WO1999004021A1

WO1999004021A1 - Dna sequence coding for a hydroxyphenylpyruvate dioxygenase and overproduction thereof in plants

Info

Publication number: WO1999004021A1
Application number: PCT/EP1998/003832
Authority: WO
Inventors: Harald Seulberger; Jens Lerchl; Ralf-Michael Schmidt; Karin Krupinska; Jon Falk
Original assignee: Basf Aktiengesellschaft
Priority date: 1997-07-14
Filing date: 1998-06-23
Publication date: 1999-01-28
Also published as: AU8216998A; ZA986188B; BR9811006A; AR014893A1; CA2296840A1; CN1270636A; EP1009841A1; DE19730066A1

Abstract

The invention relates to a method for producing plants with an increased yield of vitamin E from biosynthesis by overexpression of a plant HPPD gene from barley.

Description

DNA sequence coding for a Hydroxyphenylpyruvatdioxygenase and its overproduction in plants

description

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.

An important goal of plant molecular genetic work is the production of plants with an increased content of sugars, enzymes and amino acids. It would also be economically interesting to develop plants with an increased vitamin content, e.g. the increase in the vitamin E content.

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

Publishing company, Chapter 4., 478-488, Vitamin E). The first group (la - d) comes from Tocol, the second group consists of tocotrienol derivatives (2a - d):

la, α -tocopherol: R ¹ = R ² = R ³ = CH ₃ lb, ß-tocopherol [148 - 03 - 8]: R ¹ = R ³ = CH ₃ , R ² = H lc, γ - tocopherol [54 - 28 - 4]: R ¹ = H, R ² = R ³ = CH ₃ ld, δ - tocopherol [119 - 13 - 1]: R ¹ = R ² = H, R ³ = CH ₃

2a, α-tocotrienol [1721-51-3]: R ¹ = R ² = R ³ = CH ₃ 2b, β-tocotrienol [490-23-3]: R ¹ = R ³ = CH ₃ , R ² = H

2c, γ-tocotrienol [14101-61-2]: R ¹ = H, R ² = R ³ = CH ₃

2d, δ-tocotrienol [25612-59-3]: R ¹ = R ² = H, R ³ = CH ₃

Α-Tocopherol is of great economic importance.

There are limits to the development of crops with increased vitamin E content through tissue culture or seed mutagenesis and natural selection. On the one hand, the vitamin E content must already be detectable in tissue culture and, on the other hand, only those plants can be manipulated using tissue culture techniques that can be regenerated into whole plants from cell cultures. In addition, after mutagenesis and selection, crop plants can show undesirable properties which have to be eliminated by backcrossing, in some cases repeatedly. The increase in the vitamin E content by crossing would also be restricted to plants of the same type.

For these reasons, the genetic engineering procedure to isolate an essential biosynthetic gene coding for the vitamin E synthesis performance and to transfer it specifically in cultivated plants is superior to the classic breeding method. This method assumes that biosynthesis and its regulation are known and that genes that influence biosynthesis performance are identified.

The tocopherol biosynthesis in plants and algae is known to proceed as follows:

4-hydroxyphenylpyruvate ho ogentisic acid (3) (4)

(5) (6)

\, δ-tocotrienol (2d) δ-tocopherol (ld)

! .

ß- or Y "tocotrienol ß- or Y- tocopherol (2b or 2c) (lb or lc)

α-tocotrienol (2a) α-tocopherol (la)

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) . 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 δ - tocotrienol or to δ - tocopherol. Some of these biosynthetic enzymes have been isolated. 5

In the search for Arabidopsis mutants that have defects in carotenoid biosynthesis, a mutant with a white phenotype was identified that cannot form active HPPD. If the mutant designated pds2 is in the presence of homogentisic acid

10 is attracted, it forms like the wild type carotenoids and turns green (Norris et al., Plant Cell (1995) 7: 2139-2149). This investigation shows that the activity of HPPD is a prerequisite for the formation of photosynthetically active chloroplasts. Without this enzyme, no plastoquinones are formed, which are accepted as

15 gates are required for released reduction equivalents during carotenoid biosynthesis (phytodesaturation). The key role of HPPD in plastid metabolism makes it an interesting target for herbicides. Sulcotrione effectively inhibit the activity of the enzyme (Schultz et al., FEBS Lett. (1993)

20 318: 162-166).

Sequences of HPPD-specific genes are already known from the organisms mentioned below:

25

30

5

0 Furthermore, the following sequences with clear homology to HPPD sequences can be found in the databases:

PEA3_MOUSE: Mus muscula (mouse) PEA3 polypeptide, AC X63190;

5 MELA_SHECO: Shewanella colwelliana, melA protein, AC M5 289, WO 96/38567 describes the HPPD DNA sequence from Arabidopsis thaliana and Daucus carota.

Knowledge of the 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.

Surprisingly, both tasks were solved by the overexpression of an HPPD gene in the plants.

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).

During leaf senescence, there is a significant increase in the vitamin E content in the leaves (Rise et al., Plant Physiol. (1989) 89: 1028-1030). The monocotyledon of the barley represents a gradient of cells of different ages, since the leaf has a basal meristem from which new cells gradually separate. Thus, the oldest cells are at the top of the leaf and the youngest at the base. 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. The plants were grown in a daily light / dark change (L / D) or cut off after 6 days to induce senescence and incubated for 2 days in the dark (2 nD). A "Northern blot" analysis using RNA differentiated areas of the primary leaf of the barley (see Fig. 2) 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). Finally, the content of the 1600 nt long transcript is highest in senescent areas of the primary leaf (IV). In addition, 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.

