WO2002034924A2 - Cassettes d'expression permettant l'expression transgenique d'acides nucleiques dans l'epiderme vegetal embryonnaire et dans la fleur - Google Patents

Cassettes d'expression permettant l'expression transgenique d'acides nucleiques dans l'epiderme vegetal embryonnaire et dans la fleur Download PDF

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WO2002034924A2
WO2002034924A2 PCT/EP2001/012444 EP0112444W WO0234924A2 WO 2002034924 A2 WO2002034924 A2 WO 2002034924A2 EP 0112444 W EP0112444 W EP 0112444W WO 0234924 A2 WO0234924 A2 WO 0234924A2
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expression
promoter
plant
transgenic
nucleic acid
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PCT/EP2001/012444
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WO2002034924A3 (fr
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Andreas Reindl
Friedrich Bischoff
Chiara Tonelli
Katia Petroni
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Basf Plant Science Gmbh
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Publication of WO2002034924A3 publication Critical patent/WO2002034924A3/fr

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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • 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
    • C12N15/823—Reproductive tissue-specific promoters
    • 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
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm

Definitions

  • Promoters with specificities for the anthers, ovaries, flowers, leaves, stems, roots and seeds are described. The stringency of the specificity as well as the expression activity of these promoters is very different. Promoters that ensure leaf-specific expression are to be mentioned, such as the promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) of the Rubisco (ribulose-1, 5-bisphosphate carboxylase) or the ST- Potato LSI promoter (Stockhaus et al., EMBO J. 8 (1989), 2445-245).
  • promoters are, for example, promoters with specificity for tubers, storage roots or roots, such as, for example, the patatin promoter class I (B33), the promoter of the cathepsin D inhibitor from potato, the promoter of the starch synthase (GBSS1) or the sporamine promoter, fruit-specific promoters, such as the fruit-specific promoter made of tomato (EP-A 409625), fruit-ripening-specific promoters such as the fruit-specific promoter made of tomato
  • flower-specific promoters such as the phytoene synthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).
  • Promoters are also known which control expression in seeds and plant embryos.
  • the promoters of genes that are used for storage proteins have been identified. encode different dicotyledons.
  • Seed-specific promoters are, for example, the promoter of phaseoline (US 5,504,200, Bustos MM et al., Plant Cell. 1989; 1 (9): 839-53), of the 2S albuming gene (Joseffson LG et al., J Biol Chem 1987, 262 : 12196-12201), leguminum (Shirsat A et al., Mol Gen Genet. 1989;
  • Expression is rarely cell type specific. Differences in the expression pattern and the expression strength of a particular promoter can be caused by different host plants or by different insertion sites in the genome of the host plant (Goossens A et al., Plant Phys 1999, 120: 1095-1104).
  • a promoter activity with a specificity for certain cells within the seed has only been described for the seed coat:
  • a cryptic promoter with specificity for the seed coat was identified by "T-DNA tagging" in tobacco (Fobert PR et al., Plant Journal 19946 (4): 567-77; US 5,824,863; WO 99/53067).
  • Cryptic promoters or pseudopromotors are inherently inactive sequences within the genome, which, however, acquire expression-regulating functionality when positioned near genes.
  • a lectin-like protein that accumulates in the shoot tip epider is the garden pea (Pisum safcivum) has been described (Maiti et al., Planta 1993, 190, 241-246). Sterk et al. describe a lipid transfer protein that is strongly expressed in the epidermal cells of carrot shoot tips (Sterk et al., Plant Cell 1991, 3, 907-921). Clark et al. (Clark et al., Plant Cell 1992, 4, 1189-1198) describe a lipid transfer protein that is expressed in the epidermis of Pachyphy turn.
  • epidermis-specifically expressed genes such as the Chalcon synthase and phenylalanine ammonia gene families have been reported (Schmelzer et al., Proc. Natl. Acad. Sci. USA 1988, 85, 2989-2993; Schmelzer et al., Plant Cell 1989, 1, 993-1001).
  • Goodrich et al. describes enzymes of anthocyanin biosynthesis in the snapdragon flower, which are expressed in specialized, epidermal cells for a limited period during the development of the flower bud (Goodrich et al., Cell 1992, 68, 955-964). Wyatt et al.
  • Promoters are described with tissue specificity for the mesophyll and the pallisade cells in leaves (Broglie et al., Science 1984, 234, 838-845), the dividing shoot and the root meristem (Atanassova et al., Plant J. 1992 , 2, 291-300), pollen (Guerrero et al., Mol. Gen. Genet. 1990, 224, 161-168), seed endosperm (Stalberg et al., Plant Mol. Biol. 1993, 23, 20 671-683 ), Root epidermis (Suzuki et al., Plant Mol. Biol 1993, 21, 109-119), as well as for the root meristem, root vascular tissue and root nodules (Bogusz et al., Plant Cell 1990, 2, 633-641).
  • Epidermis-specific promoters are also known with activity in flowers (Koes et al., Plant Cell 1990, 2, 379-392) or after wound injuries (Liang et al., Proc. Natl. Acad. Sei. USA 1989, 86 , 9284-9288).
  • No. 5,744,334 describes a plant promoter (Blec promoter) with specificity for the epidermis of the shoot tip.
  • no promoter has been described that specifically ensures expression in the plant embryonic epidermis.
  • genes are regulated in all organisms via transcription factors. These also show development and
  • the task was therefore to identify corresponding promoters.
  • This task was solved by providing the promoter for the Arabidopsis thaliana mybll gene.
  • the promoter of the Arabidopsis thaliana mybll gene has an expression-regulating specificity which shows expression in the flower bud, the flower, the withering flower, the green pods and in the embryonic epidermis on day 4 of germination (compare FIGS. 1 to 6).
  • the mybll gene belongs to the R2R3-MYB family of transcription factors.
  • the MYB11 gene is known (Genbank Acc.-No: AL162651). However, neither its function nor its expression pattern are described. To date, no MYB transcription factor has been described or identified that is expressed both in flowers and in the embryonic epidermis or in the embryonic epidermis alone.
  • the flower bud and the flower of the plant is a sensitive organ, especially against stress factors such as cold, and
  • the plant embryo is particularly sensitive and susceptible to biotic and abiotic stress factors, especially in the first days of embryogenesis.
  • the epidermis of the plant embryo has a special function because it is the immediate barrier and contact point with the environment.
