DE4423022C1 - Transgenic plants with increased content of resistance factor - Google Patents

Abstract

Transgenic plants with at least one heterologous DNA sequence have an increased secondary substance content. Also disclosed is a process for producing the same and their use in forestry, grazing lands, meadows, ornamental and useful plant cultivation. In particular, the heterologous DNA sequence codes for a polypeptide with enzymatic activity that enzymatically generates at least one secondary substance in sufficient concentration to have an anti-viral, bactericide, fungicide, insecticide and/or insect repellent effect.

Description

This invention relates to a transgenic plant with at least a heterologous DNA sequence with the features of claim 1. This is said to have an increased content have certain secondary substances. The invention further relates to a method for their Production and their use in forest, pasture, meadow, Ornamental or crop cultivation.

Herbal secondary substances play a role in plant defense against pathogens, predators or parasites play an important role (see, e.g., Rhodes, Plant Molecular Biology 24 (1994), 1-20; Chasan, Plant Cell 6 (1994), 3-9).

The published patent application DE-OS 41 17 747 describes transgenic Plants, the new Kaffeoyl-CoA 3-O- isolated from plants Contain methyltransferase genes. The expression product Kaffeoyl-CoA 3-O-methyltransferase catalyzed these genes methylation of Kaffeoyl-CoA in a biosynthetic pathway, which leads from trans-4-coumaroyl-CoA to trans-feruloyl-CoA. The pathogen-resistant effect is not based on the Generation of potentially toxic metabolites in the transgenic plants, but it is the synthesis of over weighing insoluble, antibiotic ineffective compounds strive to act as a physical barrier or a pathogen-induced, enzymatic lysis of cell wall To prevent polysaccharides by acylation. The Of DE-OS 41 30 986 describes transgenes Plants, the new genes isolated from plants for pino contain sylvine synthase. Especially come as plants, from which Pinosylvinsyhthase genes can be isolated, all single or double germ plants in question. The Of Application WO 93/15 599 A1 describes transgenes Plants that come from angiosperms ("bedecktsamer") Phenoloxidase genes contain. Which occur in angiosperms the polyphenol oxidases catalyze the oxidation of pheno len to quinones with excess of O₂. The disclosure DE-OS 42 34 131 describes transgenic plants that mind contain at least two different genes, their genes products the enzymes "Chitinase" and "Glucanase", the protein "Protein synthesis inhibitor" or the polypeptide "antifunga les protein ". These gene products are themselves for the pathogen-resistant effect responsible and produce none Secondary substances.

So far there are none Process known, the content of secondary substances via a increase significantly over a longer period of time and thereby a increased pathogen resistance and / or increased resistance against predators or parasites. Currently crop protection is mainly based on the treatment of Plants with various pesticides, their Toxicology and environmental effects in many cases are problematic.

The invention is therefore based on the object, per se pathogen resistant and / or against predators or parasites to provide resistant plant to the use of Restrict plant protection products at least partially and thus crop protection from toxicological and ecological To improve your point of view.

This object is achieved by the provision a transgenic plant with at least one heterologous DNA Sequence necessary for at least one polypeptide with enzymatic Encodes activity in which the polypeptide is expressed in  enzymatically active form and at least one Secondary substance in an antiviral and / or bactericidal and / or fungicidal and / or insecticidal and / or acting as a repellent Concentration generated enzymatically, dissolved, the heterologous DNA sequence comes from bacteria.

The term "transgenic plant" or "plant" includes the whole plant as such and its parts, such as root, Stem, leaf, organ-specific tissue or cells, the reproductive material, in particular seeds, and their Seedlings.

This term also includes Cymnosperinae, Monocotyledoneae and Dicotyledoneae, especially useful plants, such as cereals, for example rye, corn, wheat, corn, barley, rice, oats and millet; Starch bulbs and roots, for example Potato, batate and cassava; Sugar plants, for example Sugar cane and sugar beet; Legumes, for example Beans, peas and chickpeas; Oil and fat fruits, for example soybean, peanut, sunflower, olive tree, rapeseed and coconut; Vegetables, e.g. tomato, cabbage, onion, Cucumber, carrot and lettuce; Fruit, for example grapes, Citrus, banana, apple, pear, peach and pineapple; nut-like fruits, for example walnut, hazelnut, almond and cashew nut; Luxury plants, for example tobacco, Coffee, tea, cocoa; Types for vegetable fibers, for example cotton, jute and flax; Types of forest Use, for example spruce, oak and poplar; and Plant types for the extraction of raw materials, for example Hevea brasiliensis (rubber) and jojoba.