Using the so-called "differential display" method, 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 λ-ZAP-II) .

Schematic representation of the cDNA subclone HvSD 36 from the λ-ZAP-II bank:

T ₇ → XhoI SaU Hindm EcoRI Ncol PstI EcoRI PstI BamHI Xbal * - T ₃

14 bp 759 bp 14 bp

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 λ-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. For this purpose 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.

A 1565 bp long cDNA clone could be isolated, see sequence listing: Sequence ID N0: 1: and 2.

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%.

To find a genomic clone which contains the complete gene sequence of the HPPD, 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. After restriction digestion of this recombinant phage with PstI and Sacl, fragments with sizes of 5400, 3800 and 1800 bp could then be isolated, which can be detected in a "Southern" blot hybridization with the HvSD36 probe. These subfragments are cloned in the bluescript vector. Figure 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.

The expression cassettes according to the invention also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. According to a preferred embodiment, 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. The 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).

For example, 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):

- HPT: hygromycin phosphotransferase - OCS: octopine synthase terminator

- PNOS: nopaline synthase promoter

- In addition, such restriction interfaces are shown that only intersect the vector once.

In principle, any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette according to the invention. In particular, 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). As is known, 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. Such promoters 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.

Furthermore, 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).

With the help of a seed-specific promoter, a foreign protein could be stably expressed up to a proportion of 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio / Technology 10 (1995), 1090-1094). 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. 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).

Particularly preferred are 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. In general, 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. When preparing an expression cassette, 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. To connect the DNA fragments to one another, 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. As a rule, the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites. In general, 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. Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as transitions and trans versions are possible, vifcro mutagenesis, "primer repair", restriction or ligation can be used. With suitable manipulations, such as restriction, "chewing-back" or filling in overhangs for "blunt ends", complementary ends of the fragments can be made available for the ligation.

Of importance for the success according to the invention can i.a. the attachment of the specific ER retention signal SEKDEL (Schouten, A. et al. Plant Mol. Biol. 30 (1996), 781-792), the average expression level is thus tripled to quadrupled. Other retention signals, which occur naturally in plant and animal proteins located in the ER, can also be used to construct the cassette.

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-

Retention signal included. 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.

To transform a host plant with a DNA coding for an HPPD, 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).

Using the recombination and cloning techniques cited above, 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.

Depending on the choice of the promoter, 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.

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. Plant Molec. Biol. 42 (1991) 205-225). 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.

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. Thus, for example, 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.

Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment. In addition, 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). However, 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.

In the context of the present invention, 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. However, it is obvious that 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. In addition, constitutive expression of the exogenous HPPD gene is advantageous. On the other hand, 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. In addition, 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.

As already mentioned, HPPD is a suitable target for herbicides of the sulcotrione type. In order to find even more efficient inhibitors of HPPD, it is necessary to provide suitable test systems with which inhibitor-enzyme binding studies can be carried out. For this purpose, for example, the complete cDNA sequence of the barley HPPD is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli.

The 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.

For this purpose, 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.

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.

Overexpression of the gene sequence coding for an HPPD Seq ID NO: 1 in a plant results in increased resistance to inhibitors of HPPD. The transgenic plants produced in this way are also the subject of the invention.

Further objects of the invention are:

- Process for transforming a plant characterized in that an expression cassette according to the invention is introduced into a plant cell, into callus tissue, an entire plant or protoplasts of plants.

Using a plant to make herbal HPPD.

Use of the expression cassette according to the invention for the production of plants with increased resistance to inhibitors of HPPD by increased expression of a DNA sequence according to the invention.

- Use of the expression cassette according to the invention for the production of plants with an increased vitamin E content by expression of a DNA sequence according to the invention in plants.

- Use of the expression cassette according to the invention for the production of a test system for the identification of inhibitors of HPPD.

The invention is illustrated by the following examples, but is not limited to these:

General cloning procedures

The 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

10 strain LBA4404 (Clontech) or other suitable strains can be used. For cloning, the vectors pUC19 (Yanish-Perron, Gene 33 (1985), 103-119) pBluescript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711-8720) and pBinAR (Höfgen and Will-

15 mitzer, Plant Science 66 (1990) 221-230) can be used.

Sequence analysis of recombinant DNA

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).

Production of plant expression cassettes

25

In the plasmid pBinl9 (Bevan et al., Nucl. Acids Res. (1984) 12, 8711), a 35S CaMV promoter was inserted as an EcoRI-Kpnl fragment corresponding to nucleotides 6909-7437 of the Cauliflower mosaic virus (Franck et al. Cell 21 (1980) 285). The polyad

30 nylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835), 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

35 plasmid pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990) 221-230).

