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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—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
  • C12N15/8243—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 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • 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
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

• the present invention relates to a method for producing plants with an increased vitamin E content by expressing an exogenous or endogenous HPPD gene in plants or parts of plants.
• the invention further relates to the use of the corresponding nucleic acids coding for an HPPD gene in transgenic plants in order to make them resistant to inhibitors of HPPD, and to the use of the DNA sequence coding for an HPPD for the production of a test system for the identification of inhibitors the HPPD.
• the eight naturally occurring compounds with vitamin E activity are derivatives of 6-chromanol (Ulimann's Encyclopedia of Industrial Chemistry, Vol. A 27 (1996), VCH
• the first group (la - d) comes from Tocol
• the second group consists of tocotrienol derivatives (2a - d):
• ⁇ -Tocopherol is of great economic importance.
• the tocopherol biosynthesis in plants and algae is known to proceed as follows:
• the precursor of the aromatic ring of the tocopherols is p-hydroxyphenylpyruvate (3), which is converted enzymatically with the help of the enzyme hydroxyphenylpyruvate dioxygenase (HPPD) into homogentisic acid (4), which reacts with phytylpyrophosphate to eliminate CO 2 to the precursor (6) .
• HPPD hydroxyphenylpyruvate dioxygenase
• the tocotrienol biosynthetic pathway starts with the condensation of homogentisic acid (4) with geranyl geranyl pyrophosphate to the precursor (5).
• the enzymatic cyclization of precursors 5 or 6 leads to ⁇ - tocotrienol or to ⁇ - tocopherol.
• PEA3_MOUSE Mus muscula (mouse) PEA3 polypeptide, AC X63190;
• MELA_SHECO Shewanella colwelliana, melA protein, AC M5 289, WO 96/38567 describes the HPPD DNA sequence from Arabidopsis thaliana and Daucus carota.
• HPPD-DNA sequences is an essential prerequisite both for use in crop protection for the production of herbicide-resistant plants and for increasing the vitamin E synthesis in plants - for example for the production of feed with increased vitamin E content .
• the object of the present invention was to develop a transgenic plant with an increased vitamin E content.
• Another object of the present invention was to develop a transgenic plant which is resistant to inhibitors of HPPD.
• An additional object of the present invention was to develop a test system for identifying inhibitors of HPPD.
• This object was achieved by expressing an HPPD gene from barley in a plant or a microorganism and then testing chemicals for inhibiting the HPPD enzyme activity.
• a first object of the present invention relates to the cloning of the complete HPPD gene from barley via the isolation of the cDNA specific for the HPPD gene (HvSD36).
• Fig. 1 shows a schematic drawing of the primary barley leaf on different days after sowing. The determined total length of the leaves can be seen on the scale on the left. The differently differentiated leaf areas of the primary leaf selected for the analysis of the gene expression are drawn in and designated I-IV.
• RNA differentiated areas of the primary leaf of the barley indicate a development-dependent expression of the barley HPPD.
• a strong accumulation of the approx. 1600 nt long transcript takes place in the meristatic area at the base of the primary sheet (I).
• the content of this transcript decreases with increasing age of the tissue (Ha and Ilb) and increases again in the fully differentiated cells with mature chloroplasts (III).
• the content of the 1600 nt long transcript is highest in senescent areas of the primary leaf (IV).
• an approx. 3100 nt long transcript can only be detected in the meristematic cells at the base of the primary leaf. This transcript is also no longer detectable as the tissue matures.
• a 207 bp cDNA fragment was first isolated, the corresponding transcript of which is accumulated in the primary leaf of the barley in the case of dark-induced senescence.
• This fragment (sequence listing: sequence ID NO: 1: nucleotide position 1342-1549) was then used as a probe in order to isolate a cDNA clone with a larger insert from barley senescent flag leaves in a cDNA library (in ⁇ -ZAP-II) .
• the cDNA fragment (sequence listing: sequence ID NO: 1: nucleotide position 771-1529) was cloned into the EcoRI site of pBluescript (SK "). At both ends of the cDNA there is also a 14 bp adapter sequence which is used for ligation in ⁇ -ZAP-II was required, and selected restriction sites of the vector and the cDNA itself are shown.
• the 759 bp cDNA fragment was used as a probe for another attempt to obtain a complete sequence of HvSD 36.
• a cDNA bank from RNA of the meristematic range of 5 day old barley seedlings was available.
• the Lambda Phage ExCell Eco was used for this cDNA bank RICIP from Pharmacia (Freiburg) (product number: 27-5011, 45.5kb) is used.
