KR19990036251A - DNA Structures - Google Patents

Abstract

a first promoter derived from the alc R gene and functionally linked to a regulatory sequence that encodes a regulatory protein, and a protein that induces metabolic pathways that produce insect metabolites that damage insects or whose expression damages plants A chemically-inducible plant gene expression cassette comprising an inducible promoter functionally linked to an encoding target gene.

Description

DNA Structures

The present invention relates to a DNA construct and a plant incorporating the same. In particular, the present invention relates to promoter sequences and their use in the expression of genes that impart pesticidal activity to plants.

The development of plant biotechnology has resulted in the production of transgenic plants that are protected against the larvae's eating of insects.

Many organisms produce proteins that are harmful to insects, among them Bacillus thuringiensis, which produces the crystal-associated protein δ endotoxin, which digests and kills larvae. However, this creature is not harmful to animals. Many strains of B. thuringiensis are active against insect plagues and the genes encoding the insect endotoxins have been characterized. B. Turingiensis δ endotoxin is particularly pesticidal for Lepidopteran larvae (CryI-type protein), specifically pesticidal for Coleopteran larvae (CryIII-type protein), and Lepidotera and Includes specificity to both choloptera (CryV). Chimeric proteins, including at least a portion of B. thuringiensis, have also been proposed for the purpose of improving the properties of endotoxins in some way, such as by increasing the insecticidal rate. Transgenic plant expression genes that encode the insecticidal endotoxin are also known.

Other methods of damaging insects include stimulating plant metabolic pathways that produce insecticidal metabolites.

The present applicant proposes a system in which a gene encoding an active pesticidal protein, such as B. thuringiensis endotoxin, is expressed in an inducible manner, depending on the application of specific activating chemicals. Alternatively, induction of pathways that produce metabolites that damage insects can be achieved. This approach has many advantages, including the following:

Plant constitutive expression of insect resistant genes, such as B. thuringiensis endotoxin, significantly increases the pressure of choice for insect resistant species. Inducible regulation of insect resistant genes may reduce the risk of developing a resistant plague. Decrease. For example, insecticidal gene expression can be induced only at the time of growth season in need of protection. Additionally, switchable insect resistance can be used as part of an integrated pest management system in which chemical treatments that induce pesticidal gene expression can be used alternately to standard pesticidal pesticide treatments.

2. Overexpression from a strong intrinsic promoter risks a fatal effect on plant growth, resulting in excessive germination and poor yield. Inducible expression can reduce the risk of fatal effects, since the transgene can be expressed in a short period of time, thereby avoiding a sensitive point in development.

3. Switch chemicals are added to standard pesticidal agents to confer both chemical and genetic effects, killing insects by two independent mechanisms.

The present application has developed an inducible gene regulatory system (gene switch) based on alcR regulatory proteins from Aspergillus nidulans. Aspergillus nidulans are derived from the alcA promoter in the presence of certain alcohols and ketones. Activates gene expression. This system is described in WO93 / 21334, which is incorporated herein by reference.

The alcA / alcR gene activation system from the fungus Aspergillus nidulans is also well characterized. A. The ethanol use pathway in nidulans is responsible for the degradation of alcohols and aldehydes. Three related genes are known in the ethanol pathway. The genes alcA and alcR are present in close proximity to linking group VII and aldA is located in linking group VIII (Pateman JH et al., 1984, Proc. Soc. Lond., B 217: 243-264; Sealy-Lewis HM And Lockington RA, 1984, Curr. Genet. 8: 253-259). The gene alcA encodes ADHI in A. nidulans and aldA encodes AldDH, which causes AldDH enzymes to utilize ethanol. Expression of both alcA and aldA is induced by ethanol and many other inducers (Creaser EH et al., 1984, Biochemical J., 255: 449-454) via the transcription activator alcR. The alcR gene and co-inducers contribute to alcA and aldA expression, because many mutations and deletions in alcR result in the multifaceted loss of ADHI and aldDH (Felenbok B et al., 1988, Gene, 73: 385-). 396; Fatman et al., 1984; Sealy-Lewis & Lockington, 1984). The ALCR protein activates expression from alcA by binding to three specific sites in the alcA promoter (Kulmberg P et al., 1992, J. Biol. Chem, 267: 21146-21153).

