DE19525034A1 - DNA sequences and their use - Google Patents

Abstract

The invention concerns novel deoxyribonucleic acid sequences (DNA sequences) and their use or the use of given plants containing these DNA sequences for detecting agents which can induce defence mechanisms in plants.

Description

The invention relates to new deoxyribonucleic acid sequences (DNA sequences) as well as their use or the use of certain plants that these Contain DNA sequences for finding agents which are plant-based Defense mechanisms can induce.

Most plant protection products known today develop an immediate we against the pests to be controlled. Many plants have about own defense mechanisms, more or less pronounced, with one Infestation by pests, in particular by the spoilage that occurs plant tissue. This natural induction of Plant defense mechanisms are usually not sufficient in the case of pest infestation to prevent major damage, since their effects often only with delay tion occurs after significant damage has already occurred. Targeted plant Protective measures are therefore not in use in agriculture and horticulture mandatory. However, since efforts are being made to strengthen the natural defense mechanisms in plant protection, it is necessary to use chemical substances (synthe tables or natural low molecular weight substances), which after a ge targeted and timely application, the plant's own defense mechanisms (e.g. formation of substances that are toxic to the pests or repelling the pests or of substances that produce tissue necrosis and thus prevent the further spread of the pests) in a sufficient To induce dimensions and quickly enough without any undesirable effects to evoke. The discovery of such suitable chemical substances has so far been difficult proven to be difficult and expensive.

There is therefore a strong need to to develop test methods that are to be carried out in a specialized manner to detect chemical substances in to test extensively for their corresponding induction ability. It's be already known to insert certain DNA sequences into the genome of plants, which has a promoter which is influenced by chemical substances and a Se quenz, which encode for an enzyme that is easily detectable, and the  plants thus obtained on the inducibility of the promoters by chemical Testing substances (cf. EP-A 0 332 104). The test systems described above ver however, all so-called PR ("pathogen related") promoters apply, which, except on pathogens, not only on chemical substances but also on other abiotic ones Stimuli (such as wounding, warmth or exposure, light / dark cycles, Heavy metals) react. These test systems are therefore relatively prone to failure and can to a large extent lead to misinterpretations and thus to un lead to correct results.

The new DNA sequence (hereinafter referred to as "DNA sequence I") has now been found, which consists of the following components, which are in direct 5′-3′-orientation follow each other:

• 1. A promoter that can be activated in plants by the agents that plant systemic defense mechanisms can be activated can;
• 2. a subsequent one for phosphinothricin-N-aceryltransferase (im following also called "PAT") coding DNA sequence: and
• 3. the subsequent termination sequence of the nopaline synthase Agrobacterium tumefaciens gene ("nos").

Agents that can induce plant defense mechanisms are chemical substances or microorganisms, their metabolites and Extracts from microorganisms, preferably chemical substances.

Among chemical substances in connection with the present inven synthetic or naturally occurring organic compounds preferably low molecular weight organic substances are understood, especially prefers those which do not naturally appear in plants as elicitors (i.e. as Ab military substances or defense mechanisms inducing substances) occur and especially highlighted are those that do not naturally occur in plants. Synthetic organic low molecular weight substances are particularly preferred do not occur in nature.  

As promoters that can be activated in plants, which plant defense mechanisms can induce systemically, plant promoters are preferred Originated. Such promoters are preferably plant-based Origin, which are suitable for induced systemic Lead resistance and at the same time through chemical resistance inducers (e.g. Na salicylate, dichlorisonicotinic acid or probenazole) can be activated.

A large number of promoters which are known from the literature induced systemic resistance (see e.g. EP-A 0 648 839, EP-A 0 516 958, EP-A 0 533 010, EP-A 0 309 862, EP-A 0 480 236, EP-A 0 464 461 and EP-A 0 412 381).

By the specialist it is with the help of general specialist knowledge and general my usual methods easily possible among these known promoters, to select those promoters which are induced by resistance inducers (e.g. Na salicylate, Dichlorisonicotinic acid or probenazole) are inducible and for carrying out of the present invention are suitable.

Such promoters of plant origin are preferably used, which plant defense mechanisms can induce systemically, however not by other abiotic stimuli (such as wounding, warmth, Exposure or light / dark cycles) can be activated.

According to the invention, the promoters are preferably used as component 1 of the DNA sequence I used which is described in the European Offen documents are described. Furthermore, it is also preferred known prp1-1 promoter from potato used. The pro A section of the CaMV 35S RNA can also be used to increase the effect Promotors (preferably the position -46 to +8) are connected.

According to the invention, promoters (component 1 of the DNA sequence 1) a region of the Vst1 promoter from Vitis vinifera (position +72 to -552) or a region of the prp1 promoter from potato from position -402 to -130 used, wherein in a particularly preferred embodiment, the prp1-Pro motor region the region with the position -46 to +8 of the CaMV 35S RNA Promotors is connected downstream.  

The Vst1 promoter originating from Vitis vinifera, or this promoter Genes containing are known from EP-A 0 464 461 and on the plasmids pVSt1, pVSt2 and pVSt12t3 included.

The Eschericha coli strain E. coli Fier 1 pVSt1 contains the plasmid pVSt1. Of the Escherichia coli strain E. coli Fier 2 pVSt2 contains the plasmid pVSt2. Of the Escherichia coli strain E. coli Fier pVSt12t3 contains the plasmid pVSt12t3. This Strains were in connection with the aforementioned EP application to the German Collection of Microorganisms (DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Federal Republic of Germany in accordance with the Budapest Treaty on the international recognition of the deposit of Microorganisms deposited for the purposes of patent proceedings (Filing date: E. coli Fier 1 pVSt1 and Fier 2 pVSt2: June 18, 1990 and E. coli Fier pVSt12t3: February 11, 1991).

The E. coli Fier 1 pVSt1 strain received the accession number DSM 6002, the Strain E. coli Fier 2 pVSt2 received the accession number DSM 6003 and the Strain E. coli Fier pVSt12t3 received the accession number 6346.

