direct the expression of the transgenic gene of interest. In order to implant traits, stable transgenic strains must be made with different combinations of genes and genetic elements to avoid gene silencing.

N-phosphonomethylglycine, also known as glyphosate, is a well-known herbicide that has activity in a wide spectrum of plant species. Glyphosate is the active ingredient in Roundup® (Monsanto Co.), a safe herbicide with a desirably short half-life in the environment. When applied to a plant surface, glyphosate moves systemically through the plant. Glyphosate is phytotoxic due to its inhibition of the shikimic acid pathway, which provides a precursor for the synthesis of aromatic amino acids. Glyphosate inhibits the enzyme 5-enolpyruvyl-3phosphochiquime synthase (EPSPS).

The glyphosate tolerance can also be obtained by the expression of EPSPS variants that have lower affinity for glyphosate and therefore retain their catalytic activity in the presence of glyphosate (US Patent No. 5,633,435 ^w, incorporated herein by reference). Enzymes that degrade glyphosate in plant tissues ^(s US Patent 5,463,175) are also capable of conferring cellular tolerance to glyphosate. Such genes are used to produce transgenic crops that are tolerant to glyphosate, thereby allowing glyphosate to be used for effective weed control with minimal concern for crop damage. For example, glyphosate tolerance has been genetically engineered maize (US Patent No. 5,554,798 ^and), wheat (US Patent Application 20020062503 C ^Q), soybean (US Patent Application 20020157139 ^and C) and canola (WO 9204449) all of which are incorporated by reference. Transgenes for glyphosate tolerance and transgenes for tolerance to other herbicides, for example, the bar gene, (Toki et al. Plant Physiol., 100: 1503 to 1507, 1992; Thompson ef al. EMBO J. 6: 2519 to 2523, 1987, fosfinothricin acetyltransferase, BAR gene isolated from Streptomyces; DeBlock et al. EMBO J., 6: 2513-2522, 1987, glufosinate herbicide) are also useful as selectable markers or recordable markers and may provide a useful phenotype for the selection of plants linked with other agronomically useful traits.

What is needed in the art are methods of planning genes for expression in plants to improve agronomically useful traits «· ········

J-Λ to avoid gene silencing when multiple copies are inserted and recombination with endogenous plant genes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Accumulated comparison of polynucleotide sequence changes in two versions of artificial rice EPSPS (OsEPSPS_AT, OsEPSPS_ZM) and a native rice EPSPS (OsEPSPS_Nat) the polypeptide of each modified to be resistant to glyphosate.

Figure 2. Accumulated comparison of the polynucleotide sequences of a native corn EPSPS (ZmEPSPS_Nat) and an artificial one (ZmEPSPS_ZM) the polypeptide of each modified to be resistant to glyphosate.

Figure 3. Cumulative comparison of the polynucleotide sequences of a native soybean EPSPS (GmEPSPS_Nat) and an artificial version (GmEPSPS_GM) of the polypeptide of each modified to be resistant to glyphosate.

Figure 4. Accumulated comparison of the polynucleotide sequences of a native BAR gene (BAR1_Nat) and two artificial versions with the codon of choice for Zea mays (BAR1_ZM) and Arabidopsis thaliana (BAR1_AT).

Figure 5. Cumulative comparison of native CTP2 and CP4EPSPS polynucleotide sequences (CTP2CP4_Nat) and artificial versions (CTP2CP4_AT, CTP2CP4_ZM and CTP2CP4_GM).

Figure 6. Plasmid map of pMON54949.

Figure 7. Plasmid map of pMON54950.

Figure 8. Plasmid map of pMON30151.

Figure 9. Plasmid map of pMON59302.

Figure 10. Plasmid map of pMON59307.

Figure 11. Plasmid map of pMON42411.

Figure 12. Plasmid map of pMON58400.

Figure 13. Plasmid map of pMON58401.

Figure 14. Plasmid map of pMON54964.

1.3

Figure 15. Plasmid map of pMON25455.

Figure 16. Plasmid map of pMON30152.

Figure 17. Plasmid map of pMON54992.

Figure 18. Plasmid map of pMON54985.

Figure 19. Plasmid map of pMON20999.

Figure 20. Plasmid map of pMON45313.

Figure 21. Plasmid map of pMON59308.

Figure 22. Plasmid map of pMON59309.

Figure 23. Plasmid map of pMON59313.

Figure 24. Plasmid map of pMON59396.

Figure 25. Plasmid map of pMON25496.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ ID NO: 1 OsEPSPS_TIPS A protein sequence of

EPSPS of rice modified to be resistant to glyphosate, with chloroplast transit peptide.

Polynucleotide sequence of an EPSPS polynucleotide native to rice modified to encode a glyphosate resistant protein.

Polynucleotide sequence of an artificial rice EPSPS polynucleotide using the Arabidopsis codon usage table and the methods of the present invention and further modified to encode a glyphosate resistant protein.

SEQIDNO: 2 OsEPSPS Nat

SEQ ID NO: 3 OsEPSPS AT ·· · »····· *

SEQ ID NO: 4

OsEPSPS ZM

SEQ ID NO: 5

GmEPSPSJKS

SEQ ID NO: 6

GmEPSPS Nat

SEQ ID NO: 7

GmEPSPS_GM

SEQ ID NO: 8

ZmEPSPS_TIPS

SEQ ID NO: 9

ZmEPSPSJMat ··· »· ···· • · · · · 9 · · · · · · · · · · · · ··· ·· ··

Polynucleotide sequence of an artificial rice EPSPS polynucleotide using the Zea mays codon usage table and the methods of the present invention and further modified to encode a glyphosate resistant protein.

A sequence of EPSPS protein from soy modified to be resistant to glyphosate, with chloroplast transit peptide.

Polynucleotide sequence of a native EPSPS polynucleotide from soy modified to encode a glyphosate resistant protein.

Polynucleotide sequence of an EPSPS polynucleotide from artificial soybean using the Glycine max codon usage table and the methods of the present invention and further modified to encode a glyphosate resistant protein.

A sequence of EPSPS protein from corn modified to be resistant to glyphosate, with chloroplast transit peptide.

Polynucleotide sequence of an EPSPS polynucleotide native to corn modified to encode a glyphosate resistant protein.

• · »·» ·· ·

SEQ ID NO; 10

ZmEPSPS ZM

SEQ ID NO: 11

CTP2 ··· ·· ···· · · · · · · · · · · · · · · · · · · · · · ·

SEQ ID NO: 12

SEQ ID NO: 13

CTP2 Nat

CTP2 AT

SEQ ID NO: 14

CTP2 ZM

SEQ ID NO: 15

CP4EPSPS

SEQ ID NO: 16

CP4EPSPS_Nat

Polynucleotide sequence of an EPSPS polynucleotide from artificial corn using the Zea mays codon usage table and the methods of the present invention and further modified to encode a glyphosate resistant protein.

Protein sequence of the chloroplast transit peptide 2 of the EPSPS gene of Arabidopsis.

Polynucleotide sequence of Arabidopsis EPSPS chloroplast transit peptide. Polynucleotide sequence of an artificial polynucleotide encoding CTP2 using the Arabidopsis codon usage table and the methods of the present invention.

Polynucleotide sequence of an artificial polynucleotide encoding CTP2 using the Zea mays codon usage table and the methods of the present invention.

The protein sequence of the glyphosate-resistant EPSPS protein of the CP4 strain of Agrobacterium.

Polynucleotide sequence of the native polynucleotide encoding the CP4EPSPS protein (US Patent No. ⁹ 5,633,435).

• · · · · ··

CP4EPSPS_AT • · ·· · · · · • ···· »···

SEQ ID NO: 17

SEQ ID NO: 18

SEQ ID NO: 19

SEQ ID NO: 20

SEQ ID NO: 21

CP4EPSPS_ZM

BAR1

BAR1 Nat

BAR1 AT

SEQ ID NO: 22

SEQ ID NO: 23

SEQ ID NO: 24

BAR1 ZM

CP4EPSPS_Syn

CP4EPSPS_AT_p1

Polynucleotide sequence of an artificial polynucleotide encoding the CP4EPSPS protein using the Arabidopsis codon usage table and the methods of the present invention.

Polynucleotide sequence of an artificial polynucleotide encoding the CP4EPSPS protein using the Zea mays codon usage table and the methods of the present invention.

The protein sequence of a phosphinothricin acetyltransferase. Polynucleotide sequence of the native polynucleotide isolated from Streptomyces that encodes phosphinothricin acetyltransferase. Polynucleotide sequence of an artificial polynucleotide encoding phosphinothricin acetyltransferase using the Arabidopsis codon usage table and the methods of the present invention.

Polynucleotide sequence of an artificial polynucleotide encoding phosphinothricin acetyltransferase using the Zea mays codon usage table and the methods of the present invention. Polynucleotide sequence of an artificial polynucleotide with a dicotyledon codon of choice.

Diagnosis of DNA primer molecule for the polynucleotide CP4EPSPS_AT.

V

SEQ ID NO: 25

SEQ ID NO: 26

SEQ ID NO: 27

SEQ ID NO: 28

SEQ ID NO: 29

SEQ ID NO: 30

SEQ ID NO: 31

SEQ ID NO: 32

SEQ ID NO: 33

SEQ ID NO: 34

SEQ ID NO: 35

CP4EPSPS_AT_p2

CP4EPSPS_ZM_p1

CP4EPSPS_ZM_p2

CP4EPSPS_Nat_p1

CP4EPSPS_Nat_p2

CP4EPSPS_Syn_p

CP4EPSPS_Syn_p

ZmAdhl initiator

ZmAdhl initiator2

GNAGIAMKS

CTPEPSPSCP4_G

M

Diagnosis of DNA primer molecule for the polynucleotide CP4EPSPS_AT.

Diagnosis of DNA primer molecule for the polynucleotide CP4EPSPS_ZM.

Diagnosis of DNA primer molecule for the polynucleotide CP4EPSPS_ZM.

Diagnosis of DNA primer molecule for the polynucleotide CP4EPSPS_Nat.

Diagnosis of DNA primer molecule for the polynucleotide CP4EPSPS_Nat.

Diagnosis of a DNA primer molecule for the polynucleotide CP4EPSPS_Syn.

Diagnosis of a DNA primer molecule for the polynucleotide CP4EPSPS_Syn.

Diagnosis of control primer 1 for the endogenous corn Adh1 gene.

Diagnosis of control primer 2 for the endogenous corn Adh1 gene.

Reason that provides resistance to glyphosate to a vegetable EPSPS. Polynucleotide sequence of an artificial polynucleotide encoding the CP4EPSPS protein using the Glycine max codon usage table.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for designing an artificial polynucleotide sequence that encodes a protein of interest, wherein the artificial polynucleotide is substantially divergent ·· ········

A polynucleotide naturally occurring in a plant or a polynucleotide that has been introduced as a transgene into a plant and the artificial polynucleotide and polynucleotide encode a substantially identical polypeptide.

The artificial polynucleotides of the present invention that encode proteins that provide agronomically useful phenotypes to a transgenic plant containing a DNA construct that comprises the artificial polynucleotide. Agronomically useful phenotypes include, but are not limited to: drought tolerance, increased yield, cold tolerance, disease resistance, insect resistance and herbicide tolerance.

Another aspect of the present invention is artificial polynucleotides that encode an herbicide-resistant EPSPS protein, a phosphinothricin acetyltransferase protein, a chloroplast transit peptide protein. In preferred embodiments of the present invention, the artificial polynucleotide molecule is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 35.

The present invention provides DNA constructs comprising: a promoter molecule that functions in plants, operably linked to an artificial polynucleotide molecule of the present invention, wherein the artificial polynucleotide molecule is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 35, operably linked to a transcription termination region.

The present invention further provides DNA constructs that comprise: a promoter molecule that functions in plants, operably linked to an artificial polynucleotide molecule encoding a chloroplast transit peptide, operably linked to a heterologous glyphosate resistant EPSPS, operably linked to a transcription termination signal region, where the artificial polynucleotide is substantial • V ········ ··· ··· · *> ··· • · · * • · · · · · · • «· · · • ·· ·· ·· ee divergent in the polynucleotide sequence of known polynucleotides that encode an identical chloroplast transit peptide.

The present invention provides DNA constructs that comprise at least two expression cassettes, the first expression cassette comprising a promoter molecule that functions in plants, operably linked to an artificial polynucleotide molecule of the present invention, operably linked to a signal region of transcription termination and the second expression cassette comprising a promoter molecule that functions in plants, operably linked to a polynucleotide molecule that encodes a substantially identical polypeptide like said artificial polynucleotide and is less than eight-five percent similar in sequence polynucleotide to said artificial polynucleotide, operably linked to a transcription termination signal region.

The present invention provides plant cells, plants or their progeny that comprise a DNA construct of the present invention. Of particular interest are the plants of your progeny selected from the group consisting of wheat, corn, rice, soybeans, cotton, potatoes, canola, lawn, forest trees, sorghum grains, vegetable crops, ornamental plants, forage crops and crops of fruits.

A method of the present invention reduces gene silencing during the generation of transgenic plants comprising the steps of:

a) construct an artificial polynucleotide that is substantially divergent from known polynucleotides that encode a substantially identical protein, and

b) constructing a DNA construct containing said artificial polynucleotide molecule; and

c) transforming said DNA construct into a plant cell; and

d) regenerating said plant cell in a transgenic plant; and

e) crossing said transgenic plant with a fertile plant, wherein said fertile plant contains a polynucleotide molecule that encodes a protein substantially identical to a protein encoded by said molecule12

··· • * • · ··· ·· • · · • · · • • • ·· ····· »·· ·· · · · · • · * · ·· ··· • · · • · · ··· ·· • • · ·· • · · · • · · · ··· ···· · * • ·

artificial polynucleotide cuvette and wherein said artificial polynucleotide molecule and said polynucleotide molecule are substantially divergent.

Another aspect of the invention is a transgenic plant cell comprising two polynucleotides, wherein at least one of the polynucleotides is a transgene and the two polynucleotides encode a substantially identical protein and are less than eight-five percent similar in the polynucleotide sequence.

Another aspect of the present invention in a method for reducing gene silencing during the production of transgenic plants comprises the steps of:

a) constructing an artificial polynucleotide that is substantially divergent from known polynucleotides that encode a substantially identical protein, and

b) constructing a first DNA construct containing said artificial polynucleotide molecule; and

c) transforming said DNA construct into a plant cell; and

d) regenerating said plant cell in a transgenic plant; and

e) retransforming a cell from said transgenic plant with a second DNA construct comprising a polynucleotide molecule that encodes a protein substantially identical to said artificial polynucleotide and said polynucleotide and artificial polynucleotide are substantially divergent in the polynucleotide sequence; and

f) regenerating said cell of step d in a transgenic plant that comprises both said artificial polynucleotide and said polynucleotide.

There are also provided by the present invention methods for the selection of a plant transformed with a DNA construct of the invention that comprises the steps of:

a) transforming a plant cell with a DNA construct of the present invention; e • · ········ dl

··· ··· ·· ···· • · • • • • 4 · • · • • · • ··· • t · • • • • • · ··· ·· ·· «·

b) cultivate said plant cell in a selective medium containing a herbicide selected from the group consisting of: glyphosate and glyphosinate, to selectively kill cells that have not been transformed with said DNA constructs; and

c) regenerate said plant cell in a fertile plant.

Another aspect of the invention is a method of detecting an artificial polynucleotide in a plant cell, plant or its transgenic progeny that comprises the steps;

a) contact a DNA sample isolated from said plant cell, plant or its progeny with a DNA molecule, wherein said DNA molecule comprises at least one DNA molecule from a pair of DNA molecules that when used in a reaction of nucleic acid amplification produces an amplicon that is diagnostic for said artificial polynucleotide molecule selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 35.

(a) carrying out a nucleic acid amplification reaction, thereby producing amplicon; and (b) detecting the amplicon.

The reagents provided to perform the above detection method include, but are not limited to: DNA molecules that specifically hybridize to an artificial polynucleotide molecule selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21 and SEQ ID NO : 22; and isolated DNA molecules selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

The present invention provides plants and progeny comprising a DNA molecule selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:

• · • · · · · · <ÀL

21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

The present invention provides pairs of DNA molecules selected from the group comprising: a first DNA molecule and a second DNA molecule, where the first DNA molecule is SEQ ID NO: 24 or its complement and the second DNA molecule is SEQ ID NO: 25 or its complement and the pair of DNA molecules when used in a DNA amplification method produces an amplicon and a first DNA molecule and a second DNA molecule, where the first DNA molecule is SEQ ID NO: 26 or its complement and the second DNA molecule is SEQ ID NO: 27 or its complement and the pair of DNA molecules when used in a DNA amplification method produces an amplicon, where the amplicon is diagnosis for the presence of an artificial polynucleotide of the present invention in the genome of a transgenic plant.

The present invention provides a plant and its progeny identified by a DNA amplification method to contain in its genome a DNA molecule selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 , SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

The present invention provides and considers DNA detection kits that comprise: at least one DNA molecule of sufficient length that is specifically homologous or complementary to an artificial polynucleotide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21 and SEQ ID NO: 22, wherein said DNA molecule is useful as a DNA probe or DNA primer; or at least one DNA molecule homologous or complementary to a DNA primer molecule selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

»· · · · · I

The present invention further provides a method of detecting the presence of an artificial polynucleotide that encodes a glyphosate-resistant EPSPS in a DNA sample, the method comprising:

(a) extracting a DNA sample from a plant; and (b) contacting the DNA sample with a labeled DNA molecule of sufficient length that is specifically homologous or complementary to an artificial polynucleotide selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 18, wherein said labeled DNA molecule is a probe of DNA; and (c) subjecting the sample and the DNA probe to the conditions of severe hybridization; and (d) detecting the DNA probe hybridized to the DNA sample.

The present invention provides an isolated polynucleotide that encodes an EPSPS enzyme, the EPSPS enzyme contains the SEQ ID NO: 34 motif. The present invention provides a DNA construct containing a polynucleotide that encodes the EPSPS enzyme with the SEQ motif ID NO: 34. A plant cell, plant or its progeny that is tolerant to glyphosate as a result of the expression of an EPSPS enzyme that contains the SEQ ID NO: 34 motif is an aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided to better define the present invention and to guide those skilled in the art in the practice of the present invention. Unless otherwise mentioned, the terms are to be understood according to conventional usage by those skilled in the relevant technique. Definitions of common terms in molecular biology may also be found in Rieger et al, Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag:. New York, (1991); and Lewin, Genes V, Oxford University Press: New York, (1994). The nomenclature for the DNA bases as presented in 37 CFR § 1,822 is used. The standard one- and three-letter nomenclature for amino acid residues is used.

Amino acid substitutions, amino acid variants, are preferably substitutions from a single amino acid residue to another amino acid residue at any position within the protein. Substitutions, deletions, insertions or any combination of these can be combined to arrive at a final construct.

An artificial polynucleotide as used in the present invention is a DNA sequence designed according to the methods of the present invention and created as an isolated DNA molecule for use in a DNA construct that provides expression of a protein in host cells and for the purposes of cloning into appropriate constructs or other uses known to those skilled in the art. Computer programs are available for these purposes, including but not limited to the BestFit or Gap programs from the Sequence Analysis Software Package, Genetics Computer Group (GCG), Inc., University of Wisconsin Biotechnology Center, Madison, W 53711. The artificial polynucleotide can be created by one or more methods known in the art, which include, but are not limited to: overlay PCR. An artificial polynucleotide of the present invention is substantially divergent from other polynucleotides that encode the identical or nearly identical protein.

The term chimeric refers to a sequence of nucleic acid or fusion protein. A chimeric nucleic acid coding sequence is comprised of two or more sequences joined in the matrix that encode a chimeric protein. A chimeric gene refers to multiple genetic elements derived from heterologous sources that comprise a gene.

The phrases coding sequence, open reading matrix and structural sequence refer to the region of continuous sequential nucleic acid triplets that encode a protein, polypeptide or peptide sequence.

Codon refers to a sequence of three nucleotides that specify a particular amino acid.

Use of codon or codon of choice refers to frequency ··· ··· ·· ···· · ·· ········ ·· ··· · · · · · · • ·· · · · · · · · · ······· · · • ··· ·· ·· ······· · · · of use of codons that encode amino acids in the coding sequences of organisms.

Complementarity and complement when referring to nucleic acid sequences, refer to the specific binding of adenine to thymine (uracil in RNA) and cytosine to guanine in the opposite strands of DNA or RNA.

Construct refers to the heterologous genetic elements operably linked together making up a recombinant DNA molecule and can comprise elements that provide the expression of a DNA polynucleotide molecule in a host cell and elements that provide maintenance of the construct.

C-terminal region refers to the region of a peptide, polypeptide or protein chain from its center to the end that carries the amino acid having a free carboxyl group.

