CA2333897A1 - Process to collect metabolites from modified nectar by insects - Google Patents

Abstract

The invention relates to a recombinant double-stranded DNA molecule comprising an expression cassette comprising the following constituents: 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. The invention further relates to a process for producing a recombinant gene product from honey, comprising: i) producing a transgenic plant by introducing in a plant cell a recombinant double-stranded DNA molecule, regenerating plants from the transgenic cell, and selecting modified plants exhibiting excretion of the recombinant gene product in nectar, ii) allowing insects, preferably bees, to collect nectar from the transgenic plants and to process the nectar into honey, and iii) isolating and purifying the gene product from the honey.

Description

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 variati-on between nectars of flowers from different species mainly being the concentration and ratio of glucose, fructose and sucrose (Baker a:nd 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 (RUE.E 2S~

WO 00/04176 PCT/Nh99/00453 _ Z _ (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 to 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 honey-cambs 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 u.p 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) processed nectar is temporarily stored in a honeycomb cell and taken out later far 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 concentra-ted to an average water content of 20%. Most nectars and honeys only contain traces of protein (<0.2%). However, CaZZuna 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 digesti-on 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 produc tion system for proteins in nectar and honey was estab fished.
It is an object of the present invention to show that recombinant proteins can be secreted in nectar of transge-nic plants, that this nectar is collected by honeybees and that the bees process this nectar into honey that contains the unaltered protein in a concentrated form.
Defi,niti.ons Honev: A substance that contains approximately 80% sugar and varying amounts of other components and that is produ-ced by insects, preferably bees, that collect and process SUBSTITUTE SHEET (RULE 26) _ g _ nectar from floral or extrafloral nectaries, from honeydew, other plant exudates or artificial sugar solutions.
MADS box crepe: a gene coding for a transcription factor having a region of 5& amino acids which is homologous to a similar region a_n the Arabidopsis AGAMOUS protein and Antirrhinum DEFICIENS protein. This region is called the 'MADS box'. At least 500 of the amino acids in this region should be identical to the amino acid composition in the MADS boxes of AGA1!~OUS and/or DEFICIENS.
Nectarv: 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 necta-ries. Nectar can also contain substances like minerals, amino acids, proteins, organic acids, volatiles, alkaloids etc.
Recombinant urotein: the gene product of a recombinant DNA
molecule.
Recombinant DNA molecule: A DNA molecule in which sequences 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 the enzyme to the correct transcriptional start site.
Summary of the invention 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 SUBSTITUTE 5HEET (RULE 26) developed. With the availability of efficient transformati-on techniques fox 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 obtai-ned after extended, and therefore expensive, purification steps.
l0 The present invention provides a method to produce metab-olites, 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. Tf one hectare of rapeseed yields 100-500 kilo honey in one season, a yield of 2 to l0 kilo protein can be obtained. Tn addition, the present invention provides a method to collect metabolites from honey that is derived from non-transgenic 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 barbatum and of Piptanthus nepalens.is (Martini et al., 1990).
This invention provides a gene from petunia, NEC1, that is highly expressed in the nectaries of petunia and weakly expressed in the stamens. It also provides another gene from petunia, FBP15, that encodes a MARS box protein and which is specifically expressed in the nectaries of petu-nia. Further, it provides the isolated DNA sequences of the promoters of the NEC1 and the FBPlS genes. Furthermore, this invention provides an isolated DNA sequence expressed in nectaries encoding a signal peptide that is translation-SUBSTITUTE SHEET (RULE 26) W~ 00/04176 PCT/NL99/00453 -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 vulgar.is). This invention gives proof that protein-contai-ning 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 qualitati-ve alterations. Tlzis invention also proofs that a recombi-nant protein can be produced in nectar of transgenic plants to 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 sequen-ce listing hereafter or homologs of NEC1. A homolog of NEC1 is predominantly expressed in nectaries and/or has at least 60% homology with the amino acid sequence given in SEQ ID
NO:1. Further this invention provides an isolated DNA
2o sequence which encodes a protein indicated FBP15 and having the amino acid sequence given in SEQ ID N0:2 of the sequen-ce listing hereafter or a homolog of FBP15. A homolog of FBP15 is specifically expressed in nectaries and belonging to the MARS box family. Furthermore, a homolog is also a gene sequence that has at least 80% homology within the MARS box region and a 60 0 overall homology with the amino acid sequence given in SEQ ID N0:2. Further this invention provides the characterisation and the isolation of a DNA
sequence which encodes a signal peptide indicated "CVSP"
(Calluna v_ulgaris 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 characteri-sed in that they comprise the NEC1 gene and the FBPI5 gene having the nucleotide sequences given in SEQ ID N0:4 and SUBSTITUTE SHEET (RULE 26) sEQ ID N0:5 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 TD NO:4 and 5 respectively.
