AU772119B2 - Process to collect metabolites from modified nectar by insects - Google Patents

Description

WO 00/04176 PCT/NL99/00453 Process to collect metabolites from modified nectar by insects.

Field of the invention The present invention relates to isolated, purified DNA sequences which can act as promoters in eukaryotic cells.

More specifically, the present invention is related to such DNA sequences which act as promoters to express genes in nectaries of plants. The present invention also relates to chimerical gene constructs comprising a structural or a synthetic gene under the control of a promoter that effects expression of said genes in nectaries. This invention also relates to a process for producing metabolites in honey by allowing insects, preferably bees, to collect and process nectar from plants that excrete said metabolites in nectar or other exudates. Further, this invention relates to plant cells, plants or derivatives therefrom, that express the said chimerical gene.

Background of the invention Nectaries are nectar secreting organs or tissues that can be located inside (floral) or outside (extrafloral) the flower. The main component of nectar is sugar, the variation between nectars of flowers from different species mainly being the concentration and ratio of glucose, fructose and sucrose (Baker and Baker, 1982). In addition, depending on the plant species, varying amounts of polysaccharides, lipids, organic acids, volatiles, minerals, phosphates, alkaloids, amino acids and proteins have been detected SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 2 (Baker and Baker, 1982). Being a specialised sink organ, the nectaries are supplied with sucrose by phloem unloading (Davis et al., 1985, Hagitzer and Fahn, 1992).

The mechanisms of sugar accumulation and nectar secretion have been described for several plant species (Fahn et al., 1979) Sugar transport to the nectaries is achieved by active transport mechanisms and/or osmotic and chemical gradients. In the nectaries of many plants sucrose is converted to glucose and fructose, resulting in a hexose dominant nectar. Part of the hexoses are converted to starch, which is hydrolysed prior to anthesis and nectar secretion. Cell to cell transport of nectar in the nectary parenchyma tissue is mainly symplastic, as demonstrated by the presence of many plasmodesmata between these cells (Fahn et al., 1979). Nectar is secreted from secretory cells via the cell membrane (eccrine secretion) or via the Golgi and endoplasmatic reticulum vesicles (granulocrine secretion). Research on the molecular regulation of nectary development and nectary biochemistry has not been reported.

The main function of floral nectar is to reward pollinating insects. Insects collect nectar to meet their short-term energy requirements. Colony-living honeybees process large quantities of nectar into honey, which is stored in honeycombs of the beehive and is used as food supply during the winter period. Within the bee colony different classes of worker bees cooperate in the honey production process.

Foraging bees collect pollen and nectar from the flowers and bring it to the hive. On returning to the hive they give most of it up to household bees. Pollen is used as a protein source, especially to feed the brood. Adult nurse and worker bees use little protein, their capacity to digest proteins being very low (Crailsheim et al., 1993).

Honey processing takes place by repeated swallowing and bringing up of the nectar from the honey stomach. In the first process 15% of the water content is lost. This semi- SUBSTITUTE SHEET (RULE 26) 3 processed nectar is temporarily stored in a honeycomb cell and taken out later for further processing. The final process includes filtering the honey to discard small particles like pollen grains. Sugar metabolising enzymes (invertase, amylase) are added and the honey is concentrated to an average water content of 20%. Most nectars and honeys only contain traces of protein However, Calluna vulgaris (heather) honey can contain up to 1.8% protein, giving it thixotropic properties (Butler, 1962).

It is known that bees add enzymes like invertase to nectar during the honey processing. Therefore, the probability that proteases are also added is very low. Protein digestion does not take place in the honey stomach but in the intestine of the honeybee. However, the ability of adult worker bees to digest proteins is very low, their main requirement being energy which they obtain from nectar.

Until now, it was not established which proteins are present in heather honey and whether these originate from floral heather nectar or are added to honey by honeybees.

In the present invention it was established that heather honey contains two unique proteins that originate from floral nectar of heather. Based on these results a production system for proteins in nectar and honey was established.

The present invention attempts to show that recombinant proteins can be secreted in nectar of transgenic plants, that this nectar is collected by honeybees and that the 30 bees process this nectar into honey that contains the unaltered protein in a concentrated form.

Definitions S 35 Honey: A substance that contains approximately 80% sugar and varying amounts of other components and that is produced by insects, preferably bees, that collect and process H:\RBe11\Keep\50706-99.doc 24/10/03 4 nectar from floral or extrafloral nectaries, from honeydew, other plant exudates or artificial sugar solutions.

MADS box gene: a gene coding for a transcription factor having a region of 56 amino acids which is homologous to a similar region in the Arabidopsis AGAMOUS protein and Antirrhinum DEFICIENS protein. This region is called the 'MADS box'. At least 50% of the amino acids in this region should be identical to the amino acid composition in the MADS boxes of AGAMOUS and/or DEFICIENS.

Nectary: secretory organ or secretory tissue of plants, located in the flowers (floral nectaries) or outside the flower (extrafloral nectaries) that excrete nectar.

Nectar: sugar containing fluid that is secreted by nectaries. Nectar can also contain substances like minerals, amino acids, proteins, organic acids, volatiles, alkaloids etc.

Recombinant protein: the gene product of a recombinant DNA molecule.

S. Recombinant DNA molecule: A DNA molecule in which sequences 25 which are not naturally contiguous have been placed next to each other by in vitro manipulations.

Promoter: The DNA region, usually upstream to the coding sequence of a gene, which binds RNA polymerase and directs S 30 the enzyme to the correct transcriptional start site.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any 35 reference constitutes prior art. The discussion of the references states what their authors assert, and the H:\RBell\Keep\50706-99.doc 24/10/03 4a applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Summary of the invention The present invention provides an isolated DNA sequence from the promoter region upstream of a nectary-specific expressed sequence, which nectary-specific expressed sequence encodes a protein comprising the amino acid sequence given in SEQ ID NO: 1, or a protein that has at least 60% homology to the amnino acid seqeuence given in SEQ ID NO:1.

The present invention further provides an isolated DNA sequence encoding a protein comprising the amino acid sequence given in SEQ ID NO:l, or a protein having at least 60% homology with the amino acid sequence given in SEQ ID *NO:I, which protein, when ectopically expressed, plays a role in sugar metabolism, the expression of the DNA sequence being predominantly confined to the nectaries of a plant.

The present invention further provides a process for producing a metabolite from honey, comprising: H \RBell\Keep\s50706-99.doc 24/10/03 4b i) producing a plants that excretes this metabolite in nectar and which plants has benn produced by current breeding and selection methods; ii) allowing insections to collect nectar from the selected plants and to process that nectar into honey; and iii) isolating and purifying the metabolite from the honey.

The present invention further provides honey obtained from transgenic plants, which nectar comprises a recombinant gene product.

The production of recombinant proteins for pharmaceutical purposes is a growing market. Until now, mainly bacterial and yeast systems have been used for bulk production of proteins. Recently animal production systems have also been .*0 *oeo eo o *ooo eO* e *o *o o* e *ooo* H:\RBell\Keep\50706-99.doc 24/10/03 WO 00/04176 PCT/NL99/00453 5 developed. With the availability of efficient transformation techniques for plants, procedures to use plants for the production of proteins are now in progress. In plants, the recombinant proteins are targeted to sink organs like tubers and seeds. A serious draw-back of these production methods is that the recombinant protein can only be obtained after extended, and therefore expensive, purification steps.

The present invention provides a method to produce metabolites, preferably recombinant proteins in honey, which is manufactured by insects, preferably honeybees, that collect floral nectar of transgenic plants. Harvesting of honey is very simple and purification of the protein is very straight forward and requires no advanced purification steps. To give an estimation of the protein yield in a crop like rapeseed, we suggest an average protein production of 2% in honey, as has been found in honey of heather. If one hectare of rapeseed yields 100-500 kilo honey in one season, a yield of 2 to 10 kilo protein can be obtained. In addition, the present invention provides a method to collect metabolites from honey that is derived from nontransgenic plants that secrete these metabolites in nectar.

An example are secondary metabolites like acetylandromedol, a diterpine compound, that is excreted in nectar of Rhododendron arboreum and Rhododendron barbaturn and of Piptanthus nepalensis (Martini et al., 1990) This invention provides a gene from petunia, NECI, that is highly expressed in the nectaries of petunia and weakly expressed in the stamens. It also provides another gene from petunia, FBPI5, that encodes a MADS box protein and which is specifically expressed in the nectaries of petunia. Further, it provides the isolated DNA sequences of the promoters of the NEC1 and the FBP15 genes. Furthermore, this invention provides an isolated DNA sequence expressed in nectaries encoding a signal peptide that is translation- SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 6 ally fused to a germin-like protein (Lane et al., 1993, Dumas et al., 1995), having the function to target the mature germin-like protein to nectar of heather (Calluna vulgaris). This invention gives proof that protein-containing sugar solution is collected by honeybees to produce honey that has a higher protein content than the sugar solution itself, the protein having undergone no qualitative alterations. This invention also proofs that a recombinant protein can be produced in nectar of transgenic plants and that this protein is present in honey produced by honeybees that collected this nectar.

Accordingly, this invention provides an isolated DNA sequence which encodes a protein indicated NEC1 and having the amino acid sequence given in SEQ ID NO:1 of the sequence listing hereafter or homologs of NEC1. A homolog of NEC1 is predominantly expressed in nectaries and/or has at least homology with the amino acid sequence given in SEQ ID NO:1. Further this invention provides an isolated DNA sequence which encodes a protein indicated FBP15 and having the amino acid sequence given in SEQ ID NO:2 of the sequence listing hereafter or a homolog of FBP15. A homolog of is specifically expressed in nectaries and belonging to the MADS box family. Furthermore, a homolog is also a gene sequence that has at least 80% homology within the MADS box region and a 60% overall homology with the amino acid sequence given in SEQ ID NO:2. Further this invention provides the characterisation and the isolation of a DNA sequence which encodes a signal peptide indicated "CVSP" (Calluna vulgaris signal peptide), wherein the information contained in the DNA sequence permits, upon translational fusion with a DNA sequence encoding a protein that is expressed in nectaries, targeting of the protein to nectar.

The DNA sequences of the invention can also be characterised in that they comprise the NECI gene and the FBP15 gene having the nucleotide sequences given in SEQ ID NO:4 and SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 7 SEQ ID NO:5 respectively, or a functionally homologous gene or an essentially identical nucleotide sequence or part thereof or derivatives thereof which are derived from said sequences by insertion, deletion or substitution of one or more nucleotides, said derived nucleotide sequences being obtainable by hybridisation with the nucleotide sequences given in SEQ ID NO:4 and 5 respectively.

Furthermore, the DNA sequences of the invention can also be characterised in that they comprise signal sequence CVSP having the nucleotide sequence given in SEQ ID NO:6, or an essentially identical nucleotide sequence or part thereof or derivatives thereof which are derived from said sequences by insertion, deletion or substitution of one or more nucleotides.

Further, this invention provides an isolated DNA sequence from the promoter region upstream of a nectary-specific expressed sequence, which nectary-specific expressed sequence encodes a protein comprising the amino acid sequence given in SEQ ID NO:1, or a homologous protein.

Furthermore, this invention provides an isolated DNA sequence from the promoter region upstream of a nectaryspecific expressed sequence, which nectary-specific expressed sequence encodes a protein comprising the amino acid sequence given in SEQ ID NO:2, or a homologous protein.

In a further aspect, the invention provides a protein encoded by any of the above defined DNA sequences. Further, the invention provides processes of producing transgenic plants exhibiting excretion of recombinant proteins in nectar, the expression of the chimerical genes and the targeting of the recombinant proteins being under the control of promoter sequences and a signal sequence as described in this invention. Still further, the invention provides processes of producing transgenic plants that produce recombinant proteins in nectar, the expression of SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 8 the chimerical genes being under the control of promoter regions upstream of other genes that are expressed in nectaries. Still further, the invention provides processes of producing transgenic plants that produce recombinant proteins in nectar, the expression of these proteins being under the control of any signal peptide that affects targeting of a protein in nectar.

Also, the invention provides recombinant double stranded DNA molecules comprising expression cassettes to be used in the above process. Further, the invention provides transgenic bacteria, transgenic plants producing recombinant proteins in nectar, and also plant cells, tissue culture, plant parts or progeny plants derived from said transgenic plants. Finally, the invention provides a process to produce recombinant gene products in honey, produced by bees that collect nectar from transgenic plants and process this nectar into honey.