• the 434 amino acid protein sequence has the highest homology to the sequence of the HPPD from Arabidopsis thaliana among the sequences in the databases with 58%.
• a Lambda FIXII bank of the barley was obtained from the company Stratagene (Heidelberg, product number 946104). DNA from etiolated leaves of winter barley cv was used to produce the bank. Igri. The DNA was partially digested with Sau3AI. Before cloning into the Xhol site of the vector, the fragment ends and the phage arms were filled with nucleotides. Screening the bank for 200,000 pfu in the first round revealed only one clone that hybridized with the cDNA HvSD36.
• FIG. 3 shows the schematic structure of the barley HPPD gene.
• the invention relates in particular to expression cassettes, the sequence of which codes for an HPPD or its functional equivalent, and their use for producing a plant with an increased vitamin E content.
• the nucleic acid sequence can e.g. be a DNA or a cDNA sequence. Coding sequences suitable for insertion into an expression cassette according to the invention are, for example, those which code for HPPD and which give the host the ability to overproduce vitamin E.
• an expression cassette according to the invention also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell.
• an expression cassette according to the invention comprises upstream, ie at the 5 'end of the coding sequence, a promoter and downstream, ie at the 3' end, a polyadenylation signal and, if appropriate, further regulatory elements which are in between coding sequence for the HPPD gene are operatively linked.
• An operational link is understood to mean the sequential arrangement of the promoter, coding sequence, terminator and possibly other regulatory elements such that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended.
• sequences preferred but not limited to the operative linkage are targeting sequences to ensure subcellular localization in the apoplast, in the vacuole, in plastids, in the mitochondrion, in the endoplasmic reticulum (ER), in the cell nucleus, in oil corpuscles or other compartments and Translation enhancers such as the 5 'guiding sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987) 8693-8711).
• the plant expression cassette can be built into the tobacco transformation vector pBinAR-Hyg.
• Fig. 4 shows the tobacco transformation vectors pBinAR-Hyg with 35S promoter (A) or pBinAR-Hyg with seed-specific promoter Phaseolin 796 (B):
• any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette according to the invention.
• a plant promoter or a plant virus-derived promoter is preferably used.
• the CaMV 35S promoter from the cauliflower mosaic virus is particularly preferred (Franck et al., Cell 21 (1980) 285-294).
• this promoter contains different recognition sequences for transcriptional effectors, which in their entirety lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J. 8 (1989) 2195-2202).
• the expression cassette according to the invention can also contain a chemically inducible promoter, by means of which the expression of the exogenous HPPD gene in the plant can be controlled at a specific point in time.
• a chemically inducible promoter as for example the PRPl promoter (Ward et al., Plant. Mol. Biol. 22 (1993), 361-366), a promoter induced by salicylic acid (WO 95/19443), a promoter induced by benzenesufonamide (EP- A 388186), one that can be induced by tetracycline (Gatz et al., (1992) Plant J. 2, 397-404), one that can be induced by abscisic acid (EP-A 335528) or that by ethanol or cyclohexanone.
• inducible (WO 93/21334) promoter can be used inter alia.
• promoters are particularly preferred which ensure expression in tissues or parts of plants in which the biosynthesis of vitamin E or its precursors takes place. Promoters that ensure leaf-specific expression should be mentioned in particular.
• the promoter of the cytosolic FBPase from potatoes or the ST-LSI promoter from potatoes should be mentioned (Stockhaus et al., EMBO J. 8 (1989) 2445-245).
• the expression cassette according to the invention can therefore, for example, be a seed-specific promoter (preferably the phaseolin promoter (US 5504200), the USP- (Baumlein, H. et al. Mol. Gen. Genet. (1991) 225 (3), 459-467) or LEB4 promoter (Fiedler and Conrad, 1995)), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
• a seed-specific promoter preferably the phaseolin promoter (US 5504200), the USP- (Baumlein, H. et al. Mol. Gen. Genet. (1991) 225 (3), 459-467) or LEB4 promoter (Fiedler and Conrad, 1995)
• the structure of such a cassette is shown schematically as an example in Figure 4.
• An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable HPPD-DNA sequence and preferably a DNA inserted between the promoter and HPPD-DNA sequence, which codes for a chloroplast-specific transit peptide, and a polyadenylation signal according to common recombination and cloning techniques, such as for example in T. Maniatis, EF 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. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
• sequences which ensure targeting in the apoplasts, plastids, the vacuole, the mitochondrium, the endoplasmic reticulum (ER) or, due to the lack of corresponding operative sequences, ensuring that the resulting compartment, the cytosol, remains (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423).
