MXPA98001008A - Constructs of - Google Patents

Abstract

The present invention describes a chemically inducible gene expression cassette in plants, comprising a first promoter operably linked to a regulatory sequence that is derived from the alcR gene, and encodes a regulatory protein, and an inducible promoter, linked in a manner operative to an objective gene that encodes a protein that is harmful to insects, or whose expression induces a metabolic pathway that produces a metabolite that is harmful to insects, the inducible promoter is activated by the regulatory protein in the presence of an effective exogenous inducer , whereby the application of the inducer causes the expression of the target gene

Description

DNA CONSTRUCTS DESCRIPTION OF THE. INVENTION The present invention relates to DNA constructs, and to plants that incorporate them. In particular, it relates to promoter sequences and their use in the expression of genes that confer insecticidal activity on plants. Advances in plant biotechnology have resulted in the generation of transgenic plants that are protected against the larvae of insects that feed on them. Many organisms produce proteins that are harmful to insects, and among these is the organism Bacillus thuringiensis, which produces a crystal-associated protein d-endotoxin, which kills insect larvae with its ingestion. However, it is not toxic to mammals. It is thus very useful as an agricultural insecticide. Many strains of B. thuringiensis are active against pests or insect pests, and the genes that encode endotoxins against insects have been characterized. The d-endotoxins of B. thuringiensis include those specifically insecticides for larvae of Lepidoptera (such as Cryl-type proteins), those specifically insecticidal for larvae of Coleoptera (such as CrylIIJ type proteins and those with dual specificity for Lepidoptera and Coleoptera) (such as CryV). Chimeric proteins comprising at least part of an endotoxin of B. thuringiensis have also been proposed in order to improve the properties of the endophoxin in some way, for example an improved kill rate. Transgenic plants that express genes that code for insecticidal endotoxins are also known. Other ways to harm insects include stimulating the metabolic pathways of plants that produce metabolites that are insecticides. We propose a system where the genes encoding active insecticidal proteins such as B. thuringiensis endotoxins would be expressed in an inducible manner, dependent on the application of a specific activating chemical compound. Alternatively, induction of pathways that produce harmful metabolites to insects could be achieved. This approach has a number of benefits, including the following: 1. Constitutive expression in plants of insect resistance genes, such as B. thuringiensis endotoxins, will result in a significant increase in selection pressure for resistant insect species. . The inducible regulation of insect resistance genes will reduce the risk of development of resistant pests. For example, the expression of insecticidal genes can be induced only to the point in the growing season where protection is required. In addition, the insect's switchable tolerance can be used as part of an integrated pest management system, in which chemical treatments to induce the expression of the insecticide gene can be alternated with treatments with standard pesticide insecticides. 2 . There is a risk that overexpression, from strong constitutive promoters, could lead to detrimental effects on the development of the plant, resulting in aberrant germination, or penalties in flowering or yield. The inducible expression would reduce the risk of detrimental effects, since the transgene could be expressed for a short period, avoiding sensitive points in the development. 3. The chemical switch could be added to standard insecticidal formulations, to give both a chemical and a genetic effect, thus killing insects by two independent mechanisms. We have developed an inducible genetic regulation system (genetic switch) based on the alcR regulatory protein of Aspergillus nidulans, which activates gene expression from the alcA promoter, in the presence of certain alcohols and ketones. This system is described in our International Patent Publication No. 093/21334, which is incorporated herein by reference. The activation system of the alcA / alcR gene of the fungus Aspergillus nidulans is also well characterized. The way of The use 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 the alcA and alcR genes are located very close to one another on the binding group VII, and aldA is on the map of the binding group VIII (Pate an, JH et al., 1984, Proc. Soc. Lond ., B217: 243-264; Sealy-Lewis, HM and Lockington, RA 1984, Curr. Genet., 8: 253-259). The alcA gene encodes 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, E. H. et al., 1984, Biochemical J., 255: 449-454) via the alcR activator of transcription. The alcR gene and a co-inducer 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 &Lockington, 1984). The ALCR protein activates the expression of alcA that binds to three specific sites in the alcA promoter (Kul berg, P. et al., 1992, J. Biol. Chem., 267: 21146-21153). The alcR gene was cloned (Lockington, R. A. et al., 1985, Gene, 33: 137-149) and the sequence was determined (Felembok et al., 1988). The expression of the alcR gene is inducible, self-regulated and subject to glucose repression mediated or CREA repressor (Bailey, C. and Arst, 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: 457-459; Dowzer, CEA and Kelly, JM, 1991, Mol. Cell. Biol. 11: 5701-5709). The regulatory protein of ALCR contains 6 cysteines near its N-termini, coordinated in a binuclear zinc cluster (Kulmberg, P. et al., 1991, FEBS Letts., 280: 11-16). This cluster is related to highly conserved DNA binding domains, found in transcription factors of other ascomycetes. It has been shown that the transcription factors GAL4 and LAC9 have binuclear complexes that have a trefoil-like structure containing two Zn (II) atoms (Pan, T. and Coleman, JE, 1990, Biochemistry, 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 loop of 16 residues between Cis-3 and Cis-4. ALCR positively activates the expression of itself, joining two specific sites in its promoter region (Kulmberg, P. et al., 1992, Mol.Cell. Biol., 12: 1932-1939). The regulation of the three genes, alcR, alcA and aldA, involved in the ethanol utilization pathway is at the level of transcription (Lockington et al., 1987; Gwyne, D. et al., 1987, Gene, 51: 205 -216; Pickett et al., 1987, Gene, 51: 217-226).