The alcR gene was cloned (Lockington RA et al., 1985, Gene, 33: 137-149) and sequenced (Felenbok et al., 1988). Expression of the alcR gene can be induced, autoregulated and directed to glucose inhibition mediated by CREA repressors (Bailey C and Arst HN, 1975, Eur. J. Biochem. 51: 573-577; Lockington RA et al., 1987, Mol Microbiolgy, 1: 275-282; Dowzer CEA and Kelly JM, 1989, Curr. Genet. 15: 457-459; Dowzer CEA and Kelly JM, 1991, Mol. Cell. Biol. 11: 5701 -5709). The ALCR regulatory protein contains six cysteines near the N terminus coordinated in a zinc binuclear cluster (Kulmberg P et al., 1991, FEBS Letts., 290: 11-16). This cluster is related to a highly conserved DNA binding domain found in other Streptococcus transcription factors. Transcription factors GAL4 and LAC9 are known to be binuclear complexes with a cloverleaf type 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 Cys-3 and Cys-4. ALCR positively activates its own expression by binding to two specific sites in the 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; Gwynne D et al., 1987, Gene, 51: 205-216; Pickett et al., 1987, Gene, 51: 217-226).

A. Nidulans has two different alcohol dehydrogenases. ADHII is present in mycelia grown in non-derived medium and is inhibitable by the presence of ethanol. ADHII is encoded by alcB and is also under the control of alcR (Sealy-Lewis & Lockington, 1984). Tertiary alcohol dehydrogenase was also cloned by supplementation of adh-strains of S cerevisiae. This gene alcC is located in linking group VII but not in alcA and alcR.

The gene alcC encodes ADHIII and uses very little ethanol (McKnight GL et al., 1985, EMB0. J., 4: 2094-2099). ADHIII has been shown to be involved in the survival of A. nidulans during the period of anaerobic stress. The expression of alcC is not inhibited by the presence of glucose, suggesting that it will not be under the control of alcR (Roland LJ and Stromer JN, 1986, Mol. Cell. Biol. 6: 3368-3372).

In summary, A. nidulans express dehydrogenase I (ADHI) enzyme encoded by the alcA gene only when grown in the presence of various alcohols and ketones. This induction is followed through and essentially expressed through regulatory proteins encoded by alcR. In the presence of an inducer (alcohol or ketone), the regulatory protein activates the expression of the alcA gene. Regulatory proteins also stimulate their own expression in the presence of inducers. This means that high levels of ADH enzyme are produced under inducible conditions (eg when alcohols or ketones are present). In contrast, the alcA gene and its product, ADHI, are not expressed in the absence of inducers. Expression of alcA and production of its enzymes are also inhibited in the presence of glucose.

Therefore, the alcA gene promoter is an inducible promoter and is activated by an alcR regulatory protein (eg, by said protein / alcohol or protein / ketone combination) in the presence of an inducer. The alcR and alcA genes (including the respective promoters) were cloned and sequenced (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).

Alcohol dehydrogenase (adh) genes have been studied in certain plant species. In maize and other grains, these genes are switched by anaerobic conditions. The promoter region of the adh gene from corn contains 300 bp regulatory elements for expression under anaerobic conditions. However, no equivalent to alcR regulatory protein was found in any plant. Therefore, a gene regulation system of alcR / alcA type is unknown in plants. Essential expression of alcR in plant cells does not result in activation of endogenous adh activity.

According to a first aspect of the invention, a first promoter derived from the alcR gene and functionally linked to a regulatory sequence encoding a regulatory protein and a metabolite that damages insects or whose expression damages plants is produced. There is provided a chemically-inducible plant gene expression cassette comprising an inducible promoter functionally linked to a target gene that encodes a protein that induces a metabolic pathway, said inducible promoter regulated in the presence of an effective exogenous inducer. Activated by a protein, wherein the inducer causes expression of the target gene.

When the target gene encodes an insect-damaged protein, it is advantageous that such protein is orally active. An example of an orally active pesticidal protein is B. thuringiensis δ endotoxin, and therefore, the target gene can encode at least a portion of B. thuringiensis δ endotoxin. have.

We have found that our alcA / alcR switch is particularly suitable for encoding B. thuringiensis δ endotoxins for at least the following reasons.

The alcA / alcR switch was developed to control high levels of gene expression. In addition, the regulatory protein alcR is preferably manipulated from a strong intrinsic promoter such as polyubiquitin. High levels of induced transgene expression can be achieved, comparable to that from a strong intrinsic promoter such as 35 CaMV.

1 shows the time course of marker gene expression (CAT) after application of induction chemicals. This study shows a sharp increase in CAT expression (2 hours) after application of inducible chemicals. That is, the immediate, initial kinetics of induction occurs by the expression of the regulatory genes in an intrinsic manner, and therefore there is no time delay while synthesis of transcription factors is taking place. In addition, the applicant chose a simple two component that does not depend on the complex signal transduction system.

