CA2007091A1 - Wound-inducible and potato tuber specific transcriptional regulation - Google Patents
Wound-inducible and potato tuber specific transcriptional regulationInfo
- Publication number
- CA2007091A1 CA2007091A1 CA002007091A CA2007091A CA2007091A1 CA 2007091 A1 CA2007091 A1 CA 2007091A1 CA 002007091 A CA002007091 A CA 002007091A CA 2007091 A CA2007091 A CA 2007091A CA 2007091 A1 CA2007091 A1 CA 2007091A1
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- Prior art keywords
- inhibitor
- proteinase
- gene
- dna sequence
- potato
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8237—Externally regulated expression systems
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8114—Kunitz type inhibitors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—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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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT
There is described a new DNA sequence of an expression cassette in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in the potato tubers are localised, as well as the transference of these in vectors containing the DNA
sequence in the plant genome, by using Agrobacterium tumefaciens as transfer microorganism. The transferred DNA
sequence is concerned both for the regulation of endogenous products as well as for the production of heterologous products. THe method is especially useful in combination with the expression of pest-resistant genes in leaves, stems and tubers and following such a use for the production of useful proteins in potato tubers.
There is described a new DNA sequence of an expression cassette in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in the potato tubers are localised, as well as the transference of these in vectors containing the DNA
sequence in the plant genome, by using Agrobacterium tumefaciens as transfer microorganism. The transferred DNA
sequence is concerned both for the regulation of endogenous products as well as for the production of heterologous products. THe method is especially useful in combination with the expression of pest-resistant genes in leaves, stems and tubers and following such a use for the production of useful proteins in potato tubers.
Description
200709~
The present invention relates to a new DNA sequence of an expression cassette in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in the potato tubers are localised, as well as the transference of these in vectors containing the DNA sequence in the plant genome, by using Agrobacteria as transfer microorganisms. The transfer DNA
sequence is concerned both for the regulation of endogenous products as well as for the production of heterologous products. Heterologous products can be for example toxic proteins that can be used for combating plant pests.
Because of the continual increasing need for food and raw materials due to the growth in world population, and because of the long-term reduction in areas of land su~table for growing crops, it is becoming increasingly the task for biological research to to increase the yields of crops and their food content. An increase of yields can be achieved amongst other methods by increasing the resistance of crops against plant pests and plant diseases and/or poor soils. An increase of the resistance could achieved for example in such a way in that the plants induce and give rise to an increased formation of protective substances. For this, the metabolism of the plants must be manipulated. This can be achieved amongst other ways by changing the DNA contained in the cell nuclei. It would be desirable to act on in those DNA areas which are responsible for transcription in one or more of the parts of the plant or during a specified period in the plant growth cycle. For this there is a great interest in ,identifying the DNA sequence in the plant genome ,.. . .. .
200709~
responsible for the transcription or expression of endogenous plant products. In order to find such DNA
sequences, products first have to be sought which appear at a specific time in the cell growth cycle or in a specific part of the plant. If the gene belonging to this is to be identified and isolated, a careful investigation of the sequence, and above all the identification and isolation of the desired transcriptional regulatory regions, is necessary. Suitable models must then be provided whose ~unctions must established through experiments. Identifying such DNA sequences is a challenging project which is subject to substantial pitfalls and uncertainty. There is however substantial interest in the possibility of genetically modifying plants, which justifies the substantial expenditure and efforts necessary in identifying transcriptional sequences and manipulating them to determine their utility.
Processes for genetic modification of dicotyledonous and monocotyledonous plants are known (EP 267159), as well as th~ following publications of Crouch et al., in: Molecular Form and Function of the Plant Genome, eds. van Vloten-Doting, Groots and Hall, Plenum Publishing Corp, 1985, pp 555-566; Crouch and Sussex, Planta (1981) 153:64-741 Crouch et al., J. Mol. Appl. Genet (1983) 2:273-283; and Simon et al., Plant Molecular Biology ~1985) 5: 191-201, in which various forms of storage proteins in Brassica napus are described and by Beachy et al., EMBO. J. (1985) 4:3047-3053; Sengupta-~opalan et al., Proc. Natl. Acad.
Sci. USA (1985) 82:3320-3324; Greenwood and Chrispeels, Plant Physiol. (1985) 79:65-71 and Chen et al., Proc.
Natl. Acad. Sci. USA (1986) 83:8560-8564, in which studies concerned with seed storage proteins and genetic manipulation are described and by Eckes et al., Mol. Gen.
Genet. (1986) 205:14 - 22 and Fluhr et al., Science (1986) 35 , 232:1106-1112, in which genetic manipulation of light ,.........
:, .
200709~
inducible plant genes are described.
There is now provided a new DNA sequence of an expression cassette in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in the potato tubers are localised, as well as the transference of these in vectors containing the DNA
sequence in the plant genome, by using Agrobacteria as transfer microorganisms. The DNA sequence contains the sequence of the regulatory transcriptional starter region for the wound induction and the potato tuber specificity.
Downstream from the starter region can be a sequence which contains the information for the modification of the phenotype of the cell tissues concerned and the formation, as well as quantitative distribution, of endogenous products or the formation of heterogenous expression products for a new function. Conveniently, the transcription and termination regions in the direction of transcription should be provided by a linker or polylinker which contains one or more restriction positions for the insertion of this sequence. As a rule, the linker has 1-10, usually 1-8, preferably 2-6 reaction positions. In general the linker has a size of less than 100 bp, usually less than 60 bp, but is however at least 5 bp. The transcriptional starter region can be native or homologous to the host or foreign or heterologous to the host plants.
Of special interest are the transcriptional starter regions which are associated with potatoes (Solanum tuberosum) proteinase-inhibitor II-gene, that during the total potato tuber development from the formation of the stolon up to the ripe tuber, is expressed. The expression of the proteinase-inhibitor II-gene cannot be shown in other plant parts before injury (for example by biting insects) which induce the expression of the 200709~
proteinase-inhibitor II-gene in leaves and stems. This wound-inducing expression is not separated only on the injured parts of the plants. A systemic induction leads to an accumulation of proteinase-inhibitor II, both in wounded and also in intact parts of the wounded plants.
The transcription cassette contains in the 5'-3' transcription direction, a region representative for the plants for the transcription and the translation, a desired sequence and a region for the transcriptional and translational termination. The termination region used is a homologue of the participating proteinase-inhibitor II-gene. If a subfragment of the proteinase-inhibitor II-gene regulator region is fused to a heterologous promoter, termination area seems to be exchangeable. The ~NA sequence could contain all possible open reading frames for a desired peptide as well as also one or more introns. Examples include sequences for enzymes; sequences that are complementary (a) to a genome sequence whereby the genome sequence can be an open reading frame; (b) to an intron; (c) to a non-coded leading sequence; (d) to ea~h sequence, which inhibits through complementarity, the transcription mRNA processing (for example splicing) or the translation. The desired DNA sequence can be synthetically produced or extracted naturally, or can contain a mixture of synthetic or natural DNA content. In general, a synthetic DNA sequence with codons is produced, which is preferred by the plants. This preferred codon from the plants can be specified from the codons with the highest protein frequency which can be expressed in the most interesting plant species. In the preparation of the transcription cassettes, the different DNA fragments can be manipulated in order to contain a DNA sequence, which leads generally in the correct direction and which is equipped with the correct reading frame. For the connections of the DNA fragments to each other, adaptors , .. ...
200709~
or linkers can be introduced on the fragment ends. Further manipulations can be introduced which provide the suitable restriction positions or separate the excess DNA or restrictio~ positions. Where insertions, deletions or substitutions, such as for example transitions and transversions, are concerned, in vitro mutaganese, primer repair, restriction or ligation can be used.
In suitable manipulations, such as for example restriction, "chewing-back" or filling up of overhangs for "blunt-ends", complementary ends of the fragments for the fusing and ligation could be used. For carrying out the various steps which serve to ensure the expected success of the intervention, a cloning is necessary for th~
increase of the DNA amounts and for the DNA analysis.
A large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 332, pUC series, M13 mp series, pACYC 184 etc. In such a way, the sequence can be introduced into a suitable restriction position in the vector. The contained plasmid is used for the transformation in E. coli. The E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis there is generally used a sequence analysis, a reætriction analysis, electrophor~sis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each plasmid sequence can be cloned in the same or different plasm~d.
After each introduction method of the desired gene in the plants further DNA sequences may be necessary. If for example for the transformation, the Ti- or Ri-plasmid of .- -'' 200709~
the plant cells is used, at least the right boundary andoften however the right and the left boundary of the Ti-and Ri-plasmid T-DNA, as flanking areas of the introduced gene, can be connected. The use of T-DNA for the transformation of plant cells is being intensi~ely studied and is well described in EP 120 516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.8., Alblasserdam, 1985, Chapter V; Fraley, at al., Crit. Rev.
Plant Sci., ~:1-46 und An et al., EMBO J. (1985) 4:277-284.
When the introduced DNA is first integrated once in thegenome, it is then also relatively stable and as a rule no more comes out. It normally contains a selection marker which passes on to the transformed plant cells, resistance against a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or chloramphenicol, amongst others. The particular marker employed should be one which will allow for selection of transformed cells compared to cells lacking the DNA which has been introduced.
A variety of techniques are available for introduction of DNA into a plant host cell. These techniques include transformation with T-DNA using Aqrobacterium tumefaciens or A~robacterium rhizogenes as transformation agent, the fusion, the injection or the electroporation as well as further possibilities. If Agrobacteria are used for the transformation, the introduced DNA must be cloned in special plasmid and either in an intermediary vector or a binary vector. The intermediary vectors which are based on sequences which are homologous with sequences in the T-DNA
can be integrated through homologous re-combination in the Ti- or Ri- plasmid. These contain also the necessary -Vir-region for the transfer of the T-DNA. Intermediary vectors cannot be replicated in Agrobacteria. By means of helper-plasmid, the intermediary vector of Agrobacterium tumefaciens can be transferred (conjugation). Binary vectors can be replicated in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed from the right and left T-DNA border regions. They can be transformed directly in the agrobacteria (Holsters et al., Mol. Gen.
