MXPA01004359A - Genes and vectors for conferring herbicide resistance in plants - Google Patents

Abstract

Genomic and cDNA sequences and plant expression vectors encoding for an eukaryotic AHAS small subunit protein are disclosed. The DNA sequences and vectors are used to transform plants to produce transgenic plants which possess elevated levels of tolerance or resistance to herbicides, such as imidazolinones.

Description

GENES AND VECTORS TO CONFER HERBICIDAL RESISTANCE! IN PLANTS i REFERENCE TO RELATED REQUESTS This application claims the benefits, in accordance with the code of E.U.A. 35 §119 (e), of the provisional patent application E.U.A. Serial No. 60 / 106,239 filed on October 29, 1998.

BACKGROUND OF THE INVENTION Herbicides are extensively used in agronomy to control weeds and other unwanted plants. Due to their phytotoxicity, herbicides also eliminate or significantly inhibit desirable plant growth and development. Some plants, for example Arabidopsis, inherently possess or develop resistance to certain herbicides upon repeated exposure to herbicides with the same mode of action. There has been a goal of plant biotechnology to identify, isolate and clone plant genes that confer resistance to herbicides and use these genes to transform desirable plants such as crops to make them resistant to herbicides.

Various methods for generating or identifying herbicide-resistant plants are known, for example, U.S. Patent Nos. 5,719,046, 5,633,444 and 5,597,717 describe a plant gene resistant to sulfonamide and methods for transforming plant cells whose growth is inhibited by sulfonamides, with vectors containing this gene.US Patent No. 5,405,765 describes a method for producing transgenic wheat plants.This method consists of administering a heterologous DNA to a stem of Type C embryonic wheat in a suspension culture by an accelerated particle bombardment method US Patent No. 5,539,092 discloses polynucleotides encoding a cyanobacteria acetyl-CoA carboxylase and plants This patent describes the process for increasing resistance to herbicides of monocotyledonous plants, which comprises transforming the plant with a DNA molecule encoding a polypeptide of herbicide resistance that has the ability to catalyze the carboxylation of acetyl-CoA. The patent further discloses that the transgenic plants produced are resistant to herbicides such as arylphenoxypropionates and cyclohexanediones. The patent of E.U.A. No. 5,304,732 describes the methods for isolating herbicide-resistant plants. The patent describes the use of in vitro cell culture methods to isolate plant cell lines that are resistant to herbicides such as imidazolinones and sulfonamides. *? - ^ - ^^ 'riÜÍ? IMTAH The trait for a specific resistance to herbicides is more frequently associated with a particular enzyme. One such enzyme that has been of interest in its association to confer resistance to herbicides in plants is acetohydroxy-synthase acid ("AHAS"), also known as acetolactate synthase ("ALS," E.C. 4.1.3.18). It is an essential enzyme in plants and many microorganisms, and in most plants the enzyme is sensitive to herbicides. The AHAS enzyme catalyzes the first step in the biosynthesis of branched chain amino acids, isoleucine, leucine and valine, and its activity is allosterically inhibited by these amino acids. The AHAS activity is also inhibited by various classes of herbicides, including imidazolinone compounds such as (PURSUIT®, American Cyanamid, Parsipanny, NJ); compounds based on sulfonylureas such as methylsufromanurone (OUST®, E.l. du Pont de Nemours and Company, Wilmington, DE); triazolopyrimidine sulfonamides (Broadstrike ™, Dow EJanco, see, Gerwick et al., Pestic.Sci. 290: 357-364, 1990); sulfamoylureas (Roda \ ^ vay et al., Mechanism of Selectively of Ac 322.140 in Paddy Rice, Wheat and Barley, Proc Brighton Crop Protec. Conf., Weeds, 1993): pyrimidyl-oxy-benzoic acids (STABLE®, Kumiai Chemical Industry Co., du Pont de Nemours and Company), and sulfonylcarboxamides (Alvarado et al., US Patent No. 4,883,914). The inhibition of AHAS activity can lead to the inability of the plant to make branched amino acids or the accumulation of toxic metabolites and, therefore, plant death.

The genes encoding the AHAS enzymes have been isolated from enteric bacteria, including Escherichia coli, and Salmonella typhimurium. The Patent of E.U.A. No. 5,643,779 discloses a nucleic acid sequence encoding an α-AHAS enzyme from Lactococcus and vectors containing the same from transformed microorganisms. The transgenic microorganisms produce an improved amount of the AHAS enzyme. Japanese Patent No. JP08214882 discloses a nucleic acid sequence for a major subunit and a minor subunit of AHAS from Rhodobacter capsulatus. The gene sequences are used to transform photosynthetic microorganisms to improve the production of the AHAS enzyme for the synthesis of amino acids. In eukaryotes, a gene encoding a polypeptide homologous to the major subunit of the bacterial AHAS enzyme has been identified in the yeast Saccharomyces cerevisiae. The genes encoding the mutant major subunit of AHAS from several plants have also been isolated, cloned, and used to create transgenic plants that are resistant to herbicides. The patents of E.U.A. Nos. 5,605,011, 5,013,659, 5,141, 870 and 5,378,824 describe fragments of nucleic acids encoding a mutant ALS plant protein associated with herbicide resistance. The major subunit of the ALS mutant protein confers resistance to herbicides for sulfonylurea compounds in plants. Nucleic acid fragments encoding this mutant major subunit protein are used in vectors to transform plants that are normally sensitive to sulfonylureas herbicides. The transgenic plants resulting from said transformation are resistant to sulfonylureas herbicides. The patent of E.U.A. No. 5,633,437 describes a gene of the major subunit and an enzyme isolated from burdock, Xanthium sp, which confers resistance to several structurally unrelated classes of herbicides in plants, plant tissues and seeds. The patent describes herbicides that normally inhibit AHAS activity. The genes of the major subunit of AHAS resistant to I herbicides have also been rationally designated. WO 96/33270, the patents of E.U. A. No. 5,853,973 and 5,928,937 describe structure-based modeling methods for the preparation of AHAS variants, including those that exhibit selective increased resistance to herbicides such as imidazoline and herbicides that inhibit AHAS. This document describes isolated DNA encoding said variants, vectors containing DNA, methods for producing variable polypeptides, and herbicide-resistant plants that contain specific mutations of the AHAS gene. The AHAS enzymes of prokaryotes exist as! two different protein subunits, but physically associated. In prokaryotes, the two polypeptides, a "major subunit" and a "minor subunit", are expressed from separate genes. The major AHAS enzymes, designated I, II, and III, have all minor and major subunits, and have been _ kú * adti --- ii > α-u ** a identified in enteric bacteria. In prokaryotes, the AHAS enzyme has been shown to be a regulatory enzyme in the branched-chain amino acid biosynthetic pathway (Miflin, B.J. Arch Biochm Biophys., 146: 542-550,1971), and only the major subunit has been observed that | It has catalytic activity. From the studies of the AHAS enzymes of microbial systems, two roles have been described for the minor subunit: 1) The minor subunit is involved in the inhibition of the allosteric feedback of the catalytic major subunit when it is in the presence of isoleucine, leucine or valine or combinations thereof; 2) The minor subunit improves the activity of the major subunit in the absence of isoleucine, leucine or valine. The minor subunit has been shown to increase the stability of the active conformation of the major subunit (Weinstock et al J. Bacteriol 174: 5560-5566, 1992). Expression of the minor subunit may also increase expression of the major subunit as seen for AHAS I of E. coli (Weinstock et al., J. Bacteriol, 174: 5560-5566, 1992). In these microbial systems, the major subunit alone in vitro exhibits a basal level of activity that can not be inhibited by feedback by the amino acids isoleucine, leucine, or valine. When the minor subunit is added to the same reaction mixture containing the major subunit, the specific activity of the subunit maypr increases. The protein of the major subunit of AHAS has been identified in plants and has been isolated and used to transform plants. A miutant allele isotype of the major subunit of the AHAS protein, AHAS3, which has the tryptophan at position 557 replaced with leucine has been found in a cell line of Brassica napus (Hattori et al., Mol.; Gen. Genet 246: 419-425, 1995). The product of the mutant protein of this gene confers resistance to the cell line to sulfonylurea, imidazolinone and triazolopyridine. When expressed in transgenic plants, this mutant allele also confers resistance to these herbicides. An AHAS resistant to herbicides has also been identified, with a double mutant allele of the major subunit of Arabidopsis thaliana. { Plant 196: 64-68, 1995). The gene, csrl-4, encodes an AHAS enzyme that alters the kinetics that make it resistant to Clorsulfuron, Mazapyr, and Triazolopyromidine. When expressed in plants, the csrl-4 gene affects the growth of plants in response to the addition of L-valine and L-leucine. Until recently, there was no direct evidence that the subunit protein of AHAS existed in eukaryotic organisms. Recently, other groups, through the use of Tag expression sequences (EST), have identified sequences homologous to the minor subunit of the microbial AHAS gene in a eukaryote, the Arabidopsis plant. They showed that a randomly isolated Arabidopsis cDNA sequence had sequence homology to the sub-unit sequence of the AHAS of the microbial systems. Since then, ESTs have been described from the genes of the minor subunit of other eukaryotes such as yeast and red algae (Duggleby 1997, Gene 190: 245). Duggleby describes three EST sequences, two from Arabidopsis and one from rice, which have homology to the gene sequences of the minor subunit of prokaryotes. More recently, WO 98/37206 describes the use of a cDNA sequence of the minor subunit of ALS from Nicotiana plumbaginifolia for selection of herbicides that inhibit the holoenzyme. Until the present invention, however, the complete genomic sequence of the eukaryotic AHAS minor subunit protein had not been determined, nor had the minor subunit protein of eukaryotic AHAS been produced nor had it been isolated from Arabidopsis.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides DNA sequences that encode a biologically functional protein of the minor subunit of eukaryotic AHA and for functional variants thereof. According to the invention, the gene of the minor subunit of Arabidopsis AHAS has been cloned and sequenced. The expression vectors containing the DNA sequences encode a protein of the minor subunit of eukaryotic AHAS that is provided to transform plants. Expression vectors containing genes encoding both the protein and proteins of the major and minor subunit of AHAS are also provided. The vector can be used in methods for producing transgenic plants of interest, such as dicotyledonous and monocotyledonous plant crops, including] wheat, rye, rice, sugar cane, cotton, corn, soybean, sulfur beet, canola and the like. The transgenic plants thus produced will possess a high level of tolerance for certain herbicides, such as imidazolinones. The invention also relates to methods for constructing DNA vectors including plasmids containing the protein genes of the minor subunit of AHAS. The vectors of the invention are suitable for transforming a wide range of plants and can also be designed to contain a DNA sequence encoding a larger subunit of the AHAS protein. The vectors containing the complementary protein gene of the minor eukaryotic subunit, for example, the gene derived from Arabidopsis or corn, can be used to transform a tolerant plant to Midazolinone to improve resistance to herbicides by a secondary mechanism. In a specific embodiment of the invention, expression vectors are provided which contain an ADI ^ J sequence that encodes the proteins of the major and minor subunits of AHAS and the promoters for the major and minor subunits of AHAS as expression systems. coordinately regulated in plants. For certain monocotyledonous crops, it has been preferred to use a gene of the minor subunit of AHAS of monocots and an I promoter such as those derived from rice or corn. These genes are useful for applications involving the development of transgenic monocotyledonous plants that exhibit resistance to the herbicide midazolinone or to other herbicides that inhibit AHAS. > In one embodiment, the invention relates to a method for creating crops of transgenic plants that exhibit high levels of tolerance or resistance to the herbicide imidazolinone. The method comprises introducing a DNA construct, such as a plasmid vector containing a mutant herbicide resistance gene of the major subunit of AHAS and a gene of the minor subunit of AHAS, into a plant that is normally sensitive or partially resistant to imidazolinone. Once the vector is introduced into the plant tissue, the vectors use the endogenous mechanisms of the plant to express the major and minor subunit proteins. The increased production of the major and minor exogenous subunits of the AHAS enzyme confers improved resistance of midazolinone to the plant. This increased resistance to midazolinone results from an increase in the catalytic activity, stability, resistance to degradation or resistance to inhibition of the major subunit protein and the presence of larger amounts of the lower subunit protein in the plant. . In a preferred embodiment, the genes of the major and minor subunits of the AHAS enzyme are presented in a single plasmid which is integrated into the genome of the transformed plants, and is segregated. as a single locus for easier cross-linking of herbicide-resistant crops. In another embodiment of the invention, the DNA or vector construct comprises a gene of the AHAS subunit that is herbicides and a minor AHAS subunit protein fused within a single gene, operatively linked to a form expressing a unique promoter. The protein of the minor subunit of AHAS produced by the present vectors can also be used as a new target site! for k 10 herbicides or used in combination with the major subunit to select for putative inhibitors of the major subunit. The invention also relates to methods for using the minor subunit DNA sequences as selection tools to identify mutations of the AHAS enzyme which confers resistance to herbicides in plants. In this aspect of the invention, the organisms that coexpress the minor subunit and the major subunit of AHAS are selected from mutations that confer resistance to herbicides in plants. The mutant gene products are isolated and tested in vivo for the effects of herbicides including imidazolinones. Thus, the forms reintents to mutant herbicides are isolated.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the construction of the pUC19 plasmid plant expression vector containing genomic DNA sequences from the minor subunit of Arabidopsis AHAS of the invention. Figure 2 depicts a gene map of the minor subunit of Arabidopsis AHAS. Figure 3 depicts a plasmid expression vector, pHUWE82 of the invention, which contains the minor subunit gene of Arabidopsis AHAS without the first three codons. Figure 4 depicts a plasmid expression vector, pHUWE83 of the invention, which contains the gene of the minor subunit of AHAS of Arabidopsis minus the nucleotide sequence encoding the first 98 amino acids. Figure 5 is a bar graph showing the in vitro activity and protein stability of the major subunit of AHAS | of the AHAS enzyme from Arabidopsis in the presence of the phosphate pH regulator (MTPBS) and dithiothreitol (DTT). Figure 6 is a bar graph showing the in vitro activity of the major subunit protein of the Arabidopsis AHAS enzyme in the presence of phosphate salt pH regulator (MTPBS).