There are two other alcohol dehydrogenases present in A. nfdulans. ADHII is present in mycelium grown in non-induced medium, and may be repressible by the presence of ethanol. 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 adh strain of S. cerevisiae. This alcC gene is located on the map of the binding group VII, but is not bound to alcA and alcR. The gene, alcC, encodes ADHII, and uses ethanol extremely weakly (McKnight, G. L. et al., 1985, EMBO J., 4: 2094-2099). It has been shown that ADHII is involved in the survival of A. nidulans during periods of anaerobic stress. The expression of alcC is not repressed by the presence of glucose, suggesting that it may not be under the control of alcR (Roland, L.J. and Stromer, J.N., 1986, Mol.Cell. Biol., 6: 3368-3372). In summary, A. nidulans expresses the enzyme alcohol dehydrogenase I (ADHI) encoded by the alcA gene only when it is grown in the presence of various alcohols and ketones. The induction is regulated through a regulatory protein encoded by the alcR gene, and expressed constitutively. In the presence of the inducer (alcohol or ketone), the regulatory protein activates the expression of the alcA gene. The regulatory protein also stimulates the expression of itself in presence of an inductor. This means that high levels of the ADHI enzyme are produced under induction conditions (ie, when alcohol or ketone is present). Conversely, the alcA gene and its product, ADHI, 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. Thus, the promoter of the alcA gene is an inducible promoter, activated by the alcR regulatory protein in the presence of inducer (ie, by the protein / alcohol or protein / ketone combination). The alcR and alcA genes (including the respective promoters) have been cloned and the sequence determined (Lockington, RA et al., 1985, Gene, 33: 137-149, Felenbok, B. et al., 1988, Gene , 73: 385-396; Gwynne et al., 1987, Gene, 51: 205-216). The genes of alcohol dehydrogenase have been investigated (adh) in certain plant species. In corn and other cereals, they are activated by anaerobic conditions. The promoter region of maize adh genes contains a regulatory element of 300 bp necessary for its expression under anaerobic conditions. However, no equivalent to the regulatory protein of alcR has been found in any plant. Hence, the type of alcR / alcA gene regulatory system is not known in plants. The constitutive expression of alcR in plant cells does not result in the activation of endogenous adh activity.