Genet.(1978) 163: 181-187). The Agrobacterium serving as host cells should contain a plasmid that carries the Vir-region, which is necessary for the transfer of the T-DNA in the plant cells whereby additional T-DNA can be contained. The bacterium so transformed is used for the transformation of plant cells. For the transfer of DNA in the plant cells, plant explanates can be cultivated in suitable manner with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (for example leaf bits, stem segments, roots as well as protoplasts or suspensions of cultivated cells), whole plants can then be regenerated in a suitable medium which can contain antibiotics or biocides for the selection, wh~ch then can be tested for the presence of introduced DNA. In the injection and e~ectroporation, no special requirements on the plasmid are needed and a simple plasmid, for example pUC derivative can be used. For the introduction of foreign genes into the plants a variety of possibilities can be used. Especially interesting, however, is the expression of genes which introduce pesticidal resistance, which after wounding of the plant, give to the plant a reduced food quality of the plant to the pests or a toxicity to the pest. Therefore excess insect eating is avoided which leads to far higher yields of crop. The corresponding genes can be toxin-genes that code, for example for B. thurinqiensis ~-endotoxin, or can be genes which code for insect hormones which lead to a change in growth of insect larvae (for axample ecdysone).
,, , .. ... .
Alternatively, genes for the most different of starting materials can be used, including mammalian products, such as for example blood factors; lymphokines; colony stimulation factors; interferons; plasminogen activators;
enzymes, such as for example superoxide-dismutase or chymosin; hormones; thioesterase-2 from rats milk;
phospholipid-acyl-desaturase which takes part in the synthesis of cicosapentaenoic acid; or human serum albumin. A further possibility is increasing the amounts of tuber proteins, especially mutated tuber proteins, which show an optimised amino acid composition (essential amino acids) and in this way the nutritive value of the tubers can be increased. Should the amounts of specified endogenous products b~ reduced, the expression of the gene or parts of this gene in the wrong orientation to the promoter is also conceivable, which leads to synthesis of an RNA, which is complementary to a total or to parts of an endogenous gene and thus the transcription of this gene or the processing and/or translation of the endogenous mRNA can be inhibited.
The transformed cells grow within the plants in the usual way tsee also McCormick et al., Plant Cell Reports t1986) 5, 81-84). These plants can be grown normally and crossed with plants, that possess the same transformed gene or Z5 other genes. The resulting hybridised individuals have the corresponding phenotypic properties. Two or more generations should be grown, in order to secure that the phenotypic state remains stable and will be passed on, especially if seeds are to be harvested, in order to ensure that the corresponding phenotype or other individual characteristics are included. As host plants for the wound inducible expression, any plant type can be used that is of economic interest.
,, .... .
200~09~
For tuber specific expression Solanum tuberosum is suitable. The identification of necessary transcriptional starter regions can be achieved in a number of ways. As a rule the mRNAs can be used, which are isolated from specified parts of the plants ~tubers) or during certain conditions of the plants (non-wounded/wounded). For the additional increase in concentration of the mRNA specific to the cells or associated with plant conditions, cDNA can be prepared whereby non-specific cDNA from the mRNA or the cDNA from other tissues or plant conditions (for example wounded/non-wounded) can be drawn off. The remaining cDNA
can then be used for probing the genome for complementary sequences using a suitable plant DNA library. Where the protein is to be isolated, it can be partially sequenced so that a probe for direct identification of the corresponding sequences in a plant ~NA library can be produced. The sequences that are hybridised with the probe can then be isolated and manipulated. Further, the non-translated 5'-region, that is associated with the coded area, can be isolated and used in expression cassettes for the identification of the transcriptional activity of the non-translated 5'-regions. The expression cassettes obtained which use the non-translated 5'-regions can be transformed in plants (see above), in order to prove their functionability with a heterologous structure gene ~other than the open reading frame of the wild types which are associated with the non-translated 5- region) as well as the tuber and wound spacificity. In this way, specific sequences can be identified which are necessary for the tuber and wound specific transcription. Expression cassettes that are of special interest contain transcriptional initiation positions of the protein-inhibitor II-gene.
,,, . ~ . . .
200709~
identification of necessary transcriptional starter regions can be achieved in a number of ways. As a rule the mRNAs can be used, which are isolated from specified parts of the plants (tubers) or duri~g certain condi~ions of the plants (non-wounded/wounded).~For the additional increase in concentration of the mRNA specific to the cells or associated with plant conditions, cDNA can be prepared whereby non-specific cDNA from the mRNA or the cDNA from other tissues or plant conditions (for example wounded/non-wounded) can be drawn off. The remaining cDNA
can then be used for probing the genome for complementary sequences using a suitable plant DNA library. Where the protein is to be isolated, it can be partially sequenced so that a probe for direct identification of the corresponding sequences in a plant DNA library can be produced. The sequences that are hybridised with the probe can then be isolated and manipulated. Further, the non-translated 5'-region, that is associated wi~h the coded area, can be isolated and used in expression cassettes for the identification of the transcriptional activity of the non-translated 5'-regions. The expression cassettes obtained which use the non-translated 5'-regions can be transformed in plants (see above), in order to prove their functionability with a heterologous structure gene (other than the open reading frame of the wild types which are associated with the non-translated 5- region) as well as the tuber and wound specificity. In this way, specific sequences can be identified which are necessary for the tuber and wound specific transcription. Expression cassettes that are of special interest contain transcriptional initiation positions of the protein-inhibitor II-gene.
zoo~o9~
lo Expressions & Abbreviations Abbreviations:
bp = Base pairs cDNA = A copy of a mRNA produced by reverse S transcriptase.
mRNA = Messenger ribonucleic acid.
T-DNA = Transfer-DNA (localised on the Ti-plasmid from Agrobacterium tumefaciens) Terms: 0 Blunt ends = DNA ends in which both DNA strands are exactly the same length.
Chewing-back = Enzymatic removal of nucleotides of a DNA strand which is longer than the complementary strand of a DNA
molecule.
Electrophoresis = A biochemical process of separation for separating nucleic acids fro~
proteins according to size and charge.
En~onuclease = An enzyme that splits independently of the chain length.
Exonuclease = An enzyme that can attack only at the end of a polynucleotide chain.
Expression = Activity of a gene.
Gene = Genetic factor; a unit of inheritance, carrier of part information for a particular specified characteristic.
Genes consist of nucleic acids (eg DNA, RNA).
Genome = Totality of the gene localised in the chromosomes of the cell.
20070~3~
Genome-sequence = The DNA sequence of the genome whereby three nucleotide bases lying within it form a codon which code again for a specific amino acid.
RNA splicing = A gene does not always show up as a colinear unity but can contain non-coded sequences ~introns) which must be spliced from the mRNA (splicing).
Heterologous gene(s) or ~NA = Foreign genes or foreign DNA.
Homologous gene(s) or DNA = Gene or DNA derived from the same species.
Klenow enzyme = Fragments of DNA polymerase I of a size 76,000 D ob~ained by splitting with a subtilisin. Possess 5' - 3' polymerase and 3' - 5' exonuclease activity but not the 5' - 3' exonuclease activity of the holoenzyme.
20 Clone = Cell population that is derived from one of its own mother cells.
Descendants are genotypically the same. By cloning, the homogeneity of cell lines can be increased further.
25 Ligation = Enzymatic formation of a phosphodiester bond between 5'-phosphate groups and 3'-hydroxy groups of the DNA.
Linker, Polylinker = Synthetic DNA sequence that contains one or more (polylinker) restriction cutting regions in direct sequence.
Northern blots, = Transfer and fixing of Southern blots, electrophoretically separate RNA or DNA
on a nitrocellulose or nylon membrane.
, .. ...
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Phenotype = A sum of characteristics which expressed in an organism as opposed to its genotype.
Plasmid = Additional extrachromosomal DNA gene carrier in bacteria cells (possibly also in eukaryons) which reduplicate themselves independently of the bacterial chromosomes. The plasmid can be integrated in other DNA hosts.
10 Primer = Starting piece; polynucleotide strand on which further nucleotides can be attached.
Promoter = Control sequence of the DNA expression which realises the transcription of homologous or heterologous DNA gene sequences.
Regulator protein = Proteins that stand with DNA in exchange activity and steer the gene expression.
Re~lication = Doubling of the DNA sequence.
Restriction enzymes = Restriction endonucleases that are in sub-units of the endo DNA's (for example EcoRI
(specificity G AATTC and EcoRII CC (AT) GG, from E.coli) show themselves through a high specificity of the substrate knowledge ( = splitting position3.
Restriction positions = A splitting position which is produced specifically by restriction enzymes.
Termination = A last stage of the protein and/or the RNA synthesis.
., .
,~......... .
20070~
Transformation = Introduction of exogenous DNA of a bacterial species which is in a receiver cell.
Transcription = Overwriting on an RNA the genetic information contained in the DNA.
Translation = Translation of the genetic information which is memorised in the form of a linear sequence of bases in nucleic acids. The product of the translation is a polypeptide that comprises a sequence of amino acids.
Transition = Base pair exchange: purine-pyrimidine to purine-pyrimidine e.g. A-T
exchanging G-C. 5 Transversion = Base pair exchange: purine-pyrimidine to pyrimidine-purine e.g. A-T
replacing T-A.
Deletion = Removal of one or more base pairs;
Insertion = Introduction of one or more base pairs;
Transition, Transversion, Deletion and Insertion are point mutations.
Transposo~ = A unity comprising resistance gene (S) and two IS elements (IS = integrated segments).
IS elements or IS sequences are DNA
sequences that can influence the expression of adjacent genes. Deletions can be induced and possess the feature of being able to extend themselves in the bacteria genome in various positions singly and also repeatedly.
,, :
~ :
200709~
Vectors = Host specific replicatable structures, that take up genes and carry these into other cells. Plasmid can also be used as vertors.
On 16.12.1988 the following microorganism was deposited at the German Collection for Microorganisms (DSM) in Braunschweig, Germany (deposit number):
Agrobacterium tumefaciens A. tum. M 14, containing the vector pM 14 (DSM 5088) Description of the Fiqures 10 Figure 1 shows the vector pM 14 on which the 3.4 kb long EcoRI/HindIII DNA sequence is localised. This DNA sequence contains the 1.3 kb long ScaI/HindIII fragment of the proteinase-inhibitor II-promoter, the 1.8 kb long BamHI/Sst~ fragment of the ~-glucuronidase and the a . 26 kb long SphI/SphI fragment of the proteinase-inhibitor II.
There are further, records of the cutting positions as we~1 as positions of the transcription starts.
~,.~, ,., :
200709~
For a better understanding of this invention the following examples are given. An explanation for these experiments is given as follows:
1. Cloning Vectors For cloning, the vectors pUC18/19 (Yanisch-Perron et al Gene (1985), 33, 103-119) were used.
For plant transformations, the gene structures were cloned either in the intermediate vector pMPK110 (Eckes, Doktorarbeit (1985) - Standort der Arbeit -Universitatsbibliothek Koln) or the binary vector BIN19 (Bevan, Nucl Acids Research (1984), 12, 8711-8720).