  • a first subject of the invention therefore relates to an expression cassette for the transgenic expression of nucleic acids in the embryonic epidermis and / or containing the flower
  • b) a functional equivalent or equivalent fragment of a) which have essentially the same promoter activities as a), where a) or b) are functionally linked to a nucleic acid sequence to be expressed transgenically.
  • Expression cassette for the transgenic expression of nucleic acids means all such constructions which have been obtained by genetic engineering methods and in which either
  • nucleotide residues are not in their natural, genetic environment (i.e. at their natural chromosomal locus) or have been modified by genetic engineering methods, the modification being, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.
  • the expression of a specific nucleic acid by a promoter with specificity for the embryonic epidermis and / or the flower can lead to the formation of sense RNA or anti-sense RNA.
  • the sense RNA can be translated into certain polypeptides.
  • the expression of certain genes can be downregulated with the anti-sense RNA.
  • a functional link is understood to mean, for example, the sequential arrangement of the promoter with specificity for the embryonic epidermis and / or the flower, the nucleic acid sequence to be expressed transgenically and if necessary.
  • further regulatory elements such as a terminator such that each of the regulatory elements can fulfill its function in the transgenic expression of the nucleic acid sequence, depending on the arrangement of the nucleic acid sequences to sense or anti-sense RNA. This does not necessarily require a direct link in the chemical sense.
  • Genetic control sequences such as, for example, enhancer sequences, can also perform their function on the target sequence from more distant positions or even from other DNA molecules.
  • nucleic acid sequence to be expressed transgenically is positioned behind the sequence functioning as a promoter, so that both sequences are covalently linked to one another.
  • the distance between the promoter sequence and the transgene is preferably to be expressed.
  • Nucleic acid sequence less than 200 base pairs, particularly preferably less than 100 base pairs, very particularly preferably less than 50 base pairs.
  • An expression cassette according to the invention is preferably produced, for example, by direct fusion of a nucleic acid sequence acting as a promoter with specificity for the embryonic epidermis and / or the flower with a nucleotide sequence to be expressed.
  • Common recombination and cloning techniques such as those described in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W.
  • the expression cassette consisting of a linkage of promoter and nucleic acid sequence to be expressed, can preferably be integrated in a vector according to the invention and inserted into a plant genome by, for example, transformation.
  • An expression cassette is also to be understood as such constructions in which the promoter, without having been functionally linked to a nucleic acid sequence to be expressed, is introduced, for example via targeted homologous recombination or a random insertion into a host genome, there regulatory control over then takes over functionally linked nucleic acid sequences and controls their transgenic expression.
  • Vectors according to the invention also contain the expression cassettes described above.
  • nucleic acid sequences contained in the expression cassettes or vectors according to the invention can be functionally linked to further genetic control sequences in addition to the promoter with specificity for the embryonic epidermis and / or the flower.
  • genetic control sequences is to be understood broadly and means all those sequences which have an influence on the formation or the function of the expression cassette according to the invention. Genetic control sequences modify, for example, transcription and translation in prokaryotic or eukaryotic organisms.
  • the expression cassettes according to the invention preferably comprise 5 'upstream of the respective nucleic acid sequence to be expressed transgenically, the promoter with specificity for the embryonic epidermis and / or the flower and 3' downstream a terminator sequence as an additional genetic control sequence, and, if appropriate, further customary regulatory elements, in each case functionally linked to the nucleic acid sequence to be expressed transgenically.
  • Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties.
  • tissue-specific expression can also depend on certain stress factors.
  • Corresponding elements are, for example, for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991; 266 (26): 17131 -17135) and heat stress (Schöffl F et al., Molecular & General Genetics 217 (2-3 ): 246-53, 1989).
  • promoters can be functionally linked to the nucleic acid sequence to be expressed, which enable expression in other plant tissues or in other organisms, such as E. coli bacteria.
  • all promoters described above can be used as plant promoters.
  • a promoter for example the promoter with specificity for the embryonic epidermis and / or the flower
  • a promoter for example the promoter with specificity for the embryonic epidermis and / or the flower
  • a promoter for example the promoter with specificity for the embryonic epidermis and / or the flower
  • a promoter for example the promoter with specificity for the embryonic epidermis and / or the flower
  • the same nucleic acid sequence is translated by another promoter with another Specificity in another tissue is transcribed to anti-sense RNA and the corresponding protein is downregulated.
  • This can be achieved by means of an expression cassette according to the invention, in that one promoter is positioned in front of the nucleic acid sequence to be expressed transgenically and the other
  • Genetic control sequences also include the 5'-untranslated region, introns or the non-coding 3 'region of genes, preferably the mybll gene from Arabidopsis thaliana. It has been shown that these can play a significant role in regulating gene expression. It has been shown that 5 'untranslated sequences can increase the transient expression of heterologous genes. They can also promote tissue specificity (Rouster J et al., Plant J. 1998, 15: 435-440.). Conversely, the 5 'untranslated region of the opaque-2 gene suppresses expression. Deletion of the corresponding region leads to an increase in gene activity (Lohmer S et al., Plant Cell 1993, 5: 65-73). The nucleic acid sequence given under SEQ ID N0: 1 contains the section of the mybll gene which represents the promoter and the 5 'untranslated region up to the ATG start codon of the MYBll transcription factor.
  • the expression cassette can advantageously one or more so-called “enhancer sequences” functionally linked to the
  • preferred but not restricted sequences are further targeting sequences different from the transit peptide, to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrium, in the endoplasmic reticulum (ER), in the cell nucleus, in oil corpuscles or other compartments; and translation enhancers such as the 5 'leader sequence cn ö tQ ⁇ J 0 tr CD H ö ⁇ J z WJ t ⁇ TJ rn 3 tr «tQ N ö ⁇ ⁇ td ⁇ rt I ⁇ rt H s: ⁇ > _ ⁇ .
