# Patent

## Title
Expression cassette for nucleic acids in plant tissue containing starch

## Abstract

The invention relates to methods for the transgenic expression of nucleic acid sequences predominantly in starch-comprising tissues of plants, preferably in fruits, roots, seeds or tubers, wherein the nucleic acid sequence is expressed under the control of a promoter of a starch synthase 3. Furthermore in accordance with the invention are transgenic expression cassettes and vectors comprising a promoter of a starch synthase 3, and the use thereof for the production of foodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals.

## Background
RELATED APPLICATIONS
This application is a national stage application (under 35 U.S.C. 371) of PCT/EP2004/000241 filed Jan. 15, 2004 which claims benefit to German application 103 02 324.0 filed Jan. 20, 2003.
SEQUENCE LISTING SUBMISSION
The Sequence Listing associated with this application is filed in electronic format via EPS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is SequenceListing—13173—00015. The size of the text file is 219 KB, and the text file was created on Apr. 25, 2008.
The invention relates to methods for the transgenic expression of nucleic acid sequences predominantly in starch-comprising tissues of plants, preferably in fruits, roots, seeds or tubers, wherein the nucleic acid sequence is expressed under the control of a promoter of a starch synthase 3. Furthermore in accordance with the invention are transgenic expression cassettes and vectors comprising a promoter of a starch synthase 3, and the use thereof for the production of foodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals.
The aim of plant biotechnology work is the generation of plants with improved properties, for example for increasing agricultural productivity. Transcriptional regulatory sequences or promoters which regulate the expression of genes in plants are essential elements of plant biotechnology. Various promoters which have been used successfully for the expression of heterologous genes in plants are available; they comprise not only plant promoters (such as, for example, cauliflower heat shock protein hsp80 promoters; U.S. Pat. No. 5,612,472), but also promoters from other nonplant sources such as, for example, promoters of plant viruses (for example the cauliflower mosaic virus 35S promoter) or of plant-infecting bacteria (for example the promoter of the agrobacterial octopine synthase; Leisner and Gelvin (1988) Proc Natl Acad Sci USA 85(8):2553-2557).
Frequently, what are known as constitutive promoters, which regulate, in the plant, the expression of a gene product largely at any time and in any tissue are employed for the expression of heterologous nucleic acid sequences in transgenic plants. A directed expression of genes in specific plant parts or at specific points in time of development is not possible using these promoters. Thus, the protein to be expressed transgenically is expressed at locations and at times where it is not required, which, for example, unnecessarily consumes energy, causes metabolic modifications and can thus have an adverse effect on plant growth. For reasons of product licensing and product acceptance too, it is desirable to express a transgenic protein only where it is required owing to its intended effect.
Tissue- and development-specific promoters are of great interest for this purpose. Various such promoters are known. Thus, the promoter of the Vicia faba “sucrose-binding-protein-like gene” (SBP) mediates strong and specific expression in seeds of oilseed rape and other plants (WO 00/26388).
Fruits, seeds, beets/swollen tap roots or tubers, being important storage organs of plant organisms, are of great agronomical relevance. They serve for the storage of proteins, oils and carbohydrates (in particular starch). As a rule, such tissues are photosynthetically inactive and are also referred to as sink tissues or sink organs. They rely on the import of photoassimilates from the photosynthetically active plant parts (source organs or source tissues). Both traditional breeding and biotechnological methods have been used for improving specific aspects of fruit and tuber quality. High-quality, mature fruits are the result of a number of coordinated biochemical and metabolic modifications which can occur not only during maturation, but also during fruit development. These modifications determine the final quality and the quantity of the fruits. Examples of modified properties, for example in the case of tomato fruits, are increased sucrose import, conversion of starch, accumulation of various organic acids, modifications of pigments and modifications in fungicidal and insecticidal compounds. Such results can be achieved by means of the overexpression of genes/proteins or by inhibition by means of double-stranded RNA, antisense RNA or cosuppression. Since sink tissues act as storage site of the most important plant raw materials, promoters which make possible a selective expression in these tissues are of particular interest in plant biotechnology since they permit a directed modification of these tissues and of their constituents.
The skilled worker is familiar with a variety of promoters which can be used for the expression of nucleic acid sequences in fruits, seeds or tubers. The promoter of the tomato genomic clone 2A11 must be mentioned (Pear et al. (1989) Plant Mol Biol 13:639-651; WO 91/19806). However, the 2A11 promoter governs expression during the very early stages and is relatively weak. The tomato ethylene-inducible E4 and E8 promoters (U.S. Pat. No. 5,859,330; Deickmann et al. (1988) EMBO J. 7:3315-3320) and the polygalacturonase promoter (U.S. Pat. No. 6,127,179) have likewise been described as being fruit-specific. The abovementioned promoters, however, show expression only during the late phases of fruit development, and their use is therefore only limited. The promoters TFM7 and TFM9 (U.S. Pat. No. 5,608,150) are active during fruit development in green and yellow stages. The fruit-specific regulation of the kiwi fruit actinidin promoter has been detected for expression in petunia (Lin et al. (1993) Plant Mol Biol 23:489-499). Thi-1, MADS2 and a promoter fusion between Thi-1 and the melon actin promoter regulate the expression of heterologous genes specifically in apples (WO 00/66610).
Further promoters are, for example, promoters with specificity for tubers, storage roots or other roots such as, for example, the tuber-specific patatin class I promoter (Bevan et al. (1986) Nucl Acids Res 14:4625-4638), the potato cathepsin D inhibitor promoter (Herbers et al. (1994) Plant Mol. Biol. 26: 73-83), the starch synthase (GBSS1) promoter or the sporamin promoter. Other genes with specific high activity in tubers are, for example, the promoter of the ADP-glucose pyrophosphorylase gene (Müller-Röber et al. (1990) Mol Gen Genet 224:136-146), of sucrose synthase (Salanoubat and Belliard (1987) Gene 60:47-56; Salanoubat and Belliard (1989) Gene 84:181-185), the promoters of the 22 kD protein complex and of the proteinase inhibitor (Hannapel (1990) Plant Physiol 94:919-925) and the other class I patatins (B33) (EP-A1 0 375 092; Rocha-Sosa et al. (1989) EMBO J. 8:23-29). A disadvantage of the patatin 1 promoter is that it is induced by high sucrose concentrations, also in tissues other than the tuber (Jefferson R et al. (1990) Plant Mol Biol 14:995-1006).
The promoters described in the prior art have one or more of the following disadvantages:

    1) The promoters do not show the desired expression level and/or are active in a few plant species only.
    2) The promoters are only active very early or very late during fruit or tuber development.
    3) The expression pattern does not agree with what has been expected, i.e. for example undesired expression activities in other tissues are found.
    4) The expression of many of the abovementioned promoters is ethylene-dependent.


Moreover, the number of existing promoters is greatly limited. This may become a limiting factor, in particular in approaches which require the expression of more than one heterologous nucleic acid sequence. The expression, under the same promoter, of different heterologous sequences, in one plant organism, can result in “switching off” (“epigenic silencing”) of the transgenic expression cassettes in question (Mette et al. (1999) EMBO J 18:241-248).
A multiplicity of biosynthetic enzymes such as, for example, the starch synthases (EC 2.4.1.21) are involved in the synthesis of starch, which takes place in the plastids of higher plants. Starch synthases serve for extending the chain of α-1,4-glucans by means of transferring glucosyl units from ADP-glucose to the non-reducing ends of existing α-1,4-glucans. In addition to the granule-bound starch synthase (GBSS), three further classes of starch synthases in plants are described: SS1 (wheat: Li et al. (1999) Theor Appl Genet 98:1208-1216; GenBank Acc. No.: U48227; rice: Baba et al. (1993) Plant Physiol 103:565-573; potato: Genbank Acc. No.: Y10416), SS2 (pea: Dry et al. (1992) Plant J 2:193-202; potato: Edwards et al. (1995) Plant J 8:283-294; maize: Harn et al. (1998) Plant Mol Biol 37:639-649; GenBank Acc. No. U66377) and SS3 (potato: Abel et al. (1996) Plant J 10:981-91, Marshall et al. (1996) Plant Cell 8:1121-1135; maize: Gao et al. (1998) Plant Cell 10:399-412).
Various publications describe the isolation and characterization of soluble starch synthases from the potato tuber, of which three isoforms in the soluble fraction of potato tuber extracts are known. For example, known are the isoform 3 (Marshall et al. (1996) Plant J 10:981-991; GenBank Acc. No.: X95759; EP 0779363-A3), the SS1 isoform (GenBank Acc. No.: Y10416; Kossmann et al. (1999) Planta 208:503-11), which is expressed predominantly in leaves and only a little in tubers. Furthermore, there are described the soluble starch synthase 3 from potato (GenBank Acc. No.: X94400; Abel et al. (1996) Plant J 10:981-91), maize (GenBank Acc. No.: AF023159, A93359), rice (GenBank Acc. No.:D16202), wheat (GenBank Acc. No.: AJ292522 Triticum aestivum; AF258609 Aegilops tauschii), from the asparagus bean (Vigna unguiculata; GenBank Acc. No.: AJ225088), and Arabidopsis (GenBank Acc. No.: AC007296). Abel et al. teach that the potato SSS3 is predominantly expressed in the tubers, but also in the sink-and-source leaves. Promoters of the SSS3 genes or transgenic expression constructs comprising them are not described.
It was therefore an object to provide novel promoters and transgenic expression cassettes derived therefrom, which ensure a transgenic expression of nucleic acid sequences during the entire development time of the tubers from the youngest stages to the storage of the tubers and which have a high specificity for starch-comprising tissues. This object is achieved by the present invention.
A first subject of the invention relates to methods for the directed, transgenic expression of nucleic acid sequences in at least one starch-comprising tissue of a plant, which comprises the following steps

    1. introducing, into plant cells, a transgenic expression cassette, where the transgenic expression cassette comprises at least the following elements
    
        a) at least one promoter sequence of a gene encoding a starch synthase 3, and
        b) at least one further nucleic acid sequence,
        where at least one of said promoter sequences and a further nucleic acid sequence are functionally linked with one another and said further nucleic acid sequence is heterologous with regard to the promoter sequence, and
    
    
    2. selection of transgenic cells which comprise said expression cassette stably integrated into the genome, and
    3. regeneration of intact plants from said transgenic cells, where at least one of the further nucleic acid sequence is expressed in a starch-comprising tissue in a directed manner.


A further subject of the invention therefore relates to an isolated nucleic acid sequence comprising the promoter of the starch synthase 3 from potato. Preferably, said isolated nucleic acid sequence comprises a nucleic acid sequence selected from

    1. the sequences as shown in SEQ ID NO: 1 or 44 or the sequences which are complementary thereto,
    2. fragments of at least 25 contiguous nucleotides, preferably at least 50 contiguous nucleotides, especially preferably at least 100 contiguous nucleotides, of the sequence as shown in SEQ ID NO: 1 or 44 or the sequences which are complementary thereto,
    3. sequences which have at least 50%, preferably 70%, by preference at least 80%, especially preferably at least 90%, very especially preferably at least 95%, most preferably 99%, homology with the sequence as shown in SEQ ID NO: 1 or 44 or the sequences which are complementary thereto, where the homology extends over a length of at least 100 base pairs, preferably at least 200 base pairs, especially preferably at least 300 base pairs, very especially preferably at least 400 base pairs, most preferably over the entire length of the sequence as shown in SEQ ID NO: 1 or 44.


In the context of the present invention, homology between two nucleic acids is understood as meaning the identity of the nucleic acid sequence over the sequence length indicated in each case, which is calculated by comparison with the aid of the GAP program algorithm (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:
















Gap Weight: 12
Length Weight: 4



Average Match: 2.912
Average Mismatch: −2.003










For example, a sequence which has at least 50% homology with the sequence SEQ ID NO: 1 on nucleic acid basis over the entire length of the sequence as shown in SEQ ID NO:1 is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 1 by the above program algorithm with the above set of parameters has at least 50% homology.
A further subject relates to transgenic expression cassettes as they can be employed for example in the method according to the invention. Preferably, the transgenic expression cassettes for the directed, transgenic expression of nucleic acid sequences in at least one starch-comprising tissue of a plant comprise

    a) at least one promoter sequence of a gene coding for a starch synthase 3, and
    b) at least one further nucleic acid sequence,

where at least one promoter sequence and one further nucleic acid sequence are functionally linked with one another and the further nucleic acid sequence is heterologous with regard to the promoter sequence.



The expression cassettes according to the invention may comprise further genetic control sequences and/or additional functional elements. Preferably, the transgenic expression cassettes can make possible, owing to the nucleic acid sequence to be expressed transgenically, the expression of a protein encoded by said nucleic acid sequence and/or the expression of a sense RNA, antisense RNA or double-stranded RNA encoded by said nucleic acid sequence.
A further subject of the invention relates to transgenic expression vectors which comprise one of the expression cassettes according to the invention.
A further subject of the invention relates to transgenic organisms which comprise one of the expression cassettes or expression vectors according to the invention. The organism can be selected from the group consisting of bacteria, yeasts, fungi, nonhuman animal and plant organisms or cells, cell cultures, parts, tissues, organs or propagation material derived therefrom; preferably, the organism is selected from the group of the agricultural useful plants. In said plants, the expression of the nucleic acid sequence to be expressed transgenically is preferably higher in at least one starch-comprising tissue, (for example the potato tuber, beet/swollen tap root or other root, the seed or the tomato fruit) than in another tissue.
A further subject relates to the use of the isolated nucleic acid sequences, transgenic expression vectors or transgenic organisms according to the invention for the transgenic expression of nucleic acids and/or proteins. Especially preferred is the use of said transgenic organisms or of cells, cell cultures, parts, tissues, organs or propagation material derived therefrom for the production of foodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals, the fine chemicals preferably being enzymes, vitamins, amino acids, sugars, saturated or unsaturated fatty acids, natural or synthetic flavorings, aroma substances or colorings. Furthermore comprised by the invention are methods for the production of said foodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals using the transgenic organisms according to the invention or cells, cell cultures, parts, tissues, organs or propagation material derived therefrom.
A particular advantage of the starch synthase 3 promoters according to the invention is their activity in starch-comprising tissues, preferably during the entire development and storage of potato tubers, and its high activity in green tomato fruits.
The expression of the natural starch synthase 3 from potato (i.e. the expression activity of the nontransgenic, homologous combination of the starch synthase 3 promoter and the coding region of the starch synthase 3 gene) also shows significant expression in the leaves (Abel et al. (1996) Plant J 10:981-91). In contrast, the heterologous transgenic expression cassettes provided within the scope of the present invention show no relevant expression in leaves, which is of great importance for a number of biotechnological applications.
The starch synthase 3 (SSS3) promoters provided within the scope of the present invention, in particular the potato SSS3 promoter, mediate not only high expression during all of the tuber development right into the late storage phase, but also high tissue specificity, which underlines the significant value for the exploitation in transgenic plants. An analysis in tomato has revealed that the SSS3 promoter likewise brings about strong expression in the fruits, predominantly in the green fruits. Weak secondary activities were only found in pollen and seed. Of particular interest is a pronounced activity post-harvest and during the storage of the transgenic plants (in particular tubers). This makes the promoters according to the invention suitable for what are known as post-harvest applications.
Owing to its high expression activity and specificity, the SSS3 promoter according to the invention is of particular value for plant biotechnology. Owing to the function of the soluble starch synthase 3 in the starch metabolism and the expression pattern which has been found, it can be expected that the promoter is active in all starch-comprising tissues, preferably in fruits, seeds, beets/swollen tap roots and tubers, of other plants. Very specially advantageous in this context is the use in approaches which serve for the modification of the carbohydrate and/or starch metabolism. Owing to their fruit or tuber specificity, the promoters according to the invention can be employed in particular for improving the quality of the developing fruit or tuber or for influencing the maturation process. In this context, it is preferred to transgenically express, under the control of the promoters according to the invention, nucleic acid sequences which influence a modulation of the sugar or starch metabolism, a modification of constituents, an improvement of nutritional values, a modification of the sink-source relations, flavor components, pathogen resistance, tissue consistency and the like. In the tomato fruits, for example, an SSS3 promoter can be employed for the modification of flavanoids and carotenoids. Specific use forms are listed hereinbelow. The genes which are regulated by the promoter according to the invention can be plant genes, fruit- and tuber-specific genes or heterologous genes whose expression in the fruits or tubers is desired.
“Promoter of a starch synthase 3 gene” or “starch synthase 3 promoter” (hereinbelow also SSS3 promoter) generally refers to the natural regulatory region of a gene coding for a starch synthase 3. The starch synthase 3 is also known as soluble starch synthase or soluble starch synthase 3. Analogous names are to be understood synonymously. Preferred promoters are those which comprise a sequence region of at least 250 base pairs, preferably at least 500 base pairs, especially preferably 1000 base pairs, most preferably at least 2000 base pairs in 5′ direction upstream of the ATG start codon of said genomic sequences coding for a starch synthase 3.
For the purposes of the present invention, “starch synthase” means any enzymatically active peptide, polypeptide, oligopeptide, protein or enzyme molecule which is at least capable of transferring one glucosyl unit from ADP-glucose to an α-1,4-glucan molecule, and fragments of said enzymatically active molecules.
“Starch synthase 3”, “soluble starch synthase 3” or “SSS3” means a starch synthase as defined above which furthermore has one or more of the following characteristics:

    1. it is encoded by a nucleotide sequence comprising at least 20 nucleotides of a sequence as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15 or 17;
    2. it is encoded by a nucleotide sequence comprising a sequence which has at least 85% or more homology with a sequence as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15 or 17;
    3. it comprises an amino acid sequence with at least 85% homology with one of the sequences as shown in SEQ ID NO: 6, 8, 10, 12, 14, 16 or 18;
    4. it comprises a sequence of at least 10 contiguous amino acids, preferably at least 20 contiguous amino acids, especially preferably at least 50 contiguous amino acids, of a sequence as shown in SEQ ID NO: 6, 8, 10, 12, 14, 16 or 18;

and wherein the amino acid sequence encoding for the starch synthase comprises at least one sequence motif selected from the group consisting of:













(a)
NE(P/S)DVXI(K/M)GAFN,
(SEQ ID NO: 19)



 