The present invention relates to a new DNA sequence of an expression cassette in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in the potato tubers are localised, as well as the transference of these in vectors containing the DNA sequence in the plant genome, by using Agrobacteria as transfer microorganisms. The transfer DNA
sequence is concerned both for the regulation of endogenous products as well as for the production of heterologous products. Heterologous products can be for example toxic proteins that can be used for combating plant pests.
Because of the continual increasing need for food and raw materials due to the growth in world population, and because of the long-term reduction in areas of land su~table for growing crops, it is becoming increasingly the task for biological research to to increase the yields of crops and their food content. An increase of yields can be achieved amongst other methods by increasing the resistance of crops against plant pests and plant diseases and/or poor soils. An increase of the resistance could achieved for example in such a way in that the plants induce and give rise to an increased formation of protective substances. For this, the metabolism of the plants must be manipulated. This can be achieved amongst other ways by changing the DNA contained in the cell nuclei. It would be desirable to act on in those DNA areas which are responsible for transcription in one or more of the parts of the plant or during a specified period in the plant growth cycle. For this there is a great interest in ,identifying the DNA sequence in the plant genome ,.. . .. .
200709~
responsible for the transcription or expression of endogenous plant products. In order to find such DNA
sequences, products first have to be sought which appear at a specific time in the cell growth cycle or in a specific part of the plant. If the gene belonging to this is to be identified and isolated, a careful investigation of the sequence, and above all the identification and isolation of the desired transcriptional regulatory regions, is necessary. Suitable models must then be provided whose ~unctions must established through experiments. Identifying such DNA sequences is a challenging project which is subject to substantial pitfalls and uncertainty. There is however substantial interest in the possibility of genetically modifying plants, which justifies the substantial expenditure and efforts necessary in identifying transcriptional sequences and manipulating them to determine their utility.
Processes for genetic modification of dicotyledonous and monocotyledonous plants are known (EP 267159), as well as th~ following publications of Crouch et al., in: Molecular Form and Function of the Plant Genome, eds. van Vloten-Doting, Groots and Hall, Plenum Publishing Corp, 1985, pp 555-566; Crouch and Sussex, Planta (1981) 153:64-741 Crouch et al., J. Mol. Appl. Genet (1983) 2:273-283; and Simon et al., Plant Molecular Biology ~1985) 5: 191-201, in which various forms of storage proteins in Brassica napus are described and by Beachy et al., EMBO. J. (1985) 4:3047-3053; Sengupta-~opalan et al., Proc. Natl. Acad.
Sci. USA (1985) 82:3320-3324; Greenwood and Chrispeels, Plant Physiol. (1985) 79:65-71 and Chen et al., Proc.
Natl. Acad. Sci. USA (1986) 83:8560-8564, in which studies concerned with seed storage proteins and genetic manipulation are described and by Eckes et al., Mol. Gen.
Genet. (1986) 205:14 - 22 and Fluhr et al., Science (1986) 35 , 232:1106-1112, in which genetic manipulation of light ,.........
:, .
200709~
inducible plant genes are described.
There is now provided a new DNA sequence of an expression cassette in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in the potato tubers are localised, as well as the transference of these in vectors containing the DNA
sequence in the plant genome, by using Agrobacteria as transfer microorganisms. The DNA sequence contains the sequence of the regulatory transcriptional starter region for the wound induction and the potato tuber specificity.
Downstream from the starter region can be a sequence which contains the information for the modification of the phenotype of the cell tissues concerned and the formation, as well as quantitative distribution, of endogenous products or the formation of heterogenous expression products for a new function. Conveniently, the transcription and termination regions in the direction of transcription should be provided by a linker or polylinker which contains one or more restriction positions for the insertion of this sequence. As a rule, the linker has 1-10, usually 1-8, preferably 2-6 reaction positions. In general the linker has a size of less than 100 bp, usually less than 60 bp, but is however at least 5 bp. The transcriptional starter region can be native or homologous to the host or foreign or heterologous to the host plants.
Of special interest are the transcriptional starter regions which are associated with potatoes (Solanum tuberosum) proteinase-inhibitor II-gene, that during the total potato tuber development from the formation of the stolon up to the ripe tuber, is expressed. The expression of the proteinase-inhibitor II-gene cannot be shown in other plant parts before injury (for example by biting insects) which induce the expression of the 200709~
proteinase-inhibitor II-gene in leaves and stems. This wound-inducing expression is not separated only on the injured parts of the plants. A systemic induction leads to an accumulation of proteinase-inhibitor II, both in wounded and also in intact parts of the wounded plants.
The transcription cassette contains in the 5'-3' transcription direction, a region representative for the plants for the transcription and the translation, a desired sequence and a region for the transcriptional and translational termination. The termination region used is a homologue of the participating proteinase-inhibitor II-gene. If a subfragment of the proteinase-inhibitor II-gene regulator region is fused to a heterologous promoter, termination area seems to be exchangeable. The ~NA sequence could contain all possible open reading frames for a desired peptide as well as also one or more introns. Examples include sequences for enzymes; sequences that are complementary (a) to a genome sequence whereby the genome sequence can be an open reading frame; (b) to an intron; (c) to a non-coded leading sequence; (d) to ea~h sequence, which inhibits through complementarity, the transcription mRNA processing (for example splicing) or the translation. The desired DNA sequence can be synthetically produced or extracted naturally, or can contain a mixture of synthetic or natural DNA content. In general, a synthetic DNA sequence with codons is produced, which is preferred by the plants. This preferred codon from the plants can be specified from the codons with the highest protein frequency which can be expressed in the most interesting plant species. In the preparation of the transcription cassettes, the different DNA fragments can be manipulated in order to contain a DNA sequence, which leads generally in the correct direction and which is equipped with the correct reading frame. For the connections of the DNA fragments to each other, adaptors , .. ...
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or linkers can be introduced on the fragment ends. Further manipulations can be introduced which provide the suitable restriction positions or separate the excess DNA or restrictio~ positions. Where insertions, deletions or substitutions, such as for example transitions and transversions, are concerned, in vitro mutaganese, primer repair, restriction or ligation can be used.
In suitable manipulations, such as for example restriction, "chewing-back" or filling up of overhangs for "blunt-ends", complementary ends of the fragments for the fusing and ligation could be used. For carrying out the various steps which serve to ensure the expected success of the intervention, a cloning is necessary for th~
increase of the DNA amounts and for the DNA analysis.
A large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 332, pUC series, M13 mp series, pACYC 184 etc. In such a way, the sequence can be introduced into a suitable restriction position in the vector. The contained plasmid is used for the transformation in E. coli. The E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis there is generally used a sequence analysis, a reætriction analysis, electrophor~sis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence. Each plasmid sequence can be cloned in the same or different plasm~d.
After each introduction method of the desired gene in the plants further DNA sequences may be necessary. If for example for the transformation, the Ti- or Ri-plasmid of .- -'' 200709~
the plant cells is used, at least the right boundary andoften however the right and the left boundary of the Ti-and Ri-plasmid T-DNA, as flanking areas of the introduced gene, can be connected. The use of T-DNA for the transformation of plant cells is being intensi~ely studied and is well described in EP 120 516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters B.8., Alblasserdam, 1985, Chapter V; Fraley, at al., Crit. Rev.
Plant Sci., ~:1-46 und An et al., EMBO J. (1985) 4:277-284.
When the introduced DNA is first integrated once in thegenome, it is then also relatively stable and as a rule no more comes out. It normally contains a selection marker which passes on to the transformed plant cells, resistance against a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or chloramphenicol, amongst others. The particular marker employed should be one which will allow for selection of transformed cells compared to cells lacking the DNA which has been introduced.
A variety of techniques are available for introduction of DNA into a plant host cell. These techniques include transformation with T-DNA using Aqrobacterium tumefaciens or A~robacterium rhizogenes as transformation agent, the fusion, the injection or the electroporation as well as further possibilities. If Agrobacteria are used for the transformation, the introduced DNA must be cloned in special plasmid and either in an intermediary vector or a binary vector. The intermediary vectors which are based on sequences which are homologous with sequences in the T-DNA
can be integrated through homologous re-combination in the Ti- or Ri- plasmid. These contain also the necessary -Vir-region for the transfer of the T-DNA. Intermediary vectors cannot be replicated in Agrobacteria. By means of helper-plasmid, the intermediary vector of Agrobacterium tumefaciens can be transferred (conjugation). Binary vectors can be replicated in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed from the right and left T-DNA border regions. They can be transformed directly in the agrobacteria (Holsters et al., Mol. Gen.
Genet.(1978) 163: 181-187). The Agrobacterium serving as host cells should contain a plasmid that carries the Vir-region, which is necessary for the transfer of the T-DNA in the plant cells whereby additional T-DNA can be contained. The bacterium so transformed is used for the transformation of plant cells. For the transfer of DNA in the plant cells, plant explanates can be cultivated in suitable manner with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (for example leaf bits, stem segments, roots as well as protoplasts or suspensions of cultivated cells), whole plants can then be regenerated in a suitable medium which can contain antibiotics or biocides for the selection, wh~ch then can be tested for the presence of introduced DNA. In the injection and e~ectroporation, no special requirements on the plasmid are needed and a simple plasmid, for example pUC derivative can be used. For the introduction of foreign genes into the plants a variety of possibilities can be used. Especially interesting, however, is the expression of genes which introduce pesticidal resistance, which after wounding of the plant, give to the plant a reduced food quality of the plant to the pests or a toxicity to the pest. Therefore excess insect eating is avoided which leads to far higher yields of crop. The corresponding genes can be toxin-genes that code, for example for B. thurinqiensis ~-endotoxin, or can be genes which code for insect hormones which lead to a change in growth of insect larvae (for axample ecdysone).
,, , .. ... .
Alternatively, genes for the most different of starting materials can be used, including mammalian products, such as for example blood factors; lymphokines; colony stimulation factors; interferons; plasminogen activators;
enzymes, such as for example superoxide-dismutase or chymosin; hormones; thioesterase-2 from rats milk;
phospholipid-acyl-desaturase which takes part in the synthesis of cicosapentaenoic acid; or human serum albumin. A further possibility is increasing the amounts of tuber proteins, especially mutated tuber proteins, which show an optimised amino acid composition (essential amino acids) and in this way the nutritive value of the tubers can be increased. Should the amounts of specified endogenous products b~ reduced, the expression of the gene or parts of this gene in the wrong orientation to the promoter is also conceivable, which leads to synthesis of an RNA, which is complementary to a total or to parts of an endogenous gene and thus the transcription of this gene or the processing and/or translation of the endogenous mRNA can be inhibited.