  • CD CD O 0 0 et CD CD ⁇ tQ ⁇ ⁇ ( Q 0 0 0 O 0 ⁇ ⁇ 0 X 3 ⁇ ⁇ o 0 cn ⁇ - 0 0 J ⁇ cn rt ⁇ o H CD ⁇ 0 C ⁇ 0 ⁇ rr 0 ⁇ ⁇ TJ o ⁇ - - ⁇ ⁇ ⁇ tQ ⁇ - P>
  • CD 3 CD X 0 0 tr 0 C ⁇ ro ⁇ - ⁇ 0 p. ß cn et P- ⁇ et ⁇ o 0 tQ P ) 0 0 tr 0 tr ⁇ - 0 ⁇ ⁇
  • CD PJ CD ⁇ 0 3. H J ⁇ ⁇ CD ⁇ - rt J ⁇ ⁇ - 0 "-
  • CD CD 0 3 CD 0: cn TJ ⁇ 3 ⁇ ⁇ 3 Z ⁇ ⁇ * - 0 ⁇ ⁇ ⁇ ⁇ - ⁇ rt tö
  • CD CD PJ B TJ ö 0 CD ⁇ - rt ⁇ NN 0 o rt CD tQ ⁇ - 0 3 ⁇ - ⁇ ⁇ - p ) ⁇ 0 ⁇ rt ⁇ ⁇ - L ⁇ tr ⁇ - ⁇ ⁇ - CD ⁇ - ⁇ CD ö ⁇ - rr CD CD tr P> w 0. -. ⁇ tr 0 0 ⁇ 0 et CD CD CD td 0 rt 0 2 ⁇ TJ ⁇ 0 ⁇ ⁇ ⁇ TJ ⁇ - rt TJ X C ⁇ 0 ⁇
  • Selection markers which are resistant to a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456), antibiotics or biocides, preferably herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinotricin etc. to lend.
  • a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456)
  • antibiotics or biocides preferably herbicides, such as, for example, kanamycin, G 418, bleomycin, hygromycin, or phosphinotricin etc. to lend.
  • reporter genes which code for easily quantifiable proteins and which, by means of their own color or enzyme activity, ensure an assessment of the transformation efficiency or of the expression site or time.
  • Reporter proteins Schoenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (l): 29-44) such as "green fluorescence protein” (GFP) (Chui WL et al ., Curr Biol 1996, 6: 325-330; Leffel SM et al., Biotechniques.
  • Replication origins which are an increase in the expression cassettes or vectors according to the invention in, for example
  • E.coli examples include ORI (origin of DNA replication), pBR322 ori or P15A ori (Sa-brook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) ,
  • a selectable marker which gives the successfully recombined cells resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-deoxyglu cose-6-phosphate (WO 98/45456) or an antibiotic.
  • a biocide for example a herbicide
  • a metabolism inhibitor such as 2-deoxyglu cose-6-phosphate (WO 98/45456) or an antibiotic.
  • the selection marker permits the selection of the transformed cells from untransformed ones (McCormick et al., Plant Cell Reports 5 (1986), 81-84).
  • Functional equivalents initially mean DNA sequences which hybridize under standard conditions with the nucleic acid sequence described by SEQ ID NO: 1 or the nucleic acid sequence complementary to it and which have essentially the same promoter activities as the nucleic acid sequence described by SEQ ID N0: 1.
  • Standard hybridization conditions are to be understood broadly and mean stringent as well as less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al. , in Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. described.
  • the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with approximately 2X SSC at 50 ° C.) and those with high stringency (with approximately 0.2X SSC at 50 ° C., preferably at 65 ° C) (20X SSC: 0.3M sodium citrate, 3MNaCl, pH 7.0).
  • the temperature during the washing step can be raised from low stringent conditions at room temperature, about 22 ° C, to more stringent conditions at about 65 ° C. Both parameters, salt concentration and temperature, can be varied simultaneously, one of the two parameters can also be kept constant and only the other can be varied. Denaturing agents such as for example amide or SDS can also be used during the hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C.
  • 6X SSC at 68 ° C, 100 ⁇ g / ml denatured fish sperm DNA, or (iv) 6X SSC, 0.5% SDS, 100 ⁇ g / ml denatured, fragmented salmon sperm DNA at 68 ° C, or
  • nucleic acids in the expression cassettes or from vectors derived therefrom which have the double-stranded DNA sequence described by SEQ ID NO: 1 or fragments thereof, such as, for example, the double-stranded DNA sequences described by SEQ ID NO: 2 and 15 to 19 DNA sequences hybridize under the conditions defined above and have essentially the same promoter activity as.
  • Sequence fragments derived from SEQ ID NO: 1 are particularly preferred, which comprise base pairs 1700 to 2700 of the sequence with SEQ ID NO: 1.
  • a functional equivalent is also understood to mean, in particular, natural or artificial mutations of the mybll promoter described under SEQ ID NO: 1 and its homologs from other plant genera and plant species, which continue to initiate expression in the plant embryonic epidermis and / or flowers can. Mutations include substitutions, additions, deletions, inversions or insertions of one or more nucleotide residues.
  • the present invention also encompasses those nucleotide sequences which are obtained by modification of the mybll promoter nucleotide sequence in accordance with SEQ ID NO: 1.
  • the aim of such a modification can be, for example, the further limitation of the sequence contained therein to certain regulatory elements or, for example, the insertion of further restriction enzyme interfaces, the removal of superfluous DNA or the addition of further sequences, for example further regulatory sequences.
  • the sequence can also be limited to specific, essential regulatory regions with the aid of a search routine for searching promoter elements. Certain promoter elements are often present in abundance in the regions relevant to promoter activity. This analysis can be carried out, for example, using computer programs such as the PLACE program ("Plant Cis-acting Regulatory DNA Elements") (K.Higo et al., (1999) Nuclear Acids Research 27: 1, 297-300).
  • insertions, deletions or substitutions e.g. Transitions and transversions
  • techniques known per se such as in vitro mutagenesis, primer repair, restriction or ligation, can be used.
  • PCR polymerase chain reaction
  • GAP World Health Organization
  • Gap Weight 12 Length Weight: 4
  • Average Match 2,912 Average Mismatch: -2, 003
  • Functional equivalents derived from one of the promoter sequences used in the expression cassettes or vectors according to the invention, for example by substitution, insertion or deletion of nucleotides, have a homology of at least 20%, preferably 40%, preferably at least 60%, particularly preferably at least 80%, very particularly preferably at least 90%, and are distinguished by essentially the same transcription activity compared to the mybll promoter sequence according to SEQ-ID NO: 1.
  • promoter sequences used in the expression cassettes or expression vectors according to the invention can be found, for example, from various organisms whose genomic sequence is known, for example from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Helianthinum, by comparing homology easy to find from databases.
  • Functional equivalents also include those promoter variants whose function is weakened or enhanced compared to the starting promoter.
  • Suitable functionally equivalent expression cassettes are sequences which express a fusion protein under the control of the promoter sequence, one component of the fusion protein the nucleic acid sequence to be expressed, the other a regulatory protein sequence, e.g. is a signal or transit peptide that directs the gene product to the desired site of action.
