MXPA98000861A - Resistance inducible to herbic - Google Patents

Abstract

The invention relates to DNA constructs that are capable of conferring on a plant inducible resistance to a herbicide. The inducible effect can be achieved by the use of gene change such as the change of alcA / alcR derived from A. Nidulas. The invention relates in particular to the resistance inducible to the herbicide N-phosphonomethylglycine (glyphosate) and its salt

Description

RESISTANCE INDUCED TO HERBICIDE The present invention relates to DNA builders and plants that incorporate it. In particular, they refer to promoter sequences for the expression of genes that confer resistance to the herbicides on the plants. Recent advances in the biotechnology of plants have resulted in the generation of transgenic plants resistant to the application of herbicides. Tolerance to herbicides has been achieved using a scale of different transgenic strategies. A well-documented example is the use of the phosphino tricine acetyl transferase (PAT) of the bacterial xenobiotic toxi ficant gene of Streptomyces hydroscopicus. Mutated genes the origin of the plant, for example the acetolactate synthase (ALS) modifier of the altered object site gene of Arabidopsis, have been used successfully to generate resistance of transgenic plants to the herbicide application. The genes of PAT and ALS have been expressed under the control of a strong consti tive promoter. A system is proposed in which the genes that confer tolerance to herbicide are expressed in an inducible form depending on the application of a specific chemical activator compound. This approach has a number of benefits for the farmer including the following: 1. The control inducible to herbicide tolerance would alleviate any risk of performance penalties associated with high levels of constitutive expression of herbicide resistance genes. This can be a particular problem since the early stages of growth where high levels of the transgene product can directly interfere with normal development. Alternatively, high levels of expression of the herbicide resistance genes can cause a "metabolic wasting of plant resources." 2. The expression of herbicide resistance genes in an inducible manner allows the herbicide in question to be used for control volunteers if the activating chemical compound is omitted during treatment 3. The use of an inducible promoter to induce herbicide resistance genes will reduce the risk of resistant weed species becoming a major problem. On the weed species from related crops, control could still be achieved with the herbicide in the absence of the inducing chemical.This would be particularly important if the tolerance gene confirmed resistance to a total vegetative control herbicide that would be used (without chemical inducer) before planting the crop and potentially after the crop has been harvested For example, it can be contemplated that herbicide resistance in cereals such as wheat, can be introduced by crossing wild grass oats or that resistance to herbicide in turnip or canola of oil seed can be transferred to wild brassica thus conferring herbicide resistance to those weeds that somehow already cause problems. A further example is that the inducible expression of herbicide resistance in sugar beet will reduce the risk of wild sugar beet becoming a problem. Several gene regulation systems (gene changers) are known and can be used to confer inducible herbicide resistance on plants. Many gene exchangers are described in the review by Gatz (Current Opinion in Biotechnology (1996) 7, 168-172) and include systems such as the tetracycline repressor gene exchanger, the Lac repressor system, copper inducible systems such as that based on in ACE1, inducible promoters of salicylic acid including the PR-la system and systems based on steroid hormones such as the glucocorticoid, progesterone and estrogen receptor systems. Modifications of glucocorticoid receptor systems that include the GAL4 ligation domain from yeast and the VP16 activator are described by Aoyama et al. The Plant Cell, (1995) 7, 1773-1785 and it is contemplated that similar systems may be based on, for example, insect steroid hormones rather than mammalian steroid hormones. In fact, a system based on the ecdysone receptor of Heliothis virescens has recently been described. The benzenesulfonamide gene exchanger systems are also known (Hershey et al, Plant Mol. Biol., 17, 679-690 (1991)) since they are systems based on the alcR protein of Aspergillus nidulans and glutathione S-trans ferase promoters. Various genes conferring resistance to herbicides are also known, for example, a herbicide for which the resistance genes have been described and which is extremely widely used is N-fos fonomet i lgl ic ina (glyphosate) and its agriculturally acceptable salts including the salts of isopropylamine, trimethulphonium, sodium, potassium and ammonium In a first aspect of the present invention there is provided a cassette for expression of chemically inducible plant genes comprising an inducible promoter operatively linked to An objective gene that confers resistance to a herbicide Any gene for herbicide resistance can be used, but the genes that confer resistance to N-phosphonomethyl glycogen The salts or derivatives thereof are especially preferred. Various inducible promoters can be used to confer inducible resistance and these include any of those listed above. However, a gene exchanger particularly useful for use in this area is based on the regulatory protein alcR of A spel rgi l ls Ni du l an s that activates gene expression of the alcA promoter in the presence of certain alcohols and ketones. This system is described in International Patent Publication No. WO 93/21334 which is incorporated herein by reference. The activation system of alcA / alcR genes from the fungus Aspergillus nidulans is also well characterized. The trajectory of utilization-of ethanol in A. Nidulans is responsible for the degradation of alcohols and aldehydes. It has been shown that three genes are involved in the path of ethanol utilization. It has been shown that alcA and alcR of the genes reside very close together in the binding group VII and the aldA maps to the binding group VIII (Pateman JH et al, 1984, Proc. Soc. Lond, B217: 243-264: sealy -Lewis HM and Lockington RA, 1984, Curr. Genet, 8: 253-259). The gene alcA codes for ADHI in A. Nidulans and aldA codes for AldDH, the second enzyme responsible for the use of ethanol. The expression of both alcA and aldA is induced by ethanol and a number of other inducers (Creaser EH et al, 1984, Biochemical J, 255: 449-454) through alcR transcriptional activator. The alcR gene and a leader are responsible for the expression of alcA and aldA since a number of mutations and deletions in alcR result in the pleiotropic loss of ADHI and aldDH (Felenbok B et al, 1988, Gene, 73: 385- 396: Pateman et al, 1984; Sealy-Lewis & Lockinton, 1984). The ALCR protein activates the expression of alcA by binding to three specific sites on the alsA promoter (Kulmberg P et al. 1, 1992, J. Biol. Chem, 267: 21146-21153). The alcR gene was cloned (Lockinton RA et al., 1985, Gene, 33: 137-149) and sequenced (Felenbok et al., 1988). The expression of the alcR gene is inducible self-regulated and subject to glucose repression mediated by the CREA repressor (Bailey C and Arts HN, 1975, Eur. J. Biochem, 51: 573-577; Lockington RA et al, 1987, Mol. Microbiology, 1: 275-281; Dowzer CEA and Kelly JM, 1989, Curr, Genet, 15: 487-459; Dowzer CEA and Kelly J, 1991, Mol. Cell. Biol, 11: 5701-5709). The regulating protein ALCR contains 6 cysteines near its coordinate of N-terminus in binuclear zinc cluster (Kulmberg P et al, 1991, FEBS Letts, 280: 11-16). This cluster is related to the highly conserved DNA binding domains found in the transcription factors of other ascomycetes. The GAL4 and LAC9 of the transcription factors have been shown to have binuclear complexes that have a trefoil crossing type structure containing two Zn (II) atoms (Pan T and Coleman JE, 1990, Biocheministry, 29: 3023-3029; Halvorsen; YDC et al, 1990. J. Biol. Chem, 265: 13283-13289). The structure of ALCR is similar to this type except for the presence of an asymmetric bond of 16 residues between Cys-3 and Cys-4. The ARCL positively activates self-expression by binding to two specific sites in its promoter region (Kulmberg P et al., 1992, Molec.Cell. Biol, 12: 1932-1939). The regulation of the three alcR genes, alcA and aldA, involved in the path of ethanol utilization is at the level of transcription (Lockinton et al, 1987, Gwynne D et al, 1987, Gene, 51: 205-216, Pickett et al. , 1987, Gene, 51: 217-226). There are two other dehydrogenases of alcohol present in A. Ni du l an s. ADHII is present in the growth of mycelia in non-induced media and is responsible for the presence of ethanol. The ADHII is encoded by alcB and is also under the control of alcR (Sealy-Lewis &Lockington, 1984). A third alcohol dehydrogenase has also been cloned by complementation with an S adh strain. Cerevi s i a e. This alcC of the gene is mapped to a linkage group VII but is unlinked to alcA and alcR. The gene, alcC, encodes ADHIII and uses ethanol in an extremely weak form (McKinight GL et al., 1985, EMBO J, 4: 2094-2099). It has been shown that ADH III is involved in the survival of A. No du l an s during the periods of anaerobic resistance. The expression of alcC is not expressed by the presence of glucose, suggesting that it may not be under the control of alcR (Roland LJ and Stromer JN, 1986, Mol Cell Biol, 6: 3368-3372). In summary, A. -Ni dül an s expresses enzyme alcohol dehydrogenase I (ADH1) encoded by the gene alcA only when it is grown in the presence of various alcohols and ketones. The induction is transmitted through a regulatory protein encoded by the alcR gene and constitutively expressed. In the presence of the inductor (alcohol or ketone), the regulatory protein activates the expression of the alcA gene. The regulator protein also stimulates the expression of itself in the presence of the inducer. This means that high levels of the ADH1 enzyme are produced under induction conditions (ie when alcohol or ketone are present). Conversely, the alcA gene and its ADH1 product are not expressed in the absence of inducer. The expression of alcA and the production of the enzyme are also repressed in the presence of glucose. Therefore the promoter of the alcA gene is an inducible promoter activated by the protein of the alcR regulator in the presence of the inducer (ie, by the protein / alcohol or protein / ketone combination). The genes of alcR and alcA (including the respective promoters) have been cloned and sequenced (Lockington RA et al, 1985, Gene, 33: 187-149, Felenbok B et al, 1988, Gene, 73: 385-396, Gwynne et al, 1987, 51: 205-216). The alcohol dehydrogenase (adh) genes have been investigated in certain plant species. .-In corn and other cereals have been exchanged by anaerobic conditions. The promoter region of the adh genes from maize contains a regulatory element of 300 bp necessary for expression under anaerobic conditions. However, no equivalent to the protein of the alcR regulator has been found in any plant. Therefore, the alcR / alcA type of the gene regulator system is not known in plants. The constitutive expression of alcR in plant cells does not result in the activation of the endogenous adh activity.