(b)
PK(E/Q)AY(R/K)XDFVFFNG,
(SEQ ID NO: 20)


 


(c)
DWVFADGP,
(SEQ ID NO: 21)


 


(d)
FL(V/L)SQK(H/D)(V/I)VYTEPL,
(SEQ ID NO: 22)


 


(e)
YNP(A/S)NT(V/N)L(N/T)GKPE
(SEQ ID NO: 23)



(V/I)WFRXSFN,


 


(f)
DAYMMDFVFSE,
(SEQ ID NO: 24)


 


(g)
KVGGL(G/A)DVVTS,
(SEQ ID NO: 25)


 



preferably AKVGGL(G/A)DVVTSLS
(SEQ ID NO: 26)



RA,


 


(h)
HCHDWSSAPV(A/S)WL,
(SEQ ID NO: 27)


 


(i)
FTIHNLEFGA,
(SEQ ID NO: 28)


 


(j)
NGIDPDIWDP,
(SEQ ID NO: 29)


 



preferably GI(L/V/I)NGIDPDIWDP
(SEQ ID NO: 30)



(Y/L) (T/N)D(N/K)FIP,


 


(k)
VG(I/V)ITRLT(A/H)QKG,
(SEQ ID NO: 31)


 



preferably VG(I/V)ITRLT(A/H)
(SEQ ID NO: 32)



QKGIHLIKHA,


 


(l)
NGQVVLLGSA,
(SEQ ID NO: 33)


 



preferably TLERNGQVVLLGSAPD,
(SEQ ID NO: 34)


 


(m)
LTYDEPLSHLIY,
(SEQ ID NO: 35)


 


(n)
DFI(L/I)VPSIFEPCGLTQL,
(SEQ ID NO: 36)


 



preferably AG(S/A)DFI(L/I)
(SEQ ID NO: 37)



VPSIFEPCGLTQL(V/I)AMRYG,


 


(h)
DTVFDVD(H/N)DK,
(SEQ ID NO: 38)


and


 


(i)
VMEQDWSWNRP
(SEQ ID NO: 39)






Further motifs which are characteristic of SSS3 proteins can be deduced readily from a comparison with known SSS3 protein or nucleic acid sequences (cf. FIG. 7).
Homology between two polypeptides is understood as meaning the identity of the amino acid sequence over the sequence length in each case, which is calculated by comparison with the aid of the GAP program algorithm (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting the following parameters:
















Gap Weight: 8
Length Weight: 2



Average Match: 2.912
Average Mismatch: −2.003










For example, a sequence which has at least 85% homology with the sequence SEQ ID NO: 6 on protein basis over the entire length is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 6 by the above program algorithm with the above set of parameters has at least 85% homology.
In particular, SSS3 promoter means nucleotide sequences which comprise a nucleic acid sequence selected from among

    1. the sequences SEQ ID NO: 1, 2, 3, 4 or 44, or the sequences which are complementary thereto,
    2. fragments of at least 25 contiguous nucleotides, preferably at least 50 contiguous nucleotides, especially preferably at least 100 contiguous nucleotides, of a sequence as shown in SEQ ID NO: 1, 2, 3, 4 or 44 or the sequences which are complementary thereto,
    3. sequences which have at least 50%, preferably 70%, by preference at least 80%, especially preferably at least 90%, very especially preferably at least 95%, most preferably 99%, homology with one of the sequences as shown in SEQ ID NO: 1, 2, 3, 4 or 44 or the sequences which are complementary thereto, where the homology extends over a length of at least 100 base pairs, preferably at least 200 base pairs, especially preferably at least 300 base pairs, very especially preferably at least 400 base pairs, most preferably at least 500 base pairs.


Most preferably, an SSS3 promoter comprises the promoter of the potato SSS3 as shown in SEQ ID NO: 1 or 44 or fragments thereof with a length of at least 50 nucleotides, preferably 100 nucleotides.
Especially preferably, an SSS3 promoter has essentially the same promoter activity as one of the promoters as shown in SEQ ID NO: 1, 2, 3, 4 or 44, preferably as the promoter as shown in SEQ ID NO: 1 or 44. In this context, the expression level can deviate down or else up in comparison with a comparative value. Preferred in this context are those sequences whose expression level, measured with reference to the transcribed mRNA or the subsequently translated protein, differs quantitatively under otherwise unaltered conditions by not more than 50%, preferably 25%, especially preferably 10%, from a comparative value obtained with the promoters described by SEQ ID NO: 1 or 44. Especially preferred are those sequences whose expression level, measured with reference to the transcribed mRNA or the subsequently translated protein, exceeds quantitatively under otherwise unaltered conditions by more than 50%, preferably 100%, especially preferably 500%, very especially preferably 1000% a comparative value obtained with the promoter described by SEQ ID NO: 1 or 44.
A promoter activity is referred to as “essentially the same” with regard to an SSS3 promoter when the transcription of a certain nucleic acid sequence to be expressed transgenically under the control of said functionally equivalent promoter under otherwise unaltered conditions shows a directed expression in at least one starch-comprising tissue.
“Starch-comprising tissue” means tissues which, at least at one point in time of their development have a starch content which can be detected by means of a starch test. Preferred as starch test is staining with Lugol's solution (Lugol's solution: for example: dissolve 2 g of KI in 5 ml of water, therein dissolve 1 g of iodine, and add 300 ml of water). The staining procedure is carried out until a blue coloration is visible (approximately 15 min at RT) and can be stopped by washing with water. The starch content may also be determined photometrically by means of the method described by Hajirezaei et al. (1994, Planta 192: 16-30).
Especially preferably, “starch-comprising tissue” means storage tissue of a plant tuber, beet/swollen tap root or fruit such as, for example, the potato tuber, beetroot or tomato fruit. “Starch-comprising tissue” also comprises “sink tissues” of starch-producing plants. “Sink-tissue” means tissues which are net importers of photosynthetically fixed carbon dioxide and, as a rule, are not photosynthetically active. Examples of sink tissues which may be mentioned are: fruits, beets/swollen tap roots and other roots, tubers and seed kernels.
The expression under the control of one of the promoters according to the invention in a carbohydrate-storing, -synthesizing or -metabolizing sink tissue or a starch sink tissue amounts to preferably at least twice, very especially preferably at least five times, most preferably at least ten times that in another tissue, for example a source tissue.
“Directed” with regard to the expression in a starch-comprising tissue preferably means that the expression under the control of one of the promoters according to the invention in a first tissue amounts to preferably at least ten times, very especially preferably at least fifty times, most preferably at least hundred times that of a second tissue, the starch content in the first tissue (for example as determined by staining with Lugol's solution) amounting to at least twice, preferably at least five times, especially preferably at least ten times, most preferably at least fifty times, the starch content in the second tissue. For example “directed” means that the expression under the control of one of the promoters according to the invention in a starch-comprising “sink” tissue such as the tubers amounts to preferably at least ten times, especially preferably at least fifty times, most preferably at least hundred times that in a “source tissue”, such as the leaves.
“Otherwise unaltered conditions” means that the expression which is initiated by one of the transgenic expression cassettes to be compared is not modified by combination with additional genetic control sequences, for example enhancer sequences. Unaltered conditions furthermore means that all external conditions such as, for example, plant species, developmental stage of the plants, culture conditions, assay conditions (such as buffer, temperature, substrates and the like) are kept identical between the expressions to be compared.
Preferably, the expression within at least one particular starch-comprising tissue is essentially constant over all developmental stages. “Essentially constant” means in this context preferably that the standard deviation of the expression between the individual point in time of the development of the tissue in question is less than 50%, preferably 20%, especially preferably 10%, very especially preferably 5%, based on the statistic mean of the expression over all the points in time of the developments.
Nucleic acid sequences which are preferably employed in the context of the determination of the expression level are nucleic acid sequences in functional linkage with the promoter to be tested which code for readily quantifiable proteins. Very especially preferred in this context are reporter proteins (Schenborn E, Groskreutz D (1999) Mol Biotechnol 13(1): 29-44) such as “green fluorescence protein” (GFP) (Chui W L et al. (1996) Curr Biol 6:325-330; Leffel S M et al. (1997) Biotechniques 23(5):912-8), chloramphenicol transferase, luciferase (Millar et al. (1992) Plant Mol Biol Rep 10:324-414), β-glucuronidase or β-galactosidase. β-Glucuronidase (Jefferson et al. (1987) EMBO J. 6:3901-3907) is very especially preferred.
The invention furthermore relates to the use of at least one nucleic acid sequence or a part thereof in methods for the identification and/or isolation of promoters of genes which code for said nucleic acid sequence, where said nucleic acid sequence codes for an amino acid sequence which comprises at least one sequence motif as shown in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 or a variation stated for these sequences. Preferably, said nucleic acid sequence codes for an amino acid sequence comprising a sequence as shown in SEQ ID NO: 6, 8, 10, 12, 14, 16 or 18. Especially preferably, said nucleic acid sequence comprises a sequence as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15 or 17. “Part” with regard to the nucleic acid sequence preferably means a sequence of at least 15 bases, preferably 25 bases, especially preferably 50 bases, most preferably 100 bases.
Also encompassed by the invention are methods for the identification and/or isolation of promoters of SSS3 genes, wherein at least one nucleic acid sequence or part thereof is employed in the identification and/or isolation, where said nucleic acid sequence codes for an amino acid sequence which comprises at least one sequence as shown in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 or a variation stated for these sequences. Preferably, said nucleic acid sequence codes for an amino acid sequence comprising a sequence as shown in SEQ ID NO: 6, 8, 10, 12, 14, 16 or 18. Especially preferably, said nucleic acid sequence comprises a sequence as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15 or 17. “Part” with regard to the nucleic acid sequence preferably means a sequence of at least 15 bases, preferably 25 bases, especially preferably 50 bases, most preferably 100 bases. In a preferred embodiment, the method according to the invention is based on the polymerase chain reaction, where said nucleic acid sequence or part thereof is employed as primer.
The skilled worker is familiar with various methods in order to identify and isolate the promoter of the gene in question, starting from a nucleic acid sequence (for example a gene transcript such as, for example, a cDNA). In principle, for example all methods for the amplification of flanking chromosomal sequences are available for this purpose. The two methods which are most frequently used are inverse PCR (“iPCR”) and thermal asymmetric interlaced PCR (“TAIL PCR”).
For “iPCR”, genomic DNA of the organism from which the functionally equivalent promoter is to be isolated is digested completely with a given restriction enzyme, and the individual fragments are subsequently religated, i.e. linked with themselves to give a circular molecule, in a dilute mixture. Among the multiplicity of resulting circular DNA molecules are also those which comprise the known sequence (for example the sequence coding for the homologous protein). Starting therefrom, it is possible to amplify the circular molecule by means of PCR by using a primer pair where both primers are capable of annealing with the known sequence segment.
The “TAIL PCR” is based on the use of, firstly, a set of successively truncated highly specific primers which anneal with the known genomic sequence (for example the sequence coding for the homologous protein) and, secondly, a set of shorter random primers with a low melting point, so that a less sequence-specific annealing with genomic DNA which flanks the known genomic sequence takes place. Using such a primer combination, the annealing reaction of the primers onto the DNA to be amplified can be designed in such a way that a specific amplification of the desired target sequence is made possible.
A further possibility of amplifying unknown sequence segments is offered by what is known as the genome walking technology (Clontech). Here, what are known as uncloned, adaptor-ligated libraries of genomic DNA fragments are generated. Using gene-specific and adaptor-specific primers, the desired PCR products which comprise unknown DNA sequences are then amplified in a plurality of PCR steps.
Examples of the promoter sequences employed in the transgenic expression cassettes or transgenic expression vectors according to the invention can be found readily, for example in a variety of organisms whose genomic sequence is known, such as, for example, Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Helianthium annuus, Linum sativum by homology comparisons in databases. To this end, it is preferred to be able to start from the coding regions of the genes whose promoters are described by SEQ ID NO: 1, 2, 3, 4 or 44. Starting from, for example, the cDNA sequences of these genes described by SEQ ID NO: 5, 7, 9 or 11 or the protein sequences derived therefrom, described by SEQ ID NO: 6, 8, 10 or 12, the corresponding homologous genes in other plant species can be identified readily in the manner with which the skilled worker is familiar by screening databases or genetic libraries (using suitable gene probes).
The invention furthermore relates to methods for the generation of a transgenic expression cassette, with specificity for starch-comprising tissues, comprising the following steps:

    I. isolation of a promoter with specificity for starch-comprising tissues, wherein at least one nucleic acid sequence or part thereof is employed in the isolation, where said nucleic acid sequence codes for an amino acid sequence which comprises at least one sequence as shown in SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 or a variation stated for these sequences.
    II. functional linkage of said promoter with a further nucleic acid sequence, where said nucleic acid sequence is heterologous with regard to the promoter.


Said nucleic acid sequence preferably codes for an amino acid sequence comprising a sequence as shown in SEQ ID NO: 6, 8, 10, 12, 14, 16 or 18. Said nucleic acid sequence especially preferably comprises a sequence as shown in SEQ ID NO: 5, 7, 9, 11, 13, 15 or 17. “Part” with regard to the nucleic acid sequence preferably means a sequence of at least 15 bases, preferably 25 bases, especially preferably 50 bases, most preferably 100 bases. In a preferred embodiment, the method according to the invention is based on the polymerase chain reaction, where said nucleic acid sequence or part thereof is employed as primer. For the purposes of functional linkage, methods with which the skilled worker is familiar, such as, for example, ligation and the like, may be employed (see hereinbelow).
Suitable SSS3 promoters also comprise natural or artificial mutations of the promoter sequences described under SEQ ID NO: 1, 2, 3, 4 or 44. “Mutation” means substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Thus, for example, the present invention also encompasses those nucleotide sequences which are obtained by modifying an SSS3 promoter as shown in SEQ ID NO: 1, 2, 3, 4 or 44. The purpose of such a modification may be the further delimitation of the sequence which is present therein, or else, for example, the introduction or removal of restriction endonuclease cleavage sites, the removal of excess DNA or the addition of further sequences, for example further regulatory sequences. Where insertions, deletions or substitutions, such as, for example, transitions and transversions, are suitable, techniques known per se such as in vitro mutagenesis, primer repair, restriction or ligation may be used. Transition means a base pair substitution of a purine/pyrimidine pair by another purine/pyrimidine pair (for example A-T by G-C). Transversion means a base pair substitution of a purine/pyrimidine pair by a pyrimidine/purine pair (for example A-T by T-A). Deletion means the removal of one or more base pairs. Insertion means the introduction of one or more base pairs. Complementary ends of the fragments may be made available for ligation by manipulations such as, for example, restriction, chewing-back or filling in overhangs for blunt ends. Analogous results may also be obtained by using the polymerase chain reaction (PCR) using specific oligonucleotide primers.
The skilled worker is familiar with methods for inducing mutations in nucleic acid sequences; by way of example, they include the use of oligonucleotides with one or more mutations in comparison with the region to be mutated (for example in what is known as “site-specific mutagenesis”). Typically, primers with approximately 15 to approximately 75 nucleotides or more are employed, where preferably approximately 10 to approximately 25 or more nucleotide residues are located on both sides of the sequence to be modified. Details and procedure of said mutagenic methods are known to the skilled worker (Kunkel et al. (1987) Methods Enzymol 154:367-382; Tomic et al. (1990) Nucl Acids Res 12:1656; Upender et al. (1995) Biotechniques 18(1):29-30; U.S. Pat. No. 4,237,224). A mutagenesis can also be performed by treatment of, for example, transgenic expression vectors which comprise one of the nucleic acid sequences according to the invention with mutation-inducing agents such as hydroxylamine.
As an alternative, it is possible to delete nonessential sequences of a promoter according to the invention, without this having a significant adverse effect on the abovementioned essential characteristics. Such deletion variants constitute fragments with functional equivalence to the promoters described by SEQ ID NO: 1, 2, 3, 4 or 44. The delimitation of the promoter sequence to certain essential regulatory regions can be effected for example with the aid of search routines for the search of promoter elements. Frequently, certain promoter elements occur in greater quantities in the regions which are relevant for the promoter activity. This analysis can be carried out for example with computer programs such as the program PLACE (“Plant Cis-acting Regulatory DNA Elements”; Higo K et al. (1999) Nucl Acids Res 27(1): 297-300), the BIOBASE database “Transfac” (Biologische Datenbanken GmbH, Brunswick; Wingender E et al. (2001) Nucleic Acids Res 29(1):281-3) or database PlantCARE (Lescot M et al. (2002) Nucleic Acids Res 30(1):325-7). Furthermore, individual fragments of said promoters can be used for example to generate or, advantageously, to optimize, synthetic promoters in combination with other regulatory elements.
Said fragments according to the invention preferably comprise at least 25 contiguous nucleotides, preferably at least 50 contiguous nucleotides, especially preferably at least 100 contiguous nucleotides, of one of the sequences as shown in SEQ ID NO: 1, 2, 3, 4 or 44.
Functionally equivalent fragments of one of the promoters according to the invention—for example the promoters described by SEQ ID NO: 1, 2, 3, 4 or 44—comprise at least 200 base pairs, very especially preferably at least 500 base pairs, most preferably at least 1000 base pairs of the 3′ terminus of the respective promoter according to the invention—for example of the promoters described by SEQ ID NO: 1, 2, 3, 4 or 44—, the length being calculated upstream from the transcription start (“ATG” codon) in 5′ direction.
Suitable SSS3 promoters furthermore comprise DNA sequences which hybridize under standard conditions with one of the nucleic acid sequences as shown in SEQ ID NO: 1, 2, 3, 4 or 44 or the nucleic acid sequences which are complementary thereto and which have essentially the same promoter characteristics.
The term standard hybridization conditions is to be understood broadly and means both stringent and less stringent hybridization conditions. Such hybridization conditions are described inter alia in Sambrook J, Fritsch E F, Maniatis T et al., in Molecular Cloning—A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the conditions during the washing step can be selected from the range of conditions limited by those of low stringency (with approximately 2×SSC at 50° C.) and of high stringency (with approximately 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). In addition, the temperature during the washing step can be raised from low-stringency conditions at room temperature, approximately 22° C., to more stringent conditions at approximately 65° C. Both parameters, the salt concentration and the temperature, can be varied simultaneously, and it is also possible for one of the two parameters to be kept constant and only the other to be varied. It is also possible to employ denaturing agents such as, for example, formamide or SDS during the hybridization. Hybridization in the presence of 50% formamide is preferably carried out at 42° C. Some exemplary conditions for hybridization and washing steps are given below:
(1) Hybridization Conditions with for Example


    
    
        a) 4×SSC at 65° C., or
        b) 6×SSC, 0.5% SDS, 100 μg/ml denatured fragmented salmon sperm DNA at 65° C., or
        c) 4×SSC, 50% formamide at 42° C., or
        d) 2× or 4×SSC at 50° C. (low-stringency condition), or
        e) 2× or 4×SSC, 30 to 40% formamide at 42° C. (low-stringency condition), or
        f) 6×SSC at 45° C., or,
        g) 0.05 M sodium phosphate buffer pH 7.0, 2 mM EDTA, 1% BSA and 7% SDS.

