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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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  • 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
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/8223—Vegetative tissue-specific promoters
  • C12N15/8225—Leaf-specific, e.g. including petioles, stomata
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  • 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
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/8223—Vegetative tissue-specific promoters
  • C12N15/8226—Stem-specific, e.g. including tubers, beets
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  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • C12N15/8239—Externally regulated expression systems pathogen inducible

Definitions

• the invention relates to wound-stimulated DNA from Solanum tuberosum and parts thereof, their use for the expression of gene products in higher plants when wounded or
• DNA transmission vectors containing them and plants or parts of plants containing them.
• the invention accordingly relates to DNA sequences from Solanum tuberosum which are stimulated by injury and / or infection with pathogenic germs, and to their parts, in particular the promoter part and the structural part.
• the invention further relates to the use of these DNA sequences for the expression of gene products in higher plants in the case of injury and / or pathogenic attack.
• the invention further relates to DNA transmission vectors with DNA sequences inserted therein, as defined above.
• the invention relates to plant or plant materials which contain such a DNA sequence.
• active region of a gene is understood to mean those nucleotide sequences which are absolutely necessary in the promoter part of the gene for the activation of the structural part and in the structural part for the expression of an effective or active gene product.
• Structural genes is understood not only to mean that in the homologous system of the DNA sequence described here from Solanum tuberosum, but also structural genes from other origins, that is to say from heterologous systems.
• Any suitable DNA molecules that can be introduced into the ultimately receiving bacterium can be used as transmission vectors. These are usually plasmids as described in more detail in the materials section. The insertion techniques are known to the person skilled in the art.
• Bacteria of the genus Agrobacterium in particular Agrobacterium tumefaciens and Agrobacterium rhizogenes, can be used as bacteria for the absorption and transmission of the transmission vector.
• EP-A 122 791 describes a DNA transfer vector which comprises T-DNA with a plant gene inserted therein.
• This plant gene consists of a plant promoter and a plant structural gene, the plant promoter being adjacent to the 5 'end of the plant structural gene and the plant structural gene being located behind the plant promoter in the direction of transcription.
• EP-A 122 791 describes in detail methods and measures for inserting genetic material into DNA transmission vectors, to which express reference is made here.
• a cDNA library was set up on the basis of purified polyA RNA from wounded tubers of the potato variety Granola, which were also incubated with Erwinia.
• Differential colony hybridization of 4000 cDNA clones against radioactively labeled RNA from unwound and Erwinia-wounded tubers identified 2 clones, here called wun1 and wun2, whose complementary mRNA in tubers from various tetraploid potato varieties and the haploid variety AM 80/5793 were wounded be induced.
• Wunl mRNA accumulates 30 minutes after the mechanical wounding of a tuber and is therefore involved in wound-induced primary processes, whereas wun2 is not induced until 3.5 hours later.
• Wun1 mRNA accumulates in high amounts in wounded potato tubers (quantitatively comparable to proteinase inhibitor llmRNA in unwound potato tubers, see Sanchez-Serrano et al., Mol. Gen. Genet. 203, 15 (1986)), but is in unwound tubers undetectable. It already appears 30 Minutes after wounding with a maximum from the 4th to 24th hour after wounding. In smaller quantities, it was still detectable after 48 hours.
• wun1 and wun2 mRNA after wounding are not limited to the tuber, but also takes place in leaves, stems and roots of various tetraploid potato plants with similar kinetics and intensity.
• Wunl mRNA is also induced in leaves without wounding if these are sprayed with compatible Phytophtora infestans spores. This result shows that wun1 can be induced not only by mechanical wounding, but also by the presence of (fungal) pathogens.
• Wunl mRNA was expressed higher in aerobically wounded tubers than in the corresponding anaerobic approach.
• Fig. 1 shows the relative size of the clones used for sequencing.
• the clone wun1-25A2 exists in two orientations in the Pstl interface, which are referred to as 25A2 * MP19 and 25A2MP19 due to the location of the asymmetrical Xhol interface.
• 25A2 * MP19 and 25A2MP19 due to the location of the asymmetrical Xhol interface.
• the size of the cDNA clone wun1-25A2 is 711 bp.
• a 14 bp poly A tail can be found at the 3 end (position 697-711).