According to a second aspect of the invention, there is provided a cassette for expression of chemically inducible genes comprising a first promoter operably linked to an alcR regulatory sequence that encodes an alcR regulatory protein and an inducible promoter operably linked to a target gene which confers resistance to the herbicide, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer by which the application of the inducer causes the expression of the target gene. The inducible promoter preferably derives alcA, but may alternatively be derived from alcR, aldA or other genes induced by alcR. It has been found that the alcA / alcR exchange is particularly suitable for inducing herbicide tolerance genes for at least the following reasons. 1. The change of alcA / alcR has been developed to induce high levels of gene expression. In addition, alcR of the regulatory protein is preferably induced from a strong constitutive promoter such as polyubiqui t in. High levels of induced transgene expression, comparable to that of a strong constitutive promoter, such as 35 CaMV, can be achieved. 2. If a gene exchange is to be used in a situation where the activating chemical is applied simultaneously with the herbicide, a rapid rise in the levels of the herbicide resistance gene is required. Figure 1 reveals a time course of marker gene (CAT) expression after application of the inducing chemical. This study shows a rapid increase (2 hours) of CAT expression after foliar application of the chemical inducer. The immediately early kinetics of induction originates expressing the regulatory protein in a constitutive form, therefore no delay is found while the synthesis of the transcription factors is carried out. In addition, a simple two-component system has been chosen that does not depend on the complex signal translation system. The specificity of the alcA / alcR system has been tested with a scale of solvents used in agronomic practice. A hydroponic seedling system revealed that ethanol, bunan-2-ol and cyclohexanone all produce high levels of induced reporter gene expression (Figure 2). In contrast when the alcohols and ketones listed in Table 1 in which they are used in agronomic practice were applied as a foliar spray only ethanol produced high levels of induced reporter gene activity (Figure 3).