Furthermore, the DNA sequences of the invention can also be characterised in that they comprise signal sequence CUSP
having the nucleotide sequence given in SEQ ID N0:6, or an essentially identical nucleotide sequence or part thereof or derivatives thereof which are derived from said sequen ces 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: I, or a homologous protein.
Furthermore, this invention provides an isolated DNA
sequence from the promoter region upstream of a nectary specific expressed sequence, which nectary-specific expres sed sequence encodes a protein comprising the amino acid sequence given in SEQ ID N0:2, or a homologous protein.
In a further aspect, the invention provides a protein encoded by any of the above defined DNA sequences. Further, 3o 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 anal 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 2B) _ g -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 1o DNA molecules comprising expression cassettes to be used in the above process. Further, the invention provides transge-nic bacteria, transgenic plants producing recombinant proteins in nectar, and also plant cells, tissue culture, plant parts or prcogeny 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 Apl1-AP15 on cDNA samples of pistils without nectaries (two independent samples), nectaries (two independent samples), leaves and a mixture of sepals (s), petals (p) and stamens (a). Bold arrow depicts the cloned fragment DD18.
Figure 2 is the DNA sequence of the Differential Display RT-PCR clone DDl8a. The primers prat 122 and prat lI9 that were used for 5' RACE PCR reactions are underlined.
SUBSTITUTE SHEET (RULE 26) _ g _ Figure 3 is the DIVA sequence of clone RC8, obtained by RACE
PCR with gene specific primers prat 122 and prat 119 (Fig.
2) in combination with adapter primers. Primex 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 NECI cDNA. The translation start (ATG) and translation stop (TAA} are depicted bold.
Figure 5 shows the expression of NEC1 (A) and FBP.I5 (B) 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 pDDlBa was used as a probe. For blot B the full length cDNA of FBP15 was used as a probe.
2o Figure 6 Expression of NEC1 by in situ localisation of NEC1 transcripts (A) and activity of the NECI promoter in the nectaries (B) and the stamen (C) as shown by GUS
expression driven by the NECI promoter. The GUS assay used for the stamens was incubated overnight without modificati-ons to prevent di:Efusion (example 8}. The GUS assay for the nectaries was incubated far 5 hrs, using an assay mixture to prevent diffusion (example 8). For in situ localisation longitudinal sections of flowers of Petunia hybrida were hybridised with digoxigenin-labeled antisense NEC1 RNA
Figure 7 is the DNA sequence from the promoter region upstream of a sequence encoding the NEC1 protein. Underli-ned is the translation start of NEC1 cDNA.
Figure 8 depicts a schematic presentation of the T-DNA
region between the borders of the binary vector pBNEPI, containing the NECI promoter (Figure 7), the GUS reporter SUBSTITUTE SHEET {RULE 26) gene and the nos terminator in pBINPLUS. This vector was used to generate transgenic plants to study the expression of the NEC1 promoter.
Figure 9 shows the SDS-PAGE separation of proteins that are present in commercial honey samples from different flowers.
M= marker, lane :I: 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, RHlOx) and heather (HH2x, HHlOx) and of nectar samples of rapeseed (RN2x, RNlOx) and heather (HN2x, HNlOx). M
molecular weight marker. Two (2x) or ten (10x) fold diluti-ons were used.
Figure 11 shows the SDS-PAGE separation of proteins present in dilutions of the sugar/BSA feeding solution (A) and of honey from bees that had collected the sugar/BSA solution (B). The dilutions of the sugar/BSA and honey/BSA solution was the same far both gels: 1~ 15x, 2= 30x, 3= &Ox, 4= 75x, 5= 75x, 6= 90x, 7w 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 3o protein sequence of CVH29. The degenerated primers prat 176 and prat 177 are underlined (A). 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 axe underlined.