The term "heterologous DNA sequence" means a DNA Sequence derived from a prokaryotic source, for example Escherichia coli, Origin, including archaebacteria. The coding region of the heterologous DNA sequence can coding ("exons") and non-coding ("introns")  Sections included. Furthermore, the heterologous DNA sequence regulatory sections such as promoters, enhancers and Termination sequences included. One is preferred heterologous DNA sequence that contains no introns.

This term also includes chimeric DNA sequences as well as synthetic or semi-synthetic DNA sequences.

In a preferred embodiment, the DNA sequence contains the ubiC gene from Escherichia coli; see. Fig. 1.

The term "polypeptide with enzymatic activity" means a polypeptide derived from the heterologous defined above DNA sequence is encoded, the coding sequence can be degenerate according to the genetic code.

The primary transcript can be translated directly or generated by "splicing" into a translatable mRNA become. Furthermore, the primary translation product can be used for Formation of the enzymatically active polypeptide post-translational be modified, for example by splitting off one Signal sequence, by enzymatic cleavage of the inactive "Precursors" for conversion into the enzymatically active form or by modifying the side chains of the polypeptide, for example by phosphorylation or glycosylation.

Preferred examples of these polypeptides are the enzyme chorismate pyruvate lyase from Escherichia coli, the primary sequence of which is shown in FIG. 2, and the enzyme isochorismate pyruvate lyase (= salicylate synthase).

According to the invention, these generate polypeptides using of substrates present in the transgenic plant enzymatic secondary substances, preferably also in the wild type occur in small quantities ("plant-specific Secondary substances "). These secondary substances accumulate surprisingly in the transgenic invention Plant in a biologically effective concentration, whereby enrichment by either anabolic or catabolic Subsequent reactions or by depositing in certain Cell compartments.  

The concentration of transgenic in the invention Plant-enriched secondary substances shows a clear antiviral and / or bactericidal and / or fungicidal and / or insecticidal and / or antiperspirant effect. Preferably is this biologically effective concentration of Secondary substances (unit: µg secondary substance / g fresh weight of Plant) at least 10 times, more preferably at least that 50 times, most preferably at least 100 times on the concentration of these substances in the wild-type plants.

The term "secondary substance" includes ingredients from plants, which belong to the secondary metabolism, as well as their derivatives, either by subsequent reactions in the plant cell, for example hydroxylation, methylation, glycosylation, Etherification, esterification or polymerization, or from that The polypeptide defined above is generated directly enzymatically become. Thus, either the one produced according to the invention Secondary substance (or its derivative) per se the above have defined biological effects or by Subsequent reactions generated derivative (s).

Examples of secondary substances are alkaloids, isoprenoids, Phenol derivatives, phenylpropane, quinones, coumarins, lignin and Flavonoids. Phenol derivatives, in particular p- Hydroxybenzoic acid and its derivatives.

Another object of the present invention relates a method for producing the transgenic invention Plant in which a plant cell through stable integration the heterologous DNA sequence in the genetic material is transformed and the transformed plant cell to transgenic plant is regenerated.

Processes for the production of transgenic plants are in the prior art known in the art. For example, the Ti plasmid or a binary plasmid system in Agrobacterium tumefaciens as Vector for the stable integration of the heterologous DNA sequence in the genetic material of the transgenic invention  Plant can be used. Furthermore, the heterologous DNA Sequence z. B. also by the Ri plasmid from Agrobacterium rhizogenes, by direct gene transfer by means of Polyethylene glycol, by electroporation or by Particle bombardment in the genetic material of the transgenic Plant are introduced.

Another object of the present invention relates the use of the transgenic plant according to the invention as pathogen resistant and / or against predators and / or Parasite resistant plant in the forest, pasture, meadow, Ornamental and useful plants.

As examples of pathogens, the tobacco mosaic Virus and the cauliflower mosaic virus, as Erwinia bacteria amylovora, Pseudomonas syringae, Corynebacterium michiganense and Xanthomonas campestris, as fungi Phytophtora infestans, Claviceps Purpurea, Botiytis cinerea and Ustilago maydis be listed. Can be predators and parasites especially nematodes, aphids, beetles and Caterpillars are listed.

Furthermore, the present invention relates to the use of The heterologous DNA sequence for production defined above the transgenic plant according to the invention.