Table 1 1. I sobut ilmet i l acetone 13 acetonyl acetone 2. Fenconea 14 JF5969 (cyclohexanone) 3. 2-Heptanone 15 N-methylpyrrolidone 4. Di-isobutyl ketone 16 Polyethylene glycol 5. 5 -Met il -2 -hexanone 17 Propylene glycol 6. 5 -Met ilpentan- 2,4-diol 18 acet of enone 7. Ethemethyl ilketone 19 JF4400 (methylcyclohexanone) 8. 2-Pentanone 20 Propa? - 2 - ol 9. Glycerol 21 But an - 2 - or 1 10.? -Butyrolactone 22. Acetone 11. Diacetone alcohol 23 Ethanol 12. Tetrahydro-24 dH20 furfuryl alcohol This is important now that the illegitimate induction of the transgenes was not found by chance exposure to the formulation solvents. Ethanol is not a common component of agrochemical formulations and therefore with proper spray control it can be considered with a specific inducer of the alcA / R gene exchange in a field situation. 4. A scale of biotic and abiotic resistance such as pathogenic infection, heat, cold, drought, winds, floods have all failed to induce alcA / alcR change. In addition a scale of non-solvent chemical treatments such as salicylic acid, ethylene, absisic acid, auxin, gibberellic acid, various agrochemicals have all failed to induce the alcA / alcR system. The first promoter can be constitutive or tissue specific, programmed in a developed or even inducible form. The regulatory sequence, the alcR gene is obtainable from Aspergi l l u s n i dul ans and encodes the alcR regulatory protein. The inducible promoter is preferably the promoter of the alcA gene obtainable from Aspergi llusni du l an or a "chimeric" promoter derived from the regulatory sequences of the alcA promoter and the core promoter region of a gene promoter that operates in plant cells ( including any plant gene promoter). The alcA promoter or a related "chimeric" promoter is activated by the protein of the alcR regulator when an alcohol or ketone inducer is applied. The inducible promoter can also be derived from the promoter of the aldA gene, the promoter of the alcD gene or the promoter of the alcC gene obtainable from Aspergi l lus ni du l ans. The inducer can be any effective chemical (such as an alcohol or ketone) suitable chemistries for use with a cassette derived from alcA / alcR includes those listed by Creaser et al (1984, Biochem J, 225, 449-454) such as butan-2-one (et i lme ti Ice tona), cyclohexanone, acetone, butan-2-ol, 3-oxobutyric acid, propan-2-ol, ethanol. The gene expression cassette responds to an applied exogenous chemical inducer that allows external activation of the expression of the target gene regulated by the cassette. The expression cassette is highly regulated and suitable for general use in plants.

The two parts of the expression cassette can be from the same constructor or separate components. The first part comprises the regulatory cDNA or the gene sequence subcloned into an expression vector with a plant-operating promoter that induces its expression. The second part comprises at least part of an inducible promoter that controls the expression of a downstream target gene. In the presence of a suitable inducer the regulator protein produced by the first part of the cassette will activate the expression of the target gene by stimulating the inducible promoter in the second part of the cassette. In practice, the constructor or constructors comprising the expression cassette of the invention will be inserted into a plant by transformation. The expression of the target genes in the construct, being under the control of the chemically interchangeable promoter of the invention, can then be activated by the application of a chemical inducer to the plant. Any suitable transformation method for the target plant or plant cells can be employed, including infection by Agro-acter-üu-n t um fa ci ens containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplasts, transformation of microproyec ti 1 and pollen tube transformation. Transformed cells can be regenerated in suitable cases to whole plants in which the new nuclear material is stably incorporated into the genome. Plants can be obtained in this form both as monocots and lodones as well as transformed dicotyledons. Examples of genetically modified plants that can be produced include field crops, cereals, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, soybeans, corn, wheat, barley, rice, sorghum, tomato, mangos, peaches, apples, pears, strawberries, bananas, melons, potatoes, carrots, lettuce, pumpkins, onions. The invention further provides a plant cell containing a gene expression cassette according to the invention. The gene expression cassette can be stably incorporated into the plant genome by transformation. The invention also provides a plant tissue or a plant comprising such cells or plants or seeds derived therefrom.