(2) Washing Steps with for Example

        a) 0.1×SSC at 65° C., or
        b) 0.1×SSC, 0.5% SDS at 68° C., or
        c) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C., or
        d) 0.2×SSC, 0.1% SDS at 42° C., or
        e) 2×SSC at 65° C. (low-stringency condition), or
        f) 40 mM sodium phosphate buffer pH 7.0, 1% SDS, 2 mM EDTA.
    
    


Methods for preparing functional equivalents of the invention preferably comprise the introduction of mutations into one of the promoters as shown in SEQ ID NO: 1, 2, 3, 4 or 44. Mutagenesis may be random, in which case the mutagenized sequences are subsequently screened for their properties by a trial-and-error procedure. Particularly advantageous selection criteria comprise for example the level of the resulting expression of the introduced nucleic acid sequence in a starch-comprising tissue.
“Expression” means the transcription of the nucleic acid sequence to be expressed transgenically, but can—in the case of an open reading frame in sense orientation—also include the translation of the transcribed RNA, of the nucleic acid sequence to be expressed transgenically, into a corresponding polypeptide.
“Transgenic” means—for example regarding a transgenic expression cassette, a transgenic expression vector, a transgenic organism or method for the transgenic expression of nucleic acids all —those constructs which are the result of transgenic methods, or all methods using them, in which either

    a) an SSS3 promoter (for example a promoter as shown in SEQ ID NO: 1, 2, 3, 4 or 44), or
    b) the nucleic acid sequence to be expressed transgenically, in functional linkage with the SSS3 promoter of a), or
    c) (a) and (b)

are not located in their natural genetic environment or have been modified by transgenic methods, where the modification can be for example a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Preferably, the SSS3 promoter sequence according to the invention which is present in the expression cassettes (for example as shown in SEQ ID NO: 1, 2, 3, 4 or 44) is heterologous with regard to the further nucleic acid sequence which is linked functionally with it and which is to be expressed transgenically. In this context, “heterologous” means that the further nucleic acid sequence does not encode the gene which is naturally under the control of said promoter.



“Natural genetic environment” means the natural chromosomal locus in the organism of origin or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, very especially preferably at least 5000 bp. A naturally occurring expression construct—for example the naturally occurring combination of the potato SSS3 promoter of SEQ ID NO: 1 or 44 and the coding sequence of the potato SSS3 gene becomes a transgenic expression construct when this combination is modified by nonnatural, synthetic (“artificial”) methods such as, for example, an in-vitro mutagenesis. Such methods have been described (U.S. Pat. No. 5,565,350; WO 00/15815; see also hereinabove).
“Transgenic” with regard to an expression (“transgenic expression”) preferably means all those expressions which have been carried out using a transgenic expression cassette, transgenic expression vector or transgenic organism, as defined hereinabove.
In transgenic expression cassettes according to the invention, at least one of the promoters according to the invention (for example described by SEQ ID NO: 1, 2, 3, 4 or 44) is in functional linkage with at least one nucleic acid sequence to be expressed transgenically.
A functional linkage means, for example, the sequential arrangement of one of the promoters according to the invention (for example described by SEQ ID NO: 1, 2, 3, 4 or 44) with a nucleic acid sequence to be expressed transgenically and, if appropriate, further genetic control sequences such as, for example, a terminator or a polyadenylation sequence in such a way that the promoter can fulfill its function under suitable conditions in the transgenic expression of the nucleic acid sequence and that expression of the nucleic acid sequence (i.e. transcription and, if appropriate, translation) takes place. “Suitable conditions” in this context preferably means the presence of the expression cassette in a plant cell, preferably a plant cell comprised by a starch-comprising tissue of a plant.
Preferred arrangements are those in which the nucleic acid sequence to be expressed transgenically is positioned behind one of the promoters according to the invention (e.g. described by SEQ ID NO: 1, 2, 3, 4 or 44) so that both sequences are linked covalently with one another. Preferably, the distance between the promoter sequence and the nucleic acid sequence to be expressed transgenically is less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
A functional linkage or a transgenic expression construct can be produced by means of conventional recombination and cloning techniques as are described, for example, in Maniatis T, Fritsch E F and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and in Silhavy T J, Berman M L and Enquist L W (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and in Ausubel F M et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience. A method which is suitable for this purpose is, for example, the GATEWAYTM cloning technology (Invitrogen Inc.), which is based on recombination.
However, it is possible, between promoter and nucleic acid sequence to be expressed transgenically, to position further sequences which have, for example, the function of a linker with certain restriction enzyme cleavage sites, or of a signal peptide. Also, the insertion of sequences may lead to the expression of fusion proteins.
A transgenic expression cassette according to the invention is prepared for example by fusing one of the promoters according to the invention as shown in SEQ ID NO: 1, 2, 3, 4 or 44 with a nucleotide sequence to be expressed transgenically, if appropriate with a sequence encoding a transit peptide, preferably a chloroplast-specific transit peptide, which is preferably arranged between the promoter and the nucleotide sequence in question, and optionally with a terminator or polyadenylation signal. Preferably, the transgenic expression construct, consisting of a linkage of promoter and nucleic acid sequence to be expressed, may be integrated in a vector and inserted into a plant genome by, for example, transformation.
However, an expression cassette also means those constructs in which one of the promoters of the invention (for example described by SEQ ID NO: 1, 2, 3, 4 or 44) is, without necessarily having been functionally linked beforehand to a nucleic acid sequence to be expressed, introduced into a host genome, for example by directed homologous recombination or random insertion, where it undertakes regulatory control over endogenous nucleic acid sequences then functionally linked thereto, and governs the transgenic expression thereof. Insertion of the promoter—for example by a homologous recombination—in front of a nucleic acid encoding for a particular polypeptide results in an expression cassette of the invention which governs the expression of the particular polypeptide selectively in a starch-comprising tissue. Also, for example, the natural promoter of an endogenous gene can be exchanged for one of the promoters according to the invention (for example described by SEQ ID NO: 1, 2, 3, 4 or 44) and the expression behavior of the endogenous gene can be modified thus.
Furthermore, the insertion of the promoter may also take place in such a way that antisense RNA to the nucleic acid coding for a particular polypeptide is expressed. This selectively downregulates or switches off the expression of the particular polypeptide in the starch-comprising tissue.
Analogously, it is also possible to place a nucleic acid sequence to be expressed transgenically—for example by homologous recombination—behind the sequence coding for one of the promoters according to the invention (for example described by SEQ ID NO: 1, 2, 3, 4 or 44), which is located in its natural chromosomal context so that an expression cassette according to the invention is obtained which controls the expression, in the starch-comprising tissue, of the nucleic acid sequence to be expressed transgenically.
The transgenic expression cassettes according to the invention may comprise further genetic control sequences. The concept of the genetic control sequences is to be understood broadly and means all those sequences which have an effect on the origin or the function of a transgenic expression cassette according to the invention. Genetic control sequences modify, for example, the transcription and translation in prokaryotic or eukaryotic organisms. Preferably, the transgenic expression cassettes according to the invention comprise a terminator sequence 3′-downstream from the particular nucleic acid sequence to be expressed transgenically as additional genetic control sequence, and, if appropriate, further customary regulatory elements, in each case functionally linked with the nucleic acid sequence to be expressed transgenically.
Genetic control sequences also comprise further promoters, promoter elements or minimal promoters which are capable of modifying expression-controlling properties. It is thus possible, by means of genetic control sequences, that for example tissue-specific expression takes place in addition in dependence on certain stress factors. Suitable elements are described, for example, for water stress, abscisic acid (Lam E and Chua N H (1991) J Biol Chem 266(26):17131-17135) and heat stress (Schoffl F et al. (1989) Mol Gen Genetics 217(2-3):246-53).
It is furthermore possible that further promoters which make possible transgenic expression in further plant tissues or in other organisms such as, for example, E. coli bacteria, are linked functionally with the nucleic acid sequence to be expressed. Suitable promoters are, in principle, all plant-specific promoters. Plant-specific promoters means in principle any promoter which is capable of controlling the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissue or plant cultures. In this context, the expression can be for example constitutive, inducible or development-dependent. Preferred are constitutive promoters, tissue-specific promoters, development-dependent promoters, chemically-inducible, stress-inducible or pathogen-inducible promoters. Such promoters are generally known to the skilled worker.
Further advantageous control sequences can be found for example in the promoters of gram-positive bacteria, such as amy and SPO2, or in the yeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
It is possible in principle for all natural promoters with their regulatory sequences like those mentioned above to be used for the method of the invention. It is additionally possible also for synthetic promoters to be used advantageously.
Genetic control sequences further comprise also the 5′-untranslated regions, introns or noncoding 3′ regions of genes such as, for example, the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (generally: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994)), preferably of the Arabidopsis thaliana genes with the gene locus At2g46720, At3g01980 and At1g63140. It can be shown that these regions may play a significant function in the regulation of gene expression. It has thus been shown that 5′-untranslated sequences are able to enhance transient expression of heterologous genes. An example of a translation enhancer which may be mentioned is the 5′ leader sequence from tobacco mosaic virus (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the like. They may in addition promote tissue specificity (Rouster J et al. (1998) Plant J 15:435-440). Conversely, the 5′-untranslated region of the opaque-2 gene suppresses expression. A deletion of the region in question leads to an increase in the gene activity (Lohmer S et al. (1993) Plant Cell 5:65-73).
In transgenic rice cells, the use of the Act1 intron in combination with the 35S promoter resulted in a ten-fold increased expression rate in comparison with the isolated 35S promoter (McElroy et al. (1991) Mol Gen Genet 231(1):150-160). An optimization of the sequence environment of the translation initiation site of the GUS reporter gene resulted in a four-fold increase in the GUS expression in transformed rice cells. A combination of the optimized translation initiation site and the Act1 intron resulted in a 40-fold increase in the GUS expression by the CaMV35S promoter in transformed rice cells; similar results were obtained using transformed maize cells. In total, the conclusion of the above-described studies was that the expression vectors based on the Act1 promoter are suitable for controlling a sufficiently strong and constitutive expression of foreign DNA in transformed cells of monocotyledonous plants.
The promoter sequences shown in SEQ ID NO: 2, 3 and 4 comprise the segment of the respective SSS3 genes which represents the promoter and the 5′-untranslated region up to before the ATG start codon of the SSS3 protein. The promoter sequences shown in SEQ ID: 1 and 44 comprise the promoter including 75 bp of the 5′-untranslated region of the cDNA.
The transgenic expression construct may advantageously comprise one or more so-called enhancer sequences, which make increased transgenic expression of the nucleic acid sequence possible, functionally linked to the promoter. Additional advantageous sequences such as further regulatory elements or terminators can also be inserted at the 3′ end of the nucleic acid sequences to be expressed transgenically. The nucleic acid sequences to be expressed transgenically may be present in one or more copies in the gene construct.
Polyadenylation signals suitable as control sequences are plant polyadenylation signals, preferably those which correspond essentially to T-DNA polyadenylation signals from Agrobacterium tumefaciens. In an especially preferred embodiment, the transgenic expression cassette comprises a terminator sequence which is functional in plants. The term terminator sequences which are functional in plants generally means those sequences which are capable of bringing about, in plants, the termination of the transcription of a DNA sequence. Examples of suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator. However, plant terminator sequences are especially preferred. The term plant terminator sequences generally means those sequences which are part of a natural plant gene. Especially preferred in this context are the terminator of the potato cathepsin D inhibitor gene (GenBank Acc. No.: X74985) or the terminator of the storage protein gene VfLE1B3 (GenBank Acc. No.: Z26489) from the field bean. These terminators are at least equivalent to the viral terminators or T-DNA terminators described in the prior art.
Control sequences additionally mean those which make homologous recombination or insertion into the genome of a host organism possible or allow deletion from the genome. In homologous recombination for example the natural promoter of a particular gene can be replaced by one of the promoters according to the invention. One of the promoters according to the invention—as described Above—can be placed, by homologous recombination, upstream of an endogenous target gene to be expressed transgenically by linking the promoter with DNA sequences which are homologous for example to endogenous sequences which precede the reading frame of the target gene. Such sequences are to be understood as genetic control sequences. Methods such as cre/lox technology allow tissue-specific, and in some circumstances inducible, deletion of the transgenic expression cassette from the genome of the host organism (Sauer B (1998) Methods (Duluth) 14(4):381-92). In this case, particular flanking sequences are attached to the target gene (lox sequences) and make later deletion by means of cre recombinase possible. To select cells which have successfully undergone homologous recombination or else transformation, it is, as a rule, necessary additionally to introduce a selectable marker (see hereinbelow). Homologous recombination is a relatively rare event in higher eukaryotes, especially in plants. Random integrations into the host genome predominate. One possibility of removing the randomly integrated sequences, and thus to accumulate cell clones with a correct homologous recombination, consists in the use of a sequence-specific recombination system as described in U.S. Pat. No. 6,110,736.
The transgenic expression, of the proteins encoded by the nucleic acid sequences, under the control of an SSS3 promoter is possible in any desired cell compartment such as, for example, the endomembrane system, the vacuole and the chloroplasts. Utilizing the secretory pathway, desired glycosylation reactions, specific folding patterns and the like are possible. The signal peptide sequences required as genetic control sequences for this purpose can either be provided in individual transgenic expression cassettes or else be introduced into the transgenic expression cassette by using a suitable cloning strategy together with the nucleic acid sequence to be expressed transgenically. Signal or transit peptide sequences which can be utilized are both homologous or heterologous sequences. Additional heterologous sequences which are preferred for functional linkage, but not restricted thereto, are further targeting sequences for ensuring the subcellular localization in the apoplast, in the vacuole, in plastids, in mitochondria, in the endoplasmic reticulum (ER), in the nucleus, in oil bodies or in other compartments; and also translation enhancers such as the tobacco mosaic virus 5′ leader sequence (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the like. The method for the directed transport into plastids of proteins which per se are not localized in the plastids is described (Klosgen R B and Weil J H (1991) Mol Gen Genet 225(2):297-304; Van Breusegem F et al. (1998) Plant Mol Biol 38(3):491-496). Preferred sequences are:

    a) the transit peptide of the SSS3 protein,
    b) the transit peptide of the small subunit (SSU) of the ribulose-bisphosphate carboxylase (Rubisco ssu) from, for example, pea, maize or sunflower,
    c) transit peptides derived from genes of plant fatty acid biosynthesis, such as the transit peptide of the plastidic “acyl carrier protein” (ACP), the stearyl-ACP desaturase, the β-ketoacyl-ACP synthase or the acyl-ACP thioesterase,
    d) the transit peptide of the GBSSI (“granule-bound starch synthase I”)
    e) the transit peptide of the LHCP II genes
    f) the transketolase transit peptide (EP-A1 0 723 017).