• the largest open reading frame spans 105 amino acids (positions 121-438). Also located 5'-further are two short open reading frames which code for position 3 for 3 amino acids and code for position 8 for 8 amino acids (underlined). However, the last two open reading frames listed differ from the reading frame, which encodes the 105 amino acids.
• the reading frame coding for 105 amino acids is to be regarded as that of the wun1 protein.
• the translation starting point of the wun1 protein TTTTTGATGCAA is only slightly correct with that of Joshi, Nuc. Acids Res. 15, 6643, (1987) determined consensus sequence for plant translation starts TAAACAATGGCT.
• the wun1 cDNA-equivalent fragment in wun1-85 has a size of 4 Kb. It thus corresponds exactly to that in wun1 cDNA hybri fragmenting fragment size of EcoRI-digested DNA from the haploid potato. Due to the asymmetric Xhol cleavage in the 5 'region of the cDNA clone, the orientation of the gene in the 4 Kb fragment could be determined. Accordingly, there is an approximately 1 Kb promoter region in front of the wun1 gene, while an approximately 2 Kb non-homologous part is located 3 'of the gene. The additional 8 Kb EcoRI fragment contained in wun1-85 could not be used for a closer analysis of the wun1 gene. Due to the total digestion of the potato DNA with EcoRI, it is likely that during the subsequent ligation two fragments which do not belong together were brought into the EMBL4 vector and therefore cannot be functionally related to one another.
• Figure 4 shows the arrangement of wun1 in the genomic clone wun1-85.
• the 4 kb EcoRI fragment of the genomic clone wun1-85 contains 1.0 kb of the wun1 promoter, 0.8 kb of the wun1 gene and 2.0 kb of the 3 'end.
• the 4 Kb fragment of wun1-85 was ligated in EcoRI-cut pUC8 for further analysis (see FIG. 4).
• the 4 Kb fragment was further cloned into the EcoRI site of M13mp18. Its orientation was determined by means of control digestions at the asymmetrically located Xhol interface.
• the clones 85 * mp18 and 85mp18 represent both orientations of the
• FIG. 5 shows the result of the deletion analysis of the clone wun1-85 in schematic form.
• the resulting deletion clones with different fragment sizes were used for sequencing. Overall, it was possible to sequence the entire wun1 gene bidirectionally, as well as to analyze approximately 400 bp of the 3 'end unidirectionally.
• FIG. 6 shows the nucleotide sequence of the wun1 promoter and gene from wun1-85.
• the CAAT box, TATA box and poly-A signal are highlighted, transcription start and stop are marked by arrows.
• the Sl nuclease mapping method was used to identify the exact start of transcription of the wun1 gene.
• Xhol cleavage region from pLS000 with wun1 mRNA leads to a 162-179 bp long DNA-RNA hybrid (TU) which is protected from the single-stranded Sl nuclease.
• the transcription start begins 179 bp upstream from the Xhol site, i.e. with the sequence ACCATAC.
• This sequence agrees in the central area (CAT) with that of Joshi et al. (1987) determined the consensus sequence for the start of transcription CTCATCA. Further information results from the position of the transcription start (see FIG. 6):
• CAAT box in the range between -60 and -80 can be found in the wun1 promoter in position -58 (CAAACT).
• CAAACT CAAT box in the range between -60 and -80
• the gene encoding wun1 mRNA comprises 794 bp, which corresponds very precisely to the size determination of wun1 mRNA based on the "Northern blot" analyzes.
• the cDNA clone wun1-25A2 lacks 97 bp of the 5'-untranslated region in the wun1 gene.
• the wun1 gene does not have introns.
• FIG. 8 shows the arrangement and position of important regions in the wun1 gene.
• the wun1 promoter is marked in black, the wun1 gene is hatched.
• Important recognition sequences are framed;
• the mRNA is represented by a serpentine line, the protein coding region by crosses.
• the size of the individual areas is given in base pairs (bp).
• Figure 9 shows a sequence comparison between the cDNA clone wun1-25A2 and the genomic clone wun1-85. 2368 bp of the genomic clone wun1-85 were compared with 711 bp of the cDNA clone wun1-25A2. Homologous base pairings are identified by a vertical line. The absence of nucleotides is indicated by a dot. The two arrows document the sequence of two 10 bp direct repeats in the cDNA clone.
