EP0455667A1 - Gene switch - Google Patents
Gene switchInfo
- Publication number
- EP0455667A1 EP0455667A1 EP90901863A EP90901863A EP0455667A1 EP 0455667 A1 EP0455667 A1 EP 0455667A1 EP 90901863 A EP90901863 A EP 90901863A EP 90901863 A EP90901863 A EP 90901863A EP 0455667 A1 EP0455667 A1 EP 0455667A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- gst
- gene
- promoter
- inducer
- maize
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
- C12N9/1088—Glutathione transferase (2.5.1.18)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8237—Externally regulated expression systems
- C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
Definitions
- This invention relates to a gene promoter sequence which is responsive to an applied exogenous chemical inducer enabling external control of expression of the gene controlled by the promoter.
- the sequence of the invention is conveniently referred to as a gene switch.
- European Patent Application 332,104 (published 18th September 1989) describes chemically regulatable DNA sequences which are capable of regulating transcription of an associated DNA sequence in plants or plant tissue.
- the particular source of the sequences is described as being the PR (pathogenesis-related) protein gene.
- GST II glutathione-S-transferase
- the invention also provides a chemically switchable gene constructs which include the maize GST II gene promoter operatively linked to a foreign gene oir a series of foreign genes whereby expression of said foreign gene or said series of genes may be controlled by application of an effective exogenous inducer.
- the chemically inducible promoter of the invention will be inserted as a promoter sequence in a recombinant gene construct destined for use in a plant. The construct will then be inserted into the plant by transformation. Expression of protein encoding genes in the construct, being under control of the chemically switchable promoter of the invention, may be controlled by the application of a chemical inducer to the plant.
- the method employed for transformation of the plant cells is not especially germane to this invention and any method suitable for the target plant may be employed.
- Transgenic plants are obtained by regeneration from the transformed cells. Numerous transformation procedures are known from the literature such as agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts, microprojectile transformation and pollen tube transformation, to mention but a few. Reference may be made to the literature for full details of the known methods. Neither is the plant species into which the chemically inducible promoter is inserted particularly germane to the invention.
- Dicotyledonous plant are rel;atively simple to transform whereas the monocotyledonous plants (which include the major cereal crops such as maize) are rather more difficult to transform.
- this invention may be applied to any plant for which transformation techniques are, or become, available. Therefore, the invention further provides a plant having a recombinant gene construct which includes the chemically inducible promoter of the invention stably incorporated in its genome by transformation.
- the invention also provides an effective promoter/inducer combination wherein the promoter is the GST II promoter aforesaid and the inducer is N,N-diallyl-2,2-dichloroacetamide (common name: dichloramid) or benzyl-2-chloro-4-(trifluoromethyl) -5-thiazole-carboxylate (common name: flurazole)
- the present invention comprises a recombinant DNA construct including a gene promoter inducible by application of an exogenous chemical inducer operatively linked to control expression of a target gene.
- Suitable promoters and specific inducers may be identified by the procedures exemplified hereinbelow in respect of the GST II gene promoter and its inducer.
- the invention comprises the use of a gene promoter which is inducible by externally applied chemical to control expression of a gene sequence within a plant genome.
- Chemical inducers which are potential inducers of the GSTII 27kd subunit expression include compounds such as:
- Glutathione-S-transferases are a family of enzymes which catalyse the conjugation of glutathione via the sulphydryl group to a large range of hydrophobic, electrophilic compounds. The conjugation results in detoxification of these compounds and in insects and mammals, removal from tissue.
- GST enzymes have been identified in a range of crop plants including maize, wheat, sorghum and peas. GST's comprise from 1 to 2% of the total soluble protein in etiolated maize seedlings.
- the major isoform of GST can be distinguished in maize tissue.
- GST I is constitutively expressed and is capable of conjugating glutathione with the pre-emergent herbicides alachlor and atrazine.