• Proven particularly beneficial for the amount of protein accumulation in transgenic plants has a localization in the ER (Schouten et al., Plant Mol. Biol. 30 (1996), 781-792).
• the invention also relates to expression cassettes whose DNA sequence encodes an HPPD fusion protein, part of the fusion protein being a transit peptide which controls the translocation of the polypeptide.
• Particularly preferred for the chloroplasts are specific transit peptides which, after translocation of the HPPD gene into the chloroplasts, are cleaved off enzymatically from the HPPD part.
• Particularly preferred is the transit peptide derived from the plastidic transketolase (TK) or a functional equivalent of this transit peptide (e.g. the transit peptide of the Rubisco small subunit or the ferredoxin NADP oxidoreductase).
• the inserted nucleotide sequence coding for an HPPD can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components.
• synthetic nucleotide sequences with codons are generated which are preferred by plants. These codons preferred by plants can be determined from codons with the highest protein frequency, which are expressed in most interesting plant species.
• various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
• adapters or linkers can be attached to the fragments.
• the promoter and terminator regions according to the invention can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
• the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
• the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges.
• the promoter according to the invention can be both native or homologous and foreign or heterologous to the host plant.
• the expression cassette according to the invention contains the promoter according to the invention, any DNA sequence and a region for the transcriptional termination in the 5 '-3' transcription direction. Different termination areas are interchangeable.
• Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 ff) or functional equivalents.
• An expression cassette according to the invention can, for example, contain a constitutive promoter (preferably the CaMV 35 S promoter), the LeB4 signal peptide, the gene to be expressed and the ER-
• the amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is preferably used as the ER retention signal.
• the fused expression cassette which codes for an HPPD gene is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens.
• Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular crop plants, such as tobacco plants, for example, by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
• the transformation of plants by agrobacteria is known, inter alia, from FF White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press, 1993, pp. 15-38 transformed cells of the wounded leaves or leaf pieces can be regenerated in a known manner transgenic plants which contain a gene integrated into the expression cassette according to the invention for the expression of an HPPD gene.
• an expression cassette according to the invention is inserted as an insert into a recombinant vector whose vector DNA contains additional functional regulation signals, for example sequences for replication or integration.
• Suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology” (CRC Press), Chap. 6/7, pp. 71-119 (1993).
• the expression cassettes according to the invention can be cloned into suitable vectors which enable their multiplication, for example in E. coli.
• suitable cloning vectors include pBR332, pUC series, M13mp series and pACYC184.
• Binary vectors which can replicate both in E. coli and in agrobacteria are particularly suitable.
• the invention further relates to the use of an expression cassette according to the invention for transforming plants, cells, tissues or parts of plants.
• the aim of the use is preferably to increase the vitamin E content of the plant.
• the expression can take place specifically in the leaves, in the seeds or in other parts of the plant.
• Such transgenic plants, their reproductive material and their plant cells, tissue or parts are a further subject of the present invention.
• the expression cassette according to the invention can also be used to transform bacteria, cyanobacteria, yeast, filamentous fungi and algae with the aim of increasing vitamin E production.
• transformation The transfer of foreign genes into the genome of a plant is called transformation.
• the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun - the so-called particle bombardment method, electroporation, and incubation dry embryos in DNA-containing solution, microinjection and gene transfer mediated by agrobae.
• the methods mentioned are described, for example, in B. Jenes et al. , Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press (1993) 128 - 143 and in Potrykus Annu. Rev. Plant Physiol.
• the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agro acterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
• Agrobacteria transformed with an expression cassette according to the invention can also be used in a known manner for transforming plants, in particular crop plants, such as cereals, maize, oats, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomato , Rapeseed, alfalfa, lettuce and the various tree, nut and wine species can be used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
• crop plants such as cereals, maize, oats, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomato , Rapeseed, alfalfa, lettuce and the various tree, nut and wine species can be used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
• Functionally equivalent sequences which code for an HPPD gene are, according to the invention, those sequences which, despite a different nucleotide sequence, still have the desired functions.
• Functional equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. Artificial nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a plant.
• a functional equivalent is also understood to mean, in particular, natural or artificial mutations of an originally isolated sequence coding for an HPPD, which furthermore show the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
• the present invention also encompasses those nucleotide sequences which are obtained by modifying this nucleotide sequence. The aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also be the insertion of further restriction enzyme interfaces.