• the gene codes for a 12,000 dalton protein, the size of which could also be approximately confirmed in hybrid-released translation experiments.
• Computer evaluations regarding the amino acid composition of the wun1 protein showed that it is a very hydrophilic protein.
• the amino acid sequence results from FIGS. 2 and 6.
• PR The size of the wun1 protein, its hydrophilic properties, the inducibility of wounding in various tissues and the inducibility by pathogens suggest that it belongs to the PR proteins.
• PR or "pathogenesis-related" protein is used to summarize proteins from different plants which can be induced, inter alia, by pathogenic attack and in some cases have chitinase or glucanase activity, ie the activity of enzymes which can destroy the cell walls of fungi .
• plants transformed with 3850 K m have the ability to synthesize nopaline, a property that distinguishes them from untransformed plants (Zambryski et al, EMBO J. 1 147-152
• Kanamycin-resistant and nopalin-positive LS1 plants were transferred to the greenhouse and analyzed at the DNA and RNA levels.
• Figure 10 shows the construction of wun1-wun1 and wun1-CAT fusions that were used for expression studies based on transient expressions and stable transformations.
• the 4 Kb fragment which contains the wun1 promoter, the wun1 gene and 2 Kb of the 3 'end, was cloned into the vector pMPK110 via the EcoRI sections (pLS001 ).
• This pMPK110 derivative allows the transfer of the wun1-wun1 construction to the Agrobacterium 3850 km .
• the wun1 promoter and 179 BP of the 5'-untranslated wun1 gene region were fused to the CAT gene including the 3 'end via the Xhol interface (pLS010).
• a cloning over PstI in the vector pMPK110 (pLS011) has the advantage that this plasmid can be used on the one hand for transient studies in protoplasts and on the other hand also acts as a mobilization plasmid for the transfer of the wun1-CAT construction into the Agrobacterium 3850 km .
• RNA level A functional analysis of the wun1-wun1 DNA of positive LS1 plants was possible at the RNA level. There was only a very weak cross hybridization against a wun1 cDNA sample in both wounded and unwound untransformed tobacco plants. In addition, isolated RNA from untransformed tobacco plants was raised against the 5 'region of the wun1 gene, which is the
• An object of the invention is to construct and isolate genes capable of expressing fused proteins as required. For this it is necessary that the wun1 promoter is solely responsible for the expression of the downstream structural gene, which need not always be the case (eg proteinase inhibitor II).
• Transcriptional fusions consisting of the wun1 promoter and 178 bp of the S'-untranslated gene were fused with various structural genes (wun1-CAT, wun1-NPT, wun1-GUS), the detection of which was possible using radioactive or fluorometric methods.
• GUS stands for ⁇ -glucuronidase (Jefferson et al, Proc. Nat. Acad. Sci. 83, 8447-8451 (1987)).
• the construction wun1-CAT was tested for its transient functionality in potato protoplasts. This method makes it possible to analyze promoter activities within a few days by transferring highly purified plasmid DNA, which contains the constructions, to protoplasts using known methods, where they are stable and can also be read. A prerequisite for expression is that the promoter is functional and that the inducing factors are present in protoplasts treated in this way.
• the wun1 promoter including 179 bp of the 5'-untranslated region was transcriptionally fused with the chloramphenicol acetyl transferase gene (CAT).
• CAT chloramphenicol acetyl transferase gene
• CAT analysis of such protoplasts transformed with pLS011 DNA revealed high CAT activities.
• a parallel test approach with a CAMV-35S-CAT construction (35S promoter from the Cauliflower mosaic virus (CAMV)) (pRT101-CAT) brought about comparable CAT activity, whereas in a control approach without addition of DNA no CAT activity was recognizable.
• the CAMV promoter is considered to be the strongest known promoter in plants.
• LS2 plants grown in the greenhouse showed slightly increased CAT activity in their unwounded leaves compared to the background activity of untransformed tobacco leaves. In wounded leaves, an approximately 4-fold higher CAT activity was found compared to unwounded leaves. If, however, an excess of boiled potato tuber extract was added to the wound, the increase was 6 times higher than in the unsealed condition. This effect was independent of the condition of the tubers before extraction (unwound or wounded tubers or tubers freshly removed from the soil (unstored) or stored). Each extract had the same inducing effect, which indicates an inductor present in the extract.