The invention further provides a method for controlling the expression of plant genes comprising transforming a plant cell with a chemically inducible plant gene expression cassette having a first promoter operably linked to an alcR regulator sequence encoding a alcA regulatory protein, and an inducible promoter operably linked to a target gene that confers resistance to the herbicide, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer by which the application of the inducer causes the expression of the target gene. This strategy of inducible expression of herbicide resistance can be achieved with a pre-asperción chemical activator or in the case of slow-acting herbicides, for example N-phosphonomet ilglicina (commonly known as glyphosate), the chemical inducer can be added as a mixture of tank simultaneously with the herbicide, This strategy can be adopted for any gene that confers resistance / corresponding herbicide combination. For example, the change of the alcA / alcR gene can be used with: 1. Maize glutathione S-transferase gene (GST-27) (see International Patent Publication No. WO 90/08826), which confers resistance to herbicides of chloroacetanilide such as acetochlor, metolachlor and alachlor. 2. Acetyl transferase of f inotr icine (PAT), which confers resistance to the herbicide. commonly known as glufosinate. 3. Maize acetolactate synthase gene mutants (see International Patent Publication No. WO 90/14000) and other genes, which confer resistance to sulfonylurea and imidazolonone. Genes that confer resistance to glyphosate. Such genes include the glyphosate oxidoreductase (GOX) gene (see International Patent Publication No. WO 92/00377 in the name of the Monsanto Company); genes encoding 5-enolpiruvi 1-3-phosphosichemical acid synthase (EPSPS), including class I and class II EPSPS, genes encoding mutant EPSPS, and genes encoding EPSPS fusion peptides such as those comprising a chloroplast transit peptide and EPSPS (see for example EP 218 571, EP 293 358, WO 91/04323, WO 92/04449 and WO 92/06201 in the name of the company Monsanto); and the genes that are involved in the expression of CPLyase. Various aspects and additional preferred embodiments of the present invention will now be described in the non-limiting examples set forth below and with reference to the drawings in which: Figure 1 illustrates the time course of marker gene (CAT) expression after the application of a chemical inducer; Figure 2 illustrates the levels of reporter gene expression induced on root soaking with a variety of solvents; Figure 3 illustrates the levels of reporter gene activity induced when the chemicals listed in Table 1 were applied as a foliar spray; Figure 4 illustrates the production of the 35S regulatory construct by ligating alcR cDNA in pJR1; Figure 5 illustrates the production of the reporter builder; Figure 6 is a summary of specific cassettes and transformation constructs; Figure 7 illustrates the chloroplast transit sequence 1 from Arabidopsis RUBISCO (CPT 1); Figure 8 shows the sequence of pMJB1 plasmid; Figure 9 is a pJRI1 map of the plasmid; Figure 10 illustrates CTP2 of the chloroplast transit sequence from the EPSPS class I gene of hybrid petunia; Figure 11 is a map of pUB-1 plasmid; Figure 12 is a map of pMF6 plasmid; Figure 13 is a map of pIE109 plasmid in which the numbers are in base pairs (not to scale) and the following abbreviations are used: ADHX Corn dehydrogenase alcohol; Pat Acet i 1 transf erasa of phosphinotri- cine (Basta resistance gene); AMP Ampicillin resistance gene; CaMV35S Promoter 35S cauliflower mosaic virus; nosPolyA Region A nopalin polisintasa orí origin of colEl of replication from pUC; Figure 14 is a map of pMVl plasmid in which the numbers are in base pairs (not drawn to scale) and the abbreviations are as for Figure 13 with the following additional abbreviations: UUBBQQpD - corn ubiquitin promoter; UBQi intron of corn ubiquitin; we terminator of nopalin synthase 3 '; CZP1 GOX --- glyphosate c-loroplas t-oxidase transit peptide sequence; CZP2 GPSPS peptide sequence of chloroplasto-synthetase transit of EPSPS; Figure 15 shows the preparation of plasmid pUC4 by ligating pAr3 and pBSSK; Figure 16 is a pMV2 map of plasmid in which the numbers are in base pairs (not drawn to scale) and the abbreviations are as for Figure 14 with the following additional abbreviations: AlcA alcA promoter from Aspergillus nidulans; AlcR alcR promoter from Aspergillus nidulans; Figure 17 is a map of pDVl-pUC plasmid; Figure 18 is a map of pDV2-pUC plasmid; Figure 19 is a map of pDV3-bin plasmid; Figure 20 is a map of pDV4-Bin plasmid; and Figure 21 is a western blot showing the expression of transformed EPSPS and GOX.

EXAMPLES It has been chosen to exemplify the exchange of alcA / alcR genes with genes that confer resistance to glyphosate. The change will be used to induce the inducible expression of glyphosate oxidase (GOX) in plants. Interchangeable GOX has been expressed alone or in conjunction with the constitutive expression of 5-inol-pyruvilsikymate 3-phosphate (EPSPS) CP4. The builders have been optimized for the expression in species of monocotyledonous and dicotyledonous cultures.

EXAMPLE 1 Production of the AlcR Regulator Builder. The alcR genomic DNA sequence has been published, allowing the isolation of a sample of alcR cDNA. The alcR cDNA was cloned into the expression vectors pJR1 (pUC). PJRl contains the 35S promoter cauliflower mosaic virus. This promoter is a constitutive plant promoter and will continuously express the regulator protein. The polyadenylation signal of nos is in * the expression vector. Figure 4 illustrates the production of the 35S regulator builder by cDNA ligation from alcR to pJR1. Partial restriction of the cDNA clone of alcR with BamHI was followed by electrophoresis on an agar gel and excision and purification of a 2.6 Kb fragment. The fragment was then ligated into the pJR1 vector that had been restricted with BamHI and phosphatased to prevent recircularization. The alcR gene is thus placed under the control of the CaMV 35S promoter and the non-s 3 'polyadenylation signal in this "35S-alcR" construct.

EXAMPLE 2 Production of the Reporter Builder of alcA-CAT Containing the Chimeric Promoter. The plasmid pCaMVCN contains the bacterial chloramphenicol transferase reporter (CAT) gene between the 35S promoter and the transcription terminator nos (the "35S-CAT" builder). The alcA promoter was subcloned into vector pCaMVCN to produce an "alcA-CAT" constructor. The fusion of part of the promoter of alcA and part of the 35S promoter created a chimeric promoter that allows the expression of genes under its control. Figure 5 illustrates the production of the reporter builder. The alcA promoter and the 35S promoter have identical TATA boxes that were used to link the two promoters together using a recombinant PCR technique: a 246 bp region of the alcA promoter and the 5 'end of the CAT gene of pCaMVCN (containing part from the core region 70 of the 35S promoter) were amplified separately and then pooled using PCR. The recombinant fragment was then digested by restriction with Ba Hl and HindIII. The pCaMVCN vector was partially digested with BanHI and HindIII, then subjected to electrophoresis in order to isolate the correct fragment and bind it to the recombinant fragment. The ligation mixtures were transformed into E co l i and plated onto media with high agar content. The plasmid DNA was isolated by minipreparation of the resulting colonies and the recombinant clones were recovered by size electrophoresis and restriction mapping. The ligation linkages were sequenced to verify that the correct recombinants had been recovered.