The transformed cells grow within the plants in the usual way tsee also McCormick et al., Plant Cell Reports t1986) 5, 81-84). These plants can be grown normally and crossed with plants, that possess the same transformed gene or Z5 other genes. The resulting hybridised individuals have the corresponding phenotypic properties. Two or more generations should be grown, in order to secure that the phenotypic state remains stable and will be passed on, especially if seeds are to be harvested, in order to ensure that the corresponding phenotype or other individual characteristics are included. As host plants for the wound inducible expression, any plant type can be used that is of economic interest.
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For tuber specific expression Solanum tuberosum is suitable. The identification of necessary transcriptional starter regions can be achieved in a number of ways. As a rule the mRNAs can be used, which are isolated from specified parts of the plants ~tubers) or during certain conditions of the plants (non-wounded/wounded). For the additional increase in concentration of the mRNA specific to the cells or associated with plant conditions, cDNA can be prepared whereby non-specific cDNA from the mRNA or the cDNA from other tissues or plant conditions (for example wounded/non-wounded) can be drawn off. The remaining cDNA
can then be used for probing the genome for complementary sequences using a suitable plant DNA library. Where the protein is to be isolated, it can be partially sequenced so that a probe for direct identification of the corresponding sequences in a plant ~NA library can be produced. The sequences that are hybridised with the probe can then be isolated and manipulated. Further, the non-translated 5'-region, that is associated with the coded area, can be isolated and used in expression cassettes for the identification of the transcriptional activity of the non-translated 5'-regions. The expression cassettes obtained which use the non-translated 5'-regions can be transformed in plants (see above), in order to prove their functionability with a heterologous structure gene ~other than the open reading frame of the wild types which are associated with the non-translated 5- region) as well as the tuber and wound spacificity. In this way, specific sequences can be identified which are necessary for the tuber and wound specific transcription. Expression cassettes that are of special interest contain transcriptional initiation positions of the protein-inhibitor II-gene.
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identification of necessary transcriptional starter regions can be achieved in a number of ways. As a rule the mRNAs can be used, which are isolated from specified parts of the plants (tubers) or duri~g certain condi~ions of the plants (non-wounded/wounded).~For the additional increase in concentration of the mRNA specific to the cells or associated with plant conditions, cDNA can be prepared whereby non-specific cDNA from the mRNA or the cDNA from other tissues or plant conditions (for example wounded/non-wounded) can be drawn off. The remaining cDNA
can then be used for probing the genome for complementary sequences using a suitable plant DNA library. Where the protein is to be isolated, it can be partially sequenced so that a probe for direct identification of the corresponding sequences in a plant DNA library can be produced. The sequences that are hybridised with the probe can then be isolated and manipulated. Further, the non-translated 5'-region, that is associated wi~h the coded area, can be isolated and used in expression cassettes for the identification of the transcriptional activity of the non-translated 5'-regions. The expression cassettes obtained which use the non-translated 5'-regions can be transformed in plants (see above), in order to prove their functionability with a heterologous structure gene (other than the open reading frame of the wild types which are associated with the non-translated 5- region) as well as the tuber and wound specificity. In this way, specific sequences can be identified which are necessary for the tuber and wound specific transcription. Expression cassettes that are of special interest contain transcriptional initiation positions of the protein-inhibitor II-gene.
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lo Expressions & Abbreviations Abbreviations:
bp = Base pairs cDNA = A copy of a mRNA produced by reverse S transcriptase.
mRNA = Messenger ribonucleic acid.
T-DNA = Transfer-DNA (localised on the Ti-plasmid from Agrobacterium tumefaciens) Terms: 0 Blunt ends = DNA ends in which both DNA strands are exactly the same length.
Chewing-back = Enzymatic removal of nucleotides of a DNA strand which is longer than the complementary strand of a DNA
molecule.
Electrophoresis = A biochemical process of separation for separating nucleic acids fro~
proteins according to size and charge.
En~onuclease = An enzyme that splits independently of the chain length.
Exonuclease = An enzyme that can attack only at the end of a polynucleotide chain.
Expression = Activity of a gene.
Gene = Genetic factor; a unit of inheritance, carrier of part information for a particular specified characteristic.
Genes consist of nucleic acids (eg DNA, RNA).
Genome = Totality of the gene localised in the chromosomes of the cell.
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Genome-sequence = The DNA sequence of the genome whereby three nucleotide bases lying within it form a codon which code again for a specific amino acid.
RNA splicing = A gene does not always show up as a colinear unity but can contain non-coded sequences ~introns) which must be spliced from the mRNA (splicing).
Heterologous gene(s) or ~NA = Foreign genes or foreign DNA.
Homologous gene(s) or DNA = Gene or DNA derived from the same species.
Klenow enzyme = Fragments of DNA polymerase I of a size 76,000 D ob~ained by splitting with a subtilisin. Possess 5' - 3' polymerase and 3' - 5' exonuclease activity but not the 5' - 3' exonuclease activity of the holoenzyme.
20 Clone = Cell population that is derived from one of its own mother cells.
Descendants are genotypically the same. By cloning, the homogeneity of cell lines can be increased further.
25 Ligation = Enzymatic formation of a phosphodiester bond between 5'-phosphate groups and 3'-hydroxy groups of the DNA.
Linker, Polylinker = Synthetic DNA sequence that contains one or more (polylinker) restriction cutting regions in direct sequence.
Northern blots, = Transfer and fixing of Southern blots, electrophoretically separate RNA or DNA
on a nitrocellulose or nylon membrane.
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Phenotype = A sum of characteristics which expressed in an organism as opposed to its genotype.
Plasmid = Additional extrachromosomal DNA gene carrier in bacteria cells (possibly also in eukaryons) which reduplicate themselves independently of the bacterial chromosomes. The plasmid can be integrated in other DNA hosts.
10 Primer = Starting piece; polynucleotide strand on which further nucleotides can be attached.
Promoter = Control sequence of the DNA expression which realises the transcription of homologous or heterologous DNA gene sequences.
Regulator protein = Proteins that stand with DNA in exchange activity and steer the gene expression.
Re~lication = Doubling of the DNA sequence.
Restriction enzymes = Restriction endonucleases that are in sub-units of the endo DNA's (for example EcoRI
(specificity G AATTC and EcoRII CC (AT) GG, from E.coli) show themselves through a high specificity of the substrate knowledge ( = splitting position3.
Restriction positions = A splitting position which is produced specifically by restriction enzymes.
Termination = A last stage of the protein and/or the RNA synthesis.
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Transformation = Introduction of exogenous DNA of a bacterial species which is in a receiver cell.
Transcription = Overwriting on an RNA the genetic information contained in the DNA.
Translation = Translation of the genetic information which is memorised in the form of a linear sequence of bases in nucleic acids. The product of the translation is a polypeptide that comprises a sequence of amino acids.
Transition = Base pair exchange: purine-pyrimidine to purine-pyrimidine e.g. A-T
exchanging G-C. 5 Transversion = Base pair exchange: purine-pyrimidine to pyrimidine-purine e.g. A-T
replacing T-A.
Deletion = Removal of one or more base pairs;
Insertion = Introduction of one or more base pairs;
Transition, Transversion, Deletion and Insertion are point mutations.
Transposo~ = A unity comprising resistance gene (S) and two IS elements (IS = integrated segments).
IS elements or IS sequences are DNA
sequences that can influence the expression of adjacent genes. Deletions can be induced and possess the feature of being able to extend themselves in the bacteria genome in various positions singly and also repeatedly.
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Vectors = Host specific replicatable structures, that take up genes and carry these into other cells. Plasmid can also be used as vertors.
On 16.12.1988 the following microorganism was deposited at the German Collection for Microorganisms (DSM) in Braunschweig, Germany (deposit number):
Agrobacterium tumefaciens A. tum. M 14, containing the vector pM 14 (DSM 5088) Description of the Fiqures 10 Figure 1 shows the vector pM 14 on which the 3.4 kb long EcoRI/HindIII DNA sequence is localised. This DNA sequence contains the 1.3 kb long ScaI/HindIII fragment of the proteinase-inhibitor II-promoter, the 1.8 kb long BamHI/Sst~ fragment of the ~-glucuronidase and the a . 26 kb long SphI/SphI fragment of the proteinase-inhibitor II.
There are further, records of the cutting positions as we~1 as positions of the transcription starts.
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For a better understanding of this invention the following examples are given. An explanation for these experiments is given as follows:
1. Cloning Vectors For cloning, the vectors pUC18/19 (Yanisch-Perron et al Gene (1985), 33, 103-119) were used.
For plant transformations, the gene structures were cloned either in the intermediate vector pMPK110 (Eckes, Doktorarbeit (1985) - Standort der Arbeit -Universitatsbibliothek Koln) or the binary vector BIN19 (Bevan, Nucl Acids Research (1984), 12, 8711-8720).
2. Bacterial Species For the pUC-and M13 vectors the E. coli species BMH71-18 (Nessing et al, Proc. Nat. Acad. Sci. USA
(1977), 24, 6342-6346) or TB1 was used. For the vectors pMPK110 and BIN19, the species TBl was exclusively used. TB1 is a recombinant, negative, tetracyclines resistant derivative of the species JM101 (Yanisch-Perron et al., Gene (1985), 33, 103-119). The genotype of the TB1 species is (Bart Barrel, personal communication): F'(traD36, proAB, lacl, lacZ~M15), ~(lac, pro), SupE, thiS, recA, Srl::TnlO( TCR ) .
As helper species for the conjugative transfer of the pMPK plasmid from the TB1 cells in Agrobacterium tumefaciens, the E. coli species GJ23 (Van Haute et al., EMBO J. (1983), 2, 411-417) can be used.
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The plant transformation was carried out with the help of the Aqrobacterium tumefaciens species GV3850kan (Jones et al., EMBO J. (1985) 4, 2411-2418, pMPK plasmid or GV2260 (Deblaere et al Nucl. Acids Res. (1985), 13, 4777-4788; Binl9-derivative).
Medium YT-Medium: 0.5% Yeast extract, 0.5% NaCl;. 0.8%
bacto-trypton, if necessary in 1.5%
agar.
YEB-Medium: 0.5% beef extract, 0.1% yeast extract, 0.5~ peptone, 0.5% saccharose, 2 mM
MgS04, if necessary in 1.5% agar.