Brief description of the figures: Figure 1 shows a polyacrylamide gel with PCR products after Differential Display mRNA amplification. PCR reactions were performed with the oligo-dT primer T12MG in combination with 5 different random primers Apll-AP15 on cDNA samples of pistils without nectaries (two independent samples), nectaries (two independent samples), leaves and a mixture of sepals petals and stamens Bold arrow depicts the cloned fragment DD18.

Figure 2 is the DNA sequence of the Differential Display RT-PCR clone DD18a. The primers prat 122 and prat 119 that were used for 5' RACE PCR reactions are underlined.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 9 Figure 3 is the DNA sequence of clone RC8, obtained by RACE PCR with gene specific primers prat 122 and prat 119 (Fig.

2) in combination with adapter primers. Primer prat 129 (underlined) is used in the next step together with primer prat 122 to amplify the coding region of the NECI cDNA.

Figure 4 is the full length sequence of NEC1 cDNA. The translation start (ATG) and translation stop (TAA) are depicted bold.

Figure 5 shows the expression of NECI and FBP15 in wild type petunia plants (line W115) as determined by Northern blot analysis. Blot A contains total RNA, while blot B is enriched for mRNA. The tissues are indicated as: 1= leaf, 2= sepal, 3= petal, 4= stamen, 5= pistil, 6= nectary. For blot A the HindIII/EcoRI fragment of pDD18a was used as a probe. For blot B the full length cDNA of was used as a probe.

Figure 6 Expression of NECI by in situ localisation of NECI transcripts and activity of the NECI promoter in the nectaries and the stamen as shown by GUS expression driven by the NEC1 promoter. The GUS assay used for the stamens was incubated overnight without modifications to prevent diffusion (example The GUS assay for the nectaries was incubated for 5 hrs, using an assay mixture to prevent diffusion (example For in situ localisation longitudinal sections of flowers of Petunia hybrida were hybridised with digoxigenin-labeled antisense NECI RNA Figure 7 is the DNA sequence from the promoter region upstream of a sequence encoding the NEC1 protein. Underlined is the translation start of NECI cDNA.

Figure 8 depicts a schematic presentation of the T-DNA region between the borders of the binary vector pBNEP1, containing the NECI promoter (Figure the GUS reporter SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 10 gene and the nos terminator in pBINPLUS. This vector was used to generate transgenic plants to study the expression of the NECI promoter.

Figure 9 shows the SDS-PAGE separation of proteins that are present in commercial honey samples from different flowers.

M= marker, lane 1: wattle bark, lane 2: flower mixture, lane 3: heather, lane 4: clover, lane 5: rapeseed.

Figure 10 shows the SDS-PAGE separation of proteins that are present in commercial honey samples of rapeseed (RH2x, and heather (HH2x, HH0Ox) and of nectar samples of rapeseed (RN2x, RNlOx) and heather (HN2x, HN1Ox). M= molecular weight marker. Two (2x) or ten (10x) fold dilutions were used.

Figure 11 shows the SDS-PAGE separation of proteins present in dilutions of the sugar/BSA feeding solution and of honey from bees that had collected the sugar/BSA solution The dilutions of the sugar/BSA and honey/BSA solution was the same for both gels: 1= 15x, 2= 30x, 3= 60x, 4= 75x, 6= 90x, 7= 105x, 8= 120x, 9= 135x. M= marker Figure 12 shows the sequence homology of the N-terminal protein sequence of CVH29, a unique protein present in heather honey and nectar, with a germin-like protein GER1 from a gene bank homology search (BLAST).

Figure 13 shows the deduced DNA sequence of the N-terminal protein sequence of CVH29. The degenerated primers prat 176 and prat 177 are underlined The DNA sequence of the PCR product obtained with prat 176 and prat 177 performed on genomic DNA of heather is shown in B. The gene-specific primers prat 207 and prat 206 used to perform 5'RACE PCR reactions on cDNA from heather flowers are underlined.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 11 Figure 14 shows the DNA sequence of four independent clones obtained by 5'RACE PCR with prat 207 and prat 206 on cDNA of heather flowers. The ATG translation start of the putative signal sequence is boxed. The end of the putative signal sequence and'the start of the mature protein are indicated by arrows.

Figure 15 is the sequence of the synthetically produced DNA molecule encoding the signal sequence CVSP (boxed) with linkers.

Figure 16 is the schematic representation of the plasmid pCVl. Not all restriction sites are indicated.

Figure 17 is the schematic representation of the plasmid pCV2. Not all restriction sites are indicated.

Figure 18 is the schematic representation of the plasmid pCV3. Not all restriction sites are indicated.

Figure 19 is the DNA sequence of the full length cDNA of The translation start (ATG) and translation stop (TAA) are boxed. The MAD-box and K-box region are underlined.

Detailed description of the invention This invention provides processes of producing transgenic plants that produce recombinant proteins in nectaries and nectar that is collected by foraging honeybees. This invention gives evidence that honeybees process protein containing nectar into honey that contains the unaltered protein in a concentrated form. Subsequently, the desired protein can be purified from the honey.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 12 To express recombinant proteins in nectaries of transgenic plants, a translational fusion of an isolated DNA sequence from a promoter region upstream of a sequence encoding a protein that is expressed in nectaries with a sequence encoding the recombinant protein has to be carried out.

Preferably, the isolated DNA sequence from a promoter region is upstream of a sequence that is specifically or highly expressed in nectaries.

The invention relates to a DNA sequence isolated from Petunia hybrida that encodes a protein indicated NEC1 or a homologous protein or part thereof. A homologous protein has at least 65% homology with the amino acid sequence given in SEQ ID NO:1. The cDNA sequence of the NEC1 gene is given in Fig. 4 and in SEQ ID NO:4. The deduced amino acid sequence of the NEC1 gene is given in SEQ ID NO:1. The NECI gene shows strong expression in the nectaries and in a very localised region of the anther filaments of Petunia hybrida. The deduced amino acid sequence of NECI predicts a membrane bound protein. The precise function of the gene has not been elucidated yet, but considering the phenotype of transgenic plants that ectopically express NEC1 in the leaves, a role in sugar metabolism of NECI is apparent.

The present invention also relates to homologous DNA sequences that can be isolated from other organisms, preferably plants, using standard methods and the already known DNA sequence of the NECI gene. More precisely, it is also possible to use DNA sequences which have a high degree of homology to the DNA sequence of the NECI gene, but which are not completely identical, in the process according to the invention. The use of sequences having homologies between 85 and 100 is to be preferred. DNA sequences can also be used which result from the sequence shown in SEQ ID NO:4 by insertion, deletion or substitution of one or more nucleotides. This includes naturally occurring variations or variations introduced through targeted mutagenesis or SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 13 recombination. The DNA sequence shown in SEQ ID NO:4 can also be produced by using DNA synthesis techniques.

The invention also relates to a DNA sequence isolated from Petunia hybrida that encodes a MADS box protein indicated or a homologous protein or part thereof. The cDNA sequence of FBP15 is given in SEQ ID NO:5. FBP25 shows exclusively expression in the nectaries of Petunia hybrida.

The function of FBP15 is unknown.

The present invention also relates to homologous DNA sequences that can be isolated from other organisms, preferably plants, using standard methods and the already known DNA sequence of FBP15. More precisely, it is also possible to use DNA sequences which have a high degree of homology to the DNA sequence of FBP25, but which are not completely identical, in the process according to the invention. The use of sequences having homologies between and 100 is to be preferred. DNA sequences can also be used which result from the sequence shown in SEQ ID NO:5 by insertion, deletion or substitution of one or more nucleotides. This includes naturally occurring variations or variations introduced through targeted mutagenesis or recombination. The DNA sequence shown in SEQ ID NO:5 can also be produced by using current DNA synthesis techniques.

Further, this invention provides an isolated DNA sequence from the promoter region upstream of a nectary-specific expressed sequence, which nectary-specific expressed sequence encodes a protein comprising the amino acid sequence given in SEQ ID NO:1, or a homologous protein that is expressed in nectaries. Furthermore, this invention provides an isolated DNA sequence from the promoter region upstream of an isolated DNA sequence from the promoter region upstream of a nectary-specific expressed sequence, which nectary specific sequence encodes a protein compri- SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 -14 sing the amino acid sequence given in SEQ ID NO:2, or a homologous protein that is expressed in nectaries.

More specifically this invention provides an isolated DNA sequence from the promoter region upstream of a nectaryspecific expressed sequence, which nectary-specific expressed sequence has: a) a nucleotide sequence given in SEQ ID NO:4, or b) a nucleotide sequence obtainable by hybridisation with the nucleotide sequence of or 'with a fragment of (a) In a more specific embodiment this invention provides an isolated DNA sequence from the promoter region upstream of a nectary-specific expressed sequence, obtained from a plant of Petunia hybrida, the sequence consisting essentially of the sequence given in SEQ ID NO:7, or a functional fragment thereof having promoter activity.

In a further aspect, the invention provides an isolated DNA sequence from the promoter region upstream of a nectaryspecific expressed sequence, which nectary-specific expressed sequence has: a) a nucleotide sequence given in SEQ ID NO:5, or b) a nucleotide sequence obtainable by hybridisation with the nucleotide sequence of or with a fragment of In a more specific embodiment this invention provides an isolated DNA sequence from the promoter region upstream of a nectary-specific expressed sequence, obtained from a plant of Petunia hybrida, the sequence consisting essentially of the sequence given in SEQ ID NO:8 or a functional fragment thereof having promoter activity.

Further, this invention provides an isolated DNA sequence comprising the coding region for a signal peptide, wherein the information contained in the DNA sequence permits, upon translational fusion with a DNA sequence encoding a protein SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCTINL99/00453 15 that is expressed in nectaries, targeting of the protein to nectar. More specifically, the DNA sequence comprises the nucleotide sequence given in SEQ ID NO:6 obtained from a plant of Calluna vulgaris, or a nucleotide sequence obtainable by hybridisation with the nucleotide sequence given in SEQ ID NO:6. The use of sequences having homologies between 95 and 100 is to be preferred. DNA sequences can also be used which result from the sequence shown in SEQ ID NO:6 by insertion, deletion or substitution of one or more nucleotides. This includes naturally occurring variations or variations introduced through targeted mutagenesis or recombination. The DNA sequence shown in SEQ ID NO:6 can also be produced by using DNA synthesis techniques. The signal peptide CVSP was isolated from nectar of Calluna vulgaris flowers and from honey processed by honeybees that collected the nectar. The function of CVSP in heather nectaries is to target the germin-like protein to nectar.

The DNA sequence CVSP can also be used to target other proteins to nectar in plant species.

A subject of the present invention is the use of DNA sequences for producing recombinant proteins in nectar of plants, wherein the protein is produced in nectaries and targeted to nectar, and wherein expression in nectaries is achieved by using a DNA sequence consisting of the promoter region upstream of a DNA sequence that is expressed in nectaries, and wherein secretion in nectar is achieved by using a DNA sequence that encodes a signal sequence that targets the recombinant protein to nectar. In a further aspect the present invention relates to processes wherein a recombinant protein is expressed in other plant tissues than the nectaries and wherein the biochemical composition of nectar is changed as a consequence of the recombinant gene expression. The present invention also relates to processes wherein a recombinant protein is expressed in nectaries of a transgenic plant, wherein the biochemical composition of nectar or the nectar secretion is changed as SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 16a consequence of this protein expression. In particular, it relates to processes where the recombinant protein is an enzyme that interferes with the sugar metabolism in nectaries.

The production of a recombinant protein in nectaries and nectar is achieved by integrating into the genome of the plants a recombinant double-stranded DNA molecule comprising an expression cassette having the following constituents and expressing it: i) a promoter functional in nectaries of plants, ii) a DNA sequence encoding a protein which is fused to the promoter, iii) a DNA sequence encoding a signal peptide that targets the recombinant protein to nectar, which is translationally fused to the DNA sequence encoding the recombinant protein, and optionally iv) a signal sequence functional in plants for the transcription termination and polyadenylation of an RNA molecule.

Such DNA molecules are also subject of the invention. The present invention provides an example of such a DNA molecule that contains the described expression cassettes in the form of plasmid pCV3 (Fig. 18), which comprises the promoter region of the NECI gene from petunia, the signal sequence CVSP from heather, the coding region of the reporter gene GUS and the NOS terminator. In principle, any promoter that is active in the nectaries of plants can be used as promoter. The promoter is to ensure that the chosen gene is expressed in nectaries. Also, in principle, any signal sequence that targets the expressed protein to nectar can be used as a signal sequence. The signal sequence is to ensure that the protein is excreted in nectar.