SUBSTITUTE SHEET (RULE 26) 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 I5 is the sequence of the synthetically produced DNA molecule encoding the signal sequence CUSP (boxed} with l0 linkers.
Figure 16 is the schematic representation of the plasmid pCVI. 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 FBP15. The translation start (ATG) arid translation stop (TAA) are boxed. The MAD-box and K-box region are underli-ned.
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~

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 N0:4. The deduced amino acid sequence of the NEC.I gene is given in SEQ TD NO:1. The NEC1 gene shows strong expression in the nectaries and in a very localised region of the anther filaments of Petunia hybri-da. The deduced amino acid sequence of NEC1 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 NEC1 gene. More precisely, it is also possible to use DNA sequences which have a high degree of homology to the DNA sequence of the NEC1 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
N0:4 by insertion, deletion or substitution of one or more nucleotides. This includes naturally Occurring variations or variations introduced through targeted mutagenesis or SU8ST1TUTE SHEET (RULE 26) recombination. The DNA sequence shown in SEQ ID N0:4 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 FBP15 or a homo7.ogous protein or part thereof. The cDNA
sequence of FBP1S is given in SEQ ID N0:5. FBP15 shows exclusively expression in the nectaries of Petuzxia hybrida.
The function of F'BP15 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 FBP1S. More precisely, it is also possible to use DNA sequences which have a high degree of homology to the DNA 'sequence of FBP15, but which are not completely identical, in the process according to the invention. The use of sequences having homologies between 85 and 100 °s is t:o be preferred. DNA sequences can also be used which result from the sequence shown in SEQ ID N0:5 by insertion, deletion or substitution of one or more nucleo-tides. This includes naturally occurring variations or variations introduced through targeted mutagenesis or recombination. The DNA sequence shown in SEQ ID N0:5 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 N0:1, 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) sing the amino acid sequence given in SEQ ID N0:2, 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 nectary-specific expressed sequence, which nectary-specific expres-sed sequence has:
a) a nucleotide sequence given in SEQ ID N0:4, or b) a nucleotide sequence obtainable by hybridisation with l0 the nucleotide sequence of (a) 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 hybrids, the sequence consisting essenti-ally of the sequence given in SEQ ID N0:7, or a functional fragment thereof having promoter activity.
In a further aspect, the invention provides an isolated DNA
2o sequence from the promoter region upstream of a nectary-specific expressed sequence, which nectary-specific expres-sed sequence has:
a) a nucleotide sequence given in SEQ ID N0:5, or b) a nucleotide sequence obtainable by hybridisation with the nucleotide sequence of (a) 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 hybrids, the sequence consisting essenti-ally of the sequence given in SEQ ID N0:8 , 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) _ ~5 that is expressed. in nectaries, targeting of the protein to nectar. More specifically, the DNA sequence comprises the nucleotide sequence given in SEQ ID N0:6 obtained from a plant of Calluna vulgaris, or a nucleotide sequence obtai-noble by hybridisation with the nucleotide sequence given in SEQ ID N0:6. 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
N0:6 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 N0:6 can also be produced by using DNA synthesis techniques. The signal peptide CVSP was isolated tram 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 SUHSTiTUTE SHEET (RULE 2f) a consequence of this pratein expression. In particular, it relates to processes where the recombinant protein is an enzyme that interferes with the sugar metabolism in necta-ries.
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 compri sing an expression cassette having the following constitu ents 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 enco ding the recombinant protein, and optionally iv) a signal sequence functional in plants for the transcription termination and polyadenylation of ari RNA molecule.
Such DNA molecules are also subject of the invention. The present invention provides an example of such a DNA molecu-le that contains the described expression cassettes in the form of plasmid pCV3 (Fig. 18), which comprises the promo-ter region of the NEC.Z 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 sequen-ce 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) sequences that encode proteins to be used for pharmaceuti-cal 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 possi-ble 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 necta-ries, resulting in changed levels of compounds that are already present in nectar, or the formation of new com-pounds in nectar.
In addition, the present invention also relates to expres sion cassettes that contain the above mentioned DNA sequen ces, 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 recombi-nant 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-transge-nic 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) A preferred embodiment of the process discussed above provides that the expression cassette is transformed to a plant species that produces nectar. Preferably, the recom-binant 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 Z5 conditions are greenhouses or field facilities where transgenic plants can be grown according to the safety rules that are required. Preferably, the controlled condi-tions are such that bee colonies that perform normal foraging behaviour can be maintained in the same compart-2o ment during the flowering period. Preferred plants origina-te from the Brassicaceae family, in particular Brassica napus.