• the transient system was also used to test which plant species the wun1 promoter is active in and
• this promoter is not only active in potato, tobacco and parsley protoplasts, but also in rice protoplasts shows an expression level which corresponds to the activity of a homologous system.
• the wun1 promoter including 179 bp of the 5'-untranslated region was cloned via EcoRI-Xhol-blunt in front of an NPT-II gene (neomycin phosphotransferase-II).
• NPT-II neomycin phosphotransferase-II
• the wun1-NPT-II fusion was cloned into a highly replicating vector (pLS 020).
• NPT-II activity was found in tobacco and in parsley protoplasts.
• wun1-dependent NPT-II activity could also be detected in rice protoplasts (Oryza sativa japonica cv Taipai), the intensity of which was comparable to that of wun1 -NPTII activity in potato protoplasts.
• Comparative transient expression of wun1-NPTII and NOS-NPTII (pGV1103) in rice protoplasts also showed similarly high values. In this together, reference is also made to the CAMV-35S-CAT-like transient expression in potato protoplasts.
• wun1 promoter has a very high activity in both homologous and heterologous systems. Neither in parsley nor in tobacco bones nor in rice protoplasts could be found with wun1-hybridizing mRNA. Factors that stimulate the wun1 promoter must be assumed, which occur equally in all plant species tested.
• the fusion of the wun1 promoter with the known TR promoter from the Agrobacterium tumefaciens TR-DNA may be mentioned as an example.
• this TR promoter is responsible for the expression of mannopine synthase and has the following characteristics: 1. It is bidirectionally active, ie one fuses
• tumefaciens mannopine synthase genes is regulated by plant growth hormones. Proc. Natl. Acad. Be. USA, 86: 3219-3223.
• the orientation of the TR promoter can be found with l 'and 2'
• the TR promoter can be induced by wounds.
• beta-galactosidase leads to a higher beta-gal activity in wounded transgenic tobacco plants than in unwounded ones.
• the activity of the TR2 'promoter is mainly found in the leaf veins, that is, in the vascular bundle system.
• TR1 'promoter 4.
• the properties of the TR1 'promoter have so far not been investigated so intensively. However, it is known that the TR1 'promoter is about 4-7 times weaker than the TR2' promoter, but otherwise has similar properties.
• the TR promoter was fused to the wun1 promoter, namely in the
• TR1'-wun1 Orientation TR1'-wun1 (Fig. 12).
• the marker gene GUS beta-glucoronidase
• TR1'-wun1-GUS was used to demonstrate the promoter activity.
• Agrobacterium tumefaciens transformation system this construction was used in tobacco
• GUS activity is mainly found in the epidermis of leaves and stems.
• TR1'-wun1-GUS transgenic tobacco the GUS activity is mainly localized in the lead bundle system. So there is the possibility, depending on the scientific question, of being able to direct gene products to different parts of the tissue. In contrast, the wound induction behavior of TR1'-wun1-GUS transgenic tobacco plants is comparable to the behavior of wun1-GUS transgenic tobacco plants.
• TR1'-wun1 promoter among the strongest promoters available for expression in plants.
• the wun1 promoter according to the invention can be fusioned with the TR1 'promoter in the following
• Leaf or Stenge1 epidermis of transgenic tobacco is shifted into the lead bundle system by the fusion with the TR1 'promoter;
• the TR1'-wun1 promoter in transgenic tobacco leaves and stems is about 10 times stronger than the wun1 promoter alone;
• the TR1'-wun1 promoter is about 30 times stronger than the wun1 promoter in transient experiments with a representative of the monocotyledonous plants (rice).
• the invention e.g. increase the expression of the wun1 promoter by fusing it with an enhancer element of the CamV-35S promoter.
• an enhancer element of the CamV-35S promoter For example, the fusion of the enhancer element of the CamV-35S promoter to the 5 'end of the wun1 promoter leads to an increased wun1 promoter activity in transgenic tobacco and potato.
• Figure 12 shows the organization of the clones wun1-GUS, TR1'-GUS and TR1'-wun1GUS.