The target sequences can be linked with other targeting sequences which differ from sequences coding for the transit peptide in order to ensure a subcellular localization in the apoplast, in the vacuole, in plastids, in mitochondria, in the endoplasmic reticulum (ER), in the nucleus, in oil bodies or other compartments.
The transgenic expression cassettes according to the invention and the transgenic expression vectors derived from them may comprise further functional elements. The term functional element is to be understood broadly and means all those elements which have an effect on the generation, multiplication or function of the transgenic expression cassettes according to the invention or on transgenic expression vectors or organisms derived from them. The following may be mentioned by way of example, but not by limitation:
1) Selection Markers
The term “selection marker” comprises not only positive selection markers, which confer a resistance to an antibiotic, herbicide or other biocide, but also negative selection markers, which confer a sensitivity to precisely the abovementioned, and also markers which confer a growth advantage to the transformed organism (for example by expression of key genes of cytokine biosynthesis; Ebinuma H et al. (2000) Proc Natl Acad Sci USA 94:2117-2121). In the case of positive selection, only those organisms which express the selection marker in question thrive, while precisely these organisms die in the case of negative selection. The use of a positive selection marker is preferred in the generation of transgenic plants. Furthermore preferred is the use of selection markers which confer growth advantages. Negative selection markers can be used advantageously when the task at hand consists in eliminating certain genes or genome segments from an organism (for example for the purposes of a hybridization process).
i) Positive Selection Markers:
The selectable marker introduced with the transgenic expression cassette confers resistance to a biocide, for example a herbicide (such as phosphinothricin, glyphosate or bromoxynil), a metabolism inhibitor (such as 2-deoxyglucose-6-phosphate; WO 98/45456) or an antibiotic (such as, for example, tetracyclins, ampicillin, kanamycin, G 418, neomycin, bleomycin or hygromycin) to the successfully transformed cells. The selection marker permits the selection of the transformed cells from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84). Especially preferred selection markers are those which confer resistance to herbicides. Selection markers which may be mentioned by way of example are:

    
    
        DNA sequences which encode phosphinothricin acetyltransferases (PAT) (also referred to as Bialophos® resistance gene (bar)), which acetylate the free amino group of the glutamine synthase inhibitor phosphinothricin (PPT) and thus detoxify the PPT (de Block et al. (1987) EMBO J. 6:2513-2518; Vickers J E et al. (1996) Plant Mol Biol Reporter 14:363-368; Thompson C J et al. (1987) EMBO J. 6:2519-2523). The bar/PAT gene can be isolated for example from Streptomyces hygroscopicus or S. viridochromogenes. Such sequences are known to the skilled worker (Streptomyces hygroscopicus GenBank Acc. No.: X17220 and X05822; Streptomyces viridochromogenes GenBank Acc. No.: M22827 and X65195; U.S. Pat. No. 5,489,520). Synthetic genes are further described for expression in plastids (GenBank Acc. No.: AJ028212).
        5-Enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes), which confer resistance to Glyphosat® (N-(phosphonomethyl)glycine). The nonselective herbicide glyphosate has 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) as molecular target. This enzyme has a key function in the biosynthesis of aromatic amino acids in plants (Steinrucken H C et al. (1980) Biochem Biophys Res Commun 94:1207-1212; Levin J G and Sprinson D B (1964) J Biol Chem 239:1142-1150; Cole D J (1985) Mode of action of glyphosate; A literature analysis, pp. 48-74. In: Grossbard E and Atkinson D (eds.). The herbicide glyphosate. Buttersworths, Boston). Glyphosate-tolerant EPSPS variants are preferably used as selection markers (Padgette S R et al. (1996). New weed control opportunities: development of soybeans with a Roundup Ready gene. In: Herbicide Resistant Crops (Duke S O, ed.), pp. 53-84. CRC Press, Boca Raton, Fla.; Saroha M K and Malik V S (1998) J Plant Biochemistry and Biotechnology 7:65-72). The EPSPS gene of Agrobacterium sp. strain CP4 has a natural tolerance to glyphosate which can be transferred to corresponding transgenic plants. The CP4 EPSPS gene has been cloned from Agrobacterium sp. strain CP4 (Padgette S R et al. (1995) Crop Science 35(5):1451-1461). 5-Enolpyruvylshikimate-3-phosphate synthases which are glyphosate-tolerant, such as, for example, those described in U.S. Pat. No. 5,510,471; U.S. Pat. No. 5,776,760; U.S. Pat. No. 5,864,425; U.S. Pat. No. 5,633,435; U.S. Pat. No. 5,627,061; U.S. Pat. No. 5,463,175; EP 0 218 571, are preferred, the sequences described in the patents in each case also being deposited in GenBank. Further sequences are described under GenBank Accession X63374. The aroA gene (GenBank Acc. No.: M10947) is furthermore preferred.
        the gox gene (glyphosate oxidoreductase), which encodes the Glyphosat®-degrading enzymes. GOX (for example the glyphosate oxidoreductase from Achromobacter sp.) catalyzes the cleavage of a C—N bond in glyphosate, which is thus converted into aminomethylphosphonic acid (AMPA) and glyoxylate. GOX can thereby confer resistance to glyphosate (Padgette S R et al. (1996) J Nutr 126(3):702-16; Shah D et al. (1986) Science 233:478-481).
        the deh gene (encoding a dehalogenase which inactivates Dalapon®; WO 99/27116; GenBank Acc. No.: AX022822, AX022820)
        bxn genes, which encode Bromoxynil®-degrading nitrilase enzymes, for example the Klebsiella ozanenae nitrilase. Sequences can be found in GenBank for example under the Acc. Nos: E01313 and J03196.
        Neomycin phosphotransferases (npt) confer resistance to antibiotics (aminoglycosides) such as neomycin, G418, hygromycin, paromomycin or kanamycin, by reducing their inhibitory action by means of a phosphorylation reaction. Especially preferred is the nptII gene (GenBank Acc. No.: AF080390; AF080389). Moreover, the gene is already a component in a large number of expression vectors and can be isolated from them using methods with which the skilled worker is familiar (such as, for example, polymerase chain reaction) (GenBank Acc. No.: AF234316 pCAMBIA-2301; AF234315 pCAMBIA-2300, AF234314 pCAMBIA-2201). The NPTII gene encodes an aminoglycoside 3′-O-phosphotransferase from E. coli, Tn5 (GenBank Acc. No.: U000004 position 1401-2300; Beck et al. (1982) Gene 19 327-336).
        the DOGR1 gene was isolated from the yeast Saccharomyces cerevisiae (EP 0 807 836) and it encodes a 2-deoxyglucose-6-phosphate phosphatase, which confers resistance to 2-DOG (Randez-Gil et al. (1995) Yeast 11:1233-1240; Sanz et al. (1994) Yeast 10:1195-1202; GenBank Acc. No.: NC001140 position 194799-194056).
        Sulfonylurea- and imidazolinone-inactivating acetolactate synthases, which confer resistance to imidazolinone/sulfonylurea herbicides. Examples which may be mentioned of imidazolinone herbicides are the active substances imazamethabenzmethyl, imazzamox, imazapyr, imazaquin and imazethapyr. Examples of sulfonylurea herbicides which may be mentioned are amidosulforon, azimsulfuron, chlorimuronethyl, chlorsulfuron, cinosulfuron, imazosulforon, oxasulforon, prosulforon, rimsulforon, sulfosulforon. The skilled worker is familiar with a large number of further active substances from the abovementioned classes. The sequence for the Arabidopsis thaliana Csr 1.2 gene (EC 4.1.3.18), which has been deposited under the GenBank Acc. No.: X51514, is suitable for example (Sathasivan K et al. (1990) Nucleic Acids Res. 18(8):2188). Acetolactate synthases, which confer resistance to imidazolinone herbicides, are furthermore described under the GenBank Acc. Nos: AB049823, AF094326, X07645, X07644, A19547, A19546, A19545, 105376 (EP 0 257 993), 105373 (EP 0 257 993), AL133315.
        Hygromycin phosphotransferases (e.g. GenBank Acc. No.: X74325) which confer resistance to the antibiotic hygromycin. The gene is a component of a large number of expression vectors and can be isolated from them using methods with which the skilled worker is familiar (such as, for example, polymerase chain reaction) (GenBank Acc. No.: AF294981 pINDEX4; AF234301 pCAMBIA-1380; AF234300 pCAMBIA-1304; AF234299 pCAMBIA-1303; AF234298 pCAMBIA-1302; AF354046 pCAMBIA-1305; AF354045 pCAMBIA-1305.1)
        genes for resistance to
        
            a) chloramphenicol (chloramphenicol acetyltransferase),
            b) Tetracyclin; various resistance genes have been described, for example with GenBank Acc. Nos: X65876, X51366. In addition, the gene is already a component of a large number of expression vectors and can be isolated from these using methods known to the skilled worker (such as, for example, polymerase chain reaction)
            c) Streptomycin; various resistance genes have been described, for example with the GenBank Acc. No.: AJ278607.
            d) Zeocin; the corresponding resistance gene is a component of a large number of cloning vectors (for example GenBank Acc. No.: L36849 cloning vector pZEO) and can be isolated from these using methods known to the skilled worker (such as, for example, polymerase chain reaction).
            e) Ampicillin (β-lactamase gene; Datta N, Richmond M H. (1966) Biochem J 98(1):204-9; Heffron F et al. (1975) J. Bacteriol 122:250-256; the Amp gene was first cloned for generating the E. coli vector pBR322; Bolivar F et al. (1977) Gene 2:95-114). The sequence is a component of a large number of cloning vectors and can be isolated from them using methods known to the skilled worker (such as, for example, polymerase chain reaction).
        
        
        Genes such as the isopentenyl transferase from Agrobacterium tumefaciens (strain:PO22) (GenBank Acc. No.: AB025109). The ipt gene is a key enzyme of cytokine biosynthesis. Its overexpression facilitates the regeneration of plants (for example selection on cytokine-free medium). The method for utilizing the ipt gene has been described (Ebinuma H et al. (2000) Proc Natl Acad Sci USA 94:2117-2121; Ebinuma H et al. (2000) Selection of marker-free transgenic plants using the oncogenes (ipt, rol A, B, C) of Agrobacterium as selectable markers, In Molecular Biology of Woody Plants. Kluwer Academic Publishers).
        Various other positive selection markers which confer a growth advantage to the transformed plants over nontransformed plants, and methods for their use, have been described, inter alia, in EP-A 0 601 092. Examples which may be mentioned are β-glucuronidase (in conjunction with, for example, cytokinin glucuronide), mannose-6-phosphate isomerase (in conjunction with mannose), UDP-galactose 4-epimerase (in conjunction with, for example, galactose), with mannose-6-phosphate isomerase in conjunction with mannose being especially preferred.

ii) Negative Selection Markers

    
    


Negative selection markers make possible for example the selection of organisms with successfully deleted sequences which comprise the marker gene (Koprek T et al. (1999) The Plant Journal 19(6):719-726). When carrying out a negative selection, a compound which otherwise has no disadvantageous effect on the plant is converted into a compound which is disadvantageous, for example owing to the negative selection marker introduced into the plant. Genes which have a disadvantageous effect per se are furthermore suitable. Negative selection markers which may be mentioned by way of example, but not by limitation, are TK thymidine kinase (TK), diphtheria toxin A fragment (DT-A), the coda gene product encoding a cytosine deaminase (Gleave A P et al. (1999) Plant Mol Biol 40(2):223-35; Perera R J et al. (1993) Plant Mol Biol 23(4): 793-799; Stougaard J (1993) Plant J 3:755-761), the cytochrome P450 gene (Koprek et al. (1999) Plant J 16:719-726), genes encoding a haloalkane dehalogenase (Naested H (1999) Plant J 18:571-576), the iaaH gene (Sundaresan V et al. (1995) Genes & Development 9:1797-1810) or the tms2 gene (Fedoroff N V & Smith D L (1993) Plant J 3:273-289).
2) Reporter Genes
Reporter genes encode readily quantifiable proteins which, via their color or enzyme activity, allow an assessment of the transformation efficiency, the site or time of expression (see also Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1):29-44). Examples which may be mentioned are:

    
    
        “green fluorescent protein” (GFP) (Chui W L et al. (1996), Curr Biol 6:325-330; Leffel S M et al. (1997) Biotechniques 23(5):912-8; Sheen et al. (1995) Plant J 8(5):777-784; Haseloff et al. (1997) Proc Natl Acad Sci USA 94(6):2122-2127; Reichel et al. (1996) Proc Natl Acad Sci USA 93(12):5888-5893; Tian et al. (1997) Plant Cell Rep 16:267-271; WO 97/41228).
        Chloramphenicol transferase (Fromm et al. (1985) Proc Natl Acad Sci USA 82:5824-5828),
        Luciferase (Millar et al. (1992) Plant Mol Biol Rep 10:324-414; Ow et al. (1986) Science 234:856-859); allows detection via bioluminescence.
        β-Galactosidase, encodes an enzyme for which a variety of chromogenic substrates are available.
        β-Glucuronidase (GUS) (Jefferson et al. (1987) EMBO J. 6:3901-3907) or the uida gene, which encodes an enzyme for a variety of chromogenic substrates.
        R-Locus gene product: protein which regulates the production of anthocyanine pigments (red coloration) in plant tissue and thus makes possible the direct analysis of the promoter activity without addition of further auxiliary substances or chromogenic substrates (Dellaporta et al. (1988) In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium, 11:263-282).
        Tyrosinase (Katz et al. (1983) J Gen Microbiol 129:2703-2714), an enzyme which oxidizes tyrosine to DOPA and dopaquinone, which subsequently form melanin, which can be detected readily.
        Aequorin (Prasher et al. (1985) Biochem Biophys Res Commun 126(3):1259-1268), can be used in the calcium-sensitive bioluminescence detection.

3) Replication Origins

    
    