Applicants tested the specificity of alcA / alcR using a wide range of solvent systems used in agricultural practice. Hydroponic seedling systems have found that ethanol, butan-2-ol and cyclohexanone all induce high levels of reporter gene expression (FIG. 2). Although various alcohols and ketones listed in Table 1 and used in agricultural practice have been applied as sprays, only ethanol induces high levels of reporter protein activity (FIG. 3). This is important because improper induction of transgenes does not have an opportunity to be exposed to the formulation solvent. Ethanol is a common component of agrochemical formulations and, therefore, with proper spray control, is considered a specific inducer of alcA / alcR gene switches in field situations.

TABLE 1

1. Isobutyl methyl ketone

2. Fenchone

3. 2-heptanone

4. Di-isobutyl ketone

5. 5-Methyl-2-hexanone

6. 5-Methylpentane-2,4-diol

7. Ethyl Methyl Ketone

8. 2-pentanone

9. Glycerol

10.γ-butyrolactone

11.Dacetone alcohol

12. Tetrahydrofurfuryl Alcohol

13. Acetonyl Acetone

14.JF5969 (cyclohexanone)

15.N-methyl pyrrolidone

16. Polyethylene Glycol

17. Propylene Glycol

18. Acetophenone

19.JF4400 (Methylcyclohexanone)

20. Propan-2-ol

21.Butan-2-ol

22. Acetone

23. Ethanol

24. dH ₂ O

Extensive biological and abiotic stresses such as pathogen infection, fever, cold, drought, wounds, and floods have failed to induce alcA / alcR switches. In addition, a wide range of non-solvent chemical treatments, such as salicylic acid, ethylene, asisic acid, auxin, gibberellic acid, and various agricultural chemicals all fail to induce the alcA / alcR system. It was.

The present invention is not limited to specific endotoxins, but is also applicable to chimeric endotoxins.

The first promoter is essentially or tissue-specific, developmentally-programmed or even inducible. The regulatory sequence, alcR gene, can be obtained from Aspergillus nidulans and encodes alcR regulatory protein.

The inducible promoter preferably comprises an alcA gene promoter obtainable from Aspergillus nidulans, or a core promoter region derived from the regulatory sequence of the alcA promoter and a gene promoter operating in plant cells (any plant gene promoter) ) Is a "virtual" promoter. The alcA promoter or related hypothetical promoter is activated by the alcR regulatory protein when an alcohol or ketone inducer is applied.

Inducible promoters can also be derived from the aldA gene promoter, alcB gene promoter or alcC gene promoter, which can be obtained from Aspergillus nidulans.

The inducer may be any effective chemical (such as alcohol or ketone). Suitable chemicals for use with alcA / alcR-derived cassettes include butan-2-one (ethyl methyl ketone), cyclohexanone, acetone, butan-2-ol, 3-oxobutyric acid, propan-2-ol, Such as those listed by Creaser et al. (1984, Biochem. J., 225, 449-454), such as ethanol.

Gene expression cassettes are responsive to applied exogenous chemical inducers that allow external activation of expression of target genes regulated by the cassette. The expression cassette is highly regulated and suitable for general use in plants.

Two portions of the expression cassette may be on the same construct or on separate constructs. The first portion contains a regulatory cDNA or gene sequence subcloned into an expression vector with a plant-acting promoter that controls its expression. The second portion includes at least a portion of the inducible promoter that controls expression downstream of the target sequence. In the presence of a suitable inducer, the regulatory protein produced by the first portion of the cassette activates expression of the target gene by stimulating an inducible promoter in the second portion of the cassette.

In practice, the construct or construct comprising the expression cassette of the invention will be inserted into the plant by transformation. Expression of the target gene in the construct is under the control of the chemically switchable promoter of the invention, which can then be activated by applying a chemical inducer to the plant.

Any transformation method suitable for the target plant or plant cell may be used, including infection, electrophoresis, microinjection of cells and protoplasts, microinjection traits with Agrobacterium tumefaciens containing recombinant Ti plasmids Conversion and pollen tube transformation.

Examples of commonly modified plants that can be produced include outdoor crops, cereals, fruits and vegetables, such as: canola, sunflower, tobacco, sugar beets, cotton, soybeans, corn, wheat, barley, rice, sorghum, tomatoes, mangoes. Contains peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage and onion.

The invention further provides plant cells containing the 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 plant tissues or plants comprising such cells, and plants or seeds derived therefrom.