Furthermore, any sequence that encodes a recombinant protein in nectaries can be used in the present invention.

Preferably, the subject of this invention relates to DNA SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 17sequences that encode proteins to be used for pharmaceutical purposes. It is also possible to use the invention to produce proteins for other purposes, e.g. enzymes for biotests or antioxidants for food additives. Furthermore, it is possible to use the invention to produce metabolites in nectar that attract predators of pest insects or that kill or repel pest insects. In another aspect it is possible to use the invention to produce metabolites in nectar that modify the attractiveness of the plant for pollinating insects or improve the health of pollinating insects.

It is also possible to use DNA sequences that encode proteins that modify the nectar composition or the sink strength of nectaries. This means that the recombinant protein interferes with metabolic pathways in the nectaries, resulting in changed levels of compounds that are already present in nectar, or the formation of new compounds in nectar.

In addition, the present invention also relates to expression cassettes that contain the above mentioned DNA sequences, except for a signal sequence. The recombinant protein is then only expressed in the nectaries, but not targeted to the nectar. Consequently, the expression of the recombinant protein in the nectaries can still affect nectar composition.

In a further aspect, the present invention also relates to expression cassettes that contain DNA sequences coding for a protein that is expressed in other tissues than the nectaries. The expression of the recombinant protein affects changes in the biochemical composition of nectar or in nectar secretion.

Finally, the present invention also relates to non-transgenic plants that produce metabolites in nectar that can be harvested and purified from honey that is produced by honeybees that collect this nectar. Examples for these metabolites are alkaloids, terpines, amino acids, proteins, pigments and volatiles.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 18 A preferred embodiment of the process discussed above provides that the expression cassette is transformed to a plant species that produces nectar. Preferably, the recombinant protein is produced in nectar of plants that are visited by honeybees that collect the nectar. Honeybees collect floral as well as extrafloral nectar. The present invention relates to plants that produce recombinant proteins in floral or extrafloral nectar. In addition, the present invention also relates to plants that produce recombinant proteins in other plant organs, said plant organs producing an exudate that is collected by insects, preferably bees, and processed into honey. A particularly preferred embodiment of the present invention are plants that can be grown under controlled conditions. Controlled conditions are greenhouses or field facilities where transgenic plants can be grown according to the safety rules that are required. Preferably, the controlled conditions are such that bee colonies that perform normal foraging behaviour can be maintained in the same compartment during the flowering period. Preferred plants originate from the Brassicaceae family, in particular Brassica napus.

Examples Example 1: Cloning of NECI The NECI cDNA was isolated using the mRNA Differential Display system (Genhunter Corporation, Brookline USA). The isolation of total RNA from nectaries, sepals, petals, stamens and pistils from open flowers and from young leaves of Petunia hybrida was done according to Verwoerd et al.

(1989). Two independent RNA isolations were performed on nectaries as well as on pistils. A DNase treatment was carried out on each RNA sample, using the RNA MessageClean

T

Kit (Genhunter Corporation Brookline USA, cat. No. M601). A SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 19 reverse transcription reaction was carried out on 0.1 Ag RNA of each sample, using the oligo-dT primer T12MG from the Genhunter Kit. Following the protocol, PCR reactions were carried out using the arbritary primers AP11-AP15 in combination with primer T12MG from the Kit. The PCR products were loaded on a sequencing gel and after electrophoresis the gel was blotted on 3M paper, dried and exposed to X-ray film (Figure Two adjacent nectary-specific bands were cut out from the blot and the DNA was purified according to the manual. Reamplification of the fragment was carried out using the oligo-dT primer T12MG and the arbritary primer APl5. After electrophoresis, the PCR product was extracted from the agarose gel by freezing the isolated fragment in liquid nitrogen, followed by centrifugation.

DNA was precipitated by adding 1/10 volume 1% HAc, 0.1M MgCl 2 and 2.5 volume of 96% ethanol to the supernatant. The pellet was dissolved in 10 pl TE buffer. The fragment, now called DD18a, was cloned into a PMOSBlue T-vector (RPN 1719, Amersham Little Chalfont UK) giving the vector pDDl8a.

The nucleotide sequence of this 3' cDNA clone was determined by the dideoxynucleotide chain termination method (ABI PRISMTM Ready Reaction DyeDeoxy T M Terminator Cycle Sequencing Kit, P/N 402078, Perkin Elmer) and is shown in Figure 2. The DNA fragment has a length of 460 nucleotides. The missing 5' part of the cDNA was isolated using the Marathon TM cDNA Amplification Kit of Clontech (catalog K1802-1) and following the procedure as described in the manual. Briefly, Poly A+ RNA was isolated from nectaries of Petunia hybrida flowers. After double stranded cDNA synthesis, adapters were ligated and a 5'RACE reaction was carried out using the adapter primer AP1 supplied in the kit and a gene-specific primer prat 122. The nucleotide sequence of prat 122 is: 5'-gtgggaaggctatgctacaagc-3' (Figure The PCR product was diluted 10x and 1 Il was used in a second RACE reaction with the nested adapter primer supplied by the kit (AP2) and the nested gene-specific primer prat 119 SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 20 (Figure The nucleotide sequence of prat 119 is: ccttctccatggactgcaatgcg-'3 .After gel electrophoreses a fragment of +850 bp was obtained that hybridised with clone DD18a. The fragment, now called RC8, was extracted from the gel, purified and cloned into a PMOSBlue T-vector as described above. The sequence is shown in Figure 3. The combined (overlapping) sequences of clones DD18a and RC8 are shown in Figure 4, comprising the full length cDNA of a gene called NECI hereafter. The NEC1 clone has a length of 1205 nucleotides and encodes for a polypeptide of 265 amino acid residues. Based on the deduced amino acid sequence, high homology was found with a cDNA that is associated with Rhizobium-induced nodule development in the legume Medicago trunculata (MtN3, gene bank number: gnl/PID/e274341). The percentages of identity and similarity are 47% and 72% respectively. Analysis of the predicted protein, using the CAOS/CAMM programme (Protein analysis 1991, Genetics Computer Group inc., Wisconsin USA), shows that the putative protein structure resembles membrane proteins, having six evenly spaced hydrophobic loops that traverse the cell membrane. In addition, a signal sequence is predicted at the N-terminus, while the C-terminus is highly hydrophilic.

Highest homology with MtN3 is found in the N-terminal signal sequence, the first two membrane-spanning loops and the last two membrane-spanning loops. The C-terminal hydrophilic part shows the lowest homology (28% identity, similarity). The function of NECI has not yet been determined.

Example 2: Cloning of Petunia MADS box cDNA clones were isolated from a cDNA library made from nectaries of Petunia hybrida flowers. The cDNA library was constructed using the lambda ZAP cloning vector (Stratagene, La Jolla USA, catalog nr. 200400- SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 21 200402). The library was screened under low stringency hybridisation conditions with a mixed probe comprising the MADS box regions of Floral binding protein gene FBP2, FBP6 and pMADS3 (Angenent et al., 1993, 1994, Tsuchimoto 1993).

The hybridizing phage plaques were purified using standard techniques. Using the in vivo excision method, E.coli clones which contain a double-stranded Bluescript SKplasmid with the cDNA insertion between the EcoRI and XhoI cleavage site of he polylinker were generated. Crosshybridisation of the purified clones revealed 3 independent clones that did not cross hybridise with previously isolated FBP cDNA's and which were designated FBPI5, FBP16 and FBP17. The nucleotide sequence of FBPI5 was determined by the dideoxynucleotide-mediated chain termination method and is depicted in SEQ ID NO:5. The FBP15 cDNA clone has a length of 1157 nucleotides and encodes a peptide of 222 amino acid residues. All characteristics of a MADS box protein are present in FBP15: a N-terminal located MADS box region which shows a high degree of similarity with other MADS box proteins, and a K-box in the middle of the protein with an alpha helical structure. FBPI5 is most similar to the tobacco MADS box protein NAG1, which is an Agamous homolog and expressed in whorl 3 and 4 (Huang et al., 1996, Mizukami et al., 1996).

Example 3: Expression of Expression of FBPI5 was determined by standard Northern blot hybridisation experiments. A DNA fragment comprising the complete cDNA of FBPI5 was used as a probe. High stringency hybridisation and washing conditions were used.

Using 10 Ag of total RNA from various petunia tissues, expression of FBPI5 was only detectable in nectaries. Using 10 g of mRNA from various tissues, prepared by using the kit and protocol of the Quickprep Micro mRNA Purification SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 22 Kit (Pharmacia Biotech), expression of FBP25 was only detectable in nectaries as shown in Figure The expression in the ovary and nectaries was determined by in situ hybridisation using a DIG labelled antisense RNA probe corresponding to the full length cDNA of FBP15. In vitro antisense RNA transcripts were made using T7 RNA polymerase. A standard protocol for in situ hybridisation was used as described by Canas et al., 1994. A hybridizing signal was observed evenly strong in all cells of the nectary tissue.

Example 4: Expression of NEC1 The RNA expression of NEC1 was determined by standard Northern blot hybridisation experiments. A DNA fragment comprising the complete sequence of the Differential Display clone DD18 (Figure 2) was used as a probe. Using Ag of total RNA from various petunia tissues, strong expression of NECI was detectable in nectaries and weak expression in anthers. No expression was detectable in other floral organs, in leaves or in roots (Figure The expression in the ovary and nectaries was determined by in situ hybridisation using a DIG labelled antisense RNA probe corresponding to the nucleotides 79 to 1036 of NECI cDNA, comprising the coding region and part of the 3' untranslated region. A clone containing this sequence was obtained by PCR on adapter-ligated cDNA, using two genespecific primers prat 122 and prat 129 (Figure The nucleotide sequence of prat 122 is: agc-3'", comprising the nucleotides 1015 to 1036 of the NECI cDNA. The nucleotide sequence of prat 129 is: gggatccatqqcqcaattacqtqctqatg-3', comprising the nucleotides 79 to 100 of the NEC1 cDNA. The gene-specific region of the primers is underlined. The primer contains an extra SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 23 BamHI and NcoI site at the 5'end. A PCR fragment of 958 nucleotides was obtained and cloned into a PMOSBlue vector.

The fragment was subcloned in a vector containing the T7 promoter and in vitro antisense RNA transcripts were made using T7 RNA polymerase. A standard protocol for in situ hybridisation was used as described by Cands et al., 1994.

Strong hybridizing signals were observed in the outer cell layers of the nectaries (Figure 6A) Example Isolation of NEC1 promoter fragment The promoter fragment of NECI was cloned using the genome walker protocol (PT3042-1) and kit as provided by Clontech Laboraties. Briefly, genomic DNA from Petunia hybrida was digested with 5 different blunt cutting restriction enzymes. GenomeWalker adapters were ligated and PCR reactions were carried out on each GenomeWalker "library" with a gene specific, reversed primer prat 148 and the adapter primer from the kit (AP1). The nucleotide sequence of prat 148 is: 5'-ccaagaaggccaaatatgaaagac-3' comprising the nucleotides 105 to 128 of the NECI cDNA (Figure PCR products were subjected to a second round of PCR, using the nested adapter primer AP2 and the nested gene specific, reversed primer prat 149. The nucleotide sequence of prat 149 is: 5'-aagtcatcagcacgtaattgcgcc-3', comprising the nucleotides 81 to 104 of the NECI cDNA. From the second PCR a 2 kb fragment was isolated from the StuI library, which was cloned in the PMOSBlue T-vector, yielding the construct pMA5-10. Figure 7 (SEQ ID NO:7) shows the DNA sequence of the NECI promoter in the construct pMA5-10, including the translation start of NECI cDNA.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 24 Example 6: Construction of NECI promoter-GUS A PCR reaction was performed on pMA5-10 (example using the forward vector primer U19 of pMOSBlue and the genespecific primer prat 169. The nucleotide sequence of prat 169 is: -cgctgcagcgccatqqttttttttaqtqaaqcccc-3'. The gene-specific region is underlined. The primer contains an NcoI and BglII restriction site at the 3' end. The PCR product was digested with KpnI and NcoI and ligated into a pBluescriptderived vector (pMO4) that contains the NTM19 promoter (Custers et al., 1997), the reporter gene GUS and the nos terminator. The KpnI/NcoI NTM19 promoter fragment was replaced, resulting in a NECl-promoter/GUS translational fusion. The resulting plasmid pNEP1 was digested with SmaI to release the NEC1 promoter/GUS/nos fragment and this fragment was ligated into a derivative of the binary plasmid pBIN (Bevan, 1984) yielding the binary plasmid pBNEP1 (Figure pBNEP1 was introduced into Agrobacterium tumefaciens strain LBA4404 or C58pMP90 by electroporation.