• Figure 13 shows Northern blot analysis of wounded and wounded transgenic tobacco leaves. Unwound (NW) and wounded (W) leaves of the transgenic tobacco plants wun1-GUS, TR1'-wun1-GUS No. 5 and No. 11 and 35S-GUS were used to isolate RNA. 50 ⁇ g of total RNA each was on a
• Plant media The media used are derived from that of Murashige and
• MSC10 MS + 2% sucrose, 500 ⁇ g / ml claforan,
• MSC15 MS + 2% sucrose, 500 ⁇ g / ml claforan,
• MS15 MS + 2% sucrose, 100 ⁇ g / ml kanamycin sulfate
• Plasmi de: pUC8 (Vieira and Messing, Gene
• plasmid DNA, transformation of bacteria, etc. are as described in Maniatis et al. (1982).
• Potato bulb material was cut into 3mm thick slices
• Leaf, stem, root and tuber material was processed immediately after its removal from the living plant or frozen in liquid nitrogen and stored at -70 ° C.
• the densitometric evaluations of the wun1 hybridization strength showed a 4-fold increase in the amount of wun1 mRNA just two hours after treatment of the leaves with water and spores of the incompatible phytophore species. From the 4th to the 8th hour, the amount of expression remained approximately the same before it gradually decreased to 1.5 to 2 times by the 30th hour. Wun1-mRNA showed the same expression behavior when using water without spores (control).
• Potato tubers were homogenized without adding buffers at 4 ° C. and centrifuged for 10 minutes at 10,000 rpm in the SS34 rotor. The supernatant was boiled for 10 minutes and centrifuged again. The clear supernatant was added in a ratio of 1:10 to 20 mM phosphate buffer, pH 7.0 and the tissue to be tested was incubated therein under the normal conditions.
• RNA isolation The isolation of RNA from various organs of the potato and tobacco was carried out as in Logemann et al. in anal. Biochem., 163: 16-20 (1987).
• RNA was electrophoresed on a 1.5% formaldehyde agarose gel (Lehrach et al., Biochemistry 16, 4743, (1977)). As with Willmitzer et al., EMBO J. 1, 139-146,
• RNA was then transferred to nitrocellulose, fixed and hybridized against 32 P * radiolabeled cDNA, washed and exposed. DNA isolation
• Nuclear DNA was obtained from potato leaves by the method of Bedbrook, PMB Newsletter II, 24 (1981) and used for cloning in LambdaPhagen EMBL4. DNA from transformed tissue was purified after the combined disruption with Triton X 100, SDS, and Proteinase K (Wassenegger, dissertation, Cologne (1988)).
• DNA was electrophoretically separated on 0.8 to 1.2% agarose gels, transferred to nitrocellulose and fixed
• Example 2 "Hybrid-releasec-translation" experiments The 800 bp fragment of the cDNA clone wun1-25A2 and the 700 bp fragment of the cDNA clone wun2-29C12 were obtained via Pstl
• Reticulocyte lysate (Pelham and Jackson, Eur. J. Biochem. 67, 247 (1976)) translated in the presence of 35 S-methionine.
• the one- or two-dimensional electrophoretic separation on a polyacrylamide gel was carried out according to Mayer et al. (1987).
• Leaf DNA of the haploid line AM isolated according to the Bedbrook (1981) method was used as the genomic potato DNA
• This DNA was digested completely with EcoRI.
• EMBL4-DNA was cut into three fragments with EcoRI, using gel electrophoretic separation followed by Frag ment isolation the two vector arms could be separated from the middle fragment.
• commercially available and cleaned EMBL4 arms were used (Amersham).
• the packaging material comes from a "Lambda in vitro packaging kit” from Amersham.
• the genomic library was created with a concentration of 25,000 plagues per plate
• Chloroform was added to 20 ml of a C600-lysed bacterial culture in order to obtain a bacteria-free supernatant after subsequent centrifugation.
• the phages were sedimented from the supernatant by centrifugation at 10,000 rpm for 4 hours.
• the phage sediment was taken up in 500 ⁇ l phage buffer (10 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 ) and treated with DNase and RNase. After extraction of the DNA by repeated phenolization, the phage DNA was EtOH-precipitated, washed with 70% EtOH and taken up in TE.
• the approximately 711 bp insert of the clone wun1-25A2 was cloned into the M13mp19 vector via the PstI sites, the orientation of the insert being determined via the asymmetric Xhol site.