The present invention further provides a method of controlling plant gene expression, wherein the foregoing method comprises: a first promoter derived from the alcR gene and functionally linked to a regulatory sequence encoding a regulatory protein and / or damaging an insect, Or a chemically-inducible plant gene expression cassette comprising an inducible promoter whose expression is functionally linked to a target gene that encodes a protein that induces a metabolic pathway that produces a damaging metabolite. Transforming the cells.

Various preferred features and embodiments of the invention will be described by the following non-limiting examples and figures, wherein:

1 shows the time course of induction of AR10 separation population with 7.5% ethanol;

FIG. 2 shows CAT activity in the AR 10-30 homozygous line for roots moistened with various chemicals; FIG.

FIG. 3 shows CAT activity in the AR 10-30 homozygote line for roots moistened with various chemicals; FIG.

4 shows the production of a 35 S regulating structure;

5 shows the production of the reporter structure;

6 shows a switchable insect resistance vector;

7 shows the sequence of an optimized Cry IA (c) gene;

8 shows restriction sites of the optimized Cry IA (c) gene;

9 shows the sequence of a Cry V gene;

10 shows vector 5129 base pairs containing Cry V gene;

11 shows the sequence of a vector pMJB1; And

12mss is a map of the vector pJRIi.

Example 1

Preparation of alcR Regulating Structures

The alcR genomic DNA sequence is known, allowing the isolation of a sample of alcR cDNA.

alcR cDNA was cloned into the expression vector, pJR1 (pUC). pJR1 contains the cauliflower mosaic virus 35S promoter. This promoter is an intrinsic plant promoter and continuously expresses regulatory proteins. nos polyadenylation signal is used in the expression vector.

4 shows the production of 35S regulatory constructs by ligation of alcR cDNA into pJR1. Subsequent restriction with BamHI of the alcR cDNA clone was followed by electrophoresis, cleavage, and separation of 2.6 Kb fragments in agarose gel. The fragments were then ligated into BJHIl and phosphorylated into a phosphorylated pJR1 vector to prevent recycling. Therefore, the alcR gene was placed under the control of the CaMV 35S promoter and the nos 3 ′ polyadenylation signal in this “35S alcR” construct.

Example 2

Production of alcT-CAT Reporter Structure Containing Virtual Promoter

Plasmid pCaMVCN contains the bacterial chloramphenicol transferase (CAT) reporter gene between the 35S promoter and the nos transcription terminator construct (“35S-CAT”).

The alcA promoter was subcloned into the vector pCaMVCN to produce an "alcA-CAT" construct. Fusion of the alcA promoter moiety and the 35S promoter moiety created a virtual promoter that allows gene expression under its control.

5 shows the production of the reporter structure. The alcA promoter and the 35S promoter have the same TATA box, which is used to link two promoters together using recombinant PCR technology: the 246 base pair region from the alcA promoter and the 5 ′ end of the CAT gene from pCaMVCN (of the 35S promoter Containing a portion of the -70 core region) were amplified separately and then conjugated together using PCR. Recombinant fragments were then digested using BamHI and HindIII. The pCaMVCN vector was partially constrained with BamHI and HindIII and then electrophoresed so that the correct fragment was separated and ligated into the recombinant fragment.

The ligation mixture was transformed into E. coli and cultured on abundant agar media. Plasmid DNA was isolated by miniprep from the resulting colonies and recombinant clones were recovered by size electrophoresis and restriction mapping. Ligation junctions were sequenced to check that correct recombination was recovered.

Example 3

Gene structure

The applicant has created the following structure, summarized in FIG.

Vector 1 contains a 35S CaMV promoter fused to a tobacco mosaic virus omega sequence translational enhancer (TMV) Bacillus thuringiensis Cry I A (c) gene and a nopoline synthase (NOS) terminator.

Vector 2 is the same as Vector 1 except that the Bacillus thuringiensis Cry I A (c) gene is replaced with the B. thuringiensis Cry V gene.

Vector 3 contains alcR regulatory protein gene from Aspergillus nidulans, alcA promoter region, TMV Enhancer CryIA (c) and nos terminator, piloted from the 35S CaMV promoter.

Vector 4 is the same as Vector 3, except that the Cry I A (c) gene is replaced with the CryV gene.

The Cry I A (c) gene is an optimized Lepidotera specific synthetic sequence that encodes Bacillus thuringiensis and is shown in FIGS. 7 and 8. The sequence was obtained from Pamela Green Laboratories, University of Michigan.

The Cry V gene is a novel Bacillus thuringiensis endotoxin that is pesticidal to coleopteran and lepidopteran larvae, which is described in International Patent Publication No. WO90 / 13651. The Cry V gene is a modulated synthetic sequence and is optimized for plant code use and has an RNA instability region that is removed. This is shown in FIGS. 9 and 10.