Examples of use

40 Example 1

Isolation of HPPD-specific cDNA sequences

With the help of the DDRT-PCR method published by Liang and Pardee (Science (1992) 257, 967 - 45 972), the composition of the RNA population from primary leaves was changed in nine days in an L / D change (16 h light / 8 h Dark) grown barley plants compared to that of primary leaves of 11-day-old barley plants in which senescence was subsequently induced after two days of dark treatment (Humbeck and Krupinska, J. Photochem. Photobiol. 36 (1996), 321-326). In each case 0.2 μg of the total RNA was converted into cDNA with the enzyme "Superscript RT" (Gibco BRL, Eggenstein). In addition to the RNA, the reaction mixtures (20 μl) also contained 20 μM dNTPs, 10 μM DTT, 1 × RT buffer and 1 μM (dT) 12VN primer each. The anchor primers required for these reactions were synthesized on the basis of Liang and Pardee:

1.5 '-TTTTTTTTTTTTAG-3'

2.5 '-TTTTTTTTTTTTCA-3'

3.5 '-TTTTTTTTTTTTAC-3' 4. 5'-ττTTTTTTTTTTGT-3 '

After synthesis of the cDNAs, the corresponding sequences were amplified in ten batches each, which differ by using the random “primers” given below:

1.5 '-TACAACGAGG-3' 2.5 '-GGAACCAATC-3'

3.5 '-AAACTCCGTC-3' 4.5 '-TGGTAAAGGG-3'

5.5 '-CTGCTTGATG-3' 6.5 '-GTTTTCGCAG-3' 7.5'-GATCTCAGAC-3 '8.5' -GATCTAACCG-3 '

9.5 '-GATCATGGTC-3 '10.5' -GATCTAAGGC-3 '

The PCR reaction mixtures each contained 20 μl of lxPCR buffer, 2 μM dNTPs, 2.5 μCi (α ³³ P) -dATP, 1 μM (dT) ι 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:

1. 94 ° C 2 min

2. 94 ° C 30 s

3. 40 ° C 2 min

4. 72 ° C 30 s

5. 72 ° C 5 min

6. 4 ° C storage until further processing

Steps 2, 3 and 4 were carried out 40 times in succession. This resulted in approximately 100 cDNA bands per reaction and "primer" combination. In a departure from Liang and Pardee's instructions, 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).

Each 3.5 μl of each PCR mixture was mixed with 2 μl sample buffer (dye II, Sambrook et al., 1989) and then applied to the gel. To determine the reproducibility of the cDNA band patterns

(Fig. 5), two independent 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). After the gel had dried at 50 ° C., an X-ray film was applied. cDNA bands that only appeared in samples 11 and 11 'in the autoradiogram were cut out of the dry gel using a scalpel and the DNA was eluted by boiling in 100 μl of 1 × TE buffer. The DNA precipitated with ethanol was resuspended in 10 μl water for the further investigations. After reamplification with the "primers" previously used for this approach, the DNA could be cloned and sequenced and also used as a probe for Norther blot hybridizations.

In order to check whether the corresponding cDNA fragment actually represents a transcription that occurs in a senescence-specific manner, hybridizations were carried out with RNA from leaves of various developmental stages:

A. 1. RNA from primary leaves of 9 days in L / D change grown plants

A. 3. RNA from primary leaves of 10 day old plants, when grown on day 10, the light phase failed

A. 4 RNA from primary leaves of 11-day-old plants that had no light phase on days 10 and 11 A. 5 RNA from primary leaves of 12 day old plants that have experienced a light phase again after 2 days of darkness

The samples for the RNA analysis were harvested in the middle of the original night phase.

B. 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.

To hybridize a filter with the RNA samples described, in addition to the HPPD probe, a specific probe for the r cS gene, which codes for the small subunit of ribulose-1,5-bisphosphate carboxylase, was used for comparison. Figure 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. As can be seen from Figure 6, 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. In the case of the flag leaves, 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. 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 ™ 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 α. Since the fragment, due to its method, represents the 3 'end of the associated transcript, the sequence information was initially insufficient to find clear homology with a sequence in the databases. In order to isolate a longer associated cDNA, a lambda ZAPII bank (Stratagene, Heidelberg) from RNA senescent flag leaves was screened using the 230 bp fragment as a probe. For this step, 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).

In the case of the probe described here, 150,000 pfu was checked. 39 phage plaques gave a positive signal. Of these, 12 phage populations were further processed. After a phage preparation, the inserted fragments could be enriched by PCR and separated electrophoretically. By means of ether blot hybridization with the HvSD36 probe, those were selected from the 12 phage populations treated in this way which had the largest "inserts" with a positive signal. After plating again, the phages were subjected to a further hybridization. Sporadic phage plaques were excised and, after elution using a helper phage, subjected to an in vivo excision according to the Stratagene protocol (Exassist ™ interference-resistant helper phage with SOLR ™ strain). The so-called "phagemids" resulting from this treatment contain the cDNA cloned in the pBLuescript (SK-).

After a subsequent plasmid preparation, 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. After sequencing, there was an exactly 759 bp long sequence flanked on the sides by a 14 bp adapter sequence. These adapter sequences were used in the production of the cDNA bank to ligate the cDNA fragments with the arms of the phage lambda ZAPII (Stratagene, Heidelberg).

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.

Another cDNA bank was examined to complete the cDNA. With the help of "Dynabeads" (Dynal, Hamburg), mRNA was isolated from the basal meristematic area of 5-day-old barley seedlings and overwritten in cDNA with the "Time Saver cDNA Synthesis Kit" (Pharmacia, Freiburg). This was followed by a ligation of EcoRI / Notl adapters (Pharmacia, Freiburg) to the cDNA with subsequent ligation in the Lambda ExCell vector (Pharmacia, Freiburg). Finally, the recombinant phage DNA was packed into phage proteins using the "Gigapack II Gold Set" (Stratagene, Heidelberg). 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 α 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).