Plasmid DNA from the Agrobacterium transformants was isolated and the structure of the binary vector was verified by restriction analysis and PCR.

Example 7: Generation of transgenic Petunia plants Agrobacterium strain LBA4404 transformants were used to transform Petunia hybrida using leaf discs as described by Horsch et al. (1985). After shoot and root induction on kanamycin selection media, plants were transferred to soil in the greenhouse.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 25 Example 8: Histochemical GUS assay Different plant parts of Kanamycin-resistant plants transformed with the pBNEP1 construct were analysed for the distribution of /-glucuronidase activity (GUS) using the method described by (Jefferson et al., 1987). In transgenic plants with high expression levels diffusion of reaction products to other tissues was observed. To avoid this spreading a modified GUS assay was used. Briefly, tissues were pre-treated with 90% cold acetone at -20 0 C for 1 h, then rinsed three times 20' with 100 mM phosphate buffer containing 1 mM potassium ferricyanide. After this treatment the standard GUS assay was performed with the modification that ferricyanide was excluded from the reaction mixture.

Example 9: Results histochemical GUS assay In very young flowers cm) no blue staining was observed, in flowers of 2-4 cm weak blue staining of the nectaries was observed. In flowers of (4-6 cm) strong blue staining was observed in the nectaries (figure 6B) and in a very restricted region of the upper part of the anther filaments (Figure 6C). GUS expression was highest in the outer cell layers of the nectary parenchyma. In cross sections of the anther filaments GUS expression was observed in all cells except in the xylem of the inner vascular bundle.

Example Protein analysis of heather honey and nectar Samples of pure heather honey, together with samples of rapeseed, clover, wattle bark and lavender honey were diluted, dialysed and loaded on a 12% SDS page gel (Laemmli, SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 26 1970). All honey samples showed several identical high molecular weight protein bands. Heather honey contained 2 unique protein bands of 29 and 50 kDa (Figure 9) The proteins were named CVH29 and CVH50 (CVH stands for Calluna vulgaris honey). To determine the origin of the proteins, nectar and honey samples of rapeseed and heather were prepared and loaded on a 12% SDS page gel. The high molecular weight protein bands of around 70 kDa that are present in all honey samples were not observed in rapeseed or heather nectar (Figure 10). These proteins are added by honeybees during honey processing. Proteins CVH29 and are present in heather honey and heather nectar, but not in nectar of rapeseed. Therefore, it was concluded that CVH29 and CVH50 are secreted in nectar of heather and can be recovered from honey derived from this nectar. The protein concentration in the heather honey we tested was around Example 11: N-terminal sequence analysis of CVH29 and Honey samples were loaded on an SDS PAGE gel and after electrophoreses the gel was blotted on a PVDF membrane.

After staining the CVH29 and CVH50 bands were cut out from the blot and N-terminal sequencing was performed on both proteins. The N-terminal sequence of CVH50 is: SVLDFCVADPS- LPDGPAGYSCTEPSTVTSQDF. The N-terminal sequence of CVH29 is: SVLDFCVADPSLPDGPAGYSCKEPAKVTVDDFVFHGLGTA. A gene bank homology search (BLAST) showed high amino acid sequence homology with germin-like proteins isolated from Arabidopsis (Figure 12).

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 27 Example 12: Identification signal sequence of CVH29 Because the germin-like protein CVH29 is excreted in heather nectar it was expected that part of the cDNA encodes a signal sequence. Based on the N-terminal amino acid sequence, degenerated primers were designed. The sequence of the forward primer prat 176 is: gygtngcngaycc-3' c or t, n= c, t, a or The sequence of the reversed primer prat 177 is: ccrtgraanacraartcrtc g or A PCR reaction performed on genomic DNA of heather yielded a 99 bp DNA fragment. The fragment was sequenced and two reversed, gene-specific 5' primers were designed to clone the 5' cDNA by "Marathon cDNA racing" using the kit and protocol of Clontech laboratories (protocol PT1115-1, Clontech Palo Alto USA) The sequence of gene-specific primer prat 207 that was used is: ggtgactttagagggctccttgc-3', the sequence of gene-specific nested primer prat 206 is: 5'-gctccttgcaggagtagcctgc-3' (Figure 13). RNA was isolated from open flowers of heather and mRNA was prepared using the Pharmacia quickprep micro rRNA kit. After cDNA synthesis and adapter ligation a PCR reaction was performed, using the adapter primer AP1 and the gene-specific primer prat 207. The PCR product was used for a second PCR, using adapter primer AP2 and the nested gene-specific primer prat 206. A single fragment of around 300 nucleotides was obtained and cloned in a PMOSBlue T-vector. Four clones were sequenced. Figure 14 shows that three clones were identical and one clone had two different nucleotides in the untranslated 5' region. A putative signal sequence of 17 amino acids was identified between the ATG start codon and the first codon of the mature protein CVH29 that was identical in all four clones. The nucleotide sequence of the putative signal sequence (SEQ ID NO:6) is: 3 SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 28 Example 13: Construction of an expression cassette for excretion of proteins in nectar To clone the NECI promoter into a PMOSBlue vector a PCR reaction was carried out on pMA5-10 (example 5) using the forward primer prat 247 and the reversed primer prat 248 (Fig. Prat 247 contains an extra Pstl restriction site.

The NdeI restriction site of prat 248 coincides with the ATG translation start of NECI. The nucleotide sequence of prat 247 is: 5'-ggctgcagqaqtqttctttgataqaatq-3', the nucleotide sequence of prat 248 is: tatgtttttttatqgaaqcccc-3'. Gene-specific regions are underlined. A 1,8 kb promoter fragment was obtained and cloned into a pMOSBlue vector, yielding the plasmid pNECP.

A DNA molecule encoding the signal sequence CVSP as depicted in SEQ ID NO:6 was produced by synthesis and subsequent annealing of two oligo molecules prat 245 and prat 246. The sequence of prat 245 is: cttcttttctcttcttctcatqcttctgttcttgatttc'3, the sequence of prat 246 is: qaqaaataqtqaaaaqaattqqaaqqaaca'3. The region encoding the signal sequence CVSP is underlined. To ensure correct cleavage of the signal peptide, the linkers were extended with the coding region for the first five amino acids of the mature germin-like protein (Fig. 13). The codon usage of the signal peptide sequence was optimised for Arabidopsis. By addition of a BamHI restriction site at the 3' end, 2 extra amino acids were linked in frame to the mature protein. The resulting DNA molecule is shown in Figure The fragment was ligated into a Ndel/BamHl cut PMOSBlue vector, yielding the plasmid pCVSP.

pNECP was digested with NdeI and PstI to release the NECI promoter fragment which was cloned into the Pstl/Ndel cut SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 29 pCVSP, yielding the plasmid pCV1. A schematic representation of pCV1 is given in Figure 16.

A 250 bp long fragment containing the NOS terminator sequence (NOST) was obtained by PCR, using the forward primer prat 251 and the reversed primer prat 252 on DNA of pRAP 33, which is a pUC 19 derived plasmid. Prat 251 adds a SacI and XhoI site, prat 252 adds a SmaI and EcoRI site.

The sequence of prat 251 is: catttqqcaataaaq-3'. The sequence of prat 252 is: cccgggatctaqtaacataqatqacac-3'. The NOST-specific regions are underlined. The PCR product was cloned into pCR-ScriptTM Amp Cloning Kit (Catalog 21188-21190, Stratagene La Jolla USA), yielding the plasmid pCR-NOST. pCR-NOST was digested with Sad and EcoRl and the resulting fragment was cloned into the pUC 19 (ClonTech), derived plasmid pUCAP yielding the plasmid pCVNOS.

The plasmid pGUSN358 was purchased from Clontech (catalog 6030-1) containing the reporter gene GUS in pUC 119, modified to destroy the N-linked glycosylation site within the 1.814 Kb GUS coding sequence. A PCR reaction was carried out with gene-specific primers prat 249 and prat 250, yielding a fragment that contains the GUS gene coding region and a BamH1 restriction site at the 5'end and a Sad restriction site at the 3'end. The sequence of prat 249 is: 5'-ccggatccatqttacqtcctqtaqaaacc-3' The sequence of prat 250 is: 5'-gggagctcccaccqaqqctqtaq-3'. The GUS specific regions are underlined. Subsequently, the PCR fragment was digested with BamHI and SacI and ligated into the BamHI/SacI cut plasmid pCVNOS, yielding the plasmid pCV2. A schematic representation of pCV2 is given in Figure 17.

pCV1 is digested with PstI and BamHI and the resulting fragment is cloned into the PstI/BamHI cut plasmid pCV2, yielding the plasmid pCV3. A schematic representation of pCV3 is given in Figure 18. pCV3 is digested with AscI and SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 30 SmaI and the resulting fragment is cloned into a derivative of the binary plasmid pBIN, yielding the binary plasmid pBCV3. pBCV3 was transferred from Escherichia coli to the Agrobacterium tumefaciens strain LBA4404 and C58pMP90 by electroporation. The transformed Agrobacterium strain was used to transform Arabidopsis and petunia.

Example 14: Protein production in nectar Using the GUS reporter gene, GUS activity in nectar of transgenic plants was measured according to the method as described by Jefferson et al., (1987). Briefly, the assay was carried out by measuring the amount of methyl umbelliferone (MU) produced by GUS fluorometrically by emission of light of 455 nm. The absolute emission was corrected for artificial quenching using an internal standard of InM MU (Angenent et al., 1993).

Example Feeding experiments with honeybees In September 1996 a beehive located outside was supplied with a 25% sucrose solution supplemented with 2% BSA (bovine serum albumin). After 3 weeks the bees had consumed litters of the feeding solution and honey was harvested from the hive. Although the flowering season had mostly past, bees still foraged on flowers to collect nectar outside. Therefore, the honey produced during this period is derived from a mixture of the feeding solution and nectar from flowers. An SDS page protein gel was loaded with dialysed honey samples and sugar/BSA solutions. Figure 11 shows that the protein band of BSA was present in all the samples tested and no qualitative changes were observed in the honey samples compared to the sugar/BSA solutions.

The BSA concentration in honey was 1.5 times higher than in the feeding samples, demonstrating that protein is concen- SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 -31trated in honey. Honeybees that foraged on the sugar/BSA solution did not show any aberrant behaviour and the colony developed normally.

Example 16: Process of honey production from transgenic plants Twohundred and fifty transgenic plants that each produce recombinant protein in nectar were grown in a greenhouse of square meters. The facilities were adjusted according to the safety rules according to European law, including safety measures to prevent in- or outflow of insects. A beehive adjusted for small populations, containing around 200 worker honeybees and a queen, was placed in the greenhouse when the plants were flowering. When a queen is present, she will start laying eggs and larvae will come out. The presence of brood stimulates the bees to collect nectar and process it into honey. After 2-3 weeks bees processed the nectar into honey and stored in sealed cells of the honeycomb. Under the described conditions the amount of honey that can be harvested is 250-1000 grams.

Example 17: Ablation of nectaries By introducing the highly sensitive Rnase BARNASE in plant cells, under the control of a tissue-specific promoter, cell ablation can be achieved in very specific tissues or organs. Ablation of nectaries can be applied to decrease the attractiveness of plants for pest insects that forage on the nectar that is secreted by nectaries. In addition, plants without nectaries can be obtained that are more resistant to bacterial and fungal infections. An example is given for the ablation of nectary tissue by expressing bacterial BARNASE in nectaries, using the NECI promoter.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 32 Plasmid DNA of pNEP1 (example 6) was digested with KpnI and NcoI to release the 1800 bp NEC1 promoter fragment. The purified promoter fragment was ligated into a pWP90 derived vector, upstream of the BARNASE-BARSTAR bacterial operon construct (Hartley, 1988). The construct contains a terminator of polyA signal cauliflower mosaic virus terminator sequence downstream of the BARNASE-BARSTAR operon.

The resulting plasmid pWP126 was digested with KpnI/ XhoI to release the NEC1-promoter/BARNASE-BARSTAR/CamVpolyA fragment and this fragment was ligated into a pBIN-derived vector pBIN Plus. The recombinant vector was transferred via Agrobacterium tumefaciens (LBA4404) to petunia variety W115. Transgenic petunia plants were selected with flowers without nectaries or underdeveloped nectaries.