Fig. 7 is a graph showing the in vitro activity of the protein of the major subunit of the Arabidopsis AHAS enzyme in the presence of incremental amounts of bovine serum albumin (BSA). Figure 8 is a graph showing the in vitro activity of the wild-type, larger subunit protein of the AHAS enzyme in incremental amounts of the menqr subunit protein of Arabidopsis. Figure 9 is a graph showing the in vitro activity of a herbicide resistant mutant (Metí 24 His) of the Arabidopsis AHAS major subunit protein in the presence of the lower subunit protein of Arabidopsis AHAS. Figure 10 is a plant transformation vector, PHUWE67 of the invention which contains a 5.6 kb DNA fragment containing the genomic DNA of the minor subunit of AHAS. Figures 11A-11E illustrate the plant transformation (expression) vectors of the invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the cloning and sequencing of a gene of the minor subunit protein of Arabidopsis AHAS: SEQ ID NO.:1 in the sequence listing containing the nucleotide sequence of the cDNA of the subunit of AHAS of Arabidopsis The sequence of J-Att ^ - I corresponding amino acids of the encoded polypeptide of the lower AHAS subunit is shown in SEQ ID NO: 2 in the sequence listing. The genomic DNA sequence of the Arabidopsis AHAS minor subunit gene is shown in SEQ ID NO: 3 of the sequence listing. The genomic DNA sequence of the Arabidopsis AHAS minor subunit gene is shown in SEQ ID NO: 3 of the sequence listing. > The invention also relates to an isolated DNA sequence encoding an acetohydroxy-synthase eukaryotic acid, sub-unit AHAS protein. Specifically, the invention relates to a DNA sequence encoding the minor subunit protein of plant AHAS, which can be obtained from dicotyledonous plants such as Arabidopsis, or from monocotyledonous plants such as rice or corn. The gene sequences of the cloned minor subunit of Arabidopsis AHAS can be used in constructions of the DNA vector to transform plant crops which are normally sensitive or partially resistant to herbicides such as imidazolinone. The transgenic plants obtained following the transformation show increased resistance to herbicides that inhibit AHAS such as imidazolinone. The expression vector systems of the present invention can be used under suitable conditions to transform virtually any plant cell. Transformed cells can be regenerated into whole plants so that when a gene is expressed in intact plants, it imparts resistance to herbicides to the transgenic plant.

'• - "---' - *« - »- II The DNA sequence encoding the minor subunit of AHA I can be used in vectors to transform plants so that the plants created" have improved resistance to herbicides, particularly imidazolinones. . The DNA sequence encoding the minor subunit protein of AHAS can be used in vectors alone or in combination with a DNA sequence encoding the major subunit of the AHAS enzyme to confer resistance to herbicides in plants. The invention also relates to a plant expression vector comprising a eukaryotic promoter and a DNA sequence encoding a protein of the minor subunit of eukaryotic AHAS. The eukaryotic motor for use in the expression vector must be a high level expression promoter, such as the minor subunit promoter of Arabidopsis AHAS. The AHAS ijnenor subunit gene sequence preferably used in the expression vector is a DNA sequence encoding the minor subunit protein of Arabidopsis. In another embodiment, the plant expression vector comprises a promoter for the eukaryotic AHAS major subunit protein; a DNA sequence encoding the protein of the major subunit of AHAS eukaryote; a promoter for the subunit protein of the AHAS; and a DNA sequence encoding a protein of the minor subunit of AHAS eukaryote. In yet another embodiment, the plant expression vector for I expressing a heterologous AHAS gene in a plant comprises a promoter! plant and a DNA sequence encoding a fusion protein comprising a major subunit and a minor subunit of a protein AHAS eukaryote. In one embodiment, the plant expression vector comprises the promoter of the minor subunit of Arabidopsis AHAS and a sequence of DNA that encodes a protein for the minor subunit of AHA ^ of Arabidopsis In another embodiment, the plant expression vector comprises in series a promoter that is expressed in plant cells, a DNA sequence encoding a transit polypeptide of the major subunit of AHAS; a DNA sequence encoding a mature wild type protein of the major subunit of AHAS, or a variant; and a sequence of AD ^ that encodes a transcript of the adapter polypeptide; a sequence of ADÑ that encodes a mature protein of the minor subunit of AHAS eukaryote; Y a terminator plant sequence. In this embodiment, the promoter, the DNA sequence encoding the transit protein of the major subunit of AHAS and the DNA sequence encoding the mature protein of the major subunit of AHAS which are preferably derived from dicotyledonous plants, particularly of Arabidopsis. Alternatively, the The plant expression vector may comprise a promoter, a DNA sequence encoding the transit protein of the major subunit of AI ^ IAS, and a DNA sequence encoding the mature protein of the higher subunit of AHAS derived from monocotyledonous plants. such as corn.

In another embodiment, the plant expression vector comprises a promoter suitable for expression in plants, a DNA sequence encoding a fusion protein comprising a major subunit and a lower I subunit of a eukaryotic AHAS protein. In another embodiment, the plant expression vector comprises a DNA sequence to improve gene expression, such as introjnes and leader sequences. In this aspect of the invention, the plant expression vector comprises a DNA sequence to regulate the expression of the AHAS gene. The intronic sequences can also be a sequence of, 10 heterologous introns from an intron such as the Adh1 intron of maize and the first intron of the locus shrunkent. In addition, the DNA sequence for improving gene expression can be a leader sequence such as the W sequence from the tobacco mosaic virus. The invention also relates to a minor subunit protein of AHAS isolated from eukaryotes. The subunit protein of AHAS has the amino acid sequence corresponding to SEQ ID NO: 2 in the sequence listing. This protein can be purified from, for example, Arabidopsis and can be used in compositions. Also, gene I can be used to express the minor subunit protein of Arabidopsis 20 in a microbe such as E coli and purified from extracts of E. coli. The invention also relates to a method for creating a transgenic plant which is resistant to herbicides, which comprises transforming a plant with a plant expression vector comprising --t - aili - i ---- I a DNA sequence that encodes a protein of the minor subunit of AHAS eukaryote. The invention also relates to a method for imparting herbicide resistance to a plant cell, which comprises cotransforming the plant cell with a first plant expression vector comprising a first plant expression promoter, and a DNA sequence encoding the subunit of the AHAS protein and a second plant expression vector comprising a second plant expression promoter and a DNA sequence encoding the minor subunit of a eukaryotic AHAS protein. The invention further relates to a method for improving resistance to herbicides from transgenic plants expressing a gene encoding the protein of the major subunit of AHAS or a mutant or variant thereof, which comprises transforming the transgenic plant with a DNA sequence encoding the minor eukaryotic subunit of the AHAS protein or a mutant or variant thereof. The invention also relates to a method for improving the herbicidal resistance in the progeny of the plant or plants, which comprises crossing the plant somatically or sexually with a transgenic plant whose genetic complement comprises a sequence encoding a herbicide-resistant mutant of the major subunit of an eukaryotic AHAS protein, and a DNA sequence that encodes the subunit less than one i- * - G, ', ** a¡im ±! AHAS protein eukaryote; and select for those plants that exhibit resistance to herbicides. The invention also relates to transgenic plants and the production of progeny by the methods of the invention, which plants exhibit high resistance to imidazolinone and other herbicides. The invention also relates to a transgenic plant whose genetic complement comprises a gene that is expressed in plants comprising a promoter for expression in plants, a DNA sequence encoding a fusion protein comprising a major subunit and a minor subunit. of AHAS protein eukaryote and a termination sequence that works in plant cells. The invention also relates to a method for identifying mutations in plant AHAS genes that confer resistance to herbicide, which comprises the exposure of an organism to a herbicidal compound, which organism possesses a heterologous vector comprising a gene for the subunit protein minor of AHAS. In another aspect of this embodiment, the heterologous vector may comprise the minor and major subunit of AHAS. This method is also useful as a screening system to test the effects of herbicides on mutant forms of the AHAS enzyme. The invention also relates to a method for identifying mutations in the plant AHAS gene (s) which alters the allosteric feedback inhibition characteristics of the enzyme. Mutations that alter the feedback characteristics of the enzyme, whether in the genes of the minor or greater subunit of AHAS, are used to alter the levels of amino acids in plants, particularly in branched chain amino acids. The method comprises: transforming a microbial strain I I which is deficient in enzymatic activity of AHAS with a vector of! I plasmidic expression comprising a mutant of the minor subunit gene of plant AHAS. A suitable microbial strain lacking AHAS activity is MI262 from E.coli. The mutant genes of the major and minor subunit of AHAS can be randomly generated or rationally designed from protein structural models using the previous methods} described (Ott et al., J. Mol. Biol. 263: 359-368, 1996). Once the microbial strain is transformed, they are selected in minimal medium in the presence of one or more, but not of three branched chain amino acids, and then the microbial strain that grows in the minimum medium is identified. Vectors containing the minor subunit gene of AHAS can be incorporated into plant or bacterial cells using conventional recombinant DNA technology. The plants are grown from transformed plant cells and the second generation of plants can be obtained from the seeds of the transgenic plants. Alternatively, the vectors for transforming plants can be recombinant viral vectors in plants containing a pa expression gene to the proteins of the minor subunit of AHAS. In this modality, the viral vetores are able to systematically infect the plants of the harvest ^ MMM? M «M» Í¿É. HRIHÉÉ white and are able to express the protein of the minor subunit of AHAS in the host plant without altering the genome of the host. The invention also relates to the DNA sequences of the promoter of the subunit gene of AHAS. In this aspect of the invention, the promoter sequence of the minor subunit of AHAS can be used to express heterologous polypeptides. Alternatively, the promoters of the minor and major subunits of AHAS can also be used as gene systems co-ordinated to express heterologous multi-subunit proteins or to overexpress a single gene.

Identification, cloning and sequencing of the minor AHUN subunit gene The putative EST sequence of the ijnenor subunit protein of Arabidopsis thaliana, designated P 12197 in GenBank, was used to clone the gene of the entire lower subunit. AHAS The synthetic primers of the polymerase chain reaction (PCR) were specifically dissected to correspond to the DNA sequences of the minor subunit of Arabidopsis AHAS corresponding to the ESTj sequence of the minor subunit of AHAS deposited in GenBank. The primers were synthesized using standard techniques (U.S. Patent 4,683,202; Sambrook et al., Molecular Cloning, 2nd Ed., Cold Spring Harbor).