Examples Example l:
Cloning of NEC1 The NEC.I cDNA was isolated using the mRNA Differential 3o Display system (Genhunter Corporation, Brookline USA). The isolation of total RNA from nectaries, sepals, petals, stamens and pistils from open flowers and fram young leaves of Petunza hybrids 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 MessageCleanTM
Kit (Genhunter Corporation Brookline USA, cat. No. M6o1). A
SUBSTITUTE SHEET (RULE 2fi) reverse transcription reaction was carried out on 0.1 ~,g RNA of each samp7.e, 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 pro-ducts were loaded on a sequencing gel and after electropho-resis the gel was blotted on 3M paper, dried and exposed to x-ray film (Figure 1). Two adjacent nectary-specific bands were cut out from the blot and the DNA was purified accor-1o ding to the manual. Reamplification of the fragment was carried out using the oligo-dT primer T12MG and the arbri-tary primer AP15. 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
MgCl2 and 2.5 volume of 96% ethanol to the supernatant. The pellet was dissolved in 10 ~.1 TE buffer. The fragment, now called DDlBa, was cloned into a PMOSBlue T-vector {RPN
1719, Amersham Little Chalfont UK) giving the vector pDDlBa.
The nucleotide sequence of this 3' cDNA clone was determi-ned by the dideoxynucleot,ide chain termination method (ABI
PRISMT'" Ready Reaction DyeDeoxy~M Terminator Cycle Sequen-cing 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. Brief-ly, Poly A~- RNA was isolated from nectaries of Petunia hyhrida flowers. After double stranded cDNA synthesis, adapters were ligated and a 5'RACE reaction was carried out using the adapter primer API supplied in the kit and a gene-specific primer prat 122. The nucleotide sequence of prat 122 is: 5'-gtgggaaggctatgctacaagc-3' (Figure 2). The PCR product was diluted lOx and 1 ~.1 was used in a second 5' 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 OOI04176 PCTINL99l00453 (Figure 2). The nucleotide sequence of prat 119 is: 5'-ccttctccatggactgcaatgcg-'3 . After gel electrophoreses a fragment of ~850 by was obtained that hybridised with clone DDl8a. 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 DDl8a and RC8 are shown in Figure 4, comprising the full length cDNA of a gene called NEC.2 :hereafter. The NEC1 clone has a length of 1205 nucleotides and encodes for a polypeptide of 265 amino acid residues. Based an the deduced amino acid sequence, high homology was found with a cDNA that is associated with Rhizobium-induced nodule development in the legume Med.icago trunculata (MtN3, gene bank number: gnl/PID/e274341). The I5 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 putati-ve 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, 30% similarity). The function of NEC1 has not yet been determined.
Example 2:
Cloning of FBP15 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) 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 (Angensnt 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 SK-plasmid with the cDNA insertion between the EcoRI and Xhol cleavage site of he polylinker were generated. Cross-to hybridisation of the purified clones revealed 3 independent clones that did not cross hybridise with previously isola-ted FBP cDNA's and which were designated FBP.IS, FBP16 and FBP17. The nucleotide sequence of FBP15 was determined by the dideoxynucleotide-mediated chain termination method and is depicted in SEQ ID N0:5. 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 axe present in FBP15: a N-terminal located MADS box region which shows a high degree of similarity with other MARS box proteins, and a K-box in the middle of the protein with an alpha helical structure, FBPIS is most similar to the tobacco MADS box protein NAGl, which is an Agamous homolog and expressed in whorl 3 and 4 (Huang et al., 1996, Mizukami et al., 1996).
Example 3:
Expression of FBP:~5 Expression of FBP15 was determined by standard Northern blot hybridisation experiments. A DNA fragment comprising the complete cDNA of FBP15 was used as a probe. High stringency hybridisation and washing conditions were used.
Using 10 ~g of total RNA from various petunia tissues, expression of FBPaS 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 SU6STiTUTE SHEET tRULE 26) Kit (Pharmacia Biotech), expression of FBP15 was only detectable in nectaries as shown in Figure 5B.