The promoter can be identified by the person skilled in the art on the basis of simple routine measures the microorganisms and the plasmids contained therein are isolated.

The complete Vst1 gene was isolated from Vitis vinifera (R. Hain et al. (1993) Nature 361: 153-156; W. Wiese et al. (1994) Plant Mol Biol 26: 667-677) and his inducible expression in the homologous and heterologous system examined. In the Vst1 promoter region used in DNA sequence 1 was determined using the PCR technique amplified and then as described below with that for PAT coding region merged.

The prp-1 promoter is from the publications by J. Taylor et al. (Mol. Plant- Microbe Interactions (1990) 3: 72-77) and N. Martini et. al. (Mol. Gen. Genet. (1993) 236: 179-186). The Cauliflower Mosaic Virus (CaMV) 35S RNA Promoter is also known from the literature (see Benfey & Chua (1990) Science 250: 959-960).

The Escherichia coli strain E. coli GSpETVgus27-1 contains the plasmid pETVgus27-1, which contains the prp-1 promoter from potatoes (N. Martini et  al. (1993) Mol. Gen. Genet. 236: 179-186 and WO 93/19 188). This E. coli Strain became at the German Collection for Microorganisms (DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Federal Republic of Germany in Compliance with the provisions of the Budapest Treaty on the International recognition of the deposit of microorganisms for the Filed for purposes of patent proceedings. He received the deposit number DSM 9627 (filing date 12/20/94). As described below, the prp1-1 promoter region from position -402 to -130 (272 bp) together with the TATA box Region of the CaMV 35S RNA promoter (position -46 to +8) from the Plasmid pEVgus27-1 can be isolated as an EcoRI / HindIII restriction fragment.

DNA sequences which can be used according to the invention and which are suitable for phosphinothricin-N- Encoding acetyl transferase ("PAT") are known.

The sequences coding for PAT are preferably from Streptomyces viridochromogenes ("pat gene") or from Streptomyces hygroscopicus ("bar gene"), the coding sequence of the pat gene is particularly preferably used.

The sequence from S. viridochromogenes coding for PAT is from the literature known (W. Wohlleben et al. (1988) Gene 70: 25-37; W. Dröge et al. (1992) Planta 187: 142-151).

Instead of the pat gene from S. coding for phosphinothricin-N-acetyltransferase viridochromogenes can also use the bar gene from S. hygroscopicus will. The bar gene is also known from the literature (M. DeBlock et al. (1987) EMBO J. 6: 2513-2518; C. Thompson et al. (1987) EMBO J. 6: 2519-2523). The bar gene from S. hygroscopicus is also described in EP-A 0 242 246. The complete coding region of the bar gene from S. hygroscopicus is in the vector pBG39 as a 1.65 kb PstI / BamHI fragment in pUC 19. pBG39 was according to EP-A 042 246 on December 12, 1985 at the German Collection of Microorganisms (DSM) in Göttingen (Germany) with the Filing number DSM 3607.

The coding region of the bar gene consists of 184 amino acids with the help the PCR technique amplified according to generally known methods and with corresponding promoter and termination sequences can be fused.  

It should be noted that the bacterial gene is the start codon for translation GTG has, instead of the ATG start codon used in eukaryotes. Consequently the start code for expression in plants must be changed accordingly. For this purpose, the oligo used for the PCR amplification nucleotides (primers) chosen accordingly (see Example Bc, below). The express sion of both genes mediates resistance to phosphinothricin in plants.

The pat gene from S. viridochromogenes, which is also in the German patent Message DE-A-36 28 747 is described, for. B. from the total DNA of S. viridochromogenes according to DSM 40736 (general collection of the Germans Collection for microorganisms (DSM) Mascheroder Weg 1 b, D-38124 Braun Switzerland, Federal Republic of Germany or DMS 4112 (deposited under Buda Fixed contract with the German Collection for Microorganisms (DSM) Masche roder Weg 1b, D-38124 Braunschweig, Federal Republic of Germany (see DE-A-36 42 829 A1 and EP-A 0 275 957).

The termination sequence of the to be used as component 3 of the DNA sequence Nopaline synthase gene ("nos gene") from Agrobacterium tumefaciens is known or available according to generally known methods (cf. A. Depicker (1982) J. Mol. Appl. Genet. 1: 561-574).

According to the invention, DNA sequence I, which consists of the following constituents exist, which are in direct 5'-3'-orientation to each other following, and which is called "DNA Sequence II" below:

• 1. A region of the Vst1 promoter from Vitis vinifera from position +72 to -552 (624 bp);
• 2. a subsequent one for phosphinothricin-N-acetyltransferase (PAT) coding region of the pat gene from Streptomyces viridochromo genes; and
• 3. the subsequent termination sequence of the nopaline synthase Agrobacterium tumefaciens gene ("nos").

Furthermore, DNA sequence I is particularly preferred according to the invention, which consists of the following components, which are in direct 5'-3'-orientation successive and which is called "DNA sequence III" in the following:

• 1. A region of the prp1-1 potato promoter from position -402 to -130 (272 bp) and
  an adjoining region (TATA box region) of the CaMV 35S RNA promoter (positions -46 to +8);
• 2. the subsequent one for phosphinothricin-N-acetyltransferase (PAT) coding region of the pat gene from Streptomyces viridochromo genes; and
• 3. the subsequent termination sequence of the nopaline synthase Agrobacterium tumefaciens gene ("nos").

In view of the present state of the art, it is surprising that the DNA sequence I and DNA sequences II and III according to the invention particularly advantageous way to find agents which plant defense mechanisms can induce. Both Resistance to phosphinothricin that has become known to date has been met corresponding coding sequences are always constitutively expressed, as expected leads to high resistance. However, it was surprising to the person skilled in the art and not predictable that the constructs according to the invention using inducible promoters in plants for the screening according to the invention Procedures would produce sufficient resistance to phosphinothricin.