According to a first aspect of the invention, there is provided a chemically inducible plant expression cassette, comprising a first operator, operably linked to a regulatory sequence that is derived from the aLcR gene, and encodes a regulatory protein, and an inducible promoter operably linked to a target gene, which encodes a protein that is harmful to insects, or whose expression induces a metabolic pathway that produces a metabolite that is harmful to insects, the inducible promoter is activated by the protein regulator in the presence of an effective exogenous inductor, whereby the application of the inducer causes the expression of the target gene. When the target gene encodes a protein harmful to insects, it is advantageous for that protein to be orally active. Examples of orally active insecticidal proteins are the d-endotoxins of B. thuringiensis, and therefore, the target gene can encode at least part of a d-endotoxin of B. thuringiensis. We have found that the alcA / alcR switch is particularly suitable for handling genes encoding B. thuringiensis endotoxins for at least the following reasons. The alcA / alcR switch has been developed to handle high levels of gene expression. In addition, the alcR regulatory protein is preferably handled from a strong constitutive promoter, such as polyubiquitin. High levels of transgene induced expression can be achieved, comparable to those of a strong constitutive promoter, such as 35 CaMV. Figure 1 reveals a time course of marker gene (CAT) expression following the application of a chemical induction compound. This study shows a rapid increase (2 hours) in the expression of CAT following the foliar application of the chemical compound inducer. The kinetics of immediate immediate induction are brought about to be constitutively expressing the regulatory protein, therefore a delay period is not found while the synthesis of transcription factors takes place. In addition, we have selected a simple two-component system, which does not depend on a complex signal transduction system. We have tested the specificity of the alcA / alcR system with a range of solvents used in agronomic practice. A hydroponic system of seedlings revealed that ethanol, 2-butanol and cyclohexanone all gave high levels of expression of the induced reporter gene (Figure 2). In contrast, when various alcohols and ketone listed in Table 1, and used in agronomic practice were applied as a foliar spray, only ethanol gave high levels of induced activity of the reporter gene (Figure 3). This is significant, since no illegitimate induction of transgenes will be found by chance exposure to the solvents of the formulation. Ethanol is not a common component of agrochemical formulations, and therefore with proper handling of sprays, it is considered a specific inducer of the alcA / alcR gene switch in a field situation. Table 1 1. Isobutyl methyl ketone 13. Acetonyl acetone 2. Fenchana- 14. JF5969 (cyclohexanone) 3. 2-heptanone 15. N-methyl pyrrolidone 4. Di-isobutyl ketone 16. Polyethylene glycol 5. 5-methyl-2- hexanone 17. Propylene glycol 6. 5-methylpentan-2,4-diol 18. Acetophenone 7. ethyl methyl ketone 19. JF4400 (methylcyclohexanone) 8. 2-pentanone 20. 2-propanol 9. glycerol 21. 2-butanol 10.? -butyrolactone 22. Acetone 11. diacetone alcohol 23. Ethanol 12. Tetrahydrofurfuryl alcohol 24. dH20 A range of biotic and abiotic stresses, for example infection by pathogens, heat, cold, drought, injury, or flooding has not been able to induce the alcA / alcR switch. In addition, a range of chemical treatments with substances that are not solvents, for example salicylic acid, ethylene nitric acid, auxin, gibberellic acid, various agrochemical compounds, could not induce the system, a.1 cA / alcR. . The present invention is not limited to any particular endotoxin, and is also applicable to chimeric endotoxins. The first promoter can be constitutive, or tissue specific, programmed by development, or even inducible. The regulatory sequence, the alcR gene, is available from Aspergillus nidulans, and encodes the alcR regulatory protein. The inducible promoter is preferably the promoter of the alcA gene available from Aspergillus nidulans, or a "chimeric" promoter derived from the regulatory sequences of the alcA promoter and the core region of the promoter of a gene promoter that operates in plant cells (including any promoter of the plant gene). The alcA promoter, or a related "chimeric" promoter, is activated by the alcR regulatory protein when an alcohol or ketone inducer is applied. The inducible promoter can also be derived from the aldA gene promoter, the alcB gene promoter, or the alcC gene promoter, available from Aspergilus nidulans. The inducer can be any effective chemical compound (such as an alcohol or ketone). Chemical compounds suitable for use with a cassette derived from alcA / alcR include those listed by Creaser et al (1984, Biochem. J., 225, 449-454) such as 2-butanone (ethyl methyl ketone), cyclohexanone, acetone, 2-butanol, 3-oxobutyric acid, 2-propanol, and ethanol. The cassette of expression of the gene is sensitive to an applied exogenous chemical inducer, which allows the external activation of the expression of the target gene regulated by the cassette. The expression cassette is highly regulated and is suitable for general use in plants. The two parts of the expression cassette may be on the same construct, or on separate constructs. The first part comprises the regulatory cDNA or gene sequence subcloned into an expression vector, with a plant-operating promoter directing 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 regulatory 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 construct or constructs comprising the expression cassette of the invention will be inserted into a plant by transformation. The expression of target genes in the construct, being under the control of a switchable promoter then be activated by the application of a chemical inducer to the plant. Any suitable transformation method can be used for the plant or target plant cells, including infection with Agrobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplasts, transformation with microprojectiles, and transformation by pollen tube. The transformed cells can then, in appropriate cases, be regenerated to whole plants, in which the new nuclear material is stably incorporated into the genome. Transformed plants of both types, monocotyledonous and dicotyledonous can be obtained in this way. Examples of genetically modified plants that can be produced include field crops, cereals, fruits and vegetables such as: cañola, sunflower, tobacco, beet, cotton, soybean, mai 7.,. triga, barley, rice, sorghum, tomatoes, mango, peaches, apples, pears, strawberries, bananas, melons * potatoes, carrots, lettuce, cabbage, and onions. The invention further provides a plant cell that contains 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 fabric of plant, or a plant comprising such cells, and plants or seeds derived therefrom, The invention further provides a method for controlling the expression of plant genes, which comprises transforming a plant cell with a plant gene expression cassette. chemically inducible, which has a first promoter operably linked to a regulatory sequence that is derived from the alcR gene, and encodes a regulatory protein, and an inducible promoter, operably linked to a target gene that encodes a d-endotoxin B. thuringiensis, the inducible promoter is activated by the regulatory protein in the presence of an effective exogenous inducer, whereby the application of the inducer causes the expression of the target gene. Various aspects and preferred embodiments of the present invention will now be described, by way of the following non-limiting examples and drawings, in which: Figure 1 is a graph showing the time course of induction of the population secreted by ARIO with 7.5% ethanol; Figure 2 is a graph showing the activity of CAT in a homozygous line of AR 10-30 by soaking the roots with various chemical compounds; Figure 3 is a graph showing the activity of CAT in the homogeneous line of AR 10-30 when soaking the roots with various chemical compounds; Figure 4 shows the production of a 35S regulatory construct; Figure 5 shows the production of a reporter's construct; Figure 6 illustrates switchable insect resistance vectors; Figure 7 illustrates the optimized Cryla (c) gene sequence; Figure 8 shows the restriction sites in the optimized Cryla (c) gene; Figure 9 illustrates the sequence of the Cry V gene, Figure shows the 5129 bp of the vector containing the CryV gene, Figure 11 illustrates the vector sequence pMJBl, and Figure 12 is a map of the pJRIi vector. Production of the alcR Regulator Construct The genomic DNA sequence of alcR has been published, allowing the isolation of an alcR cDNA sample.The alcR cDNA was cloned into the expression vector, pJR1 (pUC). 35S Cauliflower Mosaic Virus This promoter is a constitutive promoter of the plant, and will continuously express the regulatory protein.The signal of polyadenylation of nos is used in the expression vector.