• the clones 25A2-4-mp19 and 25A-5-mp19, which differed in the orientation of the insert, were cut with Kpnl / Xbal in order to enable bidirectional exonuclease digestion into the fragment. Deletion clones of different sizes could be obtained by successively stopping the exonuclease reaction, which was prepared as a single-stranded DNA for sequencing according to the method of Sanger et al. (1977) served.
• Starting point of the transcription is based on the fact that by hybridizing single-stranded DNA from the 5 'region of the wun1 gene with wun1-mRNA, only those regions are protected against degradation of the single-stranded nuclease S1 which, due to their homology, can form a double strand.
• the size of the protected DNA fragment can be determined on a sequence gel and the start of transcription can thus be traced back.
• the transcription start point was determined according to Berk and Sharp, Proc. Natl. Acad. Be. USA 75, -1274 (1987).
• the 1.2 kb fragment was isolated from the plasmid pLS000 with EcoRI and Xhol and dephosphorylated by treatment with phosphatase.
• the 5'OH ends were then radiolabeled by the combination of polynucleotide kinase and y- 32 -P-ATP. After denaturing the DNA fragments, they were hybridized with 50 ⁇ g total RNA (hybridization buffer: 80%. Formamide, 0.4 mM NaCl, 40 mM PIPES pH 6.4 and 1 mM EDTA).
• the protoplasts come from a potato stem suspension culture of the Datura variety.
• the DNA cloned in E. coli was prepared according to the method described by Van Haute et al. (1983) transferred the method using the helper strain GJ23 into the agrobacterial strain 3850.
• the 3850 km agrobacteria required for infection were grown in selective antibiotic medium (Zambrisky et al., 1983), sedimented by centrifugation and washed in YEB medium without antibiotics. After renewed sedimentation and absorption in YEB medium, the bacteria could be used for infection.
• NPT-II activity in transformed plants was detected according to Reiss et al., Gene 30, 217-223 (1984) and Schreier et al., EMBO J. 4, 25-32 (1985).
• Chloramphenicol acetyl transferase (CAT) activity was determined by the methods of Veiten and Schell, Nuc. Acids, Res. 13, 6981-6997 (1985) and Herrera-Estrella et al., EMBO J. 2, 987 (1983).
• Agrobacterial strains GV3101pmp90RK Koncz, C, and Schell, J. "The promotor of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vetor", (1986), Mol. Gen. Gent 204: 383-396).
• Plasmids pPCV720 (previously unpublished plasmid of
• 35S-GUS pRT99-GUS (Töpfer, R., Schell, J. and
• TR1'-GUS (Logemann et al., In press)
• RNA isolation “Northern blot” analysis, DNA isolation, transient expression in rice protoplasts, “Southern blot” analysis and DNA analysis of agrobacteria, reference is also made to Examples 1-8.
• the DNA cloned in E. coli was prepared according to that of Van Houte, E., Joos, H., Maes, M., Warren G., Van Montagu, M., Schell, J.
• the GV3101pmp90RK agrobacteria which contain the desired plasmid, were grown in selective antibiotic medium (hygromacin), sedimented by centrifugation and washed in YEB medium (Maniatis et al, 1982) without antibiotics. After renewed sedimentation and absorption in YEB medium, the bacteria could be used for infection.
• selective antibiotic medium hygromacin
• YEB medium Maniatis et al, 1982
• Dirnethylformamide are pH 50 in 50 mM phosphate buffer
• Thin tissue sections are incubated as described under "wounded plant tissue” and then stained in X-Gluc solution overnight at 37 ° C. Finally, the chlorophyll is removed by adding 100% methanol at 60 ° C.
• Thin tissue sections are incubated in X-Gluc solution, which also contains the translational inhibitor cycloheximide (1.8 mM). Incubation period and methanol treatment is identical to the wounded tissue.
• pollen grains are incubated directly in X-Gluc. without the formation of pollen tubes.
• the TR1 'promoter becomes transcriptional with the GUS gene
• TR1'-wun1-GUS
• the pA signal of the octopine synthase gene is cloned behind the 1 'promoter of the TR promoter in order to prevent transcription through the wun1 promoter.
• Behind the OCS pA signal lies the wun1 promoter (1022 bp), which (in homology to the wun1-GUS construction) over 179 bp des
• untranslated 5 'region of the wun1 gene is fused to the GUS gene transcriptionally.