  • the specificity of the expression constructs and vectors according to the invention for the plant, embryonic epidermis and the flower is particularly advantageous.
  • the plant epidermis represents both the primary barrier and the essential contact surface of the plant organism with its environment. It has an important function in protecting the plant against biotic and abiotic stress factors. Biotic stress factors can be pathogens, bite from animals etc., abiotic stress can be pathogens, bite from animals etc., abiotic stress can be pathogens, bite from animals etc.
  • the epidermis also has a function in attracting beneficial insects through pigment storage or the synthesis of volatile chemicals, it provides mechanical stability and prevents water loss.
  • the epidermis also has an essential role in the absorption of water, gases and nutrients in the semen embryo.
  • the plant's natural defense mechanisms for example against pathogens, are often inadequate.
  • the introduction of foreign genes from plants, animals or microbial sources can strengthen the immune system. Examples are protection against insect damage in tobacco by expression of the Bacillus thuringiensis endotoxin under the control of the 35 S CaMV promoter (Vaeck et al., Nature 1987, 328, 33-37) or protection of the tobacco against fungal attack by expression of a chitinase the bean under the control of the CaMV promoter (Broglie et al., Science 1991, 254, 1194-1197).
  • concentrated expression of the corresponding nucleic acid sequence to be expressed transgenically is particularly advantageous in the outermost envelope of the plant, the epidermis, or the flower.
  • Constituent expression in the entire plant can question the effect, for example by diluting it, or impair the growth of the plant or the quality of the plant product.
  • constitutive expression can lead to an increased shutdown of the transgene ("gene silencing").
  • promoters with specificity for the embryonic epidermis and / or the blossom are advantageous.
  • Nucleic acids which are particularly preferred are those for the chalcone synthase from Arabidopsis thaliana (GenBank Acc.-No .: M20308), the 6-4 photolyase from Arabidopsis
  • Nucleic acids which code for the cationic amino acid transporter from Arabidopsis thaliana (GenBank Acc.-No .: X92657) or for the monosaccharide transporter from Arabidopsis thaliana (GenBank Acc.-No .: AJ002399) or functional equivalents of the same.
  • neutraceuticals such as polyunsaturated fatty acids such as arachidonic acid or EP (eicosapentaenoic acid) or DHA (docosahexaenoic acid) by expression of fatty acid elongases and / or desaturases or production of proteins with improved nutritional value such as a high proportion of essential amino acids (eg the methionine-rich 2S albumingens of the Brazil nut).
  • Preferred nucleic acids are those for the methionine-rich 2S albumin from Bertholletia excelsa (GenBank Acc.
  • Modification of the fiber content in seed-based foods by, for example, expression of antisense transcripts of the caffeine acid-O-methyltransferase or the cinnamoyl alcohol-containing hydrogenase gene.
  • Functional equivalents here - analogous to the definition of the functional equivalents of the promoter with specificity for the embryonic epidermis and / or the flower - mean all the sequences which have essentially the same function, i.e. are capable of the function (for example a substrate conversion or a signal transduction) as well as the protein mentioned by way of example.
  • the functional equivalent may well differ in other features. For example, it may have a higher or lower activity, or it may have other functionalities.
  • Functional equivalents also means sequences which code for fusion proteins consisting of one of the preferred proteins and other proteins, for example another preferred protein or else a signal peptide sequence.
  • the person skilled in the art is also aware that he does not have to express the genes described above directly using the nucleic acid sequences coding for these genes or to repress them, for example, by antisense.
  • he can also use artificial transcription factors of the zinc finger protein type (Beerli RR et al., Proc Natl Acad Sei USA 2000; 97 (4): 1495-500). These factors are reflected in the regulatory areas of endogenous genes to be expressed or repressed and, depending on the design of the factor, cause expression or repression of the endogenous gene.
  • Another object of the invention relates to transgenic organisms, transformed with at least one expression cassette according to the invention or a vector according to the invention, as well as cells, cell cultures, tissues, parts - such as leaves, roots etc. in plant organisms - or well reproduced from such organisms.
  • Organism starting or host organisms are understood to mean prokaryotic or eukaryotic organisms, such as, for example, microorganisms or plant organisms.
  • Preferred microorganisms are bacteria, yeast, algae or fungi.
  • Preferred bacteria are bacteria of the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes or cyanobacteria, for example of the genus Synechocystis.
  • microorganisms which are capable of infecting plants and thus of transmitting the constructs according to the invention.
  • Preferred microorganisms are those from the genus Agrobacterium and in particular from the species Agrobacterium tumefaciens.
  • Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.
  • Preferred mushrooms are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or others in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995) on page 15, Table 6 described mushrooms.
  • Plants are primarily host or parent organisms preferred as transgenic organisms. Included in the scope of the invention are all genera and species of higher and lower plants in the plant kingdom. Also included are the mature plants, seeds, sprouts and seedlings, as well as parts, propagation material and cultures derived therefrom, for example cell cultures. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.
  • Liverwort Liverwort and musci (mosses); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms such as conifers, cycads, ginkgo and gnetals, - algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae
  • Plants within the scope of the invention include, by way of example and not limitation, the families of the Rosaceae such as rose, Ericaceae such as rhododendrons and azaleas, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as carnations, Solanaceae such as petunias, Gesneriaceae such as the African violet and Balsamineaaceae such as Balsamineaaceae and Balsamineaaceae , Iridaceae like gladiolus, iris, freesia and crocus, Compositae like marigold, Geraniaceae like geraniums, Liliaceae like the dragon tree, Moraceae like Ficus, Araceae like Philodendron and others.
  • Rosaceae such as rose, Ericaceae such as rhododendrons and azaleas
  • Euphorbiaceae such as poinsettias
  • leguminosae such as pea, alfalfa and soy are to be mentioned as examples but not restrictive for flowering plants; Gramineae such as rice, corn, wheat; Solanaceae such as tobacco and others; the Umbelliferae family, especially the genus Daucus (especially the species carota (carrot)) and Apium (especially the species graveolens dulce (selaria)) and others; the family of Solanacea, especially the genus Lycopersicon, especially the species esculentum (tomato) and the genus Solanum, especially the species tuberosum (potato) and melongena (eggplant) and others; and the genus Capsicum, especially the species annum (pepper) and others, - the family of the Leguminosae, especially the genus Glycine, especially the species max (soybean) and others; and the Cruciferae family, especially the Brassica genus, especially the campestris
  • the transgenic plants according to the invention are selected in particular from monocotyledonous crop plants, such as, for example, cereals such as wheat, barley, millet, rye, triticale, maize, rice or oats, and sugar cane.