Figure 4 illustrates the production of the 35S regulatory construct by binding the alcR cDNA to pJR1. Partial restriction of the alcR cDNA clone with Ba HI was followed by electrophoresis on an agarose gel, and the cut and purification of a 2.6 Kb fragment. The fragment was then ligated into a pJR1 vector that had been restricted with Ba HI, and subjected to the action of phosphatase to prevent recircularization.

The alcR gene was thus placed under the control of the 35S promoter of CaMV and the 3 'polyadenylation signal of us in this construct of 35S-alcR. "EXAMPLE 2 Production of the AlcA-CAT reporter Construct Containing the Chimeric Promoter Plasmid pCaMVCN contains the reporter gene of bacterial chloramphenicol transferase (CAT) between the promoter of 35S and the transcription terminator of us (the construct "35S-CAT"). The alcA promoter was subcloned into the pCaMVCN vector, to produce an "alcA-CAT" construct. The fusion of part of the promoter of alcA and part of the promoter of 35S created a chimeric promoter, which allows the expression of genes under its control. Figure 5 illustrates the production of the reporter's construct. The alcA promoter and the 35S promoter have identical TATA boxes, which were used to join the two promoters with one another, 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 of the core region of ~ 70 of the 35S promoter) was amplified "separately, and then they were spliced together, using PCR. The recombinant fragment was then digested with restriction, with Ba HI and HindIII. The pCaMVCN vector was partially digested with BamHI and HindIII, then subjected to electrophoresis, so that the correct fragment could be isolated and ligated to the recombinant fragment. The ligand mixtures were transformed into E. coli, and plated on agar rich medium. The plasmid DNA was isolated by miniprep from the resulting colonies, and the recombinant clones were recovered by size electrophoresis and restriction map plotting. The sequence of the binding splices was determined, to verify that the correct recombinants had been recovered. EXAMPLE 3 Gene Constructs We have generated the following constructs, summarized in Figure 6: Vector 1: contains the 35S CaMV promoter amplified, fused to the Cry I A gene of Bacillus thuringiensis of the translational amplifier of sequence or ega of the virus of thetobacco mosaic (TMV) and the nopolin synthase terminator (nos). Vector 2: is identical to vector 1, with the exception that the Cry I A gene from B. thuringiensis is replaced with the Cry V gene from B. thuringiensis. Vector 3: contains the gene for the alcR regulatory protein of Aspergilus nidulans, managed from the CaMV 35S promoter, the alcA promoter region, Cry I A (c) of the TMV amplifier and the nos terminator. Vector 4: is identical to vector 3, with the exception that the Cry I A (c) gene is replaced with the CryV gene. The Cry I A (c) gene is a synthetic optimized sequence specific for Lepidoptera, which encodes a Bacillus thuringiensis endotoxin, and is illustrated in Figures 7 and 8. The sequence was obtained from Pamela Green's Laboratory, Michigan State University. The Cry V gene is a novel endotoxin of Bacillus thuringiensis, entomocida for the larvae of Coleoptera and Lepidoptera, and is described in our International Patent Publication No. WO90 / 13651. The Cry V gene is a modified synthetic sequence, optimized for use in plant coding, and the regions of instability have been eliminated. This is illustrated in Figures 9 and 10.

EXAMPLE 4 Preparation of Vector Vector 1-Cry lA (c) Constitutive PCR primers were designed to amplify the omega sequence of TMV in pMJBl (see Figure 9) with the addition of a Sal I site adjacent to the Xhol site (see oligonucleotide forward) and destroying the Ncol site, and adding a Sal I site and Bgl II sites in the reverse oligonucleotide. Oligonucleotide Front (SEQ ID NO.1) Salt I 5 'CTACTCGAGTCGACTATTTTTACAACAATTACCAAC 3' Xhol Reverse Oligonucletotide (SEQ ID NO.2) 5 'CTAGGTACC GTCGAC GGATCCGTAAGATCTGGTGTAATTGTAAATAGTAATTG 3' Kpnl Salí BamHI BglII PCR was performed with the forward and reverse primers, using plasmid DNA of pMJBl on a template.