• amino acids are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.
• artificial DNA sequences are suitable as long as, as described above, they impart the desired property of increasing the vitamin E content in the plant by overexpressing the HPPD gene in crop plants.
• Such artificial DNA sequences can be determined, for example, by back-translating proteins constructed using molecular modeling, which have HPPD activity, or by in vitro selection. Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable. The specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other, known genes of the plant to be transformed.
• Sequences which code for fusion proteins are to be mentioned as further suitable equivalent nucleic acid sequences according to the invention, a component of the fusion protein being a plant HPPD polypeptide or a functionally equivalent part thereof.
• the second part of the fusion protein can e.g. be another polypeptide with enzymatic activity or an antigenic polypeptide sequence that can be used to detect HPPD expression (e.g. myc-tag or his-tag).
• this is preferably a regulatory protein sequence, such as e.g. a signal or transit peptide that directs the HPPD protein to the desired site of action.
• the invention also relates to the expression products and fusion proteins produced according to the invention from a transit peptide and a polypeptide with HPPD activity.
• increasing the vitamin E content means the artificially acquired ability of an increased vitamin E biosynthesis capacity by functional overexpression of the HPPD gene in the plant compared to the non-genetically modified plant for the duration of at least one plant generation.
• the biosythesis site of vitamin E is generally the leaf tissue, so that leaf-specific expression of the HPPD gene makes sense.
• the vitamin E bio-synthesis does not have to be restricted to the leaf tissue, but can also be tissue-specific in all other parts of the plant, for example in fatty seeds.
• constitutive expression of the exogenous HPPD gene is advantageous.
• inducible expression may also appear desirable.
• the effectiveness of the expression of the transgenically experimented HPPD gene can be determined, for example, in vitro by multiplication of the shoot meristem.
• a change in the type and level of expression of the HPPD gene and its effect on the vitamin E biosynthesis performance on test plants can be tested in greenhouse experiments.
• the invention also relates to transgenic plants transformed with an expression cassette according to the invention, and to transgenic cells, tissues, parts and propagation material of such plants.
• Transgenic crop plants such as e.g. Barley, wheat, rye, corn, oats, soy, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rape, alfalfa, lettuce and the various tree, nut and wine species.
• Plants in the sense of the invention are mono- and dicotyledonous plants or algae.
• HPPD is a suitable target for herbicides of the sulcotrione type.
• the complete cDNA sequence of the barley HPPD is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli.
• HPPD protein expressed with the aid of the expression cassette according to the invention is particularly suitable for the detection of inhibitors specific for HPPD.
• the HPPD can be used, for example, in an enzyme test in which the activity of the HPPD is determined in the presence and absence of the active substance to be tested. By comparing the two activity determinations, a qualitative and quantitative statement can be made about the inhibitory behavior of the active substance to be tested.
• test system With the help of the test system according to the invention, a large number of chemical compounds can be checked quickly and easily for herbicidal properties.
• the method makes it possible to selectively reproducibly select those with great potency from a large number of substances in order to use these sub- then perform further in-depth tests that are familiar to the specialist.
• the invention further relates to herbicides which can be identified using the test system described above.
• cloning steps carried out in the context of the present invention such as, for example, 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 were carried out as in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6).
• the bacterial strains used below (E. coli, XL-I Blue) 5 were obtained from Stratagene or Pharmacia in the case of NP66.
• the agrobacterial strain used for plant transformation (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850 can) was developed by Deblaere et al. in (Nucl. Acids Res. 13 (1985) 4777). Alternatively, the agrobact
• the sequencing of recombinant DNA molecules was carried out with a 20 laser fluorescence DNA sequencer from Licor (sales by MWG Biotech, Ebersbach) according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) , 5463-5467).
• nucleotides 11749-11939 was isolated as a PvuII-HindIII fragment and added after addition of Sphl linkers cloned the PvuII interface between the SpHI-HindIII interface of the vector pBmAR-Hyg. It was born
• the reaction mixtures (20 ⁇ l) also contained 20 ⁇ M dNTPs, 10 ⁇ M DTT, 1 ⁇ RT buffer and 1 ⁇ M (dT) 12VN primer each.
• the anchor primers required for these reactions were synthesized on the basis of Liang and Pardee:
• the PCR reaction mixtures each contained 20 ⁇ l of lxPCR buffer, 2 ⁇ M dNTPs, 2.5 ⁇ Ci ( ⁇ 33 P) -dATP, 1 ⁇ M (dT) ⁇ 2 VN “primers”, 1/10 vol. RT mix (Sambrook et al. Molecular Cloning - A Laboratory Manual, 1989), 1 U Taq DNA polymerase (Boehringer, Mannheim) and 1 uM 10-mer random "primer”.