Replication origins ensure the multiplication of the transgenic expression cassettes or transgenic expression vectors according to the invention in, for example, E. coli or agrobacteria. Examples which may be mentioned are ORI (origin of DNA replication), the pBR322 ori or the P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Examples of replication origins which are functional in Agrobacterium which may be mentioned are pRK2, pRi, PVS1 or pSA.
4) Border Sequences
“Border sequences” (such as, for example, the right or left border of the T-DNA) make possible an agrobacteria-mediated transfer into plant cells for the transfer and integration into the plant genome.
5) Multiple Cloning Regions (MCS) Permit and Facilitate the Insertion of One or More Nucleic Acid Sequences
Also according to the invention are transgenic expression vectors which comprise the above-described transgenic expression cassettes. Vectors generally means structures which are capable of replication and which are preferably host-specific, and which make possible the uptake of nucleic acid sequences and their transfer into other cells. Examples of vectors can be plasmids, cosmids, phages, viruses or else agrobacteria. Vectors which are particularly suitable for the purposes of plant biotechnology are described hereinbelow. The transgenic expression cassettes can be inserted into the vector (preferably a plasmid vector) via a suitable restriction cleavage site. The resulting vector can first be introduced into E. coli and then amplified. Correctly transformed E. coli are selected, grown, and the recombinant vector is obtained by methods with which the skilled worker is familiar. Restriction analysis and sequencing can serve for verifying the cloning step. Preferred vectors are those which make possible a stable integration of the expression cassette into the host genome. In the present context, host genome means all of the hereditary information of the host and comprises by way of example not only the chromosomal DNA of the nucleus, but also the DNA of the plastids and mitochondria. However, insertion into the chromosomal DNA of the nucleus is preferred.
In an advantageous embodiment, the introduction of the transgenic expression cassette into a cell or an organism is effected by means of plasmid vectors.
In the context of the invention, “introducing” comprises all methods which are suitable for introducing a nucleic acid sequence (for example an expression cassette according to the invention) directly or indirectly into an organism (for example a plant) or a cell, compartment, tissue, organ or propagation material (for example seed or fruits) of the same or generating it therein. Comprised are direct and indirect methods. The introduction may lead to a transient or else stable presence of said nucleic acid sequence. Introducing comprises for example methods such as transfection, transduction or transformation. Depending on the host organism, the organisms used in the methods are cultured or grown in the manner with which the skilled worker is familiar.
The generation of a transformed organism (or of a transformed cell or tissue) requires that the DNA in question (for example the expression vector) or the RNA in question is inserted or introduced into the host cell in question. A multiplicity of methods is available for this procedure, which is referred to as transformation (or transduction or transfection); (Keown et al. (1990) Methods in Enzymology 185:527-537). Thus, for example, the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles. Also, the cell can be permeabilized chemi0cally, for example with polyethylene glycol, so that the DNA can enter the cell by diffusion. The DNA can also be introduced by protoplast fusion with other DNA-comprising units such as minicells, cells, lysosomes or liposomes. A further suitable method for introducing DNA is electroporation, where the cells are reversibly permeabilized by means of an electrical pulse. Suitable methods are described (for example by Bilang et al. (1991) Gene 100:247-250; Scheid et al. (1991) Mol Gen Genet 228:104-112; Guerche et al. (1987) Plant Science 52:111-116; Neuhause et al. (1987) Theor Appl Genet 75:30-36; Klein et al. (1987) Nature 327:70-73; Howell et al. (1980) Science 208:1265; Horsch et al. (1985) Science 227:1229-1231; DeBlock et al. (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).
Preferred vectors for E. coli are pQE70, pQE60 and pQE-9 (QIAGEN, Inc.); pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.). A large number of vectors for the transformation of mammals and other eukaryotic and prokaryotic organisms are known to the skilled worker. In principle, methods like those for the “direct” transformation of plant cells (see hereinbelow) are to be applied for the transformation of eukaryotic or prokaryotic cells. Especially preferred are methods such as the calcium-phosphate- or liposome-mediated transformation, or else electroporation.
Various methods and vectors for inserting genes into the genome of plants and for the regeneration of plants from plant tissues or plant cells are known (Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); White F F (1993) Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1, Engineering and Utilization, ed.: Kung and Wu R, Academic Press, 15-38; Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, vol. 1, Engineering and Utilization, ed.: Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225; Halford N G, Shewry P R (2000) Br Med Bull 56(1):62-73). These include for example those mentioned above. In plants, the described methods for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used here. Suitable methods are mainly the transformation of protoplasts by polyethylene-glycol-induced DNA uptake, calcium-phosphate-mediated transformation, DEAE-dextran-mediated transformation, liposome-mediated transformation (Freeman et al. (1984) Plant Cell Physiol. 29:1353ff; U.S. Pat. No. 4,536,475), biolistic methods with the gene gun (particle bombardment method; U.S. Pat. No. 5,100,792; EP-A 0 444 882; EP-A 0 434 616; Fromm M E et al. (1990) Bio/Technology 8(9):833-9; Gordon-Kamm et al. (1990) Plant Cell 2:603), electroporation, the incubation of dry embryos in DNA-comprising solution, electroporation (EP-A 290 395, WO 87/06614), microinjection (WO 92/09696, WO 94/00583, EP-A 0 331 083, EP-A 0 175 966) or other methods for the direct introduction of DNA (DE 4 005 152, WO 90/12096, U.S. Pat. No. 4,684,611). Physical methods of introducing DNA into plant cells are reviewed by Oard (1991) Biotech Adv 9:1-11.
In the case of these “direct” transformation methods, the plasmid used does not need to meet any particular requirements. Simple plasmids such as those of the pUC series, pBR322, M13mp series, pACYC184 etc. can be used. If intact plants are to be regenerated from transformed cells, the plasmid must bear an additional selectable marker gene.
In addition to these “direct” transformation techniques, a transformation can also be carried out by bacterial infection by means of Agrobacterium (for example EP 0 116 718), viral infection by means of viral vectors (EP 0 067 553; U.S. Pat. No. 4,407,956; WO 95/34668; WO 93/03161) or by means of pollen (EP 0 270 356; WO 85/01856; U.S. Pat. No. 4,684,611).
The strains Agrobacterium tumefaciens or Agrobacterium rhizo-genes, which are most frequently used for the agrobacterium transformation, comprise a plasmid (Ti or R1 plasmid) which is transferred to the plant after infection with Agrobacterium. A part of this plasmid, referred to as T-DNA (transferred DNA), is integrated into the genome of the plant cell. The transformation is preferably carried out by means of agrobacteria which comprise disarmed Ti plasmid vectors, exploiting their natural ability of transferring genes to plants (EP-A 0 270 355; EP-A 0 116 718). As an alternative, it is also possible to transfer binary vectors (mini Ti plasmids) to plants by means of Agrobacterium and to integrate them into their genome. Various binary vectors are known and in some cases commercially available such as, for example, pBIN19 (Bevan et al. (1984) Nucl Acids Res 12:8711f.; Clontech Laboratories, Inc. USA) or pSUN derivatives (SunGene GmbH & Co.KGaA; WO 02/00900). The expression cassette according to the invention can be inserted into these binary vectors and—as described hereinbelow—integrated into the plant genome.
The use of T-DNA for transformation of plant cells has been intensely studied and described (Horsch R B et al. (1985) Science 225:1229ff.; Fraley et al. (1983) Proc Natl Acad Sci USA 80: 4803-4807; Bevans et al. (1983) Nature 304:184-187; Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Kung S D and Wu R Ed., Academic Press, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225; EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam, Chapter V; and An et al. (1985) EMBO J. 4:277-287).
The DNA is transferred into the plant cell by coculturing plant explants with Agrobacterium tumefaciens or Agrobacterium rhizo-genes. Starting from infected plant material (e.g. leaf, root or stem parts, but also protoplasts or plant cell suspensions), it is possible to regenerate whole plants by using a suitable medium which may comprise, for example, antibiotics or biocides for selection of transformed cells. The plants obtained may then be screened for the presence of the introduced DNA, in this case the transgenic expression cassette of the invention. As soon as the DNA has integrated into the host genome, the corresponding genotype is normally stable and the corresponding insertion is also found again in subsequent generations. Normally, the integrated transgenic expression cassette comprises a selection marker which imparts to the transformed plant a resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-DOG or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin etc. The selection marker allows the selection of transformed cells from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84). The plants obtained may be cultivated and crossed in the common manner. Two or more generations should be cultured in order to ensure that the genomic integration is stable and hereditary.
Agrobacterium transformation is widely used for the transformation of dicots, but also increasingly applied to monocots (Toriyama et al. (1988) Bio/Technology 6: 1072-1074; Zhang et al. (1988) Plant Cell Rep 7:379-384; Zhang et al. (1988) Theor Appl Genet 76:835-840; Shimamoto et al. (1989) Nature 338:274-276; Datta et al. (1990) Bio/Technology 8: 736-740; Christou et al. (1991) Bio/Technology 9:957-962; Peng et al. (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao et al. (1992) Plant Cell Rep 11:585-591; Li et al. (1993) Plant Cell Rep 12:250-255; Rathore et al. (1993) Plant Mol Biol 21:871-884; Fromm et al. (1990) Bio/Technology 8:833-839; Gordon-Kamm et al. (1990) Plant Cell 2:603-618; D'Halluin et al. (1992) Plant Cell 4:1495-1505; Walters et al. (1992) Plant Mol Biol 18:189-200; Koziel et al. (1993) Biotechnology 11:194-200; Vasil I K (1994) Plant Mol Biol 25:925-937; Weeks et al. (1993) Plant Physiol 102:1077-1084; Somers et al. (1992) Bio/Technology 10:1589-1594; WO 92/14828; Hiei et al. (1994) Plant J 6:271-282).
Many strains of Agrobacterium tumefaciens are capable of transferring genetic material—for example the expression cassettes according to the invention—such as, for example, the strains EHA101[pEHA101], EHA105[pEHA105], LBA4404[pAL4404], C58C1[pMP90] and C58C1[pGV2260] (Hood et al. (1993) Transgenic Res 2:208-218; Hoekema et al. (1983) Nature 303:179-181; Koncz and Schell (1986) Gen Genet 204:383-396; Deblaere et al. (1985) Nucl Acids Res 13: 4777-4788).
If agrobacteria are used, the expression cassette is to be integrated into specific plasmids, either into a shuttle, or intermediate, vector, or into a binary vector. It is preferred to use binary vectors, which are capable of replication both in E. coli and in Agrobacterium. As a rule, they comprise a selection marker gene and a linker or polylinker flanked by the right and left T-DNA border sequence. They can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181-187). The Agrobacterium which acts as host organism in this case should already comprise a plasmid with the vir region. The latter is required for transferring the T-DNA to the plant cell. An Agrobacterium transformed thus can be used for the transformation of plant cells. The use of T-DNA for the transformation of plant cells has been studied and described intensively (EP-A 0 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam, Chapter V; An et al. (1985) EMBO J. 4:277-287). A variety of binary vectors are known and in some cases commercially available such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA; Bevan et al. (1984) Nucl Acids Res 12:8711), pBinAR, pPZP200 or pPTV.
Then, the agrobacteria which have been transformed with such a vector can be used in the known manner for the transformation of plants, in particular crop plants, such as, for example, oilseed rape, for example by bathing scarified leaves or leaf segments in an agrobacterial solution and subsequently culturing them in suitable media. The transformation of plants by agrobacteria is described (White F F (1993) Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S D Kung and R Wu, Academic Press, pp. 15-38; Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol. Plant Molec Biol 42:205-225). The transformed cells of the scarified leaves or leaf segments can then be used for regenerating transgenic plants in the known manner, and these transgenic plants have integrated the above-described expression systems according to the invention.
Stably transformed cells (i.e. those which comprise the introduced DNA integrated into the DNA of the host cell) can be selected from untransformed cells when a selectable marker is component of the DNA which has been introduced. By way of example, any gene which is capable of conferring a resistance to a biocide (for example an antibiotic or herbicide (see hereinabove) can be used as marker (see hereinabove). Transformed cells which express such a marker gene are capable of surviving in the presence of concentrations of a suitable biocide which kill an untransformed wild type. The selection marker permits the selection of transformed cells from untransformed cells (McCormick et al. (1986) Plant Cell Reports 5:81-84). The resulting plants can be bred and hybridized in the customary manner. Two or more generations should be cultured in order to ensure that the genomic integration is stable and hereditary.
As soon as a transformed plant cell has been generated, an intact plant can be obtained using methods with which the skilled worker is familiar. The starting materials here are, for example, callus cultures, single cells (for example protoplasts) or leaf disks (Vasil et al. (1984) Cell Culture and Somatic Cel Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press; Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, Academic Press). The formation of shoot and root can be induced in the known manner from this as yet undifferentiated callus cell biomass. The resulting plantlets can be planted out and grown. Relevant methods are described (Fennell et al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al. (1995) Plant Cell Rep. 14:273-278; Jahne et al. (1994) Theor Appl Genet 89:525-533).
The expression efficacy of the transgenically expressed nucleic acids can be determined for example in vitro by shoot meristem propagation using one of the above-described selection methods. Moreover, the expression, of a target gene, which has been modified in terms of type and level and the effect on the phenotype of the plant can be tested in greenhouse experiments, using test plants.
Another subject of the invention relates to transgenic organisms, transformed with at least one transgenic expression cassette according to the invention or one transgenic expression vector according to the invention, and to cells, cell cultures, tissues, parts—such as, for example in the case of plant organisms, leaves, roots and the like—or propagation material derived from such organisms.
Organism, starting organisms or host organisms are understood as meaning prokaryotic or eukaryotic organisms such as, for example, microorganisms or plant organisms. Preferred microorganisms are bacteria, yeasts, algae or fungi.
Preferred bacteria are bacteria of the genus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes or cyanobacteria, for example of the genus Synechocystis. 
Especially preferred are microorganisms which are capable of infecting plants and thus of transferring the cassettes according to the invention. Preferred microorganisms are those from the genus Agrobacterium and in particular the species Agrobacterium tumefaciens. 
Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia. 
Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora, Fusarium, Beauveria or further fungi described in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995) on page 15, Table 6.
Host or starting organisms which are preferred as transgenic organisms are, above all, plant organisms. “Plant organism” or cells derived therefrom generally comprises any cell, tissue, parts or propagation material (such as seeds or fruits) of an organism which is capable of photosynthesis. Included as plant organisms in the context of the invention are all genera and species of the higher and lower plants of the plant kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred.
In the context of the present invention, “plants” means all genera and species of the higher and lower plants of the Plant Kingdom. The term includes the mature plants, seed, shoots and seedlings, and parts, propagation material (for example tubers, seeds or fruits) derived therefrom, plant organs, tissues, protoplasts, callus cultures and other cultures, for example cell or callus cultures, and any other types of groups of plant cells which give functional or structural units. Mature plants means plants of any developmental stage beyond the seedling. Seedling means a young immature plant in an early developmental stage.
Plant organisms for the purposes of the invention are furthermore other photosynthetically active organisms such as, for example, algae, cyanobacteria and mosses. Preferred algae are green algae such as, for example, algae of the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Especially preferred are Synechocystis, Chlamydomonas and Scenedesmus. 
Included as plant organisms within the scope of the invention are all genera and species of the higher and lower plants of the plant kingdom. The mature plants, seed, tubers, beets/swollen tap roots, fruits, shoots and seedlings and also parts, propagation material and cultures, for example cell cultures, derived therefrom are also included. Mature plants means plants at any developmental stage beyond the seedling. Seedling means a young immature plant in an early developmental stage.
Annual, perennial, monocotyledonous and dicotyledonous plants are preferred host organisms for preparing transgenic plants. The expression of genes is furthermore advantageous in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawns. Plants which may be mentioned by way of example but not by limitation are angiosperms, bryophytes such as, for example, Hepaticae (liverworts) and Musci (mosses); pteridophytes such as ferns, horsetail and club mosses; gymnosperms such as conifers, cycades, ginkgo and Gnetalae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae(diatoms) and Euglenophyceae. 
Preference is given to plants of the following plant families: Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, rosacea, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae, Tetragoniacea, Theaceae, Umbelliferae.
Preferred monocotyledonous plants are in particular selected from the monocotyledonous crop plants, for example of the Gramineae family, such as rice, corn, wheat, or other cereal species such as barley, malt, rye, triticale or oats, and also sugar cane and all grass species.
Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, for example

    
    
        Asteraceae such as sunflower, Tagetes or Calendula and others,
        Compositae, particularly the genus Lactuca, very particularly the species sativa (lettuce), and others,
        Cruciferae, particularly the genus Brassica, very particularly the species napus (oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli), and further cabbage species; and the genus Arabidopsis, very particularly the species thaliana, and also cress or canola, and others,
        Cucurbitaceae such as melon, pumpkin or zucchini, and others,
        Leguminosae, particularly the genus Glycine, very particularly the species max (soybean), soya and also alfalfa, pea, bean plants or peanut, and others,
        Rubiaceae, preferably of the subclass Lamiidae, such as, for example, Coffea arabica or Coffea liberica (coffee bush), and others,
        Solanaceae, in particular the genus Lycopersicon, very particularly the species esculentum (tomato), and the genus Solanum, very particularly the species tuberosum (potato) and melongena (aubergine) and the genus Capsicum, very especially the species annum (pepper), and also tobacco, and others,
        Sterculiaceae, preferably of the subclass Dilleniidae, such as, for example, Theobroma cacao (cacao bush) and others,
        Theaceae, preferably of the subclass Dilleniidae, such as, for example, Camellia sinensis or Thea sinensis (tea bush) and others,
        Umbelliferae, particulary the genus Daucus, very particularly the species carota (carrot), and Apium, very particularly the species graveolens dulce (celery), and others;
        Chenopodiaceae, preferably the genus Beta vulgaris, in particular the species Beta vulgaris ssp. vulgaris var. altissima L. (sugar beet) and others;

and also linseed, cotton, hemp, flax, cucumber, spinach and the various tree, nut and vine species, in particular banana and kiwi fruit.

    
    


Most preferred are plants of the family Solanaceae, especially the genus Lycopersicon, very especially the species esculentum (tomato), the genus Solanum, very especially the species tuberosum (potato) and melongena (aubergine), of the family Chenopodiaceae, in particular the genus Beta vulgaris, in particular the species Beta vulgaris ssp. vulgaris var. altissima L. (sugar beet) and others, of the family Leguminosae, especially the genus Glycine, very especially the species max (soybean) and alfalfa, pea, bean plants, especially the genus Vicia or peanut and others, and other plants with starch-containing seeds, tubers, beets/swollen tap roots, fruits or tissues. Preferred among these, in turn, are tomato, potato, aubergine, soybean, alfalfa, pea, field bean, fodder beet, sugar beet and peanut.
Furthermore in accordance with the invention are cells, cell cultures, parts—such as, for example, in the case of transgenic plant organisms, roots, leaves and the like—which are derived from the above-described transgenic organisms, and transgenic propagation material such as seeds, tubers, beets/swollen tap roots or fruits. Genetically modified plants according to the invention which can be consumed by humans and animals can also be used as foodstuffs or feedstuffs, for example either directly or after processing known per se.
The skilled worker is familiar with a multiplicity of nucleic acids or proteins whose expression, controlled by the transgenic expression cassettes according to the invention, is advantageous. Furthermore, the skilled worker is familiar with a multiplicity of genes which, when repressed or switched off by means of transgenic expression, for example by means of a suitable double-stranded RNA or an antisense RNA, likewise allow advantageous effects to be achieved. Especially suitable target genes for the scope of the present invention are those which play a role in the sugar or starch metabolism, in sink/source relations, in the balance of organic acids, as flavor components, in the resistance to biotic stress factors (pathogens such as, for example, viruses, insects or fungi), in the resistance to abiotic stress factors (heat, cold, drought, increased moisture, environmental toxins, UV radiation), in the consistency of the tissues, or in water/pH conditions, in the improvement of nutritional or feed properties, the improvement of the germination and/or storage characteristics, and in the improvement of the growth rate or the yield.
Increasing the starch content is of particular interest in particular for tomato and potato. A normal tomato consists to approximately 80 to 95% of water, while starch—as the actually relevant component for the production of, for example, tomato paste, ketchup—is degraded during maturation and only amounts to a small percentage. Even a minor increase in the starch content (and thus the content of solubles) would be of considerable economic importance. The starch content of tomatoes is markedly higher during the early maturation stages, amounting to 20%, but then drops during the development as the result of starch mobilization and conversion into sugars. In the case of potatoes, an increased starch content has in particular an advantageous effect on the deep-frying characteristics.
The high activity of the SSS3 promoter during the early stages of tuber development (starch synthesis) is outstandingly suitable for influencing starch quality. Processing (chips, fries, dry products) is a promising field of application for practical potato breeding. Since criticism of germination inhibitors is on the increase, the method of choice would be storage at 4° C., during which, however, the undesired reduced sugars are formed (cold sweetening). Depending on the variety and the storage time, heating then results in undesirable brown, bitter-tasting Maillard products (probably also acrylamide from asparagin and reducing sugars). This is why the breeding aim “suppressing the formation of reducing sugars” is of great interest. The SSS3 promoters according to the invention could be used for example to suppress invertase activities, which are probably involved in the manifestation of the trait cold sweetening, as described by Menendez et al. (2002) Genetics 162:1423-14349. These gene activities can be suppressed for example by means of double-stranded RNA, cosuppression, antisense RNA or by expressing an invertase inhibitor.
Nucleic acid sequences whose expression under the control of one of the promoters according to the invention has advantageous effects may be mentioned below by way of example, but not by limitation:

    1. Improved protection of the plant from abiotic stress factors such as drought, heat or chill, for example by overexpressing antifreeze polypeptides from Myoxocephalus Scorpius (WO 00/00512), Myoxocephalus octodecemspinosus, the Arabidopsis thaliana transcription activator CBF1, glutamate dehydrogenases (WO 97/12983, WO 98/11240), calcium-dependent protein kinase genes (WO 98/26045), calcineurins (WO 99/05902), casein kinase from yeast (WO 02/052012), farnesyltransferases (WO 99/06580; Pei Z M et al. (1998) Science 282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology 17:192-196), oxalate oxidase (WO 99/04013; Dunwell J M (1998) Biotechn Genet Eng Rev 15:1-32), DREB1A factor (“dehydration response element B 1A”; Kasuga M et al. (1999) Nature Biotech 17:276-286), genes of mannitol or trehalose synthesis such as trehalose-phosphate synthase or trehalose-phosphate phosphatase (WO 97/42326) or by inhibiting genes such as trehalase (WO 97/50561).
    2. Expression of metabolic enzymes for use in the food-and-feed sector, for example of phytases and cellulases. Especially preferred are nucleic acids such as the artificial cDNA which encodes a microbial phytase (GenBank Acc. No.: A19451) or functional equivalents thereof.
    3. Achieving a resistance, for example to fungi, insects, nematodes and diseases, by targeted secretion or accumulation of certain metabolites or proteins. Examples which may be mentioned are antibodies to pathogen proteins, sucrose isomerase, glucosinolates (defense against herbivores), chitinases or glucanases and other enzymes which destroy the cell wall of parasites, ribosome-inactivating proteins (RIPs) and other proteins of the plant resistance and stress reaction as are induced when plants are wounded or attacked by microbes, or chemically, by, for example, salicylic acid, jasmonic acid or ethylene, or lysozymes from nonplant sources such as, for example, T4-lysozyme or lysozyme from a variety of mammals, insecticidal proteins such as Bacillus thuringiensis endotoxin, α-amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor), lectins such as wheatgerm agglutinin, RNAses or ribozymes. Further examples are nucleic acids which encode the Trichoderma harzianum chit42 endochitinase (GenBank Acc. No.: S78423) or the N-hydroxylating, multifunctional cytochrome P-450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.: U3.2624), or functional equivalents of these.


The accumulation of glucosinolates in food as protection from pests (Rask L et al. (2000) Plant Mol Biol 42:93-113; Menard R et al. (1999) Phytochemistry 52:29-35), the expression of Bacillus thuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33-37) or the protection against attack by fungi, by expression of chitinases, for example from beans (Broglie et al. (1991) Science 254:1194-1197), is advantageous.
The expression of synthetic cryIA(b) and cryIA(c) genes, which encode lepidoptera-specific Bacillus thuringiensis Δ-endotoxins can bring about a resistance to insect pests in various plants (Goyal R K et al. (2000) Crop Protection 19(5):307-312).
Further target genes which are suitable for pathogen defense comprise “polygalacturonase-inhibiting protein” (PGIP), thaumatine, invertase and antimicrobial peptides such as lactoferrin (Lee T J et al. (2002) J Amer Soc Horticult Sci 127(2):158-164).