EXAMPLE 3 Glyphosate Resistance Builders. A summary of cassettes and transformation constructors of specific plants is shown in Figure 6. Vector 1 Dicotyledon Vector 1 is a constitutive control plasmid containing the glyphosate oxidase (GOX) gene fused to transit sequence 1 Arabidopsis RUBISCO chloroplast (CPT 1) (Figure 7) induced by the improved CaMV 35S (ES) promoter and the omega TMV (TMV) translational tnejorator sequence. Vector 1 uses the nopal terminator insintasa (nos). The synthetic GOX gene with the addition of CTP 1 was synthesized with information from the patent publication WO 92/00377 with the addition of the Ncol site in the translation start ATG, and a Kpn I at the 5 'end. The Sph I sites and the Ncol site were removed during the synthesis without change in amino acid usage. The synthesized sequence of GOX from CTP 1 was isolated as a fragment of Kpnl from Ncol and ligated using normal molecular cloning techniques in pMJB1 cut-off of NcoIKpnI, a plasmid based on pIBT211 containing the CaMV promoter with the duplicated enhancer ligated to the tobacco mosaic virus translational enhancer sequence replacing the 5'-untranslated leader of the tobacco virus and ending with the poly (A) signal of nopalinsynthase (nos) (Figure 8). A cassette containing GOX from CTP1 of the enhanced TMV 35 CaMV sequence and the nos terminator (1 pUC from the dicotyledonous vector of Figure 17) was isolated as a HindIII EcoR1 fragment and ligated to pJRli from the HindIII EcoR1 cut, a vector from transformation of the base plant of Bin 19 (Figure 9).

Vector 2 Dicotyledon The CP4 gene of synthetic EPSPS fused to CTP2 of the chloroplast transit sequence (Figure 10) of the class 1 EPSPS gene from hybrid petunia was synthesized with the data of WO 92/04449 with Ncol in translation initiation ATG. An internal Sph I site was silenced in the EPSPS CP4 gene without change of amino acid usage. An EPSPS-containing fragment of the synthetic CTP2 CP4 was isolated as an NcslSacI fragment and ligated to pMJBI. A fragment containing the CaMV promoter with a duplicated enhancer, the CTP2 transit peptide of the omega TMV sequence, EPSPS and the nos terminator was isolated as an EcoRI HindIII fragment (2pUC of the dicotyledonous vector of Figure 18) and cloned pJRli to give the pUC of the dicotyledonous vector 2 (Figure 18). After sequencing the junctions of the dicotyledonous vector 2, an additional sequence inserted between the Sacl site and the beginning of the nos terminator was identified. This was as follows: - 'AGG CTG CTT GAT GAG CTC GGT ACC CGG GGA TCC ATG GAG CCG AAT 3 • Vector 3 Dicotyledonous A control vector with both of the genes EPSPS and GOX was constructed by cutting the dicotyledonous 2 vector with EcoRI and inserting an adapter ? EcoRISph I? EcoRI. The sequence of the adapter is shown below: 5 'AAT TAG GGG CAT GCC CCT 3' The resulting vector was cut with Sphl to release the cassette B that was cloned into a Sphl site in the vector dicotyledonous, 5 'to the CaMV promoter 35. Cassettes 1 and 2 were excised as a fragment of HindIII and EcoRI from the 3-dicot vector pUC (Figure 19) and cloned into pJRli.

Vector 4 Dicotyledon An inducible GOX vector was constructed by excising the CAT gene from "palcCAT" as the PstI fragment. The vector band, containing the alcA promoter and the terminator was purified by gel and used in the ligations with a Pst I -Xhol -Kpnl-Pst I bond, the sequence of which is as follows: 5 'GCC ACT CGA GCT AGG TAC CCT GCA 3 • The orientation of this was confirmed by sequence analysis. The omega sequence of TMV and GOX of CTP1 of the dicotyledonous vector 1 were isolated as a fragment of XhoIKpnl, the vector of alcA-cc having the linkage Xhol-Kpnl-Pstl was cloned. The cassette from us alcA TMV CPT1 GOX was excised as a fragment of HindIII and cloned into the "p35-alcR" plant transformation vector, containing the terminator of cDNA of alcR under the control of CaMV of 35 to form the 4 dicotyledonous vector (Figure 20).

Vector 5 dicotyledonous The 5 dicotyledonous vector (Figure 22) containing the inducible GOX and constitutive EPSPS genes was prepared using the following cloning strategy. The vector 2 dicotyledonous (pDV2-pUC) was "modified by cloning a linkage EcoRI-Hindl II-EcoRI EcoRI site to allow the removal of the CaMV cassette in CTP2-EPSPS as a HindIII fragment.This fragment was then ligated to pDV4- HindIII cut bin The recombinants containing the three cassettes ie 35S-AlcR, CaMVen-EPSPS-nos and Al cA-CTP 1-GOX-nos were selected by hybridization with radiolabelled oligonucleotides. sequenced through all the limits.