I I Reverse transcriptase (RT) -PCR was carried out on RNA isolated from Arabidopsis. The primers were designed from the EST sequence that was able to amplify a fragment of cDNA from Arabidopsis This fragment was cloned into a TA vector of Invitrogen (Invitrogen Cat. No. K2000-01) using standard techniques. This clone was named pDGR102 and corresponded to a fragment of 450 base pairs that contained a portion of the EST sequence. The same PCR primers were used to amplify a fragment from an Arabidopsis cDNA library: s1. This confirmed that the gene of the minor subunit of AHAS was present in the library. Using a specific sense strand initiator with pDGR102, and a reverse primer that hybridized with the phagemid vector? .si, a fragment representing the 3 'half of the minor subunit gene was amplified by PCR using the total cDNA library of Arabidopsis as a template source. This approximately 800 base product was cloned into the TA vector of Invitrogen (Invitrogen, Cat. No. K2000-01) and named clone pDGR106. The clone PDGR106 was also sequenced, and its sequence of translated amino acids confirmed that the fragment represented the 3 'half of the minor subunit genj by homology to gene sequences of the known minor prokaryotic subunit. This fragment contained the stop codon and the poly A co flange 3 '. It was found that the PCR fragment contained in pDGR102 and pDGR106 represents a 5 'region of the 3' half, respectively, of the minor subunit gene and its DNA sequences that overlap by approximately 188 bp. A unique site of the Ssp I restriction enzyme located in the overlap region was used to break and ligate the fragment together with the construction of the near-total AHAS minor subunit gene (a portion of the N-terminal gene was still present). missing). The ! The resulting clone was designated pDGR115. A rapid 5 'amplification of the cDNA ends (RACE ', GIBCO / BRL Cat. No. 18374-058) was used to complete the 5' end sequence of the minor subunit gene of Arabidopsis AHAS. Initiators designed from sequence pDGR115 were used to clone and extend the sequence to the 5 'end of the minor subunit gene. The RNA extracted from the seeds of Arabidopsis was used as a mold. The sequence was extended 650 base pairs and a codon of putative initiation was identified from the N-terminal residue of methionine. The established total length sequence was used to generate a full-length cDNA clone. A genomic clone of the minor subunit gene of Arabidopsis was obtained by selecting a genomic lambda genome from Arabidopsis from Clonetech. To select the library, a 380 bp probe was generated from the PCR amplification of a 5 'region of the cDNA gene sequence of the minor subunit. The PCR product was obtained by using the primers; 5'-CAGAGATCATGTGGCTAGTTGA-3 '(SEQ ID NO: 4 in the sequence listing) and 5'-GAGCGTCGAGAATACGATGTAC-3"(SEQ ID NO: 5 in the sequence listing.) The PCR product of 380 base pairs was • daifaA- m ^^ M_ ^ s? - clustering was cloned into the TA cloning vector of Invitrogen. To mark the probe on PCR insert was cut by Eco Rl and labeled with a32PdCTP by random j "primer.The selection of the library was carried out on membranes. nylon by conventional methods. A lambda phage hybridizing to the probe was identified and isolated. The phage lambda DNA was extracted and digested with Sal I and the fragments were cloned into pUC19. A specific initiator for the cDNA sequence of the minor subunit was used for the sequencing reactions with the various cloned genomic fragments of Sal I in order to identify the clone containing the minor subunit gene. HE identified a clone containing the minor subunit sequence of AHAS within a 5.6 kb fragment of Sal I and is illustrated within the plasmid pMSg6 in Figure 1. The promoter region, the transit sequence, the sequence encoding the mature minor subunit gene, the introhes and the translational terminator were identified by sequencing the 4.9 kb genomic fragment. Through the comparison of the genomic and cDNA sequences, the start codon of the N-terminal methionine was identified. The cDNA for the minor subunit gene codes for a 491 polypeptide amino acids. Figure 2 is a map of the genomic DNA sequences of the minor subunit gene of Arabidopsis AHAS. As shown in Figure 2, and referring to SEQ ID NO: 3 in the sequence state, this gene contains a promoter that extends from nucleotide number 1 to nucleotide 757. The start codon of the gene corresponds to the nucleotides 758-760. The gene contains 11 introns and 12 exons. Exon 1 extends from nucleotide 758 to nucleotide 1006. Intron 1 extends from i f nucleotide 1007 to nucleotide 1084. Exon 2 extends from nucleotide 1085 to nucleotide 1300. Intron 2 extends from nucleotide 1301 to nucleotide 1455. Exon 3 extends from nucleotide 1456 to nucleotide 1534. Intron 3 extends from nucleotide 1535 to nucleotide 1659. Exon 4 extends from nucleotide 1660 to nucleotide 1731. Intron 4 extends from. 10 nucleotide 1732 to nucleotide 2036. Exon 5 extends from nucleotide 2237 to nucleotide 2320. Intron 5 extends from nucleotide 2321 to nucleotide 2486. Exon 6 extends from nucleotide 2487 to nucleotide 2640. Intron 6 extends from nucleotide 2641 to nucleotide 2910. Exon 7 extends from the nucleotide 2911 to nucleotide 2998. Intron 7 extends from nucleotide 2999 to nucleotide 3284. Exon 8 extends from nucleotide 3385 to nucleotide 3389. Intron 8 extends from • nucleotide 3390 to nucleotide 3470. Exon 9 extends from nucleotide 3471 to nucleotide 3592. Intron 9 extends from nucleotide 3593 to nucleotide 3891. Exon 10 extends from nucleotide 3892 to nucleotide 4042. Intron 10 extends from nucleotide 4043 to nucleotide 4285. Exon 11 extends from nucleotide 4286 to nucleotide 4351. Intron 11 extends from I nucleotide 4352 to nucleotide 4647. Exon 12 extends from nucleotide 4648 to the stop codon of nucleotide 4737. The transcriptional terminator i is located in the DNA segment between the codon of pak > in nucleotide 4737 and nucleotide 4895. The amino acid sequence of the lower subunit protein of Arabidopsis AHAS encoded by the DNA sequence ran I was described above, has high homology with the amino acid sequences of the proteins of the minor subunit of AHAS from prokaryotic organisms. Homology is particularly high in conserved regions of the minor subunit sequences of AHAS in prokaryotes. As an example, the gene sequence of the minor subunit of Arabidopsis AHAS of the present invention has 42.5% of sequence sequence to that of the gel of the minor subunit of Bacillus subtilis. This indicates that the genomic clone DNA sequences were those of the minor subunit of Arabidopsis AHAS. Figure 2 also shows the location of the sequences Genbank EST with access numbers P_12197 and P_21856. Both EST sequences had previously been identified as homologous to the genes of the minor subunit of microbial AHAS, suggesting that there are two isoenzymes in Arabidopsis. The EST sequence of P_12197 was used to clone the cDNA of the minor subunit of AHAS and genomic clones of the invention.

Analyzes of the complete AHAS sequence indicate that the gene encodes two repetitive amino acid sequences with homology to the known minor subunits of AHAS. The sequence of the AHAS gene is ordered in I tandem within a single polypeptide. After comparing the gene sequences of the minor subunit with the original AHAS sequences, it was determined that the two ESTs are part of the same gene, each corresponding to similar regions within each repeated sequence. Some specific materials and methods were used for the cloning of the minor genomic subunit gene. The genomic library of Arabidopsis - Arabidopsis genomic library was obtained from Clonetech.

Construction of Plasmids Containing the AHAS Minor Subunit Gene DNA molecules containing the subunit AHAS gene comprising the nucleotide sequence in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing , or a functional variant thereof, can be inserted into a suitable heterologous expression vector system in proper orientation and correct reading frame. The numerous vector systems, such as plasmids, bacteriophage viruses and other modified viruses, can be used to practice the invention. Suitable plasmid vectors include, but are not limited to, the cloning vectors pBR322, pUC8, pUC9, pUC18, pUC19, pBI122, pKC37, pKC101 and TA. Viral vectors such as? Gt10,? Gt11 and Charon 4 can also be used.

I Construction of F1 plasmid expression vectors. F2 F3 pHUWE82. v pHUWE83 In the present invention, the cDNA sequences of the minor subunit of AHAS were inserted into an expression vector pGEX-2T or pGEX-4T-2 of E. coli obtained from Pharmacia. Five different DNA fragments containing the gene sequences of the minor subunit of AHAS were cloned into the expression vectors pGEX-2T or pGEX-4T-2. These clones were designated F1, F2, F3, pHUWE82, and pHUWE83 all differed in the amount of coding sequence contained within the expression vector. Plasmids F1, F2 and F3 contained the DNc of the minor subunit of AHAS in the expression vector pGEX-4T-2 (from E. co // 'and are described in more detail in example 2 below. the minor subunit of AHAS in the plasmids pHUWE82 and pHUWE83 was cloned in the expression vectors pGEX-2T of E coli The vector pHUWE82 contained a gene of the minor subunit AHAS of Arabidopsis of almost total length (without the first 3 amino acids). pHUWE83 was designed to express the minor subunit gene without the putative transient sequence (without the first 98 amino acids.) A map of the plasmid pHUWE82 and pHUWE83 is shown in Figures 3 and 4, respectively. minor subunit are expressed in E coli such as the glutathionetransferase / subunit fusion protein of the AHAS.After affinity purification of the fusion protein the respective proteins are degraded by thrombin. the 5 amino acids for the thrombin cleavage site, ie Leu-Val-Pro-Arg-Gly-Ser- (SEQ ID NO: 6 in the sequence listing), and the location of the II protease cleavage, maintain an additional residue of glycine and serine at the N-terminus of the protein of subunit 1 minor. The resulting protein of the minor subunit of AHAS from pHUWE82 has the N-terminal sequence Gly-Ser-lle-Ser-Val-Ser (SEQ ID NO: 7 in the sequence listing, the first 3 amino acids Met-Ala- Ala were not incorporated into the vector), and the pHUWE83 protein has the N-terminal sequence Gly-Ser-Met-lle ^ Asn-Arg (SEQ ID NO: 8 in the sequence listing; the first 98 amino acids were not incorporated inside the vector). In both pHUWE82 and pHUWE83 the sequence of N-terminal amino acids Gly-Ser are remnants of the thrombin cleavage site.

Transformation / plant expression vector constructions Numerous plant transformation vectors and methods for transforming plants are available (An, G. et al., Plant Physiol., 81: 301-305, 1986; Fry, J. et al. Plant Cell Rep. 6: 31-325, 1987; Block, M. Theor. Appl.

Genet 76: 767-774, 1998; Hinchee, et al. Stadler Genet Symp. 203212.203-212, 1990; Cousins, et al. Aust. J. Plant Physiol. 18: 481-494, 1991; Chee, P.P. and Sighton, J.L. Gene. 118: 255-260, 1992; Christou, et al. Trends Biotechnol. 10: 239-246, 1992; D'Halluin, et al, Bio Technol 10: 309-314,; 1992; Dhir, et al. Plant Physiol, 99:81, 1992; Casas et al. Proc. Nat. Acad. Sci. USA 90: 11212-11216, 1993; Chirstou, P. In Vitro Cell. Dev. Biol-Plant; 29P: 119-124, 1993; Davies, et al. Plant Cell Rep. 12-180-183, 1993; Dong, J.Z. Y Mchughen, A. Plant Sci. 91: 139-148, 1993; Franklin, C.l. and Trieu, T.N. Plant Physiol. 102: 167, 1993; Golovkin, et al. Plant Sci. 90: 42-52, 1993; Gud Chin Sci. Bull. 38: 2072-2078, 1993; Asano, et al. Plant Cell Rep. 13, 1994; Ayeres N.M. and Park, W.D. Crit. Rev. Plant Sci. 13: 219-239, 1994; Barcelo, et al. Plant. J. 5: 583-592, 1994; Becker, et al. Plant. J. 5: 299-307, 1994; Borkowska et al. Acta. Physiol. Plant 16,225-230, 1994; Christou, P. Agro. Food Ind. Hi Tech. 5: 17-27, 1994; Eapen et al. Plant Cell Rep. 13: 582-586, 1994; Hartman, et al. f 10 Bid-Technology 12: 919923, 1994; Rítala, et al. Plant Mol. Biol. 24: 317-325, 1994; Wan. Y.C. and Lemaux, P.G. Plant Physiol. 104: 3748, 1994; Weeksj et al. J. Cell Biochem? Q, 1994). The minor subunit sequences of AHAS are inserted into any of the vectors using standard techniques. The selection of the vector depends on the transformation technique preferred and the white plant species to be transformed. In a preferred embodiment, the minor subunit gene of AHAS can be cloned together with a high level expression promoter, and is constructed • can be introduced into a plant that is easily transgenic p a plant that will be transformed with a mutant allele of herbicide resistance a higher subunit protein. In this way, the effectiveness of the g ^ n of resistance to herbicide can be improved by stabilization or activation of the protein of the greater subunit. This method can be applied to any plant species; however, it is more beneficial when applied to crops Important Methodologies for constructing plant expression cassettes and the introduction of external DNA into plants are generally described in the art. For example, DNA can be introduced into plants, using tumor induction plasmid vectors (Ti). Other methods used for the administration of external DNA involve the use of PEG-mediated protoplast transformation, electroporation, microinjection needles, and biolistic or microprojectile bombardment for direct DNA acquisition. Such methods are known in the art (U.S. Patent No. 5,405,765 to Vasil et al., Bilang et al. (1991) Gene 100: 247-250; Sjcheid et al., (1991) Mol. Gen. Genet, 228: 104-112; Guerche et al., (1987) \ Plant Science 52: 111-116; Neuhause et al., (1987) Theor. Appl. Genet 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208: 1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlock et al., (1989) \ Plant P? Ys / 'o / og 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schule and Zielinski, eds.) Academic Press, Inc. (1989). The transformation method depends on the plant cell to be transformed, the stability of the vector used, the level of expression of the gene products and other parameters. The components of the expression cassette can be modified to increase the expression of the injected gene. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. DNA sequences to improve gene expression can also be used in plant expression vectors. These include the introns of the Adh1 corn, the intron 1 gene (Callis et al, Genes and Development 1: 1183-1200, 1987), and the leader sequences, (sequence W) from the tobacco mosaic virus (TMV), the chlorotic mottle virus of corn and alfalfa mosaic virus (Gallie et al., Nucleic Acid Res. 15: 8693-8711, 1987 and Skuzeski et al., Plant Molec., Biol. 15: 65-79, 199). The first intron of the locus shrunkent-? of corn, has been found to increase the expression of genes in chimeric gene constructs. The patents of E.U.A. Nos. 5,424 and 5,593,874 describe the use of specific nerons in gene expression constructs, and Gallie et al. . { Plant Physol. 106: 929-939, 1994) has also shown that introns useful for the regulation of gene expression in bases to specific tissues! To further improve or optimize expression of the subunit AHAS gene, the plant expression vectors of the invention may also contain DNA sequences containing matrix binding regions (MAR). Plant cells transformed with said modified expression system may exhibit overexpression or constitutive expression of the subunit gene of AHAS. To obtain efficient expression of the minor subunit gene of AHAS and other genes of the present invention, a promoter must be present in the expression vector. Depending on the cell system When used, any of a number of suitable promoters can be used. Suitable promoters should be plant promoters of high expression level including ubiquitin, promoter nos, the promoter of the gene of the minor subunit of ribulose biphosphate carboxylase, the promoter of the binding polypeptide of the minor subunit of chlorophyll A / B, the 35S promoter of the cauliflower mosaic virus, the promoters for the minor and major subunit of AHAS, (OCS) 3 MAS and the promoters isolated from plants and viruses vegetables. See CE. Vallejos, et al., "Localization in the Tomato Genome of DNA Restriction Fragments Containing Sequences Homologous to the f 10 (45S), the major chlorophyll A / ßbinding Polypeptide and the Ribulose Bisphosphate Carboxylase Genes", Generated / csH 2: 93-105 (1986). Another suitable promoter for plant transformation is the actin promoter. A preferred promoter of the invention is the sub-unit promoter of AHAS for use with the subunit gene sequence of less than 15 AHAS. The expression vector can then be used to transform a plant cell. Other plant tissues suitable for transformation include leaf tissues, root tissues, meristems, cultured plant cells such as callus, and protoplasts.