- chemical safeners for example, N,N-diallyl-2,2-dichloroacetamide
- Figure 1 shows the results for total GST activity in roots and shoots obtained 23 and 44 hours after treatment with R25 as described below;
- Figure 2 shows the chromatographic separation of the isozymes GST I and GST II.
- Figure 3 shows GST I activity present in untreated anther tissue
- FIG. 4 shows the stimulation of GST II activity after treatment with R25 as described below
- Figure 5 shows the results using a stem reservoir technique
- Figure 6 shows the results with the application by spray; and, Figure 7 is a time course graph generated in the manner described below.
- N,N-diallyl-2,2- dichloroacetamide (hereinafter referred to as R25) was added to the water in the filter paper to give a range of concentrations
- Figure 1 shows the results for total GST activity in roots and shoots obtained 23 and 44 hours after treatment as described and Figure 2 shows the separation of the isozymes GST I and GST II.
- FIG. 5 shows the results using a stem reservoir technique.
- R25 was applied as a 100 ppm spray directly on to the exposed developing tassel.
- Figure 6 shows the results with the application by spray.
- Both GST proteins have a native molecular weight of approximately 50 kd.
- maize GST's are dimeric; GST I has apparently identical subunits of 29 kd, whereas GST II is a heterodimer of a 29 kd subunit similar to that found in GST I and a novel 27 kd subunit which is only present in tissue treated with safener except in seedling root where it is constitutively expressed, but still can be induced by safener treatment.
- a cDNA and a gene corresponding to the 29 kd subunit of GST I have been cloned previously and sequenced.
- a cDNA corresponding to a 26 kd subunit of a third, minor component of GST activity in maize seedlings (GST III) has been previously cloned and sequenced.
- Enzyme activity was measured spectrophotometrically at 340nm using 1-chloro- 2,4-dinitrobenzene (CDNB) as a substrate.
- the reaction buffer contained 0.1M EDTA, 0.001M CDNB and 0.0025M glutathione.
- Preparation of extracts and enzyme purification Tissue was homogenised in 0.05M Tris.HCl, pH 8.
- Sufficient protein to enable the immunisation of rabbits is obtained by pooling the isolated enzyme subunit isolated as described above from a number of separate experiments.
- the 27 kD GST II polypeptide is subsequently purified to apparent homogeneity by electroelution from polyacrylamide gel slices. Antisera are prepared against the 27 kd polypeptide.
- the immunisation of rabbits is carried out essentially according to Mayer and Walker (1978).
- amino terminal sequence of the intact 27 kd subunit of GST II or partial proteolytic cleavage products was determined by sequential Edman degradation and subsequent amino acid analysis by HPLC. Time Course
- the time course experiments revealed a peak of GST expression at 48 hours after treatement with safener. Therefore, two cDNA libraries were constructed from RNA extracted from tissue at 24 and 48 hours after safener treatment. To ensure that the induction procedure had been successful, a one gram sample of 24 hour induced tissue was taken and assayed for GST II. This experiment revealed that the tissue used to construct the cDNA library had indeed been successfuly induced as GST II accounted for 45.5% of the total GST activity.
- Double-stranded cDNA was prepared from oligo dT-cellulose-purified RNA by a method employing RNaseH and E.coli DNA polymerase I in the synthesis of the second strand, without prior purification of single-stranded cDNA (Gubler and Hoffraan,1983) . Screening cDNA libraries with antisera to GST I and GST II
- bacteriophage from the amplified cDNA library are screened with anti-maize GST enzyme serum. The clones producing the strongest signals are re-screened. Screening cDNA libraries using oligo probes
- the 5' ends of the oligonucleotides were labelled using polynucleotide kinase as described in the literature.
- phages containing cDNA were amplified on plates and transferred to nitrocellulose.
- the filters were hybridised to oligonucleotide probes at temperatures of from 2 to 5°C below the melting temperature calculated for the lowest melting point probe in the mixture.