The resulting PCR product was cloned into the vector pTAg (LigATor case, R &D Systems); This was then released with digestion with Asp 718 and Xho I, and was cloned into pMJBl digested with Xho I / Asp 718 (Figure 10), to form pMJB3. PMJBl is based on pIBT 211 that contains the CaMV35 promoter, with amplifier duplicate bound to the translational amplifying sequence of the tobacco mosaic virus replacing the untranslated 5 'guide of the tobacco etch virus, and terminated with the nopaline synthase poly (A) signal (nos). The synthetic gene of Cry I A (c) was cut as a fragment of Bgl II BamHI, and cloned into pMJB3. A fragment containing the TMV omega sequence of the amplified CaMV promoter, Cryl A (c) and the terminator nos was isolated using Hind III and EcoRI. The resulting fragment was ligated into pJRIi cut with EcoRI / Hind III (Figure 12), to generate a plant transformation vector based on Binl9. Vector 2 - Cry V Constitutive pMJB3 was cut with Hind III, and a Hind III-EcoRI-Hind III linker was inserted. The resulting vector was then cut with Bam Hl, and a fragment containing the CryV gene was inserted as a BaMHI fragment. The Cry V gene was oriented using a combination of restriction digestion and sequence analysis. An EcoRI fragment of the resulting vector, containing the amplified 35 CaMV promoter, the omega sequence of TMV, the CryV gene and the nos terminator, was transferred to JRIRiMCS, a vector based on Bin 19 containing the site of cloned multiple pUC18. Vector 3 - Cry 1 A (c) Inducible pMJB3, which contained the Cry lA (c) gene with Sal I, was excised by releasing a fragment containing the omega sequence of TMV fused to the Cry gene lA (c). The resulting fragment was cloned in palc A CAT cut with Sal I, and oriented by restriction digestion. A fragment containing the alcA promoter fused to the TMV omega sequence, the Cry lA (c) gene and the nos terminator was cut using HindIII, and transferred to p35SalcRalcAcat digested with HindIII, a vector based on Bin 19 which contained the 35 CaMV promoter fused to alcR cDNA with the alcAcat reporter cassette removed by digestion with HindIII. Vector 4 - Cry V Inducible Cut with Sal I pMJB3, which contained the Cry V gene, releasing a fragment containing the omega sequence of TMV fused to the Cry V gene. The resulting fragment was cloned into Cal I palcACAT, and was guided by restriction digestion and sequence analysis. Two fragments, which contained the Cry V gene of the ale A promoter and the nos terminator, were released by digestion with Hind III. A three-way ligature of the two fragments of Hind III was performed to insert the A Cry Vase ale cassette into p35SalcRalcAcat digested with HindIII, to remove the alccat cassette. Correct mounting of the cassette was confirmed by restriction digestion, Southern blotting and sequence analysis.

EXAMPLE 5 Transformation of plants Transformation of leaves by Agrobacterium The transformation was carried out according to the method described by Bevan in 1984. Sterile cultures of 3-4 weeks of tobacco (Nicotiana tabacum) cultivated on DM were used for the transformation. The edges of the leaves were cut, and the leaves were cut into pieces. Then, they were placed in the transformed Agrobacterium cells, which contained the pJRIRI plasmid with the insert, in suspension (strain LBA 4404) for 20 minutes. The pieces were placed on plates containing NBM medium (MS medium supplemented with 1 mg / l of 6-benzylamino purine (6-BAP), and 0.1 mg / l of naphthalene acetic acid (NAA) .After 2 days, they were transferred. tissues cultured in culture flasks containing the NBM medium supplemented with carbenicillin (500 mg / l) and kanamycin (100 mg / l) Five weeks later, 1 yolk per leaf disc was transferred over NBM medium supplemented with carbenicillin (200 mg / l) and kanamycin (100 mg / l) After 2-3 weeks, the yolks with roots were transferred to fresh medium, if required, 2 cuttings from each yolk were transferred to separate flasks. tissue culture pattern, and the other was transferred to the soil for growth in the greenhouse after rooting.

Using this transformation method, all four vectors were introduced into the tobacco, and primary transformants resistant to kanamycin were generated. There were 53 primary transformants generated for constitutive CrylA (c), 54 for constitutive CryV, 73 for CrylA (c) inducible, and 62 for inducible CryV. EXAMPLE 6 Extraction of leaf DNA for PCR reactions. Samples were taken from leaves of 3-4 week old plants, grown in sterile conditions. Leaf discs about 5 mm in diameter were ground for 30 seconds in 200 μl extraction buffer (0.5% sodium dodecyl sulfate (SDS), 250 mM in NaCl, 100 mM in Tris HCl (tris hydrochloride (hydroxymethyl) aminomethane), pH 8). The samples were subjected to centrifugation for 5 minutes at 13,000 rpm, and after that 150 μl of isopropanol was added to the same volume of the top layer. The samples were left on ice for 10 minutes, subjected to centrifugation for 10 minutes, at 13,000 rpm, and allowed to dry. Then, they were suspended again in 100 μl of deionized water. 2.5 μl was used for the PCR reaction, under the conditions described by Jepson et al., Plant Molecular Biology Report 9 (2), 131-138 (1991).