  • the transgenic plants according to the invention are selected in particular from dicotyledonous crop plants, such as, for example Brassicacae such as rapeseed, cress, arabidopsis, cabbage or canola, leguminosae such as soy, alfalfa, pea, alfalfa, bean family or peanut
  • Solanaceae such as potato, tobacco, tomato, eggplant or paprika
  • Asteraceae such as sunflower, tagetes, lettuce or calendula
  • Cucurbitaceae such as melon, pumpkin or zucchini, as well as linen, cotton, hemp, flax, red pepper, carrot, carrot, sugar beet and the various Tree, nut and wine species.
  • oilseed rape for example rape
  • Types of nuts soy, sunflower, pumpkin and peanut.
  • Plant organisms in the sense of the invention are further photosynthetically active capable organisms, such as algae or cyanobacteria, and mosses.
  • Preferred algae are green algae, such as algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella.
  • the production of a transformed organism or cell requires that the corresponding DNA be introduced into the corresponding host cell.
  • a large number of methods are available for this process, which is referred to as transformation (see also Keown et al. 1990 Methods in Enzymology 185: 527-537).
  • the DNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles.
  • the cell can also be chemically permeabilized, for example with polyethylene glycol, so that the DNA can get into the cell by diffusion.
  • the DNA can also be obtained by protoplast fusion with other DNA-containing units such as minicells, cells, lysosomes or liposomes.
  • Electroporation is another suitable method for introducing DNA, in which the cells are reversibly permeabilized by an electrical impulse.
  • Suitable methods are, above all, the protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun, the so-called particle bombardment method, electroporation, incubation of dry embryos in DNA-containing solution and microinjection.
  • a transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes. These strains contain a plasmid (Ti or Ri plasmid) which is transferred to the plant after agrobaterium infection. Part of this plasmid, called T-DNA (transferred DNA), is integrated into the genome of the plant cell.
  • Ti or Ri plasmid plasmid
  • T-DNA transferred DNA
  • the Agrobacterium -mediated transformation is best suited for dicotyledonous, diploid plant cells, whereas the direct transformation techniques are suitable for every cell type.
  • Vectors can be, for example, plasmids, cosmids, phages, viruses or even agrobacteria.
  • the expression cassette is introduced by means of plasmid vectors.
  • Preferred vectors are those which enable stable integration of the expression cassette into the host genome.
  • Transformation techniques are described for various monocot and dicotyledonous plant organisms.
  • Various possible plasmid vectors are also available for the introduction of foreign genes into plants, which generally contain an origin of replication for reproduction in E. coli and a marker gene for selection of transformed bacteria. Examples are pBR322, pUC series, M13mp series, pACYC184 etc.
  • Those expressing such a marker gene are able to survive in the presence of concentrations of an appropriate antibiotic or herbicide that kill an untransformed wild type.
  • Examples are the bar gene that confers resistance to the herbicide phosphinotricin (Rathore KS et al., Plant Mol Biol.
  • nptll gene which confers resistance to kanamycin
  • hpt gene which confers resistance to hygro ycin
  • EPSP gene which confers resistance to the herbicide glyphosate.
  • a Ti or Ri plasmid is to be used for transformation, at least the right boundary, but mostly the right and left boundary of the Ti or Ri plasmid T-DNA as the flanking region, is connected to the expression cassette to be inserted.
  • Binary vectors are preferably used.
  • ren can replicate in both E.coli and Agrobacterium. They usually contain a selection marker gene and a linker or polylinker flanked by the right and left T-DNA restriction sequences. They can be transformed directly into Agrobacterium (Holsters et al., Mol. Gen. Genet. 163 (1978),
  • the selection marker gene allows selection of transformed agrobacteria and is, for example, the nptll gene which confers resistance to kanamycin.
  • the Agrobacterium which acts as the host organism in this case, should already contain a plasmid with the vir region. This is for the transfer
  • T-DNA 40 g of T-DNA required on the plant cell.
  • An Agrobacterium transformed in this way can be used to transform plant cells.
  • T-DNA for the transformation of plant cells 45 has been intensively investigated and described (EP 120516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters BV, Alblasserdam, Chapter V; Fraley et al., Crit. Rev. Plant. Be., 4: 1-46 and An et al. , EMBO J. 4 (1985) 277-287).
  • Various binary vectors are known and some are commercially available, for example pBIN19 (Clontech Laboratories, Inc. USA).
  • plant explants are co-cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Agrobacterium tumefaciens or Agrobacterium rhizogenes Starting from infected plant material (e.g. leaf, root or stem parts, but also protoplasts or suspensions of plant cells), whole plants can be regenerated using a suitable medium, which can contain, for example, antibiotics or biocides for the selection of transformed cells.
  • the plants obtained can then be screened for the presence of the introduced DNA, here the expression cassette according to the invention.
  • the corresponding genotype is generally stable and the corresponding insertion is also found in the subsequent generations.
  • the integrated expression cassette contains a selection marker which gives the transformed plant resistance to a biocide (for example a herbicide) or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc.
  • the selection marker allows the selection of transformed cells from untransformed (McCormick et al., Plant Cell Reports 5 (1986), 81-84). The plants obtained can be grown and crossed in a conventional manner. Two or more generations should be cultivated to ensure that genomic integration is stable and inheritable.
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711).
  • a whole plant can be obtained using methods known to those skilled in the art. This is based on the example of callus cultures. The formation of shoots and roots can be induced in a known manner from these still undifferentiated cell masses. The sprouts obtained can be planted out and grown. The effectiveness of the expression of the transgenically expressed nucleic acids can be determined, for example, in vitro by sprout meristem propagation using one of the selection methods described above.
  • transgenic organisms described above cell cultures, parts - such as roots, leaves, etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits.
  • Genetically modified plants according to the invention which can be consumed by humans and animals can also be used as food or feed, for example directly or after preparation known per se.
  • Another object of the invention relates to the use of the transgenic organisms according to the invention described above and the cells, cell cultures, parts derived therefrom - such as roots, leaves etc. in transgenic plant organisms - and transgenic propagation material such as seeds or fruits for the production of food or feed, pharmaceuticals or fine chemicals.