The term divergent, as used herein, refers to the comparison of polynucleotide molecules that encode the same or nearly the same protein or polypeptide. The four-letter genetic code (A, G, C and T / U) comprises three-letter codons that direct tRNA molecules to assemble amino acids in a polypeptide from an mRNA pattern. Having more than one codon that can encode the same amino acid is referred to as degenerate. Degenerate codons are used to build substantially divergent polynucleotide molecules that encode the same polypeptide where these molecules have a nucleotide sequence of their full length in which they are less than 85% identical and there is no length of polynucleotide greater than than 23 nucleotides that are identical.

The term encoding DNA refers to chromosomal DNA, plasmid DNA, cDNA or artificial DNA polynucleotide that encodes any of the proteins discussed here.

The term endogenous refers to materials that originate from within an organism or cell.

Endonuclease refers to an enzyme that hydrolyzes <Χο DNA

double filament in indoor locations.

Exogenous refers to materials that originate from outside an organism or cell. This typically applies to nucleic acid molecules used in the production of host cells and transformed or transgenic plants.

Exon refers to the portion of a gene that is actually translated into protein, that is, a coding sequence.

The term expression refers to the transcription or translation of a polynucleotide to produce a corresponding gene product, an RNA or protein.

Fragments. A gene fragment is a portion of a full-length polynucleic acid molecule that is at least a minimum length capable of transcription into an RNA, translation into a peptide, or useful as a probe or primer in a DNA detection method .

The term gene refers to chromosomal DNA, plasmid DNA, cDNA, artificial DNA polynucleotide or other DNA that encodes a peptide, polypeptide, protein or RNA molecule and the genetic elements that flank the coding sequence that are involved in regulating expression .

The term genome as it applies to viruses encompasses all nucleic acid sequences contained within the virus capsid. The term genome as it applies to bacteria covers both the chromosome and the plasmids within a bacterial host cell. Nucleic acids encoding the present invention introduced into bacterial host cells can therefore be chromosomally integrated or located in the plasmid. The term genome as it applies to plant cells encompasses not only the chromosomal DNA found within the nucleus, but the DNA organelle found within the subcellular components of the cell. The nucleic acids of the present invention introduced into plant cells can therefore be chromosomally integrated or located in the organelle.

• · · · · ·

• · · · · · · · · ·

Glyphosate refers to N-phosphonomethylglycine and its salts, Glyphosate is the active ingredient in the herbicide Roundup® (Monsanto Co.). Glyphosate plant treatments refer to treatments with Roundup® or Roundup Ultra® herbicide formulations, unless otherwise stated. Glyphosate as N-phosphonomethylglycine and its salts (not formulated as the Roundup® herbicide) are components of synthetic culture media used for bacterial selection and plant tolerance to glyphosate or used to determine enzyme resistance in in vitro biochemical assays .

Sequence of heterologous DNA refers to a polynucleotide sequence that originates from a source or foreign species or, if from the same source, is modified from its original form.

Homologous DNA refers to DNA from the same source as that of the recipient cell.

Hybridization refers to the ability of a nucleic acid filament to join with a complementary filament through base pairing. Hybridization occurs when complementary sequences in the two nucleic acid strands are linked together. The nucleic acid probes and primers of the present invention hybridize under severe conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic event in a sample. Nucleic acid molecules or fragments thereof are able to specifically hybridize to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be able to hybridize specifically to each other if the two molecules are capable of forming a double-stranded antiparallel nucleic acid structure. A nucleic acid molecule is said to be the complement of another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit complete complementarity when each nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be minimally

Μ

complementary if they can hybridize to each other with sufficient stability to allow them to remain annealed to each other at least under conventional low severity conditions. Similarly, molecules are said to be complementary ”if they can hybridize to each other with sufficient stability to allow them to remain annealed to each other under conventional high severity conditions. Conventional severity conditions are described by Sambrook et al., 1989 and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985), incorporated herein by reference in its entirety. Deviations from complete complementarity are therefore permissible, provided that such deviations do not completely impede the ability of molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe it needs only to be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations used.

As used herein, a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to the complement of the nucleic acid sequence to which it is being compared under conditions of high severity. The term severe conditions is functionally defined with respect to the hybridization of a nucleic acid probe to a target nucleic acid (that is, to a particular nucleic acid sequence of interest) by the specific hybridization procedure discussed in Sambrook et al., 1989, on 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52, 9.56-9.58 incorporated herein by reference in its entirety; Kanehisa, (Nucl. Acids Res. 12: 203 to 213, 1984, hereby incorporated by reference in its entirety); and Wetmur and Davidson, (J. Mol. Biol. 31: 349 to 370, 1988, incorporated herein by reference in their entirety). Consequently, the nucleotide sequences of the invention can be used in terms of their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the intended application, it will be desirable to use variable hybridization conditions to ensure that the

obtain varying degrees of probe selectivity in relation to the target sequence. For applications that require high selectivity, it will typically be desirable to use relatively severe conditions to form the hybrids, for example, relatively low salt and / or high temperature conditions will be selected, as provided by about 0.02 M to about 0.15 M NaCl at temperatures from about 50 ° C to about 70 ° C. One of the severe conditions, for example, is to wash the hybridization filter at least twice with high-severity wash buffer. (0.2X SSC, 0.1% SDS, 65 ° C). The appropriate stringency conditions that promote DNA hybridization, for example, 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by a 2.0 x SSC wash at 50 ° C , are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, the saline concentration in the wash step can be selected from a low severity of about 2.0 x SSC at 50 ° C to a high severity of about 0.2 x SSC at 50 ° C. The temperature in the washing step can be increased from conditions of low severity at room temperature, about 22 ° C, to conditions of high severity at about 65 ° C. Both the temperature and the salt can be varied or the temperature or concentration of salt can be kept constant while the other variable is changed. Such selective conditions tolerate little, if any, mismatch between the probe and the target pattern or filament. Detection of DNA sequences via hybridization is well known to those skilled in the art and disclosed in the US Patents ³⁵ 4,965,188 and 5,176,995 are exemplary of the methods of hybridization analyzes.

Identity refers to the degree of similarity between two polynucleic acid or protein strings. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22: 4673 to 4680, 1994). The number of bases or paired amino acids • ·· ········ »« · · · · • · · · · · · · • · · ······· · · · is divided by the total number bases or amino acids and multiplied by 100 to obtain a percentage identity. For example, if two 580 base pair strings had 145 paired bases, they can be 25 percent identical. If the two strings compared are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there are 100 amino acids paired between a 200 and a 400 amino acid protein, they are 50 percent identical with respect to the shortest sequence. If the shortest sequence is less than 150 bases or 50 amino acids in length, the number of matches is divided by 150 (for nucleic acid bases) or 50 (for amino acids) and multiplied by 100 to obtain a percentage identity .

As described herein, a protein can be substantially identical to related proteins. These proteins with substantial identity in general comprise at least one polypeptide sequence that has at least ninety-eight percent sequence identity compared to its other related polypeptide sequence. The Gap program in WISCONSIN PACKAGE version 10.0-UNIX by Genetics Computer Group, Inc. is based on the method of Needleman and Wunsch (J. Mol. Biol. 48: 443 to 453, 1970) using the standard parameter set for comparison by the pair mode (for the comparison of amino acid sequence: Interval Creation Penalty = 8, Interval Extension Penalty = 20); or using the TBLASTN program in the BLAST 2.2.1 software suite (Altschul et al., Nucleic Acids Res. 25: 3389 to 3402), using the BLOSUM62 matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 to 10919, 1992) and the set of standard parameters for the comparison by the pair mode (interval creation value = 11, value for the interval extension = 1.). In BLAST, the E value or the probability value represents the number of different alignments with scores equivalent to or better than the raw alignment scores, S, which are expected to occur in a database search by chance. The lower the E value, the more significant the match • »··« · »··· • · · 9 • ·· ··· · · ···· t · · · · · · · · · · • · · · · · · · · · · · · · · ··· ··

Μ to. Because the size of the database is an element in E-value calculations, the E-values obtained by applying BLAST against public databases, such as GenBank, have generally increased over time for any given question / entry pairing. The percent identity refers to the percentage of identically matched amino acid residues that exist along the length of that portion of the strings that is aligned by the BLAST algorithm.

Intron refers to a portion of a gene that is not translated into protein, although it is transcribed into RNA.

An isolated nucleic acid sequence is substantially separated or purified from other nucleic acid sequences with which the nucleic acid is normally associated in the cell of the organism in which the nucleic acid naturally occurs, that is, other chromosomal or extrachromosomal DNA. The term encompasses nucleic acids that are biochemically purified in order to substantially remove contaminating nucleic acids and other cellular components. The term also encompasses recombinant nucleic acids and chemically synthesized nucleic acids.

Isolated, Purified, Homogeneous Polypeptides. A polypeptide is isolated if it has been separated from cellular components (nucleic acids, lipids, carbohydrates and other polypeptides) that naturally accompany it or that are chemically synthesized or recombinant. A monomeric polypeptide is isolated when at least 60% by weight of a sample is made up of the polypeptide, preferably 90% or more, more preferably 95% or more and most preferably more than 99%. The purity or homogeneity of the protein is indicated, for example, by the polyacrylamide gel electrophoresis of a protein sample, followed by the visualization of a single polypeptide strip in the dyeing of the polyacrylamide gel; high pressure liquid chromatography; or other conventional methods. Proteins can be purified by any means known in the art, for example as described in Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185. Academic Press, San

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Diego, 1990; and Scopes, Protein Purification: Principies and Practice, Springer Verlag, New York, 1982.

Lettering or labeled. There are a variety of conventional methods and reagents for labeling polynucleotides and polypeptides and fragments thereof. Typical labels include isotopes, ligands or radioactive ligand receptors, fluorophores, chemiluminescent agents and enzymes. Methods for labeling and guidance in choosing appropriate labels for various purposes are discussed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989) and Current Protocols in Molecular Biology, ed . Ausubel et al., Greene Publishing and Wiley-Interscience, New York, (1992).

Mature protein coding region, this term refers to the sequence of a processed protein product, that is, a mature EPSP synthase that remains after the chloroplast transit peptide has been removed.

Native, the term native in general refers to a naturally occurring polynucleic acid or polypeptide (wild type). However, in the context of the present invention, some modification of an isolated polynucleotide and polypeptide may have occurred to provide a polypeptide with a particular phenotype, for example, amino acid substitution in the glyphosate-sensitive EPSPS to provide a glyphosate-resistant EPSPS. For comparative purposes in the present invention, the isolated polynucleotide that contains a few substituted nucleotides to provide amino acid modification for herbicide tolerance is referred to as the native polynucleotide when compared to the substantially divergent polynucleotide created by the methods of the present invention. However, the native polynucleotide modified in this way is non-native with respect to the genetic elements normally found attached to a naturally occurring unmodified polynucleotide.

N-terminal region refers to a region of a peptide, polypeptide or protein chain of the amino acid having a free amino group at the center of the chain.

• · ·

Nucleic acid refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Nucleic acid codes: A = adenosine; C = cytosine; G = guanosine; T = thymidine. Codes used for the synthesis of oligonucleotides: N = equimolar A, C, G and T; I = deoxyinosine; K = equimolar G and T; R = equimolar A and G; S = equimolar C and G; W = equimolar A and T; Y = equimolar CeT.

A nucleic acid segment or a nucleic acid molecule segment is a nucleic acid molecule that has been isolated free of total genomic DNA from a particular species or that has been synthesized. Included with the term nucleic acid segment are the segments of DNA, recombinant vectors, plasmids, cosmids, phagemids, phage, viruses, et cetera.

Nucleotide Sequence Variants, using well-known methods, the person skilled in the art can easily produce nucleotide and amino acid sequence variants of genes and proteins, respectively. For example, the variant DNA molecules of the present invention are DNA molecules containing changes in an EPSPS gene sequence, that is, changes that include one or more nucleotides in the EPSPS gene sequence being deleted, added and / or replaced, such that the variant EPSPS gene encodes a protein that retains EPSPS activity. Variant DNA molecules can be produced, for example, by standard DNA mutagenesis techniques or by chemically synthesizing the variant DNA molecule or a portion thereof. Methods for the chemical synthesis of nucleic acids are discussed, for example, in Beaucage et al., Tetra. Letts. 22: 1859 to 1862 (1981) and Matteucci et al., J. Am. Chem. Soc. 103: 3185- (1981). Chemical synthesis of nucleic acids can be carried out, for example, on automated oligonucleotide synthesizers. Such variants preferably do not change the reading frame of the region encoding the nucleic acid protein and preferably encode a protein having no change or only minor reduction.

Open reading matrix (ORF) refers to a region of DNA or RNA that encodes a peptide, polypeptide or protein.

Operably Connected. A first sequence of nucleic acid is operably linked with a second sequence of nucleic acid when the first sequence of nucleic acid is placed in a functional relationship with the second sequence of nucleic acid. For example, a promoter is operably linked to a protein coding sequence if the promoter causes the transcription or expression of the coding sequence. In general, the operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the reading matrix.

Overexpression refers to the expression of an RNA or polypeptide or protein encoded by a DNA introduced into a host cell, where the RNA or polypeptide or protein is not normally present in the host cell or where the RNA or polypeptide or protein is present in the said host cell at a higher level than that normally expressed from the endogenous gene that encodes the RNA or polypeptide or protein.

The term plant encompasses any superior plant and its progeny, including monocots (for example, corn, rice, wheat, barley, etc.), dicots (for example, soybeans, cotton, canola, tomatoes, potatoes, Arabidopsis, tobacco, etc. ), gymnosperms (pines, firs, cedars, etc.) and includes parts of plants, including reproductive units of a plant (for example, seeds, bulbs, tubers, fruits, flowers, etc.) or other parts or tissues of that plant reproduced.

Plant expression cassette refers to segments of chimeric DNA that comprise the regulatory elements that are operably linked to provide expression of a transgene product in plants.

Plasmid refers to a circular, extrachromosomal, self-replicating piece of DNA.

Polyadenylation signal or polyA signal refers to a nucleic acid sequence located 3 'to a coding region that causes the addition of adenylate nucleotides to the 3' end of the mRNA transcribed from the coding region .

Polymerase chain reaction (PCR) ”refers to a method of DNA amplification that uses an enzymatic technique to create multiple copies of a nucleic acid sequence (amplicon). Copies of a DNA molecule are prepared by transporting a DNA polymerase between two amplimers. The basis of this amplification method are multiple cycles of temperature changes to denature, then anneal the amplimers (DNA-preparing molecules), followed by the extension to synthesize new DNA strands in the region located between the flanking amplimers. Nucleic acid amplification can be performed by any of the various methods of nucleic acid amplification known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in US Patents ⁸⁵ 4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Method and Applications, ed. Innis et al., Academic Press, San Diego, 1990. PCR amplification methods were developed to amplify up to 22 kb of genomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sei USA 91: 5695 to 5699, 1994). These methods as well as other methods known in the DNA amplification technique can be used in the practice of the present invention.

Polynucleotide refers to an extension of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules larger than two, which are connected to form a larger molecule.

Polypeptide fragments. The present invention also encompasses fragments of a protein that lacks at least one residue of a protein of native natural length, but which substantially maintains the activity of the protein.

The term promoter or promoter region refers to a polynucleic acid molecule that functions as a regulatory element, usually found upstream (5 ') in relation to an encoded sequence.

ra, which controls the expression of the coding sequence by controlling the production of messenger RNA (mRNA) by which it provides the recognition site for RNA polymerase and / or other factors necessary for the initiation of transcription at the correct site. As considered herein, a promoter or promoter region includes variations of derived promoters by binding to various regulatory sequences, random or controlled mutagenesis and the addition or duplication of enhancer sequences. The promoter region disclosed here and its biologically functional equivalents, are responsible for conducting the transcription of coding sequences under its control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its ability to produce mRNA.

Recombinant. A recombinant nucleic acid is made by a combination of two otherwise separated segments of sequence, for example, by chemical synthesis or by manipulating isolated segments of nucleic acids by genetic engineering techniques.

The term recombinant DNA construct ”or recombinant vector refers to any agent such as a plasmid, cosmid, virus, autonomously replicating sequence, phage or nucleotide sequence of DNA or RNA of single or double-stranded linear or circular filament, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule that one or more DNA sequences have been linked in a functionally operative way. Such recombinant DNA constructs or vectors are capable of introducing a 5 'regulatory sequence or promoter region and a DNA sequence for a selected gene product in a cell in such a way that the DNA sequence is transcribed into a functional mRNA that is translated and therefore expressed. The recombinant DNA constructs or recombinant vectors can be constructed to be able to express antisense RNAs, in order to inhibit the translation of a specific RNA of interest.

Regeneration refers to the process of growing a plant from a plant cell (for example, plant protoplasts or explants).

3 _Ύ

Reporter refers to a gene and corresponding gene product that when expressed in transgenic organisms produce a product detectable by chemical or molecular methods or produce an observable phenotype.

Resistance refers to an enzyme that is capable of functioning in the presence of a toxin, for example, glyphosate resistant class II EPSP synthases. An enzyme that is resistant to a toxin may have the function of detoxifying the toxin, for example, phosphinothricin acetyltransferase, glyphosate oxidoreductase or it may be a mutant enzyme having catalytic activity that is not affected by a herbicide that disrupts the same activity in the enzyme wild-type, for example, acetolactate synthase, mutant class I EPSP synthases.

Restriction enzyme refers to an enzyme that recognizes a specific palindromic sequence of nucleotides in double-stranded DNA and cleaves both strands; also called a restriction endonuclease. Diving typically occurs within the restriction site.

Selectable marker! refers to a polynucleic acid molecule that encodes a protein, which confers a phenotype that facilitates the identification of cells containing the polynucleic acid molecule. Selectable markers include those genes that confer resistance to antibiotics (eg, ampicillin, kanamycin), complement a nutritional deficiency (eg, uracil, histidine, leucine) or communicate a visually distinguishing characteristic (eg, color changes or fluorescence ). Selectable useful dominant marker genes include genes that encode antibiotic resistance genes (for example, neomycin phosphotransferase, aad); and herbicide resistance genes (for example, phosphinothricin acetyltransferase, class II EPSP synthase, modified class I EPSP synthase). A useful strategy for the selection of transformants for herbicide resistance is described, for example, in Vasil, Cell Culture and Somatic Cell Genetics of Plants, Vols. I-III, Laboratory Procedures and Their Applications Academic Press, New York (1984).

The specific term for (a target sequence) indicates that a DNA probe or DNA primer hybridizes under hybridization conditions given only to the target sequence in a sample that comprises the target sequence.

The term substantially purified, as used herein, refers to a molecule separate from other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation. A substantially purified molecule can be greater than 60% free, preferably 75% free, more preferably 90% free of the other molecules (solvent-only) present in the natural mixture. The term substantially purified is not intended to cover molecules present in their native state.

Tolerant or tolerance refers to a reduced effect of a biotic or abiotic agent on the cultivation and development of organisms and plants, for example, a pest or herbicide.

Transcription refers to the process of making a copy of RNA from standard DNA.

Transformation refers to a process of introducing an exogenous polynucleic acid molecule (for example, a DNA construct, a recombinant polynucleic acid molecule) into a cell or protoplasty and for the exogenous polynucleic acid molecule to be incorporated into a chromosome or be capable of autonomous replication.

Transformed or transgenic refers to a cell, tissue, organ or organism in which a foreign polynucleic acid, such as a DNA vector or recombinant polynucleic acid molecule. A transgenic or transformed cell or organism also includes the progeny of the cell or organism and the progeny produced from a breeding program that uses such a transgenic plant as a precursor at an intersection and that exhibits an increased phenotype resulting from the presence of the strange polynucleic acid.

The term transgene refers to any acidic molecule

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linucleic acid not native to a cell or organism transformed into the cell or organism. Transgene also encompasses the component parts of a native plant gene modified by the insertion of a non-native polynucleic acid molecule by targeted recombination or site-specific mutation.

Transit peptide or targeting peptide molecules, these terms generally refer to peptide molecules that when attached to a protein of interest direct the protein to a particular tissue, cell, subcellular location or cellular organelle. Examples include, but are not limited to, chloroplast transit peptides, nuclear targeting signals and vacuolar signals. The chloroplast transit peptide is of particular use in the present invention for directing the expression of the EPSPS enzyme to the chloroplast.

The term translation refers to the production of the corresponding gene product, that is, a peptide, polypeptide or protein from an mRNA.