The generated primary transgenics were tested by PCR analysis, to identify plants that contained the full-length transgene: CrylA (c) Constitutive. Two PCR reactions were performed for these extracts, using the following pairs of primers: TMV1 5 'CTA CTC GAG TCG ACT ATT TTT ACA ACA ATT ACC AAC (SEQ ID NO.3) CRY1A2R 5' CGA TGT TGA AGG GCC TGC GGT A (SEQ ID NO.4) The PCR conditions were 35 cycles of 95 ° C, 1.2 minutes; 62 ° C, 1.8 minutes; 72 ° C, 2.5 minutes; and an extension of 6 minutes at 72 ° C.

CRY1A1 5 'GCA CCT CAT GGA CAT CCT GAA CA (SEQ ID NO.5) US 5' CAT CGC AAG ACC GGC AAC AG (SEQ ID NO.6) The PCR conditions were 35 cycles of 95 ° C, 0.8 minutes; 61 ° C, 1.8 minutes; 72 ° C, 2.5 minutes; and an extension of 6 minutes at 72 ° C. Nine primary transformants gave PCR products for both sets of primers; these and two negative lines in PCR were planted in soil in 7.5-centimeter pots in the greenhouse.

Cry V Constitutive Two PCR reactions were performed for these extracts, using the following pairs of primers: TMV1 (see above) CryVIR 5 'GCT GTA GAT GGT CAC CTG CTC CA (SEQ ID NO.7) The PCR conditions were 35 cycles of 94 ° C, 0.8 minutes; 64 ° C, 1.8 minutes; 72 ° C, 2.5 minutes; and an extension of 6 minutes at 72 ° C.

CryVl 5 'TGT ACA CCG ACG CCA TTG GCA (SEQ ID NO.8) NOS (see above) The PCR conditions were 35 cycles of 94 ° C, 0.8 minutes; 58 ° C, 1.8 minutes; 72 ° C, 2.0 minutes; and an extension of 6 minutes at 72 ° C. 24 primary transformants gave PCR products for both sets of primers; these and seven negative PCR lines were planted in soil in 15.24 cm (7.5 inch) pots in the greenhouse. CrylA (c) Inducible Three PCR reactions were carried out for these extracts, using the following pairs of primers: ALCR1 5 'GCG GTA AGG CTT TCA ACA GGC T (SEQ ID NO. 9)' NOS (as above) The PCR conditions were 35 cycles of 94 ° C, 1.0 minutes; 60 ° C, 1.0 minutes; 72 ° C, 1.5 minutes; and an extension of 6 minutes at 72 ° C. The primer pairs TMV1 / CRY1A2R, CRY1A1 / NOS were used as above. Forty-five plants gave PCR products for all starter sets; these and two negative lines in PCR were planted in soil, in pots of 15.24 cm (6 inches) in the greenhouse. CryV Inducible Sixty-two primary transformants have been generated, but PCR analysis has not been carried out. EXAMPLE 7 Western Blot Analysis 120 mg of leaf of 3-4 week old plants, cultured under sterile conditions at 4 ° C in 0.06 g of polyvinylpolypyrrolidine (PVPP) were ground to adsorb the phenolic compounds, and in 0.5 my extraction buffer solution (Tris 1 M HCl, 0.5 M EDTA (ethylenediamine tetraacetic acid), DTT (dithiothreitol) 5 mM, pH 7.8). Then 200 ml more extraction buffer was added. The samples were mixed, and then subjected to centrifugation for 15 minutes at 4 ° C. The supernatant was extracted, the protein concentration by the Bradford assay, using bovine serum albumin (BSA) as standard. The samples were kept at -70 ° C until required. Samples of 25 mg of protein were loaded with 33% v / v Laemmli dye (97.5% buffer solution (62.5 mM Tris HCl, 10% w / v sucrose, 2% w / v SDS, pH 6.8), 1.5% pyronine and 1% b-mercaptoethanol) on a SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (17.7% of 30: 0.174 acrylamide: bisacrylamide), after 2 minutes of boiling. The translation products were separated electrophoretically in the following buffer: (14.4% w / v glycine, 1% w / v SDS, 3% w / v Tris Base). Then, they were transferred onto nitrocellulose (Hybond-CÓ, Amersham) using an electroblotting procedure (Biorad unit) in the following transfer buffer (14.4% w / v glycine, 3% w / v Tris base, 0.2% p / v of SDS, 20% v / v of methanol) at 40 mV overnight. Equal protein loads were verified by staining nitrocellulose subjected to fresh transfer in 0.05% CPTS (copper phthalocyanine tetrasodium tetrasodium salt of tetrasulfonic acid) and 12 mM in HCl. The dye was then stripped from the bands by 2-3 rinses in 12 mM HCl solution, and the excess dye was removed with 0.5 M NaHC03 solution for 5-10 minutes, followed by rinses in deionized water. The filters were blocked for 1 hour with TBS-Tween (2.42% w / v Tris HCl, 8% w / v NaCl, 5% Tween 20 (polyoxyethylene sorbitan monolaurate), pH 7.6) containing 5% w / v of BSA. They were then washed for 20 minutes in TBS-Tween supplemented with 2%. of BSA. Indirect immunodetections were performed with a 1: 200 dilution of a Cry IA (c) or Cry V antiserum as the first antibody, and with a 1: 1000 dilution of a rabbit anti-rabbit antiserum as a second antibody, associated with the peroxidase of Horseradish (HRP). Any excess antiserum was washed with TBS-Tween supplemented with 2% w / v BSA. Detection by ECL (enhanced chemiluminescence) was performed using the protocols described by Amersham. Any background noise was eliminated by additional washes of the membranes in the solution mentioned above. The latter was then subjected to detection by ECL. An estimate of the expression level of the B. thuringiensis gene was performed on the LKB laser densitometer 2222.-020 Ultroscan XL (Pharmacia). A helium-neon laser beam (wavelength 633 nm) scanned the autoradiography of a band 2.4 mm wide in the middle of the band corresponding to the products of the translation. Each maximum was characterized by its area, determined by the inner area of the curve of the absorbance function of the beam position.