- Hybridising plaques were selected and rescreened through two or more rounds exactly as described above but at lower densities Isolation of cDNA gene sequences by the PCR method cDNA or DNA sequences are isolated from the libraries described using oligo primers based on the amino acid sequence obtained from partial proteolytic cleavage or in the case of genomic DNA, primers based on cDNA sequence determined previously.
- the isolated cDNA is characterised and subjected to sequencing by one or more of the standard available techniques. Isolation of genomic sequences
- An existing genomic library of fragments of total maize DNA cloned into XEMBL3 is used to isolate clones that hybridise to the cDNA clones isolated as described above.
- the PCR method described above may be used to selectively amplify and clone gene fragments.
- GSTII genes and ther promoter sequences can then be isolated, and characterised using established techniques. It can be demonstrated that the GSTII promoter sequences mediate safener- induced gene activity by fusing them to marker genes like GUS and CAT, and testing then in transgenic plants.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Saccharide Compounds (AREA)
- Lock And Its Accessories (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
On utilise une séquence promotrice de gène répondant à l'application d'un inducteur chimique exogène tel qu'un agent protecteur d'herbicide végétal, comme interrupteur de gène, pour permettre la régulation externe de l'expression d'un gène placé sous la régulation d'un promoteur. Un exemple spécifique de la structure de l'interrupteur de gène est la séquence d'enzyme GSTII (isoforme II de glutathione-S-transférase) du maïs qui est inductrice par application externe de l'agent protecteur N,N-diallyle-2,2-dichloracétamide.A gene promoter sequence responding to the application of an exogenous chemical inducer such as a plant herbicide protective agent is used as a gene switch to allow the external regulation of the expression of a gene placed under the gene. regulation of a promoter. A specific example of the structure of the gene switch is the maize glutathione-S-transferase isoform II (GSTII) enzyme sequence which induces by external application of the protective agent N, N-diallyl-2, 2-dichloracetamide.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8901673 | 1989-01-26 | ||
GB898901673A GB8901673D0 (en) | 1989-01-26 | 1989-01-26 | Gene switch |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0455667A1 true EP0455667A1 (en) | 1991-11-13 |
Family
ID=10650613
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP90901863A Ceased EP0455667A1 (en) | 1989-01-26 | 1990-01-25 | Gene switch |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0455667A1 (en) |
AU (1) | AU647016B2 (en) |
CA (1) | CA2008695C (en) |
GB (1) | GB8901673D0 (en) |
WO (1) | WO1990008826A1 (en) |
ZA (1) | ZA90604B (en) |
Families Citing this family (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8901677D0 (en) | 1989-01-26 | 1989-03-15 | Ici Plc | Hybrid seed production |
US5907086A (en) * | 1991-05-01 | 1999-05-25 | Pioneer Hi-Bred International, Inc. | Plant promoter sequences |
GB9114259D0 (en) * | 1991-07-02 | 1991-08-21 | Ici Plc | Plant derived enzyme and dna sequences |
PL170326B1 (en) * | 1991-07-11 | 1996-11-29 | Int Flower Dev Pty Ltd | Genetic sequences encoding the enzymes of flavonoid path and their applications |
DE69331055T2 (en) | 1992-04-13 | 2002-06-20 | Syngenta Ltd., Haselmere | DNA CONSTRUCTIONS AND PLANTS CONTAINING THEM |