Example 4

Manufacture of vector

Vector 1-Intrinsic Cry 1A (c)

The PCR primers add the SalI site (see forward Origo nucleotide) adjacent to the XhoI site, destroy the NcoI site, and add the Sal I and Bgl II sites at the reverse oligonucleotide, while the TMV omega sequence in pMJB1 ( Designed to amplify).

Forward oligonucleotide (SEQ ID NO: 1)

Reverse oligonucleotide (SEQ ID NO: 2)

PCR was performed with forward and reverse primers using template pMJBI plasmid DNA. The resulting PCR product was cloned into pTAg vector (LigATor kit, R & D systems); It was then released into Asp718 and Xho I digestion and cloned into XhoI / Asp718 digested pMJBI (FIG. 10) to form pMJB3. pMJB1 is based on pIBT 211 containing a CaMV35 promoter with overlapping enhancers (duplicate enhancers replace the tobacco etch virus 5 ′ non-translating leader, linked to the tobacco mosaic virus translation enhancer sequence), nopalin Terminated with synthetase poly (A) signal (nos).

The Cry IA (c) synthetic gene was cleaved as a BglII Bam HI fragment and cloned into pMJB3. Fragments containing the enhanced 35S CaMV promoter TMV omega sequence, Cry IA (c) and nos terminators were isolated using Hind III and EcoRI. The resulting pieces were ligated into EcoRI / HIndIII digested pJRIi (FIG. 12) to generate Bin19 based plant transformation vectors.

Vector 2-Essential Cry V

pMJB3 was cleaved with HindIII and a HindIII-EcoRI-HindIII linker was inserted. The resulting vector was then digested with BamHI and a fragment containing the CryV gene as a BamHI fragment was inserted. CryV genes were oriented using a combination of restriction digestion and sequencing. EcoRI fragments from the obtained vector were transferred to a Bin 19 base vector containing the enhanced 35CaMV promoter, TMV omega sequence, Cryv gene and nos terminator and containing FRIRiMCS, ie, the pUC18 multiple cloning site.

PMJB3 containing Cry 1A (c) is cleaved with Sal I to generate a fragment containing TMV omega sequence fused to Cry 1A (c) gene. The resulting pieces were cloned into SalI cut palc A CAT and oriented by restriction digestion. Fragments containing the alcA promoter, Cry 1A (c) gene and nos terminator fused to TMV omega sequence were cleaved using HIndIII and, together with the alcAcat reporter cassette removed by HindIII digestion, ie alcR cDNA Is transferred to a Bin 19 based vector containing a 35CaMV promoter fused to

Vector 4-derived CryV

PMJB3 containing the Cry V gene was cleaved with SalI to generate a fragment containing the TMV omega sequence fused to the CryV gene. The pieces obtained were cloned into Sal I palcACAT and oriented by restriction digestion and sequencing. Two fragments containing the alcA promoter CryV gene and nos terminator were released by digestion with HindIII. Three types of ligation of two HidIII fragments were performed by inserting the alcACryVnos cassette into p35alcRalcAcat digested with HindIII, removing the alccat cassette. The correct set of cassettes was confirmed by restriction digest Southern blotting and sequencing.

Example 5

Plant transformation

Leaf Transformation by Agrobacterium

Transformation was performed according to the method described by Bevan 1984. Aseptic cultures of 3-4 weeks old of tobacco (Nicotiana tabacum cv Sansum) were grown on MS and used for transformation. The edges of the leaves were cut off and the leaves were torn into pieces. They were then placed into transformed Agrobacterium cells containing pJR1RI plasmid and suspended for 20 minutes. The pieces were placed on a plate containing NBM medium (MS medium supplemented with 1 mg / l 6-benzylaminopurine (6-BAP), and 0.1 mg / l naphthalene acetic acid (NAA)). After 2 days, the implant was transferred to a culture pot containing NBM medium supplemented with carbenicillin (500 mg / l) and kanamycin (100 mg / l). After 5 weeks, 1 shoot per leaf disc was transferred onto NBM medium supplemented with carbenicillin (200 mg / l) and kanamycin (100 mg / l). After 2-3 weeks, rooted shoots were transferred to fresh medium. If necessary, two cuts from each shoot were transferred to separate pots. One will be preserved as a tissue culture stock and the other will be rooted and transferred to soil for growth in the glasshouse.