    4. Expression of genes which bring about an accumulation of fine chemicals such as of tocopherols, tocotrienols, vitamin C or carotenoids. An example which may be mentioned is phytoene desaturase. Preferred are nucleic acids which encode the Narcissus pseudonarcissus phytoene desaturase (GenBank Acc. No.: X78815) or functional equivalents thereof. For example, the production of vitamin C can be modified and increased via the expression of GDP-mannose 3′,5′-epimerase (WO 02/103001). The carotenoid content in plants, for example potatoes, can be increased by expressing a protein which has the enzymatic activity of a zeaxanthin epoxidase (WO 02/103021).
    5. Production of nutraceuticals such as, for example, polyunsaturated fatty acids (for example arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid) by expression of fatty acid elongases and/or desaturases, or production of proteins with improved nutritional value such as, for example, with a high content of essential amino acids (for example the high-methionine 2S albumin gene of the Brazil nut). Preferred are nucleic acids which encode the Bertholletia excelsa high-methionine 2S albumin (GenBank Acc. No.: AB044391), the Physcomitrella patens Δ6-acyl-lipid desaturase (GenBank Acc. No.: AJ222980; Girke et al. (1998) Plant J 15:39-48), the Mortierella alpina Δ6-desaturase (Sakuradani et al. 1999 Gene 238:445-453), the Caenorhabditis elegans Δ5-desaturase (Michaelson et al. 1998, FEBS Letters 439:215-218), the Caenorhabditis elegans Δ5-fatty acid desaturase (des-5) (GenBank Acc. No.: AF078796), the Mortierella alpina Δ5-desaturase (Michaelson et al. JBC 273:19055-19059), the Caenorhabditis elegans Δ6-elongase (Beaudoin et al. 2000, PNAS 97:6421-6426), the Physcomitrella patens Δ6-elongase (Zank et al. 2000, Biochemical Society Transactions 28:654-657), or functional equivalents of these.


The overexpression of glutamate dehydrogenase (GDH), for example, results in an accumulation of free amino acids and an improved yield in transgenic potatoes (WO 03/000041). A production of polyfructans (levans) in plants can be achieved by overexpressing a bacterial sucrase in transgenic plants (U.S. Pat. No. 6,501,005). The content in, and the structure of, sterol glycosides has been modified by expressing a sterol glucosyltransferase (U.S. Pat. No. 6,498,239).

    6. Production of high-quality proteins and enzymes for industrial purposes (for example enzymes, such as lipases) or as pharmaceuticals (such as, for example, antibodies, blood clotting factors, interferons, lymphokins, colony stimulation factor, plasminogen activators, hormones or vaccines, as described by Hood E E, Jilka J M (1999) Curr Opin Biotechnol 10(4):382-6; Ma J K, Vine N D (1999) Curr Top Microbiol Immunol 236:275-92). For example, it has been possible to produce recombinant avidin from chicken albumen and bacterial β-glucuronidase (GUS) on a large scale in transgenic maize plants (Hood et al. (1999) Adv Exp Med Biol 464:127-47). The expression of monoclonal antibodies against cariogenic organisms in edible fruits of plants is described in the patent WO 02/102975.
    8. Obtaining an increased storability in cells which normally comprise fewer storage proteins or storage lipids, with the purpose of increasing the yield of these substances, for example by expression of an acetyl-CoA carboxylase. The expression of an acetyl-CoA carboxylase leads to a modification of the quality and quantity with regard to the oil and fatty acid production in plants (WO 94/17188). Preferred nucleic acids are those which encode the Medicago sativa acetyl-CoA carboxylase (accase) (GenBank Acc. No.: L25042), or functional equivalents thereof. Further examples are described in Herbers & Sonnewald (1999) Curr Opinion Biotechnol 10 163-168; Biesgen & Herbers (2000) J Plant Biotechnol 2:1-12; Biesgen et al. (2002) Phytochemistry Reviews 1:79-85.
    9. Modification of the carbohydrate metabolism:
     The SSS3 promoter can be used for example for expressing a starch synthase from wheat in order to modify the content in, or composition of, plant starch, which can be used for the optimization of foodstuffs, the development of coatings, adhesives or packaging materials, and applications in the construction industry (WO 00/66745). Further possible applications are described in DE06483010 and WO 02/086112. The SSS3 promoter is particularly suitable for reducing the accumulation of sugars as the result of the expression of a phosphofructose kinase. This can be exploited in particular during the storage of potatoes at low temperatures to suppress what is known as cold sweetening (U.S. Pat. No. 6,489,539). As the result of the expression or repression of plant sugar transporters, the carbohydrate metabolism is modulated (WO 02/0199217). Further advantageous examples are invertase inhibitor (Börnke et al. (1999) Nature Biotech 17:708-711), sucrose isomerase (Börnke et al. (2002) Planta 214:356-364, starch-related R1 protein (Lorberth et al. (1998) Nature Biotechnology 16(5):473-7, Ritte et al. (2000) Plant Journal 21(4):387-91).
    10. The SSS3 promoter can also be used for expressing proteins in starch-comprising tissues, such as, for example, potato tubers, to increase the nutritional value (Chakraborty et al. (2000) Proc Natl Acad Sci USA 97:3724-3729). As the result of the expression of an endo-1,4-β-D-galactanase, pectin can be modified (Sørensen et al. (2000) Proc Natl Acad Sci USA 97: 7639-7644). For example, the content of storage substances, such as lipids, fatty acids, starch or seed proteins, can be modulated as the result of the expression of lipid metabolism proteins (LMPS) (WO 02/099076). The starch content and the yield can be increased as the result of overexpression of the plastidic adenylate kinase in transgenic potato plants (Regierer et al. (2002) Nature Biotechnology 20:1256).
    11. The SSS3 promoter can be used for expressing xylanase to control the growth, dying down and fruit maturation of plants (U.S. Pat. No. 6,495,743).


Further examples of advantageous genes are mentioned for example in Dunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000; 51 Spec No; pages 487-96.
Furthermore, it is possible to express functional analogs of the abovementioned nucleic acids or proteins. In this context, functional analogs means all those sequences which have essentially the same function, i.e. which are capable of exerting the function (for example a substrate conversion or a signal transduction), as the protein mentioned by way of example. In this context, the functional analog may indeed differ with regard to other features. For example, it may have a higher or lower activity or else have further functionalities. Functional analogs furthermore means sequences which encode fusion proteins consisting of one of the preferred proteins and other proteins, for example a further preferred protein, or else a signal peptide sequence.
Furthermore, the skilled worker knows that the above-described genes need not be expressed directly using the nucleic acid sequences encoding these genes or repress these above-described genes, for example by antisense. It is also possible to use for example artificial transcription factors of the zinc finger protein type (Beerli R R et al. (2000) Proc Natl Acad Sci USA 97(4):1495-500). These factors attach in the regulatory regions of the endogenous genes to be expressed or to be repressed and, depending on the design of the factor, bring about an expression or repression of the endogenous gene. Thus, the desired effects can also be achieved by expressing a suitable zinc finger transcription factor under the control of one of the promoters according to the invention. By expressing certain Myb transcription factors, flavonoid biosynthesis can be modulated, for example (Moyano et al. (1996) Plant Cell 8:1519-32). Further examples of the regulation of the secondary metabolism are described (Memelink (2001) Advances in Biochemical Engineering-Biotechnology 72:103-25).
Likewise, the transgenic expression cassettes according to the invention can be employed for the reduction (suppression) of transcription and/or translation of target genes by “gene silencing”. Thus, the transgenic expression cassettes according to the invention can express nucleic acids which bring about PTGS (post-transcriptional gene silencing) or TGS (transcriptional silencing) effects and thus a reduction of the expression of endogenous genes. Said reduction can be achieved for example by expression of an antisense RNA (EP-A1 0 458 367; EP-A1 0 140 308; van der Krol A R et al. (1988) BioTechniques 6(10):658-676; de Lange P et al. (1995) Curr Top Microbiol Immunol 197:57-75, inter alia) or of a double-stranded RNA, each of which has homology with the endogenous target gene to be reduced. Also, the expression of a suitable sense RNA can bring about a reduction of the expression of endogenous genes, by means of what is known as cosuppression (EP-A1 0 465 572). Especially preferred is the expression of a double-stranded RNA for reducing the gene expression of a target gene. WO 99/32619 and WO 99/53050 describe methods for inhibiting individual target genes using an RNA with double-stranded structure, where the target gene and the region of the RNA duplex have at least partial identity (see also: Montgomery M K et al. (1998) Proc Natl Acad Sci USA 95:15502-15507; Sharp P A (1999) Genes & Development 13(2):139-141; Fire A et al. (1998) Nature 391:806-11). The method is currently also referred to as RNA interference (RNAi).
Preferred applications where the reduction (suppression) of gene expression brings about an advantageous phenotype comprise by way of example, but not by limitation:

    1. Modification of the Carbohydrate Composition


A modification of the carbohydrate composition can be achieved for example by reducing the gene expression of genes of the carbohydrate metabolism or of carbohydrate biosynthesis, for example the biosynthesis of amylose, pectins, cellulose or cell-wall carbohydrates. A multiplicity of cellular processes (maturation, starch composition, starch content and the like) can thus be influenced in an advantageous manner. Target genes which may be mentioned by way of example, but not by limitation, are phosphorylases, starch synthetases, branching enzymes, lipoxygenases (Griffiths A. et al. (1999) Postharvest Biology & Technology 17(3):163-173), debranching enzymes, and various amylases. The genes in question are described (Dunwell J M (2000) J Exp Botany 51 Spec No: 487-96; Brar D S et al. (1996) Biotech Genet Eng Rev 13:167-79; Kishore G M and Somerville C R (1993) Curr Opin Biotech 4(2):152-8). Advantageous genes for influencing the carbohydrate metabolism—in particular starch biosynthesis—are described in WO 92/11375, WO 92/11376, U.S. Pat. No. 5,365,016 and WO 95/07355 and WO 02/097101. The fusion of an SSS3 promoter with a sequence, in antisense orientation, of one or both subunits of the ADP-glucose pyrophosphorylase is capable of inhibiting the activity of the latter during the early development of fruits and tubers and of increasing the ratio between soluble sugars and starch.
In a further advantageous embodiment, a shift of the amylose/amylopectin ratio in starch can be brought about by suppression of the two isoforms of the branching enzyme which are responsible for the α-1,6-glycosidic linkage. Such procedures are described (for example by Schwall G P et al. (2000) Nat Biotechnol 18(5):551-554). Nucleic acid sequences such as that of the potato starch branching enzyme II (GenBank Acc. No.: AR123356; U.S. Pat. No. 6,169,226) or its homologs from other genera and species are preferably used for this purpose. A further application is the suppression of endogenous activities (enzymes, signal transduction, phytohormone and the like) by immunomodulation.
Especially advantageous is the reduction of starch mobilization and conversion into sugars at low temperatures (cold sweetening) by means of reducing the expression of glucan phosphorylase (systematic name: 1,4-α-D-glucan:phosphate α-D-glucosyltransferase; U.S. Pat. No. 5,998,710).

    2. Delayed Fruit Maturation


Delayed fruit maturation or a modified maturation phenotype (prolonged maturation, later senescence) can be achieved for example by reducing the gene expression of genes selected from the group consisting of polygalacturonases, pectin esterases, β-(1,4)glucanases (cellulases), β-galactanases (β-galactosidases), or genes of ethylene biosynthesis, such as 1-aminocyclopropane-1-carboxylate synthase, adenosylmethionine hydrolase (SAMase), aminocyclopropane-1-carboxylate deaminase, aminocyclopropane-1-carboxylate oxidase, genes of carotenoid biosynthesis such as, for example, genes of pre-phytoene biosynthesis or phytoene biosynthesis, for example phytoene desaturases, and O-methyltransferases, acyl carrier protein (ACP), elongation factor, auxin-induced gene, cysteine(thiol) proteinases, starch phosphorylases, pyruvate decarboxylases, chalcone reductases, protein kinases, auxin-related gene, sucrose transporters, meristem pattern gene. Further advantageous genes are described for example in WO 91/16440, WO 91/05865, WO 91/16426, WO 92/17596, WO 93/07275 or wo 92/04456. Especially preferred is the reduction of the expression of polygalacturonase for the prevention of cell degradation and mushiness of plants and fruits, for example tomatoes. Nucleic acid sequences such as that of the tomato polygalacturonase gene (GenBank Acc. No.: X14074) or its homologs are preferably used for this purpose.

    3. Improved protection against abiotic stress factors (heat, chill, drought, elevated moisture, pollutants, UV radiation). It is preferred to reduce the expression of genes which are implicated in stress reactions.
    4. Reduction of the Storage Protein Content


The reduction of the gene expression of genes encoding storage proteins (hereinbelow SPs) has numerous advantages, such as, for example, the reduction of the allergenic potential or modification regarding the composition or quantity of other metabolites, such as, for example, oil or starch content.

    5. Obtaining a Resistance to Plant Pathogens


Resistance to plant pathogens such as arachnids, fungi, insects, nematodes, protozoans, viruses, bacteria and diseases can be achieved by reducing the gene expression of genes which are essential for the growth, survival, certain developmental stages (for example pupation) or the multiplication of a specific pathogen. Such a reduction can bring about a complete inhibition of the abovementioned steps, or else a delay of same. They can take the form of plant genes which for example make possible the penetration of the pathogen, but may also be homologous pathogen genes. The transgenically expressed nucleic acid sequence (for example the double-stranded RNA) is preferably directed against genes of the pathogen. The antipathogenic agent which acts may be, in this context, the transgenically expressed nucleic acid sequence itself (for example the double-stranded RNA), but also the transgenic expression cassettes or transgenic organisms. The plants themselves, in the form of a transgenic organism, may contain the agents and pass them on to the pathogens, for example in the form of a stomach poison. Various essential genes of a variety of pathogens are known to the skilled worker (for example for nematode resistance WO 93/10251, WO 94/17194). Virus resistance can be achieved for example by reducing the expression of a viral coat protein, a viral replicase, a viral protease and the like. A large number of plant viruses and suitable target genes are known to the skilled worker.

    8. Reduction of undesired, allergenic or toxic plant constituents such as, for example, glucosinolates. Suitable target genes are described (in WO 97/16559, inter alia). The target genes which are preferred for reduction of 14-16 kDa allergenic proteins are described for example by Tada Y et al. (1996) FEBS Lett 391(3):341-345 or Nakamura R (1996) Biosci Biotechnol Biochem 60(8):1215-1221.
    9. Delayed signs of senescence. Suitable target genes are, inter alia, cinnamoyl-CoA:NADPH reductases or cinnamoyl-alcohol dehydrogenases. Further target genes are described (in WO 95/07993, inter alia).
    10. Reduction of the susceptibility to bruising of, for example, potatoes by reducing for example polyphenol oxidase (WO 94/03607) and the like.
    11. Increase of the methionine content by reducing threonine biosynthesis, for example by reducing the expression of threonine synthase (Zeh M et al. (2001) Plant Physiol 127(3):792-802).


Antisense nucleic acid firstly means a nucleic acid sequence which is fully or in part complementary to at least part of the sense strand of said target protein. The skilled worker knows that an alternative is the use of the cDNA or the corresponding gene as starting template for suitable antisense constructs. Preferably, the antisense nucleic acid is complementary to the coding region of the target protein or part of same. However, the antisense nucleic acid may also be complementary to the noncoding region or part of same. Starting from the sequence information of a target protein, an antisense nucleic acid can be of designed in the manner with which the skilled worker is familiar, taking into consideration the Watson-Crick base pair rules. An antisense nucleic acid can be complementary to all or part of the nucleic acid sequence of a target protein. In a preferred embodiment, the antisense nucleic acid is an oligonucleotide with a length of, for example, 25, 30, 35, 40, 45 or 50 nucleotides.
The antisense strategy can advantageously be combined with a ribozyme method. Ribozymes are catalytically active RNA sequences which, when linked with the antisense sequences, catalytically cleave the target sequences (Tanner N K (1999) FEMS Microbiol Rev 23(3):257-75). The efficiency of an antisense strategy may thereby be increased. The expression of ribozymes for reducing certain proteins is known to the skilled worker and described for example in EP-A1 0 291 533, EP-A1 0 321 201 and EP A1 0 360 257. Suitable target sequences and ribozymes can be identified for example as described by Steinecke (Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460) by calculating the secondary structures of ribozyme and target RNA and also by their interaction (Bayley C C et al. (1992) Plant Mol Biol 18(2):353-361; Lloyd A M and Davis R W et al. (1994) Mol Gen Genet 242(6):653-657). Examples which may be mentioned are hammerhead ribozymes (Haselhoff and Gerlach (1988) Nature 334:585-591). Preferred ribozymes are based on derivatives of the Tetrahymena L-19 IVS RNA (U.S. Pat. Nos. 4,987,071; 5,116,742). Further ribozymes with selectivity for an L119 mRNA can be selected (Bartel D and Szostak J W (1993) Science 261:1411-1418).
Also comprised is the use of the above-described sequences in sense orientation which, as the skilled worker will know, can lead to cosuppression. The expression of sense RNA to an endogenous gene can reduce or eliminate expression thereof, in a similar manner to what has been described for antisense approaches (Goring et al. (1991) Proc Natl Acad Sci USA, 88:1770-1774; Smith et al. (1990) Mol Gen Genet 224:447-481; Napoli et al. (1990) Plant Cell 2:279-289; Van der Krol et al. (1990) Plant Cell 2:291-299). In this context, the construct introduced may represent the gene to be reduced either fully or only in part. No possibility of translation is necessary.
Also very especially preferred is the use of methods such as gene regulation by means of double-stranded RNA (double-stranded RNA interference). Such methods are known to the skilled worker and described in detail (for example Matzke M A et al. (2000) Plant Mol Biol 43:401-415; Fire A. et al. (1998) Nature 391:806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). The methods and processes described in the references cited are expressly referred to. Here, a highly efficient suppression of native genes is brought about by the simultaneous introduction of strand and counterstrand.
A further subject of the invention relates to the use of the above-described transgenic organisms according to the invention and of the cells, cell cultures, parts—such as, for example, roots, leaves and the like in the case of transgenic plant organisms—and transgenic propagation material, such as seeds or fruits, derived from these transgenic organisms for the production of foodstuffs or feedstuffs, pharmaceuticals or fine chemicals.
Preference is further given to a method for the recombinant production of pharmaceuticals or fine chemicals in host organisms, in which a host organism is transformed with one of the above-described expression cassettes and said expression cassette contains one or more structural genes which code for the fine chemical of interest or catalyze the biosynthesis of the fine chemical of interest, and the transformed host organism is cultivated and the fine chemical of interest is isolated from the cultivation medium. This method is broadly applicable for fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, natural and synthetic flavorings, aromatizing substances and colorants. Particular preference is given to the production of tocopherols and tocotrienols and also carotenoids such as astaxanthin. Cultivation of the transformed host organisms and isolation from said host organisms or from the cultivation medium are carried out by means of the methods known to the skilled worker. The production of pharmaceuticals such as, for example, antibodies or vaccines is described in Hood E E & Jilka J M (1999) Curr Opin Biotechnol 10(4):382-6; Ma J K & Vine N D (1999) Curr Top Microbiol Immunol 236:275-92.
SEQUENCES