• the pA signal of the 35-S gene serves as the termination sequence for the GUS gene.
• TR1'-wun1-GUS lies in the binary
• Vector pPCV720 which can replicate itself both in E. coli and in agrobacteria (with the help of a helper plasmid).
• RNA from wounded and from unwounded leaves of various TR1'-wun1-GUS transgenic tobacco plants as well as from wun1-GUS and 35S-GUS leaves was isolated and radioactively labeled GUS-DNA hybridizes.
• RNA is higher both in TR1'-wun1-GUS plants and in wun1-GUS plants in wounded leaves than in unwounded leaves.
• the wound inducibility already known for wun1-GUS is thus also confirmed for TR1'-wun1-GUS.
• TR2'1'-wun1-GUS plants A quantitative analysis of the GUS activity of transgenic TR1'-wun1-GUS plants shows that the TR2'1'-wun1 promoter in transgenic leaves can be induced by a factor of 2 to 13 times by wounding (about 6 times on average). This roughly corresponds to the wound inducibility of the wun1 promoter in transgenic leaves (Logemann J., Lipphardt S., Lörz H., whether I., Willmitzer L. and Schell J. "5'upstream segments from the wun1-gene are responsible for gene activation by wounding in transgenic plants "The Plant Cell 1: 151-158 (1989A)).
• the promoter is 10 times stronger than the wun1 promoter.
• the TR1'-wun1 promoter is about 100 times stronger than the TR1 'promoter.
• the activity of the wun1 promoter is mainly limited to the epidermis (including trichomes) and to a lesser extent to the
• the wun1 promoter activity is thus to be regarded as epidermis-specific in leaves and stems.
• TR1'wun1-GUS plants an intense blue coloration can be seen in the guide bundle system of wounded leaf sections. In other areas (epidermis, parenchyma) the color is only weak. Unwound leaf cuts show a slight blue color in the lead bundle system and no color in the epidermis. The same results are achieved with stem cross sections.
• TR1 'wun1-GUS is a promoter which is specific in the guide bundle system and expresses in leaves and stems. A relative comparison of promoter activities in transgenic tobacco leaves and stems shows that the wun1 promoter is about 10 times stronger than the TR1 'promoter.
• the TR1'-wun1GUS is a promoter which is specific in the guide bundle system and expresses in leaves and stems. A relative comparison of promoter activities in transgenic tobacco leaves and stems shows that the wun1 promoter is about 10 times stronger than the TR1 'promoter.
• the promoter is 10 times stronger than the wun1 promoter.
• the TR1'-wun1 promoter is about 100 times stronger than the TR1 'promoter.
• the activity of the wun1 promoter is mainly limited to the epidermis (including trichomes) and to a lesser extent to the
• the wun1 promoter activity is thus to be regarded as epidermis-specific in leaves and stems.
• TR1'wun1-GUS plants an intense blue coloration can be seen in the guide bundle system of wounded leaf sections. In other areas (epidermis, parenchyma) the color is only weak. Unwound leaf cuts show a slight blue color in the lead bundle system and no color in the epidermis. The same results are achieved with stem cross sections.
• TR1 'wun1-GUS is an in
• TR1 'GUS plants Leaves and stems of a promoter expressing the guide bundle system.
• Leaf sections of TR1 'GUS plants served as a control. Because of the weak promoter activity of the TR1 'promoter (10 times weaker than the wun1 promoter), no blue coloration is discernible either in wounded or in unwounded cross sections of the leaf.
• 35S-GUS were used for transient expression studies with rice protoplasts of the variety (Oryza sativa japonica cv Taipai). As Table 1 shows, the activity of TR1'-GUS does not differ significantly from the control (GUS activity of rice protoplasts that were transformed without DNA). wun1-GUS and 35S-GUS show 2-3 times higher activity than the control. TR1'-wun1-GUS DNA, on the other hand, shows on average 57 times higher activity than the control and is therefore a highly active promoter in rice protoplasts.
• the indicated GUS activity was determined from 5 independent experiments.
• Table 1 The statement in Table 1 can be substantiated by staining the protoplasts with X-Gluc. Only protoplasts transformed with TR1'-wun1-GUS show an intense blue color.

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