• the PCR reactions were carried out in a Uno block (Biometra) according to the following program:
• Steps 2, 3 and 4 were carried out 40 times in succession. This resulted in approximately 100 cDNA bands per reaction and "primer" combination.
• the amplified cDNA fragments were separated in non-denaturing polyacrylamide gels of the following composition: 6% (w / v) acrylamide (Long Ranger, AT Biochem), 1.2 x TBE buffer, 0.005% (v / v) TEMED and 0.005% (w / v) APS (Bauer et al, Nucl. Ac. Res. (1993) 21, 4272-4280).
• RNA preparations (9 and 9 'and 11 and 11') were made from the primary leaves of the barley harvested on days 9 and 11 and used in parallel in the subsequent analysis.
• the result is shown for two different primer combinations (A and B), two differences in the band pattern between the sample from days 9 and 11 being highlighted by arrows. Only those bands that appeared equally in the two samples from senescent plants and did not appear in the two comparative samples were observed in the later analysis of the gels.
• the electrophoresis was carried out over a period of 2.5 hours at 40 watts (0.8 W / cm 3 ) in 1 x TBE buffer. After separation of the cDNA fragments, the gel was transferred to filter paper (Schleicher & Schüll, Dassel).
• the samples for the RNA analysis were harvested in the middle of the original night phase.
• RNA from flag leaves collected at seven different times in the field (Fig. 6). The leaves were fully grown on May 29 and had less than 10% of the original chlorophyll content on June 21. The beginning of the senescence processes is indicated by an arrow in Figure 6 (i.e. 17 days after reaching its full length on June 15). The day on which photosystem II efficiency decreased (Humbeck et al., Plant Cell Environment (1996) 19: 337-344) was defined as the beginning of senescence.
• FIG. 6 shows the hybridization of "Northern blots" A and B with the cDNA HvSD36 and a probe specific for the rbcS gene.
• Filter A carries RNA from barley primary leaves after 9 days of L / D change (9), after one or two days of dark incubation (10, 11) and then again for one day (12).
• Filter B contains RNA from flag leaves, the 1992 in the period from 29.05. until June 21 were harvested outdoors. The arrow indicates the beginning of the sequence on June 15th. on.
• the amount of r cS-specific RNA is high when the amount of the mRNA specific for the HPPD is relatively small.
• the mRNA specific for HPPD is not detectable in primary leaves of nine-day-old plants before transfer to the dark and clearly accumulates during the dark phase. When the plants are re-exposed, the amount of this mRNA decreases significantly.
• small amounts of the mRNA specific for the HPPD can already be detected in fully grown, non-senescent leaves. An increased expression occurs 4 days before the actual start of senescence. The highest amount of this mRNA is in senescent leaves.
• RNA species A size comparison with known RNA species showed that the transcript detected with the cDNA probe HvSD36 (S: senescence; D: dark, fragment number 36 in the DDRT gel) has a length of approximately 1.6 kb.
• DDRT-PCR independently obtained three cDNA fragments that gave this expression pattern and actually represent the same transcript based on the sequence analysis. The longest fragment was 230 bp in size.
• the 230 bp PCR product was finally cloned with the "Sure Clone TM Ligation Kit" (Pharmacia, Freiburg) according to the manufacturer's instructions into the Smal interface of the vector pUC18.
• the recombinant plasmid was transformed into competent cells of the E. coli strain DH5 ⁇ .
• the sequence information was initially insufficient to find clear homology with a sequence in the databases.
• a lambda ZAPII bank (Stratagene, Heidelberg) from RNA senescent flag leaves was screened using the 230 bp fragment as a probe.
• the probe was labeled with Dig-dUTP according to the "DNA Labeling and Detection Kit” (Boehringer, Mannheim).
• the bank was examined according to the protocol of the "ZAP-cDNA Synthesis Kit” (Stratagene, Heidelberg).
• the insert in question could be cut out of the Bluescript plasmid using EcoRI.
• the cDNA clone obtained in the case of the HvSD36 cDNA contains an "insert" with a size of approx. 800 bp.
• the cDNA was completely sequenced using the "SequiTherm Excel Long-Read DNA Sequencing Kit” (Epicenter Technologies, Biozym Diagnostic, Oldendorf) using universal "primers” labeled with IRD41, which bind to sequence regions in the Bluescript vector.