MS-Medium: According to Murashige and Skoog (Physiologia Plantarum (1962), 15, 473-497).
(1977), 24, 6342-6346) or TB1 was used. For the vectors pMPK110 and BIN19, the species TBl was exclusively used. TB1 is a recombinant, negative, tetracyclines resistant derivative of the species JM101 (Yanisch-Perron et al., Gene (1985), 33, 103-119). The genotype of the TB1 species is (Bart Barrel, personal communication): F'(traD36, proAB, lacl, lacZ~M15), ~(lac, pro), SupE, thiS, recA, Srl::TnlO( TCR ) .
As helper species for the conjugative transfer of the pMPK plasmid from the TB1 cells in Agrobacterium tumefaciens, the E. coli species GJ23 (Van Haute et al., EMBO J. (1983), 2, 411-417) can be used.
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The plant transformation was carried out with the help of the Aqrobacterium tumefaciens species GV3850kan (Jones et al., EMBO J. (1985) 4, 2411-2418, pMPK plasmid or GV2260 (Deblaere et al Nucl. Acids Res. (1985), 13, 4777-4788; Binl9-derivative).
Medium YT-Medium: 0.5% Yeast extract, 0.5% NaCl;. 0.8%
bacto-trypton, if necessary in 1.5%
agar.
YEB-Medium: 0.5% beef extract, 0.1% yeast extract, 0.5~ peptone, 0.5% saccharose, 2 mM
MgS04, if necessary in 1.5% agar.
MS-Medium: According to Murashige and Skoog (Physiologia Plantarum (1962), 15, 473-497).
3. Conjugation and/or Transformation of Aqrobacterium tumefaciens For the transfer of the recombinant pMPKllO plasmid there were mixed together 20 yl each of an overnight culture, washed under selection, of the pMPXllO
containing TBl cells (5U ug/ml spectinomycin and 25 ~g/ml streptomycin in YT-medium), of the helper species GJ23 (10 ~g/ml tetracyclines and 25 ~g/ml kanamycin in YT-medium) and of the Agrobacterium tumefaciens species GV3850Kan (50 ~g/ml erythromycin and 50 ~g/ml chloramphenicol in YEB-medium~ and incubated overnight on a YEB agar plate, without selection, at 28aC. The resulting bacterial growth was plated out on YEB plates that contained 50 ~g/ml chloramphenicol, 50 ~g/ml erythromycin, 100 ~g/ml spectinomycin and 300 ~g/ml streptomycin. The ,, 20070!9~
colonies were washed after three stage incubation at 28 C and combined by the same selection on YEB
plates. From the resulting single colony the total DNA was isolated, and this was tested after suitable restriction cleavage with help of southern blots for the integration of the recombinant DNA.
For Binl9 derivatives, the introduction of the DNA in Agrobacteria was carried out by direct transformation by the method of Holsters et al. (Mol. Gen. Genet.
(1978), 163, 181-187). The plasmid DNA transformed Agrobacteria were isolated by the method of Birnboim and Doly) Nucl. Acids Res. (1979), 7, 1513-1523) and opened up gel electrophore~ically by a suitable restriction cleavage.
containing TBl cells (5U ug/ml spectinomycin and 25 ~g/ml streptomycin in YT-medium), of the helper species GJ23 (10 ~g/ml tetracyclines and 25 ~g/ml kanamycin in YT-medium) and of the Agrobacterium tumefaciens species GV3850Kan (50 ~g/ml erythromycin and 50 ~g/ml chloramphenicol in YEB-medium~ and incubated overnight on a YEB agar plate, without selection, at 28aC. The resulting bacterial growth was plated out on YEB plates that contained 50 ~g/ml chloramphenicol, 50 ~g/ml erythromycin, 100 ~g/ml spectinomycin and 300 ~g/ml streptomycin. The ,, 20070!9~
colonies were washed after three stage incubation at 28 C and combined by the same selection on YEB
plates. From the resulting single colony the total DNA was isolated, and this was tested after suitable restriction cleavage with help of southern blots for the integration of the recombinant DNA.
For Binl9 derivatives, the introduction of the DNA in Agrobacteria was carried out by direct transformation by the method of Holsters et al. (Mol. Gen. Genet.
(1978), 163, 181-187). The plasmid DNA transformed Agrobacteria were isolated by the method of Birnboim and Doly) Nucl. Acids Res. (1979), 7, 1513-1523) and opened up gel electrophore~ically by a suitable restriction cleavage.
4. Plant Transformation A) Tobacco: 10 ml of an overnight culture of Agrobacterium tumefaciens, washed under selection was centrifuged, the supernatant discarded and the bacteria resuspended in the same volume of anabiotic-free medium. In a sterile petri dish, leaf discs of sterile plants, (ca 1 cm2), from which the middle vein had been removed, were bathed in this bacterial suspension. The leaf discs were then compactly laid down in petri dishes which contained MS-medium with 2% saccharose and 0.8% bacto-agar. After two d~ys incubation at 25 C in the dark, they were transferred to MS-medium which contained 100 mg/l kanamycin, 500 mg/l claforan, 1 mg~l benzylaminopurine (BAP), 0.2 mgJ1 naphthylacetic acid (NAA) and 0.8% bacto-agar.
Growing shoots were put into hormone-free ,, Z00709~
MS-medium with 250 mg/l claforan and tested for nopaline content (Otten et al. Biochimica et Biophysica Acta (1978), 527, 497-500). Positive shoots were put into soil after root growth.
B) Potatoes: 10 small leaves of a sterile potato culture, wounded with a scalpel, were put into 10 ml MS-medium with 2% saccharose which contained 30 to 50 ul of an overnight culture of Agrobacterium tumefaciens, washed under selection. After 3-5 minutes gentle shaking, the petri dishes were incubated at 25C in the dark.
After two days, the leaves were laid in MS-medium with 1.6% glucose, 2 mg/l zeatinribose, 0.02 mg/l naphthylacetic acid, 0.02 mg/l gibberellic acid, 500 mg/l claforan, 50 mg/l kanamycin and 0.8% bacto-agar. After one week incubation at 25C and 3000 lux the claforan concentration in the medium was reduced by half.
Growing shoots were put into hormone-free ,, Z00709~
MS-medium with 250 mg/l claforan and tested for nopaline content (Otten et al. Biochimica et Biophysica Acta (1978), 527, 497-500). Positive shoots were put into soil after root growth.
B) Potatoes: 10 small leaves of a sterile potato culture, wounded with a scalpel, were put into 10 ml MS-medium with 2% saccharose which contained 30 to 50 ul of an overnight culture of Agrobacterium tumefaciens, washed under selection. After 3-5 minutes gentle shaking, the petri dishes were incubated at 25C in the dark.
After two days, the leaves were laid in MS-medium with 1.6% glucose, 2 mg/l zeatinribose, 0.02 mg/l naphthylacetic acid, 0.02 mg/l gibberellic acid, 500 mg/l claforan, 50 mg/l kanamycin and 0.8% bacto-agar. After one week incubation at 25C and 3000 lux the claforan concentration in the medium was reduced by half.
5. AnalYsis of the Genomic DNA from Transgenic Plants The isolation of genomic plant DNA was carried out by the method of Rogers and Bendich (Plant Mol. Biol (1985), 5, 69-76).
For DNA analysis 10-20 ~g DNA was tested after suitable restriction cleavage with the aid of southern blots by integration of the DNA sequences being analysed.
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For DNA analysis 10-20 ~g DNA was tested after suitable restriction cleavage with the aid of southern blots by integration of the DNA sequences being analysed.
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;
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6. AnalYsis of the Total RNA from Transqenic Plants The isolation of the total plant RNA was carried out by the method of Longemann et al (Analytical Biochem (1987), 163, 16-2-).
For the analysis, 50 ,ug samples of total RNA were tested with the use of northern blots to determine the presence of the sought transcripts.
For the analysis, 50 ,ug samples of total RNA were tested with the use of northern blots to determine the presence of the sought transcripts.
7. CAT Test The activity of the chloramphenicol-acetyltransferase (CAT) in transgenic plants was carried out by the method of Colot et al (EMBO J (1987) 6, 3559-3564).
The Bradford protein determination was carried out however before the heat treatment (75C, 10 minutes) of the extract. For the determination of the CAT
activity, an amount of heat treated extracts was used that corresponded to a protein content of 500 ~g of the measured extracts.
The Bradford protein determination was carried out however before the heat treatment (75C, 10 minutes) of the extract. For the determination of the CAT
activity, an amount of heat treated extracts was used that corresponded to a protein content of 500 ~g of the measured extracts.
8. GUS Test The activity of the ~-glucuronidase (GUS) in transgenic plants was determined by the method of Jefferson (Plant Mol. Biol. Rep. (1987), 5, 387-405).
The protein determination was carried out by the CAT-Test according to Bradford (Anal. Biochem (1976), 72, 248-254). For the determination of the GUS
activity, 50 ,ug protein was used, in which the incubation was carried out at 37~C for 30 minutes.
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The following examples illustrate the isolation identification as well as the function and use of the wound induced and tuber specific proteinase inhibitor to promoters from potato tubers.
Example 1 Cloning and structural analysis of a proteinase inhibitor II-gene from Solanum tuberosum.
cDNA clones, that have been coded for the proteinase-inhibitor II of potato were isolated and sequenced from the potato variety Berolina (Sanchez-Serrano et al., Mol. Gen. Genet (1986), 203 15-20). These cDNA clones were used to isolate a homologous genomic proteinase-inhibitor II-clone from the monohaploid potato line AM 80/5793 (Max-Planck Institut fur Zuchtungsforschung, Koln); by restriction and sequence analysis the exact structure of the gene was determined.
Further, the transcription start could be established by es~ablished by RNase-digestion of an SP6-antisense-RNA/mRNA hybrid (Keil et al., Nucl. Acids Res (1986), 14, 5641-5650). -.
Example 2 Identification of the regulatory regions responsible for wound inducibility of the proteinase-inhibitor II-gene.
It could be shown th t the isolated proteinase-inhibitor II-gene (see Example I) in transgenic Wisconsin 38 tobacco plants which themselves contain no homologous sequences for the proteinase inhibitor II-gene, were induced by wounding leaves and stems (Sanchez-Serrano et al., EMBO J
(1987), 6, 303-306). The DNA fragment which was thus ,*~ .
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introduced into the plants, reached from a EcoRI
restriction cutting position, that was located ca. 3 kb 5' before the transcription start of the proteinase inhibitor II-gene, to an EcoRI cutting position, that was located ca. l.S kb 3' behind the polyadenylation position.