    1. SEQ ID NO: 1 Promoter of the SSS3 gene from potato (Solanum tuberosum)
    2. SEQ ID NO: 2 Promoter of the SSS3 gene from Arabidopsis thaliana 
    3. SEQ ID NO: 3 Promoter of the SSS3 gene from rice (Oryza sativa)
    4. SEQ ID NO: 4 Promoter of the SSS3 gene from wheat (Triticum aestivum)
    5. SEQ ID NO: 5 Nucleic acid sequence coding for starch synthase 3 (SSS3) from potato (Solanum tuberosum)
    6. SEQ ID NO: 6 Amino acid sequence coding for starch synthase 3 (SSS3) from potato (Solanum tuberosum)
    7. SEQ ID NO: 7 Nucleic acid sequence coding for starch synthase 3 (SSS3) from Arabidopsis thaliana 
    8. SEQ ID NO: 8 Amino acid sequence coding for starch synthase 3 (SSS3) from Arabidopsis thaliana 
    9. SEQ ID NO: 9 Nucleic acid sequence coding for starch synthase 3 (SSS3) from rice (Oryza sativa)
    10. SEQ ID NO: 10 Amino acid sequence coding for starch synthase 3 (SSS3) from rice (Oryza sativa)
    11. SEQ ID NO: 11 Nucleic acid sequence coding for starch synthase 3 (SSS3) from wheat (Triticum aestivum)
    12. SEQ ID NO: 12 Amino acid sequence coding for starch synthase 3 (SSS3) from wheat (Triticum aestivum)
    13. SEQ ID NO: 13 Nucleic acid sequence coding for starch synthase 3 (SSS3) from wheat (Aegilops tauschii)
    14. SEQ ID NO: 14 Amino acid sequence coding for starch synthase 3 (SSS3) from wheat (Aegilops tauschii)
    15. SEQ ID NO: 15 Nucleic acid sequence coding for starch synthase 3 (SSS3) from asparagus bean (Vigna unguiculata)
    16. SEQ ID NO: 16 Amino acid sequence coding for starch synthase 3 (SSS3) from asparagus bean (Vigna unguiculata)
    17. SEQ ID NO: 17 Nucleic acid sequence coding for starch synthase 3 (SSS3) from maize (Zea mays)
    18. SEQ ID NO: 18 Amino acid sequence coding for starch synthase 3 (SSS3) from maize (Zea mays)
    19-39. SEQ ID NO: 19 to 39: Sequence motifs for starch synthase 3 proteins
    40. SEQ ID NO: 40 Oligonucleotide primer R-DSS3-263 (27-mer)












5′-TGCATTGGAGACACTTGTGCAACTCAA-3′







    41. SEQ ID NO: 41 Oligonucleotide primer R-DSS3-317 (27-mer)












5′-TGTGGTTCCATGAGAGACAAACCCAAG-3′







    42. SEQ ID NO: 42 Oligonucleotide primer L-DS3 (44-mer)










5′-GTCGACTCTAGAGGAAGAAATCTTCTCTGTCTAAAAAATTGACG-3′







    43. SEQ ID NO: 43 Oligonucleotide primer R-DS3: (38-mer)












5′-CCCGGGATCCTCTCTCCCTCTCTGTATCTGTGCTGCAA-3′







    44. SEQ ID NO: 44 Promoter of the SSS3 gene from potato (Solanum tuberosum; polymorphous to SEQ ID NO: 1)
    45. SEQ ID NO: 45 Oligonucleotide primer 5′actin AC1 (23-mer)












5′-ATGGCAGACGGTGAGGATATTCA-3′







    46. SEQ ID NO: 46 Oligonucleotide primer 3′actin AC2 (23-mer)












5′-GCCTTTGCAATCCACATCTGTTG-3′







    47. SEQ ID NO: 47 Oligonucleotide primer L-SSS3-G50 (24-mer)












5′-GCAGCACAGATACAGAGAGGGAGA-3′







    48. SEQ ID NO: 48 Oligonucleotide primer R-GUS-G809 (22-mer)












5′-TGGCTGTGACGCACAGTTCATA-3′










DESCRIPTION OF THE FIGURES
1. FIG. 1:
A: Detection by means of Xgluc staining of the glucuronidase activity expressed under the control of the potato SSS3 promoter in tubers of lines 1, 15 and 17. This correlates with the presence of starch in this tissue (FIG. 1B).
B: Detection of the correlation of the glucuronidase activity expressed under the control of the potato SSS3 promoter and the starch production (X-Gluc staining: section on the left; starch staining: section on the right) of potato tubers of line 13.
2. FIG. 2: GUS activity in tubers of 5 potato lines (2, 12, 14, 15 and 17) transformed with SSS3 promoter: GUS construct. L: leaves; G: developing tubers; R2: 45 days storage at room temperature; R3: 90 days storage at room temperature.
3. FIG. 3: PCR amplification of the cDNA (primer in the 5′-untranslated region of the SSS3 promoter and in the GUS gene) H2O: Negative control; pDSSS-Bi-Hp4: plasmid DNA as positive control, cDNA after 45 days' (left) and after 90 days' (right) storage of the tubers of lines 2, 9, 12, 15 and 17; cDNA solara: wild-type control
4. FIG. 4: Green fruits of lines 1 (A), 2 (B), 6 (C), 21 (D), 30 (E), WT (F), orange fruit of line 23 (G), blue-stained seeds (H), unstained sections across petioles (I), bud with stained anthers (J) and flower (K) and stained pollen (L) of tomato plants transformed with pSun0301_SSS3
5. FIG. 5: GUS activity in the green fruits of tomato lines, transformed with pSun0301_SSS3. Error bars show in each case the standard error
6. FIG. 6: Comparison of the GUS activity in selected lines of transgenic tomato plants (white bars: green fruits, black bars: red fruits; error bars show in each case the standard error).
7. FIG. 7a-g: Alignment of SSS3 protein sequences from potato (STSSS3; SEQ ID NO: 6), Arabidopsis thaliana (AtSSS3; SEQ ID NO: 8), asparagus bean (Vigna unguiculata; VUSSS3;SEQ ID NO: 16), Aegilops tauschii (AtaSSS3; SEQ ID NO: 14), wheat (Triticum aestivum; TASSS3; SEQ ID NO: 12), rice (OS SSS3; SEQ ID NO: 49), maize (ZM SSS3; SEQ ID NO: 18). Identical amino acids are shown against a gray background and emphasized by the additionally shown consensus sequence.



EXAMPLES
General Methods
Recombinant DNA techniques were carried out as described by Maniatis et al., Molecular Cloning—A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., 1982). The enzymes employed were used as specified. The cloning vectors used were pCR2.1 and pCR-BLUNT (Invitrogen). The vectors pBI101 (Jefferson et al. (1987) EMBO J. 6:3901-3907) and pSUN3 (SunGene GmbH & Co KgaA, WO 02/00900) were employed for the transformation of plants. Strain DH5α (Hanahan D (1983) J Mol Biol 166:557-580) was used for the transformation into E. coli. The agrobacteria LBA4404 and C58C1 [pGV2260] strains were transformed directly by means of the freeze-thaw method as described by An G (1987) Mol Gen Genet 207:210-216.
Example 1
Transformation of Potato and Tomato
1.1 Potato
20 small leaves of a sterile potato culture which had been scarified using a surgical blade were placed into 10 ml of MS medium supplemented with 2% sucrose and comprising 50 μl of an Agrobacterium tumefaciens overnight culture grown under selection. After the Petri dishes had been shaken gently for 5 minutes, they were incubated at 25° C. in the dark. After two days, the leaves were plated onto MS medium supplemented with 1.6% glucose, 2 mg/l zeatin ribose, 0.02 mg/l gibberellic acid, 500 mg/l Claforan, 50 mg/l kanamycin and 0.8% Bacto agar. After incubation for one week at 25° C. and 3000 lux, the Claforan concentration in the medium was halved. Cultivation was continued for a further week under known conditions (Rocha-Sosa et al. (1989) EMBO J. 8:23-29).
1.2 Tomato
The starting explant for the transformation are cotyledons of seven- to ten-day-old seedlings of the line Microtom. The culture medium as described by Murashige and Skoog (Murashige and Skoog, 1962, Physiol. Plant. 15, 473-497) supplemented with 2% sucrose, pH 6.1, is used for the germination. Germination takes place at 21° C. under low-light conditions (20-100 μE). After seven to ten days, the cotyledons are divided transversely and placed on the medium MSBN (MS, pH 6.1, 3% sucrose+1 mg/l BAP, 0.1 mg/l NAA) which had been charged with suspension-cultured tobacco cells on the previous day. The tobacco cells are covered with sterile filter paper so that air bubbles are excluded. The explants are precultured on the above-described medium for three to five days. Thereafter the explants are infected with the Agrobacterium tumefaciens strain LBA4404, which harbors the binary plasmid with the gene to be transformed, as follows: the strain which had been cultured overnight at 28° C. in YEB medium with the antibiotic for the binary plasmid is centrifuged. The bacterial pellet is resuspended in liquid MS medium (3% sucrose, pH 6.1) and adjusted to an optical density of 0.3 (at 600 nm). The precultured explants are transferred into the suspension and incubated for 30 minutes at room temperature with gentle shaking. Thereafter, the explants are dried with sterile filter paper and returned to their preculture medium for three days of coculturing (21° C.).
After coculturing, the explants are transferred to MSZ2 medium (MS pH 6.1 supplemented with 3% sucrose, 2 mg/l zeatin, 100 mg/l kanamycin, 160 mg/l Timentin) and retained for the selective regeneration at 21° C. under low-light conditions (20-100 μE, photoperiod 16 h/8 h). The explants are transferred every two to three weeks until shoots form. Small shoots can be excised from the explant and rooted on MS (pH 6.1+3% sucrose) 160 mg/l Timentin, 30 mg/l kanamycin, 0.1 mg/l IAA. Rooted plants are transferred into the greenhouse.
Example 2
Isolation of Genomic DNA
The genomic DNA of transgenic potato and tomato plants was isolated with the aid of the DNA isolation kit from Macherey & Nagel. In a first step, the transgenic lines were identified via PCR, using gene-specific primers. The integration of the foreign DNA was analyzed by means of Southern blot analyses of 20 Mg of DNA after a suitable restriction cleavage.
The following method was applied for obtaining genomic potato DNA for isolating the SSS3 promoter: mortar and pestle were cooled with liquid nitrogen, and 5 g of young leaf material was homogenized to give a fine powder. This powder was transferred into a 50 ml centrifuge tube. The material was shaken vigorously with 15 ml of freshly prepared extraction buffer. After 1 ml of 20% SDS pH 7.2 had been added, the mixture was incubated for 10 minutes at 65° C. Thereafter, 5 ml of 5M potassium acetate was added, the mixture was incubated on ice for a further 30 minutes and subsequently centrifuged at 12 000 rpm. The supernatant was filtered through a Miracloth membrane, transferred into a fresh centrifuge tube and again centrifuged as described above. Then, 10 ml of isopropanol were added to the supernatant and the mixture was mixed, incubated for 30 minutes at −20° C. and again centrifuged at 8000 rpm. The supernatant was decanted off and the pellet was dried for 10 minutes in the tube which had been turned upside down. The pellet was then dissolved in 700 μl of 50× TE buffer and transferred into an Eppendorf tube, 5 μl RNase (10 mg/ml) were added, and the mixture was digested for 30 minutes at 37° C.
After addition of 75 μl of 3 M sodium acetate and mixing, the mixture was centrifuged for 15 minutes at 13 000 rpm, the supernatant was transferred into a fresh tube, 500 μl of isopropanol were added, and the mixture was mixed, precipitated for 5 minutes at room temperature and centrifuged for 15 minutes at 13 000 rpm. The pellet is dried for a few minutes at room temperature. The DNA is dissolved overnight at 4° C. in 400 μl of TE buffer.
Solutions:
Extraction Buffer:


    100 mM Tris-HCl pH 8.0
    500 mM NaCl
    10 mM β-mercaptoethanol
    50 mM EDTA pH 8.0

50×TE

    50 mM Tris-HCl pH 8.0
    10 mM EDTA pH 8.0

TE buffer

    10 mM Tris-HCl pH 8.0
    1 mM EDTA pH 8.0


Example 3
Isolation of the Promoter of the Soluble Starch Synthase 3 from Potato
Starting from the cDNA sequence of the soluble starch synthase isoform 3 of Solanum tuberosum cultivar Désirée, Acc. No. X94400, which has been published by Abel et al. (Plant Journal 10:981-91) 1996, primers were derived in order to isolate the desired SSS3 promoter in what is known as a genome walking experiment. The Universal Genome Walker Kit from Clontech Laboratories, (catalog No. K1807-1) was used for this experiment. This kit can be used for isolating an unknown sequence which is next to a known sequence. The first step consisted in the generation of uncloned, adaptor-ligated genomic DNA fragments, what are known as genome walker libraries, for which the genomic DNA of Solanum tuberosum cultivar Desireé was used. As described in the protocol, the enzyme HpaI was used as restriction enzyme for this purpose (“library DNA”).
The following primers were derived for the amplification of the promoter fragment:








(SEQ ID NO: 40)











Primer R-DSS3-263 (27-mer):




5′ TGC ATT GGA GAC ACT TGT GCA ACT CAA 3′



 








(SEQ ID NO: 41)











Primer R-DSS3-317 (27-mer):




5′ TGT GGT TCC ATG AGA GAC AAA CCC AAG 3′






and the PCR was carried out under the following conditions:











PCR 1:


Mix:



















Library DNA
1.4
μl



Sterile water
18.0
μl



10X PCR reaction buffer
2.5
μl



dNTP (10 mM of each)
0.5
μl



Mg(OAc)2
1.1
μl



Tth polymerase mix
0.5
μl



Primer AP1 from the kit
0.5
μl



Primer R-DSS3-263 (10 pmol)
0.5
μl










The PCR reaction was carried out using a Perkin Elmer GeneAmp PCR System 2400 Thermal Cycler.
PCR Conditions:


    7 cycles: 94° C. for 2 sec; 68° C. for 4 min
    38 cycles: 94° C. for 2 sec; 63° C. for 4 min
    1 cycle: 63° C. for 6 min; 4° C. until further use

PCR 2:

    3 μl of the PCR reaction 1 were diluted with 97 μl of water and used for the PCR reaction 2.












Mix:



















Dilute DNA from PCR 1
2.0
μl



Sterile water
36.8
μl



10X PCR reaction buffer
5.0
μl



dNTP (10 mM of each)
1.0
μl



Mg(OAc)2
2.2
μl



Tth polymerase mix
1.0
μl



Primer AP2 from the kit
1
μl



Primer R-DSS3-263 (10 pmol)
1
μl










PCR Conditions:

    7 cycles: 94° C. for 2 sec; 68° C. for 4 min
    25 cycles 94° C. for 2 sec; 63° C. for 4 min
    1 cycle 63° C. for 6 min; 4° C. until further processing


After separation on a 1% agarose (1× TAE) gel for 4 hours at 70V, a 1.2 kb fragment was obtained. This band was isolated with the “Quiagel Purification Kit” from Quiagen and cloned into the vector pCR2.1 by means of the TA cloning kit from Invitrogen. The resulting plasmid was named pDSSS3-Hp263-1. This plasmid was sequenced and verified on the basis of the agreement with the known portion of the sequence.
Example 4
Cloning the SSS3 Promoter into the Plasmid pCR-BLUNT
Primers were derived from the sequence of the amplified product, and another PCR starting from genomic DNA was carried out to ensure that it is indeed the genomic promoter region of the SSS3 gene. Using the high fidelity PfuTurbo DNA Polymerase (Stratagene) a 1.1 kb fragment was amplified under the following conditions.
The primers (derived and ordered on Mar. 8, 2000) comprise the restriction sites SalI, XbaI, SmaI and BamHI for the subsequent cloning step.
L-DS3: (5′ primer for the promoter amplification, 44-mer) 5′ GTC GAC TCT AGA GGA AGA AAT CTT CTC TGT CTA AAA AAT TGA CG 3′ (SEQ ID NO: 42; the primer starts with SalI and XbaI restriction sites; printed in bold)
R-DS3: (3′ primer for the promoter amplification, 38-mer) 5′ CCC GGG ATC CTC TCT CCC TCT CTG TAT CTG TGC TGC AA 3′ (SEQ ID NO: 43; the primer starts with SmaI and BamHI sites; printed in bold).
The 3′ end of the isolated SSS3 promoter ends at position 75 of the cDNA sequence of the SSS3 gene (Acc. No. X94400), which is 131 bp upstream of the start codon ATG. The sequence without the added cleavage sites is shown by SEQ ID NO: 1 and 44.