Under the term DNA sequence I and DNA sequences II and III are the Assembled from the above components 1. to 3. Understand DNA sequences. However, this term also includes the DNA sequences I, II and III derived DNA sequences. Derived DNA sequences are those DNA sequences that are essentially the DNA sequences I, II and III correspond and can be used according to the invention. It han This is, for example, DNA sections that contain the DNA sequences I, II or III contain such that their function according to the invention is not or not is significantly impaired. Such DNA segments can, for example  arise when the DNA sequences I, II and III from a larger piece of DNA are cut out with the help of restriction enzymes. The DNA sequences I, II and III can also have such DNA sequences at their ends wear, which are adapted for their handling (e.g. so-called "left"). Furthermore, one or. In the DNA sequences I, II or III multiple DNAs or sections of DNA by others essentially the same kende DNAs or DNA sections can be replaced, as this z. B. from the Variation of DNA resulted from the degeneracy of the genetic code can.

For the use according to the invention, DNA sequence I or DNA sequence II or III built into the genome of plants. The one here emerging transgenic plants or parts of these plants, such as leaves, stems, Roots, flowers and seeds or their parts or cells (including Protoplasts), calli, etc. are used for their inducing effectiveness investigating chemical substances brought into contact. If such inducing efficacy is due to the sequence of the pat or bar gene the enzyme phosphinothricin-N-acetyltransferase (PAT) is formed, which or its activity based on the phosphinothricin resistance of the test plants or in Enzyme test can be determined qualitatively or quantitatively. That way it is possible to set up test systems that are easy to carry out with their help even larger numbers of test substances relatively quickly and safely on their inducing properties can be tested. A particular advantage of this The test system is also based on the fact that substances with a positive result are not just that Activate VSt1 or prp1-1 promoter, but are generally suitable, also others Promoters of plant pest defense genes, in particular promoters of phytoalexin genes and PR promoters. With the help of The method according to the invention can thus be obtained from a large number of chemical Suitable compounds are quickly and reliably determined, which then if necessary, can be used specifically for further investigations. The The inventive method enables the simple optical assessment of the gen activating potency of a test substance by observing the herbicidal Effect of phosphinothricin, with which the test plants subsequently on the Treatment with the test substances are treated so that an analysis on enzymatic level is not required. The present invention relates to thus a test method according to which with the help of a simple Routine procedures from a variety of organic compounds such  can be selected that induce plant pest defense genes can and which thus as pesticides in pest control Question come. The plant defense genes to be induced are are preferably genes that are used to protect plants against attack by phytopathogenic microbial pests, preferably by fungi, bacteria or viruses, particularly preferably caused by phytopathogenic fungi.

The present invention thus also relates to the use of the DNA sequence I (or the DNA sequence II and II) and transgenic plants or Plant parts that the DNA sequence I (or the DNA sequence II or III) or contain a DNA sequence derived therefrom in their genome for detection of chemical substances that may be suitable, the plant's own defense mechanism to induce organisms against plant pests. Furthermore, the present concerns Invention also the new use of the for phosphinothricin-N-acetyltransferase coding DNA sequence as a reporter gene in connection with inducible plant promoters in plants for finding agents which plants defense mechanisms.

Furthermore, the present invention thus also relates to a method for opening find plant-specific chemicals that may be suitable to induce defense mechanisms against plant pests, which ge is characterized in that one transgenic plants or parts of plants in their Genome the DNA sequence I (or the DNA sequences II or III) or a DNA sequence derived therefrom, with those on their inducing Effectiveness in contact with chemical substances to be investigated, and the Substances that have an increased expression of phosphinothricin-N-acetyltransferase and thus cause increased resistance to phosphinothricin.

As test plants which can be used according to the invention and which contain the DNA sequence I in contained in their genome, practically all plants can be used. Before preferably one will use plants in which the found Resistance inductors are to be used for pest control. For this belong mainly to plants for agriculture and forestry, for ornamental plant cultivation, medicinal plant cultivation and plant breeding are of interest. However, such plants are particularly preferably used as test plants, which are easy to use in a test laboratory and are also quick and easy to use  let multiply. Transgenic plants are particularly preferred Arabidopsis thaliana, tomato and tobacco, especially called tobacco.

The present invention also includes new plants (including the plant parts and propagation material, such as seeds, offshoots, tubers, cells, proto plasti, calli, etc.) that have the DNA sequence I (or the DNA sequences II or III) included. Preferred new plants according to the invention are Arabidopsis thaliana, tomato, potato and tobacco, most preferably tobacco.

The present invention also relates to a method for producing the new er transgenic plants according to the invention (including the plant parts and the Propagation material), which is characterized in that one

• (a) DNA sequence I (or DNA sequences II or III) or one of them derived DNA sequence in the genome of plant cells (including Protoplasts) and, if necessary,
• (b) Plants completely transformed from the transformed plant cells regenerated and optionally increased, and if necessary
• (c) from the transgenic plants of the parent generation thus obtained or white the desired plant parts eventually propagating material wins.

The present invention also includes those described above The transgenic plants obtainable by the method.

Process steps (a), (b), and (c) can be carried out according to known processes and Me methods in the usual way.

To carry out the test method, the organic chemical to be tested Substances preferably dissolved in water. For substances that are sparingly soluble in water preferably first a solution in a physiologically acceptable orga African solvents, such as acetone, dimethylformamide, dimethyl sulfoxide, Made of methanol or ethanol. This solution is then mixed with water diluted. The test solution should preferably be less than 5% (percent by weight), particularly preferably contain less than 1% organic solvent.  