EXAMPLE 8 Northern Blot Analysis Total RNA was fractionated on a 1.2% agarose gel containing 2.2 M formaldehyde. After electrophoresis, the RNA was transferred onto a Hybond-N membrane (Amersham) by capillary transfer in 20X SSPE. The RNA was fixed to membranes using a combination of UV binding Strata (Stratagene) and baked for 20 minutes at 80 ° C. The cDNA probes cut from pBluescript SK 'by digestion with EcoRI, were labeled with a 32PdCPT, using a protocol of random initiation, described by Feinberg and Vogelstein. The prehybridizations were performed in 5X SSPE, 0.1% SDS, 0.1% Marvel (dry milk powder), 100 mg / ml denatured salmon sperm DNA, for 4 hours at 65 ° C. Hybridizations were performed in the same buffer solution, which contained a probe marked at 65 ° C for 12-24 hours. Filters were washed at 65 ° C in 3X SSC with 0.1% SDS for 30 minutes, and once at 0.5 X SSC with 0.1% SDS for 30 minutes before autoradiography at -80 ° C. Insect Feeding Tests The effectiveness of the present invention can be conveniently tested by feeding leaves of transgenic plants containing the constructs of the present invention to insect larvae, both in the presence or absence, as a control, of the inducer.

EXAMPLE 9 Primary Selection A primary selection was made by cutting leaves from the plants, and cutting a number of leaf pieces of 1 cm2. The replicas were placed separately on agar at 0.75%, and each one was infested with approximately 10 sterilized eggs of Heliotis virescens. The leaf discs were covered and incubated at 25 ° C, and 70% RH for 5 days before qualifying the effects of feeding the larvae. The leaf damage was assigned a rating in the range from 0 to 2 in increments of 0.5; 2 denotes that there was no damage to the leaf (total protection against feeding the insect), and 0 implies that the leaf disc was completely consumed. Leaves were cut from all primary transformants of the tissue culture of constitutive CryLA (c) and wild-type tobacco, and tested for the effect on Heliotis virescens as described above. The results are shown in Table 2 below: TABLE 2 15 20 In typical bioassay experiments, wild-type (wt) tobacco mainly gave an average rating of less than 0.5. EXAMPLE 10 Primary Analysis - Repetition of the Test Eleven of the plants with constitutive CryLA (c) grown in the greenhouse and the wild type tobacco were retested. This was to demonstrate that plants with constitutive CrylA (c) that had been grown on land under greenhouse conditions for three weeks after tissue culture were also showing reduced leaf damage by Heliothis virescens.

TABLE 3 EXAMPLE 11 Primary Analysis with Primary Transformants with CryV The leaves of the primary transformants were tested with constitutive CryV and wild-type tobacco by the method described above. The damage supported by the cut sheet pieces is recorded below in Table 4.

TABLE 4 EXAMPLE 12 Secondary Analysis To verify the data obtained from the primary analysis, a secondary analysis was performed on transgenic lines, on larger pieces of leaves, using third stage larvae. Tobacco leaves were cut from the plant, and stored on ice for up to one hour. Leaf discs 40 mm in diameter were cut and placed, with the cuticle down, on 3% agar in 50 mm plastic plates. Larvae of Heliothis zea of third stage, raised with artificial diet of LSU for five days at 25 ° C were weighed and infested on each leaf disc, one per disc. After the infestation, the caps were placed on the plates, and stored at 25 ° C under diffuse light. The treatments were estimated after 3 days to determine the mortality, stage of development and% leaf disk eaten. The larvae were weighed in the infestation and after 3 days. TABLE 5 EXAMPLE 13"Inducible Insecticidal Activity Forty-five PCR-positive lines of CrylA (c) inducible, two PCR-negative lines and wt tobacco in 6-inch pots were soaked into the roots with 100 ml of 5% ethanol 28 hours later, 4 replicas of small pieces of leaves were extracted and infested with Heliothis virescens eggs The results are shown below (Table 6) Of the 45 lines grown in the presence of ethanol, 66% showed a total resistance on the selection test "primary to Heliothis virescens. To demonstrate that the plants were inducible and not expressing constitutive, leaves were cut 8 days later than 7 of the lines that scored high, and were infested with eggs of Heliothis virescens. Previous data from a reporter gene driven by the 35SalcRalcA switch promoter showed that CAT protein levels were at their maximum at 24/48 hours, and was in decline after 48 hours (Figure 1). Other data (not shown) showed that CAT protein was not detected 9 days after induction. Table 7 demonstrates that in the absence of irrigation with ethanol, it was found that the mortality levels were comparable to those seen with the wild-type control.