Vector refers to a plasmid, cosmid, bacteriophage or virus that carries foreign DNA in a host organism. Polynucleotides

The methods of the present invention include designing genes that confer a trait of interest to the plant into which they are introduced. Transgenes of agronomic interest that provide beneficial agronomic traits for crop plants, for example, including, but not limited to, genetic elements that comprise herbicide resistance (US Patent N ⁹ 5,633,435; US Patent N ⁹ 5,463,175), increased yield (U.S. Patent No. ⁹ 5,716,837), insect control (U.S. Patent ^No.'s 6,063,597; US Patent No. ⁹ 6,063,756; US Patent No. ⁹ 6,093,695; US Patent No. ⁹ 5,942,664; U.S. N ^s 6,110,464), fungal disease resistance (U.S. Patent No. 5,516,671 ^{9; 9} US Patent 5,773,696; US Patent No. ⁹ 6,121,436, and US Patent 6,316,407 , and ^9th US Patent 6,506 ⁹ .962), virus resistance (US Patent N ⁹ 5,304,730 and US Patent N ⁹ 6,013,864), nematode resistance (US Patent N ⁹ 6,228,992), bacterial resistance to diseases (US Patent N ⁹ • · · •

• <

• · ·

5,516,671), starch production (U.S. Patent No. 5,750,876 and U.S. ^{9 9} No 6,476,295), modified oil production (U.S. Patent No. 6,444,876), high oil production (U.S. Patent No. 5,608 ^to .149 ^s and US Patent 6,476,295), modified fatty acid content (U.S. Patent No. ⁹ 6,537,750), high protein production (U.S. Patent No. ⁹ 6,380,466), fruit ripening (U.S. Patent No. 5,512 ⁹ .466), enhanced animal feed and human ^(s US Patent 5,985,605 and US Patent No. ⁹ 6,171,640), biopolymers (U.S. Patent No. 5,958,745 and ⁹ of US Patent Application US20030028917 ^9), resistance to environmental stress (US Patent No. ⁹ 6,072,103), pharmaceutical peptides (US Patent No. ⁹ 6,080,560), improved processing traits (US Patent No. ⁹ 6,476,295), improved digestibility (US Patent No. ⁹ 6,531,648) low raffinose ( US Patent No. ⁹ 6,166,292), production of industrial enzyme (US Patent No. ⁹ 5,543,576), improved taste (US Patent No. ⁹ 6,011,199), nitrogen fixation (US Patent No. ⁹ 5,229,114), production of hybrid seed (US Patent No. ⁹ 5,689,041) and biofuel production (US Patent No. ⁹ 5,998,700), the genetic elements and transgenes described in the patents listed above are hereby incorporated by reference.

The herbicides to which the tolerance of the transgenic plant has been demonstrated and the method of the present invention can be applied, include but are not limited to: glyphosate herbicides, glyphosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cycloananedione, protoporphyriongen oxidase inhibitors and isoxasflutol. Polynucleotide molecules that encode proteins involved in herbicide tolerance are known in the art and include, but are not limited to, a polynucleotide molecule that encodes 5-enolpyruvylshiquimato-3-phosphate synthase (EPSPS, described in US Patents No. 5,627. 061, 5,633,435. 6,040,497; Padgette et al. Herbicide Resistant Crops, Lewis Publishers, 53 to 85, 1996; and PenalozaVazquez, et al. Plant Cell Reports 14: 482 to 487, 1995; and aroA (US Patent No. ⁹ 5,094,945) for glyphosate tolerance; bromoxynil nitrilase (Bxn) for tolerance to bromoxynil (US Patent No. ⁹ 4,810,648); phytoene desaturase (crtl, Misawa et al, (1993) Plant J. 4; 833 a 840 and (1994) Plant J. 6; 481 to 489); for tolerance to norflurazon, acetohydroxy acid synthase (AHAS, aka

ALS, Sathasiivan et al. Nucl. Acids Res. 18: 2188 to 2193. 1990); and the bar gene for tolerance to glufosinate and bialaphos (DeBlock, et al. EMBO J. 6: 2513 to 2519, 1987).

Herbicide tolerance is a desirable phenotype for crop plants. N-phosphonomethylglycine, also known as glyphosate, is a well-known herbicide that has activity in a wide spectrum of plant species. Glyphosate is the active ingredient in Roundup® (Monsanto Co.), a safe herbicide with a desirably short half-life in the environment. When applied to a plant surface, glyphosate moves systemically through the plant. Glyphosate is toxic to plants by inhibiting the pathway of shikimic acid, which provides a precursor for the synthesis of aromatic amino acids. Specifically, glyphosate affects the conversion of phosphoenolpyruvate and 3-phosphochiquimic acid to 5-enolpyruvyl-3-phosphochiquimic acid by inhibiting the enzyme 5-enolpyruvyl-3-phosphochiquime synthase (hereinafter referred to as EPSP synthase or EPSPS). For the purposes of the present invention, the term glyphosate should be considered to include any herbicidally effective form of N-phosphonomethylglycine (including any salt thereof) and other forms that result in the production of the glyphosate anion in the plant.

Through plant genetic engineering methods, it is possible to produce glyphosate-tolerant plants by inserting a DNA molecule into the plant's genome that causes the production of higher levels of wild-type EPSPS (Shah et al., Science 233: 478 a 481, 1986). Tolerance to glyphosate can also be achieved by expressing EPSPS variants that have lower affinity for glyphosate and therefore retain their catalytic activity in the presence of glyphosate (US Patent No. 5,633,435). Enzymes that degrade glyphosate in plant tissues (US Patent No. ^Q 5,463,175) are also capable of conferring cellular tolerance to glyphosate. Such genes, therefore, allow the production of transgenic crops that are tolerant to glyphosate, thereby allowing glyphosate to be used for effective weed control with minimal concerns of crop damage. For example, tolerance to glyphosate was genetically engineered • · · «· ·· · •« • «• · · drained on corn (US Patent No. ^Q 5,554,798, 6,040,497), wheat (Zhou et al. Rep. 15: 159 to 163, 1995), soy (WO 9200377) and canola (WO 9204449).

Variants of the wild-type EPSPS enzyme have been isolated that are resistant to glyphosate as a result of changes in the EPSPS amino acid coding sequence (Kishore et al., Annu. Rev. Biochem. 57: 627 to 663, 1988; Schulz etal. , Arch. Microbiol. 137: 121 to 123. 1984; Sost et al., FEBS Lett. 173: 238 to 241, 1984; Kishore et al., In Biotechnology for Crop Protections ACS Symposium Series No. 379. eds. Hedlin et al., 37 to 48, 1988). These variants typically have a higher Ki for glyphosate than the wild-type EPSPS enzyme that confers the glyphosate-tolerant phenotype, but these variants are also characterized by a high K _m for PEP that makes the enzyme kinetically less efficient. For example, the apparent K _m for PEP and the apparent K for glyphosate for E. coli native EPSPS are 10 μΜ and 0.5 μΜ while for a glyphosate resistant isolate having a single amino acid substitution of an alanine in place of glycine at position 96 these values are 220 μΜ and 4.0 mM, respectively. ^And US Patent No. 6,040,497 reports that the mutation known as the TIPS mutation (substitution of an isoleucine in place of threonine at amino acid position 102 and a serine substitution in place of proline at amino acid position 106) comprises two mutations when introduced in the EPSEA polypeptide sequence of Zea mays it confers resistance to the enzyme glyphosate. Transgenic plants containing this mutant enzyme are tolerant to glyphosate. Identical mutations can be made in glyphosate-sensitive EPSPS enzymes from other sources to create glyphosate-resistant enzymes.

A variety of native and variant EPSPS enzymes have been expressed in transgenic plants in order to confer glyphosate tolerance (Singh, et al., In Biosynthesis and Molecular Regulation of Amino Acids in Plants, Amer Soc Plant Phys. Pubs., 1992) . Examples of some of these EPSPSs include those described and / or isolated according to US Patent No. ⁵ 4,940,835. ^Q US Patent 4,971,908, US Patent No. • · · · "······"

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5,145,783, US Patent No. ^s 5,188,642, US Patent 5,310,667 , and ^9th US Patent 5,312,910 ^9. They can also be derived from a structurally distinct class of non-homologous EPSPS genes, such as the class II EPSPS genes isolated from Agrobacterium sp. strain CP4 as described in US Patent 5,633,435 , and ^9th US Patent 5,627,061 ^9.

Chloroplast transit peptides (CTPs) are engineered to be fused to the N-terminus of bacterial EPSPS to target glyphosate-resistant enzymes in the plant's chloroplast. In native plant EPSPS, the chloroplast transit peptide regions are contained in the native coding sequence (for example, CTP2, Klee et al., Mol. Gen. Genet. 210: 47 to 442, 1987, hereby incorporated by reference in their totality). The native CTP can be replaced with a heterologous CTP during the construction of a transgene plant expression cassette. Many proteins located in the chloroplast, including EPSPS, are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP) that is removed during the import steps. Examples of other such chloroplast proteins include the small subunit (SSU) of Ribulose-1,5-bisphosphate carboxylase, Ferredoxin, Ferredoxin oxidoreductase, protein I and protein II of the light harvesting complex and Thioredoxin F. in vivo and in vitro that non-chloroplast proteins can be targeted to the chloroplast by using protein fusions with a CTP and that the CTP sequence is sufficient to target a protein to the chloroplast. The incorporation of a suitable chloroplast transit peptide, such as EPSPS CTP from Arabidopsis thaliana (Klee et al., Mol. Gen. Genet. 210: 437 to 442 (1987) and EPSPS CTP from Petunia hybrida ( della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83: 6873 to 6877 (1986) has been shown to target the EPSPS protein sequences heterologous to chloroplasts in transgenic plants. The production of glyphosate-tolerant plants by the expression of a fusion protein comprising an amino-terminal CTP with a EPSPS glyphosate resistant enzyme is well known to those skilled in the art (US Patent No. ⁹ 5,627,061, US Patent No. ^s 5,633,435.

·· ····

• · • · ·

US Patent No. ^Q 5,312,910, EP 0218571, EP 189707, EP 508909 and EP 924299). Those skilled in the art will recognize that various chimeric constructs can be made that use the functionality of a particular CTP to import glyphosate-resistant EPSPS enzymes into the plant cell chloroplast.

Modification and changes can be made in the structure of the polynucleotides of the invention and still obtain a molecule that encodes a functional protein or peptide with desirable characteristics. The following is a method based on replacing the codon (s) of a first polynucleotide to create an equivalent or improved secondary generation polynucleotide, where this new artificial polynucleotide is useful in transgenic gene implantation methods and enhanced expression. It is considered that codon substitutions in the secondary generation polynucleotide may in certain cases result in at least one amino acid different from that of the first polynucleotide. Amino acid substitution can provide an improved protein characteristic, for example, a glyphosate resistant EPSP synthase or it can be a conserved change that does not substantially affect the characteristics of the protein. The method provides an artificial polynucleotide created by retro-translating a polypeptide sequence into a polynucleotide using a codon usage table, followed by steps to highlight the characteristics of the artificial polypeptide that makes it particularly useful in transgenic plants.

In forms of particular embodiments of the invention, modified polypeptides encoding herbicide-resistant proteins are considered to be useful for at least one of the following: confer herbicide tolerance in a transformed or transgenic plant, to improve the expression of herbicide resistance genes in plants, for use as selectable markers for introducing other traits of interest in a plant and to prevent recombination with a similar endogenous plant gene or existing transgene while still allowing gene implantation without gene silencing.

It is known that the genetic code is degenerate. The amino acids37

and their RNA codon (s) are listed below in Table 1. TABLE 1. Amino acids and the RNA codons that encode them. Codon Amino Acid

Histidine; His; H Isoleucine; Ile; I

Full name; 3 letter code; 1 letter code Alanine; Allah; GCA GCC GCG GCU Cysteine; Cys; C UGC UGU Aspartic acid; Asp; D GAC GAU Glutamic acid; Glu; E GAA GAG Phenylalanine; Phe; FUUC UUU Glycine; Gly; G GGA GGC GGG GGU CAC CAU AUA AUC AUU

Lysine; Lys; KAAA AAG

Leucine; Read; L UUA UUG CUA CUC CUG CUU

Methionine; Met; M AUG

Asparagine; Asn; N AAC AAU

Proline; Pro; P CCA CCC CCG CCU

Glutamine; Gin; Q CAA CAG

AGA AGG CGA CGC CGG CGU AGC AGU UCA UCC UCG UCU ACA ACC ACG ACU GUA GUCGUGGUU

Arginine; Arg; R Serina; To be; S Threonine; Thr; T Valina; Go; V

Tryptophan; Trp; W UGG

Tyrosine; Tyr; Y UAC WOW

Codons are described in terms of RNA bases, for example, adenine, uracil, guanine and cytosine, it is the mRNA that is directly translated into polypeptides. It is understood that when planning a DNA polynucleotide for use in a construct, the DNA bases can be replaced, for example, thymine instead of uracil.

It is desirable to provide transgenic plants that have multiple agronomically improved phenotypes. Herbicide tolerance is often used as a selectable marker to aid in the production of

transgenic plants that may have additional genes of agronomic importance. The implantation of transgenes by traditional crossing methods or by the retransformation of a first transgenic plant with an additional plant expression cassette may include the introduction of genes or genetic elements that have identical or almost identical polynucleotide sequence. The progeny containing these implanted genes may be susceptible to loss of gene expression due to gene silencing. The method of the present invention provides a modified polynucleotide molecule that encodes an herbicide-resistant protein. Polynucleotide molecules are designed to be sufficiently divergent in polynucleotide sequence from other polynucleotide molecules that encode the same herbicide resistance protein. These molecules can then coexist in the same plant cell without concern for gene silencing.

The divergent polynucleotide sequence is created using a codon usage table constructed from known coding sequences of various plant species. For example, the codon usage tables for Arabidopsis thaliana, Zea mays and Glicina max can be used in the method for designing the polynucleotides of the present invention. Other codon usage tables from other plants can also be used by those skilled in the art.

The first step in the method for designing a new artificial polynucleotide molecule that encodes a herbicide-tolerant protein is the use of a codon usage table to determine the use of percent codon in a plant species for each amino acid in the tolerance protein. herbicide, followed by replacing at least one out of every eight contiguous codons with a different codon selected from the codon usage table and adjusting the percentage codon usage for each amino acid encoded by the polynucleotide to substantially the same percentage codon usage found in the codon use. Additional steps may include the introduction of a translational stop codon in the second and third open reading frames of the new polynucleotide sequence; eliminated

adding some translational start codons in the second and third open reading matrices; adjusting the local GC: AT ratio to about 2: 1 over a range of about 50 nucleotides; disrupting potential polyadenylation signals or potential intron junction sites; removing at least one restriction enzyme site of six contiguous or more nucleotides at least me5; and comparing the sequence identity of the new artificial polynucleotide with an existing polynucleotide that encodes the same or similar protein so that the sequence identity between the two polynucleotides is not more than 85 percent.

A back-translation of a protein sequence to a nucleotide sequence can be performed using a codon usage table, such as that found in the Genetics Computer Group (GCG) SeqLab or other DNA analysis programs known to those skilled in the analysis technique of DNA or as provided in Tables 2, 3 and 4 of the present invention. The codon usage table for Arabidopsis thaliana (Table 2), Zea mays (Table 3) and Glycine max (Table 4) are examples of tables that can be built for plant species, the codon use tables can also be built that represent the use of a monocot or dicot codon.

Table 2. Codon usage table for Arabidopsis thaliana.

Amino Acid Codon Number / 1000 Fraction Gly GGG 188335.00 10.18 0.16 Gly GGA 443469.00 23.98 0.37 Gly GGT 409478.00 22.14 0.34 Gly GGC 167099.00 9.03 0.14 Glu GAG 596506.00 32.25 0.48 Glu GAA 639579.00 34.58 0.52 Asp GAT 683652.00 36.96 0.68 Asp GAC 318211.00 17.20 0.32 Go GTG 320636.00 17.34 0.26

········

Amino Acid Codon Number / 1000 Fraction Go GTA 185889.00 10.05 0.15 Go GTT 505487.00 27.33 0.41 Go GTC 235004.00 12.71 0.19 Allah GCG 162272.00 8.77 0.14 Allah GCA 323871.00 17.51 0.27 Allah GCT 521181.00 28.18 0.44 Allah GCC 189049.00 10.22 0.16

Arg AGG 202204.00 10.93 0.20 Arg AGA 348508.00 18.84 0.35 To be AGT 260896.00 14.11 0.16 To be AGC 206774.00 11.18 0.13

Lys THE AG 605882.00 32.76 0.51 Lys AAA 573,121.00 30.99 0.49 Asn AAT 418805.00 22.64 0.52 Asn AAC 385650.00 20.85 0.48

Met ATG 452482.00 24.46 1.00 Ile ATA 235528.00 12.73 0.24 Ile ATT 404070.00 21.85 0.41 Ile ATC 341584.00 18.47 0.35

Thr ACG 140880.00 7.62 0.15 Thr A CA 291436,00 15.76 0.31 Thr ACT 326366.00 17.65 0.34 Thr ACC 190135.00 10.28 0.20 Trp TGG 231618.00 12.52 1.00 End TGA 19037.00 1.03 0.43 Cys TGT 196601.00 10.63 0.60 Cys TGC 131390.00 7.10 0.40

··· • · • · · · · • · · • · · 999 9 9 9 «· _# 99999 · 99 99 9 9 9 9 * * *. 9 9 • · · • · 9 99 · 9 • · • · · · 9 · · · ··· ·· • 9 ··· * ··· 9 · *

Amino Acid Codon Number / 1000 Fraction End TAG 9034.00 0.49 0.20 End TAA 16317.00 0.88 0.37 Tyr TAT 276714.00 14.96 0.52 Tyr TAC 254890.00 13.78 0.48 Read TTG 389368.00 21.05 0.22 Read TTA 237547.00 12.84 0.14 Phe TTT 410976.00 22.22 0.52 Phe TTC 380505.00 20.57 0.48

To be TCG 167804.00 9.07 0.10 To be TCA 334881.00 18.11 0.20 To be TCT 461774.00 24.97 0.28 To be CBT 203174.00 10.99 0.12

Arg CGG 88712.00 4.80 0.09 Arg CGA 115857.00 6.26 0.12 Arg CGT 165276.00 8.94 0.17 Arg CGC 69006.00 3.73 0.07

Gin CAG 280077,00 15.14 0.44 Gin CAA 359922.00 19.46 0.56 His CAT 256758.00 13.88 0.62 His CAC 160485.00 8.68 0.38 Read CTG 183128.00 9.90 0.11 Read CTA 184587.00 9.98 0.11 Read CTT 447606.00 24.20 0.26 Read CTC 294275.00 15.91 0.17 Pro CCG 155222.00 8.39 0.17 Pro CCA 298880.00 16.16 0.33

tso

··· • · ··· ·· • · · ···· t · «········ ·· · · · · ·· • · • · · • • · · · ··· • · · • · · • • · • · · · • · · · »·· · · • · · »·« ··· · · • *

Amino Acid Codon Number / 1000 Fraction Pro CCT 342406.00 18.51 0.38 Pro CCC 97639.00 5.28 0.11

Table 3. Zea mays codon usage table

Amino Acid Codon Number / 1000 Fraction Gly GGG 8069.00 15.19 0.21 Gly GGA 7100.00 13.37 0.18 Gly GGT 7871.00 14.82 0.20 Gly GGC 15904.00 29.94 0.41

Glu GAG 22129.00 41.67 0.68 Glu GAA 10298.00 19.39 0.32 Asp GAT 11996.00 22.59 0.41 Asp GAC 17045.00 32.09 0.59

Go GTG 13873.00 26.12 0.38 Go GTA 3230.00 6.08 0.09 Go GTT 8261.00 15.55 0.23 Go GTC 11330.00 21.33 0.31

Allah GCG 11778.00 22.18 0.24 Allah GCA 8640.00 16.27 0.18 Allah GCT 11940.00 22.48 0.24 Allah GCC 16768.00 31.57 0.34 Arg AGG 7937.00 14.94 0.27 Arg AGA 4356.00 8.20 0.15 To be AGT 3877.00 7.30 0.10 To be AGC 8653.00 16.29 0.23 Lys THE AG 22367.00 42.11 0.74 Lys AAA 7708.00 14.51 0.26 Asn AAT 6997.00 13.17 0.36

: ·· « ·· « • · ♦ • · ··· · • • ·· ········ ·· · · · · ·· • · • • · • • · · · ··· • • • · • · • • · • · · · • · · · ··· • · ·· ······· · · • ·

Amino Acid Codon Number / 1000 Fraction Asn AAC 12236.00 23.04 0.64 Met ATG 12841.00 24.18 1.00 Ile ATA 3997.00 7.53 0.16 Ile ATT 7457.00 14.04 0.31 Ile ATC 12925.00 24.34 0.53 Thr ACG 5665.00 10.67 0.22 Thr A CA 5408.00 10.18 0.21 Thr ACT 5774.00 10.87 0.22 Thr ACC 9256.00 17.43 0.35