The concentration of the test substances in the test solution can be over a wide range Range fluctuate and depends on the solubility of the test substances and the respective details of the test implementation, e.g. B. the sensitivity of the test plants or the plant parts selected for the test and can be described by the person skilled in the art simple routine attempts, e.g. B. with the help of known resistance inductors, such as e.g. B. Na salicylate, dichlorisonicotinic acid or probenazole can be determined. This Substances can also be used to test the test system on its Functionality to check and calibrate as well as with respect to the optimize certain circumstances. The test substances are preferably used in a concentration of 5 µM to 20,000 µM, particularly preferably of 100 µM to 10,000 µM and very particularly preferably from 50 µM to 5,000 µM used. In the case of strongly acidic or strongly basic compounds, it can be appropriate to adjust the pH by adding the usual buffer solutions bring a physiologically acceptable range (preferably pH 4.5 to 7.5). In general, however, such pH adjustment is not necessary. For a superficial application can improve the wetting of the vegetable mass terials can also be added to one of the usual wetting agents It is important to ensure that the wetting agent used itself has no inducing properties having.

Likewise, the concentration of phosphinothricin can vary over a wide range can be varied. Depending on the plant, plant age, plant part, genotypic as well phenotypic factors, etc., phosphinothricin concentrations can be used be the usual application rates between 0.1 kg / ha and 10 kg / ha Correspond to phosphinothricin. By simple series tests, each can optoimal phosphinothricin application rate can be easily determined.

As already explained above, the test method according to the invention can the test plants in the form of whole plants or in the form of plants Center parts, preferably in the form of leaves, leaf parts or stems or parts used by stems. The test plants, which as such or in form parts of plants or parts obtained from them (e.g. leaf pieces) should have reached at least the 4-leaf stage, i.e. at least first fully developed leaves growing after the cotyledons point. In the case of tobacco, this stage, based on seeds, becomes dependent on the conditions after about 14 to 21 days in soil at 20 to 22 ° C and reached about 10,000 lux lighting. If necessary, it can be useful  the young plants in the growing period before too much water evaporation, e.g. B. by covering to protect. The size and age of the test plants can be largely varied. It is expediently and preferably used small and young plants, which are the 4-leaf stage or the following Have reached growth stage. The test plants are particularly preferred used in the 4-leaf stage.

The dissolved test substances can be mixed with the plants or in any way be brought into contact with the plant material, e.g. B. by spraying or Brush up. The plant material can also be in the solutions of the test substances to be immersed or inserted. In addition to these methods of superficial on bring, it is also possible to record the test substances via the Wur individual plants or parts of the plant. About who the roots of the intact plants, plant stems or petioles in the test solutions for a sufficiently long time that the absorption of the test substances allowed, immersed. Application is preferably via the roots. The The exposure time is preferably between 1 and 96, particularly preferably between 2 and 72 and very particularly preferably between 24 and 48 hours. The Incubation is preferably done under normal exposure or exposure in a conventional light cabinet, which is expediently the one for the plants usual light-dark rhythm (e.g. 16 h light, 8 h dark) is observed. The incubation is preferably at temperatures between 15 and 30 ° C, be made particularly preferably between 18 and 28 ° C.

The application of the herbicide phosphinothricin can simultaneously with the Treatment of the chemical to be tested or by various large ones Time intervals after the treatment of the chemical substance to be tested. If intact plants that have been planted in soil are examined, those with the test substance or test plants treated with phosphinothricin Greenhouse conditions (20-25 ° C, approx. 10,000 lux and 60% relative humidity) incubated and irrigated exclusively from below. The evaluation takes place 3-21 Days after treatment with phosphinothricin. The resistance indu ornamental influence of the test substance based on the phosphinothricin resistance of the test plants compared to control plants treated only with phosphinothricin were judged. Here criteria such as leaf size, growth rate damage, leaf damage etc. are used. In addition, the genakti  Fourth potency of a test substance by determining the in vitro phosphino thricin-N-aceytltranferase activity can be analyzed in more detail.

In a further possible embodiment of the invention, the appli is carried out cation of the test substances via the roots of young test plants. The sub punches are taken up by the root and over the lead tissue distributed in the plant. Then the test is carried out as described and evaluated.

If there is an increase in the performance of the test method according to the invention low phosphinothricin resistance, d. H. e.g. B. increased plant growth or a reduction in plant damage caused by phosphinothricin, the Test plants treated with test substances in comparison with the control plants is measurable, there is a test substance that is accepted may have the inductive properties sought. In doing so the control plants traded with water as a test solution, which if necessary the organic solvent used and / or the surface used contains active agents in the appropriate concentration. The form of the Resistance to phosphinothricin usually correlates with the strength of the induction ver of the respective test substance.

Transgenic plants that have the DNA sequence I (or the DNA sequences II or III) contained in their genome and as for the test method according to the invention Test plants can be used according to the common practice Methods available.

A number of different methods are available, DNA sequence I (or the DNA sequences II or III) in the genetic material of plants or use plant cells. As stated earlier, DNA transfer can take place according to the generally customary known methods, the person skilled in the art identify and perform the appropriate method without difficulty can.

All percentages listed in this text refer to if nothing else is given on weight percentages.  

The test method according to the invention and the generation of the invention usable transgenic plants are intended to be non-limiting by the following acting examples are explained. In the examples, "PPT" means Phosphinothricin.

Examples I. Example A (test procedure) a) Cultivation and treatment of phosphinothricin-resistant test plants

Phosphinothricin (PPT) resistant tobacco plants are grown house conditions (22 ° C, 60% relative humidity, approx. 15,000 lux) up to Flower cultivated and self-grown to gain the branch (F1) generation. F1 generation seeds are grown in sterile culture on LS medium at 75 µg / ml Kanamycin (Sigma) preselected and the resistant F1 seedlings after Transferred to soil for 2 weeks. After another 6-14 days, the Plants with aqueous test solutions of different chemi to be tested Kalien / active ingredients sprayed (5-20 000 µM) to different sizes Intervals to be treated with PPT (0.1-10 kg / ha). As a negative control serve SR1 wild-type seedlings or tobacco seedlings, which are only the transferring plasmid with the kanamycin resistance gene (nptII), however without promoter / pat or promoter / bar fusion included. Parallel to Screening examinations with F1 plants are approx. 10 F1 plants self-grown and the F2 seed homo for identification offspring of zygotes designed on LS medium containing kanamycin. Homozygous F2 seeds are then sown directly into the earth, the tobacco seedlings pricked after about 10 days and after another 5-14 days like that F1 seedlings treated. The spraying takes place in spray booths. Here 50 ml of test or PPT solution are evenly mixed with a Pressure of 3.0 bar distributed over an area of 1 m², so that approx. 80 plants / m² can be sprayed simultaneously.