Example 2

Characterization of the genomic sequence

To find a genomic clone that contains the gene sequence of the HPPD, 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. To Restriction digestion of this recombinant phage with PstI and SacI then made it possible to isolate fragments with sizes of 5400, 3800 and 1800 bp, which can be detected in a "Southern" blot hybridization with the HvSD36 probe. These subfragments are cloned in the bluescript vector.

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).

A genomic "Southern blot" with total DNA from barley (Carina) was carried out (Fig. 7). 15 μg DNA each were digested with BamHI (B), EcoRI (E), Hindin (H) or XBAI (X) and separated in a 0.75% agarose gel. After transfer to a Hybond N + membrane (Amersham, Braunschweig), hybridization was carried out with the incomplete 759bp long cDNA probe from HvSD36 according to the manufacturer of the membrane. The following fragments could be detected:

BamHI: 6.0.3.9 and 3.0 kbp

EcoRI:> 10kbp Hindlll: 8.3.2.6.1.1 and 1.0 kbp

Xbal: 9.0,5.2 and 4.2kbp

The lengths of the fragments were estimated by comparison with a DNA size standard (Kb ladder from GibcoBRL, Eggenstein).

Example 3

Homology comparison of the protein sequence of HvSD36

A comparison of the protein sequence of HvSD36 with protein sequences in the database revealed homologies to the following previously known protein sequences:

10 20 30 40 50

HPPD_Hv MP PTPTTPAATG

HPPD_Ath MGHQNAA VSENQNHDDG

HPPD_HUMAN

HPPD_RAT

HPPD_PIG

HPPD_MOUSE

HPPD_PSESP

ME A_SHECO

PEA3_M0USE MTKSSNHNCL LRPENKPGLW GPGAQAASLR PSPATLWSS PGHAΞHPPAA 60 70 80 90 100

HPPD_Hv AAAAVTPEHA RPHRMVRFNP RSDRFHTLSF HHVEFWCADA ASAAGRFAFA

HPPD_Ath AASSPGFKLV GFSKFVRKNP KSDKFKVKRF HHIEFWCGDA TNVARRFSWG

HPPD_HUMAN M TTYSDKGAKP ERGRFLH — F HSVTFWVGNA KQAASFYCSK

HPPD_RAT YWDKGPKP ΞRGRFLH — F HSVTFWVGNA KQAASFYCNK

HPPD_PIG M TSYSDKGEKP ERGRFLH — F HSVTFWVGNA KQAASYYCSK

HPPD_MOUSΞ M TTYNNKGPKP ERGRFLH — F HSVTFWVGNA KQAASFYCNK

HPPD_PSESP AD YENP MGLMGFEFIE LASPTPNTLE

MELA_SHECO MASEQNP LGLLGIEFTE FATPDLDFMH

PEA3_M0USE PAQTPGPQVS ASARGPGPVA GGSGRMERRM KGGYL DQ RVPYTFCSKS

110 120 130 140 150

HPPD_Hv LGAPLAARSD LSTGNSAHAS QLLRSGSLAF LFT — APYAN G-CDAA

HPPD_Ath LGMRFSAKSD LSTGNMVHAS YLLTSGDLRF LFT — APYSP S-LSAGEIKP

HPPD_HUMAN MGFEPLAYRG LΞTGSREWS HVIKQGKIVF VLS — SA LNP

HPPD_RAT MGFEPLAYKG LETGSREWS HVIKQGKIVF VLC — SA LNP

HPPD_PIG IGFEPLAYKG LETGSREWS H KQDKIVF VFS — SA LNP

HPPD_MOUSE MGFEPLAYRG LΞTGSREWS HVIKRGKIVF VLC — SA LNP

HPPD_PSESP PIFEIMGFTK VATHRSKDV- HLYRQGAINL ILN — NE

MELA_SHECO KVFIDFGFSK LKKHKQKDI- VYYKQNDINF LLN — NE

PEA3_M0USE PGNGSLGEAL MVPQGKLMDP GSLPPSDSED LFQDLSHFQE TWLAEAQVPD

160 170 180 190 200

HPPD_Hv - ASLPSFS ADAARRFSAD HGIAVRSVAL RVADAAEAFR ASRRRGARPA

HPPD_Ath TTTASIPSFD HGSCRSFFSS HGLGVRAVAI EVEDAESAFS ISVANGAIPS

HPPD_HUMAN WN KEMGDHL-VK HGDGVKDIAF EVEDCDYIVQ KARERGAKIM

HPPD_RAT WN KEMGDHL-VK HGDGVKDIAF EVEDCEHIVQ KARERGAKIV

HPPD_PIG WN KEMGDHL-VK HGDGVKDIAF EVEDCDYIVQ KARERGAIIV

HPPD_MOUSE WN KEMGDHL-VK HGDGVKDIAF EVEDCDHIVQ KARERGAKIV

HPPD_PSΞSP _P HSVASYFAAE HGPSVCGMAF RVKDSQKAYK RALELGAQPI

MELA_SHECO K QGFSAQFAKT HGPAISSMGW RVEDANFAFE GAVARGAKPA

PEA3_M0USE SDEQFVPDFH SENLAFH SPTTRIKKEP QSPRTDPALS CSRKPPLPYH

210 220 230 240 250

HPPD_Hv FAPV DLGRG FAFAEVELYG —D LRFVS HP — DG — TD

HPPD_Ath SPPI VLNEA VTIAEVKLYG — DWLRYVS YKAEDT — EK

HPPD_HUMAN REP -WVEQDKFGK VKFAVLQTYG —DTTHTLVE KMN YI

HPPD_RAT REP -WVEEDKFGK VKFAVLQTYG --DTTHTLVE KIN YT

HPPD_PIG REEVC-CAAD VRGHHTPLDR AR QVWE —GT LVE KMT FC

HPPD_MOUSE REP -WVEQDKFGK VKFAVLQTYG —DTTHTLVE KIN YT

HPPD_PSESP _HI ETG _PME LNLPAIKGIG —GAPLYLID RFGEGSSIYD

MELA_SHECO _{AD EV} - _KD LPYPAIYGIG —DSLIYFID TFGDDNNIYT

PEA3_M0USE HGEQCLYSRQ IAIKSPAPGA PGQSPLQPFS RAEQQQSLLR ASSSSQSHPG

260 270 280 290 300

HPPD_Hv VPFLPGFEGV TNPDA VDYGLTRFDH WGNVP — EL -APAAAYIAG

HPPD_Ath SEFLPGFERV EDASSF P LDYGIRRLDH AVGNVP — EL -GPALTYVAG

HPPD_HUMAN GQFLPGYEAP AFMDPLLPKL PKCSLEMIDH IVGNQPDQEM -VSASEW

HPPD_RAT GRFLPGFEAP TYKDTLLPKL PSCNLEIIDH IVGNQPDQEM -ESASΞW

HPPD_PIG LDSRPQPSQT LLHRLLLSKL PKCGLEIIDH IVGNQPDQEM -ESASQW

HPPD_MOUSE GRFLPGFEAP TYKDTLLPKL PRCNLEIIDH IVGNQPDQEM -QSASEW

HPPD_PSESP IDFV — FLEG VDRHPVGA— GLKIIDH LTHNVYRGRM -A YWANF

MELA_SHECO SDF EA LDEPIITQ— -EKGFIEVDH LTNNVHKGTM -E YWSNF

PEA3_M0USE HGYLGEHSSV FQQPVDMCHS FTSPQGGGRE PLPAPYQHQL SEPCPPYPQQ

310 320 330 340 350

HPPD_Hv FT GFHEF AEFTAEDVGT TESGLNSWL ANNSEGVLLP LNEPVHGTKR

HPPD_Ath FT GFHQF AEFTADDVGT AESGLNSAVL ASNDEMVLLP INEPVHGTKR

HPPD_HUMAN YLKNLQFHRF WSVDDTQVHT EYSSLRSIW ANYEESIKMP INEPAPG-KK

HPPDJAT YLKNLQFHRF WSVDDTQVHT EYSSLRSIW ANYEESIKMP INEPAPG-RK

HPPD_PIG YMRNLQFHRF WSVDDTQIHT EYSALRSWM ANYEESIKMP INEPAPG-KK HPPD_MOUSE YLKNLQFHRF WSVDDTQVHT EYSSLRSIW TNYEESIKMP INEPAPG-RK

