CA2008695C - Gene switch - Google Patents

Abstract

1. A gene promoter sequence which responds to application of an exogenous chemical inducer such as one of the plant herbicide safeners is utilised as a gene switch to enable external control of expression of a gene placed under control of the promoter. A
specific example of the gene switch construct is the maize GSTII (glutathione-S-transferase isoform II) enzyme promoter sequence which is inducer by external application of the safener N,N-diallyl-2,2-dichloroacetamide.

Description

pS35106 Z~ 9~;

GENE SWI TCH

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 ~pplication 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.
According to the invention there is provided a chemically inducible gene promoter sequence isolated from a 27kd subunit of the maize glutathione-S-transferase (GST II) gene.
The invention also provides a chemically switchable gene constructs which include the maize GST II gene promoter operatively linked to a foreign gene or 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.
In practice 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 '~ pS35106 2~ 95 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 fos 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 procedure9 are known from the literature such as aqroinfection 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 mai~e) are rather more difficult to transform.
However, 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.

- ~535106 3 2~695 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) However, more generically, 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. In other words, 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:
1. benzyl-2-chloro-4-(trifluoromethyl)-5-thiazole-carboxylate;
2. naphthalene-1,8-dicarboxylic anhydride;
3. 2-dichloromethyl-2-methyl-1,3-dioxolane;

4. 1-(dichloroacetyl)-hexahydro-3,3,8a-trimethyl-pyrrole (1,2-a)-pyrimidin-6(2H~-one;

5. 2,2,5-trimethyl-N-dichloroacetyloxazolidine;

6. 1,3-dioxolan-2-ylmethoxyimono(phenyl)benzene acetonitrile;

7. 4,6-dichloro-2-phenyl-pyrimidine;

8. 2,2-dichloro-[N-allyl-N(1,3-dioxalano-2-methy)] acetamide;

9. 1-(cyanomethoxyimino)benzacetonitrile;

~S35106 4 20~695 10. 4'-chloro-2,2,2-trifluoroacetophenone-O-1,3-diDxolan- 2-yl methyloxime;

11. 2,2-dichloro-1-~3,4-dihydro-3-methyl-2H-1,4-benzoxazin-4-yl~ ethanone;

12. 3-dichloroacetyl-2,2-dimethyloxazolidine;

13. 4-methoxy-3,3-dim~thylbenzophenone;

14. 1-cyclohexyl-4,4-dimethyl-2-( lH-l, 2,4-triazol-l-yl) pent-1-en-3-ol;

15. 2,2-dichloro-N-(3-methyl-4-thiazolin-2-ylidene) acetamide;

16. O,O-diethyl-O-phenyl phosphorothioate;

17. 2,2-spirocyclohexyl-N-dichloroacetyl oxazolidine;

18. N-benzyl-N-ethyl-dichloroacetamide;

19. 3-chloroacetyl-4,4-cyclohexane-spiro-2,2-dimethyl-1,3- oxazolidine; and, 20. spirooxazolidine acetamide.
Glutathione-S-transferases (GST) 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, ~emoval 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 bs distinguished in maize tissue. GST I is constitutively expressed and is capable of conjugating glutathione with the pre-emergent herbicides alachlor and atrazine.
Treatment of maize tissues with chemical safeners (for example, N,N-diallyl-2,2-dichloroacetamide) - Ps35106 5 f~ 5 raises the activity of GST I which participates in the detoxification of the pre-emergent herbicides.
The invention will now be described by way of illustration in the following description.
The drawings which accompany this application are as follows:
Figure 1 Figure 1 shows the results for total GST
activity in roots and shoots obtained 23 and 44 hours after treatment with R2~ 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;
Figure 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.
Safener treatment of corn tissue For treatment of young maize seedlings, seeds were germinated on moist filter paper. After germination and growth (up to one week) the safener 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 (0.003 to 30 ppm) and the seedlings grown for a further 23 to 44 hours before harvesting of root and shoot tissue. Figure 1 shows the results for total GST activity in roots and shoots obtained 23 pS35106 6 2~369S

and ~4 hours after treatment as described and Figure 2 shows the separation of the isozymes GST I
and GST II.
For treatment of maize tassel and anther tissue, a solution of 800 ~g of R25 was injected into the node directly below the develop}ng tassel.
Uptake then continued for a further 48 to 72 hours.
Figure 3 shows that only GST I activity was present in untreated anther tissue and Figure 4 shows the stimulation of GST II activity after treatment as described.
Alternatively a 100 ppm solution of ~25 was supplied from a glass reservoir attached to the exposed stem immediately below the developing tassel. Figure 5 shows the results using a stem reservoir technique.
Additionally, 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. As in mammals, 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. In addition, a cDNA corresponding to a 26 kd subunit of a third, minor component of GST