Trp TGG 6695.00 12.61 1.00 End TGA 591.00 1.11 0.45 Cys TGT 2762.00 5.20 0.30 Cys TGC 6378.00 12.01 0.70

End TAG 411.00 0.77 0.32 End TAA 299.00 0.56 0.23 Tyr TAT 4822.00 9.08 0.31 Tyr TAC 10546.00 19.86 0.69

Read TTG 6677.00 12.57 0.14 Read TTA 2784.00 5.24 0.06 Phe TTT 6316.00 11.89 0.32 Phe TTC 13362.00 25.16 0.68 To be TCG 5556.00 10.46 0.14 To be TCA 5569.00 10.49 0.15 To be TCT 6149.00 11.58 0.16 To be CBT 8589.00 16.17 0.22 Arg CGG 4746.00 8.94 0.16

Amino Acid Codon Number / 1000 Fraction Arg CGA 2195.00 4.13 0.07 Arg CGT 3113.00 5.86 0.10 Arg CGC 7374.00 13.88 0.25 Gin CAG 13284.00 25.01 0.64 Gin CAA 7632.00 14.37 0.36 His CAT 5003.00 9.42 0.39 His CAC 7669.00 14.44 0.61

Read CTG 13327.00 25.09 0.28 Read CTA 3785.00 7.13 0.08 Read CTT 8238.00 15.51 0.17 Read CTC 12942.00 24.37 0.27

Pro CCG 8274.00 15.58 0.27 Pro CCA 7845.00 14.77 0.26 Pro CCT 7129.00 13.42 0.23 Pro CCC 7364.00 13.87 0.24

Table 4. Glycine max codon usage table

Codon Amino Acid Number / 1000 Fraction

Gly GGG 3097.00 12.82 0.18 Gly GGA 5434.00 22.49 0.32 Gly GGT 5248.00 21.72 0.31 Gly GGC 3339.00 13.82 0.20 Glu GAG 8296.00 34.33 0.50 Glu GAA 8194.00 33.91 0.50 Asp GAT 7955.00 32.92 0.62 Asp GAC 4931.00 20.40 0.38 Go GTG 5342.00 22.11 0.32 Go GTA 1768.00 7.32 0.11

Amino Acid Codon Number / 1000 Fraction Go GTT 6455.00 26.71 0.39 Go GTC 2971.00 12.29 0.18 Allah GCG 1470.00 6.08 0.08 Allah GCA 5421.00 22.43 0.31 Allah GCT 6796.00 28.12 0.38 Allah GCC 4042.00 16.73 0.23

Arg AGG 3218.00 13.32 0.28 Arg AGA 3459.00 14.31 0.30 To be AGT 2935.00 12.15 0.17 To be AGC 2640.00 10.92 0.15

Lys THE AG 9052.00 37.46 0.59 Lys AAA 6370.00 26.36 0.41 Asn AAT 5132.00 21.24 0.48 Asn AAC 5524.00 22.86 0.52

Met ATG 5404.00 22.36 1.00 Ile ATA 3086.00 12.77 0.23 Ile ATT 6275.00 25.97 0.47 Ile ATC 3981.00 16.47 0.30

Thr ACG 1006.00 4.16 0.08 Thr A CA 3601.00 14.90 0.29 Thr ACT 4231.00 17.51 0.34 Thr ACC 3562.00 14.74 0.29 Trp TGG 2866.00 11.86 1.00 End TGA 221.00 0.91 0.36 Cys TGT 1748.00 7.23 0.49 Cys TGC 1821.00 7.54 0.51

ϋΜ

Amino Acid Codon Number / 1000 Fraction End TAG 143.00 0.59 0.23 End TAA 256.00 1.06 0.41 Tyr TAT 3808.00 15.76 0.51 Tyr TAC 3667.00 15.17 0.49 Read TTG 5343.00 22.11 0.24 Read TTA 2030.00 8.40 0.09 Phe TTT 4964.00 20.54 0.49 Phe TTC 5067.00 20.97 0.51

To be TCG 1107.00 4.58 0.06 To be TCA 3590.00 14.86 0.21 To be TCT 4238.00 17.54 0.24 To be CBT 2949.00 12.20 0.17

Arg CGG 683.00 2.83 0.06 Arg CGA 964.00 3.99 0.08 Arg CGT 1697.00 7.02 0.15 Arg CGC 1538.00 6.36 0.13

Gin CAG 4147.00 17.16 0.46 Gin CAA 4964.00 20.54 0.54 His CAT 3254.00 13.47 0.55 His CAC 2630.00 10.88 0.45 Read CTG 2900.00 12.00 0.13 Read CTA 1962.00 8.12 0.09 Read CTT 5676.00 23.49 0.26 Read CTC 4053.00 16.77 0.18 Pro CCG 1022.00 4.23 0.08 Pro CCA 4875.00 20.17 0.37 Pro CCT 4794.00 19.84 0.36

• · · · · · · ·

Amino Acid Codon Number / 1000 Fraction Pro CCC 2445.00 10.12 0.19

Codon usage tables are well known in the art and can be found in gene databases, for example, Genbank database. The Codon Use Database is an extended WWW version of CUTG (Tabulated Codon Use by Genbank). The frequency of codon use in each organism is made searchable through this site on the World Wide Web (Nakamura et al. Nucleic Acids Res. 28: 292, 2000).

In various embodiments of the invention, the steps can be carried out in any order or simultaneously. Any or all of the steps can be performed in the design of an artificial polynucleotide of the invention. Each step is described in detail below.

The different codons for a particular amino acid must be distributed throughout the polynucleotide based on the use of approximate percent codon for particular species from a codon use table. Local clusters of identical codons should be avoided. At least one codon is replaced for every eight contiguous codons to provide sufficient divergence of polynucleotide sequences that encode identical or similar proteins. Except where specifically desired, for example, to provide an herbicide-tolerant enzyme, the encoded protein remains unchanged by replacing one codon in place of another codon that is translated to the same amino acid as listed in Table 1.

In embodiments of the present invention, corrections are made to the local GC: AT ratio of a polynucleotide by adjusting the local GC: AT ratio to be approximately the same ratio as the life-size poly25 nucleotide, but no greater than 2X in a range of about 50 contiguous nucleotides of the polynucleotide molecule. The range of GC: AT ratios of a polynucleotide using the codon use tables of dicotyledonous plants should be from about 0.9 to about 1.3 and for monocotyledonous plants from about 1.2 to about 1, 7. The relationship

GC: Local TA can be important in maintaining the appropriate secondary structure of RNA. Regions that comprise many consecutive A + T bases or G + C bases are predicted to be more likely to form hairpin structures due to self-complementarity. Therefore, substitution with a different codon can reduce the likelihood of forming a self-complementary secondary structure, which is known to reduce transcription and / or translation in some organisms. In most cases, adverse effects can be minimized by using polynucleotide molecules that contain no more than five consecutive A + T or G + C. The maximum length of the local GC track (without any AT nucleotide) should not be greater than 10 nucleotides. Therefore the codons encoding proteins rich in Gly, Ala, Arg, Ser and Pro can be replaced to prevent long clusters of GC nucleotides. The listed GC-rich codons can be used in combination with the AT-rich codons for the amino acids Lys, Asn, Ile, Tyr, Leu, Phe and vice versa to correct the local GC: AT ratio.

A sequence identity check using nucleotide sequence alignment tools such as the GAP program (GCG, Madison, Wl) can be done immediately after back-translation to ensure that the generated sequence has an appropriate degree of sequence diversity. The contiguous polynucleotide sequence greater than 23 nucleotides that have one hundred percent sequence identity must be eliminated by performing codon substitutions at these sequence lengths.

The translational starting codons (DNA ATG, AUG in mRNA) present in the second reading matrix (matrix b), the third reading matrix (matrix "c") and the reverse reading matrices (matrix d, e, f) . The second and third matrix start codons can initiate translation, however much less efficiently than the first. Therefore, if one or two AUGs are found near the 5 'end of an mRNA molecule that resides in matrix b or c, it may be beneficial to eliminate them in a polynucleotide region that contains at least the first three y codons.

Met in the matrix a. Also, if the protein sequence has no more than one Met in matrix a, then eliminate as much of the advanced matrices b or c as possible. To accomplish this, for example, the codons for amino acids, Asp, Asn, Tyr, His in the protein of interest followed by any of the amino acids: Gly, Glu, Asp, Vai or Ala, can be replaced to eliminate a starting codon in the second matrix. The GATGGG sequence encodes the Asp-Gly amino acids and forms an ATG in the reading matrix b. When the sequence is changed to GACGGG, the beginning ATG is eliminated and the sequence still encodes Asp-Gly. A similar strategy is used to eliminate start codons in the reading matrix c. The combination of an amino acid selected from the group of Gly, Glu, Vai, Ala, Arg, Lys, Ile, Thr, Cys, Tyr, Leu, Ser, His or Pro followed by Trp can result in ATG formation in the third reading matrix of the gene. In this situation, the first codon can be changed to have a nucleotide other than A in the third position.

The elimination of the ATG codon in the complementary DNA strand of the gene in the alternating matrices (d, e, and / or f) without changing the protein's amino acid sequence can be accomplished in a similar way. This modification reduces the likelihood of translation even if the transgene is integrated into a plant genome in an orientation that allows for the transcription of the reverse complement mRNA of a native plant promoter. The translation of any reverse reading matrix can be minimized by introducing a stop codon in all three reverse reading matrices as described below.

The creation of stop codons for all three complementary DNA arrays can be performed as follows. Codons Leu (TTA and CTA) and Ser (TCA) produce three different stop codons in the reverse complement filament. If those amino acids can be found at the C-terminus of the protein of interest, their codons can be used to generate stops in the complementary filament in the reading matrix d. To generate a stop codon in the reading and complementary filament matrix, find the amino acids Ala, Arg, Asn, Asp, Cys, Gly, His, Ile, Leu Phe, Pro,

Ser, Thr, Tyr or Vai followed by the amino acids Gin, His or Tyr of the protein of interest. For example, the GCCCAC polynucleotide sequence that encodes the Ala-His amino acids can be modified to GCTCAC. The complementary sequence, GTGAGC, will now have the TGA stop codon shown in italics. When the protein of interest has an Ala, Ile, Leu, Phe, Pro, Ser, Thr or Vai followed by an Arg, Asn, Ile, Lys, Met or Ser the reading matrix in the complementary filament can be modified to have a codon stop in the reading matrix ”f 'of the complementary filament. The ATATCT polynucleotide sequence for Ile and Ser can be modified for ATCAGT to generate stop codon in the complementary filament as shown in italics, ACTGAT. The combination of codons for Phe followed by any of the codons for the amino acids Asn, Ile, Lys, Met or Thr will always generate stop codon in the matrix of the complementary filament and or llflt

To create a stop codon in the advanced reading matrix b, the reading matrix a must end in nucleotides TA or TG. The search for the protein of interest for the amino acids Ile, Leu, Met or Vai in combination with any of the following amino acids: Ala, Arg, Asn Asp, Glu, Gly, Ile, Lys, Met, Ser, Thr or Vai. For example, if the polynucleotide sequence that encodes the Met-Ser amino acids is ATGTCT, it can be modified to ATGAGT to produce a TGA stop codon in the second reading matrix.

To be able to create a stop codon for reading matrix c, reading matrix a must have nucleotide T in the third position and the next codon must start from AA, AG or GA. To find suitable codons to modify, look for the protein of interest for any of the amino acids: Ala, Asn, Asp, Arg, Cys, Gly, His, Ile, Leu Phe, Pro, Ser, Thr, Tyr or Vai followed by anyone of the following amino acids: Arg, Asn, Asp, Glu, Lys or Ser. For example, if the nucleotide sequence for the amino acids Gly-Glu is GGAGAG, the sequence can be modified to GGTGAG to create a TGA stop codon in the third matrix of reading.

Another useful modification in the artificial polynucleotide design methods of the present invention is to eliminate unwanted restriction sites and other specific sequence patterns. The restriction sites can interfere with cloning and future gene manipulations. For example, some restriction sites commonly used in gene cloning include, but are not limited to, Type I restriction enzymes with 6 or more non-N bases listed in Table 5 below which is an excerpt from the restriction endonuclease from New England Biolabs, Inc. (Beverly, MA, USA). The search for restriction enzyme recognition sites can be done using the application of the Map function found in the GCG SeqLab or a similar application contained in other DNA analysis programs known to those skilled in the DNA analysis technique. Restriction enzymes can also be added to the sequence to facilitate cloning. For example, the Clal restriction site is placed in CP4EPSPS version AT (SEQ ID NO: 17) and ZM (SEQ ID NO: 18) to generate recombinant sequences by fragment change and to facilitate gene synthesis using nucleotide fragments that can be assembled into the entire gene. The transit peptide CTP2 polynucleotide sequence (SEQ ID NO: 12) is connected with CP4EPSPS by the Sphl restriction site to facilitate replacement of CTP2 with nucleotide versions other than CTP2 (SEQ ID NO: 13, SEQ ID NO: 14 ) or polynucleotides that encode different chloroplast transit peptides. For example, in rice EPSPS, the NgaMIV restriction site is preserved around the position of nucleotide 205 in all artificial versions to facilitate the change of the chloroplast transit peptide coding region. Also, for soybean EPSPS, the polynucleotide sequence for the chloroplast transit peptide is separated from the mature peptide by the restriction site for Sacll endonuclease.

It is understood that modification of restriction endonuclease sites is not required, but is useful for other manipulations of DNA molecules. Table 5 provides a list of restriction endonucleases, those of particular interest to the present invention are marked with

an asterisk. Other restriction endonuclease sites desirable for elimination or addition to an artificial polynucleotide of the present invention will be evident to those skilled in the art and are not limited by those listed in Table 5.

Table 5. Restriction enzyme recognition sequences

Enzymes Recognition string Aatll G_ACGT ^A C * Accl GT ^A MK_AC Acc65l G ^A GTAC_C Acell G_CTAG ^A C- Acll AA ^A CG_TT Acyl GR ^A CG_YC Afel AGC ^A GCT Aflll C ^A TTAA_G ‘Afllll A ^A CRYG_T Agel A ^A CCGG_T Ahalll TTT ^TO AAA Apal G_GGCC ^A C ApaLI G ^A TGCA_C Apol ^The R AATT_Y Ascl GG ^A CGCG_CC Asei AT ^A TA_AT AsiSI GCG_AT ^A CGC Asull TT ^A CG_AA Aval C ^A YCGR_G Avalll ATGCAT Avrll ^The C CTAG_G Bali TGG CCA ‘BamHI G ^A GATC_C Bani G ^A GYRC_C Banll G_RGCY ^A C * Bbel G_GCGC ^A C BbvCI CC ^A TCA_GC ‘Bell T ^A GATC_A Betl W ^A CCGG_W BfrBI ATG ^TO CAT

Enzymes * Bglll

BloHII

Blpi

Bme1580l

Bmgl

BpulOI

Bsal

BsaAI

BsaHI

BsaWI

Bsbl

BsePI

BseSI

Bsil

BsiEI

BsiWI

Bsml

Bsp1286l

Bsp1407l

BspEI

BspGI

BspHI

BspLU11l

BspMII

BsrBI

BsrDI

BsrFI

BsrGI

BssHII

BssSI

BstAPI

BstBI

BstEII

BstXI

BstYI

Recognition sequence A ^A GATC_T CTGCA ^A G GC ^A TNA_GC G_KGCM ^A C

GKGCCC

CC ^A TNA_GC

GGTCTCN ^A NNNN_

YAC ^A GTR

GR ^A CG_YC

W ^A CCGG_W

CAACAC

G ^A CGCG_C

G_KGCM ^A C

C ^A ACGA_G

CG_RY ^A CG

^The C GTAC_G

GAATG_CN ^A

G_DGCH ^A C

^The T GTAC_A

T ^A CCGG_A

CTGGAC

T ^A CATG_A

A ^A CATG_T

T ^A CCGG_A

CCG ^TO CTC

GCAATG_NN ^A

R ^A CCGG_Y

^The T GTAC_A

G ^A CGCG_C

C ^A ACGA_G

GCAN_NNN ^THE NTGC

TT ^A CG_AA

G ^A GTNAC_C

CCAN_NNNN ^TO NTGG

R ^A GATC_Y

Enzymes

BstZ17l

Bsu36l

Btgl Btrl Btsl Cfrl CfrlOI * Clal Dral Drall Drdll Dsal Eael Eagl Ecl136ll Eco47lll Eco NI EcoO109l ‘EcoRI‘ EcoRV Espl * Fsel * Fspl FspAI Gdill Hael Haell HgiAI HgiCI HgiJII * Hincll Hindll ll

GTA ^A TAC recognition sequence

CC ^A TNA_GG

C ^A CRYG_G

CAC ^TO GTC

GCAGTG_NN ^A

Y ^A GGCC_R

R ^A CCGG_Y

AT ^A CG_AT

TTT ^TO AAA

RG ^A GNC_CY

GAACCA

C ^A CRYG_G

Y ^A GGCC_R

C ^A GGCC_G

GAG ^TO CTC

AGC ^A GCT

CCTNN ^TO N_NNAGG

RG ^A GNC_CY

L AATT_C

GAT ^A ATC

GC ^A TNA_GC

GG_CCGG ^A CC

TGC ^TO GCA

RTGC ^TO GCAY

C ^A GGCC_R

WGG ^A CCW

R_GCGC ^A Y

G_WGCW ^A C

G ^A GYRC_C

G_RGCY ^A C

GTY ^A RAC

GTY ^A RAC

A ^A AGCT_T

GTT AAC

G ^A GCGC_C

<2> 3

Enzymes Recognition string * Kpnl G_GTAC ^A C Lpnl RGC ^A GCY Mcrl CG_RY ^A CG Mfel ^The C AATT_G * Mlul A ^A CGCG_T Mscl TGG CCA MspA11 CMG ^TO CKG Mstl TGC ^TO GCA Nael ^The GCC GGC Narl GG ^A CG_CC * Ncol C ^A CATG_G * Ndel CA ^TO TA_TG * NgoMIV G ^A CCGG_C * Nhel G ^A CTAG_C NIÍ3877I C_YCGR ^A G * Notl GC ^A GGCC_GC * Nrul TCG ^TO CGA * Nsil A_TGCA ^A T Nspl R_CATG ^A Y NspBII CMG ^TO CKG * Pacl TTA_AT ^A TAA * Easy A ^A CATG_T Pfl11081 TCGTAG ‘PfIMI CCAN_NNN ^THE NTGG PmaCI CAC ^A GTG Pmel GTTT ^TO AAAC Pmll CAC ^A GTG PpulOI A ^A TGCA_T * PpuMI RG ^A GWC_CY PshAI GACNN ^TO NNGTC Psil TTA ^TO TAA ‘PspOMI G ^A GGCC_C Pssl RG_GNC ^A CY ‘Pstl C_TGCA ^A G * Pvul CG_AT ^A CG

Enzymes * Pvull

Rsrll * Sacl * Sacll * Sall

SanDI

Sapl

Saul

Sbfl * Scal

Scil

Sdul

FriAI

Sfcl

Sfel

Sfil

Sfol

Sgfl

SgrAI * Smal

Smll

Snal * SnaBI * Spel * Sphl

Spll

Srfl

Sse232l

Sse8387l

Sse8647l * Sspl * Stul * Styl * Swal

Tatl

Recognition sequence CAG CTG

CG ^A GWC_CG

G_AGCT ^A C

CC_GC ^A GG

G ^A TCGA_C

XL GWC_CC

GCTCTTCN ^A NNN_

CC ^A TNA_GG

CC_TGCA ^A GG

AGT ^A ACT

CTC ^A GAG

G_DGCH ^A C

A ^A CCWGG_T

C ^A TRYA_G

C ^A TRYA_G

GGCCN_NNN ^TO NGGCC

GGC ^TO GCC

GCG_AT ^A CGC

CR ^A CCGG_YG

CCC ^A GGG

C ^A TYRA_G

GTATAC

TAC ^A GTA

^A CTAG_T

G_CATG ^A C

^The C GTAC_G

GCCC ^TO GGGC

CG ^A CCGG_CG

CC_TGCA ^A GG

AG ^A GWC_CT

AAT ^TO ATT

AGG ^A CCT

C ^A CWWG_G

ATTT ^TO AAAT

W ^A GTAC_W

• · ······ »· • · · · · · · · · · * φ · · · ·» (ÓA>

Enzymes Recognition string UbaMI TCCNGGA UbaPI CGAACG * Vspl AT ^A TA_AT * Xbal T ^A CTAG_A * Xhol C ^A TCGA_G Xholl R ^A GATC_Y * Xmal C ^A CCGG_G Xmalll C ^A GGCC_G Xmnl GAANN ^TO NNTTC Zral GAC ^TO GTC

A standard search can be performed to find destabilizing sequences and potential polyadenylation sites and then disrupt or eliminate them as described in US Patent N ^? 5,500,365. Certain long stretches of AT-rich regions, for example, the ATTTA sequence motif (or AUUUA, as it appears in RNA) have been implicated as a destabilizing sequence in mammalian cell mRNA (Shaw and Kamen, Cell 46: 659 to 667, 1986). Many short-lived mRNAs have untranslated 3 'regions rich in A + T and these regions often have the ATTTA sequence, sometimes present in multiple copies or as multimers (eg ATTTATTTA...). Shaw and Kamen showed that transferring the 3 'end of an unstable mRNA to a stable RNA (globin or VA1) decreased the stable RNA half life dramatically. They further showed that an Al 1A pentamer had a profound destabilizing effect on a stable message and that this signal can exert its effect if it is located at the 3 'end or within the coding sequence. However, the number of Al I IA sequences and / or the sequence context in which they occur also appears to be important in determining whether they function as destabilizing sequences. They also showed that an Al lA trimer had much less effect than a pentamer on mRNA stability and a dimer or monomer had no effect on stability. Note that ATTTA multimers such as a pentamer automatically create a region rich in

·· «· ♦ ·····

A + T. In other unstable mRNAs, the ATTTA sequence may be present in only a single copy, but is often contained in an A + T-rich region. A repeat of 11 AUUUA pentamers has been shown to target reporter transcripts for rapid degradation in plants (OhmeTakagi et al., Proc. Nat. Acad. Sci. USA 90, 11811 to 11815, 1993). The ATTTA sequence can be formed by combining codons for the amino acid Ile (ATT) and Tyr (TAT) as shown in A11 / AT. Another example can be codons ending in AT as in Asn, Asp, His or Tyr, followed by the codon TTA for Leu (for example, AATTTA). Also the codon for Phe (UUU) when placed between the codons that ends at A and starts at A will form the ATTTA motif. To eliminate this reason, usually the single nucleotide change is sufficient as in the example shown: GCA7TTAGC changes to GCATTCAGC or GCCTTTAGC. All three polynucleotides shown encode Ala-Phe-Arg.