  • This process is widely applicable to fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavors, aromas and colors.
  • the production of tocopherols and tocotrienols and carotenoids is particularly preferred.
  • the transformed host organisms and the isolation from the host organisms or from the growth medium are cultivated using methods known to those skilled in the art.
  • SEQ ID NO: 1 promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene.
  • SEQ ID NO: 2 2035bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene.
  • SEQ ID NO: 3 oligonucleotide primers, oligo (dT) primers
  • SEQ ID NO: 7 oligonucleotide primers
  • SEQ ID NO: 11 oligonucleotide primer, myBLI forward primer
  • SEQ ID NO: 12 oligonucleotide primers, myBLI reverse primer
  • SEQ ID NO: 14 oligonucleotide primers
  • SEQ ID NO: 15 888bp fragment of promoter and 5 'untranslated region of the Arabidopsis thaliana mybll gene.
  • Fig. 1 (I and II) show a section through a seed and thus through the plant embryo (Arabidopsis thaliana). "in” denotes the integument, "h” the hypocotyl, “c” the cotyledon and “r” the root.
  • Fig.l (I) shows the section without clarifying the expression region.
  • Fig.l (II) illustrates the expression region of the mybll promoter, which runs along the white, dashed line (marked by the white arrow). This region corresponds to the embryonic epidermis.
  • Fig. 3 (I and II) show a section through the ovary systems of Arabidopsis thaliana. "al” denotes the pollen sac
  • Fig. 3 (I) shows the section without clarifying the expression region.
  • Fig. 3 (II) illustrates the expression region of the mybll promoter, which can be found in the white hatched regions (marked by white arrows). These regions correspond to the egg facilities.
  • Fig. 4 (I and II) show a section through the plant flowers (Arabidopsis thaliana). "im” denotes the flower meristem (tunica). The numbers 2 and 6 describe flowers in the flower development stage 2 or 6 (Arabidopsis Atlas of Morphology, Bowman J ed ' ., Springer Verlag, New York, USA, 1995).
  • Fig. 4 (II) shows the section without clarifying the expression region.
  • Fig. 4 (I) illustrates the express sion region of the mybll promoter, which can be found in the white hatched regions. These regions correspond to the flower meristem and the flower tip (development stage 2) or the stamen plant and the petal primordia (development stage 6).
  • FIG. 6 The result of an RT-PCR is shown using different tissues to determine the expression pattern of the mybll promoter. Expression can be demonstrated in the seedling (day 4), the flower bud, flowering and withering flower, and the green pods.
  • Fig. 7 Schematic representation of the vectors pCR-TOPO-mybll (I), pGPTV-ybll:: GUS (II), pGPTVl-mybll :: GUS (III) and pGPTV2-mybll:: GUS (IV).
  • the direction of the arrow indicates the orientation of the promoter, with the arrow pointing in the direction of the start codon.
  • the abbreviations have the following meaning: mybll: promoter or promoter fragment of the mybll promoter NOS-T: terminator sequence of nopaline synthase (NOS)
  • NOS-T terminator sequence of nopaline synthase (NOS)
  • GUS reporter gene (bacterial ⁇ -glucuronidase)
  • Fig. 8 Schematic representation of the vectors pGPTV3-mybll :: GUS (V), pGPTV4-mybll:: GUS (VI), pGPTV5-mybll :: GUS
  • Fig. 9 Schematic representation of the vectors pBSK-mybll (IX) and pBM-ybll (X).
  • the direction of the arrow indicates the orientation of the promoter, with the arrow pointing in the direction of the start codon.
  • the abbreviations have the following meaning: mybll: promoter or promoter fragment of the mybll promoter Examples
  • oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
  • the cloning steps carried out in the context of the present invention such as e.g. Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA are carried out as in Sambrook et al , (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6.
  • the sequencing of recombinant DNA molecules takes place with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).
  • Example 1 Plant growth conditions for tissue-specific RT-PCR analysis
  • the seedlings are grown in a 16-hour light / 8-hour dark cycle (Philips 58W / 33 white light lamps) at 22 ° C and harvested 4 or 7 days after the start of the germination phase.
  • Roots 100 seeds for the extraction of Roots sterilized as described above, incubated for 4 days at 4 ° C and then see in 250ml bottles with MS medium (Sigma M5519) with the addition of a further 3% sucrose and 0.5 g / 1 MES (Sigma M8652), pH 5.7.
  • the seedlings are grown in a 16-hour light / 8-hour dark cycle (Philips 58W / 33 white light lamps) at 22 ° C, 120 rpm and harvested after 3 weeks.
  • the seeds are sown on uniform soil (type VM, Manna-Italia, Via S.
  • RNA is isolated from the organs of the plant described in Example 1 at various times of development as described (Prescott A, Martin C Plant Mol Biol Rep 1987, - 4: 219-224).
  • the reverse transcriptase polymerase chain reaction (RT-PCR) is used to detect the Atmybll gene transcript. All RNA samples are treated with DNasel (15 units, Boehringer, Mannheim) before the cDNA synthesis.
  • the first strand cDNA synthesis is carried out starting from 6 ⁇ g total RNA with an oligo (dT) primer and RT Superscript TM II enzyme (300 U ⁇ its) according to the manufacturer in a total volume of 20 ⁇ l (Life Technologies, Gaithersburg, MD).
  • 150 ng oligo- (dT.) are added to the RNA in a final volume of 12 ⁇ l.
  • the mixture is heated at 70 ° C. for 10 minutes and then immediately cooled on ice.
  • 4 ⁇ l of the 5X first strand buffer, 2 ⁇ l of 0. IM DTT, 1 ⁇ l of 10 mM dNTP mix (in each case 10 mM dATP, dCTP, dGTP and dTTP) and RNase inhibitor (5 units, Bschreibinger Mannheim) are added.
  • the mixture is heated to 42 ° C. for 2 minutes, RT 'Superscript TM II enzyme (300 units, Life Technologies) is added and incubated at 42 ° C. for 50 minutes.
  • the first strand cDNA is used as a template for the PCR.
  • the following primers are used for the PCR:
  • Forward primer Z17F2 5 '-GCCAATACCGTCGAGAATGCGCC-3' (SEQ ID NO: 8)
  • Reverse primer Z17R3 5 '-TCGTCAATATCCAACGGTTCTCC-3' (SEQ ID NO: 9)
  • the PCR reaction approach is composed as follows:
  • each of dATP, dCTP, dGTP and dTTP 0.5 ⁇ M of each of the oligonucleotide primers Z127F2 and Z17F3 (SEQ ID No.: 8 and 9) 2.5 units AmpliTaq (Perkin Elmer) dest. Water up to a final volume of 50 ⁇ l.