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., 3.994. A hybridizing signal was observed evenly strong in all cells of the l0 nectary tissue.
Example 4:
Expression of NECI
The RNA expression of NEC1 was determined by standard Northern blot hybridisation experiments. A DNA fragment comprising the complete sequence of the Differential 2o Display clone DD18 (Figure 2) was used as a probe. Using 10 ~g of total RNA from various petunia tissues, strong expression of NEC.1 was detectable in necta.ries and weak expression in anthers. No expression was detectable in other floral organs, in leaves or in roots (Figure 5A).
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 NEC1 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 gene-specific primers prat 122 and prat 129 (Figure 4). The nucleotide sequence of prat 122 is: 5'-gtgggaaggctatgctaca-agc-3"', comprising the nucleotides 1015 to 1036 of the NEC1 cDNA. The nucleotide sequence of prat 129 is: 5'-gggatccatgctcgcaattacQtqctgatg-3°, comprising the nucleoti-des 79 to 100 of i:he NEC1 cDNA. The gene-specific region of the primers is underlined. The primer contains an extra SU8ST1TUTE SHEET (RULE 26) BamHI and Ncol site at the 5'end. A PCR fragment of 958 nucleotides was obtained and cloned into a PMOSBIue 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 Canas et al., 1994.
Strong hybridizing signals were observed in the outer cell layers of the nectaries (Figure 5A) Example 5:
Isolation of NEC1. promoter fragment The promoter fragment of NEC.I was cloned using the genome walker protocol (PT3042-1) and kit as provided by Clontech Laboraties. Briefly, genomic DNA from Petunia hybrids was digested with 5 different blunt cutting restriction enzy-mes. GenomeWalker adapters were ligated and PCR reactions 2o 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 NEC1 cDNA (Figure 4 ) . 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 NECZ 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 pMAS -10 . Figure 'I ( SEQ ID NO : 7 ) shows the DNA sequence of the NECZ promoter in the construct pMA5-10, including the translation start. of NECI cDNA.
SUBSTITUTE SHEET (RULE 26) Example 6:
Construction of ,NEC1 promoter-GUS
A PCR reaction was performed on pMA5-10 (example 5), using the forward vector primer U19 of pMOSBIue and the gene-specific primer prat 169. The nucleotide sequence of prat 169 is:
5'-cgctgcagcgccataattttttttagtgaa~ccccc-3' The gene-speci fic region is underlined. The primer contains an Ncol and BgIII restriction site at the 3' end. The PCR product was digested with Kpnl and Ncol and ligated into a pBluescript--derived vector (pM04) that contains the NTM19 promoter (Custers et al., 1997), the reporter gene GUS and the nos terminator. The KpnT/NcoI NTM19 promoter fragment was replaced, resulting in a .NECI-promoter/GUS translational fusion. The resulting plasmid pNEPI was digested with Smal 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 pBNEPl (Figure 8). pBNEPI 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 veri-fied 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 00104176 PCTlNL99100453 Example 8:
Histochemical GUS assay Different plant parts of Kanamycin-resistant plants trans-formed with the pBNEPI construct were analysed for the distribution of a-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°C for 1 h, then rinsed three times 20' with 100 mM phosphate buffer containing 1 mM potassium ferricyanide. After this treat-went the standard GUS assay was performed with the modifi-cation that ferricyanide was excluded from the reaction mixture.
Example 9:
Results histochemical GUS assay In very young flowers (~1,4 cm) na 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 ne~tary parenchyma. In cross sections of the anther filaments GUS expression was obser ved in all cells except in the xylem of the inner vascular bundle.
Example 10:
Protein analysis of heather honey and nectar Samples of pure heather honey, together with samples of rapeseed, clover, wattle bark and lavender honey were di-luted, dialysed and loaded on a 12% SDS page gel (Laemmli, SUBSTITUTE SHEET (RULE 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 vu.Igaris 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 molecu-lar 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 CVH50 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 0.5°s.
Example 11:
N-terminal sequence analysis of CVH29 and CVH50 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 (630) with germin-like proteins isolated from Arabidopsis (Figure 12).