CA 0200869~ 1998-09-14 activity in maize seedlings (GST III) has been previously cloned and sequenced.
Enzyme Assay Enzyme activity was measured spectrophotometrically at 340nm using l-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The reaction buffer contained O.lM EDTA, O.OOlM CDNB
and 0.0025M glutathione.
Preparation of extracts and enzyme purification Tissue was homogenised in 0.05M Tris.HCl, pH
7.8; O.OOlM EDTA; O.OOlM DTT; and 7.5%
polyvinylpyrrolidone in a pestle and mortar, at 4~C, and centrifuged at 30,000g to obtain a crude extract.
Separation of the GST isoforms from the crude extract was achieved as follows: the crude extract was applied to a DEAE Sepharose* column and washed with O.OlM Tris.HCl, pH 7.8; O.OOlM EDTA; and O.OOlM DTT. The bound GST was eluted with 0.3M
potassium chloride. Fractions containing GST
activity were combined and desalted using PD10 gel filtration columns. Separation of the GST I and GST II isoforms was achieved by FPLC on a mono-Q*
column and a zero to 0.4M potassium chloride concentration gradient.
Pure samples of GST I and GST II were obtained by applying desalted fractions of GST I and GST II
from the FPLC to a glutathione-S-sepharose affinity column equilibrated with 0.05M phosphate buffer at pH 7.3. After washing with buffer, bound GST was eluted with 0.005M glutathione.
SDS-PAGE (17.5%, 30:0.174 acrylamide:
bisacrylamide) of GST I or GST II was achived by concentrating pure GST samples using Amicon * Trade-mark CA 0200869~ 1998-09-14 Centricon 10 Microconcentrations (Trade Mark), denaturing samples in mercaptoethanol containing Laemmli buffer, and staining the gels with Coomassie Blue.
Generation of antibodies to the enzyme 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), Biological Techniques Series: Immunochemical Methods in Cell and Molecular Biology N-terminal sequence analysis The 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 A time course experiment was carried out to examine the expression of GST's after safener treatment. A 30 ppm solution of R25 was applied to three-day old seedling roots and tissue harvested after various time intervals following safener treatment. Samples were tested for GST activity using the enzyme assay described above. The results of this experiment are presented graphically in Figure 7.
Synthesis of cDNA libraries The time course experiments revealed a peak of GST expression at 48 hours after treatement with CA 0200869~ 1998-09-14 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 Hoffman,1983).
Gene 25, page 263.
Screening cDNA libraries with antisera to GST I and GST II
In order to identify a cDNA clone encoding maize tassel GST enzyme, 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 Mixtures of synthetic oligonucleotides based on the amino acid sequence determined above were prepared by phosphoramidite chemical synthesis.
The 5' ends of the oligonucleotides were labelled using polynucleotide kinase as described in the literature.
Approximately 40,000 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 ~S35106 the lowest melting point probe in the mixture.
~ybridising 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 sequen5es are isolated from the libraries described using oligo primecs 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.
Characterisation and sequence analysis of GST CDNA
clones 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 ~EMBL3 is used to isolate clones that hybridise to the CDNA clones isolated as described above.
Alternatively, 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.

Claims (3)

1. A method of controlling the expression of a foreign gene of series of genes in a transgenic plant wherein a coding region is placed under the control of the promoter of a gene specifying the 27kd subunit of the maize glutathione-S-transferase gene (GST II) whereby application to the plant of an effective concentration of a chemical inducer of the promoter induces expression of the coding region.

2. A method according to claim 1 wherein the inducer is N,N-diallyl-2,2-dichloroacetamide or benzyl-2-chloro-4-(trifluoromethyl)-5-thiazole carboxylate.

3. A method acccording to claim 2 wherein the inducer is N,N-diallyl-2,2-dichloroacetamide.