Many promoters are less specific than can be concluded based on promoter/GUS expression is concluded. Because the bacterial BARNASE is highly cytotoxic at very low concentrations it can be preferred to protect other plant tissues by expression of a ribonuclease inhibitor gene under the control of a weak, constitutive promoter NOS promoter) or a tissue-specific promoter that is not active in the tissues where cell ablation is to be achieved (Mariani et al., 1992, Beals et al., 1997).

Example 18: Ectopic nectary development MADS box genes regulate floral meristem and floral organ identity. Ectopic expression of MADS box genes can change the developmental fate of floral organs or cells. Transgenic petunia plants ectopically expressing FBP11, an ovule- SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 33 specific MADS box gene, develop ovule-like structures on sepals and petals Colombo et al., 1995). FBP15 is a nectary-specific MADS box gene, involved in the molecular regulation of nectary development. In petunia nectaries develop at the base of the carpel. Ectopic expression of in petunia may result in the development of nectaries on other organs of the flower or on vegetative parts of the plant. An example is given of a gene construct that, when transformed to a plant, results in ectopic expression of was amplified using a 5' primer that hybridises with sequences just upstream of the ATG translation start site and a 3' primer that hybridises with FBP15 sequences just downstream the translation stop site. The contains a NcoI recognition site, the 3'primer contains a BamHI recognition site. After the sequence was confirmed, the amplified FBP25 fragment was inserted as a BamHI/NcoI fragment into the binary vector pCPO31. This binary vector was derived from pPCV708, as described by Florack et al.

(1994), and contains three expression cassettes with a multiple cloning site between the left and right T-DNA borders. The cDNA was cloned in sense orientation between a modified CaMV 35S promoter and the nopaline synthase terminator sequence. The chimerical gene construct was transferred via Agrobacterium GV3101 to petunia variety W115, using the transformation method as described in example 7. Transgenic petunia plants were selected that show ectopic nectary development.

Example 19: Modification of sugar composition and nectar secretion Although sugar content of nectar from different petunia W115 flowers shows some variation, the ratio between hexoses and sucrose is very stable. Down-regulation or upregulation of genes involved in the establishment of the SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 34 ratio between hexoses and sucrose in nectar will therefore modify nectar composition. An example is given for antisense expression of a petunia-derived invertase gene.

PCR primers were designed that hybridise with the cDNA of an invertase gene cloned from Solanum tuberosum. The primer 5'-AAGGACTTTAGAGAGACCCGACCACTGCTGG-3'and the 3' primer 5'-AAATGTCTTTGATGCATAATATTTCCCATAATC-3' were used for a PCR reaction on genomic DNA of petunia to yield a fragment of around 420 bp. The fragment was sequenced and cloned into a pMOSBlue vector to used as a probe to screen a petunia nectary-specific cDNA library. Hybridizing phage plaques were purified and cDNAs were retrieved by in vivo excision as described in example 2. The expression of the cDNA's was determined by Northern blotting as described in example 3 and the sequence of a nectary-specific invertase was determined as described in example 2. The invertase gene was amplified using a 5' primer that hybridises with sequences just upstream of the ATG translation start site and a 3' primer that hybridises with sequences just downstream of the translation stop site. Extra restriction enzyme recognition sites were generated to allow cloning of the cDNA in sense (overexpression) or antisense direction into the binary vector pCP031 as described in example 18.

The chimerical gene constructs are transferred via Agrobacterium GV3101 to petunia variety W115, using the transformation method as described in example 7. Transgenic petunia plants were selected that exhibit modified sugar composition in nectar.

Example Modification of plant development A DNA which is the NECI gene or a homologous gene is introduced into a plant cell, the said DNA being induced by promoter elements controlling the expression of the introduced DNA in such a way that transcription produces SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 35 sense RNA. Plants were regenerated from the transgenic cells as described in example 7. Plants that ectopically express the NECI gene exhibited modified leaf morphology and modified sugar composition. Furthermore, plants that ectopically express the NECI gene showed a delay in flowering time.

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Martini Schmid A. and Hess D. (1990). Antibiotics, and amino acids in nectar of Rhododendron and Piptanthus species from Nepal. Bot. Acta 103, 343-348.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 38 Mizukami Huang Tudor M. and Ma H. (1996). Functional domains of the floral regulator AGAMOUS: Characterisation of the DNA binding domain and analysis of dominant negative mutations. The Plant Cell 8, 831-845.

Tsuchimoto van der Krol A.R. and Chua 1993.

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant. Plant Cell 5, 843- 853.

Verwoerd Dekker, B.M.M. and Hoekema A. (1989). A small-scale procedure for the rapid isolation of plant RNAs. Nucl. Acids Res. 17, 2362.

Zer H. and Fahn A. (1992). Floral nectaries of Rosmarinus officinalis L. Structure, Ultrastructure and Nectar Secretion. Annals of Botany 70, 391-397.

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 39 Sequences: SEQ ID NO:l amino acid sequence NECi 1 MAQLPADDLS FIFGLLGNIV SFMVFLAPVP TFYKIYKRKS SEGYQAIPYM 51 VALFSAGLLL YYAYLRKNAY LIVSINGFGC AIELTYISLF LFYAPRKSKI 101 FTGWLMLLEL GALGMVMPIT YLLAEGSHRV MIVGWICA-AI NVAVFAAPLS 151 IMRQVIKTKS VEFMPFTLSL FLTLCATMWF FYGFFKKDFY IAFPNILGFL 201 FGIVQM.LYF VYKDSKRIDD EKSDPVREAT KSKEGVEIII NIEDDNSDNA 251 LQSMEKDFSR LRTSK SUBSTITUTE SHEET (RULE 26) WO 00/04176 WO 0004176PCT/NL99/00453 SEQ ID No: 2 amino acid sequence of Met 1 Arg Tyr Ser Thr Ser Leu, Gly Thr Leu 145 His Asn Tyr Gly Gin Giu.

Ser Il.e Thr Arg Glu.

Lys 130 Leu Asn Val Asp Arg Val Leu Arg Asp Ser Val.

Ser 115 Leu Phe Asn Asn Pro 195 Gly Thr Ser Gay Arg Glu Gin 100 Leu Giu Ala Asn Met 180 Arg Lys 5 Phe Val Arg Tyr Al a Ile Ser Lys Giu Gin 165 Met Asp Ile Cys Leu Leu, Lys Asn Gly Ser Gly Ile 150 Met Gly Phe Glu Lys Cys Tyr 55 Lys Thr Asn Leu Ile 135 Giu Leu Gly Phe Ile Arg Asp 40 Glu Ala Gin Leu Thr 120 Ser Tyr Arg Glu Gin Lvs Arg 25 Al a Tyr Ser Phe Gin 105 Ala Arg Met Al a Phe 185 Val Arg Asn Giu Ala S er Tyr 90 Asn Lys Ile Arg Lys 170 Giu Asn Ile Giu Gly Leu Val Ala Asn Asn Asp Ser 75 Gin Gin Ser Asn Asp Leu Arg Ser 140 Lys Arg 155 Ilie Ala Leu Met Gly Leu Asn Leu.

Leu Ser Ser Glu, Arg Lys 125 Lys Giu.

Giu Gin Gln 205 Thr Lys Ile Val Asn Ala Asn 110 Gly Lys Ile Ser Ser 190 H~is Thr Lys Val Lys Thr Al a Met Leu Asn Asp Giu 175 His Asn Asn Ala Phe Ala Gly Lys Leu Glu.

Glu.

Leu 160 Arg Pro His 200 Gin Tyr 210 Pro Arg Gin Asp Asn Met Ala Leu Gin Leu Val SUBSTITUTE SHEET (RULE 26) WO 00/04176 WO 0004176PCTIN L99/00453 41 SEQ ID NO:3 amino acid sequence CVSP

MFLPILFTISLLFSSSHA

SUBSTITUTE SHEET (RULE 26) WO 00/04176 WO 0004176PCT/NL99/00453 42 SEQ ID NO:4 Nucleotide sequence NECI 1 TCGAGCGGCC 51 TCAAALAGGGG 101 ACTTGTCTTT 151 TTCCTAGCAC 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201

AGAAGGATAT

TATTGCTATA

ATTAATGGCT

CTTTTACGCG

TAGAATTGGG

GAAGGCTCAC

TGTTGCTGTC

CAAAGAGTGT

TGTGCCACTA

TGCGTTTCCA

TATATTTTGT

CCTGTTCGAG

CATTGAAGAT

TTTCCAGACT

AAAGCTAAGG

GCTAATTAGC

TTACCTTATA

AGCGAATCTT

ACATGTTTGG

CTCAATTTGT

GCCCGGGCAG

CTTCACTAAA

CATATTTGGC

CCGTGCCAAC

CAAGCAATAC

TTATGCTTAT

TTGGATGTGC

CCCAGAAAGT

AG CC CTAGGA

ATAGAGTGAT

TTTGCTGCTC

AGAGTTCATG

TGTGGTTTTT

AATATACTGG

TTACAAGGAT

AAGCTACAAA

GATAATTCTG

GCGGACATCA

AGTTTGAAGT

AAGACTTTAG

ATTAGCTTGT

ATATATGGGA

CACTTGACTA

CACTTACTTA

GTATTCAACA

AAAAAATCAT

CTTCTTGGTA

ATTTTACAALA

CATATATGGT

CTCAGGAAGA

CATTGAATTA

CTAAGATTTT

ATGGTGATGC

GATAGTGGGA

CTTTAAGCAT

CCCTTCACTT

CTATGGGTTT

GCTTTCTATT

TCAAAGAGAA

ATCAAAAGAA

ATAACGCATT

AAATAAGCAA

AAGGCAAGGA

CAGCTTGTAA

AGCATAGCCT

AATACTTACA

TACATAGAAA

TAAGTAGCTG

AGAGTATTCA

GGCGCAATTA

ATATTGTATC

ATATATAAAA

AGCACTGTTC

ATGCCTATCT

ACATATATCT

CACAGGGTGG

CAATTACTTA

TGGATTTGTG

CATGAGGCAA

TATCTTTGTT

TTCAAGALAGG

CGGAATCGTT

TAGATGATGA

GGTGTAGAAA

GCAGTCCATG

GAAGATGATC

ACTTGACACT

TATTTAGTGT

TCCCACTAAT

CTAGTATGCA

AATTAACAAG

AATAATATAA

CCACTTGAAC

CGTGCTGATG

ATTCATGGTC

GGAAATCATC

AGCGCCGGAC

TATCGTCAGC

CTCTGTTTCT

CTGATGCTCT

TTTATTAGCA

CAGCTATCAA

GTAATAAAAA

CCTCACTCTC

ACTTTTACAT

CAAATGCTAT

AAAATCTGAT

TCATTATCAA

GAGAAGGATT

AAAAAATGAC

GAATATCTAA

TTGTGAGGTG

AATTCTGCTT

TCTTCTATAT

CATTTCTCAC

TGCAATTTTC

ACCCC

SUBSTITUTE SHEET (RULE 26) WO 00/04176 WO 0004176PCT/NL99/00453 43 SEQ ID NO:5 Nucleotide sequence 2.

51 102.