HPPD_PSESP YEKLFNFREI RYF DIKG EYTGLTSKAM TAPDGMIRIP LNE — ESSKG

MELA_SHECO YKDIFGFTEV RYF DIKG SQTALISYAL RSPDGSFCIP INE —- GKGDD

PEA3_MOUSE NFKQ-EYHDP LYEQAGQPAS SQGGVSGHRY PGAGWIKQΞ RTDFAYDSDV

360 370 380 390 400

HPPD_Hv RSQIQTFLEH HGGPGVQH-I AVASSDVLRT LRKMRARSAM GGFDFLPPPL

HPPD_Ath KSQIQTYLEH NEGAGLQH-L ALMSEDIFRT LREMRKRSSI GGFDFMPSPP

HPPD_HUMAN KSQIQEYVDY NGGAGVQH-I ALKTEDUTA IRHLRER -GLEFLSVP-

HPPD_RAT KSQIQEYVDY NGGAGVQH-I ALRTEDIITT IRHLRER -GMEFLAVP-

HPPD_PIG KSQIQEYVDY NGGAGVQH-I ALKTEDIITA IRSLRER -GVΞFLAVP-

HPPD MOUSE KSQIQEYVDY NGGAGVQH-I ALKTEDIITA IRHLRER -GTEFLAAP-

HPPD_PSESP AGQIEEFLMQ FNGEGIQH-V AFLSDDLIKT WDHLKSI -GMRFMTAPP

MELA_SHECO RNQIDEYLKE YDGPGVQH-L AFRSRDIVAS LDAMEGS -SIQTLDIIP

PEA3_MOUSE PGCASMYLHP EGFSGPSPGD GVMGYGYEKS LRPFPDDVCI VPKKFEGDIK

410 420 430 440 450

HPPD_Hv PKYYEGVRRL AGD — VLSEA QIKECQELGV LVDRDDQG— —VLL

HPPD_Ath PTYYQNLKKR VGD— LSDD QIKECEELGI LVDRDDQG— - LL

H PD_HUMAN STYYKQLREK LKTAKIKVKE NIDALEELKI LVDYDEKG— —YLL

HPPD_RAT SSYYRLLREN LKTSKIQVKE NMDVLEELKI LVDYDEKG— —YLL

HPPD_PIG FTYYKQLQEK LKSAKIRVKE SIDVLEELKI LVDYDEKG— —YLL

HPPD_MOUSE SSYYKLLREN LKSAKIQVKE SMDVLEELHI LVDYDEKG— —YLL

HPPD_PSESP DTYYEMLEGR LPN HGE PVGELQARGI LLDGSSESGD KRLLL

MELA_SHECO E-YYDTIFEK LPQ VTE DRDRIKHHQI LVDGDEDG— —YLL

PEA3_MOUSE QEGIGAFREG PPYQR -RGALQLWQF LVALLDDPTN AHFIAWTGRG

460 470 480 490 500

HPPD_Hv QIFTKPVGDR PTLFLEMIQR IGCMEKDERG EE YQKG GCGGFGKGNF

HPPD_Ath QIFTKPLGDR PTIFIEIIQR VGCMMKDEEG KA YQSG GCGGFGKGNF HPPD_HUMAN QIFTKPVQDR PTLFLEVIQR mTun -

HPPD_RAT QIFTKPMQDR PTLFLEVIQR HNHQ GFGAGNF HPPD_PIG QIFTKPMQDR PTVFLEVIQR M TUΠ ~ P Ä MP HPPD_MOUSE QIFTKPMQDR PTLFLEVIQR HPPD_PSESP QIFSETLMGP -VFFEFIQR KGDD- ^T, T? ΠN "TT

MELA_SHECO QIFTKNLFGP —IFIEIIQR KNNL- GFGEGNF

PEA3_MOUSE MEFKLIEPEE VARLWGIQKN RPAMNYDKLS RSLRYYYEKG IMQKVAGERY

510 520 530 540 550

HPPD_Hv SE LFK-SIE-DY —EKS — LEA KQSAAV-QGS

HPPD_Ath SE LFK-SIE-EY —EK — LEA KQLVG

HPPD_HUMAN NS LFK-AFEEEQ —NLRGNLTN METNG PGM

HPPD_RAT NS LFK-AFEEEQ —ALRG

HPPD_PIG NS LFK-AFEEEQ —ELRGNLTD TDPNGVPFRL

HPPD_MOUSE NS LFK-AFEEEQ —ALRGNLTD LEPNGVRSGM

HPPD_PSESP KA LFE-SIERDQ —VRRGVLST -D

MELA_SHECO KA LFE-SIERDQ --VRRGVL

PEA3_MOUSE VYKFVCEPEA LFSLAFPDNQ RPALKAEFDR PVSEEDTVPL SHLDESPAYL 560 570

HPPD_Hv

HPPD_Ath

HPPD_HUMAN

HPPD_RAT

HPPD_PIG

HPPD_MOUSE

HPPD_PSESP

MELA_SHECO PEA3_MOUSE PELTGPAPPF GHRGGYSY Explanation: HPPD_Hv: Hordeum vulgarär 4-hydroxyphenylpyruvate dioxygenase (HvSD36)

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) PEA3 polypeptides

The greatest homology was found with the Arabidopsis sequence with 58% over the entire sequence (62% over 412 As), followed by HPPD_RAT with 35% (over 365 As), HPPD_HUMAN 34% (over 365 AS), HPPD_MOUSE 34% (over 371 ace).

Example 4

Cultivation of barley (Hordeum vulgare)

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 μm-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).

Example 5 Growing Tobacco

The tobacco plants were grown using a known method. The variety of tobacco used is Nicotiana tabacum, cv. Xanthi. Example 6 Transformation of tobacco

The expression cassette according to the invention containing the HPPD gene with the sequence 1 was cloned into the vector pBinAR-Hyg

(Fig. 4). This vector was then used to transform tobacco plants according to Example 5 by a known method.

Example 7 Increasing Tocopherol Biosynthesis in Tobacco

The HPPD cDNA was provided with a CaMV35S promoter and overexpressed in tobacco using the 35S promoter. At the same time, 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 α-tocopherol content of the whole plant or the seeds of the plant was then determined. In all cases the α-tocopherol concentration was increased compared to the non-transformed plant.

SEQUENCE LOG

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: BASF AG

(B) ROAD: Carl Bosch

(C) LOCATION: Ludwigshafen

(D) FEDERAL COUNTRY: Germany

(F) POSTAL NUMBER: 67056

(G) TELEPHONE: 0621-60-52698

(ii) REGISTRATION TITLE: Barley HPPD sequence (iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPA)

(2) INFORMATION ABOUT SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1565 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: hppd from barley

(D) STAGE OF DEVELOPMENT: senescence

(vii) DIRECT ORIGIN:

(A) LIBRARY: lambda FIXII bank of barley

(B) CLON: pHvSD36.seq

(ix) FEATURES:

(A) NAME / KEY: CDS

(B) LOCATION: 9..1313

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Krupinska, Karin

(B) TITLE: Overexpression of HPPD

(C) MAGAZINE: overexpression of HPPD (G) DATE: 1998

(K) SIGNIFICANT REMAINS IN SEQ ID NO: 1: FROM 1 TO 1565

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CGCACACC ATG CCG CCC ACC CCC ACC ACC CCC GCG GCT ACC GGC GCC GCC 50 Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala 1 5 10

GCC GCG GTG ACG CCG GAG CAC GCG CGA CCG CAC CGA ATG GTC CGC TTC 98 Ala Ala Val Thr Pro Glu His Ala Arg Pro His Arg Met Val Arg Phe 15 20 25 30

AAC CCG CGC AGC GAC CGC TTC CAC ACG CTC TCC TTC CAC CAC GTC GAG 146 Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu

35 40 45

TTC TGG TGC GCG GAC GCC GCC TCC GCC GCC GGC CGC TTC GCG TTC GCG 194 Phe Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala 50 55 60