The figures show:

Fig. 1 shows the encoding 495 bp nucleic acid sequence of ubiC gene from Escherichia coli.

Fig. 2 shows the 165 aa long amino acid sequence of chorismate pyruvate lyase from Escherichia coli.

Fig. 3 is a schematic representation of the construction of transformation vectors pROK-ubiC and pROK-TP0-ubiC with the ubiC gene from Escherichia coli.

The following example illustrates the invention:

The phenol derivative p-hydroxybenzoic acid (PHB) is a key metabolite in bacteria as well as in higher plants in the biosynthesis of ubiquinone, an electron carrier in the respiratory chain (Pennock and Threlfall (1983), In: Biosynthesis of Isoprenoid Compounds (Porter and Spurgeon, ed .) Vol. 2, 191-203). In both groups of organisms, PHB arises from chorismate, but in completely different ways. In plants, chorismate is first converted to phenylalanine in the course of aromatic amino acid biosynthesis via prephenate and then converted to PHB using cinnamic acid and p-cumaric acid (Heide et al., Phytochemistry 28 (1989), 2643-2645). In contrast, in bacteria the chorismate is metabolized in a single step by the enzyme chorismate pyruvate lyase to PHB and pyruvate. The ubiC gene coding for this enzyme was cloned from Escherichia coli (Siebert et al., FEBS Letters 307 (1992), 347-350); see. Fig. 1 and 2.

1) Construction of the transformation vector with the ubiC gene from Escherichia coli

To construct a transformation vector, the ubiC gene contained in pUBIC (Siebert et al., Microbiology 140 (1994), 897-904) is cleaved with EcoRI and SalI, the ends filled in with Klenow enzyme and into the binary plant expression vector pROK1 (Bevan et al., EMBO J. 4 (1985), 1921-1926), which had previously been linearized with BamHI and whose ends were also filled in with Klenow enzyme. This vector enables selection of transgenic plants using a kanamycin resistance gene, the ubiC gene being under the control of a 35S CaMV promoter. The transformation vector produced in this way is referred to as pROK-ubiC; see. Fig. 3.

A modified ubiC gene is produced in such a way that it maintains the reading frame at the 5'-end with a plastid transit peptide (Sugita et al., Mol. Gen. Genet. 209 (1987), 247-256), that of the small Subunit of the ribulose-bisphosphate carboxylase is derived via the cleavage site KpnI, whereby the construct pTP0-ubiC is obtained. The fusion gene TPubiC contained in pTP0-ubiC is then cloned into the vector pROK1 as described above to obtain the transformation vector pROK-TP0-ubiC; see. Fig. 3.

2) Transformation of tobacco plants

For the transformation of tobacco plants (Kultivar Petite Havana SR1) leaves of sterile cultivated tobacco cut into pieces of approx. 0.5 × 0.5 cm and for at least one minute in a culture of Agrobacteriuin tumefaciens (LBA4404 with each under point (1) described transformation vectors) dipped previously centrifuged briefly and sterile in the same volume Tap water has been resuspended. The leaf pieces are placed on SHI medium (in one liter of 4.6 g of Murashige and Skoog salts (Physiol. Plans. 15 (1962), 473-497), 30 g sucrose, 1 mg 6- Benzylaminopurine, 0.1 mg naphthylacetic acid, 100 mg Inositol, 10 mg thiamine, 1 mg pyridoxine, 1 mg nicotinic acid and 7 g agarose) in Petri dishes and at 25 ° C co-cultivated with the agrobacteria for three days. Here make sure that the amount of light is not too large (Scattered light, approx. 1000 to 3000 lux).

The selection against agrobacteria and untransformed leaf parts is carried out by moving the leaf pieces to SHI _{Cef250, Kan100} medium (composition like the SHI medium with additional 250 mg / l cefotaxime and 100 mg / l kanamycin).

After 4 to 6 weeks, the regenerated shoots that have formed at the interfaces are separated and placed in _{Cef125, Kan100} medium (composition as the corresponding SHI medium, but without the addition of naphthylacetic acid, 6-benzylaminopurine, inositol, thiamine, pyridoxine and Nicotinic acid) in 450 ml mason jars for rooting. With a size of approx. 10 cm, the plants are carefully removed from the roots of the agar medium and placed in sterilized soil.