  • PCR products are separated using 2% w / v agarose gel electrophoresis, blotted onto a Hybond N + nylon membrane (Amersham, England) and hybridized with fluorescein-labeled probes.
  • the production of the probes and the detection reaction is carried out as specified in the manufacturer's protocol of the Amersham company.
  • the standardization of the method is verified using primers specifically for the Arabidopsis ACT1 gene which codes for actin (An YQ et al. Plant Cell 1996, 8: 15-30). The following primers are used for the mRNA detection of At-MYBll:
  • Forward primer Z17F2 5 '-GCCAATACCGTCGAGAATGCGCC-3' (SEQ ID NO: 8)
  • Reverse primer Z17R3 5 '-TCGTCAATATCCAACGGTTCTCC-3' (SEQ ID NO: 9)
  • the template for the in-si tu hybridization sample is prepared as described in the instructions for the Lig'nScribe TM PCR Promoter Addition Kit (Ambion, USA). For this purpose, 20 ng of the PCR fragment, which is obtained with the primers Z17F2 and Z17R3, starting from a partial cDNA clone of the AtMYBll gene (EMBL AF062863; Kranz et al., 1998 (see above), against a reading direction behind a T7 promoter adapter cloned (Ambion, USA)
  • Ligation reaction contains 1 ⁇ l of the 10X ligation buffer, 1 ⁇ l of the promoter adapter, 1 ⁇ l of T4 DNA ligase and distilled water in a total volume of 10 ⁇ l.
  • the ligation reaction is incubated for 15 min at room temperature.
  • the template for the antisense strand of the AtMYBll probe is carried out by means of PCR using part of the ligation reaction and the primers Z17F2 (SEQ ID NO: 8) and Ambion PCR Primer 1. For this, 2 ⁇ l of the ligation reaction and the primers Z17F2 and the PCR adapter primer 1
  • PCR adapter primer 1 5 '-GCTTCCGGCTCGTATGTTGTGTGG-3' (SEQ ID NO: 10)
  • the primers Z17R3 and PCR adapter primer 1 are used to produce the template for the sense strand of the AtMYBll probe.
  • the PCR conditions for producing the templates for the antisense and sense strands of the AtMYBll probes are identical and are carried out under the conditions described in Example 2. In deviation from this, 35 cycles are carried out and only a concentration of 0.25 mM of the primers used is used.
  • Sense and antisense DIG-11-UTP labeled RNA probes are synthesized using T7 RNA polymerase with reagents from Röche Diagnostics: The following components are combined in one RNase-free Eppendorf tube, which is kept on ice, mixed in the order shown. The final concentration in a reaction volume of 20 ⁇ l is given.
  • lx labeling mixture (as lOx Mix with 10MM ATP, 10MM CTP, 10MM GTP; 6.5mM UTP, 3.5mM DIG-UTP in Tris-HCl, pH 7.5), lx transcription buffer (as lOx buffer with 400mM Tris-HCl, pH 8.0, 60mM MgCl 2 / lOOmM dithioerythritol, 20mM spermidine, lOOmM NaCl, 1 unit / ml RNase inhibitor) and 40 5 units T7 RNA polymerase.
  • the mixture is shaken briefly and centrifuged for a few seconds.
  • the mixture is then incubated at 37 2 C for at least 2 hours.
  • 20 units of DNase are added and incubated at 37 S C for 15 min. 10 2 ⁇ l of the supplied EDTA solution are added to terminate the reaction.
  • the supports are then washed for 5 min in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl 2 and for 48 hours in a solution of 0.34 mg / ml nitro blue tetrazolium salt and 0.175 mg / ml 5-bromo Incubate -4-chloro-3-indolylphosphate toluidinium salt in 100mM Tris-HCl (pH 9.5), 100mM NaCl and 50mM MgCl 2 .
  • the color reaction is stopped with 10 mM Tris-HCl (pH 8), ImM EDTA and the sections are washed with gentle shaking in 95% ethanol, then with an ethanol dilution series and finally with sterile water before they are fixed with Euparal (BDH).
  • BDH Euparal
  • genomic 'DNA from Arabidopsis thaliana (ecotype Columbia) is extracted as described (Galbiati M et al Funct Integr Genomics 2000, 1:.. 25-34).
  • the isolated DNA is used as template DNA in a PCR using the following primers:
  • the PCR product is cloned into the vector pCR4-T0P0 (Invitrogen) according to the manufacturer's instructions, ie the PCR product obtained is inserted into a vector with T overhangs using its A overhangs and a topoisomerase.
  • the construct obtained is 5 pCR4-T0P0-mybll (Fig. 7, I).
  • a fine mapping of the mybll promoter i.e. The 15 nucleic acid sections relevant to its specificity are narrowed by producing different reporter gene expression vectors which contain the entire promoter region on the one hand and various fragments thereof on the other hand.
  • the entire promoter region or fragments thereof are cloned into the pGPTV-GUS-Kan 0 vector (Becker D et al., 1992 Plant Mol Biol 20: 1195-1197).
  • fragments are used for this, which are obtained by using restriction enzymes for the internal restriction sites in the full-length promoter sequence.
  • PCR fragments are used which are provided with interfaces introduced by primers.
  • pCR4-T0P0-mybll is digested with EcoRI and the 5 ca.2050 base pairs fragment isolated from a 1% agarose gel is subcloned into pBluescript SK (Stratagene).
  • the insert is obtained from the resulting vector pBSK-ybll (FIG. 9, construct IX) by digestion with HindII and Smal, isolated over a 1% agarose gel and cloned into pGPTV-GUS-Kan.
  • the construct obtained bears the designation 0 pGPTVl-mybll :: GUS (FIG. 7, construct III).
  • pBSK-mybll is digested with EcoRV and Ncol. Overhanging ends are digested with T4 DNA polymerase and the plasmid is religated with blunt ends (as described in Sambrook et al., 5, Molecular Cloning. A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press, 1989) to pBM-ybll ( Fig. 9, construct X).
  • the Ver- The shortened insert is obtained from the vector pBM-mybll by digestion with Hinllll and Smal, isolated using a 1% agarose gel and cloned into pGPTV-GUS-Kan. The construct obtained is called pGPTV2-mybll :: GUS (Fig. 7, construct IV).