CTC GGC GCG CCG CTC GCC GCC AGG TCC GAC CTC TCC ACG GGG AAC TCC 242 Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser 65 70 75

GCG CAC GCC TCC CAG CTG CTC CGC TCG GGC TCC CTC GCC TTC CTC TTC 290 Ala His Ala Ser Gin Leu Leu Arg Ser Gly Ser Leu Ala Phe Leu Phe 80 85 90

ACC GCG CCC TAC GCC AAC GGC TGC GAC GCC GCC ACC GCC TCC CTG CCC 338 Thr Ala Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro 95 100 105 110

TCC TTC TCC GCC GAC GCC GCG CGC CGG TTC TCC GCC GAC CAC GGG ATC 386 Ser Phe Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Ile 115 120 125

GCG GTG CGC TCC GTA GCG CTG CGC GTC GCA GAC GCC GCC GAG GCC TTC 434 Ala Val Arg Ser Val Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe 130 135 140

CGC GCC AGT CGT CGA CGG GGC GCG CGC CCG GCC TTC GCC CCC GTG GAC 482 Arg Ala Ser Arg Arg Arg Gly Ala Arg Pro Ala Phe Ala Pro Val Asp 145 150 155

CTC GGC CGC GGC TTC GCG TTC GCG GAG GTC GAG CTC TAC GGC GAC GTC 530 Leu Gly Arg Gly Phe Ala Phe Ala Glu Val Glu Leu Tyr Gly Asp Val 160 165 170

GTG CTC CGC TTC GTC AGC CAC CCG GAC GGC ACG GAC GTG CCC TTC TTG 578 Val Leu Arg Phe Val Ser His Pro Asp Gly Thr Asp Val Pro Phe Leu 175 180 185 190 CCG GGG TTC GAG GGC GTA ACC AAC CCG GAC GCC GTG GAC TAC GGC CTG 626

Pro Gly Phe Glu Gly Val Thr Asn Pro Asp Ala Val Asp Tyr Gly Leu 195 200 205

ACG CGG TTC GAC CAC GTC GTC GGC AAC GTC CCG GAG CTT GCC CCC GCC 674 Thr Arg Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala 210 215 220

GCA GCC TAC ATC GCC GGG TTC ACG GGG TTC CAC GAG TTC GCC GAG TTC 722 Ala Ala Tyr Ile Ala Gly Phe Thr Gly Phe His Glu Phe Ala Glu Phe 225 230 235

ACG GCG GAG GAC GTG GGC ACG ACC GAG AGC GGG CTC AAC TCG GTG GTG 770 Thr Ala Glu Asp Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Val Val 240 245 250

CTC GCC AAC AAC TCG GAG GGC GTG CTG CTG CCG CTC AAC GAG CCG GTG 818 Leu Ala Asn Asn Ser Glu Gly Val Leu Leu Pro Leu Asn Glu Pro Val 255 260 265 270

CAC GGC ACC AAG CGC CGG AGC CAG ATA CAG ACG TTC CTG GAA CAC CAC 866 His Gly Thr Lys Arg Arg Ser Gin Ile Gin Thr Phe Leu Glu His His 275 280 285

GGC GGC CCG GGC GTG CAG CAC ATC GCG GTG GCC AGC AGT GAC GTG CTC 914 Gly Gly Pro Gly Val Gin His Ile Ala Val Ala Ser Ser Asp Val Leu 290 295 300

AGG ACG CTC AGG AAG ATG CGT GCG CGC TCC GCC ATG GGC GGC TTC GAC 962 Arg Thr Leu Arg Lys Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp 305 310 315

TTC CTG CCA CCC CCG CTG CCG AAG TAC TAC GAA GGC GTG CGA CGC CTT 1010 Phe Leu Pro Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Leu 320 325 330

GCC GGG GAT GTC CTC TCG GAG GCG CAG ATC AAG GAA TGC CAG GAG CTG 1058 Ala Gly Asp Val Leu Ser Glu Ala Gin Ile Lys Glu Cys Gin Glu Leu 335 340 345 350

GGT GTG CTC GTC GAT AGG GAC GAC CAA GGG GTG TTG CTC CAA ATC TTC 1106 Gly Val Leu Val Asp Arg Asp Asp Gin Gly Val Leu Leu Gin Ile Phe 355 360 365

ACC AAG CCA GTA GGG GAC AGG CCG ACC TTG TTC CTG GAG ATG ATC CAG 1154 Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gin 370 375 380

AGG ATC GGG TGC ATG GAG AAG GAC GAG AGA GGG GAA GAG TAC CAG AAG 1202 Arg Ile Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gin Lys 385 390 395 GGT GGC TGC GGC GGG TTC GGC AAA GGC AAC TTC TCC GAG CTG TTC AAG 1250

Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys 400 405 410

TCC ATT GAA GAT TAC GAG AAG TCC CTT GAA GCC AAG CAA TCT GCT GCA 1298 Ser Ile Glu Asp Tyr Glu Lys Ser Leu Glu Ala Lys Gin Ser Ala Ala 415 420 425 430

GTT CAG GGA TCA TAGGATAGAA GCTGGTCCTT GTATCATGGT CTCATGGAGC 1350

Val Gin Gly Ser

435

AAAAGAAAAC AATGTTGTTT GTAATATGCG TCGCACAATT ATATCAATGT TATAATTGGT 1410

GAAGCTGAAG ACAGATGTAT CCTATGTATG ATGGGTGTAA TGGATGGTAG AGGGGCTCAC 1470

ACATGAAGAA AATGTAGCGT TGACATTGTT GTACAATCTT GCTTGCAAGT AAAATAAAGA 1530

ACAGATTTTG AGTTCTGCAA AAAAAAAAAA AAAAA 1565

(2) INFORMATION ABOUT SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 434 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Ala 1 5 10 15