SUBSTITUTE SHEET (RULE 2fi) Example 12:
Identification signal sequence of CVH29 Because the gerrnin-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: 5'-gayttyt-gygtngcngaycc-3' (y= c or t, n= c, t, a or g). The sequence of the reversed primer prat 177 is: ccrtgraanacraartcrtc (r= g or a). A PCR reaction performed on genomic DNA of heather yielded a 99 by 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 (proto-col PT1115-1, Clontech Palo Alto USA). The sequence of gene-specific primer prat 207 that was used is: 5'-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 mRNA kit. After cDNA synthe-sis 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 S' 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 (SEg ID N0:6) is:
5'-atgtttcttccaattctcttcaccatttccctcctcttctcctcctcccatgct-

3'.
SUBSTITUTE SHEET (RULE 26) 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 pMAS-10 (example 5) using the forward primer prat 247 and the reversed primer prat 248 (Fig. 7). Prat 247 contains an extra Pst1 restriction site.
The Ndel restriction site of prat 248 coincides with the ATG translation start of NEC.I. The nucleotide sequence of prat 247 is: 5'-ggctgcaggaqtgttctttgataqaatg-3', the nucleotide sequence of prat 248 is: 5'-cgcca-tatgtttttttatggaaqcccc-3'. Gene-specific regions are underlined. A 1,8 kb promoter fragment was obtained and 3.5 cloned into a pMOSBlue vector, yielding the plasmid pNECP.
A DNA molecule encoding the signal sequence CVSP as depic-ted in SEQ ID N0:6 was produced by synthesis and subsequent annealing of two oligo molecules prat 245 and prat 246. The sequence of prat 245 is: 5'tatattccttccaattcttttcactatttct-cttcttttctcttcttctcatgcttctgttcttgatttc'3, the sequence of prat 246 is: 5'gatccgaaatcaagaacagaagcatclaqaagaagagaaaacxaa-ctagaaataat aaaagaattggaacLqaaca'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 Arabidop-sis. 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 15.
The fragment was ligated into a Nde1/BamH1 cut PMOSBlue vector, yielding the plasmid pCVSP.
pNECP was digested with Ndel and PstI to release the NEC1 promoter fragment which was cloned into the Pstl/Nde1 cut SUBSTITUTE SHEET (RULE 26) pCVSP, yielding the plasmid pCVI. A schematic representati-on of pCV1 is given in Figure 16.
A 2S0 by long fragment containing the NOS terminator sequence (NOST} was obtained by PCR, using the forward primer prat 251 and the reversed primer prat 2S2 on DNA of pRAP 33, which is a pUC 19 derived plasmid. Prat 251 adds a SacI and Xhol site, prat 252 adds a Smal and EcoRI site.
The sequence of prat 251 is: 5'-gggagctcgagtcgttcaaa-catttctacaataaaQ-3'. The sequence of prat 252 is: 5'-cgaatt-cccgggatctaQtaacataQatgacac-3' The HOST-specific regions are underlined. The PCR product was cloned into pCR-ScriptTM
Amp SK(+) Cloning Kit (Catalog 21188-21190, Stratagene La Jolla USA}, yielding the plasmid pCR-NOST. pCR-NOST was digested with Sacl 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 BamHl restriction site at the 5'end and a Sacl restriction site at the 3'end. The sequence of prat 249 is:
5'-ccggatccatctttaca~tcctcrtagaaacc-3'. The sequence of prat 250 is: 5'-gggagctcccacccLaggctqtaq=3'. The GUS specific regions are underlined. Subsequently, the PCR fragment was digested with BamHI and Sacl and ligated into the BamHI/Sa-cI cut plasmid pCVNOS, yielding the plasmid pCV2. A schema-tic representation of pCV2 is given in Figure 17.
pCVI 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 Ascl and SUBSTITUTE SHEET (RULE 26) _ 30 Smal and the resulting fragment is cloned into a derivati-ve of the binary plasmid pBIN, yielding the binary plasmid pBCV3. pBCV3 was transferred from Eschexich.ia coli to the Agxobactexium tumefaciens strain LBA4404 and C58pMP90 by electroporation. The transformed Agrobacterium strain was used to transform Arabidopsis and petunia.
Example l4:
Protein production in nectar Using the GUS reporter gene, GUS activity in nectar of transgenicplants was measured according to the method as described by Jefferson et al., (19871. Briefly, the assay was carried out by measuring the amount of methyl umbelli-ferone (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 1nM MU
(Angenent et al., 1993).