Illustration Monocot i ledóneos Vector 1: Cásete D An EcoRI link -Not I - EcoRI (5 'AATTCATTTGCGGCCGCAAATG3') was inserted into the dicotyledonous vector PDV1. The plasmid was cut with Ncol and the 5 'projection was filled with I Klenow Fragment of DNA polymerase. The linear vector was then cut with Notl and the resulting blunt / NotI fragment containing the GOX terminator of CTP1 was ligated to a pMUBI digested vector of Smal / Notl (Figure 12) containing the promoter of pol iubiqui t in, intron. of pol iubiqui t in with an insertion of (5 'CATTTGCGGCCGCAAATGGTAC 3') link of Kpnl -Not I -Kpnl. A link of HindI 11 -Not I -HindIII (5 'AGCTTGCAGCGGCCGCTGCA 3') was inserted into the resulting construct.

Vector 1: Casete E An EcoRI -Not I-EcoRI linkage (5 'AATTCATTTGCGGCCGCAAATG 3') was inserted into the dicotyledonous vector pDV2. The plasmid was cut with Ncol and the 5 'projection was filled with I Klenow fragment of DNA polymerase. The linear vector was then cut with NotI and the resulting blunt / NotI fragment containing the CTP2 EPSPS terminator and the terminator was ligated to a pMUB / NotI digested pPUBI vector containing the polyubiquit in, polytron iubiqui t intron promoter. with an insertion of (5 'CATTTGCGGCCGCAAATGGTAC 3') of Kpnl -Not I-Kpnl link was inserted to create plasmid 1. The selectable marker cassette of PAT (promoter of CaMV 35S, intron of Adhl, gene of acetyl transferase of phosphine tricine (PAT), nos terminator) was excised from pIE108 (Figure 14) and cloned into the HindIII site on plasmid 1 to give the monocot E cassum monocot. Diagnostic restriction digestion was used to confirm that the selectable marker cassette was inserted 5 'to 3' in the same orientation as the EPSPS cassette of CTP2. A fragment containing the polyubiquit in promoter, the polubusit intron intron, the nos terminator and GOX of CTP1 was excised from cassette D with Notl and ligated to the E cassette of Notl to form the vector 1 monocot i ledon (FIG. 14). Restriction digestion was used to confirm that the two cassettes were inserted in the same orientation. The selectable marker cassette (promoter of CaMV 35, Adhl's intron, the acet iltransferase gene of fos f inot ris ina (PAT) nos) was excised from pIE108 and cloned into the HindIII site at 5) to give the cassette E monocot i ledóneo.

Vector 1 A fragment containing a promoter of pol iubiqui t in, a Gox intron of pol iubiqui t in and nos was excised from cassette D. with Notl and cloned in cassette E of the Notl cut to form vector 1 monocot i ledóneo.

Vector 2: Cassette F An EcoRl fragment from pUC4 (Figure 15), containing the cDNA sequences of alcR and the terminator of nos was filled at its shaved end with the Klenow fragment of DNA polymerase I, ligated to pUBl with the insertion of the Kpnl -Not I-Kpnl link and guided by restriction analysis. The selectable marker cassette of PAT was inserted into the HindIII site after the excision of pIE108 and oriented by restriction analysis to create vector 1. Plasmid 1 above containing the promoter of pol iubiqui t in, the intron of pol iubiqui t in, EPSPS of CTP2 and the terminator We were cut with HindIII and a linkage of? HindIII-Notl-HindIII: 5 'AGCTCGCAGCGGCCGCTGCA3' 5 'GCGTCGCCGGCGACGTTGCA3 * was inserted and sequenced to create vector 2. A Cl to I-col-Clal bond (5' CGATGCAGCCATGGCTGCAT 3 ') was inserted into Pmf6 (Figure 13) to give vector 3. An Ncol / Kpnl fragment containing GOX from CTPl was excised from pDVl and inserted into vector 3 of the Ncol / Kpnl cut to create vector 4. A fragment of Salí containing Adhl's intron of corn, GOX of CTPl was excised from vector 4 and ligated to the pUC2 of the SalI cut containing the alcA promoter and the nos terminator and was sequenced to create vector 5. A HindIII fragment of vector 5 containing the alcA promoter , the Adhl corn intron, CTPI GOX and the terminator were ligated to vector 2 of the HindIII cut and oriented by restriction digestion. A Notl fragment of the resulting constructor containing the promoter of pol iubiqui t in, intron of pol iubíqui t in, EPSPS of CTP2, the terminator nos, the promoter of alcA, the intron of Adhl of corn, GOX of CTPl and the terminator nos ligated to vector 1 of the Notl cut and was oriented by restriction analysis to create the vector 2 monocot and ledon (Figure 16).

EXAMPLE 4 Transformation of the Plant Plasmids for dicotyledonous transformation were transferred to LBA4404 using the defrosting method by Holsters et al 1978. Tobacco transformants were produced by the leaf disc method described by Bevan 1984. Sprouts were regenerated on a medium containing 100 mg / l of cannamisin. After rooting the plants were transferred to the greenhouse and cultivated under conditions of 16 hours of light / 8 hours of darkness. Transformations of oil seed turnip (Brassica napus cv westar) were carried out using the cotyledon petiole method described by Moloney et al. 1989. The selection of the transformed material was carried out on cannamisin (15 mg / l). The rooted shoots were transferred directly to a manure-based manure and were grown to maturity under controlled greenhouse conditions (day of 16 hours at 20 ° C during the day 15 ° C at night at RH of 60%). Corn transformations were carried out using the particle bombardment approach as described by Klein et al. 1988. Selections were made on biolophos of 1 mg / l. Transformation of used sugar beet was carried out the protoplast cell-guard procedure see the publication of the international patent Ne > . WO / 95/10178. The results showing details of the obtained transgenic plants are shown in Tables 2 and 3 below.