Use of the ael promoter of the minor subunit of AHAS. In a preferred embodiment, the promoter to be used to transcribe the gel of the minor subunit of AHAS must be a strong plant promoter. The native promoter of the subunit gene of AHAS in I plants can be used to express the protein gene of the minor subunit of AHAS in transgenic plants. The promoter of the minor subunit gene can also be used in vectors to express heterologous genes. The minor subunit promoter of AHAS can also be used in vectors in conjunction with the gene promoter of the AHAS major subunit. These promoters, which must direct the transcription of the two genes, code for two units of a single multimeric protein, which can be used with coordinated regulatory promoters. For example, both promoters can overregulate simultaneously at a specific time or in a specific tissue such as meristems. The advantage of two different but simultaneously active promoters is that they may not be susceptible to co-suppression. Co-suppression can occur when two genes of similar sequence are present within a transformed organism. Co-suppression causes the same level of silencing of gene expression. In addition, a transformation vector containing two genes regulated with the same promoter sequence can carry out recombination between similar sequences, thus inactivating one or both genes. The use of different promoters that are co-regulated can allow the expression of two genes without recombination problems, and facilitate the expression of multimeric proteins. The promoter for the subunit gene of AHAS can be used for additional purposes. First, the genes of the minor subunit I and greater code for polypeptides that work in concert and in physical contact with each other, the two genes can coordinate coordinately the expression. Having two promoters of the minor subunit and maypr can enable the expression of other multimeric proteins, or the overexpression of the same gene from different promoters. This is an advantage since the expression of two genes within the same promoter can cause problems due to the recombination of the homologous promoter sequences. If two different promoters are used, the genesj for multimeric proteins may not be expressed at the same time or in the same tissue. The coordinated regulation of heterologous promoters could solve these problems. In the second place, having both the promoter of the minor subuhity and that of the greater one provides tools to understand the gene regulation. These promoters can be linked to reporter genes to evaluate the determination of the level of coordination, tissue specificity, and coordination of subunit genes. Third, it is advantageous to express the gene of the minor subunit with its own promoter so that it is expressed at the appropriate time and in the tissue that have the greatest effect in improving resistance to herbicides. Finally, having two promoters that can be regulated in a coordinated manner provides a tool to analyze, and isolate regulatory factors that may be common to each of the promoters, these factors include the transcription factors that regulate the expression of both promoters of the minor subunit as the largest of AHAS. The promoter sequences may also have common motifs that may be involved in the coordination of the regulation of the two genes. f In addition, the role of introns in the genomic clone of the minor subunit may be involved in the co-regulation of the two promoters. The two promoters and the introns provide tools to elucidate the mechanism of coordinated regulation of the promoters.

Use of templates for the subunit gene of AHAS f 10 The genomic clone has several introns that can be used to regulate gene expression. It has been shown that introns regulate gene expression. For example, the intron 1 of the AdM corn significantly increases the expression of reporter genes in corn (Callis et al., 1987, Genes &Development 1: 1183-1200 by Cold Spring Harbor Laboratory 15 ISSN 0890-9369 / 87). The first locus shrunkent intron from maize has been shown to increase expression in chimeric gene constructs. The patents of E.U.A. Nos. 5,424,412 and 5,593,874 describe the use of specific introns to regulate gene expression. It has been seen that introns regulate the level of expression with base in specific tissue. Gallie et al. (Plant Physiol. 106: 929-939) show that the improvement of gene expression by introns is dependent on the cell type. Therefore, the introns of the subunit gene of AHAS can be used to express genes with particular emphasis on specific tissue types. It is also advantageous to express the gene of the minor subunit f with all its introns so that it is expressed at appropriate time and appropriate fabric so that it has the best effect on the improved resistance to herbicides.

Use of the expression vectors in the gene of the minor subunit of AHAS f ^ 10 Enzyme coupled. In this embodiment of the invention, the minor subunit of AHAS is translationally coupled to the major subunit via a transcript encoding an adapter polypeptide, such as polyglycine (PoliGly). The length of the polypeptide coupled to the adapter is variable. The positioning of the two AHAS subunits with respect to the polypeptide coupled to the adapter is the transit sequence of the major subunit followed by the coding sequence of the mature major subunit, the transcript of the adapter polypeptide, and the coding sequence of the mature subunit. . An alternative position involves the exchange of the mature coding sequence of the major and minor subunit around the transcript of the adapter polypeptide with the transit sequence of the minor subunit.

Enzymes coupled to improve the activity and resistance to I herbicides. It has been shown with the E. coli enzyme that the asation between the major and minor subunit is not firm. It was estimated that at a concentration of M for each subunit in E coli, the major subunit was only half asated as the active a2ß2 holoenzyme (Sella et al., 1993, J.

Bacteriology 175: 5339-5343). The highest activity is achieved with the molar excess protein of the subunit of AHAS. Since it has been determined that the AHAS enzyme is more stable and active when both subunits are asated (Weinstock et al 1992, J. Bacteriology) 174: 5560-5566, Sella et al. 1993, J. Bacteriology 175: 5339-5343) a highly active and stable enzyme can be created by fusing the two dehtro units of a single polypeptide. The selected polypeptides have been shown to work properly. Gllbert et al. expressed two synthetic oligonucleotide enzymes selected in E. coli to produce an enzyme that was functional, stable and soluble in vitro (Gilbert et al., Nature Biotechnology 16: 769-772, 1998). The expression of both the major and minor subunits of AHAS with a single polypeptide from a single gene also has advantages in producing crops resistant to transgenic herbicides. The use of a single gene to transform a harvest plant into the selection crop line is much easier and more advantageous than when two or more genes are used. Pair of fused enzymes. In this aspect of the invention, the AHAS minor subunit is positioned in the plant vector directly towards the 3 'end of the major subunit under the direction of a promoter.

^^^^^^^^^^^^^^^^^^^^^^^^^ Alternatively, the genes of the minor and major subunit of AHAS can be separated and placed under the direction of different promoters within a single construct. Two genes, one construction. In another aspect of the invention, in this expression vector, both the minor and larger subunits of AHAS are placed under the control of separate promoters, in a single plasmid construct. This enables the expression of both genes as separate entities; however, the tandem could behave in the plant progeny as a single locus. Two genes, a promoter. The corn stripe virus promoter is a bi-functional promoter capable of expressing genes in two directions. Using this promoter, gene transcription can be negated over genes encoded in opposite strands of the vector and in opposite directions. Therefore, the genes of the major and minor subunit of AHAS can be expressed from a single promoter. Two genes, two constructions. In another method, two separate vector constructions are made, each containing either the major or minor subunit under the direction of several promoters. This method requires that the plant be double transformed. I I The plant expression vectors can thus contain a suitable transcription terminator sequence towards the 3 'end of the gene sequence. A variety of transcriptional terminators are known for use in plant expression cassettes for the correct termination of gene transcription and polyadenylation of the transcript. Terminators for use in the invention include the i 35S terminator of CaMV, the terminator of the nopalinsintase, the pea fin terminator, the tml terminator, the terminators of the minor and greater subunits of AHAS. These terminators can be used in vectors for use in both monocotyledonous and dicotyledonous transformation. The gene products of the invention can also be directed towards the chloroplast. This is achieved by introducing a signal sequence that can be fused within the gene and therefore within the expression vector. The signal sequence corresponding to the amino terminus of the gene product (see Comai et al., J. Biol. Chem. 263: 15104-15109, 1998)) is fused to the 5 'end of the gene. It has been found that the AHAS subunit protein of the invention possesses a signal sequence, and therefore, this signal sequence can be used in the vectors of the invention. Other signal sequences are known for use in the invention, such as those from the 5 'end of the DNA of the major and subunit of AHAS, the CAB protein, the EPSP synthase, the GS2 protein, and the like. (Cheng &Jogendorf, J. Biol. Chem. 268: 2363-2367, 1993). The bacteria of the genus Agrobacterium can be used! to introduce external DNA and transform plant cells. Suitable species for such bacteria include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (ie strains LBA4404 or EHA 105) is particularly useful because of its well-known ability to transform plants. Another method for transforming plant cells with a heterologous gene involves propelling biologically active or inert particles into plant tissues and cells. The patents of E.U.A. Nos. 4,945,050; 5,036,006; and 5,100,792, all from Sanford et al., describe this technique. In summary, this method involves propelling inert or biologically active particles to the cells under conditions effective to penetrate the outer surface of the cell such that the vectors are incorporated into the interior of the cells. When the inert particles are used, the vector can be introduced into the cells by covering the particles with the vector containing the desired gene. Alternatively, the target cells may be surrounded by the vector such that the vector is carried within the cell by the force of the particle. Other biologically active particles include cells from Dry yeasts, dried bacteria, or bacteriophages, each containing the desired DNA, can also be propelled into the plant tissue. In addition, the vectors of the invention can be constructed to be • suitable for use in plastid transformation methods using standard techniques. The gene isolated from the minor subunit of AHAS of the present invention can be used to confer herbicide resistance to a wide variety of plant cells including monocots and dicots. Although the gene can be inserted into any plant cell within these broad classes, it is particularly useful in the cells of crop plants, such as those of rice, wheat, rye, barley, corn, carrot, sugar cane, tobacco , beans, peas, soybeans, sugar beets and sugarcane. The expression system of the present invention can be used to transform virtually any crop plant cell i under suitable conditions. Transformed cells can be regenerated to whole plants so that the genes of the minor subunit of AHAS impart or improve herbicide resistance to intact transgenic plants. As stated above, the expression system can be modified so that the herbicide resistance gene is expressed continuously or constitutively.

Use of the subunit gene of AHAS to improve resistance to herbicides in plants A plasmid can be constructed that contains both the genes of the major and minor subunit of AHAS. In this way, the two genes can be segregated as a single locus making the cross-linking of the herbicide-resistant crops much easier. Alternatively, the major and minor subunit can be fused into a single gene that is expressed from a single promoter. The fusion protein could also raise the levels of activity and resistance to herbicides. The major subunit of AHAS may be a wild type sequence (if resistance is intended in the presence of an independent minor subunit or fused subunit), or it may be a mutant major subunit that itself has some level of herbicide resistance. The presence of the subunit mehor 1) l I will improve the activity of the major subunit, 2) it will improve the resistance to herbicides of the major subunit, 3) it will increase the stability of the enzyme when it is expressed in vivo and 4) it will increase the resistance of the subunit greater than degradation. The minor subunit could in this way raise the resistance of the plant / crop to imidazolinone or other herbicides. The high resistance could allow the application and / or increase of the security of control proportions of herbicide pests without cytotoxicity to the transformed plant / crop. Ideally, the strength conferred will be able to raise the resistance to imidazolinone and other classes of herbicides without increasing, or increasing to a very small degree, the resistance to other AHAS inhibiting herbicides such as sulfonylureas, triazolopyrimidines, etc.

Additional aspects of herbicide resistance improved by the addition of the minor subunit gene to plants expressing a herbicide-resistant form of the major subunit gene. It has been seen in many cases that mutations in proteins can cause instability, decreased activity, and increased susceptibility to degradation. AHAS genes of resistance to herbicides, particularly those of plants, generally contain an I I I mutation that confers resistance to inhibition by the herbicide. This takes a great! level of importance in stabilizing, maintaining activity, and resistance to the degradation of these proteins. Accompaniment of the gene of the minor subunit with the gene of the major subunit can assist in these areas of susceptibility. Subunits of multisubunit proteins that I are present in the absence or absence of stoichiometric levels of the complementary subunits may preferably be degraded. Eeito has been shown for the major and minor subunits of ribulose biphosphate carboxylase / oxygenase (Spreitzer et al Proc Nati Acad Sci USA 82: 5460-5464). If the subunit gene greater for AHAS is transformed and expressed in a crop that does not have the minor complementary subunit, or which is expressed at high levels beyond the expression levels of the minor subunit gene, the protein of the major subunit may be unstable and preferably degrade. This could result in a lower level of resistance to herbicides. The association of the major and minor subunit seems to be highly specific. E. coli has three isoenzymes of the major subunit and three isoenzymes of the minor subunit. Each isoenzyme of the major subunit is specifically associated only with one of the subunits1 of the minor isoenzyme, even when all subunits are expressed in the same organism (Weinstrock et al 1992, J. Bacteriology, 174: 5560-5566).

This specificity suggests that the subunit protein AHAS At-endogenous may not stabilize or enhance activity for a greater subunit of AHAS introduced if the gene of the larger subunit is derived from different organisms or isoenzyme pairs of the minor subunit. This | It is important, for purposes of herbicide resistance, to introduce both the genes of the greater subunit as of the minor from the same organism and pair isoenzyme. The expression of a minor subunit gene can improve these problems. The minor subunit gene of AHAS in combination with the gene of the major subunit of AHAS can also be used as a f-10 marker to select transformed plant cells and tissues. Any gene of interest can be incorporated into vectors containing the genes of the minor and major subunit of AHAS. These vectors can be introduced into plant cells or tissues that are susceptible to herbicides that inhibit AHAS. Transformants containing these vectors can be selected in the presence of herbicides using standard techniques.

EXAMPLE 1 • DNA and lambda DNA isolation: 20 DNA isolation was carried out using a QIAgen equipment Spin Miniprep (50) (QIAgen Cat. No. 27104) and the standard procedures that are provided by the manufacturer. For lamba DNA isolation, the QIAgen Lambda Midi (25) kit (QIAgen Cat. No. 12543) was used following the! manufacturer's protocol. For TA cloning, the original Invltrogen i TA cloning kit (Invitrogen Cat No. K2000-01) was used and the standard protocols were followed. ! • 5 Subcloning: The lambda DNA was digested with the appropriate restriction enzymes and mixed with pUC19 with which it was digested with the same restriction eijizime. After extraction with phenol, the insert was ligated with pUC19 by adding 1 μl of llgasa DNA (4 units / mL) and incubated at 17 ° C. • 10 all night.

RACE 5 ': The RACE 5' that was used in the experiments was the system for rapid amplification of the RACE 5 'cDNA end which was obtained from GIBCO / BRL (Cat. No. 18374-058). The reactions were carried out following the standard procedure provided by the manufacturers.