More cis-acting sequences that target the transcript for rapid rotation in plants and in another system have been identified (Abler and Green, Plant Mol. Biol. 32: 63 to 78, 1997). These include the DST element which consists of three highly conserved subdomains separated by the two variable regions found downstream from the stop codon of the SAUR transcripts (Newmaan etal., Vegetal Cell 5: 701 to 714, 1993). The conserved STD sequence consists of GGAG (N5) CArAGAn'G (N ₇ ) CATTTTG7'A7 ', where highly conserved residues are shown in italics. The second and third subdomains of DST elements contain residues that are invariable between DST elements and are called ATAGAT and GTA, respectively. Both of these subdomains are required for the DST function. New artificial polynucleotide sequences are screened for the presence of conserved motifs of DST elements GGAG, ATAGATT, CATTT and CAI II IGTAT. These sequences are eliminated by codon-based substitutions that preserve the protein sequence encoded by the polynucleotide. DST sequence motifs GGAG, ATAGAT, CATTT and GTAT that appeared in clusters or patterns similar to the conserved STD sequence are also eliminated by γ substitutions

··· • * • · • · «· · • · · • · · ···· • • • ·· • «· ········ • · · · • »* • · ·· · • · · • • • · · • · · • · • • · · * · · · · ·· ··· ···· · · • ·

base.

Polynucleotide sequences that may possibly function as polyadenylation sites are eliminated in the new polynucleotide design (U.S. Patent No. 5,500,365). These polyadenylation signals may not act as appropriate polyA sites, but instead function as aberrant sites that give rise to unstable mRNAs.

The addition of a polyadenylated strand to the 3 'end of an mRNA is common to most eukaryotic mRNAs. Contained within these mRNA transcripts are signals for polyadenylation and the formation of the appropriate 3 'end. This processing at the 3 'end involves dividing the mRNA and adding polyA to the mature 3' end. By searching for consensus sequences close to the polyA tract in both plant and animal mRNAs, it was possible to identify consensus sequences that are apparently involved in the addition of polyA and the divination of the 3 'end. The same consensus strings seem to be important for both of these processes. These signals are typically a variation in the AATAAA sequence. In animal cells, some variants of this sequence that are functional have been identified; in plant cells there appears to be an extended range of functional sequences (Dean et al., Nucl Acid Res., 14: 2229 to 2240, 1986; Hunt, Annu Rev. Plant Physiol. Plant Mol. Biol 45: 47 to 60, 1994; Rothine, Plant Mol. Biol. 32: 43 to 61, 1996). All of these consensus strings are variations on AATAAA, so they are all strings rich in A + T.

Typically, to obtain sufficient expression of modified transgenes in plants, the existing structural polynucleotide coding sequence (structural gene) encoding the protein of interest is modified by removing the putative AI1A sequences and putative polyadenylation signals by site-directed mutagenesis of the DNA that comprises the structural gene. Substantially all known polyadenylation signals and ATTTA sequences are removed in the modified polynucleotide, although enhanced expression levels are often observed only with the removal of some of the polyadenyl signal sequences60

6 # tion identified above. Alternatively, if an artificial polynucleotide is prepared encoding the object protein, the codons are selected to avoid the ATTTA sequence and putative polyadenylation signals. For the purposes of the present invention putative polyadenylation signals include, but are not necessarily limited to, AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA and CATAAA.

The selected DNA sequence is scanned to identify regions with more than four consecutive adenine (A) or thymine (T) nucleotides. The A + T regions are scanned for potential plant polyadenylation signals. Although the absence of five or more consecutive A or T nucleotides eliminates most of the plant polyadenylation signals, if there is more than one of the minor polyadenylation signals identified within ten nucleotides together, then the nucleotide sequence of this region is altered to remove these signals while maintaining the original encoded amino acid sequence.

The next step is to consider the approximately 15 to about 30 or more nucleotide residues that surround the A + T-rich region. If the A + T content of the surrounding region is less than 80%, the region should be examined for signs of polyadenylation. Alteration of the region based on polyadenylation signals is dependent (1) on the number of polyadenylation signals present and (2) on the presence of a larger plant polyadenylation signal. The polyadenylation signals are removed by the base substitution of the DNA sequence in the context of codon substitution.

Two additional standards not identified in US Patent No. 5,500,365. are sought and eliminated in the embodiments of the present invention. The AGGTAA and GCAGGT sequences are consensus sequences for the 5 'and 3' intron junction sites, respectively, in monocotyledonous and dicotyledonous plants. Only GT from the 5 'junction site and AG at the 3' junction site are required to be an exact match. However, when conducting research on these consensus strings, no mismatches are allowed

Ó5 for each base.

After each step, the sequence mapping is done using the GCG MAP program to determine the location of the open reading matrices and identify sequence patterns that still need to be modified. The final step would be to perform the sequence identity analysis using, for example, the GAP program in the GCG package to determine the degree of sequence divergence and the percent identity.

Polypeptides

In general, the translated protein from the artificial polynucleotide will have the same amino acid sequence as the translated protein from the unmodified coding region. However, the replacement of codons that encode amino acids that provide a functional homolog of the protein is an aspect of the invention. For example, certain amino acids can be substituted in place of other amino acids in a protein structure without appreciable loss of the ability to interact interactively with structures such as, for example, antibody antigen binding regions or binding sites on substrate molecules . Since it is the interactive ability and nature of a protein that defines this biological functional activity of the protein, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, not despite obtaining a protein with similar properties. It is thus considered by the inventors that several changes can be made to the peptide sequences of the disclosed compositions by making changes to the corresponding DNA sequences encoding the peptides in which the peptides have not shown any appreciable loss of their biological utility or activity.

Another aspect of the invention comprises functional homologs, which differ in one or more amino acids from those of a polypeptide provided herein as the result of one or more conservative amino acid substitutions. It is well known in the art that one or more amino acids in a native sequence can be replaced with at least one other amino acid, the charge and polarity of which are similar to those of amino62

Ύ) native acid, resulting in a silent change. For example, valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native polypeptide sequence can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids, (2) basic amino acids, (3) neutral polar amino acids and (4) neutral non-polar amino acids. Representative amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; and (4) neutral (hydrophobic) non-polar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic hydroxy side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. The naturally conservative amino acid substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid and asparagine-glutamine. DNA Contracts

Exogenous genetic material can be transferred in a plant using a DNA construct designed for this purpose by methods using Agrobacterium, particle bombardment or others

• · · · · · ·> 1 methods known to those skilled in the art. The design of such a DNA construct is generally within the knowledge of the technique (Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer, New York (1997). Examples of such plants in which exogenous genetic material can be transferred , include, without limitation, alfalfa, Arabidopsis, barley, Brassica, broccoli, cabbage, Citrus, cotton, garlic, oats, rapeseed, onion, canola, flax, corn, an ornamental and perennial ornamental annual plant, peas, peanuts, peppers, potato, rice, rye, sorghum, soy, strawberry, sugar cane, beet, tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce, lentils, grape, banana, tea, grasses, sunflower, oil palm, Phaseolus, trees, shrubs, vines, etc. It is well known that agronomically important plants comprise genotypes, varieties and cultivars and that the methods and compositions of the present invention can be tested on these plants by those of ordinary skill in modern biology. lecular plant and plant crossing.

A large number of isolated DNA-promoting molecules that are active as a genetic element of a transgene in plant cells have been described. These include the promoter of nopaline synthase (P-nos) (Ebert et al., Proc. Natl. Acad. Sci. (USA) 84: 5745 to 5749, 1987), all of which is incorporated by reference), the octopine synthase promoter (P-ocs), which are transported in tumor-inducing plasmids from Agrobacterium tumefaciens, the kaolinimovirus promoters, such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol Biol. 9: 315 to 324, 1987), all of which are hereby incorporated by reference) and the CaMV 35S promoter (Odell et al., Nature 313: 810 to 812, 1985), all of which are herein incorporated by reference), the 35S promoter from Figwort mosaic virus (US Patent ^No.'s 6,018,100, the entirety of which is incorporated herein by reference), the light - inducible promoter from the small subunit of ribulose-1,5-bis phosphate carboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl. Acad. Sci. (USA) 84: 6624 to 6628, 1987), all of which are included here provided by reference), the sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. (USA) 87: 4144 a • · · · · ·

4148, 1990), all of which is incorporated herein by reference), the promoter of the R gene complex (Chandler et al., Plant Cell 1: 1175 to 1183, 1989, all of which is incorporated by reference) and the a / b chlorophyll binding protein gene promoter, etc.

A variety of promoters specifically active in vegetative tissues, such as leaves, stems, roots and tubers, can be used to express the nucleic acid molecules of the present invention. Examples of tuber-specific promoters include, but are not limited to, class I and II patatin promoters (Bevan et al., EMBO J. 8: 1899 to 1906, 1986); Koster-Topfer et al., Mol Gen Genet. 219; 390 to 396, 1989); Mignery et al., Gene 62: 27 to 44, 1988); Jefferson et al., Plant Mol. Biol. 14: 995 to 1006, 1990), hereby incorporated by reference in their entirety), the promoter for the tuber ADPGPP genes, both large and small subunits; the sucrose synthase promoter (Salanoubat and Belliard, Gene 60: 47 to 56, 1987), Salanoubat and Belliard, Gene 84: 181 to 185, 1989), hereby incorporated by reference in its entirety); and the promoter for major tuber proteins including 22 kd protein complexes and proteinase inhibitors (Hannapel, Plant Physiol. 10T. 703 to 704, 1993), hereby incorporated by reference in their entirety). Examples of specific leaf promoters include, but are not limited to, ribulose bisphosphate carboxylase promoters (RbcS or RuBISCO) (see, for example, Matsuoka et al., Plant J. 6; 311 to 319, 1994), incorporated herein by reference in its entirety); the a / b light collecting chlorophyll binding protein gene promoter (see, for example, Shiina et al., Plant Physiol. 115: 477 to 483. 1997; Casal etal., Plant Physiol. 116: 1533 a 1538, 1998, incorporated herein by reference in its entirety); and the myb-related gene promoter from Arabidopsis thaliana (Atmyb5) (Li et al., FEBS Lett. 379: 117 to 121, 1996, hereby incorporated by reference in its entirety). Examples of root-specific promoter include but are not limited to the promoter for the acid chitinase gene (Samac et al., Plant Mol. Biol. 25; 587 to 596, 1994), hereby incorporated by reference in its entirety); the root specific subdomains of the CaMV35S promoter that were

identified (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.) 86: 7890 to 7894. 1989, hereby incorporated by reference in their entirety); the ORF13 promoter of Agrobacterium rhizogenes which exhibits high activity in the roots (Hansen etal., Mol. Gen. Genet. 254: 337 to 343. 1997), incorporated herein by reference in its entirety); the promoter for the tobacco root specific gene RB7 (US Patent 5,750,386; Yamamoto et al., Plant Cell 3: 371 to 382, 1991, hereby incorporated by reference in its entirety); and the root cell-specific promoters reported by Conkling et al. (Conkling et al., Plant Physiol. 93: 1203 to 1211, 1990, incorporated herein by reference in its entirety) and the POX1 promoter (Pox1, pox1) (Hertig, et al. Plant Mol. Biol. 16: 171, 1991).

Another class of specific promoters of useful vegetative tissue are meristematic promoters (root tip and bud apex). For example, the promoters SHOOTMERISTEMLESS and SCARECROW, which are active in the development of apical sprout or root meristems (Di Laurenzio et al., Cell 86: 423 to 433, 1996; Long, Nature 379: 66 to 69, 1996); incorporated herein by reference in its entirety), may be used. Another example of a useful promoter is that which controls the expression of the 3-hydroxy-3-methylglutaryl coenzyme HMG2 reductase gene, whose expression is restricted to meristematic and floral tissues (stigma-secreting zone, mature pollen grains, vascular tissue of gynecium and fertilized eggs) (see, for example, Enjuto et al., Plant Cell, 7: 517 to 527, 1995, hereby incorporated by reference in its entirety). Another example of a useful promoter is one that controls the expression of genes related to corn knl and other species that display specific meristem expression (see, for example, Granger etal., Plant Mol. Biol. 31: 373 a 378, 1996; Kerstetter et al., Plant Cell 6: 1877 to 1887, 1994; Hake et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350: 45 to 51, 1995, incorporated herein by reference in their totalities). Another example of a meristematic promoter is the KNAT1 Arabidopsis thaliana promoter. At the bud apex, the KNAT1 transcript is located primarily in the bud apical meristem; KNAT1 expression in the bud meristem decreases during tran66 ···

floral location and is restricted to the cortex of the inflorescence stem (see, for example, Lincoln et al., Plant Cell 6: 1859 to 1876, 1994, hereby incorporated by reference in its entirety).

Suitable seed-specific and seed enhancers can be derived from the following genes: maize MAC1 (Sheridan et al., Genetics 142: 1009 to 1020, 1996, hereby incorporated by reference in its entirety); Maize Cat3 (.... ^S GenBank Accession No. L05934 Abler et al, Plant Mol Biol 22: 10131-1038, 1993 incorporated herein by reference in its entirety); vivparous-1 from Arabidopsis (Genbank No. ² U93215); Atimycl de Arabidopsis (Urao et al., Plant Mol. Biol. 32: 571-57, 1996; Conceicao et al., Plant 5: 493 to 505, 1994, hereby incorporated by reference in their entirety); napA from Brassica napus (Genbank No. ² J02798); the Brassica napus napin gene family (Sjodahl et al., Planta 197: 264 to 271, 1995, hereby incorporated by reference in its entirety).

The egg specific promoter for the BEL1 gene (Reiser et al. Cell 83: 735 to 742, 1995, Genbank N ² U39944; Ray et al, Proc. Natl. Acad. Sci. USA 91: 5761 to 5765, 1994, all which are hereby incorporated by reference in their entirety) may also be used. The egg and central cell-specific MEA (FIS1) and FIS2 promoters are also useful reproductive tissue-specific promoters (Luo et al., Proc. Natl. Acad. Sci. USA, 97: 10637 to 10642, 2000; Vielle-Calzada, et al., Genes Dev. 13: 2971 to 2982.1999; incorporated herein by reference in its entirety).

A specific corn pollen promoter has been identified in corn (Guerrero et al., Mol. Gen. Genet. 224: 161 to 168, 1990, hereby incorporated by reference in its entirety). Other genes specifically expressed in pollen have been described (see, for example, Wakeley et al., Plant Mol. Biol. 37: 187 to 192, 1998; Ficker et al., Mol. Gen. Genet 257: 132 to 142, 1998; Kulikauskas et al., Plant Mol. Biol. 34: 809 to 814, 1997; Treacy et al., Plant Mol. Biol. 34: 603 to 611, 1997; all of which are incorporated herein by reference in their entirety).

Promoters derived from genes encoding embryonic storage proteins, which include the gene encoding ar proteins67

2S storage of Brassica napus (Dasgupta et al, Gene 133: 301 to 302, 1993, hereby incorporated by reference in its entirety); the Arabidopsis- 2s seed storage protein gene family, the gene encoding Brassica napus 20kD oleosin (GenBank No. ^s M63985); the genes encoding oleosin A (Genbank N ⁹ U09118) and oleosin B (GenBank N ⁹ U09119) from soy; the gene encoding Arabidopsis oleosin (GenBank No. ⁹ Z17657); the gene encoding 18kD maize oleosin (GenBank N ⁹ J05212, Lee, Plant Mol. Biol. 26: 1981 to 1987, 1994), incorporated herein by reference in its entirety); and the gene encoding the low molecular weight sulfur rich protein of soy (Choi et al., Mol. Gen. Genet. 246: 266 to 268, 1995, hereby incorporated by reference in its entirety), may also be used.

Promoters derived from zein encoding genes (including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and gamma genes; Pedersen et al., Cell 29: 1015 to 1026, 1982, hereby incorporated by reference in their totality) can also be used. Zeins are a group of storage proteins found in the corn endosperm.

Other known promoters that work, for example, in maize, include promoters for the following genes: waxy, Brittle, Shrunken 2, Branching enzymes I and II, starch synthase, debranching enzymes, oleosins, glutellins and sucrose synthases. A particularly preferred promoter for the expression of the corn endosperm is the promoter for the rice glutelin gene, more particularly the Osgt-1 promoter (Zheng et al., Mol. Cell Biol. 13: 5829 to 5842, 1993, incorporated herein by reference in its entirety). Examples of promoters suitable for expression in wheat include those promoters for the ADPglucose pyrophosphorylase (ADPGPP) subunits, granule binding and other starch synthases, branching and debranching enzymes, abundant proteins in embryogenesis, giiadins and glutenins . Examples of such promoters in rice include those promoters for the ADPGPP subunits, granule binding and other starch synthases, branching enzymes, debranching enzymes, sucrose synthases and glutellins. A promoter

particularly preferred is the promoter for rice glutelin, Osgt-1. Examples of such promoters for barley include those for the subunits of ADPGPP, granule binding and other starch synthases, branching enzymes, debranching enzymes, sucrose synthases, hordeins, embryo globulins and specific proteins of aleurone.

A tomato promoter active during fruit ripening, senescence and abscission of leaves and, to a lesser extent, flowers can be used (Blume et al., Plant J. 12: 731 to 746, 1997, hereby incorporated by reference in its totality). Other exemplary promoters include the specific promoter of the piston SK2 gene in potatoes (Sotanum tuberosum L.), which encodes a basic pistil-specific endoquitinase (Ficker et al., Plant Mol. Biol. 35: 425 to 431, 1997, incorporated herein by reference in its entirety); the Blec4 gene of the pea (Pisum sativum cv. Alaska), active in the epidermal tissue of apexes of the vegetative and floral buds of transgenic alfalfa. This makes it a useful tool to target the expression of genes foreign to the epidermal layer of actively growing shoots. The tomato tissue-specific E8 promoter is also useful for targeting gene expression in fruits (Deikman, et al., Plant Physiology 100: 2013 to 2017, 1992).

It is also recognized that, since in most cases the exact limits of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

Promoters that are known or are found to cause transcription of DNA in plant cells can be used in the present invention. Such promoters can be obtained from a variety of sources such as plants and plant viruses. In addition to the promoters that are known to cause DNA transcription in plant cells, other promoter molecules can be identified for use in the current invention by screening a plant cDNA library for genes that are selectively or preferably expressed in target tissues or cells. and isolating the 5 'genomic region of the identified cDNAs.

The constructs or vectors may also include with the coding region of interest a polynucleic acid that acts, in whole or in part, to end the transcription of that region. For example, such sequences have been isolated including the 3 'Tr7 sequence and the 3' sequence (Ingelbrecht et al., The Plant Cell 1: 671 to 680, 1989, all of which are incorporated by reference; Bevan et al. , Nucleic Acids Res. 11: 369 to 385, 1983, all of which are incorporated by reference).