Using this transformation method, four vectors were introduced into tobacco and kanamycin-resistant primary transformants occurred. 53 primary transformants generated for intrinsic Cry1A (c), 54 transformants for intrinsic CryV, 73 transformants for inducible Cry1A (c) and 62 transformants for inducible CryV There was a sieve.

Example 6

Leaf DNA Extraction for PCR Reactions

Leaf samples were taken from 3-4 week old plants grown in sterile conditions. 5 mm diameter leaf disks were ground for 30 seconds in 200 μl extraction buffer (0.5% sodium dodecyl sulfate (SDS), 250 mM NaCl, 100 mM Tris HCl (tris (hydroxymethyl) aminomethane hydrochloride), pH8). This sample was centrifuged at 13,000 rpm for 5 minutes, and then 150 μl isopropanol was added to the same volume top layer.

The sample was placed on ice for 10 minutes, centrifuged at 13, 000 rpm for 10 minutes, and left to dry. They were then resuspended in 10 μl 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 containing full length transgenes.

Intrinsic Cry1A (c)

Two PCR reactions were performed on these extracts using the following primer pairs.

PCR conditions were 35 cycles of 95 ° C, 1.2 minutes, 61 ° C 1.8 minutes, 72 ° C 2.5 minutes, and 72 ° C 6 minutes.

Nine primary transformants provided the PCR product for each primer set; these two PCR negative lines were transplanted into soil in 7.5 "ports in the glasshouse.

Intrinsic CryV

Two PCR reactions were performed on these extracts using primer pairs of sugar.

TMV1 (see above)

PCR conditions were 35 cycles of 94 ° C 0.8 minutes, 64 ° C 1.8 minutes, 72 ° C 2.5 minutes, and 72 ° C 6 minutes.

PCR conditions were 35 cycles of 94 ° C, 0.8 minutes, 58 ° C 1.8 minutes, 72 ° C 2.0 minutes, and 72 ° C 6 minutes.

Twenty-four primary transformants provided PCR products for both primer sets; these seven PCR negative lines were implanted in soil within 7.5 "ports in the glasshouse.

Inducible Cry1A (c)

Three PCR reactions were performed on these extracts using the following primer pairs.

NOS See above

PCR conditions were 35 cycles of 94 ° C 1.0 minutes, 60 ° C 1.0 minutes, 72 ° C 1.5 minutes, and 72 ° C 6 minutes.

Three primer pairs TMV1 / CRY1A2R, CRY1A1 / NOS were used as above.

Twenty-five primary transformants provided PCR products for all primer sets; these seven PCR negative lines were transplanted into soil in 6 "ports in the glasshouse.

Inducible CryV

Although 62 primer transformants were issued, PCR analysis was not currently performed.

Example 7

Western blot analysis

120 mg of leaves from 3-4 week-old plants grown under sterile conditions were treated at 4 ° C. with polyvinylpolypyrrolidone (PVPP) and 0.5 ml extraction buffer (1M Tris HCl, 0.5 M EDTA) for adsorbing phenolic mixtures. tetraacetate), 5 mM dithiothreiol (DTT), pH 7.8). Then 200 l additional extraction buffer was added. The samples were mixed and centrifuged at 4 ° C. for 15 minutes. The supernatant was removed and the concentration of protein was measured by Bradford assay using bovine serum albumin (BSA) as standard. The sample was stored at -70 ° C until needed.

Sample of 25 mg of protein with 33% vv Laemmli dye (97.5% Laemmli buffer (62.5 mM Tris HCl, 10% w / v sucrose, 2% w / v SDS, pH 6.8), 1.5% pyronin y and 1% b-mercaptoethanol was loaded on SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel (17.7% 30: 0.174 acrylamide: bisacrylamide) and disconnected after 2 minutes.

Translation products were electrophoretically separated in the following buffer (14.4% w / v glycine, 1% w / v SDS, 3% w / v Tris Base). Then they were electroblotting at 40 mV overnight in the following blotting buffer (14.4% w / v glycine, 3% w / v Tris Base, 0.2% w / v SDS, 20% v / v methanol). Biorad unit) was transferred onto nitrocellulose (Hybond-CO, Amersham).