b) Assessment of the tobacco treated with inducing substances seedlings

Depending on the seedling age and the PPT concentrations applied plant growth is assessed after 5-20 days by the largest sheet is measured. In addition, the Leaves open after treatment with the inducing chemicals possible leaf damage examined. A multiple evaluation in  various large intervals after the PPT treatment proved to be extremely useful because the time course of gene activation or PPT resistance induction by the various chemical substances strong can vary. If a substance shows a resistance-inducing effect, can change the time course and the level of gene activation with the help a simple enzyme test can be analyzed and quantified (M. DeBlock et al. (1987) EMBO J. 6: 2513-2518 and M. DeBlock et. al. (1989) Plant Physiol. 91: 694-701).

Test results Trial 1

Tobacco plants that have the DNA sequence III with the pat gene from Strepto containing myces viridochromogenes were tested for PPT resistance Litter: Homozygous seedlings of the F2 generation became 16 days after sowing with probenazole (1 mM), water as a control test solution, and 5-chlorosalicylic acid (1 mM) treated. 24 hours later the plants were with 2 kg / ha PPT sprayed. The test treated with the resistance inducers plants showed significantly larger 1 week after PPT treatment Leaves than plants treated only with water.

Trial 2

Tobacco plants that have the DNA sequence III with the pat gene from Strepto myces viridochromogenes, were the PPT resistance test Subject: Homozygous F2 seedlings were grown 20 days after sowing treated with 10 mM sodium salicylate or water, 24 hours later 4 kg / ha PPT applied. Wild type plants and Plants that have the PPT resistance gene under the constitutive 35S Contained Cauliflower Mosaic Virus RNA promoter (W. Dröge et al. (1992) Planta 187: 142-151).

The test plants treated with Na salicylate showed considerably larger ones Leaves as the plants treated only with water. The sheet size of the with The plants treated with Na salicylate corresponded to those of the control plants contained the constitutive CaMV 35S RNA promoter. Wild type plants  were so badly damaged by 4 kg / ha PPT that they only a few days after treatment stopped and stunted.

II. Example B Cloning strategy for the production of DNA sequence II

The region of the Vitis vinifera Vst1 promoter from position +72 to -552 (624 bp) was determined using the PCR technique with the following primers of the sequences according to SEQ ID No. 1 and SEQ ID No. 2 (see also FIG. 1 and FIG. 2) amplified.

The plasmid pVSt12t3 according to EP-A 0 464 461 was used as a template, which in E. coli strain E. coli Fier pVSt12t3 according to DSM 6346 ent hold is.

The amplified fragment, which is the sequence of the Vst1 promoter from Vitis vinifera from position +72 to -552 was with the restrictions enzymes BamHI and HindIII cut. The resulting BamHI / HindIII Vst1 promoter fragment was combined with a BamHI / HindIII fragment (approx. 900 bp) from the vector pWD26.41 (W. Dröge et al. (1992) Planta 187: 142-151), which is the coding region of the pat gene from S. viridochromogenes and the polyadenylation sequence of the nopaline synthase gene from A. tumefaciens contains in the HindIII linearized binary vector pPCV002 (C. Koncz and J. Schell (1986) Mol. Gen. Genet. 204: 383-396) in the resulting Construct pVst1PAT is under the coding region of the pat gene Control of the Vst1 promoter (up to -552). pVst1PAT corresponds to the DNA sequence II.

b) Cloning strategy for the production of DNA sequence III (cf. FIG. 4)

The region of the prp1-1 promoter from Solanum tuberosum (position -402 to -130) was together with the adjoining region (TATA box region) of the CaMV 35S RNA promoter (position -46 to +8) as an EcoRI / HindIII fragment (326 bp) from the vector pETVgus27-1 (N. Martini et al. (1993) Mol. Gen. Genet. 236: 179-186) isolated and into the  multiple cloning site of the binary vector pPCV002 (C. Koncz and J. Schell (1986) Mol. Gen. Genet. 204: 383-396) added (→ pPRP1-1Pro). The coding region of the pat gene from Streptomyces viridochromogenes was together with the termination signal of the nopaline synthase gene (nos) from A. tumefaciens as BamHI / HindIII fragment (approx. 900 bp) the vector pWD26.41 (W. Dröge et al. (1992) Planta 187: 142151) cleaned. The bacterial pat gene is already in the vector pWD26.41 modified in such a way that efficient expression in plants is possible is. The 900 bp fragment was made with a HindIII / BamHI oligonucleotide linker (see below) merged with the linker oriented so that its HindIII restriction interface only by a subsequent HindIII restriction digest was generated. The resulting HindIII frag was inserted into the HindIII restriction site of the construct pPRP1-1-Pro ligated so that the coding region of the pat gene under The prp1-1 promoter is in control (→ pPRP1-PAT (= DNA sequence III)).

The HindIII / BamHI oligonucleotide linker used consisted of the oligonucleotide according to SEQ ID No. 3 (see Fig. 3).

c) Example of the preparation of the DNA sequence II with the bar gene S. hygroscopicus 1. Amplification of the coding region of the bar gene as BamHI / EcoRI (5 ′ → 3 ′) restriction fragment

The bar gene is primed with the sequences according to SEQ ID No. 4 and SEQ ID No. 5 (cf. also FIGS. 5 and 6) were amplified in a PCR reaction and then trimmed with the restriction enzymes BamHI and EcoRI.