A vector or construct can also include regulatory elements. Examples of such include the intron Adh 1 (Callis et al., Genes and Develop. 1; 1183 to 1200, 1987, all of which is incorporated by reference), the sucrose synthase intron (Vasil et al., Plant Physiol. 91: 1575 to 1579, 1989, all of which is incorporated by reference) and the element TMV omega (Gallie et al., Plant Cell 1: 301 to 311, 1989, all of which is incorporated by reference ). These and other regulatory elements can be included where appropriate.

A vector or construct can also include a selectable marker. Selectable markers can also be used to select plants or plant cells that contain exogenous genetic material. Examples of such include, but are not limited to, a neo gene (Potrykus et al., Mol. Gen. Genet. 199: 183 to 188, 1985, all of which is incorporated herein by reference) which encodes resistance to kanamycin and can be selected to use kanamycin, G418, etc .; a bar gene that provides resistance to bialafos; a mutant EPSP synthase gene (Hinchee et al., Bio / Technology 6: 915 to 922 (1988), all of which is incorporated by reference) that provides glyphosate resistance; a nitrilase gene that provides resistance to bromoxynia (Stalker et al., J. Biol. Chem. 263: 6310 to 6314 (1988), all of which is incorporated herein by reference); a mutant acetolactate synthase (ALS) gene that confers imidazolinone or sulfonylurea; and a methotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem. 263; 12500 to 12508, 1988, all of which is incorporated herein by reference).

A vector or construct can also include a triá • • ········

speed. Trieable markers can be used to monitor expression. Exemplary triple markers include a β-glucuronidase or uidA (GUS) gene that encodes an enzyme for which several chromogenic substrates are known (Jefferson, Plant Mol. Biol, Rep. 5: 387 to 405 (1987), the entire which is incorporated herein by reference; Jefferson et al., EMBO J. 6: 3901 to 3907 (1987), all of which are incorporated by reference); a R site gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., Stadler Symposium 11: 263 to 282 (1988), all of which is incorporated by reference); a β-lactamase gene (Sutcliffe et al., Proc. Natl. Acad. Sci. (USA) 75: 3737 to 3741 (1978), all of which is incorporated by reference), a gene that encodes an enzyme for which various chromogenic substrates are known (for example, PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al., Science 234: 856 to 859 (1986), all of which are incorporated by reference); an xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A.) 80: 1101 to 1105 (1983), all of which is incorporated by reference) that encodes a dioxygenase catechol that can convert chromogenic catechols; an α-amylase gene (Ikatu et al., Bio / Technol. 8: 241 to 242, 1990, all of which are incorporated herein by reference); a tyrosinase gene (Katz etal., J. Gen. Microbiol. 129: 2703 to 2714,1983, all of which is incorporated by reference) that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to melanin; and a-galactosidase.

Introduction of Polynucleotides in Plants

There are many methods for introducing transforming nucleic acid molecules into plant cells. Suitable methods are believed to include virtually any method by which nucleic acid molecules can be introduced into a cell, such as by infection with Agrobacterium or direct release of nucleic acid molecules such as, for example, by PEG-mediated transformation , by electroporation or by the acceleration of particles coated with DNA, etc. (Po • · ······· *

»· · · · · · Trykus, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205 to 225 (1991), all of which are incorporated by reference; Vasil, Plant Mol. Biol. 25: 925 to 937 (1994), all of which are incorporated by reference). For example, electroporation has been used to transform Zea mays protoplasts (Fromm et al., Nature 312: 791 to 793, 1986, all of which are incorporated by reference).

Other vector systems suitable for introducing transforming DNA into a plant host cell include but are not limited to binary artificial chromosome vectors (BIBAC) (Hamilton et al., Gene 200: 107 to 116, 1997, all of which are incorporated herein by reference) and transfection with viral RNA vectors (Della-Cioppa et al., Ann. Ν. Y. Acad. Sei. (1996), 792 pp Engineering Plants for Commercial Products and Applications, pp 57 to 61, all of which is incorporated herein by reference.

The technology for introducing DNA into cells is well known to those skilled in the art. Four general methods for releasing a gene into cells have been described: (1) chemical methods (Graham and van der Eb, Virology 54: 536 to 539, 1973, all of which are incorporated by reference); (2) physical methods such as microinjection (Capecchi, Cell 22: 479 to 488 (1980), all of which is incorporated by reference), electroporation (Wong and Neumann, Biochem. Biophys. Res. Commun. 107: 584 a 587 (1982); Fromm et al, Proc Natl Acad Sci (USA) 82:..... 5824-5828 (1985); US Patent ^No.'s 5,384,253, all of which are incorporated herein in their entirety); and the gene gun (Johnston and Tang, Methods Cell Biol. 43; 353 to 365 (1994), all of which are incorporated herein by reference); (3) viral vectors (Clapp, Clin. Perinatol. 20: 155 to 168, 1993; Lu et al., J. Exp. Med. 178: 2089 to 2096, 1993; Eglitis and Anderson, Biotechniques 6: 608 to 614, 1988, all of which are incorporated herein in their entirety); and (4) receptor-mediated mechanisms (Curiel et al., Hum. Gen. Ther. 3: 147 to 154, 1992, Wagner et al., Proc. Natl. Acad. Sci. USA 89: 6099 to 6103, 1992, all of which are incorporated by reference in their entirety).

w • ·· ·· · ·· · • · · «• · · • • · * • · · · ⁴ • ·· ·· ··

The acceleration methods that can be used include, for example, microprojectile bombardment and others. An example of a method for releasing transforming nucleic acid molecules to plant cells is microprojectile bombardment. This method was reviewed by Yang and Christou, eds., Particle Bombardment Technology for Gene Transfer, Oxford Press, Oxford, England (1994), all of which is incorporated by reference. Non-biological particles (microprojectiles) that can be coated with nucleic acids and released into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum and others.

Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because DNA can be introduced into whole plant tissues, thereby deflecting the need for the regeneration of an intact plant from a protoplast. The use of plant integration vectors measured by Agrobacterium to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et al., Bio / Technology 3: 629 to 635 (1985) and Rogers et al., Methods Enzymol. 153: 253 to 277 (1987), both of which are incorporated herein by reference in their entirety. Furthermore, the integration of Ti-DNA is a relatively precise process that results in few rearrangements. The DNA region to be transferred is defined by the end sequences and the intervening DNA is usually inserted into the plant's genome as described (Spielmann et al., Mol. Gen. Genet. 205: 34 (1986), the total of which is incorporated herein by reference).

A transgenic plant that results from Agrobacterium transformation methods often contains a single gene on a chromosome. Such transgenic plants can be alluded to as being homozygous for the added gene. Most preferred is a transgenic plant that is homozygous for the added structural gene; that is, a transgenic plant that contains two added genes, one gene at the same location on each chromosome of a pair of chromosomes. A homozygous transgenic plant can be obtained by sexually mating (self pollinating) an independent segregating transgenic plant that contains a single added gene, germinating (germinating) · germinating (germinating). some of the seeds produced and analyzing the resulting plants produced for the gene of interest.

It should also be understood that two different transgenic plants can also be mated to produce progeny that contain two independently secreted exogenous genes added. Self-pollination of appropriate progeny can produce plants that are homozygous for both added exogenous genes that encode a polypeptide of interest. Retro-crossing with a precursor plant and crossing with a non-transgenic plant are also considered, as is vegetative propagation.

The regeneration, development and cultivation of plants from a single plant protoplast transformant or from several transformed explants are well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (eds.), Academic Press, Inc. San Diego, CA, (1988), all of which are incorporated by reference). This process of regeneration and cultivation typically includes the stages of selection of transformed cells, cultivating these individualized cells through the usual stages of embryonic development to the rooted seedling stage. Embryos and transgenic seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant cultivation medium such as the soil.

The development or regeneration of plants containing the foreign, exogenous gene encoding a protein of interest is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from regenerated plants is crossed with plants developed with seed from agronomically important strains. In contrast, pollen from plants of these important strains is used to pollinate regenerated plants. A transgenic plant of the present invention contains 74

A desired polypeptide is cultured using methods well known to a person skilled in the art.

The present invention also provides parts of the plants of the present invention. The plant parts, without limitation, include seed, endosperm, egg and pollen. In a particularly preferred embodiment of the present invention, the plant part is a seed.

Methods for transforming dicots, primarily by use of Agrobacterium tumefaciens , and obtaining transgenic plants have been published, for example, cotton (US Patent No. 5,004,863 ^and. 5,159,135 US Patent ^Nos. US Patent No. 5518908 ^Q, all of which are incorporated herein by reference in its entirety), soybean (US Patent 5,569,834 ^{Ν Ω.} the entirety of which is hereby incorporated by reference) and Brassica (US Patent No. 5,463,174 ^and. the entirety of which is hereby incorporated by reference).

The transformation of monocots using electroporation, particle bombardment and Agrobacterium has also been reported. For example, plant transformation and regeneration was achieved in asparagus, barley, Zea mays (Fromm et al., Bio / Technology 8: 833 (1990), Armstrong et al., Crop Science 35: 550 to 557 (1995) , all of which are incorporated by reference in their entirety): oats; rice, rye, sugar cane; tall fescue and wheat (US Patent No. ^Q 5,631,152, all of which is incorporated by reference).

In addition to the procedures discussed above, technicians are familiar with standard resource materials that describe specific conditions and procedures for the construction, manipulation and isolation of macromolecules (eg, DNA molecules, plasmids, etc.), the generation of recombinant organisms and the screening and isolation of clones, (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989); Mailga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995 ), all of which is incorporated by reference; Birren et al., Genome Analisis: Detecting Genes, 1, Cold Spring Harbor, New York (1998), all of which is incorporated by reference; Birren et al., Genome Analysis: Analyzing DNA, 2, Cold

Í3

Spring Harbor, New York (1998), all of which are incorporated by reference; Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer, New York (1997), all of which are incorporated by reference).

Having now generally described the invention, it will be more easily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention, unless specified.

EXAMPLES

EXAMPLE 1

When an isolated native plant polynucleotide comprising a coding sequence is reconstructed as a transgene, then introduced into the plant by plant transformation methods, there is a risk that the expression of the endogenous homologous plant gene will interact negatively with the transgene. To avoid these negative interactions it may be necessary to provide a substantially divergent transgene polynucleotide in sequence with the native plant gene. An artificial polynucleotide molecule can be produced by the method of the present invention and used to reduce the occurrence of transgene silencing.

This example serves to illustrate the methods of the present invention that result in the production of a polynucleotide that encodes a modified plant EPSP synthase. The EPSPS enzyme and the native rice chloroplast transit peptide (Oryzae sativa) are used to build an artificial polynucleotide molecule that also includes codons that encode naturally occurring substituted amino acids in the EPSPS enzyme in rice. These substituted amino acids provide a glyphosate resistant rice EPSPS enzyme (OsEPSPS_TIPS, SEQ ID NO: 1).

The steps described in Table 6 are used to construct such an artificial polynucleotide sequence (OsEPSPS_AT, SEQ ID NO: 3) using an Arabidopsis codon usage table and the parameters for the construction of a substantially divergent polynucleotide molecule, which when expressed in plants encodes an EPSPS enzyme from

modified rice resistant to the glyphosate herbicide. The comparison of the native rice EPSPS gene sequence referred to as OsEPSPS_Nat (SEQ ID NO: 2) that was previously modified to encode a glyphosate-resistant enzyme with the modified polynucleotide molecule for the use of Arabidopsis codon, OsEP3PS_AT (SEQ ID NO: 3) and with the modified sequence regarding the use of Zea mays codon, OsEPSPS_ZM (SEQ ID NO: 4) by this method is shown in Figure 1. Figure 1 shows changed nucleotide bases in the modified polynucleotides compared to OsEPSPS_Nat, SEQ ID NO: 2.

Table 6. Polynucleotide planning for a modified EPSP synthase of rice (OsEPSPS_AT)

1. Substitute amino acids at positions 173 and 177 to provide a modified rice EPSPS enzyme resistant to the glyphosate herbicide shown in SEQ ID NO: 1.

2. Retro translate SEQ ID NO: 1 to generate an artificial polynucleotide sequence using the Arabidopsis thaliana codon usage table (Table 2).

3. Perform sequence alignment with the native OsEPSPS polynucleotide sequence (SEQ ID NO: 2) and the artificial polynucleotide sequence to determine the degree of sequence identity, map the open reading matrices, select patterns to search for and identify sequences of restriction enzyme recognition.

4. Make corrections to the codons used in the artificial polynucleotide sequence to obtain the desired percentage of sequence identity and avoid the formation of clusters of identical codons. This is especially important for amino acids that occur in high frequency, that is, alanine, glycine, histidine, leucine, serine and valine. Approximate distribution of codon use in the polynucleotide sequence according to the use of Arabidopsis codon, Table 2.

5. The polynucleotide sequence is inspected for local regions that have a GC: AT ratio higher than about 2 in a range of about 50 contiguous nucleotides. The polynucleotide sequence

it is adjusted as necessary, replacing the codons in these regions such that the local GC: AT ratio is less than about 2 and the entire polynucleotide composition is in the range of 0.9 to 1.3.

6. Introduce stop codons to translation matrices b, c, d, e and T. Translation stop codons are created in translation matrices b, c, d, e and by replacing one or more codons within about 130 base pairs (base pairs) of the ends of the artificial polynucleotide that creates a stop codon without changing the amino acid coding sequence of matrix one.

7. Eliminate the ATG codons from the advanced open reading matrices (matrices b and c) and reverse (matrices d, e, T). The forward and reverse reading matrices are inspected for the presence of ATG codons. Any ATG codon in matrices b and c found in the polynucleotide sequence before the third Met in matrix a of the polynucleotide is eliminated by replacing one or more codons that surround the ATG by changing one of the nucleotides without changing the amino acid coding sequence of matrix a ”. In reverse arrays, substitution of ATG or introduction of the stop codon can be done to interrupt potential reading arrays.

8. Eliminate the recognition patterns of unwanted restriction enzymes and other specific patterns (polyadenylation, RNA junction, sequence instability patterns). The polynucleotide sequence is inspected for the presence of any unwanted polynucleotide patterns and the patterns are disrupted by replacing codons in these regions.

9. Check the sequence identity between a first polynucleotide and the artificial polynucleotide created by the method of the present invention. Eliminate the sequence identity in a contiguous polynucleotide that is greater than 23 base pairs. It is desirable to eliminate the sequence identity greater than about 15 base pairs. It is useful to select from amino acids such as serine, arginine and leucine that have 6 codons or from amino acids with 4 codons to eliminate sequence identity.

«20

cia.

10. Review the artificial polynucleotide sequence that results from any of steps 1 through 9 for any of the sequence characteristics identified in steps 4 through 9 and if the sequence does not meet the conditions, perform additional codon substitutions to the sequence until the conditions of steps 4 to 9 are reached.

11. Build the artificial polynucleotide molecule by methods known in the art, for example, using PCR with a mixture of primers involved. The primers at the ends of the gene may contain convenient restriction sites to allow easy cloning of the gene into the selected vector. At the 5 'end usually Alflll, BspHI, Ncol, Ndel, Pcil or Sphl are more convenient since their sequence contains an ATG starting codon, however other enzymes can also be used if a modified polynucleotide is designed to create a fusion with a another polynucleotide segment, for example, chloroplast transit peptide and EPSPS coding sequence.

12. Perform a DNA sequence analysis of the artificial polynucleotide to confirm the resulting synthetic construct in the desired polynucleotide molecule. If errors are found, then eliminate them by mutagenesis directed to the site for which many methods are known to those skilled in the DNA mutagenesis technique.

A version of the Zea mays codon usage (Zea mays, Table 3) of the EPSPS enzyme sequence of glyphosate resistant rice (EPSPS enzyme from Oryzae sativa with TIPS mutations, SEQ ID NO: 1) is manufactured. The polynucleotide encoding this enzyme includes codons that encode substituted amino acids that do not occur naturally in the native EPSPS enzyme in rice. These substituted amino acids provide a glyphosate-resistant EPSPS enzyme in rice. The steps described in Table 7 are used to construct a modified artificial polynucleotide sequence (OsEPSPS_ZM, SEQ ID NO: 4) based on a Zea mays codon usage table that encodes a modified EPSPS enzyme from the herbicide resistant rice. glyphosate. The comparison of the sequence of • · · · · · ·

SY polynucleotide OsEPSPS_Nat (SEQ ID NO: 2) with the artificial polynucleotide sequence OsEPSPS_ZM (SEQ ID NO: 4) using the Zea mays codon is shown in Figure 2.

Table 7. Polynucleotide construction for modified EPSP synthase of rice (OsEPSPS_ZM)

1. Retro translate SEQ ID NO: 1 to generate an artificial polynucleotide sequence using the Zea mays codon usage table (Table 3).

2. Perform sequence alignment with the native OsEPSPS polynucleotide sequence (SEQ ID NO: 2) and the artificial polynucleotide sequence to determine the degree of sequence identity, map the open reading matrices, select patterns to search for and identify sequences of restriction enzyme recognition.

3. Make corrections to the codons used in the artificial polynucleotide sequence to obtain the desired percentage of sequence identity and avoid the formation of clusters of identical codons. This is especially important for amino acids that occur in high frequency, that is, alanine, glycine, histidine, leucine, serine and valine. Approximate distribution of codon usage in the polynucleotide sequence according to Zea mays codon usage, Table 3.

4. The polynucleotide sequence is inspected for local regions that have a GC: AT ratio greater than about 2 in a range of about 50 contiguous nucleotides. The polynucleotide sequence is adjusted as necessary, by replacing codons in these regions such that the local GC: AT ratio is less than about 2 and the entire polynucleotide composition is in the range of 1.2 to 1.7.

5. Follow steps 6 through 12 in Table 6.

• · · ·····

Table 8. Percentage sequence identity among OsEPSPS polynucleotides.

OsEPSPS ZM OsEPSPS AT OsEPSPS Nat OsEPSPS ZM 100.00 73.51 71.58 OsEPSPS AT 100.00 74.03 OsEPSPS Nat 100.00

Table 9. The nucleotide composition and the GC: AT ratio of the polynucleotide sequences modified to the EPSPS gene sequence of rice.

THE Ç G T GC.AT OsEPSPS. _AT 377 336 444 391 1.02 OsEPSPS. ZM 365 381 470 332 1.22

The two artificial EPSPS polynucleotide sequences in rice (SEQ ID NO: 3 and SEQ ID NO: 4) are modified such that the percent identity is below 75 percent compared to SEQ ID NO: 2 or in relation to one another (Table 8). The nucleotide composition and the GC: AT ratio of the polynucleotide sequences to the rice EPSPS gene sequence are shown in Table 9. These polynucleotides can be selected for use in plant expression constructs together with different regulatory elements or they can be combined into a single plant by retransformation with a DNA construct or by plant breeding methods. Problems with gene silencing and recombination are reduced when DNA constructs have reduced levels of homologous DNA.

EXAMPLE 2

Corn (Zea mays) was genetically modified to have resistance to the glyphosate herbicide (US Patent No. ^Q 6,040,497). These maize plants contain a transgene with a modified EPSP synthase from maize for glyphosate tolerance. The methods of the present invention can be used to construct a new artificial polynucleotide that encodes a corn EPSP synthase that is substantially different in percentage identity to the endogenous corn EPSP synthase gene. The arti • · · $ 3 polynucleotide from newly constructed maize EPSP synthase can be used as a selectable marker when selecting transgenic plant strains that may contain additional transgenic agronomic traits. During hybrid maize seed production, it is useful to have both glyphosate tolerant precursors using non-interfering transgenes.

Table 10. Polynucleotide construct for modified corn EPSP synthase (ZmEPSPS_ZM, SEQ ID NO: 10)

1. Retro translate SEQ ID NO: 8 to generate a polynucleotide sequence using the Zea mays codon usage table (Table 3).

2. Perform sequence alignment with the polynucleotide sequence ZmEPSPS_Nat (SEQ ID NO: 9) and the artificial polynucleotide sequence to determine the degree of sequence identity, map the open reading matrices, select patterns to search for and identify sequences of restriction enzyme recognition.

3. Make corrections to the codons used in the artificial polynucleotide sequence to obtain the desired percentage of sequence identity and to avoid the formation of clusters of identical codons. This is especially important for amino acids that occur in high frequency, that is, alanine, glycine, histidine, leucine, serine and valine. Approximate distribution of codon usage in the polynucleotide sequence according to Zea mays codon usage, Table 3.

4. The artificial polynucleotide sequence is inspected for local regions that have a GC: AT ratio greater than about 2 in a range of about 50 contiguous nucleotides. The polynucleotide sequence is adjusted as necessary, by replacing codons in these regions such that the local GC: AT ratio is less than about 2 and the entire polynucleotide composition is in the range of 1.2 to 1.7.