Equal loading of the protein was checked by staining fresh blot nitrocellulose in 0.05% CPTS (copper phtalocyanine tetrasulfonic acid, tetrasodium salt) and 12 mM HCl. The blot was then destained by rinsing in 12 mM HCl solution 2-3 times, and excess dye was removed by 0.5 M NaHCO 3 for 5-10 minutes and then rinsed with deionized water. The filter was blocked for 1 hour with TBS-Tween (2.42% w / v Tris HCl, 8% w / v NaCl, 5% Tween 20 (polyxyetheylene soritan monolaureate), pH7.6) containing 5% w / v BSA. . They were then washed for 20 minutes in TBS-Tween supplemented with 2% w / vBSA. Indirect immunodetection results in a 1: 2000 dilution of Cry IA (c) or CryV serum as primary antibody and a 1: 1000 dilution of rabbit anti-rabbit serum as secondary antibody, horseradish peroxidase. , HRP). Excess antiserum was washed with TBS-tween supplemented with 2% w / v BSA. Enhanced chemiluminescence (ECL) detection was performed using the protocol described by Amersham. All background was removed by washing the membrane in the above mentioned solution. The latter is then detected by ECL. Measurement of the expression level of the B. thuringiensis gene was performed on an LKB 2222-020 Ultroscan XL laser densitometer (Pharmacia). A helium-neon laser beam (wavelength 633 nm) is screened on a 2.4 mm wide radiographic band in the center band corresponding to the translation product. Each peak is characterized by its area and determined by internal software from a 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, RNA was transferred onto Hybond-N membranes (Amersham) by capillary blotting in 20X SSPE. RNA was fixed using a combination of UV layer linking (Stratagene) and baked at 80 ° C. for 20 minutes. CDNA probes cleaved from pBluescript SK digested with EcoRI were labeled as ³² PdCTP, using a random priming protocol described by Feinberg and Vogelstein. Prehybridization was performed at 65 ° C. for 4 hours in 5 × SSPE, 0.1% SDS, 0.1% Marvel (dry milk powder), 100 mg / ml denatured salmon sperm DNA. Hybridization was done for 12-24 hours at 65 ° C., in the same buffer containing labeled probes. The filter was washed for 30 minutes in 3 × SSC 0.1% SDS at 65 ° C. and once in −80 ° C., 0.5 × SSC 0.1% SDS for 30 minutes prior to radiography.

Insect breeding attempt

The effects of the present invention have been conveniently tested by feeding transgenic plants containing the inventive constructs to larvae of insects, both in the presence of inducers and in the absence of controls.

Example 9

Primary screen

The primary screen was performed by removing leaves from the plant and cutting them into 1 cm ² leaf pieces. Replicas were separately placed on 0.75% agar and each was infested with about 10 sterile Heliothis virescens eggs. The leaf disks were covered and incubated at 70 ° RH, 25 ° C. for 5 days before measuring the effects of larval rearing. Leaf damage was scored at a rate of 0.5 increase, ranging from 0 to 2; Two had no leaf damage (completely protected from insect feeding) and none of the leaf disks had been completely eaten.

Leaves from all essential Cry1A (c) tissue culture transformants and wild type tobacco were removed and tested for effect on heliotis nonresense as described above. The results are shown in Table 2:

In typical bioassay experiments, wild-type (wt) cigarettes mainly had an average score of less than 0.5.

Example 10

Primary screen-retest

Eleven essential Cry1A (c) plants and wild-type tobacco grown in the glasshouse were retested. This experiment also showed that the essential Cry1A (c) plants grown in soil in glasshouse conditions for three weeks after tissue culture were also tested for heliotis nonresense. It was proved that the leaf damage caused by

Example 11

Primary screen using CryV primary transformants

The leaves from the essential CryV primary transformants and wild type tobacco were tested by the method as described above. The damage inflicted by the cut leaf pieces is reported in Table 4 below.

Example 12

Secondary screen

To verify the data obtained on the primary screen, a secondary assay was performed on the transgenic line for larger leaf pieces, using a third larva.

Tobacco leaves were cut from plants and stored on ice within 1 hour. A 40 mm diameter leaf disc was cut and placed on 3% agar in a 50 mm plastic pot with the epidermis down. Third Heliothis Zea, which was grown on an LSU artificial diet for 5 days at 25 ° C., was weighed and infested with each leaf disc on each disc. Treatments were assessed for mortality, stage of development and% of leaf disk eaten after 3 days. The larvae were weighed at infestation and 3 days later.

Example 13

Inducible pesticidal activity

45 inducible Cry1A (c) PCR positive lines, 2 PCR negative lines and wild-type tobacco in 6 "pots were soaked with 100 ml of 5% ethanol. After 4 hours 4 replica small leaf fragments were removed and heliotis viresense The results were as follows (Table 5): Of the 45 lines grown in the presence of ethanol, 66% showed complete resistance in the first screen test for heliotis nonresense. The leaves were removed after 8 days from the high score line 7 and were invaded with heliotis nonresense eggs, to prove that they are intrinsic expressors dml previous data from the reporter genes seeded by the 35SalcRalcA switch promoter This peaked at 24/48 hours and decreased after 48 hours (Figure 1). It shows that quality has not been detected in nine days after the induction.