  • pCR4-T0P0-mybll is used as a template for a PCR.
  • the Hindlll and Smal interfaces are introduced using the following primers
  • Reverse primer 5 '-CCCGGGAAAATCACTCACTTCACT-3' (SEQ ID NO: 12).
  • the fragment is isolated from a 1.5% agarose gel and again cloned in pGPTV-GUS-Kan.
  • the construct obtained is called pGPTV3-mybll :: GUS (Fig. 8, construct V).
  • pCR4-T0P0-mybll is used as a template for a PCR.
  • the Hindlll and Sall interfaces are introduced using the following primers
  • pCR4-T0P0-mybll is used as a template for a PCR.
  • the Hindlll and Xbal interfaces are introduced using the following primers
  • the fragment is isolated from a 1.5% agarose gel and again cloned in pGPTV-GUS-Kan.
  • the construct obtained is called pGPTV5-mybll:: GUS-Kan (Fig. 8, construct VII).
  • the fragment is isolated from a 1.5% agarose gel and again cloned in pGPTV-GUS-Kan.
  • the construct obtained is called pGPTV6-mybll:: GUS-Kan (Fig. 8, construct VIII).
  • i-grrojbacterium tumefaciens (strain C58C1 pGV2260) is transformed with various mybll promoter-GUS vector constructs.
  • the agrobacterial strains are then used to produce transgenic plants.
  • a single transformed agrobacterium colony in a 4 ml culture (medium: YEB medium with 50 ⁇ g / ml
  • Kanamycin and 25 ⁇ g / ml rifampicin incubated overnight at 28 ° C.
  • a 400 ml culture is then inoculated with this culture in the same medium, incubated overnight (28 ° C., 220 rpm) and centrifuged off (GSA rotor, 8,000 rpm, 20 min).
  • the pellet is resuspended in infiltration medium (.1 / 2 MS medium; 0.5 g / l MES, pH 5.8; 50 g / 1 sucrose).
  • the suspension is placed in a plant box (Duchefa) and 100 ml of SILVET L-77 (heptamethyltrisiloxane modified with polyalkylene oxide; Osi Specialties Inc., Cat.
  • transgenic Arabidopsis plants can be obtained by root transformation.
  • White root sprouts from plants up to 8 weeks old are used.
  • plants are placed under sterile conditions in 1 MS medium (1% sucrose; 100mg / l inositol; l, 0mg / l thiamine; 0.5 mg / 1 pyridoxine; 0.5 mg / 1 - nicotinic acid; 0.5 g MES, pH 5, 7; 0.8% agar) are used.
  • Roots are grown on kailus-inducing medium for 3 days (Ix Gambourg's B5 medium; 2% glucose; 0.5 g / 1
  • the small, green sprouts are placed on germination medium (1 MS medium; 1% sucrose; 100 mg / 1 inositol; 1.0 mg / l thiamine; 0.5 mg / 1 pyridoxine; 0.5 mg / 1 nicotinic acid; 0.5 g MES, pH 5, 7; 0.8% agar) and regenerated into plants.
  • MS medium 1% sucrose; 100 mg / 1 inositol; 1.0 mg / l thiamine; 0.5 mg / 1 pyridoxine; 0.5 mg / 1 nicotinic acid; 0.5 g MES, pH 5, 7; 0.8% agar
  • the bacterial ⁇ -glucuronidase may be mentioned as an example (Jefferson et al., EMBO J. 1987, 6, 3901-3907).
  • the ß-glucuroni-. this activity can be determined in-planta using a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid as part of an activity staining (Jefferson, 1987, Plant Molecular Biology Reporter 5, 387-405).
  • a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid as part of an activity staining (Jefferson, 1987, Plant Molecular Biology Reporter 5, 387-405).
  • the plant tissue is cut, embedded, stained and analyzed as described (e.g. Bäumlein H et al., 1991 Mol Gen Genet 225: 121-128).
  • MUG methylumbelliferyl glucuronide
  • MU methylumbelliferone
  • glucuronic acid glucuronic acid

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Abstract

La présente invention concerne une cassette d'expression permettant l'expression transgénique d'acides nucléiques dans l'épiderme embryonnaire et/ou dans la fleur. Ladite cassette comprend le promoteur du gène myb11 issu de Arabidopsis thaliana ou équivalents fonctionnels ou fragments équivalents qui possèdent sensiblement les mêmes activités de promoteur, le promoteur étant lié d'un point de vue fonctionnel à une séquence d'acide nucléique à exprimer de manière transgénique. Cette invention concerne également des vecteurs dérivés de ces cassettes d'expression, ainsi que des végétaux génétiquement modifiés à l'aide de ces cassettes ou de ces vecteurs, des cultures, parties ou matières de multiplication transgéniques dérivées desdits végétaux, et l'utilisation de ces éléments pour produire des aliments, des aliments pour animaux, des semences, des produits pharmaceutiques ou des produits chimiques fins.
PCT/EP2001/012444 2000-10-27 2001-10-26 Cassettes d'expression permettant l'expression transgenique d'acides nucleiques dans l'epiderme vegetal embryonnaire et dans la fleur WO2002034924A2 (fr)

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DE2000153519 DE10053519A1 (de) 2000-10-27 2000-10-27 Expressionskassetten zur transgenen Expression von Nukleinsäuren in der pflanzlichen embryonalen Epidermis und der Blüte
DE10053519.4 2000-10-27

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EP2314703A1 (fr) * 2009-10-23 2011-04-27 Friedrich-Alexander-Universität Erlangen-Nürnberg Promoteur de plante spécifique à un tissu pour un endosperme et un embryon d'une graine de plante
CN116286869A (zh) * 2023-03-23 2023-06-23 石河子大学 一种羽毛针禾糖转运蛋白基因SpSWEET14在提高植物抗寒性中的应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2314703A1 (fr) * 2009-10-23 2011-04-27 Friedrich-Alexander-Universität Erlangen-Nürnberg Promoteur de plante spécifique à un tissu pour un endosperme et un embryon d'une graine de plante
CN116286869A (zh) * 2023-03-23 2023-06-23 石河子大学 一种羽毛针禾糖转运蛋白基因SpSWEET14在提高植物抗寒性中的应用
CN116286869B (zh) * 2023-03-23 2024-04-05 石河子大学 一种羽毛针禾糖转运蛋白基因SpSWEET14在提高植物抗寒性中的应用

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