151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151

TCTGAATACAAGCTGTGTGTGTAGAGAGATTTCATAGACAGCAAJACAT

CCCTTCTTTTTGTTCTGTTTTAJACTTCCCTTCTTCACCAGCTCTTTT

CCCTAGTATGAAAAGGAGTCGACAAG

GAAGATGTCAGACTCGCCTCAGAGGAAGATGGGAAGAGGAJXAGATTGAGA

TTAAGAGGATTGAAATACAACATCGTCAJAGTCACTTTCTGTAGAGA

AGAAATGGGTTGCTTAAJAGCTTATGAJCTTTCTGTTCTTTGTGATGC

TGAAGTTGCTCTCATCGTTTTCTCAAGCCGTGGCCGCCTCTATGAATATG

CTAACAACAGTGTGAAGGCACATTGATAGATATAGAGCATCCTCA

GATTCCTCCAACACTGGATCTACTTCTGAAGCTAACACTCAGTTTTATCA

ACAGAAGCTGCCAACTCCGAGTTCAGATTGGTAACTTACAGAACTCAA

ACAGGAACATGCTAGGCGAGTCTCTAGTTCTCTGACTGCAAGATCTG

AAGCTGGCAATGGAGATATG-TAGCA

AAAGAATGAACTCCTGTTTGCTGAGATTGAGTATATGCGAGGGA

TTGATTTGCACAACAACAATCAGATGCTTCGGGCAGATAGCTGAGAGT

GAAAGAAATGTGAACATGATGGGAGGAGATTTGAGCTGATGCAATCTCA

TCGAGTCAAATCTCAGGAGCTCGAAT

ATATTCCCAGCAAGGTTCATGAAGTA

AATAAAATGCATGGTTTGAAGCACTCTGATTGTGGTGGATTTGGATTATG

TATAAGGGAGTGCAGGCCATTTGCCATTATTGAGGTACTCAACAGG

AATGAAGTACTTTTCTTTTTTACAAT

TTAGCTTATGGACTCTAACAAGACTTATTTAACATATAATATAAT

TGTGTAATGCTGTTGTATTGTATGGTATGTATCCAACATTATAJCC

TATCTTTTTCTTCAATTATGTCTCCTTTGATACAAAJCTACTAACATATT

TTCTTAT

SUBSTITUTE SHEET (RULE 26) WO 00/04176 PCT/NL99/00453 44 SEQ ID NO:G6 Nucleotide sequence CVSP ATGTTTCTTCCAATTCTCTTCACCATTTCCCTC CTCTTCTCCTCCTCCCATGCT SUBSTITUTE SHEET (RULE WO 00/04176 PTN9/05 PCT/NL99/00453 45 SEQ ID NO:7 Nucleotide sequence NEC. promoter 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401

CCTAGGAGAAATCAAGCCTACTCTTAAGATGGATGACTCACTTGCCCCGA

TGGTAAGGTGAAGGATCTGTTGATTAGAGTTGGGAGTTCATGTTCTCTG

CTATTTATTGCAGAGGACAAGTCAAT

TTGAGGATTATAACGTGATATAAGAC

TGGGGAGATGACTGTGAGAGCGCATGGAGAAGGTTACTTTCAAGGTTT

ATAATAAAAAGGATCATATGGCTAAGTTTGAGAGTGTTCTTTGATAGA

TGTGTCAGACGAGAACATGAAGTACCGAAAGAGGTGTTTGAGCGGAA

TGAAAAATACCGAATATAAGTAGAAT

CACCTAAAGGAAGGAAGAAGACAAGTTCGTCGTAACAGAGGAGACGT

AAATGCTGGAAGTGAGCTTAAAGGTGTTGT

CGTACTACGACGTTAACTAA

GGCG CTTGTCGGGAGGCAACCCTAGCTTTGTATGTTGTAGTAAA AAATATATATATAGAAAAAGGAAAATACAAAAAGACTCGTGC

CGCGACGT

TAAATCAAGCGCTTGTTGGAAGGCAACCCATTTTTATTGTTTTAGTTGT

TTTACTTATTTAGTATTACGTAGTTTCTTGTTGTTTTTGTAGGGCTCGGG

ACTTTCGGAAGGTGAGGTAATTTCAAGGCATCGCGGTGTGTATTGCAGCG

AGGTAAGTGTAAGAGTTGAGTTGGAAGCGTTTGGCCAAGTGTTGCACCGT

GAGAGGCTTTCAACCTGTTGCGACACGTGAAAAATTAAGAGCCAGATCTG

CTACATTAGCACTGAAGCATCG

CTTGCCAATAGCTTGGAATGGAAGCA-A

GAATTCAAAC CAAAATCAGAAACGCCACAAGAGATGTGTCGCACACTGCA

AAGCTTTGTGCAAACTAGTGAACGCAGAAATAGAAATCCTACACCCCATG

CGTCGCTTGGCTTATGGCAGGCAGCAAAAATTCAGCAG

CAAAACAGAAAC

-GCTGCGAGAAACGCGTCGCATACGCCATAGCTTTGTGTCACAGAACGT

CCAGAAATTGAAAAGCTATAAGCCTG

CGTCCCTTGGCTCATGGCGTGCAG

ACTAGAAAAG CTCTAGCAGATGCGTCGCGTATTGTATAGCTTGGTGTGA ACAGAAAGTTCGAAACTTGGAAA.ACGATAAC

CCAGCGTCGCCTCTTCAAC

CGCGTCCAGGTAAGTTCAAGATTCTTACGGGTTGACCCATTACCCATTG

ATCGGCTGATTATAhACAATAAAACATCAC

CTTCAACTATCACATGATTT

CATAAGTTTGACCTAGGATATTTTATATATATATATATATATATACACAC

ACACACCATTTCCAGCGATCTTACCTC-ATTTTTATT

CAAACCATTTTTCT

SUBSTITUTE SHEET (RULE 26) WO 00/04176 WO 0004176PCT/NL99/00453 4-6 1452.

1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 2001 2051 2101

GCTTCAAAGTTTA-TTATTATATGATAGTCATCCATAGTCACA

GATTTTCTATACTATTTTGTCCCTTGTATTTTAAAAAAA

ATGAGCGA

TGGTAAGATACATTGTTTGCAGTGTACAATTTTAGTATATGCACC

AACGCTTCTTCTTCCAACTATCACCTAA2CTACATCATTTATGGCGGGC

GGACTAGACGTAGCCAAATATAJACGCA-TGGCCATTCAGTTCATGTC

ATTTTTATATCCTTCATCCATATATTACTCAAATTGATGTACAGTTT

GGTCTCTGATGTGCACTTTACTATACGTATACGGATTTACATTATAT

TAAGAGAACTGTTCCACTTTTTATGATTTATTAATTTACTCGG

TTACTTGTATTATTATTATTGCTGTATTTGTTTGTCATTTGAATTTGGCA

CCGCAGATTTTTGTATGCAATTAACC

CTCATATATCTTTTGGCCAA.ATAA

AGAAAAAGTCTGCATATTT

CTTGCCAAACATTTATCATACTTTACCGAT

TCTTGTTTTTTGTTTCTCTGTTGTTGTTCT

CCACTATAAATAACATTTGC

AGTGAGTAAAGTTTCTTCAGGTCTCTTTTGTAGATTCACAGAGTATTC

AGATGATAAGGCTATAAAACT

SUBSTITUTE SHEET (RULE 26) WO 00/04176 -47 SEQ ID NO:8 Nucleotide sequence FBP15 promoter PCT/NL99/00453 SUBSTITUTE SHEET (RULE 26) EDITORIAL NOTE APPLICATION NUMBER 50706/99 The following sequence listing pages 1-12 are part of the description. The claims pages follow on pages 48-54.

WO 00/04176 WO 0004176PCT/N L99/00453 SEQUENCE LISTING <110> CPRO-DLO (120> Process to collect metabolites from modified nectar by insects (130> 159782 <140> pct/n199/00453 <141> 1999-07-15 (160> <170> Patentjn Ver. 2.1 <210> <211> 212> <213> <220> <223> <220> <223> <220> <223> 265 P RT Petunia x hybrida strain: W115 tissue type: nectar gland NECi amino acid sequence <400> 1 Met Ala Gin Leu Arg 1 5 Ala Asp Asp Leu Phe Ile Phe Gly Leu Leu Gly Asn Ile Tyr Lys Ile Val Ser Phe Met Val Leu Ala Pro Val Pro Thr Phe Tyr Lys Arg Lys Ser 40 Ser Glu Gly Tyr Gln Ala Ile Pro Tyr Met Val Ala Leu Phe Ala Gly Leu Leu Tyr Tyr Ala Tyr Arg Lys Asn Ala Leu Ile Val Ser Ile Asn Gly Phe Gly Ala Ile Giu Leu Thr Tyr Ile Ser Leu Leu Phe Tyr Ala Pro Arg WO 00/04176 PTN9/05 PCT/NL99/00453 Lys Ser Lys Ile Phe Thr Gly Trp Leu Met Leu Leu Giu Leu Gly Ala Val Leu Gly Met 115 Ara Val Met Met Pro Ile Tyr Thr 120 Ile Leu Leu Ala Gly Ser His Val Ala Val Ilie Val Gly Cys Ala Ala 130 Phe Ala Ile 140 Ile Ala Pro Leu Met Arq Gin 145 ValI Val 155 Phe Lys Thr Lys Ser 160 Glu Phe Met Pro 165 Phe Thr Leu Ser Leu Thr Leu Cys Ala 175 Thr Met Trp Phe Pro Asn 195 Tyr Phe Val Tyr Gly Phe Ph e 185 Phe Lys Asp Phe Leu Gly Phe Gly Ile Val Gin 205 Glu Tyr Ile Ala 190 Met Leu Leu Lys Ser Asp Tyr Lys Asp Arg Ile Asp 210 Pro Val Asp 220 Val Arg Glu Ala 225 As n Thr 230 Asn Ser Lys Giu Gly 235 Leu Gu Ile Ile Ile 240 Lys Ile Glu Asp Asp 245 Leu Ser Asp Asn Gin Ser Met Asp Phe Ser Arg Thr Ser <210> 2 <211> 221 <212> PRT <213> Petunia x hybricia <220> <223> strain: W115 <~220> <223> tissue type: nectar gland, secretory cell <220> WO 00/04176 <223> FBP15 PCT/NL99/00453 amino acid sequence <400> 2 Met Gly 1 Arg Gly Lys 5 Ile Giu Ile Lys Ile Giu Asn Thr Thr Asn Arg Gin Vai Thr Phe Cys Lys Arg Arg 25 Asp Aia Gly Leu Leu Tyr Giu Leu Ser Ser Arg Ser Vai Leu Cys Giu Vai Ala Lys Lys Aia Ile Vai Phe Vai Lys Aia Giu Giy Arg Leu Ile Tyr 55 Lys Tyr Ala Asn As n Se r Thr Ser Asp Arq Tyr Lys 70 As n Aia Ser Ser Asp 75 Gin Ser Asn Thr Giy Thr Ser Giu Thr Gin Phe Gin Giu Ala Ala Lys Leu Arg Vai Giy Giu Ser 115 Thr Lys Leu Gin 100 Leu Giy Asn Leu Ser Asn Arq Ser Ser Leu Thr 120 Ser Lys Asp Leu Asn Met Leu 110 Giy Leu Giu Lys Asn Giu Giu Lys Giy 130 Leu Leu Ile 135 Giu Arg Ile Arq Phe Ala Giu 145 His Tyr Met Arg Lys 155 Ile Giu Ile Asp Asn Asn Asn Leu Arg Ala Ala Giu Ser Giu Arg 175 Asn Val Asn Tyr Asp Pro 195 met 180 Arq Giy Giy Glu Leu Met Gin Ser His Pro 190 His Asn His Asp Phe Phe Gin 200 Asn Gly Leu Gin Tyr Pro Arg Gin Asp Asn Met Ala Leu Gin Leu Val 210 215 220 <210> 3 WO 00/04176 PTN9/05 PCT/NL99/00453 <211> 18 <212> PRT <213> Caliuna vuigaris <220> <223> tissue type: flower <220> <223> Caliuna vuigaris signal peptide <400> 3 Met Phe Leu Pro Ile Leu Phe Thr Ile Ser Leu Leu Phe Ser Ser Ser 1 5 10 His Ala <210> 4 <211> 1205 <212> DNA <213> Petunia x hybrida <220> <221> CDS <222> (79)..(873) <220> <223> strain: W115 <220> <223> tissue type: nectar gland <22 0> <223> NECi <400> 4 tcgagcggcc gcccgggcag gtattcaaca agagtattca ccacttgaac tcaaaagggg cttcactaaa aaaaaatc atg gcg caa tta cgt gct gat gac ttg tct ttc ill Met Ala Gin Leu Arg Ala Asp Asp Leu Ser Phe 1 5 ata ttt ggc ctt ctt ggt aat att gta tca ttc atg gtc ttc cta gca 159 Ile Phe Gly Leu Leu Gly Asn Ile Val Ser Phe Met Val Phe Leu Ala 20 WO 00/04176 WO 0004176PCT/NL99/00453 000 gtg cca aca Pro Val Pro Thr ttt tac aaa ata Phe Tyr Lys Ile tat aaa agg aaa Tyr Lys Arg Lys tca gaa gga Ser Giu Gly tat caa Tyr Gin gca ata cca tat Aia Ile Pro Tyr gta gca ctg ttc Vai Aia Leu Phe agc Ser gcc gga cta ttg Aia Gly Leu Leu tat tat qct tat Tyr Tyr Ala Tyr agg aag aat gco Arg Lys Asn Aia tat Tyr ott atc gtc ago Leu Ile Vai Ser att Ile 255 303 351 aat ggc ttt gga Asn Giy Phe Giy goc att gaa tta Aia Ile Glu Leu tat atc tct ctg Tyr Ile Ser Leu ttt Otc Phe Leu ttt tao gcg Phe Tyr Aia tta gaa ttg Leu Giu Leu 110 aga aag tot aag Arg Lys Ser Lys att Ile 100 tto aoa ggg tgg Phe Thr Giy Trp ctq atg oto Leu Met Leu 105 tat tta tta Tyr Leu Leu gga gcc ota qga Giy Aia Leu Giy atg Met 115 gtg atg coa att Val Met Pro Ile act Thr 120 gca gaa Aia Giu 125 ggo toa oat aga Gly Ser His Arg atg ata qtg gga Met Ile Vai Gly att tgt gca got Ile Cys Ala Ala at 0 Ile 140 aat gtt got gto ttt got got cot tta Asn Vai Aia Val Phe Aia Aia Pro Leu 145 ago Ser 150 ato atg agg oaa Ile Met Arg Gin gt a Val1 155 543 ata aaa aoa aag Ile Lys Thr Lys gta gag tto atg Vai Giu Phe Met tto act tta tot Phe Thr Leu Ser ttg tto Leu Phe 170 oto aot oto Leu Thr Leu gao ttt tao Asp Phe Tyr 190 goc act atg tgg Aia Thr Met Trp ttt Phe 180 tto tat ggg ttt Phe Tyr Giy Phe ttc aag aag Phe Lys Lys 185 att gog ttt oca Ile Aia Phe Pro ata otg ggo ttt cta tto gga ato Ile Leu Giy Phe Leu Phe Giy Ile 200 gtt oaa atg ota tta tat ttt gtt tao aag gat Val Gin Met Leu Leu Tyr Phe Vai Tyr Lys Asp toa Ser 215 aag aga ata gat Lys Arg Ile Asp WO 00/04176 WO 0004176PCT/NL99/00453 gat Asp 220 gt a Val gaa aaa tct gat Giu Lys Ser Asp gaa atc att Gu Ile Ile atc Ile 240 cag tcc at< Gin Ser Met taagcaagaa tgacactqaa tgaggtgtta gaatcttata ttgactatac gtagctgaat ;gag aag g Giu Lys A 255 gatgatcaaa tatctaagct ccttataatt tatgggaaat atagaaaaat aatataatgc at sp aa aa ag ac ta aa gtt cga gaa gct aca Val Arg Giu Ala Thr 230 att gaa gat gat aat Ile Giu Asp Asp Asn 245 ttt tcc aga ctg cgg Phe Ser Arg Leu Arg 260 atgacaaa gctaaggagt ttagcaag actttagcag cttgtagc atagccttcc ttacacta gtatgcatct acaagcat ttctcacctc ttttcacc cc aaa tca aaa Lys Ser Lys tct gat aac Ser Asp Asn aca tca aaa Thr Ser Lys 265 ttgaagtaag cttgtaatat t cactaataat t tctatataca t aatttgtcac t gaa ggt Glu Gly 235 gca ttq Ala Leu 250 ~caaggaact t agt gt tt g :ctgcttagc ;gtttggcac tact tata a 783 831 933 993 1053 1113 1173 1205 <210> <211> 265 <212> PRT <213> Petunia x hybrida <223> NECi <400> Met Ala Gin Leu Arg Ala 1 5 Gly Asn Ile Val Ser Phe Asp Asp Leu Phe Ile Phe Gly Leu Leu Met Val Phe Ser Aia Pro Val Tyr Lys Ile Tyr Met Val Lys Arg Lys Ala Leu Phe Ser Ala Ile Giu Gly Tyr Pro Thr Phe Ala Ile Pro Tyr Ala Tyr Leu Arg Lys Asn Ala Gly Leu Leu Val Ser Ile 75 Leu Phe Leu Leu Asn Ala Gly Phe Gly Cys Arg Ile Glu Leu Thr Ile Ser 6 Phe Tyr Ala Pro WO 00/04 176 PTN9/05 PCT/NL99/00453 Lys Ser Lys Leu Gly Met 115 Phe Thr Gly Trp, Leu 105 Met Leu Leu Giu Leu Gly Ala 110 Val Met Pro Ile Tyr Leu Leu Ala Giu 125 Giy Ser His Arg Val 130 Met Ile Val Gly Ile Cys Ala Ala Asn Val Ala Val Phe Ala Ala Pro Leu.