• Selection library: The genomic lambda library of Arabidopsis from Clontech is sowed at a density of 30,000 plates / 150 mm dish as described in the protocol supplemented by the manufacturer. The nucleic acid transfer membranes Hybond ™ -N + from Amersham (disk: 0.137 m DAY, removal rate: 0.45 um). The transfer membranes of I I I nylon were carefully placed on the surface of the dish, and the membranes and the agar were labeled using a sterile needle. The first membranes were removed after three minutes and the duplicated membrane was placed on the surface of the dish and removed after 8 minutes. The membranes were placed, with the colonies upwards, on a tissue of absorbent paper filter soaked in denaturing buffer (f LAOH I 0.5N, NaCl 1.5N) for 5 minutes, then each membrane was placed, with the colony up, on absorbent filter paper cloth soaked in neutralizing solution (Tris-HC1 1 M, NaCl 1.5N) for 5 minutes.] The membranes were briefly washed in 2X SSC, and transferred to a dry filter paper. The samples were fixed to the membrane by crosslinking by I UV and baked under vacuum at 80 ° C for one hour. The membranes were then prehybridized in a buffer containing 50% formamide, 2X SSC, 5X Denhardt's solution, 1% sodium dodecylsulfate (SDS), 0.05 mg / ml denatured salmon sperm DNA, and 0.05% NaPPi. at 42 ° C for 2 hours. The DNA was digested with restriction enzyme and fractionated on a 1% agarose gel. The DNA fragment containing the minor subunit gene of AHAS was purified using a QIAquick gel extraction kit (50) (QIAquick Cat. No. 28704). The DNA marking system of Random Primers GIBCO / BRL Life Technologies (Cat. No. 18187-013) was used to label the DNA with the following modification. 125 ng of the DNA probe was dissolved in 55 μL of distilled water in a microcentrifuge tube and denatured on heating for 5 minutes in a boiling water bath, and ^^^^^ immediately cooled on ice. Then, dATP, dGTP, dTTP, [a-32P] dCTP and Klenow were added to the denatured DNA, and the mixture was incubated at 25 ° C for one hour. A volume of formamide f was added to the mixture. and the reaction was heated to 65 ° C for 30 minutes. The reaction that contained The labeled DNA was added to the prehybridization solution and the membrane was hybridized at 42 ° C for 20 hours with slow agitation. After hybridization, the membranes were washed twice with 0.4X SSC buffer containing 1% SDS at room temperature for 10 minutes, followed by a single wash in 0.2X SSC buffer containing 0.1% SDS at 65 ° C. 30 or 10 minutes. The membranes were exposed to an X-ray film overnight. Plates containing DNA that hybridized to the DNA probe on the membrane duplicates yielded a positive result, and these plates were isolated. The procedure doubled until a single isolate could be collected. Sequencing: A The sequencing reaction was carried out using the ABI PRISM DNA sequencing kit following the protocol provided by ABI. After precipitation of ethanol, the DNA was dissolved in ABI PRISM template suppressive reagent and denatured at 90 ° C for 5 minutes. Then, the samples were loaded onto an ABI 310 sequencer.

EXAMPLE 2 Preparation of plasmid DNA containing the protein genes of the greater subunit of AHAS The genes of the wild type major subunit (pAC774) and the Met92His mutant (pAC786) of AHAS were constructed from Arabidopsis on pGEX-4T-2 Expression Vector from Pharmacia E. coli The genes were constructed to express a glutathionetransferase / subunit fusion protein greater than AHAS to aid in purification, similarly as described for the plasmids pHUWES82 and pHUWE83 as described above. A five amino acid protease cleavage site was encoded at the junction of the two proteins so that they could break after purification! This vector construct contained the cDNA sequences for the minor subunit gene of Arabidopsis AHAS that were made using the PGEX-4T-2 expression vector. These were designated F1, F2 and F3, and all three differed in the amount of peptide sequence they contained within the gene. The N-terminal amino acid sequence of the peptide encoded by the ADI ^ c of AHAS of the F1 plasmid is Gly-Ser-Pro-Lys-lle-Ala-Leu-Arg- (SEQ ID NO: 9 in the sequence listing). The N-terminal amino acid sequence of the peptide encoded by the AHAS cDNA of the F2 peptide is Gly-Ser-Leu-Asp-Ala-HIs-Trp- (SEQ NO: 10 in the sequence listing). The N-terminal amino acid sequence of the peptide encoded by the AHAS cDNA of plasmid F3 is '-', ~ - * »*«? * Gly-Ser-Val-Glu-Pro-Phe-Phe- (SEQ ID NO: 11 in the sequence listing). The N-terminal Gly-Ser for all three peptides are remanentf from the thrombin break. i Expression of the proteins of the minor and large subunit of AHAS of Arabidopsis. The DH5a competent cells (Gibco BRL) were transfused using the plasmids of the major subunits pAC774 and pAC786, as well as the plasmids of the minor subunits F1, F2 and F3. The cells were thawed on ice. 1 μl of a 1: 5 dilution of the plasmid DNA was added to 75 μl of cells, which were placed on ice for 30 minutes. The cells were made a thermal shock at 42 ° C in a bath leave for 90 seconds and then placed on ice for two minutes. 800 μl of Luria-Bertani medium (LB) was added to each tube containing the transformed cells which then grew for one hour in a shaker at 37 ° C. the cells were centrifuged for 2 minutes and the excess medium was aspirated. The cells in the pellet were resuspended in 100 μl of LB and seeded on LB containing 100 μg of carbenicillin overnight at 37 ° C. Single colonies were inoculated into 50 ml of LB medium containing 375 μg / ml carbenicillin and grown overnight in a shaker at 37 ° C. The aliquots of 700 μl were taken and added to 300 μl of 50% glycerol, and then frozen in liquid nitrogen and kept at -80 ° C for cellular storage. - * '--- - - • • - - - • - • »***» - ---' ^ ^^^ u ^^ - Mrito.

I Purification of the AHAS gene from the major subunit i An overnight culture of 50 ml of transformed E. coli containing the gene of the major subunit in the expression vector PGEX-2T was inoculated within one liter of YT 2X with 2% glucose, 375 μg / ijnl of carbenicillin. The cells were grown for 5 hours in an incubator / shaker to 37 ° C and then they were induced with mT IPTG and then a stirrer was placed at 30 ° C for another 2.5 hours. The cells were harvested by centrifugation in a JA10 rotor at 8000 rpm at 4 ° C for 10 minutes. The cells were stored as a tablet at -20 ° C until purification. f 10 The cellular tablet of 1 liter cell culture was resuspended in ml of MTPBS (150 mM NaCl, 16 mM Na2HP04, 4 mM NaH2P04), p.H 7.3 (Smith and Johnson Gene 67: 31-40, 1998) and 100 μg /? l of lysozyme. The suspension was adjusted to 5 mM dithiothreitol. Triton X- | 100 was added to a final concentration of 1% by the addition of 20% Triton X-100 in MTPBS solution. The cells were gently shaken at 30 ° C for 15 minutes and then cooled to 4 ° C on ice and sonicated by 8-10.

To seconds using a microtip probe, with a 70% cycle, maximum output control for the microtip pulsor. The sonicate was centrifuged twice in a J20 rotor, 4 ° C 17,000 rpm, 10 minutes, to remove the insoluble material. The lysate was added to 150 mg (dry weight) of agarose glutathione (balanced and hydrated in MTPBS) and inverted for 30 minutes at 4 ° C. The agarose was settled by centrifugation at 500 rpm for 5 minutes and washed with ice-cooled MTPBS by repeated centrifugation cycles until A2β or washing reached that of MTPBS. The agarose was transferred to a suitable column for elution. The fusion proteins were eluted with 50 mM I Tris-HCl, 5 mM reduced glutathione in 1 ml fractions. The A28o for Each fraction was checked for protein content and fractions appropriate (A28 or 0.100) were grouped. To the pooled samples were added 5 units of bovine thrombin per milliliter of projtein solution. I The sample was dialyzed against MTPBS, 3 mM dithiothreitol for 15 hours at room temperature to allow proteolytic cleavage of the prostate! of fusion and the removal of Tris-HCl and the reduction of glutathione. The dialyzed sample A 10 was passed twice more through a balanced glutathione agarose column to remove the protein fragmented by GST and to remove the non-fragmented fusion protein. The purified samples were stored at 4 ° C with or without 0.02% sodium azide.

Purification of the protein from the minor subunit of AHAS The transformed DH5 cells were cultured and harvested from Jfc similar to those used for the collected cells that express the major subunit of AHAS. The cell pellet from one liter of culture was resuspended in 10 ml of STE (150 mM NaCl, 10 mM Tris-HCl pH 8.0, EDTA 1 mM), and then 100 μg / ml lysozyme was added. This was put on ice for 15 minutes. The dithiothreitol was added at 5mM and the N-I lauroyl sarcosinate was added to a final concentration of 1.5% for a 10% storage solution in STE. The cells were mixed for 10 seconds.

The lysate is sonicated on ice for 8-10 seconds using a microtip probe. With a 70% cycle, with maximum output control for the microtip pulsor. The sonicate was centrifuged twice in a JA20 I rotor at 4 ° C at 17,000 rpm for 10 minutes, to remove the insoluble material. To the supernatant were added 200 mg (dry weight) of agarose glutathione (balanced and hydrated in MTPBS) and inverted for 20 minutes at 4 ° C. The agarose was settled by centrifugation at 500 rpm for 5 minutes, and the supernatant was aspirated. The agarose was washed with MTPBS cooled in ice by repeated centrifugation cycles until the A28o wash was equal to that of MTPBS. The agarose was transferred to an appropriate column for elution. The fusion proteins were eluted with 50 mM Tris-HCl pH 8.0 reduced glutathione 10 mM, 5 mM dithiothreitol, and 2% N-octylglucoside, and the 1 ml fractions were collected. The absorbance was checked at 280 nmi for each fraction, and the appropriate fractions were pooled (A28 or 0.100). The sample was dialysed for 15 hours against MTPBS, 3 mM dithiothreitol, at 4 ° C to remove the reduced glutathione, Tris-HCl, and N-octylglucoside. The SDS was added at 0.005% to form a 1% storage solution in H20. Five units of bovine thrombin were added per ml of protein solution, and the sample was gently stirred at room temperature for 30-45 minutes to allow proteolytic cleavage of the fusion protein. The sample was immediately passed through a column of affinity detergent EtractiGel-D (Pierce) to remove SDS. The fragmented sample was stored at 4 ° C or passed twice through a re-balanced glutathione agarose to remove GST and uncut fusion protein. The purified sample was stored at 4 ° C.

Determination of protein concentration An aliquot of protein solution was adjusted to 5% trichloroacetic acid and placed on ice for 20 minutes to allow precipitation of the protein. The aliquot was centrifuged for 10 minutes at high speed and 4 ° C to concentrate the precipitate. The pellet was resuspended in an equivalent volume of 3% sodium carbonate (w / v), 0.1 N NaOH. A Pierce BCA protein assay was used to determine the concentration of the protein solution. The bovine serum albumin standard was prepared in 3% sodium carbonate, 0.1 N NaOH.

AHAS assays. The AHAS assays were carried out with slight modification as described by Singh er al. . { Singh et al., 1988.}. .

AHAS activity was assayed in AHAS 1X assay buffer (HEPES 50 Mm pH 7.0, 100 mM pyruvate, 10 mM MgCl 2, 1 mM TPP, and 50 μM FAD). A final concentration was obtained for the 1X assay buffer either by diluting the enzyme extraction in 2X assay buffer or adding enzyme to make a final concentration of AHAS 1X assay buffer. All the assays containing mazetapyr and the associated controls contained a final concentration of 5% DMSO due to the addition of imazetapyr to assay the mixtures as an I-solution. 50% DMSO. The tests were carried out in a final volume of 250 μL at 37 ° C in microtitre plates.

EXAMPLE 3 Effects of dithiothreitol The effect of dithiothreitol (DTT) on phosphate buffer salt (MTPBS) on the enzyme activity of the large subunit of AHAS was measured. The experiments were carried out with or without 3 mM DTT in the purified protein solution of the major subunit. The tests were conducted as previously mentioned. The results are presented in figures 5 and 6. As can be seen in figure 5 when conjugated with figure 6, the catalytic activity of the greater subunit of AHAS is dramatically improved by DTT. Furthermore, in the absence of DTT, the activity of the major subunit degrades over a period of 4 days (Figure 6). The major subunit of Arabidopsis was found to be very stable in the presence of 3 mM DTT over a period of one month when it was stored at 4 ° C. Other sulfhydryl reducers were tested, but DTT appeared to stabilize and activate the enzyme better than glutathione reductase and β-mercaptoethanol. Due to the activation of the enzyme by DTT all the experiments directed to the activation of the enzyme of the subunit greater than AHAS by the enzyme of the minor subunit were carried out either in the absence of DTT or other reducing agents of the enzyme. sulfhydryl or at a constant concentration of 3 mM DTT.

Effects on bovine serum albumin. To determine whether the improvement of the subunit may r was specific to the minor subunit, another non-specific control was run to test the effects of bovine serum albumin. To fix a larger subunit amount, an incremental amount of one sojoution of 0.25 mg / ml BSA was added. The results are shown in Figure 7. As seen in Figure 7, the addition of BSA to the test sample caused a slight, but not significant, increase in the plateau in the catalytic activity of the prptein of the higher subunit of AHAS. .

EXAMPLE 4 The following experiments use the purified proteins of the minor and major subunit as described in the aforementioned example 2. Plasmid F1 containing the gene of the minor subunit of Arabidopsis AHAS was expressed in E coli and partially purified. The protein concentration of the sample was determined and aliquots of increasing concentration were added to a constant amount of the purified major subunit of Arabidopsis.

. ^. Ma ^ AMaM-í *.