In another experiment, the proteinase-inhibitor II-gene was introduced after deletion of the single intron by exchange of the intron-containing genomic sequence through the corresponding cDNA sequence in tobacco (Wisconsin 38).
In this case a HindII cutting position 1.5 kb 5' in front of the transcription start was used. The EcoRI cutting position 1.5 kb 3' behind the gene was retained. Analysis of the mRNA that resulted from this construction of the transgenic tobacco plants resulted in a wound inducibility of the intronless proteinase-inhibitor II-gene which was comparable with ~he corresponding intron containing gene.
It thus appears that the intron of the proteinase inhibitor II-gene does not contain any necessary re~ulatory elements for the wound inducibility..
Example 3 Promoter deletion constructions.
From the results of Example 2, chimeric constructions of the deletions, as well as the restriction fragments of the proteinase inhibitor II-gene with the bacterial chloramphenicol-acetyltransferase-gene (CAT) resulting from the exonuclease III digestion and of the 3' region of the proteinase inhibitor II-gene, were produced.
The deletion fragments resulted from the sequence analysis of the proteinase-inhibitor II-gene. They were cloned with the help of an EcoRI cutting position that was located 5' - . .
-:
zoo~o~
before the corresponding promoter deletion in the polylinker of the phage vector M13mpl9, and a ScaI cutting position 32 bp 3' of the transcription start and 18 bp 5' of the ATG start codon of the proteinase-inhibitor II-gene S in the EcoRI/SmaI splitting intermediary vector pMPK110.
The promoter fragment resulting from this had a length of 700, 514, 210 and 150 bp 5' of the transcription start of the proteinase-inhibitor II-gene.
On the one hand, a fragment served as a restriction fragment that spread from a HindIII cutting position 1.3 kb 5' in front of the transcription start up to the above mentioned ScaI cutting position (pM 11) but also a 441 bp long SspI/ScaI fragment of the promoter region that respectively were cloned in the Smai cutting position of pMPK 110. The HindIII cutting position of the first fragment first had to be filled with T4-DNA polymerase.
The CAT gene was cloned as 800 bp long BamHi fragments tVelten et al., Nucl Acids Res (1985). 13, 6981-699~), .. -re~pectively 3' behind the promoter fragment of the proteinase-inhibitor II-gene in the BamHI cutting position of the polylinker of pMPKllO.
The 260 bp long RsaI/SspI fragment of the proteinase-inhibitor II-gene that was supplied with SphI
link, was cloned in the corresponding restriction cutting position of the polylinker of pMPKllO, as polyadenylation signal. The RsaI cutting position was located 11 bp in front of the TGA stop codon and the SspI cutting position was located 74 bp 3' behind the polyadenylation position of the proteinase inhibitor II-gene.
The construction was introduced using the Aqrobacterium tumefaciens transformation system in the tobacco variety ..
20070~
Wisconsin 38. From the resulting transgenic tobacco plants were unwounded and wounded leaves were tested in the presence of CAT-mRNA and/or for the activity of the CAT
enzymes. The results showed that for maximal wound inducibility, the area of 700 - 1300 bp 5' before the transcription start is necessary (pM 11). After deletion of this region only a very small wound inducibility was observed, if at all. Interestingly no CAT activity was observed with a construction that contained the total promoter of the proteinase inhibitor I~-gene up to 1500 bp 5' before the transcription start, but not the 3'-end of the proteinase-inhibitor II-gene. In this construction, 3' behind the promoter a 1000 bp long SalI eye fragment was cloned in the corresponding cutting position of the pMPK110 polylinXer which contained, 3' behind the CAT
gene, the polyadenylation signal of the gene 7 of the T-DNA of Aqrobacterium tumefaciens (Velten et al., Nucl Acids Res (1985), 13, 6981-6998). This polyadenylation signal is functional in plant cells.
To summarise: the regulatory region of the proteinase inhibitor II-gene functions in a specific manner also in combination with a heterologous bacterial gene, in this case the chloramphenicol acetyltransferase. For maximal inducibility of the chimeric gene an enhancer is necessary that is located in the region of 700 - 1300 bp 5' in front of the transcription start and also ~ 260 bp long fragment of the 3'-region of the proteinase-inhibitor II-gene which stretches up to 74 bp 3' behind the polyadenylation position.
. :-200709~
Example 4 Fusion of a proteinase inhibitor II promoter fragment with a heterologous promoter.
Should the proteinase inhibitor II promoter actually contain an enhancer, this must be able to activate an inactive promoter that contains only the TATA and CAAT
steering element.
For this, first, a CAT construction with an inactive promoter was prepared. The 35 S-promoter of the cauliflower mosaic virus constitutively and very strongly expressed in plant cells was cloned as 550 bp long EcoRI/RpnI fragments in the corresponding restriction cutting positions in front of the 1000 bp long SalI-fragment of the CAT gene with the gene 7-polyadenylation signal in pUC18 (pDHCAT1). The KpnI
cutting position was located on position +10 in relation to the transcription start of the 35 S-promoter. The EcoRV
cu~ting position on position -90 in relation to the transcription starts of the 35 S-promoter and the HincII
cutting position 3' behind the gene 7-polyadenylation signal was usecl, in order to clone the resulting (-90)35S/CAT/g7pA-fragment in the HincII cutting position of pMPK110 (pMP35SCAT1). For this a partial digestion of pDHCAT1 with HincII was necessary since a second HincII
cutting position was present in front of the CAT gene that must remain uncleaved. In order to be able to use the SmaI
cutting position located 5' in front of the (-90)35 S-promoter, for the insertion of DNA fragments, the SmaI
cutting position was eliminated immediately 5' in front of the CAT-gene. For this pMP(-90)35SCAT1 was cleaved with SalI, the cutting position was filled up with Rlenow enzyme and then cleaved with PstI. The ligation with . .
200709~
SmaI/PstI cleaved pDHCAT1 gave pMP (-90)35SCAT10, in which the SalI and the SmaI cutting positions, which are located between the (-90)35 S-promoter and the CAT gene, were fused and could thus not be used again. This construction was introduced in Wisconsin 38 tobacco plants whereby an activity of the (-90)35 S-promoter could not be demonstrated either in unwounded nor in wounded leaves of these transgenic plants.
In order to be able to answer the question whether the proteinase-inhibitor II-promoter can activate this inactive (-90)35 S-promoter, a deletion fragment 5', which was cloned in M13mpl9, was cloned in front of this promoter with the resulting sequence analysis of the proteinase-inhibitor II-gene. The deletion fragment which stretched from position -195 to -1300 in relation to the transcription start of the proteinase inhibitor II-gene, could be split off by a Eco~I/HindIII cleavage from the M13mpl-vector. After filling the cutting positions with T4-DNA polymerise this fragment was inserted in the SmaI
cu~Eting position of pMP(-90)35SCAT10. The resulting vectors pM21 and pM22 contained the promoter fragment of the proteinase-inhibitor II-gene in both orientations.
The analysis of the transgenic tobacco plants containing this construction gave both in northern blot and also in the CAT test a definite wound inducibility of the CAT
genes in leaves.
These results show that the proteinase-inhibitor II-promoter actually possesses an enhancer, which can specifically activate in both orientations an inactive promoter. Further, the region from +32 to -195 of the proteinase inhibitor II-gene is clearly not necessary for the wound inducibility. Interestingly, in the combination -:
200709~
of the (-1300/-195)-promoter fragment of the proteinase-inhibitor II-gene with the (-90)35 S-promoter, the 3' region of the proteinase-inhibitor II-gene is not necessary in order to have wound inducibility of the CAT
gene, as is typical for the total proteinase-inhibitor II-promoter from position 1300 to +32 in relation to the transcription start.
ExamPle 5 Fusions of the regulatory regions of the proteinase inhibitor II-gene with another bacterial gene.
In order to show that the regulatory region of the proteinase inhibitor II-gene functions also in combination with another gene, there was constructed not only a fusion with the gene for the bacterial chloramphenicol-acetyltransferase but also a fusion with a gene which iscoded for the bacterial ~-glucuronidase (GUS).
The GUS gene was first cut from the vector pBI101 (Jefferson et al., EMBO J (1987), 6, 3901-3907), together with the polyaclenylation signal of the nopaline synthase gene (Nos) of Aqrobacterium tumefaciens, by an EcoRI/SmaI
cleavage and after Klenow filling, the restriction cutting positions in the HincII cutting position of pUC18, was cloned ~pGUS; Meike Koster, personal communication).
Through a BamHI/PstI cleavage, the CAT gene was cleaved from pM11. Instead of this, a 1800 bp long BamHI/SstI
fragment from the plasmid pGUS, which contains a gene for the ~-glucuronidase without the Nos-3' end, was ligated.
For this, the PstI and the SstI cutting positions had to previously be filled with T4-DNA polymerase. The resulting vector pM14 (see Figure 1) now contained the GUS gene 3' ,.. ,.............. - ~
behind the proteinase-inhibitor II-promoter and 5' in front of the 3' end of the proteinase inhibitor II-gene (see Figure 1). Since the potato transformation with the binary vector system functions efficiently as with intermediary vectors, this chimeric gene was cloned round as 3.4 kb long Eco~I/HindIII fragments in the corresponding cutting positions of the binary vector BINlg (pS9). These constructions were introduced both in tobacco Wisconsin 38 (pM14) and also in the potato variety Berolina (pS9).
The analysis of the resulting transgenic tobacco and potato plants with northern blots showed a definite activity of the GUS gene in wounded leaves, but in non-wounded leaves none or only a small activity of the GUS
gene could be shown. The analysis of the mRNA and the GUS
activity in potato tubers showed interestingly around a 10 times greater activity of the gene in comparison to that in wounded leaves. Therefore the proteinase-inhibitor II-promoter not only contained the steering element for the wound induction but also that for expression in potato tubers.
Example 6 Expression of genes which are a code for toxic proteins under control of regulatory regions of the protein inhibitor II-gene.
A use for regulatory regions of the proteinase-inhibitor II-gene is the wound specific expression of toxic proteins in leaves for combating plant pests such as insects or certain microorganisms. Thus a gene which codes for the toxic thionine was kept under the control of the proteinase-inhibitor II-promoter. Thionine was normally . .
200709~
expressed in the endosperm of various cereals but also in the leaves of barley seedlings (Bohlmann und Apel, MGG
(1987), 207, 446-454). Under control of the proteinase-inhibitor II-promoter on the other hand in transgenic tobacco plants, a wound specific accumulation of thionine mRNA in leaves could be demonstrated.
.. .,., ~, ..