PCR Mix:



















Genomic potato DNA (~100 ng)
1.0
μl



Sterile water
37.8
μl



10X PCR Pfu polymerase mix
5.0
μl



dNTP (10 mM of each)
1.0
μl



MgCl2 (25 mM)
2.2
μl



Pfu polymerase mix
1.0
μl (2.5 U)



Primer R-DS3 (10 pmol)
1.0
μl



Primer L-DS3 (10 pmol)
1.0
μl










PCR Conditions for the Amplification of Genomic Potato (Désirée) DNA:

    1 cycle: 94° C. for 1 min
    10 cycles: 94° C. for 30 sec; 70° C. for 4 min
    40 cycles: 94° C. for 30 sec; 66° C. for 4 min
    1 cycle: 66° C. for 4 min; 4° C. until further processing


The PCR reaction was separated on a 1% strength agarose gel, and the 1.1 kb PCR fragment was isolated as described above. This fragment was cloned into the vector pCR-Blunt (Invitrogen, Zero blunt PCR Cloning Kit, #K2700-20). The sequence data showed that two promoters which differ slightly from one another were isolated. The corresponding plasmids were named pDSSS-Hp4 (Seq ID NO: 1) and pDSSS-Hp6 (SEQ ID NO: 44). In addition to some base pair substitutions and minor deletions, they differ from each other by an additional XbaI site (SEQ ID NO: 44) in the plasmid pDSSS-Hp6.
Example 5
Cloning the Promoters into the Binary Vector pBI101
The binary vector pBI101 (Jefferson et al. (1987) EMBO J. 6:3901-3907) was used for the transformation into potato, and the promoters were cloned before the GUS gene.
The plasmid pDSSS-Hp4 was cut with XbaI and BamHI. The 1.1 kb promoter fragment was isolated and ligated into pBI101, which had been cut with the same enzymes. The resulting plasmid was named pDSSS3-Bi-Hp4 and transformed into the agrobacterial strain C58C1[pGV2260].
The plasmid pDSSS-Hp6 was cut with restriction enzymes SalI and BamHI. The 1.1 kb promoter fragment was isolated and ligated into pBI101, which had been cut with the same enzymes. The resulting plasmid was named pDSSS3-Bi-Hp6 and transformed into the agrobacterial strain C58C1[pGV2260]. The agrobacterial colonies were selected on Km50/Amp50/Rif25 (μg/ml). These strains were used for the transformation into potato.
Example 6
Detection of the Tissue-Specific Expression
To determine the characteristics of the promoter, it is necessary to place the promoter before what is known as a reporter gene, which makes possible a determination of the expression activity. An example which may be mentioned is the bacterial β-glucuronidase (Jefferson et al. (1987) EMBO J. 6:3901-3907). The β-glucuronidase activity can be determined in planta by means of a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) in connection with an activity staining. To examine the tissue specificity, the plant tissue is prepared, treated with the X-Gluc reaction solution and stained at 37° C.
A second assay permits a quantitative determination of the GUS activity in the test tissue. β-Glucuronidase MUG (4-methylumbelliferyl-β-D-glucuronide) is used as substrate for the quantitative activity determination; the former is cleaved into MU (methylumbelliferone) and glucuronic acid.
Example 7
Results of the GUS Analysis for the Tissue Specificity of the Transgenic Potato Plants
High-level expression of glucuronidase in the tubers was detected in all transgenic potato plants which had been transformed with the plasmid pDSSS3-Bi-Hp4. The tubers of the best lines showed a blue staining after as little as 30 minutes after the beginning of the enzyme detection (X-Gluc) at room temperature. Surprisingly, the staining was visible even during the very early stages of tuber development. Glucuronidase activity was detected in tubers only 5 mm in length (FIG. 1A). This correlates with the occurrence of starch in this tissue (FIG. 1B). No staining was observed in the potato skin.
A detailed analysis of the GUS expression of the SSS3-Hp4 promoter in the potato plant was carried out by means of the above-described histochemical method, using X-Gluc. To this end, the different tissue types were incubated with the X-Gluc solution, and the blue staining was recorded. The analyses showed that the promoter mediates expression exclusively in starch-comprising tissues. A moderate activity was observed in sections across the internodes, limited to the region below the leaf bud. A weak activity was also shown by the ovaries and the pollen of the flowers of individual lines. Staining was never observed in roots, shoot axis or petioles. In some lines, weak (6 of 18) to moderate (2 of 18) GUS staining was also found in the leaves. Quantitative analyses showed that the activities in the leaves are only very low in comparison with the highly pronounced activities in the tubers.
The analyzed transgenic plants which had been transformed with the plasmid pDSSS3-Bi-Hp6 showed an activity exclusively in the tubers.
Example 8
Quantitative Determination of the Strength of the SSS3-Hp4 Promoter
The quantitative analysis of the activity of the SSS3-Hp4 promoter of these lines was carried out by means of the fluorimetric GUS assay. The data of 10 lines are shown in Table 1.







TABLE 1







GUS activity in developing tubers and mature leaves of


selected SSSIII::GUS lines. The lines were obtained by transformation


with pDSSS-BiHp4. The means of in each case 4 measurements


are shown. The data are shown in picomol MU/mg protein/min;


line 10 is a negative control.










SSSIII::GUS
GUS activity in developing
GUS activity in
Ratio tuber/


line
tubers
mature leaves
leaf













1
1033 ± 180
 55 ± 2
19


2
3884 ± 33
162 ± 19
24


9
3089 ± 165
 56 ± 6
55


10
  6 ± 9
 7 ± 4
—


11
2175 ± 53
 46 ± 5
47


12
2550 ± 280
217 ± 11
12


13
1316 ± 73
 63 ± 1
21


14
3252 ± 86
272 ± 38
12


15
6339 ± 437
 88 ± 8
72


16
 862 ± 32
 23 ± 11
37


17
3166 ± 117
−58 ± 53
—









Growing tubers 10 g in weight and leaf material (source leaves from a point in time where the amount of starch should have reached a maximum) were used for the analysis. The data agree with histochemical data. In mature leaves, the activity varies greatly, if measurable in the first place, but, if so, is approximately 50 times lower than in the tubers.
Example 9
Analysis of the Activity of the SSS3 Promoter During Tuber Storage
The activity of the SSS3 promoter during tuber storage was determined mined by means of the above-described fluorimetric GUS assay. To do so, samples were taken from developing tubers and 45 and 90 days after putting the tubers into storage. The results of the best 5 lines are shown in FIG. 2. For comparison reasons, the data for the adult leaves are also shown. In most lines, the GUS activity in the tubers which had been stored for 45 days was approximately twice as high as in the developing tubers. During further storage, the GUS activity in the tubers continued to rise slightly.
The line with the highest activity is line 15. During tuber growth, a GUS activity of 6339±437 pM MU/min mg protein was measured. After storage for 45 days at room temperature, the GUS activity was 10843±329 and after 90 days 12344±681. Thus, this activity was twice as high as in growing tubers and 140× higher than in leaves.
Example 10
Nalysis of the Expression of the SSS3 Promoter by RT-PCR
To rule out that the high GUS activity in the tubers is caused by the storage and the high stability of glucuronidase, the glucuronidase mRNA was detected via RT-PCR.
The cDNA of the potato tubers was isolated via the method of Bauer et al. (Plant Physiol. 105: 585-592, 1994). The actin gene, which was amplified by means of the primers stated hereinbelow, acted as the positive control.








(SEQ ID NO: 45)











5′actin AC1 (23-mer)




5′ ATG GCA GAC GGT GAG GAT ATT CA 3′



 








(SEQ ID NO: 46)











3′actin AC2 (23-mer)




5′ GCC TTT GCA ATC CAC ATC TGT TG 3′
















Mix:



















H2O
37
μl



cDNA
1
μl



10x PCR buffer
5
μl



dNTP (2.5 mM of each)
4
μl



Primer AC1 (10 pmol)
1.25
μl



Primer AC2 (10 pmol)
1.25
μl



Takara Taq polymerase
0.5
μl (2.5 U)










PCR Conditions:

    1 cycle: 95° C. for 5 min
    35 cycles: 95° C. for 5 sec; 52° C. for 40 sec; 72° C. for 70 sec
    1 cycle. 72° C. for 3 min; 4° C. until further processing


The mRNA as negative control to verify the amplification of genomic DNA and the cDNA of the tubers, isolated from lines 2, 9, 12, 15 and 17, in each case after 45 and 90 days, are shown. The amplificates (1.1 kb) show that the method works and that the expression of the actin gene is comparably high in all of the transgenic lines and that no genomic DNA is present.
Primers were deduced in order to detect the expression of the GUS gene mediated by the SSS3 promoter. The 5′ primer L-SSS3-G50 corresponds with the 5′-untranslated region of the SSS3 gene, which is also present in the binary plasmid pDSSS3-Bi-Hp4. The 3′ primer R-GUS-G809 was derived from the GUS gene. The PCR mix and the PCR conditions were as described for the amplification of the actin gene, with the exception that the PCR reaction was carried out in 25 μl with 0.125 μl TaKaRa Taq polymerase and 1 μl of primer.








(SEQ ID NO: 47)











Primer L-SSS3-G50 (24-mer):




5′ GCA GCA CAG ATA CAG AGA GGG AGA 3′



 








(SEQ ID NO: 48)











Primer R-GUS-G809 (22-mer):




5′ TGG CTG TGA CGC ACA GTT CAT A 3′






To detect the glucuronidase mRNA, 0.1 μg of the cDNA of the tubers of lines 2, 9, 12, 15 and 17 and, as negative control, of the wild type were used. To rule out contamination of the mRNA with genomic DNA, 0.2 μg of DNase-treated mRNA were also used as template. cDNA was isolated from tubers of each line after 45 days and after 90 days. The left side shows the result of the amplification of the mRNA or cDNA after 45 days and the right after 90 days. The expected 0.5 kb PCR product became apparent after separation on a 1% strength agarose gel. The result showed that the amount of the cDNA was similar for each preparation and that no contamination with genomic DNA was discernible (FIG. 4).
Example 11
Cloning the SSS3 Promoter into the Binary Vector pSUN0301 for Analyzing the Expression Pattern in Tomato
The plasmid pDSSS-Hp4 was cut with the restriction enzymes BamHI and EcoRI, and the resulting 1 kb fragment was cloned into the binary plasmid pSUN0301 (derivative of pSUN; SunGene GmbH & Co KgaA, WO 0200900) before the GUS gene which was present therein. The resulting plasmid was named pSUN0301_SSS3 and verified via a sequence analysis. After transformation into the agrobacterial strain LBA4404, a tomato transformation was carried out by means of the above-described protocol.
Example 12
Tissue-Specific Analysis of the Transgenic Lines
Using X-Gluc staining, a staining exclusively in the anthers, pollen and seeds and a very pronounced staining in the green tomato fruits was observed in the transgenic tomato plants. The staining is weaker in mature fruits than in the green fruits (FIG. 5). GUS staining was never observed in leaves, roots, petals and calyces, in petioles and shoot axes.
Example 13
Quantitative Determination of the Strength of the SSS3 Promoter in Green Fruits of Transgenic Tomato Plants
FIG. 6 shows the evaluation of the quantitative analysis of the GUS activity in the green fruits of tomato lines which had been transformed with the construct pSUN0301_SSS3. The diagram shows the high expression level of the SSS3 promoter, which is also suitable for expressing other genes at a high level, specifically in the green fruits.
Example 14
Comparison of the Gus Expression in Green Immature and Red Mature Fruits of Transgenic Tomato Plants
As can be seen from FIG. 7, the GUS activity in the immature green fruits of the tomatoes, transformed with the construct pSun0301_SSS3 is markedly higher than in the mature red fruits.

## Claims

1. A method for directing transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, which comprises
I. introducing, into plant cells, a transgenic expression cassette, wherein the transgenic expression cassette comprises at least the following elements:
a) the promoter sequence of the gene encoding SEQ ID NO: 6, or a fragment thereof having the same promoter activity, and
b) at least one further nucleic acid sequence,

wherein said promoter sequence or the fragment thereof and the at least one further nucleic acid sequence are functionally linked with one another, and the further nucleic acid sequence is heterologous with regard to the promoter sequence,
II. selecting transgenic cells which comprise said expression cassette stably integrated into the genome, and
III. regenerating intact plants from said transgenic cells, wherein the at least one further nucleic acid sequence is expressed in a starch-comprising tissue in a directed manner.

2. The method as claimed in claim 1, wherein the promoter sequence comprises a nucleic acid sequence selected from the group consisting of
i) the sequence as shown in SEQ ID NO: 1 or 44,
ii) a fragment of the sequence as shown in SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant; and
iii) a promoter sequence comprising a nucleic acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant.

3. An isolated nucleic acid sequence comprising
i) the nucleic acid sequence of SEQ ID NO: 1 or 44,
ii) a fragment of the nucleic acid sequence of SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, or
iii) a nucleic acid sequence having at least 95% identity with the sequence as shown in SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant.

4. A method for the generation of a transgenic expression cassette having specificity for at least one-starch-comprising tissue of a plant, comprising:
I. isolating a promoter with specificity for at least one starch-comprising tissue of a plant, wherein at least one nucleic acid sequence or part thereof is employed in the isolation and wherein said nucleic acid sequence comprises the sequence as shown in SEQ ID NO: 5 or codes for an amino acid sequence comprising the sequence as shown in SEQ ID NO: 6 or a part thereof and wherein the promoter comprises at least one nucleic acid sequence of claim 3; and
II. functionally linking said promoter with a further nucleic acid sequence, where said nucleic acid sequence is heterologous with regard to the promoter.

5. The method according to claim 4, wherein the method is carried out using the polymerase chain reaction and said nucleic acid sequence or a part thereof is employed as primer.

6. A transgenic expression cassette for directing transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, comprising
a) the promoter sequence of the gene encoding SEQ ID NO: 6, or a fragment thereof having the same promoter activity, and
b) at least one further nucleic acid sequence,
wherein said promoter sequence or the fragment thereof and the at least one further nucleic acid sequence are functionally linked with one another, and the further nucleic acid sequence is heterologous with regard to the promoter sequence.

7. A transgenic expression cassette comprising the isolated nucleic acid sequence according to claim 5.

8. The transgenic expression cassette according to claim 7, wherein
a) the nucleic acid sequence to be expressed is linked functionally with further genetic control sequences, or
b) the expression cassette comprises additional functional elements, or
c) a) and b) apply.

9. The transgenic expression cassette according to claim 7, wherein the nucleic acid sequence to be expressed transgenically makes possible
a) the expression of a protein encoded by said nucleic acid sequence, or
b) the expression of a sense RNA, antisense RNA or double-stranded RNA encoded by said nucleic acid sequence.

10. A transgenic expression vector comprising the expression cassette according to claim 7.

11. A transgenic cell or organism transformed with the expression cassette according to claim 7 or with an expression vector comprising said expression cassette.

12. The transgenic cell or organism according to claim 11, selected from the group consisting of bacteria, yeasts, fungi, nonhuman animal and plant organisms, or transgenic cells, cell cultures, parts, tissues, organs or propagation material derived therefrom.

13. The transgenic cell or organism according to claim 11, wherein the cell is from or the organism is an agricultural crop plant.

14. A method for the production of foodstuffs, feedstuffs, seed, pharmaceuticals or fine chemicals comprising growing or culturing the transgenic cell or organism according to claim 11 or transgenic cells, cell cultures, parts, tissues, organs or propagation material derived therefrom.

15. A method for the production of pharmaceuticals or fine chemicals in the transgenic cell or organism according to claim 11 or in transgenic cells, cell cultures, parts, tissues, organs or propagation material derived therefrom, comprising growing or culturing the transgenic cell or organism of claim 11 or transgenic cells, cell cultures, parts, tissues, organs or propagation material derived therefrom and isolating the desired pharmaceutical or fine chemical.

16. A method for producing a transgenic plant cell, comprising transforming a plant cell with the nucleic acid of claim 5, an expression cassette comprising the nucleic acid, or a vector comprising the expression cassette.

17. A method for identifying and/or isolating a promoter sequence which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, comprising
a) providing the nucleic acid sequence of SEQ ID NO: 1 or 44;
b) obtaining variants of the nucleic acid sequence of SEQ ID NO: 1 or 44;
c) testing the variants obtained for directing expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant; and
d) identifying and/or isolating a variant which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant and wherein the variant has the tissue specificity of the promoter sequence described by SEQ ID NO: 1 or 44.

18. The isolated nucleic acid of claim 5, wherein the isolated nucleic acid sequence comprises a nucleic acid sequence having at least 99% identity with the sequence as shown in SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant.

19. A method for directing transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, which comprises
I. introducing, into plant cells, a transgenic expression cassette, wherein the transgenic expression cassette comprises at least the following elements:
a) a promoter sequence comprising a nucleic acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1 or 44 which direct transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, and
b) at least one further nucleic acid sequence,
wherein said promoter sequence and the at least one further nucleic acid sequence are functionally linked with one another, and the further nucleic acid sequence is heterologous with regard to the promoter sequence,
II. selecting transgenic cells which comprise said expression cassette stably integrated into the genome, and
III. regenerating intact plants from said transgenic cells, wherein the at least one further nucleic acid sequence is expressed in a starch-comprising tissue in a directed manner.

20. The transgenic expression cassette according to claim 6, wherein the promoter sequence comprises a nucleic acid sequence selected from the group consisting of
i) the sequence as shown in SEQ ID NO: 1 or 44;
ii) a fragment of the sequence as shown in SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant; and
iii) a promoter sequence comprising a nucleic acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant.

21. A method for identifying and/or isolating a promoter sequence which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, comprising
a) preparing fragments of the nucleic acid sequence of SEQ ID NO: 1 or 44,
b) testing the fragments obtained for directing expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, and
c) identifying and/or isolating a fragment which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant.

22. An expression cassette for directing transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, comprising at least one promoter sequence, wherein the promoter sequence directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant and comprises a fragment obtained by the method of claim 21.

23. The expression cassette of claim 7, further comprising at least one further nucleic acid sequence which is functionally linked to the promoter sequence and is heterologous with regard to the promoter sequence.

24. The method of claim 2, wherein the isolated nucleic acid sequence comprises a nucleic acid sequence having at least 99% identity with the sequence as shown in SEQ ID NO: 1 or 44 which directs trans genie expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant.

25. The method of claim 19, wherein the isolated nucleic acid sequence comprises a nucleic acid sequence having at least 99% identity with the sequence as shown in SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant.

26. The transgenic expression cassette of claim 20, wherein the promoter sequence comprises a nucleic acid sequence having at least 99% identity with the sequence of SEQ ID NO: 1 or 44 which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant.

27. A method for identifying and/or isolating a promoter sequence which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant, comprising
a) providing a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 6;
b) using the nucleic acid of a) as a gene probe to obtain a homologue of the nucleic acid;
c) obtaining the promoter sequence of the homologue;
d) testing the promoter sequence of c) for directing expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant; and
e) identifying and/or isolating a promoter, or fragment thereof having the same promoter activity, which directs transgenic expression of a nucleic acid sequence in at least one starch-comprising tissue of a plant and which has the tissue specificity of the promoter sequence described by SEQ ID NO: 1 or 44.