Figure I shows the activation of the major subunit of wild-type Arabidopsis AHAS by the addition of the γ protein of the minor subunit of Arabidopsis. The AHAS assay was carried out as described above and the results are shown in Figure 8 which indicates that the protein of the minor subunit improves the level of enzyme activity of the major catalytic subunit. The activation of the subunit protein greater than AHAS is shown in both the wild type of the major subunit and in the herbicide-resistant mutant of the major subunit. In another experiment, a herbicide-resistant mutant of the major subunit enzyme (substitution mutation at position 124, methionine substituted for histidine) was used. The results shown in Figure 9 demonstrate that the enzyme activity is also improved in a herbicide-resistant form of the major subunit. '15 EXAMPLE 5 • The plant transformation vector, pHUWE67 illustrated in Figure 10 contains the genomic DNA of the minor subunit of AHAS 20 Arabidopsis and was constructed as follows. The pUC19 vector shown in Figure 1 containing 5.6 kb genomic fragment of the minor subunit gene of AHAS Arabidopsis (pMSg6) was cut with Sal I. The 5.6 kb fragment containing the gene of the minor subunit of Arabidopsis AHAS (see figure 2), includes the promoter and introns that were separated from the vector by agarose gel electrophoresis. The fragment was cut from the agarose gel and purified using QIAquick gel extraction equipment (Cat. No. 213706) following the procedures provided by the manufacturer. The Agrobacterium-based transformation vector, pBIN19, was cut with Sal I. The vector was purified with extractions of phenol chloroform. The purified vector was dephosphorylated by treatment with calf intestinal alkaline phosphatase and re-extracted with phenol chloroform. The vector and the genomic insert were ligated and the ligation mixture containing the construct was used to transform E coli strain DH5a. E. coli was selected on kanamycin boxes and the plasmids were extracted from transformed E. coli. The construction of the vector was verified by generation of a PCR product and sequencing of the product using the sequencing procedures described in example i. 1. The vector designated pHUWE67 (FIG. 10) thus contained a 5.6 kb fragment comprising the AHAS genomic DNA containing the AHAS promoter, the open reading frame (ORF), and the 3 'terminator fused to the plasmid pBIN19. This vector construct was used for the transformation of plants based on Agrobacterium using standard techniques. The plant transformation vectors illustrated in Figures 11A-11 E are similarly constructed as the aforementioned vector pHUWE67, following standard cloning procedures. In Figures 11A-11E, the vector may comprise a cDNA of the minor subunit of AHAS, fragment, genomic fragment or mutant. In Figure 1 (1B) the vector further comprises a promoter of the minor subunit of AHAS II operably linked to the 5 'end of the gene insert In this embodiment, the minor subunit gene of Arabidopsis includes the terminator. In Figure 11C, the construction of the vector contains the genes of the major and minor subunit of AHAS in tandem, with the gene of the subunit greater towards the 3 'end from the gene of the minor subunit, both I genes are regulated by the promoter of the minor subunit of AHAs which is located towards the 5 'end of the insert of the gene The gene of the major subunit of AHAS is the mutant allele resistant to herbicide In figure 11 D the plant expression vector contains genes of the minor and major subunit under the control of their own promoter Transcription termination signals are provided by the terminator of the minor subunit of AHAS The gene of the greater subunit of AHAS confers resistance to herbi such as imidazolinone. The vector in Figure 11 E is similar to the vector shown in Figure 11C. in this vector, however, the genes of the minor subunit and i major AHAS are in a reverse position with respect to the construction. The genes of the AHAS major subunit in this example are towards the 5 'end of the minor subunit gene. The gene for the AHAS major subunit is an I I mutant allele resistant to herbicides. • ttÉÜl-É ^ -ihÉÉ ^ -tlI I I Based on the techniques of the present invention and following the techniques of severe hybridization for DNA, the sequences of the gene of the minor subunit homologous to AHAS can be obtained from a variety of plant species, such as rice, corn , wheat, rye and similar II. Therefore, these AHAS menpr subunit gene sequences are also useful in the present vectors and in the methods! to transform plants.