3) Analysis of the transgenic tobacco plants for PHB content

Sheet material of the transgenic obtained under point (2) Plants are pounded under liquid nitrogen. For the extraction of the PHB freely available in the plants the powdered sheet material is dissolved in 0.75 M sodium acetate Solution pH 4.0 suspended and extracted with ethyl acetate. The organic phase is removed to dryness evaporated, in methanol / water / formic acid (30: 69.3: 0.7) recorded and examined by HPLC chromatography.

To determine the bound PHB (e.g. esters, glucosides) the powdered plant material in 1 M HCl Hydrolyzed at 80 ° C for hours, extracted with ethyl acetate and analyzed as described above.

Transformed plants show in comparison to the wild type Plant a content of free that is increased by a factor of 50 PHB (2.3 µg / g fresh weight) and one by the factor 1150 increased bound PHB content. The PHB is located in 50% of the transgenic plants are glucosidically bound in front. Their PHB glucoside content is approx. 0.3 mg / g Fresh weight. In addition, phenolic substances continue to be used enriched.  

4) Biological effect of the increased concentration of certain Secondary substances in the transgenic tobacco plants on tobacco Mosaic virus (TMV)

A suspension of the virus is used to test for TMV resistance with diatomaceous earth under light pressure with a brush the leaves of transgenic and wild-type tobacco plants (Kultivar Petite Havana SR1) applied.

Because the virus is in the Petite Havana plants spreads systemically and does not form local lesions, the leaves of the tobacco plants become after about 10 days mortared and the extract as described above Leaves of the tobacco cultivar Nicotiana tabacuin cv. Xanthi applied. After about three days they will be with them Plants observed local lesions.

As a result, (i) shows that the untransformed control plants of N. tabacum cv. Petite Havana can establish a high TMV titer, (ii) that the TMV propagation in transgenic with pROK-TP0-ubiC transformed plants is significantly reduced and (iii) that transformed in transgenic with pROK-ubiC Plants essentially no TMV is detectable.

In summary it can be said that the inventive transgenic tobacco plants that have the ubiC gene from Escherichia coli, a significantly increased Show pathogen resistance in comparison with wild-type plants and no new ingredients with unknown effects and Form toxicology, but surprisingly they contain an increased amount of p-hydroxybenzoic acid (one in all Organisms naturally occurring substance with known Properties) and their derivatives, in particular p- Hydroxybenzoic acid glucoside, which is responsible for pathogen resistance are responsible or play an important role.

Claims (11)

1. Transgenic plant with at least one heterologous DNA Sequence necessary for at least one polypeptide with enzymatic Encodes activity in which the polypeptide is expressed in enzymatically active form and at least one Secondary substance in an antiviral and / or bactericidal and / or fungicidal and / or insecticidal and / or acting as a repellent Concentration generated enzymatically, the heterologous DNA sequence comes from bacteria. 2. Transgenic plant according to claim 1, wherein the heterologous DNA sequence comes from Escherichia coli. 3. The transgenic plant according to claim 1 or 2, wherein the heterologous DNA sequence contains the following sequence: 4. Transgenic plant according to one of claims 1 to 3, wherein the polypeptide is the enzyme chorismate pyruvate lyase or the enzyme isochorismate pyruvate lyase (= salicylate synthase) is. 5. Transgenic plant according to one of claims 1 to 4, where the secondary substance is a plant-owned substance. 6. Transgenic plant according to one of claims 1 to 5, the secondary substance being a phenol derivative.   7. Transgenic plant according to one of claims 1 to 6, the secondary substance being p-hydroxybenzoic acid and / or its Is derivative (s). 8. Transgenic plant according to one of claims 1 to 7 wherein the concentration of the secondary substance is at least that 10 times based on the concentration of the secondary substance in the Wild type. 9. Method of producing a transgenic plant according to one of claims 1 to 8, wherein a plant cell by stable integration of the heterologous DNA sequence into the genetic material is transformed and the transformed Plant cell is regenerated to the transgenic plant. 10. Use of the transgenic plant according to one of the Claims 1 to 8 as pathogen-resistant and / or against Predators and / or parasite resistant plants in forest, Willow, meadow, ornamental or crop production. 11. Use of a heterologous DNA sequence that is suitable for min least encoded a polypeptide with enzymatic activity for Production of a transgenic plant with at least one plant-derived secondary substance produced by the polypeptide in an antiviral and / or bactericidal and / or fungicidal and / or insecticidal and / or acting as a repellent Concentration.