5. Follow steps 6 through 12 in Table 6.

• · · • ·

I · »· · ·

Table 11. Percentage sequence identity among ZmEPSPS polynucleotides.

ZmEPSPS ZM ZmEPSPS Nat ZmEPSPS ZM 100.00 74.81 ZmEPSPS Nat 100.00

The nucleotide sequence of the EPSPS gene in corn is also modified to reduce identity between the synthetic and native gene and maintain the typical global GC: AT ratio for monocots. The GC: AT ratio for the ZmEPSPS_ZM sequence is 1.38. The sequence identity is reduced to about 75% between the native (ZmEPSPS_Nat, SEQ ID NO: 9) and the synthetic (ZmEPSPS_ZM, SEQ ID NO: 10).

The comparison of native polynucleotides encoding EPSPS indicates that the chloroplast transit peptide is the most divergent fragment of the gene. The nucleotide sequence similarity of mature peptides is greater than 88% for corn and rice enzymes and some conserved regions have sequence identities as long as 50 base pairs. Post-transcriptional gene silencing has been observed for sequences as small as 60 polynucleotides (Sijen et al., Plant Cell, 8: 2277 to 2294. 1996; Mains, Plant Mol. Biol. 43: 261 to 273, 2000).

EXAMPLE 3

Soy (Glycine max) was genetically modified to be glyphosate tolerant by the expression of a class II EPSPS isolated from Agrobacterium (Padgette et al. Crop Sci. 35: 1451 to 1461, 1995). An EPSPS gene sequence from native soybean was identified and an artificial polynucleotide sequence designed using the method of the present invention. The artificial polynucleotide encodes a protein sequence that is modified to produce a glyphosate-resistant EPSPS enzyme (GmEPSPSJKS, SEQ ID NO: 5) by replacing the amino acids T for I, R for K and P for S within the GNAGTAMRP motif, resulting in in a soybean EPSPS enzyme modified with the motif GNAGIAMKS (SEQ ID NO: 34), also referred to as the IKS mutant. The expression of an enzyme

of EPSPS modified in the cells of a plant by transformation with a transgenic plant expression cassette, which contains a polynucleotide that encodes the EPSPS modified with the GNAGIAMKS motif will confer glyphosate tolerance to plants. Additional amino acid substitutions for arginine (R) in the motif can also include asparagine (N). Table 12. Polynucleotide construct for the modified soybean EPSP synthase gene (GmEPSPS_GM, SEQ ID NO: 7).

1. Retro translate SEQ ID NO: 5 to generate an artificial polynucleotide sequence using the Glycine max codon usage table (Table 4).

2. Perform sequence alignment with the polynucleotide sequence GmEPSPS_Nat (SEQ ID NO: 6) and the artificial polynucleotide sequence to determine the degree of sequence identity, map the open reading matrices, select the patterns to search for and identify the restriction enzyme recognition sequences.

3. Make corrections for the codons used in the artificial polynucleotide sequence to obtain the desired percentage of sequence identity and to avoid the formation of clusters of identical codons. This is especially important for amino acids that occur in high frequency, that is, alanine, glycine, histidine, leucine, serine and valine. Approximate distribution of codon usage in the polynucleotide sequence according to the use of Glycine max codon, Table 4.

4. The polynucleotide sequence is inspected for local regions that have a GC: AT ratio greater than about 2 in a range of about 50 contiguous nucleotides. The polynucleotide sequence is adjusted as necessary, by replacing codons in these regions such that the local GC: AT ratio is less than about 2 and the entire polynucleotide composition is in the range of 0.9 to 1.3.

5. Follow steps 6 through 12 in Table 6.

»·« • · · · · »··· · ·

Table 13. Comparison of the percentage sequence identity of the modified GmEPSPS at the level of the polynucleotide sequence.

GmEPSPS GM GmEPSPS Nat GmEPSPS GM 100.00 72.43 GmEPSPS Nat 100.00

The EPSPS gene of native soybeans is modified using a soy codon table (Table 4) and the conditions of the method of the present invention. The relative ratio of GC: AT is not changed in the modified gene, however the sequence identity between the two is reduced by 72%. EXAMPLE 4

The aroA gene native polynucleotide isolated from Agrobacterium strain CP4 (US Patent No. 5,633,435 ^are incorporated herein by reference in its entirety) that encodes a glyphosate resistant EPSP synthase (SEQ ID NO: 15) may be modified by this method invention to provide a polynucleotide using the codon of Arabidopsis, Zea mays or Glycine max. For the proper expression of CP4EPSPS to confer tolerance to glyphosate in plants, a chloroplast transit peptide is necessarily fused to the CP4EPSPS coding sequence to target the enzyme accumulation in chloroplasts. The CTP2 chloroplast transit peptide is commonly used for the expression of this gene in transgenic plants (Nida et al., J. Agric. Food Chem. 44: 1960 to 1966, 1996). The CP4EPSPS sequence together with the CTP2 polynucleotide (SEQ ID NO: 11) were modified by the method of the present invention. Other chloroplast transit peptides known in the art can be fused to CP4EPSPS to target the enzyme to chloroplasts.

Table 14. Polynucleotide construction for the coding sequence for the EPSP synthase of aroA: CP4 (CP4EPSPS_AT, CP4EPSPS_ZM or CP4EPSPS_GM)

1. Place the CTP2 transit peptide sequence (SEQ ID

NO: 11) in front of CP4EPSPS (SEQ ID NO: 15) as a fusion polypeptide. Retro translate the fusion polypeptide to produce an artificial polynucleotide sequence using the Arabidopsis thaliana codon usage table (Table 2), or the Zea mays codon usage table (Table 3) or the codon usage table Max glycine (Table 4).

2. Perform sequence alignment with the native CTP2 polynucleotide (SEQ ID NO: 12) and native CP4EPSPS (SEQ ID NO: 16) and the artificial polynucleotide sequence to determine the degree of sequence identity, map the reading matrices open, select patterns to search for and identify restriction enzyme recognition strings.

3. Make corrections to the codons used in the artificial polynucleotide sequence to obtain the desired percentage of sequence identity and to avoid the formation of clusters of identical codons. This is especially important for amino acids that occur in high frequency, that is, alanine, glycine, histidine, leucine, serine and valine. Approximate distribution of codon usage in the polynucleotide sequence according to the use of Arabidopsis thaliana codon, Table 2 or the Zea mays codon usage table (Table 3) depending on the table in use.

4. The artificial polynucleotide sequence is inspected for local regions that have a GC: AT ratio greater than about 2 in a range of about 50 contiguous nucleotides. The polynucleotide sequence is adjusted as necessary, by replacing codons in these regions such that the local GC: AT ratio is less than about 2 and the entire polynucleotide composition being in the range of 0.9 to 1.3 is the Table 2 that is used and from 1.2 to 1.7 if Table 3 is used.

5. Follow steps 6 through 12 in Table 6.

Table 15. Comparison of percentage sequence identity of artificial CP4EPSPS polynucleotides.

CTP2CP GM CTP2CP4 _AT CTP2CP4 _ZM CTP2CP4 _Syn CTP2CP4 _NAT GM CTP2CP4 100.00 75.66 74.12 75.15 74.37 CTP2CP4 AT 100.00 76.13 74.56 * 72.93 CTP2CP4 ZM 100.00 77.76 * 82.58 CTP2CP4 Syn 100.00 82.70 CTP2CP4 NAT 100.00

* Α percentage of identity concerns CP4EPSPS and does not include the transit peptide.

Table 16. The nucleotide composition and the GC: AT ratio of the artificial polynucleotide sequences to the CP4EPSPS gene sequence.

THE Ç G T GC: AT CTP2CP4_GM 382 375 442 397 1.05 CTP2CP4_AT 369 408 469 350 1.22 CTP2CP4 ZM 312 487 577 290 1.65

The polynucleotide sequence CTP2_Nat (SEQ ID NO: 12) plus CP4EPSPS_Nat (SEQ ID NO: 16) designated as CTP2CP4_Nat is compared in Table 15 with the artificial polynucleotide sequences designated as CTP2CP4_AT (CTP2_AT, SEQ ID NO: 13 fused to CP4, fused to CP4 SEQ ID NO: 17) and CTP2CP4_ZM (CTP2_AT, SEQ ID NO:

14 fused to CP4EPSPS_ZM, SEQ ID NO: 18) produced by the method of the present invention. The polynucleotide sequence that is the most divergent from the native CTP2CP4_NAT and CTP2CP4EPSPS_Syn sequences is CTP2CP4_AT having about 73% and 75% sequence identity, respectively. The polynucleotide sequence CTP2CP4_ZM compared to CTP2CP4_Nat and CP4EPSPS_Syn has about 83% and 78% identity with these two sequences, respectively.

A primary criterion for selecting transgenes to combine in a plant is percent identity. Table 15 can be used to select a CP4EPSPS polynucleotide molecule for the construction of the plant expression cassette when it is known that the recipient plant will contain more than one CP4EPSPS polynucleotide. The GC: AT ratio in native CP4EPSPS is about 1.7. The artificial version with the Zea mays codon of choice is produced to have a very similar GC: AT ratio. In the codon version of Arabidopsis, the GC: AT ratio is decreased by about 1.2.

Gene expression is also a criterion for the selection of transgenes to be expressed. The expression of a transgene can vary in different crop plants, therefore having several coding sequences ·· ♦ ·······

crop and an artificial polynucleotide as87 available for testing on plants of different genotypes, varieties or cultivars is an advantage and aspect of the invention.

EXAMPLE 5

The polynucleotide bar sequence (SEQ ID NO: 20) encoding a phosphinothricin acetyl transferase protein (SEQ ID NO: 19) was used to genetically modify plants for resistance to the glufosinate herbicide. Two new bar polynucleotide sequences were designed using the method of the present invention. The alignment of BAR1_Nat with the two new artificial BAR1 polynucleotides is shown in Figure 4. Table 17. Polynucleotide gene construction for BAR1_AT (SEQ ID NO: 21) and BAR1_ZM (SEQ ID NO: 22)

1. Retro translate SEQ ID NO: 19 to generate a polynucleotide sequence using the Arabidopsis thaliana codon usage table (Table 2) or the Zea mays codon usage table (Table 3)

2. Perform sequence alignment with the native BAR1_Nat polynucleotide sequence (SEQ ID NO: 20) and the artificial polynucleotide sequence to determine the degree of sequence identity, map open reading structures, select patterns to search for and identify the restriction enzyme recognition strings.

3. Make corrections to the codons used in the artificial polynucleotide sequence to obtain the desired percentage of sequence identity and to avoid the formation of clusters of identical codons. This is especially important for amino acids that occur in high frequency, that is, alanine, glycine, histidine, leucine, serine and valine. Approximate distribution of codon usage in the polynucleotide sequence according to the use of Arabidopsis thaliana codon, Table 2 or the Zea mays codon usage table (Table 3) depending on the table in use.

4. The artificial polynucleotide sequence is inspected for local regions that have a GC: AT ratio greater than about 2 in a range of about 50 contiguous nucleotides. The polynucleotide sequence is adjusted as necessary by replacing codons in these regions • · ····· ♦ ·· ··· ··· ·· • · · · · · · · · ··· · · · • · · · Ions such that the local GC.AT ratio is less than about 2 and the entire polynucleotide composition is in the range of 0.9 to 1.3 if Table 2 is used and 1.2 to 1.7 if Table 3 is used.

5. Follow steps 6 through 12 in Table 6.

The sequence identity of artificial BAR polynucleotides is in the range of 73 to 77% (Table 18). The native polynucleotide is highly rich in GC. The artificial version (BAR1_ZM) with the Zea mays codon of choice reduced the GC: AT ratio to about 1.3 and the artificial version (BAR1_AT) with the Arabidopsis codon of choice the ratio is about 1.0 (Table 19).

Table 18. Percentage sequence identity between bar genes at the polynucleotide sequence level.

BAR1 ZM BAR1 AT BAR1 Nat BAR1 ZM 100.00 77.35 76.99 BAR1 AT 100.00 73.73 BAR1 Nat 100.00

Table 19. Nucleotide composition and the GC: AT ratio of artificial polynucleotide sequences to the bar gene sequence.

BAR_AT 139 130 144 139 1.01 BAR ZM 122 156 154 120 1.28

EXAMPLE 6

This example serves to illustrate the DNA constructs for the expression of the artificial polynucleotides of the present invention in plants. A transgenic DNA plant expression cassette comprises regulatory elements that control the transcription of an mRNA from the cassette. A plant expression cassette is constructed to include a plant-functioning promoter that is operably linked to a leader 5 'region that is operably linked to a DNA sequence of interest operably linked to a 3' end region. These cassettes are constructed in plasmid vectors, which can then be transferred to plants by Agrobacterium-mediated transformation methods or other methods known to those skilled in the art of plant transformation. The »······· γ

The following plasmid vector constructs are illustrated to provide examples of plasmids containing plant expression cassettes that comprise the artificial polynucleotide molecules of the present invention and are not limited to these examples.

The artificial polynucleotide molecules of the present invention, for example, CP4EPSPS_AT and CP4EPSPS_ZM are synthesized using primers involved. The full-size product is then amplified with specific gene primers containing projections with Sphl (advanced primer) and EcoRI (reverse primer). The genes are cloned into the pCRIl-TOPO vector (Invitrogen, CA). The resulting plasmids pMON54949 (CP4EPSPS_AT, Figure 6) and pMON54950 (CP4EPSPS_ZM, Figure 7) contain the artificial polynucleotide and these polynucleotides are sequenced using DNA sequencing methods to confirm that the modifications designed by the method of the present invention are contained in the artificial polynucleotides. In the next step, the artificial polynucleotide encoding the CTP2 chloroplast transit peptide is attached to the 5 'end of the CP4EPSPS polynucleotides. The CaMV 35S promoter with a duplicate enhancer (P-CaMVe35S) and a rice actin 1 intron (l-OsAct1) derived from pMON30151 (Figure 8) by digestion with Sphl and Hindlll linked to the CTP2CP4EPSPS polynucleotides to create the pMON59302 (CTP2PS2) plasmids (CTP2PS2) , Figure 9) and pMON59307 (CTP2CP4EPSPS_ZM, Figure 10).

For the expression of the new artificial polynucleotides in monocotyledonous plants, the genes are placed in the plant expression cassettes containing at the 5 * end of the polynucleotide, a promoter and an intron, an untranslated region 5 'and at the 3' end of the polynucleotide a signal of transcription termination. For this purpose, pMON42411 (Figure 11) containing P-CaMV35S: en, I-HSP70, CTP2CP4_Nat and NOS 3 'are digested with restriction enzymes Ncol and EcoRI. PMON59302 (Figure 9) and pMON59307 (Figure 10) are digested with the same restriction enzymes. The fragments are gel purified using the Qiagen gel purification kit and ligated to form pMON58400 (CP4EPSPS_AT, Figure • ·· ··· «• · · · · · · ·» · «· · ··· <

12) and pMON58401 (CP4EPSPS_ZM, Figure 13). The additional vector pMON54964 (Figure 14), containing P-OsAct1 / l-OsAct1 is manufactured by substituting P-e35S / l-Hsp70 from pMON58400 (Figure 12) using the Hindlll / Ncol fragment of pMON25455 (Figure 15). To create a monocot expression vector containing the P-FMV promoter, pMON30152 (Figure 16) is digested with Nhel, the ends are made abrupt with T4DNA polymerase in the presence of 4 dNTP-s (200 μΜ) and Ncol. The DNA fragments CPT2CP4_AT or CTP2CP4_ZM are isolated from pMON59302 (Figure 9) or pMON59307 (Figure 10), respectively by digesting with EcoRI, abruptly using T4 DNA polymerase and digesting with Ncol. The gel-purified DNA fragments are ligated and new plasmids pMON54992 (CTP2CP4_AT, Figure 17) and pMON54985 (CTP2CP4_ZM, Figure 18) are created. In each case, successful plasmid construction is confirmed by restriction endonuclease digestion, using among others Clal (introduced in both artificial polynucleotides) and Pst I (introduced in CP4EPSPS_ZM). The CP4EPSPS_Nat present in the precursor vectors has both Clal and two Pstl restriction sites in the coding region in a different location than in the artificial polynucleotides.

For the expression of artificial CP4EPSPS polynucleotides in dicotyledonous plants, two precursor vectors are used: pMON20999 (P-FMV / CTP2CP4_Syn / 3'E-9, Figure 19) and pMON45313 (Pe35S / CTP2CP4_Syn / 3'E9, Figure 20). In each plasmid, a DNA fragment containing the polynucleotide CTP2CP4_Syn is replaced with CTP2CP4_AT or CTP2CP4_ZM. To create pMON59308 (PCaMVe35S / CTP2CP4_AT, Figure 21) or pMON59309 (PCaMVe35S / CTP2CP4_ZM, Figure 22), pMON45313 is digested with Ncol and EcoRI and the DNA restriction fragments derived from digestions with Ncol / EcoRI_ of CM2 ) or pMON59307 (CTP2CP4_ZM, Figure 10) are connected, respectively. To create pMON59313 (P-FMV / CTP2CP4_AT / 3'E9, Figure 23) and pMON59396 (PFMV / CTP2CP4_ZM / 3'E9, Figure 24) the precursor plasmid pMON20999 is digested with Ncol and BamHI to remove CTP2CP4_Syn and fragments

··· • · • · ··· ··· ·· • · · • · · • · · • · · ···· • • • • · • ·· ········ ·· · · · · • · · · • · · · • · · · ·· ··· ·· ·· ······· · · •

Ncol / BamHI restriction measures derived from pMON59308 (CTP2CP4_AT, Figure 21) or pMON59309 (CTP2CP4_ZM, Figure 22) are linked, respectively.

EXAMPLE 7

Artificial polynucleotides are tested to determine the effectiveness of giving transgenic plants tolerance to glyphosate. Five different expression cassettes (Table 20) with the new artificial CP4EPSPS polynucleotides are transformed into maize and the resulting transgenic maize plants compared to the commercial standard (Roundup Ready® Corn 603, Monsanto Co.). The plasmid pMON25496 (Figure 25) contained in the commercial standard has two copies of the polynucleotide CP4EPSPS_Nat, the expression directed by the promoters P-CaMVe35S (P-CaMVe35S) and POsActl, respectively. Plasmids containing the new artificial CP4EPSPS polynucleotides contain only a single copy of the polynucleotide to be tested. The expression of these polynucleotides is directed by the P-CaMVe35S promoter with the heat shock protein intron 1-Hsp70 or the P-FMV promoter with a rice sucrose synthase (1-OSSS) intron. Plasmid pMON54964 contains the rice actin 1 promoter with the first native intron (US Patent No. ^Q 5,641,876, incorporated herein by reference in its entirety).

These plasmids are transformed into corn cells by a method mediated by Agrobacterium and the transgenic maize strains regenerated in selection with glyphosate. Transgenic corn plants can be produced by an agrobacterium-mediated transformation method. An unarmed Agrobacterium cepa C58 (ABI) that houses a binary construct of the present invention is used. This is transferred to Agrobacterium by a triparental mating method (Ditta etal., Proc. Natl. Acad. Sci. 77: 7347 to 7351). Liquid cultures of Agrobacterium are initiated from glycerol stocks or from a freshly scratched plate and grown overnight between 26 ° C and 28 ° C with agitation (approximately 150 rpm) until the hemi-log cultivation phase in liquid LB medium, pH 7.0 containing the appropriate antibiotics. Agrobacterium cells

they are resuspended in the inoculation medium (liquid CM4C) and the density is adjusted to OD ₆ 6o out of 1. The newly isolated immature HillxLH198 and Hill Type li embryos are inoculated with a construct containing Agrobacterium and co-cultured for several days in the dark at 23 ° C. The embryos are then transferred to the delay medium and incubated at 28 ° C for several or more days. All subsequent cultures are maintained at this temperature. The embryos are transferred to a first selection medium containing 500 / 0.5 mM glyphosate carbenicillin). Two weeks later, the surviving tissue is transferred to a second selection medium containing 500 / 1.0 mM glyphosate carbenicillin). Subcultivate the surviving callus every 2 weeks until events can be identified. This can take about 3 subcultures in 1.0 mM glyphosate. Once the events are identified, swell the tissue to be regenerated. The seedlings are transferred to the MSOD medium in a culture vessel and maintained for two weeks. Then the plants with roots are transferred to the soil. Those skilled in the art of corn processing can modify this method to provide substantially identical transgenic corn plants containing the DNA compositions of the present invention.