Val Thr Pro Glu His Ala Arg Pro His Arg Met Val Arg Phe Asn Pro 20 25 30

Arg Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu Phe Trp 35 40 45

Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Leu Gly 50 55 60

Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala His 65 70 75 80

Ala Ser Gin Leu Leu Arg Ser Gly Ser Leu Ala Phe Leu Phe Thr Ala

85 90 95

Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro Ser Phe 100 105 110

Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Ile Ala Val 115 120 125 Arg Ser Val Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe Arg Ala 130 135 140

Ser Arg Arg Arg Gly Ala Arg Pro Ala Phe Ala Pro Val Asp Leu Gly 145 150 155 160

Arg Gly Phe Ala Phe Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu 165 170 175

Arg Phe Val Ser His Pro Asp Gly Thr Asp Val Pro Phe Leu Pro Gly 180 185 190

Phe Glu Gly Val Thr Asn Pro Asp Ala Val Asp Tyr Gly Leu Thr Arg 195 200 205

Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala Ala Ala 210 215 220

Tyr Ile Ala Gly Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala 225 230 235 240

Glu Asp Val Gly Thr Thr Glu Ser Gly Leu Asn Ser Val Val Leu Ala 245 250 255

Asn Asn Ser Glu Gly Val Leu Leu Pro Leu Asn Glu Pro Val His Gly 260 265 270

Thr Lys Arg Arg Ser Gin Ile Gin Thr Phe Leu Glu His His Gly Gly 275 280 285

Pro Gly Val Gin His Ile Ala Val Ala Ser Ser Asp Val Leu Arg Thr 290 295 300

Leu Arg Lys Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp Phe Leu 305 310 315 320

Pro Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Leu Ala Gly 325 330 335

Asp Val Leu Ser Glu Ala Gin Ile Lys Glu Cys Gin Glu Leu Gly Val 340 345 350

Leu Val Asp Arg Asp Asp Gin Gly Val Leu Leu Gin Ile Phe Thr Lys 355 360 365

Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met Ile Gin Arg Ile 370 375 380

Gly Cys Met Glu Lys Asp Glu Arg Gly Glu Glu Tyr Gin Lys Gly Gly 385 390 395 400

Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile 405 410 415 Glu Asp Tyr Glu Lys Ser Leu Glu -Ala Lys Gin Ser Ala Ala Val Gin 420 425 430

Gly Ser

Claims

claims

1. DNA sequence SEQ ID N0: 1 and thus hybridizing DNA 5 sequences coding for an HPPD.

2. Expression cassette containing a promoter and a DNA sequence according to claim 1.

10 3. Expression cassette according to claim 2, containing the CaMV35S promoter.

4. Expression cassette according to claim 2, containing the seed-specific phaseolin promoter.

15

5. Expression cassette according to claim 2, wherein the DNA sequence according to claim 1 is functionally linked to another protein so that a common translation product is formed.

20th

6. Use of the expression cassette according to claim 2 for the transformation of plants.

7. The method for transforming a plant is characterized in that an expression cassette according to claim 2 is introduced into a plant cell, into callus tissue, an entire plant or protoplasts of plant cells.

8. Process for the transformation of plants, characterized in that 30

1) the expression cassette according to claim 2 is transferred into an agrobacterial strain,

2) the resulting recombinant clones are isolated and 35 3) these are used to transform plants.

9. The method according to claim 8, wherein the transformation is carried out with the help of the strain Agrobacterium tumefaciens.

40 10. A method for transforming plants according to claim 7, characterized in that the transformation is carried out with the aid of electroporation.

45

(Sign.

11. A method for transforming plants according to claim 7, characterized in that the transformation is carried out using the particle bombardment method.

5 12. Plant with increased vitamin E content containing an expression cassette according to claims 2 to 5.

13. Plant according to claim 12, selected from the group of soy, barley, oats, wheat, rapeseed, corn or sunflower.

10

14. A method for producing plants with an increased vitamin E content, characterized in that a DNA sequence according to claim 1 is expressed in plants.

15 15. The method according to claim 14, characterized in that the DNA sequence is expressed in a tobacco plant.

16. The method according to claim 14 and 15, characterized in that the expression in the leaves or the seeds of the plant

20 takes place.

17. Use of expression cassettes according to claims 2 to 5 for the production of plants with an increased vitamin E content by expression of a DNA sequence according to claim 1

25 in plants.

18. Use of the expression cassette according to claim 2 for the production of a test system for the identification of inhibitors of HPPD.

30

19. Test system based on the expression of an expression cassette according to claim 2 for the identification of inhibitors of HPPD.

35 20. Herbicidal active ingredients, identifiable by means of a test system according to claim 19.

21. Use of a plant according to claim 12 for the production of plant HPPD.

40

22. Use of the expression cassette according to claim 2 for the production of plants with increased resistance to inhibitors of HPPD by increased expression of a DNA sequence according to claim 1.

45

23. A process for the production of plants with increased resistance to inhibitors of HPPD by increased expression of a DNA sequence according to claim 1.

24. Plant with increased resistance to inhibitors of HPPD containing an expression cassette according to claims 2 to 5.