GB9223332D0 (en) * | 1992-11-06 | 1992-12-23 | Ici Plc | Production of polyhydroxyalkanoate in plants |
GB9223454D0 (en) * | 1992-11-09 | 1992-12-23 | Ici Plc | Novel plants and processes for obtaining them |
US6066456A (en) * | 1993-12-30 | 2000-05-23 | Zeneca Limited | Plant-derived enzyme and DNA sequences and uses thereof |
US5723765A (en) * | 1994-08-01 | 1998-03-03 | Delta And Pine Land Co. | Control of plant gene expression |
EG23907A (en) * | 1994-08-01 | 2007-12-30 | Delta & Pine Land Co | Control of plant gene expression |
MX9709156A (en) | 1995-05-26 | 1998-03-31 | Zeneca Ltd | A gene switch comprising an ecdysone receptor. |
US5859338A (en) * | 1995-06-06 | 1999-01-12 | California Institute Of Technology | Plant clavata1 nucleic acids, transformed plants, and proteins |
EP0859850B1 (en) * | 1995-09-22 | 2006-10-18 | Syngenta Limited | Plant glutathione s-transferase promoters |
US6610828B1 (en) | 1996-05-24 | 2003-08-26 | Syngenta Limited | Heliothis ecdysone receptor |
PL338605A1 (en) * | 1997-07-30 | 2000-11-06 | Zeneca Ltd | Method of exercising genetics-based control over germination |
PT1054985E (en) | 1998-02-20 | 2012-07-03 | Syngenta Ltd | Hybrid seed production |
WO1999053050A1 (en) | 1998-04-08 | 1999-10-21 | Commonwealth Scientific And Industrial Research Organisation | Methods and means for obtaining modified phenotypes |
EP1029923A1 (en) | 1999-01-27 | 2000-08-23 | D.J. Van Der Have B.V. | Method for conveying BNYVV resistance to sugar beet plants |
US7749751B2 (en) | 1999-12-16 | 2010-07-06 | Cropdesign N.V. | Optimized T-DNA transfer and vectors therefor |
EP2119786A1 (en) | 2008-05-13 | 2009-11-18 | Expressive Research B.V. | Increased production of health-promoting compounds in plants |
US20130055471A1 (en) | 2009-12-15 | 2013-02-28 | Edwin Henricus Antonius HOLMAN | Transgenic Ozone-Resistant Plants |
NL2004624C2 (en) | 2010-04-28 | 2011-11-01 | Stichting Dienst Landbouwkundi | A new glycosyltransferase protein and its role in the metabolism of phenylpropanoid volatiles in tomato. |
JP6133275B2 (en) | 2011-05-02 | 2017-05-24 | ボード・オブ・リージェンツ・オブ・ザ・ユニヴァーシティ・オブ・ネブラスカBoard Of Regents Of The University Of Nebraska | Plants with useful characteristics and related methods |
US9612235B2 (en) * | 2012-04-05 | 2017-04-04 | Koch Biological Solutions, Llc | Herbicidal compound screening |
US10767188B2 (en) | 2013-09-25 | 2020-09-08 | Nutech Ventures | Methods and compositions for obtaining useful plant traits |
CN110036114B (en) | 2016-08-26 | 2023-09-29 | 勒萨弗尔公司 | Enhanced itaconic acid production |
EP3533878A1 (en) | 2018-02-28 | 2019-09-04 | Dutch DNA Biotech B.V. | Process for producing citramalic acid employing aspergillus |
-
1989
- 1989-01-26 GB GB898901673A patent/GB8901673D0/en active Pending
-
1990
- 1990-01-25 WO PCT/GB1990/000101 patent/WO1990008826A1/en not_active Application Discontinuation
- 1990-01-25 EP EP90901863A patent/EP0455667A1/en not_active Ceased
- 1990-01-25 AU AU49476/90A patent/AU647016B2/en not_active Ceased
- 1990-01-26 ZA ZA90604A patent/ZA90604B/en unknown
- 1990-01-26 CA CA002008695A patent/CA2008695C/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
---|
See references of WO9008826A1 * |
Also Published As
Publication number | Publication date |
---|---|
GB8901673D0 (en) | 1989-03-15 |
WO1990008826A1 (en) | 1990-08-09 |
ZA90604B (en) | 1990-11-28 |
CA2008695C (en) | 1999-03-16 |
AU4947690A (en) | 1990-08-24 |
CA2008695A1 (en) | 1990-07-26 |
AU647016B2 (en) | 1994-03-17 |
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Legal Events
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