2. Fusion of the bar gene with a polyA signal

The resulting BamHI / EcoRI (5 ′ → 3 ′) restriction fragment (556 B sp.) with the polyadenylation signal of the 35S RNA from Cauliflower Mosaic Virus fused, which was previously an EcoRI / HindIII (5 ′ → 3 ′) fragment (approx. 250 bp) from the vector pRT104 (R. Töpfer et al. (1987) Nucl. Acids Res. 15: 5890) was isolated.  

3. Fusion of the bar gene with the Vst1 promoter from wine

The resulting BamHI / HindIII (5 ′ → 3 ′) fragment, consisting of the bar- Gene and a polyA sequence is generated using common methods cleaned and in the next cloning step together with the Vst1 promoter, which is present as a HindIII / BamHI (5 ′ → ′ 3 ′) fragment (cf. clone Development strategy for the production of DNA sequence II with pat gene from S. viridochromogenes), in the HindIII site of the binary vector pPCV002 (C. Koncz and J. Schell (1986) Mol. Gen. Genet. 204: 383-396) ligated. The coding is in the resulting construct pVST1BAR Region of the bar gene under the control of the Vst1 promoter (up to -552; cf. DNA sequence II).

d) Example of the preparation of the DNA sequence in with the bar gene from S. hygroscopicus 1. Amplification of the coding region of the bar gene as HindIII / BamHI (5 ′ → 3 ′) restriction fragment

The bar gene is primed with the sequences according to SEQ ID No. 6 and SEQ ID No. 7 (cf. also FIGS. 7 and 8) amplified in a PCR reaction and then cut with the restriction enzymes HindIII and BamHI.

2. Fusion of the bar gene with a polyA signal

The resulting HindIII / BamHI (5 ′ → 3 ′) fragment (558 base pairs) is with the polyadenylation signal of the 35S RNA from Cauliflower Mosaic Virus fused, which was previously a BamHI / HindIII (5 ′ → 3 ′) fragment (approx. 250 bp) from the vector pRT104 (R. Töpfer et al. (1987) Nucl. Acids Res. 15: 5890) was isolated.

3. Fusion of the bar gene with the potato prp1-1 promoter

The resulting HindIII fragment, consisting of the bar gene and one polyA sequence, is cleaned using generally customary methods and  in the next cloning step into the HindIII restriction site of Vector pPRP1-1Pro (see Fig. 4) ligated. In the resulting construct pPRP1-1BAR controls the coding region of the bar gene of the prp1-1 promoter from S. tuberosum (cf. DNA sequence III).

III. Example C (Generation of Transgenic Plants) I. Tobacco Transformation a) Culture of tobacco sprouts and isolation of tobacco protoplasts

Nicotiana tabacum (Petit Havana SR1) is grown as a sterile shoot culture on Hor mon-free LS medium (Linsmaier and Skoog 1965) increased. In intervals From about 6-8 weeks, shoot sections are placed on fresh LS medium implemented. The shoot cultures are in 12 h light (1000-3000 lux) in kept in a culture room at 24-26 ° C.

Approx. 2 g leaves (approx. 3-5 cm long) with a fresh razor blade into small pieces (0.5 cm × 1 cm) cut. The leaf material is in 20 ml enzyme solution consisting of K3 Medium (Nagy and Maliga 1976), 0.4 m sucrose, pH 5.6, 2% Cellulase R10 (Serva), 0.5% Macerozym R10 (Serva) for 14-16 h at Incubated at room temperature. After that, the protoplasts by filtration Over 0.30 mm and 0.1 mm steel sieves separated from cell debris. The filtrate is centrifuged at 100 x g for 10 minutes. During this centrifuge gation float intact protoplasts and collect in a band top of the enzyme solution. The pellet from cell debris and the enzyme solution is aspirated with a glass capillary. The pre-cleaned proto plastic with fresh K3 medium (0.4 M sucrose as Osmotic) made up to 10 ml and floated again. The washing medium is aspirated and the protoplasts are used for culture or following Infection with agrobacteria (coculture) diluted to 1-2 × 10⁵ / ml. The Protoplast concentration is determined in a counting chamber.  

b) Transformation of regenerating tobacco protoplasts by co-culture with Agrobacterium tumefaciens

The method of Marton et al. 1979 with little ones Changes used. The protoplasts are isolated as described and in a density of 1-2 × 10⁵ / ml in K3 medium (0.4 M sucrose, 0.1 mg / l NAA, 0.2 mg / l kinetin) 2 days in the dark and one or two Incubated for days under low light (500 lux) at 26 ° C. As soon as the first division of the protoplasts occur, 30 µl one Agrobacterium suspension of the strain GV3 101 C58CI pMP90RK: prp1-1-PAT or Vst1-PAT or other suitable agrobacteria strain containing prp1-1-PAT or Vst1-PAT in Minimal A (Am) Medium (density approx. 10⁹ agrobacteria / ml) to 3 ml regenerating proto given plastic. The coculture time is 3-4 days at 20 ° C in Dark. Then the tobacco cells are placed in 12 ml centrifuge tubes filled, diluted to 10 ml with sea water (600 mOsm / kg) and at 60 × g Pelleted for 10 minutes. This washing process will be 1-2 times again picks up to remove most of the agrobacteria. The cell suspensions sion is in a density of 5 × 10⁴ / ml in K3 medium (0.3 in Saccha rose) with 1 mg / l NAA (naphthyl-1-acetic acid), 0.2 mg / l kinetin and 500 mg / l of the cephalosporin antibiotic cefotaxime cultivated. The cell suspension is diluted every week with fresh K3 medium and the Gradually increase the osmotic value of the medium by 0.05 m sucrose (approx. 60 mOsm / kg) per week. Selection with kanamycin (100 mg / l Kanamycin sulfate) is agarose 2-3 weeks after coculture "Bead type culture" (Shillito et al. 1983) started. Kanamycin resistant Colonies can emerge 3-4 weeks after the start of selection from the rear to be distinguished on the basis of remaining colonies.

c) Direct transformation of tobacco protoplasts with DNA. Calcium nitrate PEG transformation