. . ,
The protein determination was carried out by the CAT-Test according to Bradford (Anal. Biochem (1976), 72, 248-254). For the determination of the GUS
activity, 50 ,ug protein was used, in which the incubation was carried out at 37~C for 30 minutes.
' ~
~00709~
The following examples illustrate the isolation identification as well as the function and use of the wound induced and tuber specific proteinase inhibitor to promoters from potato tubers.
Example 1 Cloning and structural analysis of a proteinase inhibitor II-gene from Solanum tuberosum.
cDNA clones, that have been coded for the proteinase-inhibitor II of potato were isolated and sequenced from the potato variety Berolina (Sanchez-Serrano et al., Mol. Gen. Genet (1986), 203 15-20). These cDNA clones were used to isolate a homologous genomic proteinase-inhibitor II-clone from the monohaploid potato line AM 80/5793 (Max-Planck Institut fur Zuchtungsforschung, Koln); by restriction and sequence analysis the exact structure of the gene was determined.
Further, the transcription start could be established by es~ablished by RNase-digestion of an SP6-antisense-RNA/mRNA hybrid (Keil et al., Nucl. Acids Res (1986), 14, 5641-5650). -.
Example 2 Identification of the regulatory regions responsible for wound inducibility of the proteinase-inhibitor II-gene.
It could be shown th t the isolated proteinase-inhibitor II-gene (see Example I) in transgenic Wisconsin 38 tobacco plants which themselves contain no homologous sequences for the proteinase inhibitor II-gene, were induced by wounding leaves and stems (Sanchez-Serrano et al., EMBO J
(1987), 6, 303-306). The DNA fragment which was thus ,*~ .
~ ` ~
200709~
introduced into the plants, reached from a EcoRI
restriction cutting position, that was located ca. 3 kb 5' before the transcription start of the proteinase inhibitor II-gene, to an EcoRI cutting position, that was located ca. l.S kb 3' behind the polyadenylation position.
In another experiment, the proteinase-inhibitor II-gene was introduced after deletion of the single intron by exchange of the intron-containing genomic sequence through the corresponding cDNA sequence in tobacco (Wisconsin 38).
In this case a HindII cutting position 1.5 kb 5' in front of the transcription start was used. The EcoRI cutting position 1.5 kb 3' behind the gene was retained. Analysis of the mRNA that resulted from this construction of the transgenic tobacco plants resulted in a wound inducibility of the intronless proteinase-inhibitor II-gene which was comparable with ~he corresponding intron containing gene.
It thus appears that the intron of the proteinase inhibitor II-gene does not contain any necessary re~ulatory elements for the wound inducibility..
Example 3 Promoter deletion constructions.
From the results of Example 2, chimeric constructions of the deletions, as well as the restriction fragments of the proteinase inhibitor II-gene with the bacterial chloramphenicol-acetyltransferase-gene (CAT) resulting from the exonuclease III digestion and of the 3' region of the proteinase inhibitor II-gene, were produced.
The deletion fragments resulted from the sequence analysis of the proteinase-inhibitor II-gene. They were cloned with the help of an EcoRI cutting position that was located 5' - . .
-:
zoo~o~
before the corresponding promoter deletion in the polylinker of the phage vector M13mpl9, and a ScaI cutting position 32 bp 3' of the transcription start and 18 bp 5' of the ATG start codon of the proteinase-inhibitor II-gene S in the EcoRI/SmaI splitting intermediary vector pMPK110.
The promoter fragment resulting from this had a length of 700, 514, 210 and 150 bp 5' of the transcription start of the proteinase-inhibitor II-gene.
On the one hand, a fragment served as a restriction fragment that spread from a HindIII cutting position 1.3 kb 5' in front of the transcription start up to the above mentioned ScaI cutting position (pM 11) but also a 441 bp long SspI/ScaI fragment of the promoter region that respectively were cloned in the Smai cutting position of pMPK 110. The HindIII cutting position of the first fragment first had to be filled with T4-DNA polymerase.
The CAT gene was cloned as 800 bp long BamHi fragments tVelten et al., Nucl Acids Res (1985). 13, 6981-699~), .. -re~pectively 3' behind the promoter fragment of the proteinase-inhibitor II-gene in the BamHI cutting position of the polylinker of pMPKllO.
The 260 bp long RsaI/SspI fragment of the proteinase-inhibitor II-gene that was supplied with SphI
link, was cloned in the corresponding restriction cutting position of the polylinker of pMPKllO, as polyadenylation signal. The RsaI cutting position was located 11 bp in front of the TGA stop codon and the SspI cutting position was located 74 bp 3' behind the polyadenylation position of the proteinase inhibitor II-gene.
The construction was introduced using the Aqrobacterium tumefaciens transformation system in the tobacco variety ..
20070~
Wisconsin 38. From the resulting transgenic tobacco plants were unwounded and wounded leaves were tested in the presence of CAT-mRNA and/or for the activity of the CAT
enzymes. The results showed that for maximal wound inducibility, the area of 700 - 1300 bp 5' before the transcription start is necessary (pM 11). After deletion of this region only a very small wound inducibility was observed, if at all. Interestingly no CAT activity was observed with a construction that contained the total promoter of the proteinase inhibitor I~-gene up to 1500 bp 5' before the transcription start, but not the 3'-end of the proteinase-inhibitor II-gene. In this construction, 3' behind the promoter a 1000 bp long SalI eye fragment was cloned in the corresponding cutting position of the pMPK110 polylinXer which contained, 3' behind the CAT
gene, the polyadenylation signal of the gene 7 of the T-DNA of Aqrobacterium tumefaciens (Velten et al., Nucl Acids Res (1985), 13, 6981-6998). This polyadenylation signal is functional in plant cells.
To summarise: the regulatory region of the proteinase inhibitor II-gene functions in a specific manner also in combination with a heterologous bacterial gene, in this case the chloramphenicol acetyltransferase. For maximal inducibility of the chimeric gene an enhancer is necessary that is located in the region of 700 - 1300 bp 5' in front of the transcription start and also ~ 260 bp long fragment of the 3'-region of the proteinase-inhibitor II-gene which stretches up to 74 bp 3' behind the polyadenylation position.
. :-200709~
Example 4 Fusion of a proteinase inhibitor II promoter fragment with a heterologous promoter.
Should the proteinase inhibitor II promoter actually contain an enhancer, this must be able to activate an inactive promoter that contains only the TATA and CAAT
steering element.
For this, first, a CAT construction with an inactive promoter was prepared. The 35 S-promoter of the cauliflower mosaic virus constitutively and very strongly expressed in plant cells was cloned as 550 bp long EcoRI/RpnI fragments in the corresponding restriction cutting positions in front of the 1000 bp long SalI-fragment of the CAT gene with the gene 7-polyadenylation signal in pUC18 (pDHCAT1). The KpnI
cutting position was located on position +10 in relation to the transcription start of the 35 S-promoter. The EcoRV
cu~ting position on position -90 in relation to the transcription starts of the 35 S-promoter and the HincII
cutting position 3' behind the gene 7-polyadenylation signal was usecl, in order to clone the resulting (-90)35S/CAT/g7pA-fragment in the HincII cutting position of pMPK110 (pMP35SCAT1). For this a partial digestion of pDHCAT1 with HincII was necessary since a second HincII
cutting position was present in front of the CAT gene that must remain uncleaved. In order to be able to use the SmaI
cutting position located 5' in front of the (-90)35 S-promoter, for the insertion of DNA fragments, the SmaI
cutting position was eliminated immediately 5' in front of the CAT-gene. For this pMP(-90)35SCAT1 was cleaved with SalI, the cutting position was filled up with Rlenow enzyme and then cleaved with PstI. The ligation with . .
200709~
SmaI/PstI cleaved pDHCAT1 gave pMP (-90)35SCAT10, in which the SalI and the SmaI cutting positions, which are located between the (-90)35 S-promoter and the CAT gene, were fused and could thus not be used again. This construction was introduced in Wisconsin 38 tobacco plants whereby an activity of the (-90)35 S-promoter could not be demonstrated either in unwounded nor in wounded leaves of these transgenic plants.
In order to be able to answer the question whether the proteinase-inhibitor II-promoter can activate this inactive (-90)35 S-promoter, a deletion fragment 5', which was cloned in M13mpl9, was cloned in front of this promoter with the resulting sequence analysis of the proteinase-inhibitor II-gene. The deletion fragment which stretched from position -195 to -1300 in relation to the transcription start of the proteinase inhibitor II-gene, could be split off by a Eco~I/HindIII cleavage from the M13mpl-vector. After filling the cutting positions with T4-DNA polymerise this fragment was inserted in the SmaI
cu~Eting position of pMP(-90)35SCAT10. The resulting vectors pM21 and pM22 contained the promoter fragment of the proteinase-inhibitor II-gene in both orientations.
The analysis of the transgenic tobacco plants containing this construction gave both in northern blot and also in the CAT test a definite wound inducibility of the CAT
genes in leaves.
These results show that the proteinase-inhibitor II-promoter actually possesses an enhancer, which can specifically activate in both orientations an inactive promoter. Further, the region from +32 to -195 of the proteinase inhibitor II-gene is clearly not necessary for the wound inducibility. Interestingly, in the combination -:
200709~
of the (-1300/-195)-promoter fragment of the proteinase-inhibitor II-gene with the (-90)35 S-promoter, the 3' region of the proteinase-inhibitor II-gene is not necessary in order to have wound inducibility of the CAT
gene, as is typical for the total proteinase-inhibitor II-promoter from position 1300 to +32 in relation to the transcription start.
ExamPle 5 Fusions of the regulatory regions of the proteinase inhibitor II-gene with another bacterial gene.
In order to show that the regulatory region of the proteinase inhibitor II-gene functions also in combination with another gene, there was constructed not only a fusion with the gene for the bacterial chloramphenicol-acetyltransferase but also a fusion with a gene which iscoded for the bacterial ~-glucuronidase (GUS).
The GUS gene was first cut from the vector pBI101 (Jefferson et al., EMBO J (1987), 6, 3901-3907), together with the polyaclenylation signal of the nopaline synthase gene (Nos) of Aqrobacterium tumefaciens, by an EcoRI/SmaI
cleavage and after Klenow filling, the restriction cutting positions in the HincII cutting position of pUC18, was cloned ~pGUS; Meike Koster, personal communication).
Through a BamHI/PstI cleavage, the CAT gene was cleaved from pM11. Instead of this, a 1800 bp long BamHI/SstI
fragment from the plasmid pGUS, which contains a gene for the ~-glucuronidase without the Nos-3' end, was ligated.