About 30 transgenic maize strains for each plasmid construct are tested and the transformation efficiency and expression levels of the CP4EPSPS enzyme are shown in Table 20. Transgenic maize strains are treated with glyphosate at a rate of 64 oz / acre (1.8 kg / acre) as young plants, surviving plants are assayed by CP4EPSPS ELISA (Padgette et al. Crop Sci. 35: 1451 to 1461, 1995) to determine the levels of protein expression of EPSPS CP4 (% exp CP4) shown in Table 20 and the level of expression is compared with the commercially available standard glyphosate tolerant corn plant (Roundup Ready® Com 603, Monsanto Co., St. Louis, MO) as a percentage of the amount of protein expression determined in the commercial standard. Overall, more than 50% of maize strains survive spraying with 1.8 kg / acre (64 oz / acre) of glyphosate. The surviving plants are shown to have a high level of CP4EPSPS expression that

range from about 75 to 86% of commercial standard 603.

Table 20. Transformation efficiency (TE), CP4 expression (average%) derived from the transformation of different CP4-alt constructs.

pMON Promoter / lntron # TE (%) CP4 exp (%) * 58400 (CP4 AT) P-CaMVe35S / IHsp70 5.4 75.5 58401 (CP4 ZM) P-CaMVe35S / IHsp70 7.2 84.7 54964 (CP4 AT) P-OsAct1 8.2 78.1 54985 (CP4 ZM) P-FMV / IOsSS 11.5 85.7 54992 (CP4 AT) P-FMV / IOsSS 11.5 78.2 nk603 (control) P-OsAct1 / P-e35S: - 100

* CP4EPSPS expression is calculated as the percentage of control (603) made on plants that survived spraying with glyphosate (1.8 kg / acre (64 oz / acre)).

EXAMPLE 8

Three plasmid constructs are evaluated in transgenic cotton plants (Table 21). The control construct (pMON20999) contains

P-FMV / CP4EPSPS_Syn this expression cassette is contained in the commercially available glyphosate-tolerant 1445 line of cotton (Roundup Ready® cotton, Monsanto Co., St. Louis, MO). The plasmid constructs, pMON59313 and pMON59396 containing the polynucleotides CP4EPSPS_AT and CP4EPSPS_ZM, respectively, are tested for transformation efficiency and CP4EPSPS enzyme levels in relation to the commercial glyphosate tolerant expression cassette. About fifty lines of transgenic cotton are evaluated for each construct. The artificial polynucleotide CP4EPSPS_AT driven by the PFMV promoter gives a higher percentage of plants with a single insert and an increase in the expression level of the CP4EPSPS enzyme compared to the pMON20999 expression cassette as measured by the ELISA.

Table 21. Transformation efficiency (TE), average CP4EPSPS expression in RO cotton lines derived from the transformation of different CP4EPSPS constructs.

pMON District Attorney TE (%) CP4 Exp (%) 20999 (CP4EPSPS Syn) P-FMV 15.0 100.0 59313 (CP4EPSPS AT) P-FMV 15.0 116.4 59396 (CP4EPSPS ZM) P-FMV 16.1 52.0

EXAMPLE 9

The constructs containing the artificial CP4EPSPS polynucleotides, CP4EPSPS_AT and CP4EPSPS_ZM are evaluated on soybeans (Table 22). The plasmid constructs all contain the P-FMV promoter to direct the expression of the new CP4EPSPS polynucleotides and are compared to the P-FMV / CP4EPSPS_Syn contained in pMON20999. About 25 to 30 transgenic soy plants are produced for each construction. Transformation efficiency and CP4EPSPS enzyme levels are measured. A surprisingly high expression level of CP4EPSPS protein is measured in soybean plants containing the CP4EPSPS_ZM coding sequence (Table 22).

Table 22. Transformation efficiency (TE), average CP4 expression derived from the transformation of different CP4EPSPS constructs.

pMON District Attorney TE (%) CP4Exp (%) 20999 (CP4 Syn) P-FMV 0.55 100.0 59313 (CP4 AT) P-FMV 0.40 66.6 54996 (CP4 ZM) P-FMV 0.29 242.5

EXAMPLE 10

Tobacco cells are transformed with three plasmid constructs containing different and regenerated CP4EPSPS polynucleotide sequences in plants. About twenty transgenic strains are evaluated for each construct. The expression of each of the CP4EPSPS polynucleotides is driven by the duplicate enhancer promoter P-CaMVe35S (Table 23). The transformation efficiency and the expression level of the CP4EPSPS enzyme is measured. The CP4EPSPS polynucleotide constructs

different are shown to perform approximately the same in transgenic tobacco in terms of transformation and expression efficiency.

Table 23. Transformation efficiency (TE), expression of average CP4 in RO tobacco lines derived from the transformation of constructs of

EPSPS of different CP4.

pMON District Attorney TE (%) CP4 exp. (%) 59308 CP4EPSPS AT P-CaMVe35S 35 100.0 59309 CP4EPSPS ZM P-CaMVe35S 35 91.0 54313 CP4EPSPS_Syn P-CaMVe35S 35 100.0

EXAMPLE 11

Arabidopsis thaliana is transformed with four plasmid constructs by vacuum infiltration (Bechtold N, et al., CR Acad Sei Paris Sciences di Ia vie / life Sciences 316: 1194 to 1199, (1993) and progeny V1 evaluated to compare effectiveness of different CP4EPSPS polynucleotide sequences and different promoters for use in glyphosate plant selection (Table 24), about 30 transgenic V1 plants (+) are produced for each construct. Constructs targeted by PCaMVe35S with duplicate enhancer (pMON45313, pMON59308 and pMON59309) show no substantial difference in the level of expression in leaves as determined by the ELISA.The plants are transformed with pMON26140 which contains CP4EPSPS_Syn directed by the P-FMV promoter, these plants show the highest level of expression, the levels of expression detected from the plants of the test constructs are compared to pMON26140.

Table 24. Evaluation of different CP4 expression cassettes in Arabidopsis

pMON District Attorney/ Plants produced CP4 exp. (%) 45313 (CPEPSPS4 Syn) P-CaMVe35S + 82.1 59308 (CP4EPSPS AT) P-CaMVe35S + 79.3 59309 (CP4EPSPS ZM) P-CaMVe35S + 77.3 26140 (CP4EPSPS Syn) P-FMV + 100.0

EXAMPLE 12

Wheat plants transformed with the new CP4EPSPS polynucleotides are compared for transformation efficiency and the expression of the CP4EPSPS enzyme determined by the ELISA (Table 25). CP4EPSPS_ZM provides CPEPSPS enzyme expression at least seven times higher than CP4EPSPS_AT. The average expression of CP4EPSPS in the leaves of wheat plants containing the polynucleotide CP4EPSPS_ZM is about 64% of that found in glyphosate resistant wheat that contains a double cassette construct, pMON30139: Pe35S / l-Hsp70 / CP4EPSPS_Nat and P-OsAct1 / l-OsAct1 / CP4EPSPS_Nat (WO / 0022704).

Table 25. Performance of different CP4EPSPS polynucleotides in wheat

pMON Promoter / lntron TE (%) CP4 Exp. (%) 58400 CP4EPSPS AT P-e35S / l-Hsp70 0.25 9.2 58401 CP4EPSPS ZM P-e35S / l-Hsp70 0.35 64.0 30139 CP4EPSPS Nat P-e35S: P-OsAct1 - 100.0

EXAMPLE 13

This example serves to illustrate the detection of different artificial polynucleotides in transgenic plants, specifically CP4EPSPS_AT and CP4EPSPS_ZM. The other artificial polynucleotides, OsEPSPS_AT, OsEPSPS_ZM, GmEPSPS_GM, ZmEPSPS_ZM, CTP2_AT, CTP2_ZM, Bar1_AT and Bar1_ZM can all be specifically detected in transgenic plants by the methods that provide a DNA amplicon to a DNA sample or by hybridization of a plant sample. Those skilled in the technique of DNA detection can easily design preparatory molecules from the artificial polynucleotide sequences provided in the present invention to enable a method that will specifically detect the artificial polynucleotide in a plant sample. The use of a method or kit that provides DNA primers or probes homologous or complementary to the artificial polynucleotides described herein is an aspect of the present invention.

A method of detecting DNA (polyme chain reaction • · · · · · · · · · ·

rase, PCR) is designed to detect artificial CP4EPSPS polynucleotides in transgenic plants. The unique sets of DNA primers shown in Table 26 are designed to amplify a specific CP4EPSPS polynucleotide and to provide distinctly dimensioned amplicons. Amplicons differ sufficiently in polynucleotide length between the various CP4EPSPS polynucleotides to make separation of amplicons easy by standard agarose gel electrophoresis. The presence of more than one of the artificial polynucleotides can be detected in a plant using a multiplex PCR method.

Table 26. Sequence of primers used to detect different CP4 genes in transgenic plants.

Initiator pair Gene specificity PCR product (bps) SEQ ID NOs: 24 and 25 CP4EPSPS AT 938 (940) SEQ ID NOs: 26 and 27 CP4EPSPS ZM 595 (600) SEQ ID NOs: 28 and 29 CP4EPSPS Nat 712 (710) SEQ ID NOs: 30 and 31 CP4EPSPS Syn 443 (440)

The DNA primer pairs (Table 26) are used to produce an amplicon diagnosis for a specific CP4EPSPS polynucleotide in a transgenic plant. These primer pairs include, but are not limited to SEQ ID NO: 24 and SEQ ID NO: 25 for polynucleotide CP4EPSPS_AT; SEQ ID NO: 26 and SEQ ID NO: 27 for CP4EPSPS_ZM; SEQ ID NO: 28 and SEQ ID NO: 29 for CP4EPSPS_Nat and SEQ ID NO: 30 and SEQ ID NO: 31 for the polynucleotide molecule CP4EPSPS_Syn. In addition to these preparer pairs, any preparer pair derived from SEQ ID

NO: 17 or SEQ ID NO: 18 that when used in a DNA amplification reaction produces a diagnosis of DNA amplicon for the respective polynucleotide CP4EPSPS is an aspect of the present invention.

The amplification conditions for this analysis are illustrated in Table 27 and Table 28, however, any modification of these conditions including the use of fragments of the DNA molecules of the present invention or their complements as initiator molecules, producing a diagnosis of amplicon DNA molecule for artificial polynucleotides ···· ····

described here is within the vesatility of the technique. The DNA molecules of the present invention include at least SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO : 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 35. DNA molecules that function as primer molecules in a DNA amplification method to detect the presence of artificial polynucleotides include, but are not limited to, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31.

In a method for determining the presence of polynucleotides of the present invention, the analysis of a plant tissue DNA extract sample should include a known positive control containing the artificial polynucleotide and a negative DNA extract control from a plant that is not transgenic or it does not contain the artificial polynucleotide and a negative control that does not contain any pattern in the DNA extract.

Additional DNA primer molecules of sufficient length can be selected from SEQ ID NO: 17 and SEQ ID NO: 18 and conditions optimized for the production of an amplicon which may differ from the methods shown in Table 27 and Table 28, but result in a amplicon diagnosis for artificial polynucleotides. The use of these DNA preparation sequences homologous or complementary to SEQ ID NO: 17 and SEQ ID NO: 18 used with or without the modifications for the methods of Tables 27 and 28 are within the scope of the invention. The assay for the CP4EPSPS_AT and CP4EPSPS_ZM amplicons can be performed using a Stratagene Robocycler, MJ Engine, PerkinElmer 9700 or Eppendorf Mastercycler Gradient thermocycler as shown in Table 28 or by the methods and apparatus known to those skilled in the art.

. ··. ···; ···:.

Table 27. Procedure for DNA amplification and reaction mixture for confirmation of artificial EPSPS polynucleotide CP4EPSPS_AT in corn plants.

Stage Reagent Amount comments 1 Nuclease-free water add up to a final volume of 20 μΙ - 2 10X reaction buffer (with MgCI ₂ ) 2.0 μΙ Final concentration of 1X buffer, final concentration of 1.5 mM MgCl ₂ 3 10 mM dATP, dCTP, dGTP and dTTP solution 0.4 μΙ Final concentration of 200 μΜ of each dNTP 4 Initiator (SEQ ID NO: 24) (resuspended in 1X TE buffer or nuclease-free water at a concentration of 10 μΜ) 0.4 μΙ Final concentration of 0.2 μΜ 5 Initiator (SEQ ID NO: 25) (resuspended in 1X TE buffer or nuclease-free water at a concentration of 10 μΜ) 0.4 μΙ Final concentration of 0.2 μΜ 6 Control initiator (SEQ ID NO: 32) (resuspended in 1X TE buffer or nuclease-free water to a con- 10 μΜ centering) 0.2 μΙ Final concentration of 0.1 μΜ

100

Stage Reagent Amount comments 7 Control initiator (SEQ ID NO: 33) (resuspended in 1X TE buffer or nuclease-free water to a con- 10 μΜ centering) 0.2 μΙ Final concentration of 0.1 μΜ 8 RNase, free DNase (500 ng / μΙ) 0.1 μΙ 50 ng / reaction 9 REDTaq DNA polymerase (1 unit / μΙ) 1.0 μΙ (recommended to shake the pipettes before the next step) 1 unit / reaction 10 Extracted DNA (standard): • Samples to be analyzed • individual sheets • collated sheets (maximum 50 sheets / collation) • Negative control • Negative control • Positive control • 10 to 200 ng of genomic DNA • 200 ng of DNA genomic • 50 ng of genomic DNA from non-transgenic plant • no DNA standard • 5 ng plasmid DNA

Table 28. Suggested PCR parameters for different thermal cyclers

Gentle mixing and, if necessary (no hot top in the thermal cycler), add 1 to 2 drops of mineral oil on top of each reaction. Process with PCR in a Stratagene Robocycler, MJ Engine,

Perkin-Elmer 9700 or Eppendorf Mastercycler Gradient using the following cycling parameters.

Note: MJ Engine or Eppendorf Mastercycler Gradient thermal cyclers must be driven in the calculated mode. Drive the Perkin-Elmer 9700 thermal cycler with the lifting speed set to maximum.

101

A Λ <······· * ··· 9 · • · · · · • · · • · · · • ·: ·: · • · 9 · • · · • • · · · ·· · • · · • · · • · · · · • • · • · ζ · · · ······ * · · • ·

No. of Cycles Adjustments: Stratagene Robocycler 1 94 ° C 3 minutes 38 94 ° C 1 minute 60 ° C 1 minute 72 ° C 1 minute 30 seconds 1 72 ° C 10 minutes

N ⁹ of Cycles Settings: MJ Engine or Perkin-Elmer 9700 1 94 ° C 3 minutes 38 94 ° C 60 ° C 72 ° C 10 seconds 30 seconds 1 minute 1 72 ° C 10 minutes

No. of Cycles Settings: Eppendorf Mastercycler Gradient 1 94 ° C 3 minutes 38 94 ° C 60 ° C 72 ° C 15 seconds 15 seconds 1 minute 1 72 ° C 10 minutes

All of the compositions and methods described and claimed herein can be made and performed with consideration of the present description. Although the compositions and methods of this invention have been described, it will be apparent to those of skill in the art that variations can be applied to the compositions and methods and in the steps or sequence of steps of the methods described here without departing from the concept, spirit and scope of the invention. invention. More specifically, it will be evident that certain agents that are both chemically and physiologically related can be substituted in place of the agents described herein while the same or similar results can be obtained. All such substitutes and similar modifications evident to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined by the appended claims.

All publications and patent applications cited herein are incorporated by reference in their entirety to the same degree as if each

102 publication or individual patent application was specific and individually indicated as being incorporated by reference.

Claims (48)

1. Method to reduce the silencing of transgene in transgenic plants, characterized by the fact that it comprises the steps of: a) obtaining a DNA construct comprising an artificial polynucleotide that encodes a chloroplast transit peptide operably linked to SEQ ID NO: 18; b) transforming said DNA construct into a plant cell comprising SEQ ID NO: 16; and c) regenerate said plant cell in a transgenic plant 10 fertile. 2. Method according to claim 1, characterized by the fact that said artificial polynucleotide is expressed in said fertile transgenic plant. 3. Method according to claim 2, characterized by the fact that said artificial polynucleotide provides an agronomically useful phenotype selected from the group consisting of: herbicide tolerance and resistance to diseases. 4. Artificial polynucleotide molecule, characterized by the fact that it comprises SEQ ID NO: 18. 20 5. DNA construct, characterized by the fact that it comprises: a promoter molecule that works in plants, operably linked to the said artificial polynucleotide molecule as defined in claim 4. 6. Method of detecting an artificial polynucleotide in a ve25 getal cell, plant or its progeny that comprises an artificial polynucleotide molecule, as defined in claim 4, characterized by the fact that it comprises the steps: (a) contacting a DNA sample isolated from said plant cell, plant or its progeny with a DNA molecule, in which said 30 DNA molecule comprises at least one DNA molecule from a pair of DNA molecules that when used in a nucleic acid amplification reaction produces an amplicon that is diagnostic for that moleculePetition 870160006724, of 02/26/2016, p. 4/10 an artificial polynucleotide cuve, as defined in claim 4, wherein the DNA molecule comprises a fragment of SEQ ID NO: 18; (b) carrying out a nucleic acid amplification reaction, thereby producing amplicon; and (C) detecting the amplicon. 7. DNA detection kit, characterized by the fact that it comprises at least one DNA molecule of sufficient length to be specifically homologous or complementary to an artificial polynucleotide, as defined in claim 4, in which the DNA molecule comprises 10 a fragment of SEQ ID NO: 18 and the DNA molecule is not complementary to SEQ ID NO: 16, wherein said DNA molecule is useful as a DNA probe or DNA primer. 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CD • • You CD • ο LO σ> Ll. <c X ç CD <C X <r CD <c X <c CD 1 1 1 1 1 1 1 1 1 ^ 1 " * xt • st xt- xt xt • = 4 · O_ O_ Q_ Q_ Q_ Q_ O_ O_ O- Q_ Q_ O_ CJ CJ CJ O CJ O O O O O O CJ X X X C \ J X X X X X * = 4 · X X Ο- Ο- Ο- Q_ Ο- Ο- α- O_ Ο- Ο- Ο- Ω- Ι— Ι— Ι— P ρ- Ι— μ- Ι- Ι— Ι— Ι— μ- CJ CJ CJ O ο O ο Ο O O O ο 1> M 33/52 • · · ·: ·. ··. ····· • · ..... ····: • · ·. · • · • · • · ·· · s *· * ·. • · . · Í: • · · · · · • · · Sphl1765 Fig 6 34/52 ·· · ·: ·. ··. ····· ......: ···: ·: ·: ·. · • · • · • · • · · : · ’·. :. * ’·: ... ···· · • · EcoRI 325 Fig 7 35/52 • · · · · 1¼ Fig 8 36/52 . ·· ···! • * · · • · • · · ·: ·. ··. ·· ”· ·: · ’· • • · ; · · • · · The • · · • · · 1 · · ♦ · '·' · «·· ···· »·» * EcoRI396 indlll 3174 * Pstl 3172 CP4EPSPS AT ΡΜΟΝ59302 5413bp Ncol1992 CTP2 l-0sAct1 P-ü35SL-TaCAB rrnxc Fig 9 37/52 ···. · Ί ·· »· bYl EcoRI396 Clal 1049 NcoI1992 HindlH3174 _stl3172 Fig 1 θ 38/52 • ···· í> 5 Hindll 10 BamHI1477 Ncol1491 EcoRI3O96 Fig 11 39/52 Ou Fig 12 40/52 Hindlll458 Fig 13 41/52 lindlll 13 EcoRl399 BamH11110 Ncol 1424 Sphl 1656 pMON54964 CP4EPSPS_AT 8877bp EcoRl3020 Sphl 3457 Fig 14 42/52 1 · 8Λ Fig 15 43/52 Sphl 5629 Nhel 5872 Ncol 6672 Hindlll 8563 Sphl 197 Fig 16 44/52 Sphl 9174 Hindlll 9367 Sphl 3742 Ncol 1891 Sphl 2123 Fig ’17 1¾ 45/52 ........... • · • · · ·: ····· • · · · • ·: ’: ·. · • · • · • · * • · · • • : * ·: • · · • · · • · * ··· ·· • · • · • Ϊ ····· · Sphl 9174 Hindlll 9367 Sphl 3742 Ncol 1891 Sphl 2123 Fig 18 ·· ··. - · · 46/52 Sphl212 ^Hindl11369 Kpnl1787 EcoRI2657 KpnI2673 BamHI2675 Ncol1050 Sphl1282 Nhel1400 Fig 19 ·· ···· 47/52 Iwi Sphl507 Fig 20 48/52 ΚρηΙ8601 Fig 21> · · ·· ·· 49/52 BamHI 8591 Kpnl 8601 EcoRI 8609 Hindlll 2321 Sphl 2486 j— Sphl 1372 Ncol 1596 Fig 22 50/52 Hi Fig 23 • ·. · ···· 51/52 Fig 24 V Kpnl 159 Hindlll 161 Hindlll193 Sphl5189