LISTING DB SEQUENCES < 110 > Kakefuda, Genichi Costello, Colleen I Sun, Ming 'Hu, eiming, < 120 > GENES AND VECTORS TO CONFER RESISTANCE TO HERBICIDES RESISTANCE IN PLANTS < 130 > 008103/195497 < 140 > 09 / 426,568 < 141 > 1999-10-22 < 150 > 60 / 106,239 < 151 > 1998-10-29 < 160 > 11 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 1673 < 212 > DNA < 213 > Arabidopsis sp. < 220 > < 221 > CDS < 222 > (42) .. (1514) '< 223 > Peptide Maduro < 400 > 1 gtcttcttca gtagcaaaaa accttcggct tcgtctcgtc to atg gcg gcc att tet 56 Met Ala Ala lie Ser 1 5 gta agt tet tca cea tet att cgc tgc ttg aga tcg gca tgt tec gat 104 Val Ser Ser Ser Pro lie lie Arg Cys Leu Arg Ser Ala Cys Be Asp 10 15 20 tet tet cct gct ctt gta tec tcg acg cgt gta tcg tcc gcg aagí 152 Ser Ser Pro Ala Leu Val Ser Ser Thr Arg Val Ser Phe Pro Wing Lysl 25 30 35 att tca tat etc tec ggt ata tet tcg cgt ggc gat gaa atg ggt 200 Be Ser Tyr Leu Be Gly lie Ser Gly Gly Asp Glu Met Gl 40 45 50 aag aga aga gaga gga ttc gtt aga gtc gat ggg aag atc tet gat 248 Lys Arg Met Glu Gly Phe Val Arg Ser Val Asp Gly Lys lie Ser Aspi 55 60 65 gcg tet ttc tec gaa gct tca tet gcg act cea aaa tcg aag gtg agg 296 Wing Ser Phe Ser Glu Wing Ser Ser Wing Thr Pro Lys Ser Lys Val Arg, 70 75 80 85 | aag falls here att tca gta ttt gtt gga gac gaa age gga atg att aat 344 Lys His Thr lie Ser Val Phe Val Gly Asp Glu Be Gly Met lie Asnl 90 95 100 'agg att gca gga gtg ttt gca agg aga gga tac aat att gag agt ctti 392 Arg lie Wing Gly Val Phe Wing Arg Arg Gly Tyr Asn lie Glu Ser Leu 105 105 115 gct gtt ggt ctg aac aga gac aag gct cta ttc acc ata gtt gtc tgti 440 Wing Val Gly Leu Asn Arg Asp Lys Wing Leu Phe Thr lie Val Val Cys! 120 125 130 gga act gaa agg gta ctt cag cag gtc atc gag caa etc cag aag ctci 488 Gly Thr Glu Arg Val Leu Gln Gln Val lie Glu Gln Leu Gln Lys Leu 135 140 145 gtt aat gtt cta aag gtt gaa gat atc tca agt gag ccg ca gtg gagí 536 Val Asn Val Leu Lys Val Glu Asp lie Ser Glu Pro Gln Val Glu 150 150 160 165 cgt gag ctg atg ctt gta aaa gtg aat gca cat cea gaa tec agg gcaí 584 Arg Glu Leu Met Leu Val Lys Val Asn Ala His Pro Glu Ser Arg Ala¡ 170 175 180 gag atc tg tgg cta gtt gac here ttc aga gca aga gtt gta gat atai 632 Glu lie Met Trp Leu Val Asp Thr Phe Arg Wing Arg Val Val Asp lie 185 190 195 gcg gaa cat gca ttg act atc gag gta act gga gat cct gga aaa atg¡ 680 Wing Glu His Wing Leu Thr lie Glu Val Thr Gly Asp Pro Gly Lys Met 200 205 210 att gct gta gaa aga aat ttg aaa aag ttt cag atc aga gag att gtaj 728 lie Wing Val Glu Arg Asn Leu Lys Lys Phe Gln lie Arg Glu lie Val1 215 220 225 agg here gga aag ata gca ctg aga agg gaa aag atg ggt gca act gct (776 Arg Thr Gly Lys lie Ala Leu Arg Arg Glu Lys Met Gly Ala Thr Ala 230 235 240 245 cea ttt tgg tga ttt tca gca gca tec tat cea gat etc aag gag caa, 824 Pro Phe Trp Arg Phe Ser Ala Ala Ser Tyr Pro Asu Leu Lys Glu Gln 250 255 260 gcg cct gtt agt gtt ctt cga agt age aaa aaa gga gcc att gtc cct1 872 Ala Pro Val Ser Val Leu Arg Ser Ser Lys Lys Gly Ala lie Val Pro 265 270 275 caa aag gaa here tca gca ggg gga gat gtt tat ccc gtt gag cea ttti 920 Gln Lys Glu Thr Ser Wing Gly Gly Asp Val Tyr Pro Val Glu Pro Phe 280 285 290 ttt gac ccc aag gta cat cgt att etc gac gct falls tgg gga ctt ctci 968 Phe Asp Pro Lys Val His Arg lie Leu Asp Ala His Trp Gly Leu Leu 295 300 305 ^^^ ¡and ^ aii ^ ÉÁÉtfMÍi. aag falls here att tca gta ttt gtt gga gac gaa age gga atg att aat 344 Lys His Thr lie Ser Val Phe Val Gly Asp Glu Ser Gly Met lie Asn 90 95 100 agg att gca gga gtg ttt gca agg aga gga tac aat att gag agt ctt 392 Arg lie Wing Gly Val Phe Wing Arg Arg Gly Tyr Asn lie Glu Ser Leu 105 110 115 gct gtt ggt ctg aac aga gac aag gct cta ttc acc ata gtt gtc tgt 440 Wing Val Gly Leu Asn Arg Asp Lys Ala Leu Phe Thr lie Val Val Cys 120 125 130 gga act gaa agg gta ctt cag cag gtc atc gag caa etc cag aag etc 488 Gly Thr Glu Arg Val Leu Gln Gln Val lie Glu Gln Leu Gln Lys Leu 135 140 145 gtt aat gtt cta aag gtt gaa gat atc tca agt gag ccg ca gtg gag 536 Val Asn Val Leu Lys Val Glu Asp lie Ser Glu Pro Gln Val Glu 150 155 160 1651 cgt gag ctg atg ctt gta aaa gtg aat gca cat cea gaa tec agg gca 584 Arg Glu Leu Met Leu Val Lys Val Asn Ala His Pro Glu Ser Arg Ala f 10 170 175 180 gag atc atg tgg cta gtt gac here ttc aga gca aga gtt gta gat ata 632 Glu lie Met Trp Leu Val Asp Thr Phe Arg Ala Arg Val Val Asp lie 185 190 195 gcg gaa ca t gg ttg act atc gag gta act gga gat cct gga aaa atg 680 Wing Glu His Wing Leu Thr lie Glu Val Thr Gly Asp Pro Gly Lys Met 200 205 210 att gct gta gaa aga aat ttg aaa aag ttt cag atc aga gag att gta 728 lie Wing Val Glu Arg Asn Leu Lys Lys Phe Gln lie Arg Glu lie Valj 15 215 220 225 agg here gga aag ata gca ctg aga agg gaa aag atg ggt gca act gct 776 Arg Thr Gly Lys lie Ala Leu Arg Arg Glu Lys Met Gly Ala Thr Ala1 230 235 240 245 • cea ttt tgg cga ttt tca gca gca tec tat cea gat etc aag gag caa 824 Pro Phe Trp Arg Phe Ser Ala Ala Ser Tyr Pro Asp Leu Lys Glu Glni 250 255 260 gcg cct gtt agt gtt ctt cga agt age aaa aaa gga gcc att gtc cct | 872 Wing Pro Val Ser Val Leu Arg Ser Ser Lys Lys Gly Ala lie Val Pro] 20 265 270 275 caa aag gaa here tca gca ggg gga gat gtt tat ccc gtt gag cea ttt 920 Gln Lys Glu Thr Ser Wing Gly Gly Asp Val Tyr Pro Val Glu Pro Phe 280 285 290 ttt gac ccc aag gta cat cgt att etc gac gct drops tgg gga ctt etc 968 Phe Asp Pro Lys Val His Arg lie Leu Asp Ala His Trp Gly Leu Leu 295 300 305 I act gac gaa gat acg agt gga cta cgg tcg cat act cta tca ttg ctt; 1016 Thr Asp Glu Asp Thr Ser Gly Leu Arg Ser His Thr Leu Ser Leu Leu 310 315 320 325 gta aat gat att cea gga gtt ctt aat att gtg act ggt gtt ttc gct: 1064 Val Asn Asp Lie Pro Gly Val Leu Asn lie Val Thr Gly Val Phe Ala1 330 335 340 j cga agg gga tac aat atc cag age ttg gcc gta gga cat gct gaa acc 1112 • Arg Arg Gly Tyr Asn lie Gln Ser Leu Wing Val Gly His Wing Glu Thr 345 350 355 aag ggc att tca cgc att here gtt ata cct gca here gat gaa tcg 1160 Lys Gly lie Ser Arg lie Thr Thr Val lie Pro Ala Thr Asp Glu Ser 360 365 370 gtc age aaa ttg gtg cag ca ta ctt tac aaa etc gta gat gtg cat gag 1208 Val Ser Lys Leu Val Gln Gln Leu Tyr Lys Leu Val Asp Val His Glu 375 380 385 gtc cat gat ctt act cat ttg cea ttt tet gaa aga gaa ctg atg ctg 1256 Val His Asp Leu Thr His Leu Pro Phe Ser Glu Arg Glu Leu Met Leu 390 395 400 405 • 10 att aag att gcc gtg aac gct gct gct aga aga gat gtc ctg gac att 1304 lie Lys lie Wing Val Asn Wing Wing Wing Arg Arg Asp Val Leu Asp lie 410 415 420 gct agt att ttc agg gct aaa gct gtt gac gta tet gat fall here att 1352 Ala Ser lie Phe Arg Ala Lys Ala Val Asp Val Ser Asp His Thr He 425 430 435 act ttg cag ctt act ggg gat cta gac aag atg gtt gca ctg caa agg 1400 Thr Leu Gln Leu Thr Gly Asp Leu Asp Lys Met Val Ala Leu Gln Arg 440 445 450 15 tta ttg gag ccc tat ggt ata tgt gag gtt gca aga acc ggt cgt gtg 1448 Leu Leu Glu Pro Tyr Gly He Cys Glu Val Wing Arg Thr Gly Arg Val 455 460 465 gca ttg gct cgt gaa tcg gga gtg gac tec aag tac ctt cgt gga tac 1496 Wing Leu Ala Arg Glu Ser Gly Val Asp Ser Lys Tyr Leu Arg Gly Tyr 470 475 480 485 tec ttt ctt tta here ggc taaaccgttg cagagtgcat ccatcgaaca i 1544 Ser Phe Leu Leu Thr Gly 20 490 tcagaaactt tggaaggtaa aagttteatt acacagtcta tgaacetcaa agacagacag 1604 I agagactgcg tegatatatg tttgtgactt tgtttatgaa acaattaget gattttgggc 1664 ttcatttcg 1673 < 210 > 2 < 211 > 491 < 212 > PRT < 213 > Ara ± iidopsis sp. < 400 > - 2 Met Ala Ala Be Ser Val Ser Be Ser Pro Pro Be Arg Cys Leu Arg, 1 5 10 15 Be Ala Cys Ser Asp Ser Ser Pro Ala Leu Val Ser Ser Thr Arg Val 20 25 30 Ser Phe Pro Ala Lys He Ser Tyr Leu Ser Gly Be Ser His Arg 35 40 45 Gly Asp Glu Met Gly Lys Arg Met Glu Gly Phe Val Arg Ser Val Asp 50 55 60 Gly Lys He Ser Asp Wing Ser Phe Ser Glu Wing Ser Ser Ala Thr Pro, 65 70 75 801 Lys Ser Lys Val Arg Lys His Thr He Ser Val Phe Val Gly Asp Glu f 10 85 90 95 Ser Gly Met He Asn Arg He Wing Gly Val Phe Wing Arg Arg Gly Tyr, 100 105 110 Asn He Glu Ser Leu Wing Val Gly Leu Asn Arg Asp Lys Ala Leu Phe 115 120 125 Thr He Val Val Cys Gly Thr Glu Arg Val Leu Gln Gln Val He Gluí 130 135 140 Gln Leu Gln Lys Leu Val Asn Val Leu Lys Val Glu Asp He Ser Ser 145 150 155 160 Glu Pro Gln Val Glu Arg Glu Leu Met Leu Val Lys Val Asn Wing His 165 170 175 Pro Glu Ser Arg Ala Glu He Met Trp Leu Val Asp Thr Phe Arg Alai • 180 185 190 i Arg Val Val Asp He Ala Glu His Ala Leu Thr He Glu Val Thr Gly1 195 200 205 Asp Pro Gly Lys Met He Wing Val Glu Arg Asn Leu Lys Lys Phe Gln 210 215 220 20 He Arg Glu He Val Arg Thr Gly Lys He Wing Wing Leu Arg Arg Glu Lys 225 230 235 240¡ Met Gly Wing Thr Wing Pro Phe Trp Arg Phe Wing Wing Being Tyr Pro 245 250 255 Asp Leu Lys Glu Gln Wing Pro Val Ser Val Leu Arg Ser Ser Lys Lys 260 265 270 A ^^ iata ^ Gly Ala He Val Pro Gln Lys Glu Thr Ser Wing Gly Gly Asp Val Tyr 275 280 285 Pro Val Glu Pro Phe Phe Asp Pro Lys Val His Arg He Leu Asp Ala 290 295 300 His Trp Gly Leu Leu Thr Asp Glu Asp Thr Ser Gly Leu Arg Ser His 305 310 315 320 Thr Leu Ser Leu Leu Val Asn Asp He Pro Gly Val Leu Asn He Val 325 330 335 Thr Gly Val Phe Ala Arg Arg Gly Tyr Asn He Gln Ser Leu Ala Val, 340. 345 350 Gly His Wing Glu Thr Lys Gly He Ser Arg He Thr Thr Val He Pro 355 360 365 Wing Thr Asp Glu Ser Val Ser Lys Leu Val Gln Gln Leu Tyr Lys Leu 370 375 380 Val Asp Val His Glu Val His Asp Leu Thr His Leu Pro Phe Ser Glu 385 390 395 400 Arg Glu Leu Met Leu He Lys He Wing Val Asn Wing Wing Wing Arg Arg 405 410 415 Asp Val Leu Asp He Wing Be He Phe Arg Wing Lys Wing Val Asp Va 420 425 430 ' Be Asp His Thr He Thr Leu Gln Leu Thr Gly Asp Leu Asp Lys Met1 435 440 445 Val Ala Leu Gln Arg Leu Leu Glu Pro Tyr Gly He Cys Glu Val Wing 450 455 460 Arg Thr Gly Arg Val Ala Leu Ala Arg Glu Ser Gly Val Asp Ser Lys 465 470 475 480 Tyr Leu Arg Gly Tyr Ser Phe Leu Leu Thr Gly 485 490 < 210 > 3 < 211 > 4895 < 212 > DNA < 213 > Arabidopsis sp. < 220 > < 221 > promoter < 222 > (1) .. (757) < 223 > Promoter region I I i < 220 > < 221 > misc_caracteristic < 222 > (717) < 223 > Transcriptional start point, < 220 > '< 221 > misc_signal < 222 > (758) .. (760) < 223 > Start codon i < 220 > i < 221 > misc_signal < 222 > (4737) .. (4739) < 223 > Stopcock < 220 > < 223 > n in position 694 can be a, c, g, or t < 400 > 3 tcgcatattg ttccggcgag gatcatgtga agcttgacgc gtgaattgac gactaagcgt 60 acgacgaagc gatccagttg agaattgtct cgagattcct cgttttagct gtcccact c 120 attcgccatg atttcgaaat ctctttctct tcttctctct ttcgtcttct tctgcgaaaa 180 aatcgaatgg ataatcacat tttctttttc tcgagaaaat tgatctggtg attatgtgag 240 atccgtctct agcgcgttgc ttatcgagaa ataattaatt ttaatttgac gggtgaagat 300 attattggcg acgtctgttt ccgattgact ttgatttgac ttttcctttc aatcattatt 360 tggcgagtcc cgcgtaaata tggactcttc ttgattgtcc cacttttttc ggtggctt 'fca 420 ccggatttaa aatcattttc ttttcctaaa ttatgaattt taccctaaac ttctcataat 480 tacaattagt tccgacgaac ccaagatact ttttagcaaa attaggaaaa tagttgactc 540 gaaaaggttg ttataacgtg gagctgacgt gttggtctta tctactcgaa gccttttggg 600 cttttcttaa agccattgat ttctaaggtc gtcaacaacc gaaccggacc ggacggtttg 660 ccaacatata accggtctaa tacgttcttt ttcnacttgc cgtttcgtcg tcgtcagtct 720 tcttcagtag caaaaaacct tcggcttcgt ctcgtcaatg gcggccattt ctgtaagt ^ c 780 ttcaccatct attcgctgct tgagatcggc atgttccgat tcttctcctg ctcttgtatc 840 ctcgacgcgt gtatcg TTCC cggcgaagat ttcatatctc tccggtatat cttcgcac g 900 atgggtaaga tggcgatgaa gaatggaagg attcgttaga agcgtcgatg ggaagatctc 960 tgatgcgtct ttctccgaag cttcatctgc gactccaaaa tcgaagcgac tgtgaataat 1020 atttgcttaa agtcgtttcc ttttggcctt tgctttgatt gattctttgt gcattaaaát 1080 cagggtgagg aagcacacaa tttcagtatt tgttggagac gaaagcggaa tgattaatag 1140 gtgtttgcaa gattgcagga ggagaggata caatattgag agtcttgctg ttggtctgaa 1200 gctctattca cagagacaag ccatagttgt ctgtggaact gaaagggtac ttcagcaggt 1260 catcgagcaa ctccagaagc tcgttaatgt tctaaaggtt gttcttttgt tagatcgcac 1320 ttattagttt ctgcatgact atagtttcat tcgcaccaac tttacgcatc agccaatttg 1380 cttattcatt atttgaagat tagatttgcg atttcctttt ccattctctt cattgactf; g 1440 gacatgaatt aggttgaaga tatctcaagt gagccgcaag tggagcgtga gctgatgctt 1500 gtaaaagtga atgcacatcc agaatecagg gcagaggtac tattccttgc ctatgggaaa 1560 ttagagttta ctgtacttgc tggttgcttc tgatttaggg cagaggtggt gttagttt ^ c 1620 tetetaaatt tgattaaget tctgttttaa tgaatteaca gatcatgtgg ctagttga ^ a 1680 catteagage aagagttgta g atatagcgg aacatgeatt gactategag gtacatetac 1740 ttattatgat ttgtgttggt cttgatattt gtttcgcact gtagcctgtg ggtttcaaga 1800 cttctgtttg aacatettac taatcgttgg aagacatcag aaatattatg gagggatcat 1860 tttaactttt atatetatta gttggatttt cgttgccttt tgaaactgat gatgatecae 1920 atgcaggact ctattatagg atgtgtatta aagtttattt gaaacttttg gtgcaactfc 1980 ttgaatttaa tataaegaga aagttattca acagtgtgct acctttgatt accctatg'ct 2040 tataatctgt attctgagtt gtattgcctg tgcaaatttc tgtgggaatg ctcagtgttc 2100 aettttgaaa gttagagaag cataacetta aatatattgt tctttttacc ttgattatga 2160 gaaagtggag taaaagaaag ggtgtctctg atttacctat tttagctctt tagtaatciat 2220 ttttaagcta ttttgcaggt aactggagat cctggaaaaa tgattgctgt agaaagaaat 2280 ttgaaaaagt ttcagatcag agagattgta aggacaggaa aggtagtgta tgtttggaat 2340 tactagattt tatggctttt gaatatcatc tagtttgtgc tatetaatgt atgtatgt'ag 2400 tttgagtgga tagttacatc cacaaaggca tagatetcag ggactttcac taatttaggg 2460 aaaatggaat gacatttttg gataacagat agcactgaga agggaaaaga tgggtgcaác 2520 tgctccattt tggcgat ttt cagcagcatc etatecagat ctcaaggagc aagcgcctgt 2580 tagtgttctt cgaagtagca aaaaaggagc cattgtccct caaaaggaaa catcageagg 2640 • ggtgtgtgct tctctgctcc ttagattgtt taaetteage ttgaagttcc tcactttcet 2700 ttcaaaaaat ttggttgcat aaattatagt aggtttggct atttgataaa gttaaacagc 2760 aactatagat gcctgtgttt ttttccctct atgtggtggc tgcctggaat caacatttga 2820 agcatgccct tttttgtttt tctccctggc tgcactgaag gatttccgag tttgctaa, 2880 tttaaaagtt tt atettatett tttaaatgta gggagatgtt tatcccgttg agecattt ^ t 2940 tgaccccaag gtacatcgta ttctcgacgc tcactgggga cttctcactg acgaagatgt 3000 aagagagttc tttgctatat atetaaette tttgctaaaa gtgccatgaa agcaatatga 3060 aaaattcaga ttgtggtttg cattacaega gttacacttg tttttccatt caagccgt ^ t 3120 attctgttaa ggcataatca tatagttaca taaatgataa atcaattgag tgttagatj: t 3180 ggagactgta tgtatttact tacaaagcag acattgaaag agttggggtt ttctttaagc 3240 tatttcgttt tatttatcac agttattctt ttttgatctt teagaegagt ggactacggt 3300 cgcatactct atcattgctt gtaaatgata ttccaggagt tettaatatt gtgactggtg 3360 ttttcgctcg aaggggatac aatatccagg catagtcctt tacacgcaca atctctctca 3420 cacagtgtgc ttaggtttac tgacacactg aaagatetec tttcttttag agcttggcpg 3480 10 taggacatgc tgaaaccaag ggcatttcac gcattacaac agttatacct gcaacagajtg 3540 aatcggtcag caaattggtg cagcaacttt acaaactcgt agatgtgcat gaggtgggat 3600 taccaaaagc tactgtcttt cttatatatt taacagtttg aatgtctttg atggccctat 3660 cattcctttg ctgtcttaga ccttttggct ttttttaaaa cgtagattag aggaagagtt 3720 tctgctaaa t ctttctggac tttectatat catttcctgg tcttgtctgt ttactcgaat 3780 gagaectett gttecagaaa gtcaaactgt acaggcttga tgaaaataat tctgaaca ^ g 3840 atttgccgca actttccaag ctgttattaa ctttgtgagg attttctgca ggtccatgat 3900 ettaetcatt tgccattttc tgaaagagaa ctgatgctga ttaagattgc cgtgaacg ^ t 3960 gctgctagaa gagatgtcct ggacattgct agtattttca gggctaaagc tgttgacgta 4020 tctgatcaca caattaettt gcaggtaaaa tacatttetc ataaatggga tttttatg ^ to 4080 gctgttattg catetcagat gagaaatcct ttcaattgga gatcttcaaa gtttcacgtc 4140 tttccatagg tcttcaactt gtttgacata ateagagtte cgtttgaaaa aaatatatga 4200 15 agctgacttg gattttccat cttaatctct tttttttgct tttgtgtttt ggatttgtgt 4260 gctgaaattt gttggctgtg ggtatagett actggggatc tagacaagat ggttgcactg 4320 caaaggttat tggagcccta tggtatatgt gaggtttgtt tegeaateta ctttcatc (tc 4380 ttagtgaatg cataaccccg tgaattetta tttcttataa tgctacccca attgctccgg 4440 ataaagtccc aaaatttagt tgtagtcttt aegaettaga aacagagtag tgaacatCjta 4500 actctctggt aaaatcaata accaaagctg gacetagtta catgaatctt cttctggtjtg 4 560 • tgtgtagaac aagaataage ttgacaagcc atgactactt tcagattatg catcgtgttg 4620 tgaacaatca aegettatta atcacacagg ttgcaagaac cggtcgtgtg gcattggctc 4680 gtgaatcggg agtggactcc aagtaccttc gtggatactc ctttctttta acaggctaaa 4740 ccgttgcaga gtgeatecat cgaacatcag aaactttgga aggtaaaagt tteattacae 4800 agtctatgaa cctcaaagac agacagagag actgcgtcga tatatgtttg tgactttgtt 4860 tatgaaacaa ttagctgatt ttgggcttca tttcg 20 <4895; 210 > 4 < 211 > 22 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the artificial Sequence: Initiator made from the cDNA sequence H ^^ ta? Tt ^^^^^^^^^^^^^ bi-á? -? Á? - É < 220 > < 221 > misc_caracteristic < 222 > (1) .. (22) '< 223 > DNA initiator < 400 > -4 cagagatcat gtggctagtt ga 22 • < 210 > 5 < 211 > 22 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of the Artificial Sequence: Initiator made from the cDNA sequence < 220 > < 221 > misc_caracteristic < 222 > (1) .. (22) < 223 > DNA initiator • 10 < 400 > 5 gagcgtcgag aatacgatgt ac 22 < 210 > 6 < 211 > 6 < 212 > PRT < 213 > Artificial sequence < 220 > < 223 > Description of the Artificial Sequence: Ig thrombin cleavage site < 400 > 6 Leu Val Pro Arg Gly Ser 1 5 • < 210 > 7 < 211 > 6 < 212 > PRT < 213 > Arabidopsis sp. 20 < 220 > < 221 > PEPTIDE < 222 > (1) .. (6) < 223 > N-terminal peptide of the minor subunit of AHAS of pHU E82 < 220 > < 221 > SITE < 222 > (1) .. (2) < 223 > Thrombin Breaking Site < 400 > 7 Gly Ser He Ser Val Ser 1 5 < 210 > "8 < 211 > 6 < 212 > PRT < 213 > Arabidopsis sp. < 220 > < 221 > PEPTIDE < 222 > (1) .. (6) < 223 N -terminal peptide of the AHAS minor subunit of pHU E83 <220> <221> SITE <222> (1) .. (2) <223> Thrombin Breaking Site < 400 > 8 10 Gly Ser Met He Asn Arg 1 5 < 210 > 9 < 211 > 8 < 212 > PRT < 213 > Arabidopsis sp. < 220 > < 221 > PEPTIDE < 222 > (1) .. (8) < | < 223 > N-terminal sequence of the peptide of the minor subunit of AHAS of the plasmid Fl < 220 > < 221 > SITE < 222 > (1) .. (2) < 223 > Thrombin Breaking Site < 400 > 9 Gly Ser Pro Lys He Wing Leu Arg 1 5 0 < 210 > 10 < 211 > 7 < 212 > PRT < 213 > Arabidopsis sp. < 220 > < 221 > PEPTIDE < 222 > (1) .. (7) < 223 > N-terminal sequence of the peptide of the minor subunit of AHAS of plasmid F2 < 220 > < 221 > SITE < 222 > - (1) .. (2) < 223 > Thrombin cleavage site • < 400 > 10 Gly Ser Leu Asp Ala His Trp 1 5 < 210 > 11 < 211 > 7 < 212 > PRT < 213 > Arabidopsis sp. < 220 > < 221 > PEPTIDE < 222 > (1) .. (7) < 223 > N-terminal sequence of the peptide of the minor subunit of AHAS of the plasmid F3 < 220 > < 221 > SITE < 222 > (1) .. (2) < 223 > Thrombin Breaking Site < 400 > 11 Gly Ser Val Glu Pro Phe Phe 1 5 •