For this, the PstI and the SstI cutting positions had to previously be filled with T4-DNA polymerase. The resulting vector pM14 (see Figure 1) now contained the GUS gene 3' ,.. ,.............. - ~
behind the proteinase-inhibitor II-promoter and 5' in front of the 3' end of the proteinase inhibitor II-gene (see Figure 1). Since the potato transformation with the binary vector system functions efficiently as with intermediary vectors, this chimeric gene was cloned round as 3.4 kb long Eco~I/HindIII fragments in the corresponding cutting positions of the binary vector BINlg (pS9). These constructions were introduced both in tobacco Wisconsin 38 (pM14) and also in the potato variety Berolina (pS9).
The analysis of the resulting transgenic tobacco and potato plants with northern blots showed a definite activity of the GUS gene in wounded leaves, but in non-wounded leaves none or only a small activity of the GUS
gene could be shown. The analysis of the mRNA and the GUS
activity in potato tubers showed interestingly around a 10 times greater activity of the gene in comparison to that in wounded leaves. Therefore the proteinase-inhibitor II-promoter not only contained the steering element for the wound induction but also that for expression in potato tubers.
Example 6 Expression of genes which are a code for toxic proteins under control of regulatory regions of the protein inhibitor II-gene.
A use for regulatory regions of the proteinase-inhibitor II-gene is the wound specific expression of toxic proteins in leaves for combating plant pests such as insects or certain microorganisms. Thus a gene which codes for the toxic thionine was kept under the control of the proteinase-inhibitor II-promoter. Thionine was normally . .
200709~
expressed in the endosperm of various cereals but also in the leaves of barley seedlings (Bohlmann und Apel, MGG
(1987), 207, 446-454). Under control of the proteinase-inhibitor II-promoter on the other hand in transgenic tobacco plants, a wound specific accumulation of thionine mRNA in leaves could be demonstrated.
.. .,., ~, ..
. . ,
Claims (29)
1. Agrobacteria containing a DNA sequence of an expression cassette, in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves, as well as for the constitutive transcriptional regulation in the potato tubers, are localised.
2. Agrobacteria according to claim 1, which is an Agrobacterium tumefaciens species.
3. Agrobacterium tumefaciens according to claim 2, that consists of the 3.4 kb long EcoRI/HindIII fragment.
4. Agrobacterium tumefaciens according to claim 3, in which the DNA sequence of the expression cassette, comprises a proteinase-inhibitor II-gene.
5. Agrobacterium tumefaciens according to claim 4, in which the proteinase-inhibitor II-gene is from Solanum tuberosum.
6. Agrobacterium tumefaciens according to claim 3, in which the DNA sequence contains the 0.26 kb long SphI/SphI-fragment of the proteinase-inhibitor II-region.
7. Agrobacterium tumefaciens according to claim 3, in which the DNA sequence contains a proteinase-inhibitor II-promoter.
8. A proteinase-inhibitor II-promoter according to claim 2, that consists of the 3.4 kb long ScaI/HindIII
fragment.
fragment.
9. Vector pM 14, consisting of a 8.1 kb long DNA
sequence.
sequence.
10. Vector pM 14 according to claim 9, that contains the 3.4 kb long EcoRI/HindIII fragment.
11. Vector pM 14 according to claim 10, in which the DNA
sequence contains the 1.3 kb long ScaI/HindIII
fragment of the proteinase-inhibitor II-promoter, the 1.8 kb long BamHI/SstI-fragment of the .beta.-glucuronidase and the 0.26 kb long SphI/SphI fragment of the proteinase-inhibitor II-region.
sequence contains the 1.3 kb long ScaI/HindIII
fragment of the proteinase-inhibitor II-promoter, the 1.8 kb long BamHI/SstI-fragment of the .beta.-glucuronidase and the 0.26 kb long SphI/SphI fragment of the proteinase-inhibitor II-region.
12. Vector pM 14 according to claim 11, in which the .beta.-glucuronidase-gene is localised 3' behind the proteinase-inhibitor II-promoter and 5' in front of the 3'-end of the proteinase-inhibitor II-gene.
13. Vector pM 14 according to claim 9, that is contained in Agrobacterium tumefaciens.
14. A plant genome of potato or tobacco.
15. Potato according to claim 14, which contains a DNA
sequence of an expression cassette in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in the potato tubers are localised.
sequence of an expression cassette in which the regulating regions for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in the potato tubers are localised.
16. Potato according to claim 15, in which the DNA
sequence of the expression cassette contains a proteinase-inhibitor II-gene.
sequence of the expression cassette contains a proteinase-inhibitor II-gene.
17. Potato according to claim 15, in which the DNA
sequence contains the 0.26 kb long SphI/SphI-fragment of the proteinase-inhibitor II-region.
sequence contains the 0.26 kb long SphI/SphI-fragment of the proteinase-inhibitor II-region.
18. Potato according to claim 15, in which the DNA
sequence contains a proteinase-inhibitor II-promoter.
sequence contains a proteinase-inhibitor II-promoter.
19. Potato according to claim 15, in which the proteinase-inhibitor II-promoter consists of the 3.4 kb long ScaI/HindIII fragment.
20. Use of the proteinase-inhibitor II-gene for the wound-inducible transcriptional regulation in the stem and in leaves as well as for the constitutive transcriptional regulation in tubers of crops.
21. Use of the proteinase-inhibitor II-gene according to claim 20, in which the crop is tobacco.
22. Use of the proteinase-inhibitor II-gene according to claim 20, in which the crop is potato.
23. Use of the proteinase-inhibitor II-gene for the wound-inducible expression of genes that encode toxic proteins in leaves, stems and tubers of crops.
24. Use of the proteinase-inhibitor II-gene according to claim 23, in which the crop is tobacco.
25. Use of the proteinase-inhibitor II-gene according to claim 23, in which the crop is potato.
26. Use of the proteinase-inhibitor II-gene according to claim 23, in which the expressing gene for the toxic protein encodes thionine.
27. Use of the proteinase-inhibitor II-gene according to claim 23, in which the toxic protein is used to combat pests.
28. Use of the proteinase-inhibitor II-gene according to claim 27, in which the toxic protein is used to combat insects and microorganisms.
29. Agrobacterium tumefaciens A tum. m 14 (DSM 5088).
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3843628A DE3843628A1 (en) | 1988-12-21 | 1988-12-21 | Wound-inducible and potato-tuber-specific transcriptional regulation |
| EP89250116A EP0375091A1 (en) | 1988-12-21 | 1989-12-18 | Wound-inducible and potato tuber-specific transcriptional regulation |
| JP1329771A JPH02283275A (en) | 1988-12-21 | 1989-12-21 | Wound inducible transcriptional control and constitutive transcriptional control for agrobacterium, proteinase inhibiter 2-promotor, vektor pm14 and vegetable genom, which have dna-arrangement of manifest cassette ; and genetic wound inducible manifestation |
| CA002007091A CA2007091A1 (en) | 1988-12-21 | 1989-12-21 | Wound-inducible and potato tuber specific transcriptional regulation |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3843628.0 | 1988-12-21 | ||
| DE3843628A DE3843628A1 (en) | 1988-12-21 | 1988-12-21 | Wound-inducible and potato-tuber-specific transcriptional regulation |
| CA002007091A CA2007091A1 (en) | 1988-12-21 | 1989-12-21 | Wound-inducible and potato tuber specific transcriptional regulation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2007091A1 true CA2007091A1 (en) | 1990-06-21 |
Family
ID=25673861
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002007091A Abandoned CA2007091A1 (en) | 1988-12-21 | 1989-12-21 | Wound-inducible and potato tuber specific transcriptional regulation |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0375091A1 (en) |
| JP (1) | JPH02283275A (en) |
| CA (1) | CA2007091A1 (en) |
| DE (1) | DE3843628A1 (en) |
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| US6229067B1 (en) | 1996-10-25 | 2001-05-08 | Basf Aktiengesellschaft | Leaf-specific gene expression in transgenetic plants |
| US6617498B1 (en) | 1999-09-03 | 2003-09-09 | Pioneer-Hi-Bred International, Inc. | Inducible promoters |
| US7601889B2 (en) | 2001-03-26 | 2009-10-13 | Napier Johnathan A | Elongase gene and production of Δ9-polyunsaturated fatty acids |
| US8664475B2 (en) | 2007-09-18 | 2014-03-04 | Basf Plant Science Gmbh | Plants with increased yield |
| US8809059B2 (en) | 2007-09-21 | 2014-08-19 | Basf Plant Science Gmbh | Plants with increased yield |
| US9029111B2 (en) | 2006-08-24 | 2015-05-12 | Basf Plant Science Gmbh | Isolation and characterization of a novel pythium omega 3 desaturase with specificity to all omega 6 fatty acids longer than 18 carbon chains |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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-
1988
- 1988-12-21 DE DE3843628A patent/DE3843628A1/en not_active Withdrawn
-
1989
- 1989-12-18 EP EP89250116A patent/EP0375091A1/en not_active Withdrawn
- 1989-12-21 JP JP1329771A patent/JPH02283275A/en active Pending
- 1989-12-21 CA CA002007091A patent/CA2007091A1/en not_active Abandoned
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6229067B1 (en) | 1996-10-25 | 2001-05-08 | Basf Aktiengesellschaft | Leaf-specific gene expression in transgenetic plants |
| US6617498B1 (en) | 1999-09-03 | 2003-09-09 | Pioneer-Hi-Bred International, Inc. | Inducible promoters |
| US7601889B2 (en) | 2001-03-26 | 2009-10-13 | Napier Johnathan A | Elongase gene and production of Δ9-polyunsaturated fatty acids |
| US9029111B2 (en) | 2006-08-24 | 2015-05-12 | Basf Plant Science Gmbh | Isolation and characterization of a novel pythium omega 3 desaturase with specificity to all omega 6 fatty acids longer than 18 carbon chains |
| US10113189B2 (en) | 2006-08-24 | 2018-10-30 | Basf Plant Science Gmbh | Isolation and characterization of a novel pythium omega 3 desaturase with specificity to all omega 6 fatty acids longer than 18 carbon chains |
| US8664475B2 (en) | 2007-09-18 | 2014-03-04 | Basf Plant Science Gmbh | Plants with increased yield |
| US8809059B2 (en) | 2007-09-21 | 2014-08-19 | Basf Plant Science Gmbh | Plants with increased yield |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0375091A1 (en) | 1990-06-27 |
| DE3843628A1 (en) | 1990-07-05 |
| JPH02283275A (en) | 1990-11-20 |
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