145 Ile Met Arg Gin Val 155 Ile Lys Thr Lys Ser 160 Vai Giu Phe Met Pro 165 Phe Thr Leu Ser Phe Leu Thr Leu Cys Ala 175 Thr Met Trp Phe Pro Asn 195 Phe Tyr Giy Phe Ph e 185 Lys Lys Asp Phe Tyr Ile Ala 190 Met Leu. Leu Ile Leu Gly Phe Phe Giy Ile Vai Gin 205 Tyr Phe 210 Vai Tyr Lys Asp Lys Arg Ile Asp Giu Lys Ser Asp Pro 225 Val Arg Giu Ala Lys Ser Lys Giu Giy 235 Val Gu Ile Ile Ile 240 Asn Ile Giu Asp Asp 245 Asn Ser Asp Asn Leu Gin Ser Met Giu Lys 255 Asp Phe Ser Leu Arg Thr Ser Lys 265 <210> 6 <211> 1157 <212> DNA <213> Petunia x hybrida <220> <221> CDS <222> (179)..(841) <220> <~223> strain: W115 WO 00/04176 <220> <223> tissue type: nectar qland <220> <223> cDNA library of nectaries from Petunia hybrida flowers <220> <223> PCT/NL99/00453 <400> 6 tctgaataca agctgtgtgt gtagagagat ttcataaaga cagcaaacat cccttctttt tgttctgttt taaaagttcc cttcttcaac cagctctttt cctcatcagg gtaagttgca 120 aataaagggg atqttccaga atcaagaaga gaagatgtca gactcgcctc agaggaaq 178 atg Met 1 gga aga gga Gly Arq Gly att gag att aag Ile Glu Ile Lys att qaa aat aca Ile Glu Asn Thr aca aat Thr Asn 226 cgt caa qtc act Arg Gin Val Thr tat gaa ctt tct Tyr Giu Leu Ser ttc tgt aag aga Phe Cys Lys Arg aat ggg ttg ctt Asn Gly Leu Leu aaa aaa gct Lys Lys Ala atc gtt ttc Ile Val Phe gtt ctt tgt Val Leu Cys gat Asp gct qaa gtt gct Ala Glu Yal Ala tca agc Ser Ser cgt ggc cgc ctc Arg Gly Arg Leu gaa tat qct aac Glu Tyr Ala Asn agt gtg aag gca Ser Val Lys Ala aca Thr att gat aga tat Ile Asp Arg Tyr aaa gca tcc tca Lys Ala Ser Ser gat Asp tcc tcc aac act Ser Ser Asn Thr tct act tct gaa Ser Thr Ser Glu aac act caq ttt Asn Thr Gln Phe caa caa gaa gct Gln Gin Glu Ala gcc aaa Ala Lys ctc cqa gtt Leu Arg Val cag Gin 100 att ggt aac tta Ile Gly Asn Leu aac tca aac agg Asn Ser Asn Arg aac atg cta Asn Met Leu 110 ggc gag tct cta agt tct ctg act gca aaa gat ctg aaa gqc ctg gag Gly Giu Ser Leu Ser Ser Leu Thr Ala Lys Asp Leu Lys Gly Leu Giu WO 00/04176 PTN9/05 PCT/NL99/00453 115 acc aaa Thr Lys 130 ctt gag aaa gga Leu Glu Lys Gly agt aga att agg Ser Arg Ile Arg tcc Ser 140 aaa aag aat gaa Lys Lys Asn Glu ctg ttt gct gag Leu Phe Ala Glu gag tat atg cga Glu Tyr Met Arg agg gaa att gat Arg Glu Ile Asp ttg Leu 160 610 658 706 cac aac aac: aat His Asn Asn Asn atg ctt cgg gca Met Leu Arg Ala aag Lys 170 ata gct gag agt Ile Ala Glu Ser gaa aga Glu Arg 175 aat gtg aac Asn Val Asn tac gat oca Tyr Asp Pro 195 atg gga gga gaa Met Gly Gly Glu gag ctg atg caa Glu Leu Met Gin tct cat ccg Ser His Pro 190 cat aat cat His Asn His aga gac ttc ttc Arg Asp Phe Phe gtg aac ggc tta Val Asn Gly Leu cag Gin 205 caa tat Gin Tyr 210 cca cgc caa gac Pro Arg Gin Asp atg gct ctt caa Met Ala Leu Gin tta gta taagtttata Leu Val 220 ataaaatgca tggtttgaag cactctgatt gtggtggatt tggattatgt ataagggagt 911 gcaggccatt tgccaattat tgaaaggtac tcaaacagga agttgaagaa gttcatcatc 971 tctctcatct atatgtctta acaaaagtct tagcttatgg actctaaaac aaagacttaa 1031 tttaacatat aaatataatt gtgtaatgct gttgtattgt atggtatgta tccaaaaaca 1091 ttaataacct atctttttct tcaaattatg tctcctttga tacaaactac taacatattt 1151 tcttat 1157 <210> 7 <211> 221 <212> PRT <213> Petunia x hybrida <223> <400> 7 Met Gly Arg Gly 1 Lys Ile Giu Ile Lys Arg Ile Giu Asn Thr Thr Asn 5 10 WO 00/04176 PTN9/05 PCT/NL99/00453 Arg Gin Val.

Tyr Giu Leu Ser Ser Arg Phe Cys Lys Arg Asn Gly Leu Leu Giu Vai Ala Leu Lys Lys Ala Ile Val Phe Vai Leu Cys Gly Arg Leu Thr Ile Tyr 55 Lys Tyr Ala Asn As n Ser Ser Val Lys Ala Ser Asn Thr Giv Asp Arg Tyr Lys Asn Ala Ser Ser Thr Ser Glu Al a Ile Thr Gin Phe Tyr 90 Asn Gin Giu Ala Ala Lys Leu Arg Val Gly Giu Ser 115 Thr Lys Leu Gly Asn Leu Ser Asn Arg Ser Ser Leu Lys Asp Leu Lys 125 Lys Asn Met Leu 110 Gly Leu Giu Lys Asn Giu Giu Lys Gly Arg Ile Arg 130 Leu Leu Ser 140 Arg Phe Ala Giu Ile 150 Met Tyr Met Arg Giu Ile Asp Asn Asn Asn Gin 165 Met Leu Arg Ala Lys 170 Glu Ala Giu Ser Giu Arg 175 Asn Val Asn Tyr Asp Pro 195 Met 180 Arg Gly Gly Giu Phe 185 Val Leu Met Gin Ser His Pro 190 His Asn His Asp Phe Phe Asn Gly Leu Gin Tyr Pro Arg Gin Asp Asn Met Ala Leu Gin Leu Val 210 215 220 <210> 8 <211> 54 <212> DNA <213> Caliuna vuigaris <220> WO 00/04176 WO 00/41 76PCT/NL99/00453 <221> CDS <222> (54) <220> <221> sigpeptide <222> (54) <400> 8 atg tit ctt cca att Met Phe Leu Pro Ile 1 5 ctc ttc acc att tcc ctc ctc ttc tcc tcc tcc Leu Phe Thr Ile Ser Leu Leu Phe Ser Ser Ser 10 cat gct His Ala <210> 9 <211> 18 <212> PRT <213> Caliuna vuigaris <400> 9 Met Phe Leu Pro Ile Leu Phe Thr Ile Ser Leu Leu Phe Ser Ser Ser 1 5 10 His Ala <210> <211> 2141 <212> DNA <213> Petunia x hybrida <220> <223> <220> <223> strain: W115 NECI promoter <400> cctaggagaa aaggatctgt tatgaagagg gcaattattg ttcaaggttt tgtgtcagac atcaagccta tgattagagt accaagaagc acatqgaact ataataaaaa gagaacatga ctcttaagat tgggaagttc tccaataatt tggggagatg ggatcatatg aagtaaaccq ggatgactca atgttctctg ttgggaagag actgtgagag gctaagtttg aaagaggtgt cttgccccga ctgattttat cattcttaat cgcatggaga aagagtgttc ttgaqcggaa tggtaaggtg tattctagac cacatcgatg aaaggttact tttgatagaa tgtagaacaa WO 00/04176 agtgaccacg acaaaaqttc cgtactacga taaaagtaaa taaatcaagc tagtattacg tttcaaggca ttggccaagt gccagatctg gaattcaaac caaactagtg gcagcaaaaa ctttgtgtca tggcgtgcag acagaaagtt taagttcaag aaaacatcac atatatatat ccatttttct gattttctat aacattgttt tcacctaaaa atggccattc tgtacagttt taaagagaac tattattatt ttaaccctca tttatcatac taacatttgc agcacttgaa PCT/NL99/00453 gcacaataat gtcgtaacaa cgttaactaa aaatatatat gcttgttgga tagtttcttg tcgcggtgtg gttgcaccgt ctacattagc caaaatcaga aacgcagaaa ttcagcagca aacagaacgt actagaaaag cgaaacttgg attcttacgg cttcaactat atatacacac gcttcaaaag actattttgt gcaagtgtac ctacatcatt agttcatqtc ggtctctgat tgttccacta gctgtatttg tatatctttt tttaccgaat agtgagtaaa ctcaaaaggg tgacaagttg gaggagacgt ggcgcttgtc atagaaaaag aggcaaccca ttqtttttgt tattgcagcg gagaggcttt actgaagcat aacgccacaa tagaaatgct aaacagaaac ccagaaattg ctctagcaga aaaacgataa gttgacccat cacatgattt acacaccatt tttaaattat cccttgtaat aattttagta tatggcgggc atttttatat qtgcacttta aattttaatg tttgtcattt ggccaaataa tcttgttttt gtttcttcag gcttcactaa aaggaaaatt aaatgctgga gggaggcaac gaaaatacaa atttttattg agggctcggg aggtaagtgt caacctgttg cgcttggcca gagatgtgtc acagcccatg gctgcgagaa aaaagctata tgcgtcgcqt cccagcgtcg t aa cc catt g cataagtttg tccagcgatc taatatgata tttaaaaaaa tatgcaaacc ggactagacg ccttcatcca ctatacgtaa atttaattaa gaatttggca agaaaaagtc tgtttctctg gtctcttttg aaaaaatcat cacctaaagg agtgagctta cctagctttg aaagagtcgt ttttagttgt actttcggaa aagagttgag cgacacgtga atagcttgga gcacactgca cgtcgcttgg acgcgtcgca agcctgcgtc at tgt atagc cctcttcaac atcggctgat acctaggata ttacctcatt agtcatccat aaatgaqcga aacgcttctt tagccaaata ataatattac tacggaattt tttaactcgg ccgcagattt tgcatatttc ttgttgttct tagattcaac g aaggaagaag aaggtgttgt tatgtaaatg gccgcgacgt t tta cttat t ggtgaggtaa ttggaagcgt aaaattaaga atggaagcaa aagctttgtg cttatggcag tacgccatag gcttggctca ttggtgtgaa cgcgtccagg tataaacaat ttttatatat tttattcaaa agtcaaacaa tggtaagata cttccaacta taaaaacgca tcaaaattga acattataat ttacttgtat ttgtatgcaa ttgccaaaca ccactataaa aagagtattc 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2141