• the DNA fragments were detected using the infrared laser of the 4000L automatic sequencer from Licor.
• the protein sequence HvSD36 which has a total of 180 amino acids, has the highest homology to the sequence of human HPPD of 41% of the sequences in the databases. In view of the length of the transcript detected in the "Northern blot" (approx. 1600 nt), it can be assumed that the cDNA still lacks 850-900 bp.
• 400,000 pfu were checked with the 759 bp long HvSD36 probe, 5 phages being detected by the probe. Excision of the "phagemids" from the phage was carried out in vivo using the bacterial strain NP66 according to the information from Pharmacia (Freiburg). The recombinant pExCell plasmids were isolated from individual bacterial colonies and transferred to the bacterial strain D115 ⁇ for propagation.
• the longest cDNA clone HvSD36 isolated in this way has a length of 1565 bp and has been completely sequenced (see sequence listing).
• a Lambda FiXII bank of the barley was obtained from the company Stratagene (Heidelberg). DNA from etiolated leaves of winter barley cv was used to produce the bank. Igri. The DNA was partially digested with Sau3AI. Before cloning into the Xhol site of the vector, the fragment ends and the phage arms were filled with nucleotides. Screening the bank for 200,000 pfu in the first round revealed only one clone that hybridized with the cDNA HvSD36.
• the bank was screened according to the instructions given for the HybondN membrane.
• the labeling of the probe for the screening of the bank and for "Southern” blot hybridizations was carried out by "random priming" with 32 P-dATP using the Klenow enzyme (Sambrook et al., (1989) Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory, New York).
• the lengths of the fragments were estimated by comparison with a DNA size standard (Kb ladder from GibcoBRL, Eggenstein).
• HPPD_PSESP PIFEIMGFTK VATHRSKDV- HLYRQGAINL ILN — NE
• PEA3_M0USE PGNGSLGEAL MVPQGKLMDP GSLPPSDSED LFQDLSHFQE TWLAEAQVPD
• HPPD_PSESP IDFV FLEG VDRHPVGA— GLKIIDH LTHNVYRGRM -A YWANF
• HPPD_Ath Arabidopsis thaliana 4-hydroxyphenylpyruvate dioxygenase
• HPPD_HUMAN H. sapiens 4-hydroxyphenylpyruvate dioxygenase
• HPPD_PIG Pig 4-hydroxyphenylpyruvate dioxygenase
• HPPD_RAT Rat F alloantigen
• HPPD_MOUSE Mouse 4-hydroxyphenylpyruvate dioxy- genese
• MELA_SHECO S. colwelliana melA protein
• HPPD_PSESP Pseudomonas sp. (strain P.J.874) 4-hydroxyphenylpyruvate dioxygenase
• PEA3_M0USE Mus musculus (mouse)
• Barley seedlings (Hordeum vulgäre L., cv.Carina, Ackermann Saatzucht, Irbach, Germany) were grown over a period of 15 days under controlled conditions in the climatic chamber in so-called Mitscherlich pots in soil, the 4 g per liter Osmocote 5M (Urania, Hamburg , Germany), attracted. To ensure uniform growth, the seeds were germinated on moist filter paper in the dark for 2 days at 4 ° C and 1 day at 21 ° C and only those seedlings that showed the same length growth of the primary root were used. After transferring these seedlings to earth, they were covered with 1.5 cm of sieved earth.
• the plants were then incubated for 9 days with 16 hours of light (120 ⁇ m-m- 2 -s _1 ) and 8 hours of darkness in conjunction with a temperature chart (21 ° C. during the day, 16 ° C. at night). To induce senescence, the plants are kept in the dark at the above temperature after 9 days for 2 days (days 10 and 11).
• the tobacco plants were grown using a known method.
• the variety of tobacco used is Nicotiana tabacum, cv. Xanthi.
• the expression cassette according to the invention containing the HPPD gene with the sequence 1 was cloned into the vector pBinAR-Hyg
• the HPPD cDNA was provided with a CaMV35S promoter and overexpressed in tobacco using the 35S promoter.
• the seed-specific promoter of the phaseolin gene was used to specifically increase the tocopherol content in the tobacco seed.
• Tobacco plants transformed with the appropriate constructs were grown in the greenhouse.
• the ⁇ -tocopherol content of the whole plant or the seeds of the plant was then determined. In all cases the ⁇ -tocopherol concentration was increased compared to the non-transformed plant.

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