TABLE 6 TABLE 7 Several lines were selected for secondary analysis, to test the effect of induction on insect feeding, together with line 10 of constitutive CrylA (c) and wt tobacco as controls. Ten pieces of leaf were cut for each line of primary transformants 12 days after they had been induced by soaking roots with 100 ml of 5% ethanol, and placed on 3% agar in 50 mm plates with lids, and were incubated overnight at 25 ° C and 60% humidity. It was expected that the expression of the CrylA protein (c) would be low or not detectable after 12 days. The plants were then flooded in the roots with 100 ml of 5% ethanol. Leaves were cut 22 hours later, and 10 pieces of 40 mm sheet were trimmed and placed on 3% agar in 50 mm plates with lid. Five uninduced and 5 ethanol induced leaf disks were infested with Heliothis zea third instar larvae, and 5 of each infested with Heliothis virescens were cultured as described above. Table 8 demonstrates that wild-type controls in the presence or absence of ethanol show a high percentage of eaten leaf disc, while 35S controls show good insect control under both chemical regimes. The transgenic lines containing the constructions of Ale Cry lA (c) showed poor insect control in the absence of ethanol treatment. Table 8 shows that induction with ethanol gives an insect control comparable to that seen in the control of 35S Cry I A (c).

TABLE 8 LIST OF SEQUENCES GENERAL EDUCATION: (i) APPLICANT: (A) NAME: ZENECA LIMITED (B) ADDRESS: 15 Stanhope Gate (C) CITY: London (E) COUNTRY: RU (F) POSTAL CODE: W1Y 6LN (ii) TITLE OF THE INVENTION: DNA CONSTRUCTS (iii) NUMBER OF SEQUENCES: 9 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMMING ELEMENTS: Patentln Relay # 1.0, Version # 1.30 (EPO) (vi) DATA - OF THE PRIOR APPLICATION: (A) NUMBER OF THE APPLICATION: GB 9516241.8 (B) DATE OF SUBMISSION: August 08, 1995 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: CATCTCGAGT CGACTATTTT TACAACAATT ACCAAC 36 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: CTAGGTACCG TCGACGGATC CGTAAGATCT GGTGTAATTG TAAATAGTAA TTG 53 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid 4 (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CTACTCGAGT CGACTATTTT TACAACAATT ACCAAC 36 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: CGATGTTGAA GGGCCTGCGG TA 22 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GCACCTCATG GACATCCTGA ACA 23 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TIPOi nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CATCGCAAGA CCGGCAACAG 20 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear ( ii) TYPE OF. MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: GCTGTAGATG GTCACCTGCT CCA 23"(2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: TGTACACCGA CGCCATTGGC A 21 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GCGGTAAGGC TTTCAACAGG CT 22 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (12)

1. CLAIMS 1. A chemically inducible gene expression cassette in plants, characterized in that it comprises a first promoter operably linked to a regulatory sequence that is derived from the alcR gene, and encodes a regulatory protein, and an inducible promoter, linked in a manner operative to an objective gene that encodes a protein that is harmful to insects, or whose expression induces a metabolic pathway that produces a metabolite that is harmful to insects, the inducible promoter is activated by the regulatory protein in the presence of an exogenous inducer effective, with which the application of the inducer causes the expression of the target gene.
2. 2. A chemically inducible plant gene expression cassette according to claim 1, characterized in that the target gene encodes an orally active insecticidal protein.
3. 3. An inducible-inducible plant gene cassette according to claim 2, characterized in that the orally active insecticidal protein is at least part of a Bacillus thuringiensis d-endotoxin.
4. 4. A cassette for the expression of plant genes according to any of claims 1 to 3, characterized in that the inducible promoter is derived from the promoter of the alcA gene.
5. 5. A cassette for the expression of plant genes according to any of claims 1 to 4, characterized in that the inducible promoter is a chimeric promoter.
6. 6. A plant cell characterized in that it contains a cassette for expression of plant genes according to any of the preceding claims.
7. 7. A plant cell according to claim 6, characterized in that the expression cassette of plant genes is stably incorporated in the genome of the plant.
8. 8. A plant tissue characterized in that it comprises a plant cell according to any of claims 6 and 7.
9. 9. A plant characterized in that it comprises a plant cell according to any of claims 6 and 7.
10. 10. A plant characterized in that it is derived from a plant according to claim 9.
11. 11. A seed characterized in that it is derived from a plant according to any of claims 9 and 10.
12. 12. A method for controlling insects, characterized in that it comprises transforming a plant cell with the expression cassette of plant genes according to any of claims 1 to 5.