Approx. 106 protoplasts in 180 µl K3 medium are mixed in a Petri dish 20 ul aqueous DNA solution containing 0.5 ug / l of plasmid prp1-1-PAT or contains Vst1-PAT, mixed gently. Then 200 µl Fusion solution (0.1 M calcium nitrate, 0.45 M mannitol, 25% polyethylene Glycol (PEG 6000), pH 9) carefully added. After 15 minutes 5 ml washing solution (0.275 M calcium nitrate pH 6) added and after white After 5 minutes, the protoplasts are placed in a centrifuge tube transferred and pelleted at 60 × g. The pellet comes in a small amount  K3 medium recorded and as described in the next section cultured. Alternatively, the protoplasts according to Hain et al. 1985 be transformed.

d) Culture of protoplasts incubated with DNA and selection of kanamycin resistant calli

For the culture and selection described below Kanamycin-re resistant colonies becomes a modified "Bead Type culture" technique (Shillito et al. 1983). A week after treatment of the Protoplasts with DNA (see c) are 3 ml of the cell suspension with 3 ml K3 Medium (0.3 M sucrose + hormones; 1.2% (Seaplaque) LMT Agarose (low melting agarose, marine colloids) in 5 cm petri dishes mixes. For this purpose, agarose is autoclaved dry and after Add K3 medium briefly boiled in the microwave. After The agarose disks ("beads") solidify with the embedded tobacco micro calli for further culture and selection in 10 cm Petri dishes are transferred and 10 ml K3 medium (0.3 M sucrose, 1 mg / l NAA, 0.2 mg / l kinetin) and 100 mg / l kanamycin sulfate added. The Liquid medium is changed every week. The osmotic Value of the medium gradually reduced.

The exchange medium (K3 + Km) increases by 0.05 M per week Sucrose (approx. 60 mOsm) reduced.  

Scheme of selection of kanamycin-resistant tobacco colonies after DNA transformation

e) Regeneration of kanamycin-resistant plants

As soon as the kanamycin-resistant colonies have a diameter of approx. 0.5 cm, half will be on regeneration medium (LS medium, 2% sucrose, 0.5 mg / l benzylaminopurine BAP) and at 12 h Light (3000-5000 lux) and 24 ° C kept in the culture room. The other Half is used as a callus culture on LS medium with 1 mg / l NAA, 0.2 mg / l Kinetin, 0.1 mg / l BAP and 100 mg / l kanamycin sulfate. If the regenerated shoots are approx. 1 cm in size, they are cut off and on 1/2 LS medium (1% sucrose, 0.8% agar) without growth regulators set for rooting. The rungs are set to 1/2 MS medium rooted with 100 mg / l kanamycin sulfate and later in soil implemented.

The F1, F2 and F3 generations of these regenerates are used as a test plants used.  

f) transformation of leaf disks by Agrobacterium tumefaciens

For the transformation of leaf disks (Horsch et al. 1985) approx. 2-3 cm long leaves of sterile shoot cultures in 1 cm slices Punched diameter and with a suspension of appropriate agro bacteria, GV3101 C58CIpMP90RK :: prp1-1-PAT or Vst1-PAT, (approx. 10⁹ / ml) (cf. b) in Am medium, see below) for about 5 minutes. The infected leaf pieces are on MS medium (see below) without Hormones kept at about 24 ° C for 3-4 days. During this time Agrobacterium grows the leaf pieces. The leaf pieces are on finally washed in MS medium (0.5 mg / ml BAP, 0.1 mg / ml NAA) and on the same medium (0.8% agar) with 500 g / ml cefotaxime and 100 g / ml kanamycin sulfate. After two weeks, the medium should to be renewed. Transformed rung will be after another 2-3 Weeks visible. The regeneration of sprouts should also take place in parallel Selection printing. The regenerated rung must then by biochemical tests e.g. B. on nopaline synthase or PAT shares be tested for transformation. In this way 1-10% obtained transformed rung.

Biochemical detection method of transformation

In the plant cells and plants (Ta bak) was the presence of DNA sequence I (or II and III) by Northern Blot analysis, Southern blot analysis, phosphinothricin-N-acetyltransferase- Activity (M. DeBlock et al. (1987) EMBO J. 6: 2513-2518 and M. DeBlock et al. (1989) Plant Physiol. 91: 694-701) and phosphinothricin tolerance Phosphinothricin-containing nutrient medium confirmed.

In the following, some of the Media used in plant cells are described.  

Am medium

3.5 g K₂HPO₄
1.5 g KH₂PO₄
0.5 g Na₃ citrate
0.1 g MgSO₄ × 7H₂O
1 g (NH₄) ₂SO₄
2 g glucose
ad 11

Medium for sterile shoot culture of tobacco

Macro elements 1/2 of the concentration of MS salts
Micro elements 1/2 of the concentration of MS salts

K3 medium

On the culture of Nicotiana tabacum petit Havana SR1, Nicotiana tabacum Wiscon sin 38, and Nicotiana plumbaginifolia protoplasts (Nagy and Maliga, 1976)

Linsmaier and Skoog Medium (Linsmaier and Skoog 1965)

For the culture of regenerating protoplasts and for tissue culture of tabaktu moren and callus. Linsmaier and Skoog (LS) Medium is Murashige and Skoog Medium (Murashige and Skoog, 1962) with the following modifications:

• - Thiamine is weighed in a higher concentration instead of 0.4 mg / l 0.1 mg / l;
• - Glycine, pyridoxine and nicotinic acid are missing.

The following literature can be used to transform plants or plant cells be listed:

Fromm ME, Taylor LP, Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319: 791-793.

Hain, R., Stabel, P., Czernilofsky, A.Pp., Steinbiß, H.H., Herrera-Estrella, L., Schell, J. (1985) Uptake, integration, expression and genetic transmission of a selectable chimeric gene by plant protoplasts. Molec Gen Genet 199: 161-168.

Herrera-Estrella L., De Block M., Messens E., Hernalsteens JP., Van Montagu M., Schell J. (1983) EMBO J. 2: 987-995.  