Table 7 demonstrates that mortality levels in the absence of ethanol irrigation were found to be comparable to those seen in wild-type controls.

Several lines were selected on the second screen to test the effect of induction of insect breeding on essentially Cry1A (c) line 10 and wild type tobacco as a control. Ten leaf pieces for each line were removed from the primary transformants 12 days after induction by root wetting with 100 ml of 5% ethanol and placed on 3% agar in a 50 mm pot with lid, at 25 ° C. Incubate overnight at 60% humidity. The expression of the Cry1A (c) protein was expected to be low or undetectable after day 12. The plant was then moistened with 10 ml, 5% ethanol. Five non-derived and five ethanol derived leaf discs were infested with third-age heliothio zea, five were heliothio nonresense and bred as above.

Sequence list

(1) General Information:

(i) Applicant:

(A) Name: ZENECA LIMITED

(B) Distance: 15 Stanhope Gate

(C) city: London

(E) Country: United Kingdom

(F) ZIP Code: W1Y 6LN

(ii) Name of invention: DNA construct

(iii) Number of sequences: 9

(iv) form of computer reading:

(A) Medium form: floppy disk

(B) Computer: IBM PC Compatibility

(C) operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.30 (EPO)

(v) previous application data:

(A) Application number: GB 9516241.8

(B) Application date: August 8, 1995.

(2) Information about SEQ ID NO: 1:

(i) Sequence Properties:

(A) Length: 36 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence number 1:

CATCTCGAGT CGACTATTTT TACAACAATT ACCAAC

(2) Information about sequence number 2:

(i) Sequence Properties:

(A) Length: 53 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence 2:

CTAGGTACCG TCGACGGATC CGTAAGATCT GGTGTAATTG TAAATAGTAA TTG

(2) Information about SEQ ID NO: 3

(i) Sequence Properties:

(A) Length: 36 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence 3:

CTACTCGAGT CGACTATTTT TACAACAATT ACCAAC

(2) Information about sequence number 4:

(i) Sequence Properties:

(A) Length: 22 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence 4:

CGATGTTGAA GGGCCTGCGG TA

(2) Information about SEQ ID NO: 5

(i) Sequence Properties:

(A) Length: 23 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence 5:

GCACCTCATG GACATCCTGA ACA

(2) Information about SEQ ID NO: 6

(i) Sequence Properties:

(A) Length: 20 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence 6:

CATCGCAAGA CCGGCAACAG

(2) Information about sequence seven:

(i) Sequence Properties:

(A) Length: 23 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence 7:

GCTGTAGATG GTCACCTGCT CCA

(2) Information about SEQ ID NO: 8

(i) Sequence Properties:

(A) Length: 21 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence 8:

TGTACACCGA CGCCATTGGC A

(2) Information about SEQ ID NO: 9

(i) Sequence Properties:

(A) Length: 22 base pairs

(B) Form: Hexane

(C) Chain: Japanese chain

(D) topology: linear

(ii) Molecular type: DNA

(xi) Description of sequence 9:

GCGGTAAGGC TTTCAACAGG CT

Claims (12)

a first promoter derived from the alc R gene and functionally linked to a regulatory sequence that encodes a regulatory protein, and a protein that induces metabolic pathways that produce insect metabolites that damage insects or whose expression damages plants A chemically-inducible plant gene expression cassette comprising an inducible promoter functionally linked to an encoding target gene. The chemically-inducible plant gene expression cassette of claim 1, wherein the target gene encodes an orally active pesticidal protein. 3. The chemically-inducible plant gene expression cassette of claim 2 wherein the orally active pesticidal protein is at least Bacillus thuringiensis δ endotoxin. 4. The chemically-inducible plant gene expression cassette of claim 1, wherein the inducible promoter is derived from an alc A gene promoter. 5. The chemically-inducible plant gene expression cassette of claim 1, wherein the inducible promoter is a chimeric promoter. 6. A plant cell comprising the plant gene expression cassette according to any one of the preceding claims. The plant cell of claim 6, wherein the plant gene expression cassette is stably incorporated into the plant genome. A plant tissue comprising the plant cell according to claim 6. A plant comprising a plant cell according to claim 6. A plant derived from the plant according to claim 9. Seed derived from a plant according to claim 9. A method for controlling insects comprising transforming plant cells with a plant gene expression cassette according to any one of claims 1 to 5.