Table 2 - Details of Tobacco Transformation Vector Species Removed rootstocks Rooted pDVl Tobacco 150 57 pDV2 Tobacco 150 60 pDV3 Tobacco 270 77 pDV4 Tobacco 350 135 pDV5 Tobacco 150 75 Table 3 - Transformation Details of Oil Seed Turnip Ve c tor Species Calli Enrai zados pDVl OSR 14 Retoños from 14 pDV2 OSR 13 Retoños from 13 pDV3 OSR 18 Retoños from 18 pDV4 OSR 20 Retoños from 20 pDV5 OSR 19 Retoños from 18 Analysis of Transgenic Plant Polymerase Chain Reaction (PCR) Genomic DN was prepared for PCR analysis of transgenic plants according to the method described by Edwards et al 1992. PCR is performed using the conditions described by Jepson et al. Plant Molecular Biology Repórter, 9 (2), 131-138 (1991). Load assemblies were designed for each of the introduced cassettes. The plants were analyzed using the following oligonucleotide combinations: PDVl TMV1 + G0X1, GOX3 + nosl pDV2 TMV1 + EPSPS1, EPSPS3 + nosl pDV3 EPSPS3 + GOX1, pDV4 35S + AlcRl, AlcA2 + GOX1 pDV5 35S + AlcRl, AlcA2 + GOX1, TMV1 + EPSPS1 The oligonucleotide sequences are given inuac ion cont: TMV1 5 'CTCGAGTATTTTTACAACAATTACCAAC GOX1 5' AATCAAGGTAACCTTGAATCCA GOX3 5 * ACCACCAACGGTGTTCTTGCTGTTGA NOS1 5 'GCATTACATGTTAATTATTACATGCTT EPSPS1 5 • 5 • TACCTTGCGTGGACCAAAGACTCC EPSPS3 GTGATACGAGTTTCACCGCTAGCGAGAC 35S 5' GTCAACATGGTGGAGCACG AlcRl 5 'GTGAGAGTTTATGACTGGAGGCGCATC AlcA2 5' GTCCGCACGGAGAGCCACAAACGA Selection On Glyphosate Curves of Samsun and Brassica napus var Wetstar for tobacco Kill on glyphosate Both species were tested on a glyphosate concentration scale by inserting in the case of tobacco a segment of 5-5 mm stem bearing a leaf node and in the case of oil seed turnip growing point plus two leaves in the middle of MS containing glyphosate at 0, 0.0055, 0.011, 0.0275, 0.055 and 0.01 mM .. of glyphosate isopropylamine salt. The results were recorded after two weeks of culture as given in Table 4 below.

Table 4 Westar Tobacco Concentration 0 Good stem growth, as OSR 4 to 5 new leaves, roots up to 5 cm 0.005 without stem growth, only one new leaf, roots cm are i -. • • • • * mient o- in any organ 0. 011 without stem growth without new leaves, roots of 0.5 cm " 0. 0275 without growth of stem, without new leaves, roots of 2 mm " 0. 055 without growth in any organ, ends of rods of oil "0.01 such for 0.055 mM" The selection for glyphosate-tolerant transformants was carried out on glyphosate concentrations of 0.01 and 0.05 mM.

Constitutively Tolerant Plants After the data obtained on wild-type plants, the primary transformants of PDV1, 2 and PCR + ve were screened on MS medium containing glyphosate at the levels described above. For tobacco this was done by inserting three or four inserts of the stem per transformant in the middle and using Samsun not transformed as the control. The record was based on the behavior of the majority. Plants that show tolerance to higher concentration of the herbicide were grown at maturity in the greenhouse, for seed collection.

Segregation test Seeds were sterilized in 10% bleach for 10 minutes. After several washes in sterile water, 200 seeds were seeded on an MS medium (2.3 g / 1 of MS salt, 1.5% sucrose, 0.8% Bactoagar, pH of 5.9) containing 100 mg / l of cannamisin. Seeds were cultured at 26 ° C with 16 hours / 8 hours light / dark before registration.

Western Analysis Generation of GOX Antibodies and EPSPS protein were over expressed in E. coli using a pET expression system. After induction of IPTG the GOX and EPSPS were elect rolavados of the growth cell paste in shake flask and used to immunize rabbits (two animals per clone).

Preparation of Tissue Extracts for Immunoblotting 120 mg of leaf tissue plus 60 mg of PVPP and 500 μl saline extraction solution (50 mM Tris-HCL pH 8, 1 mM EDTA, 0.3 mM DDT) were moved with a mixer during several minutes After homogenization the extract was centrifuged at 15,000 rpm for 15 minutes. The supernatant was stored at -80 ° C until required. The protein concentrations of the extract were measured according to Bradford.

SDS-PAGE E Immunostaining 25 μg of protein was separated by SDS-PAGE. The current saline solution was Glycine at 14.4% weight on volume, SDS at 1% weight on volume and Tris base at 3% weight on volume. The samples were loaded according to Laemmli. After the SDS-PAGE proteins were read, they were broadened overnight with 40 mA on nitrocellulose (Hybond C, Amersham) using an e-test unit from Biorad. The membrane was stained in 0.05% CTPS dissolved in 12 mM HCL. The stains were rinsed in 12 M HCL and stained for 5 to 10 minutes in 0.5 mM NaHCO 3 followed by an intensive rinse with H20. The membranes were blocked after immunization was detected and washed according to the Amersham ECL kit. Indirect detections were performed with a 1: 1000 dilution of an anti-GOX or polyclonal rabbit anti-EPSPS as the first antibody and with a 1: 1000 dilution of a second anti-rabbit antibody, associated with turnip peroxidase. An additional wash was carried out overnight to eliminate the surrounding environment. The detection was performed using the ECL equipment from Amersham and the results are shown in Figure 21 in which Lane (1) is the control and the remaining pathways are transformants. Western analysis shows that some transformants are capable of expressing GOX and EPSPS.