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 277: 1229-1231.

Krens FH, Molendÿk L, Wullems GJ, Schilperoort RA (1982) in vitro transforma tion of plant protoplasts with Ti-plasmid DNA. Nature 296: 72-74.

Koncz C, Schell J (1986) The promotor of T _L -DNA gene 5 controls the tissue specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. (1986) 204: 338-396.

Linsmaier DM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100-127.

Marton L, Wullems GJ, Molendÿk L, Schilperoort PR (1979) In vitro transforma tion of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Well ture277: 1229-131.

Murashige, T. and Skoog F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiol. Plans. 15, 47.

Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Plant Physiol 78: 453-455.

Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I (1984) Direct gene transfer to plants. EMBO J 3: 2717-2722.

Shillito RD, Paszkowski J. Potrykus I (1983) Agarose plating and Bead type cul ture technique enable and stimulate development of protoplast-derived colonies in an number of plant species. Pl Cell Rep 2: 244-247.

Van den Elzen PJM, Townsend J, Lee KY, Bedbrook JR (1985) Achimaeric resistance gene as a selectable marker in plant cells. Plant Mol. Biol. 5, 299-302.

Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergenic transfer and exchange recombination of restriction fragments cloned in  pBR322: a novel strategy for the reversed genetics of Ti plasmids of / Agrobac terium tumefaciens. EMBO J 2: 411-418.

Velten J, Velten L, Hain R, Schell J (1984) Isolation of a dual plant promotor fragment from the Ti Plasmid of Agrobacterium tumefaciens. EMBO J 12: 2723-2730.

Wullems GJ, Molendÿk L, Ooms G, Schilperoort RA (1981) Differential expres sion of crown gall tumor markers in transformants obtalned after in vitro Agrobac terium tumefaciens - induced transformation of cell wall regenerating protoplasts derived from Nicotiana tabacum. Proc Natl Acad Sci 78: 4344-4348.

Zambryski P, Joos H, Genetello C, van Montagu M, Schell J (1983) Ti-plasmid vector for the introduction of DNA into plant cells without altering their normal regeneration capacity, EMBO J 12: 2143-2150.

The following published patent applications can also be listed will:

EP-A 116718
EP-A 159 418
EP-A 120 515
EP-A 120 516
EP-A 172 112
EP-A 140 556
EP-A 174 166
EP-A 122 791
EP-A 126 546
EP-A 164 597
EP-A 175 966
WO 84/02913
WO 84/02919
WO 84/02920
WO 83/01176

Sequence listing

Claims (14)

1. DNA sequence (hereinafter referred to as "DNA sequence I"), which consists of the following components which follow one another in direct 5′-3′-orientation:

• 1. A promoter which can be activated in plants and which can be activated by agents which can systemically induce plant defense mechanisms;
• 2. a subsequent DNA sequence coding for phosphinothricin-N-acetyltransferase (hereinafter also referred to as "PAT"); and
• 3. the subsequent termination sequence of the nopaline synthase gene ("nos") from Agrobacterium tumefaciens;

or a DNA sequence derived from it. 2. DNA sequence according to claim 1, which consists of the following components, which are successive in direct 5'-3'orientation, and which is called "DNA sequence II" in the following:

• 1. A region of the Vitis vinifera Vst1 promoter from position +72 to -552 (624 bp);
• 2. a subsequent region of the pat gene from Streptomyces viridochromogenes coding for phosphinothricin-N-acetyltransferase (PAT); and
• 3. the subsequent termination sequence of the nopaline synthase gene ("nos") from Agrobacterium tumefaciens;

or a DNA sequence derived from it.   3. DNA sequence according to claim 1, which consists of the following components, which follow each other in direct 5'-3 'orientation and which is called "DNA sequence III" in the following:

• 1. A region of the prp1-1 potato promoter from position -402 to -130 (272 bp) and
  an adjoining region (TATA box region) of the CaMV 35S RNA promoter (positions -46 to +8);
• 2. the region of the pat gene from Streptomyces viridochromogenes coding for phosphinothricin-N-acetyltransferase (PAT); and
• 3. the subsequent termination sequence of the nopaline synthase gene ("nos") from Agrobacterium tumefaciens,

or a DNA sequence derived from it. 4. Use of the DNA sequence I according to claim 1 or one of them derived DNA sequence, for finding agents that are suitable can, the plant's own defense mechanisms against plant pests induce. 5. Use of transgenic plants or parts of plants which DNA sequence I according to claim 1 or a DNA sequence derived therefrom in contained in their genome, for finding agents that are suitable can, the plant's own defense mechanisms against plant pests induce. 6. Plant methods for finding agents that may be suitable to induce own defense mechanisms against plant pests, which is characterized in that transgenic plants or Parts of plants which have the DNA sequence I in their genome according to claim 1 or contain a derived DNA sequence with which to indu ornamental effectiveness in contact with the agents to be investigated and the substances that have an increased expression of phosphinothricin-N-acetyl  cause transferase or an increased resistance to phosphinothricin, selects. 7. Transgenic plants (including plant parts and propagation material), the DNA sequence I according to claim 1 or contain a derived DNA sequence. 8. A method for producing the transgenic plants according to claim 7 (including plant parts and propagation material), which characterized in that (a) the DNA sequence I according to Claim 1 or a DNA sequence derived therefrom into the genome of Plant cells (including protoplasts) and (b) trans Formed plant cells regenerate completely transformed plants and optionally increased, and optionally (c) of those thus obtained transgenic plants of the parent generation or others obtained therefrom Generations of desired parts of plants (including propagation material) wins. 9. Use according to claim 4, wherein the transgenic plants or Plant parts are used according to claim 7. 10. The method according to claim 6, wherein the transgenic plants or plants parts are used according to claim 7. 11. Use of DNA encoding phosphinothricin-N-acetyltransferase as reports in connection with inducible plant promoters to find agents that may be suitable, plant-owned To induce defense mechanisms against plant pests.