Claims (29)

1. An isolated DNA sequence from the promoter region upstream of a nectary-specific expressed sequence, which nectary-specific expressed sequence encodes a protein comprising the amino acid sequence given in SEQ ID NO: 1, or a protein that has at least 60% homology to the amino acid sequence given in SEQ ID NO:1.

2. An isolated DNA sequence according to claim 1, wherein the nectary-specific .expressed sequence has: a) a nucleotide sequence given in SEQ ID NO:4; or b) a nucleotide sequence which hybridises with a) or with a fragment of a) under the following conditions: pre- hybridisation for lh at about 65 0 C in a solution of Church and Gilbert, comprising 0.5 M sodium phosphate, pH 7.2, 1 mM EDTA, 1% BSA, 7% SDS, followed by hybridisation in the same solution for 18h at about 0 C, followed by washing three times in 0.1 x SSC, 0.1 SDS at about 65 0 C for 30 min.; or c) a nucleoctide sequence that has at least 85% homology to the nuelcotide sequence of a).

3. An isolated DNA sequence according to claim 1 or claim 2, obtained from a plant of Petunia hybrida, the sequence consisting essentially of the sequence given in 25 SEQ ID NO:7, or a functional fragment thereof having promoter activity.

4. An isolated DNA sequence encoding a protein comprising the amino acid sequence given in SEQ ID NO:1, or a protein having at least 60% homology with the amino acid sequence given in SEQ ID NO:1, which protein, when ectopically i expressed, plays a role in sugar metabolism, the expression of the DNA sequence being predominantly confined to the nectaries of a plant. 35 H:\RBell\Keep\50706-9 9 .doc 27/01/04 49 An isolated DNA sequence according to claim 4, having: a) a nucleotide sequence given in SEQ ID NO:4; or b) a nucleotide sequence that hybridises with the nucleotide squence of a) or with a fragment of a) under hybridisation conditions as defined in claim 2; or c) a nuclecotide sequence that has at least 85% homology to the nucleotide sequence of a), the nucleotide sequences as defined under b) and c) having the biological activity of sequence SEQ ID NO:1.

6. An isolated DNA sequence that results from the sequence shown in SEQ ID NO:4 by insertion, deletion or substitution of one or more nucleotides, including naturally occuring variations or variations introduced by targeted mutatenesis or recombination, wherein the DNA sequence encodes a protein exhibiting the same function as the protein according to claim 4.

7. An isolated DNA sequence according to claim 4 having a nucleotide sequence given in SEQ ID NO:4, said sequence being produced by current DNA synthesis techniques.

8. An isolated DNA sequence comprising the coding region 25 for a signal peptide, wherein the information contained in the DNA sequence permits, upon translational fusion with a DNA sequence encoding a protein that is expressed in nectaries, targeting of the protein to nectar, which isolated DNA sequence has: 30 a) a nucleotide sequence given in SEQ ID NO:6 obtained from a plant of Calluna vulgaris; or b) a nucleotide sequence that hybridises with the nucleotide sequence given in under the hybridisation conditions as defined in claim 2; or 35 c) a nucleotide sequence that has at least 95% homology to the nucleotide sequence of a), the nucleotide sequences as defined under b) and c) having H:\RBell\Keep\50706-99.doc 24/10/03 the biological activity of seqeunce SEQ ID NO:6.

9. A recombinant double-stranded DNA molecule comprising an expression cassette comprising the following constituents: i) a promoter functional in plants; and ii) a DNA sequence coding for a protein as defined in any one of claims 4 to 7 which is fused to the promoter sequence in sense or antisense orientation.

10. A recombinant DNA molecule according to claim 9, further comprising a signal sequence functional in plants for the transcription determination and polyadenylation of an RNA molecule.

11. A recombinant double-stranded DNA molecule comprising an expression cassette comprising the following constituents: i) a promoter functional in nectaries of plants, wherein the promoter is that according to any one of claims 1 to 3; and ii) a DNA sequence coding for a protein which is fused to .the promoter sequence in the ,sense or antisense orientation. 25 12. A recombinant double-stranded DNA molecule according to claim 11, further comprising a signal sequence functional in plants for the transcription termination and polyadenylation of an RNA moleule. 30 13. A recombinant double-stranded DNA molecule comprising 00. an expression cassette comprising the following constituents: Si) a promoter functional in nectaries of plants, wherein the promoter is that according to any one of claims 1 35 to 3; ii) a DNA sequence encoding a protein which is fused to the promoter; and H:\RBell\Keep\50706-99.doc 24/10/03 51 iii) a DNA sequence encoding a signal peptide that targets the recombinant protein to nectar, which is translationally fused to the DNA sequence encoding the recombinant protein.

14. A recombinant double-stranded DNA molecule according to claim 13-, further comprising a signal sequence functional in plants for the transcription termination and polyadenylation of an RNA molecule. A recombinant double-stranded DNA molecule according to claim 13 or claim 14, wherein the DNA sequence encoding a signal peptide is as defined in claim 8.

16. A process for producing a transgenic plant exhibiting excretion of a recombinant protein in its nectar, comprising: i) introducing into a plant cell a recombinant double- stranded DNA molecule according to any one of claims 9 to 15, wherein the recombinant protein is excreted in nectar; ii) regenerating plants from the transgenic cell; and iii) selecting transgenic plants. 25 17. A process for producing a transgenic plant exhibiting modified nectar composition, comprising: i) introducing into a plant cell a recombinant double- stranded DNA molecule according to any one of claims 9 S• to 15, wherein the recombinant protein interferes with metabolic pathways in the nectaries; ii) regenerating plants from the transgenic cell; and iii) selecting transgenic plants. :i 18. A process for producing a transgenic plant exhibiting 35 a modified nectar sectretion, comprising: i) introducing into a plant cell a recombinant double- stranded DNA molecule accoding to any one of claims 9 H:\RBell\Keep\50706-99.doc 27/01/04 52 to 15, wherein the recombinant protein interferes with sink strength of nectaries; ii) regenerating plants from the transgenic cell; and iii) selecting transgenic plants.

19. A process for producing a transgenic plant exhibiting a modified nectary development, comprising: i) introducing into a plant cell a recombinant double- stranded DNA molecule according to any one of claims 9 to 15, wherein the recombinant protein interfers with the development of nectaries; ii) regenerating plants from the transgenic cell; and iii) selecting transgenic plants.

20. A process for producing honey from modified nectar of transgenic plants, comprising: i) producing a transgenic plant by introducing into a plant cell a recombinant double-stranded DNA molecule according to any one of claims 9 to 15, regenerating plants from the trangenic cell, and selecting modified plants exhibiting the excretion of nectar with a modified composition; and ii) allowing insects to collect nectar from the transgenic plants and to process the nectar into honey.

21. A process for producing a recombinant gene product from honey, comprising: i) producing a transgenic plant by introducing into a plant cell a recombinant double-stranded DNA molecule according to any one of claims 13 to 15, regenerating plants from the transgenic cell, and selecting modified plants exhibiting excretion of the recombinant gene product in nectar; ii) allowing insects to collect nectar from the transgenic 35 plants and to process the nectar into honey; and iii) isolating and purifying the gene product from the honey. H:\RBell\Keep\50706-99.doc 27/01/04 53

22. A process for producing a metabolite from honey, comprising: i) producing a plant according to a process of any one of claims 16 to 19, wherein the plant excretes the metabolite in nectar; ii) reproducing the plant by current breeding and selection methods; iii) allowing insects to collect nectar from the selected plant and to process that nectar into honey; and iv) isolating and purifying the metabolite from the honey.

23. A process for producing a metabolite from honey, comprising: i) producing a plant according to a process of any one of claims 16 to 19, wherein the plant excretes a metabolite in nectar; ii) allowing insects to collect nectar from the selected plant and to process that nectar into honey; and iii) isolating and purifying the metabolite from the honey.

24. A process of any one of claims 20 to 23, wherein the insects are bees. 25 25. Microorganisms containing one or more DNA sequences according to any one of claims 1 to 8.

26. Microorganisms containing one or more recombinant DNA molecules according to any one of claims 9 to

27. A plant cell or plant cell culture transformed with o .one or more DNA sequences according to any one of claims 1 to 8. 000005

28. A plant cell or plant cell culture transformed with one or more recombinant DNA molecules according to any one of claims 9 to H:\RBell\Keep\50706-99.doc 27/01/04 54

29. A plant consisting essentially of the plant cells of claim 27 or claim 28.

30. A transgenic plant obtained by a process of any one of claims 16 to 24.

31. Seeds, tissue culture, plant parts or progeny plants derived from a transgenic plant according to claim

32. Honey obtained from nectar from transgenic plants produced by a process of any one of claims 16 to 19, which nectar comprises a recombinant gene product.

33. Use of a DNA of any one of claims 1 to 15, a microorganism of claim 25 or claim 26, a cell or cell culture of claim 27 or claim 28, a plant of claim 29 or claim 30, or a seed, tissue culture, plant part of progeny plant of claim 31, in the production of a recombinant gene product.

34. Use of a DNA of any one of claims 1 to 15, a microorganism of claim 25 or claim 26, a cell or cell e culture of claim 27 or claim 28, a plant of claim 29 or claim 30, or a seed, tissue culture, plant part of progeny plant of claim 31, in the production of a metabolite from ee*honey. An isolated DNA sequence according to claim 1 or claim 4, substantially as herein described with reference to any one of the examples or figures.

36. A process according to claim 22 or claim 23, substantially as herein described with reference to any one 35 of the examples or figures. *.oo.

37. Honey according to claim 32, substantially as herein H:\RBell\Keep\50706-99.doc 27/01/04 55 described with reference to any one of the examples or figures. Dated this 27 th day of January 2004 PLANT RESEARCH INTERNATIONAL B.V. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia S H:\RBell\Keep\50706-99.doc 27/01/04