Consistently Tolerant Plants Cell extracts were prepared from each glyphosate tolerant plant and the amount of expressed protein estimated by Western analysis using appropriate antibody to the transformant. Plants that express very high levels of GOX or EPSPS were tested on higher glyphosate levels to relate the level of expression to tolerance of the herbicide.

Inducible Tolerant Plants To demonstrate the tolerance inducible to glyphosate, the positive PCR primary transformants from transformations with pDV4 and 5 were transferred directly to the greenhouse. After 2 weeks the plants were induced by root soaking in ethanol (5% solution) and left for 24 hours before performing Westenr analysis to quantify the level of GOX expression after induction. After a period of time to allow the plants to return to the non-induced state, Western analysis was repeated to allow selection of inducible tolerant plants. Plants that showed the highest levels of GOX expression after ethanol treatment were later taken for analysis over time. GOX levels were estimated at 6, 12, 18, 24, 36, 48 hours after treatment with ethanol, by Western analysis. Plants expressing highly GOX were used for both pDV4 and pDV5 in greenhouse tests to demonstrate tolerance to glyphosate. The plants were induced using a scale of ethanol concentrations (1 to 15%) by application of root soaking to potted plants. After induction of GOX the plants were sprayed with glyphosate. Controls of wild type and non-induced plants were also treated with herbicide.

Northern analysis Primary transformants containing vectors 2), 3) and 5) dicotyledonous were analyzed by Northern blot analysis an EPSPS probe of CTP2, as a fragment of Ncol Sacl. The primary transformants that contain the vectors 1). 3) . dicotyledons were analyzed by Bloting using a GOX probe of CTPl as a fragment of Ncol Kpnl. Similarly, the lines of transgenic maize containing vectors 1) and 2) monocot and ledon were analyzed using an EPSPS probe of CTP2. The transformants contained in the vector 5) dicotyledonous or vector 2) monocot and ledon were treated with a foliar application of 5% ethanol to induce GOX levels. RNA was isolated 24 hours after treatment and subjected to northern analysis with a GOX probe of CTPl. The primary transformants that were PCR positive for the appropriate cassettes and that showed transcript levels of GOX or EPSPS were taken for internal analysis.

Glyphosate Oxidoreductase Test The glyphosate oxidoreductase tests were carried out as described by Kishoré and Barry (WO 92/00377). These cause the measurement of glyphosate-oxygen-dependent uptake.

Claims (19)

1. A chemically-inducible gene expression cassette comprising an inducible promoter operably adapted to a target gene that confers resistance to a herbicide wherein the target gene confers resistance to the herbicide N-f osf onomet i lgl icine or a salt or derivative thereof.

2. A cassette of chemically inducible gene expression as claimed in claim 1, wherein the herbicide is N-phosphonomethylglycine or a salt or derivative thereof.

3. A chemically inducible gene expression cassette as claimed in claim 1 or claim 2, wherein the inducible promoter is the tetracycline repressor gene exchange, the Lac repressor system, an inducible copper system such as that based on in ACE1, inducible promoters of salicylic acid, for example the PR-1 system, a system based on a steroid hormone such as the glucocorticoid, progesterone-estrogen systems or a modification of one of these such as a glucocorticoid receptor system that includes the ligation domain of GAL4 from yeast and the activator of VP16, insect steroid hormone systems such as that based on the ecdysone receptor of Hi lio th is vi sc sc s, a gene exchange system of benzene sulfonamide, an exchange gene based on the alcR protein of Aspergi llusni du l ans or a promoter of S - t transferase glut ationa.

4. A chemically-inducible gene expression cassette comprising a first promoter operably adapted to an alcR regulator sequence encoding an alcR regulator protein, and an inducible promoter operably adapted to a target gene that confers resistance to the herbicide, the inducible promoter being activated by the regulator protein in the presence of an effective exogenous inducer by which the application of the inducer causes the expression of the target gene.

5. A gene expression cassette according to claim 4, wherein the inducible promoter is derived from the promoter of the gene induced by alcA, alcR, aldA or other gene promoter by alcR.

6. A gene expression cassette according to any of claims 4 and 5, wherein the inducible promoter is a chimeric promoter.

7. A plant cell containing a gene expression cassette according to any of the preceding claims.

8. A plant cell according to claim 7, wherein the gene expression cassette is stably incorporated into the genome of the plant.

9. A plant tissue comprising a plant cell according to any of claims 7 and 8.

10. A plant comprising a plant cell according to any of claims 7 and 8.

11. A plant derived from a plant according to claim 10.

12. A seed derived from a plant according to any of claims 10 and 11.

13. A method for controlling herbicide resistance comprising transforming a plant cell with the plant gene expression cassette of any of claims 1 to 6.

14. A method for selectively controlling seeds in a field of plants according to any of claims 10 or 11 or seeds according to claim 12, comprising applying to the field an effective amount of the herbicide and the exogenous inducer.

15. A gene expression cassette comprising a first promoter operably adapted to a first target gene by coding for a product that confers resistance to N-phosphonomethylglycine; and a second promoter operably adapted to a second target gene by coding for a product that metabolizes N-phosphonomethyl glycine; wherein the second promoter is inducible by the external application of an effective exogenous inducer, and wherein the resistance of a plant to N-fos fonomet i lgl ic ina is provided by the expression of the first and / or second target genes.

16. A gene expression cassette according to claim 15, wherein the inducible promoter is derived from the promoter of the gene induced by alcA, alcR, aldA or another gene promoter induced by alcR.

17. A gene expression cassette according to claim 15 or claim 16 wherein the first promoter is a constitutive promoter.

18. A gene expression cassette in which the first target gene codes for 5-enol-pyruvilsikyimate 3-phosphate (EPSPS) CP4.

19. A gene expression cassette in which the second target gene codes for glyphosate oxidase.