Claims (56)

NOVELTY OF THE INVENTION CLAIMS

1. - An isolated DNA sequence comprising a sequence selected from the group consisting of: a) a DNA sequence encoding the protein of the minor subunit of Arabidopsis' AHAS, b) a DNA sequence comprising a sequence set forth in SEQ. ID NO: 1; c) a DNA sequence comprising a sequence set forth in SEQ ID NO: 3; and d) a DNA sequence that hybridizes with the sequences of b) or c) under severe conditions.

2. An isolated DNA sequence comprising a sequence selected from the group consisting of: a) a sequence of DNA I encoding amino acids 169-491 of the protein of the minor subunit of Arabidopsis AHAS; b) a DNA sequence comprising nucleotides 546-1514 of the sequence set forth in SEQ ID NO; 1; c) a DNA sequence comprising nucleotides 794-4895 of the sequence set forth in SEQ ID NO; 3; and d) a DNA sequence that hybridizes with the sequence of b) or c) under severe conditions.

3. A plant expression vector comprising a promoter expressed in plant cells and a DNA sequence according to claim 1.

4. - An expression vector according to claim 3, further characterized in that the promoter is a plant promoter of high expression level.

5. A plant expression vector according to claim 3, further characterized in that the promoter is the promoter of the minor subunit of Arabidopsis AHAS.

6. The plant expression vector according to claim 3, further characterized in that the DNA sequence encodes the minor subunit protein of Arabidopsis.

7. A plant expression vector according to claim 3, further characterized in that it comprises a secondary promoter that is expressed in plant cells and a DNA sequence encoding a protein of the eukaryotic AHAS major subunit.

8. A plant expression vector according to claim 7, further characterized in that the promoter of the major subunit and the DNA sequence encoding a protein of the greater subunit of AHAS eukaryote is derived from a dicotyledonous plant.

9. A plant expression vector according to claim 8, further characterized in that the dicot plant is Arabidopsis.

10. A plant expression vector according to claim 7, further characterized in that the DNA sequence encodes a variant of the protein of the large Arabidopsis subunit which is resistant to herbicide.

11. A plant expression vector according to claim 10, further characterized in that the Arabidopsis DNA sequence encodes a mutant protein of the major subunit of Arabidopsis AHAS that is resistant to the herbicide imidazolinone.

12. A plant expression vector comprising a promoter that is expressed in a plant cell and a DNA sequence according to claim 2.

13. A plant expression vector according to claim 12, further characterized by the promoter is a plant promoter of high expression level.

14. A plant expression vector according to claim 12, further characterized in that the promoter is the promoter of the minor subunit AHAS of Arabidopsis.

15. The plant expression vector according to claim 12, further characterized in that the DNA sequence encodes the protein of the minor subunit of Arabidopsis.

16. A plant expression vector according to claim 12, further characterized in that it comprises a second promoter that is expressed in a plant cell and a DNA sequence encoding a higher subunit protein of eukaryotic AHAS. i!

17. A plant expression vector according to claim 16, further characterized in that the promoter of the major subunit and the DNA sequence encode a protein of the eukaryotic AHAS major subunit and is derived from a dicotyledonous plant.

18. A plant expression vector according to claim 17, further characterized because the dicot plant is Arabidopsis.

19. A plant expression vector according to claim 16, further characterized in that the DNA sequence cc-difies a variant of the protein of the greater subunit of AHAS which is resistant to herbicides.

20. A plant expression vector according to claim 19, further characterized in that the DNA sequence of Arabidopsis encodes a mutant protein of the major subunit of Arabidopsis AHAS which is resistant to the herbicide imidazolinone.

21. A plant expression vector for expressing an AHAS I gene in a plant, comprising a promoter and a DNA sequence encoding a fusion protein comprising a major subunit and a minor subunit of an AHAS protein.

22. A plant expression vector according to claim 21, further characterized in that the DNA sequence encoding the minor subunit of the AHAS protein is derived from eukaryotes.

23. - The plant expression vector according to claim 22, further characterized in that the vector and the DNA sequence are derived from Arabidopsis.

24. A plant expression vector comprising a series of I 5 promoters that are expressed in plant cells; a DNA sequence encoding a transit polypeptide of the major subunit of AHASj; a DNA sequence encoding a mature protein of the major subunit of AHAS; a DNA sequence encoding an adapter polypeptide; a DNA sequence encoding a protein of the minor subunit of AHAS) eukaryotic; and a terminator sequence in the plant.

25. A plant expression vector that comprises in series a promoter expressed in plant cell; a DNA sequence encoding a transit polypeptide of the minor subunit of AHAS; a DNA sequence encoding a protein of the minor subunit of AHAS; a DNA sequence encoding an adapter polypeptide; a DNA sequence a polypeptide of the major subunit of AHAS and a vegietal terminator.

26. A plant expression vector according to claim 24 or 25 further characterized in that the promoter, the DNA secuence encoding the transit protein of the AHAS major subunit and the DNA sequence encoding the mature protein. of the major subunit of AHAS are derived from a dicotyledonous plant.

27. A plant expression vector according to claim 24 or 25 further characterized in that the promoter, the DNA sequence encodes the transit protein of the major subunit of AHAS and the DNA sequence encoding the protein of the major subunit of mature AHAS are derived from Arabidopsis.

28. A plant expression vector according to claim 24 or 25 further characterized in that the promoter, the DNA sequence encoding the transit protein of the major subunit of AHAS and the DNA sequence encoding the mature protein of the Greater subunit of AHAS derive from a monocotyledonous plant.

29. A plant expression vector according to claim 24 or 25 further characterized in that the promoter, the DNA sequence I encoding the transit protein of the greater subunit of AljíAS and the DNA sequence encoding the mature protein of the major subunit of AHAS are derived from rice or corn.

30. A protein isolated from the minor subunit of AHAS eukaryote comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence comprising the sequence set forth in SEQ ID NO; b) an amino acid sequence comprising amino acids 169-491 of the sequence set forth in SEQ ID NO: 2; and c) an amino acid sequence of a mutant or a variant of a) or b).

31. A composition comprising the protein according to claim 30. i _ ^^^^

32. - A method for creating a transgenic plant is resistant to herbicides, which comprises transforming a plant with a plant expression vector I comprising a DNA sequence selected from the group consisting of: a) a DNA sequence encoding the protein of the minor subunit of Arabidopsis AHAS; c) a sequence of ADÑ comprising the sequence set forth in SEQ ID NO: 1: c) a DNA sequence comprising the sequence set forth in SEQ ID NO: 3: and d) a DNA sequence that hybridizes with the sequences of b) or under severe conditions.

33.- The method according to claim 32, further characterized in that the expression vector further comprises a high level expression promoter plant and a DNA sequence encoding a subunit protein greater than eukaryotic AHAS.

34.- The method according to claim 33, further characterized in that the promoter is a promoter of the minor subunit of AHAS eukaryote and the DNA sequences of the major subunit encodes a mutant resistant to eukaryotic herbicides of a protein of the larger subunit of AHAS.

35. The method according to claim 34, further characterized in that the promoter and the DNA sequence encoding the AHAS subunit protein are derived from Arabidopsis

36. - A method for imparting herbicide resistance to a plant cell, comprising cotransformation of the plant cell with a first plant expression vector comprising a first motor expressible in plants, and a DNA sequence encoding the major subunit of the AHAS protein and a second expression vector comprising a second plant expression promoter and a DNA sequence encoding the minor subunit of a eukaryotic AHAS protein. 37.- A plant cell produced by the method according to claim 36. 38.- A method for improving the herbicide resistance of a transgenic plant that expresses a gene that encodes a protein of the subunit greater than AHAS that comprises transforming the transgenic plant with a DNA sequence that encodes a protein of the minor subunit of AHAS eukaryote. 39.- A transgenic plant produced by the method according to claim 38. 40.- A transgenic plant produced by the method according to claim 38, further characterized in that the plant exhibits a high resistance to the imidazolinone herbicide compared to other herbicides that inhibit AHAS. 41. The transgenic plant according to claim 40, further characterized in that the AHAS inhibiting herbicides are selected from the group consisting of sulfonylureas, triazolopyrimidines, pyrimidyl-oxy-benzoic acids, sulfamoylureas and sulfonylcarboxamides. i 42.- A method to improve resistance to herbicides in the plants of a plant progeny that involves crossing the plant with a A transgenic plant whose genetic complement comprises a sequence encoding a herbicide-resistant mutant in the major subunit of a eukaryotic AHAS protein and a DNA sequence encoding the minor I subunit of a eukaryotic AHAS protein; and select for those progeny plants that exhibit resistance to herbicide. V 10 43.- The method according to claim 42, further characterized in that the crossing of the plant with a transgenic plant is carried out by sexual reproduction. 44.- A plant progeny produced by the method according to claim 42. - A progeny according to claim 42, further characterized in that the genetic complement contains a DNA sequence coding for the minor subunit of the protein. AHAS eukaryote. 46.- A herbicide-resistant plant derived from a plant In accordance with claim 44, further characterized by its genetic complement comprises a DNA sequence encoding the herbicide-resistant mutant of the major subunit of the eukaryotic protein of the DNA sequence encoding the minor subunit of the eukaryotic AHAS protein. 47. A plant that is derived from the plant according to claim 46, characterized in that it exhibits a tolerance to the imidazolinone herbicide. 48.- A transgenic plant whose genetic complement comprises a gene that is expressed in plants that comprises a promoter for expression in plants, a DNA sequence that encodes a fusion protein comprising a major subunit and a minor subunit of an AHAS protein eukaryote and a terminator sequence that works in plant cells. 49. A plant derived from the plant according to claim 47, further characterized in that its genetic complement contains a DNA sequence that encodes a minor subunit of the eukaryotic AHAS protein. 50.- A transgenic plant according to claim 44 or 45, further characterized in that it is a dicotyledonous plant. 51.- A transgenic plant according to claim 44 or 45, further characterized because it is a monocotyledonous plant. 52.- A method to identify mutations in genes AHAS plants that confer resistance to herbicides, which comprises the exposure of an organism to a herbicidal compound, whose organism possesses a heterologous vector comprising a protein gene of the subunit of AHAS. 53. The method according to claim 52, characterized further because the vector also comprises a gene for the 5 protein of the major subunit of AHAS. 54. The method according to claim 52, further characterized in that the gene of the minor subunit of AHAS has a DNA sequence derived from AHAS. 55.- A method of selection to identify herbicides! what • 10 affect the enzymatic activity of AHAS, which comprises exposing an organism to a compound, said organism possesses a heterologous expression vector comprising a gene of the subunit protein of the AHAS, and determining the effects of the herbicide. 56.- A selection method for identifying a mutation that alters the inhibition of allosteric feedback of the AHAS enzyme, which comprises: transforming a microbial strain which is deficient in AHAS enzymatic activity with a plasmid expression vector comprising a mutant of the minor subunit gene of plant AHAS; selecting the microbial strain in minimal medium in the presence of one or two branched chain amino acids; and identify the microbial strains that grow in said medium.