Example 15:
Feeding experiments with honeybees In September 1996 a beehive located outside was supplied with a 25o sucrose solution supplemented with 2% BSA
(bovine serum albumin). After 3 weeks the bees had consumed 15 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) trated 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 25 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 green-house 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 NEC1 promoter.
SUBSTITUTE SHEET (RULE 26) WO 00/0417b PCT/NL99/00453 Plasmid DNA of pNEPI (example 6) was digested with Kpnl and NcoI to release the 1800 by NECK 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 35SCaMV
terminator of pol.yA signal cauliflower mosaic virus termi-nator sequence downstream of the BARNASE-BARSTAR operon.
The resulting plasmid pWP126 was digested with Kpnl/ 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 a.re less specific than can be concluded based on promoter/GUS expression is concluded. Because the bacterial BARNASE is highly cytotoxic at very low concen-trations it can be preferred to protect other plant tissues by expression of a ribonuclease inhibitor gene under the control of a weak, constitutive promoter (e. g. NOS promo-ter) 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 MARS box genes can change the developmental fate of floral organs or cells. Transge-nic petunia plants ectopically expressing FBP11, an ovule-SUBSTITUTE SHEET (RULE 26) specific MADS box gene, develop ovule-like structures on sepals and petals ( Colombo et al., 1995). FBP15 is a nectary-specific MARS box gene, involved in the molecular regulation of nectary development. In petunia nectaries develop at the base of the carpel. Ectapic expression of FBP15-in petunia may result in the development of nectaries on other organs of the flower ar on vegetative parts of the plant . An example is given of a gene construct that , when transformed to a plant, results in ectapic expression of FBP15.
FBP3.5 was amplified using:a 5' primer that hybridises with .
FBP15 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 5'primer contains a NcoI recognition site, the 3'primer contains a BamHI recognition site. After the sequence was confirmed, the amplified FBP15 fragment was inserted as a BamHI/NcoI
fragment into the binary vector pCP03~. 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 Agrobacteriurn GV31o1 to petunia variety W115, using the transformation method as described in example 7. Transgenic petunia plants were selected that show ectopic nectary development.
Example 1.9:
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 up-regulation of genes involved in the establishment of the SUBSTITUTE SHEET (RULE 26) ratio between hexoses and sucrose in nectar will therefore modify nectar composition. An example is given for anti-sense expression of a petunia-derived invertase gene.
PCR primers were designed that hybridise with the cDNA of an invertase gene cloned from Solanum tuberoaum. The 5' primer 5'-AAGGACTTTAGAGAGACCCGACCACTGCTGG-3'and the 3' primer 5'-AAATGTC:TTTGATGCATAATATTTCCCATAATC-3' were used for a PCR reaction on genomic DNA of petunia to yield a l0 fragment of around 420 bp. The fragment was sequenced and cloned into a pM4SBlue 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 1.5 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 20 and a 3' primer that hybridises with sequences just down-stream 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.
25 The chimerical gene constructs are transferred via Agrobac-terium Gv31o2 to petunia variety W115, using the transfor-mation method as described in example 7. Transgenic petunia plants were selected that exhibit modified sugar compositi-on in nectar.
Example 20 Modification of plant development A DNA which is the IiTECI 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) sense RNA. Plants were regenerated from the transgenic cells as described in example 7. Plants that ectopically express the NEC1 gene exhibited modified leaf morphology and modified sugar composition. Furthermore, plants that ectopically express the NEC.I gene showed a delay in flowe-ring time.
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Lane B.G., Dunwell J.M., Ray J.A., Schmitt M.R. and Cuming A.C. (1993). Germin, a protein marker of early plant development, is an oxalate oxidase. The Journal of Biological Chemistry 268 (17), 12239-12242.
Mariani C., Gossele V., De Beuckeleer M., De Block M., Goldberg R.B., De Greef W. and Leemans J. (1992). a chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 357, 384-387.
Martini M., 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) ~ 02333897 2001-O1-16 Mizukami Y., Huang H., Tudor M. and Ma H. (1996). Functio-nal domains of the floral regulator AGAMOUS: Characteri-sation of the DNA binding domain and analysis of domi-nant negative ~tnutations. The Plant Cell 8, 831-845.
Tsuchimoto S., Van der Kro1 A.R. and Chua N.-H, 1993.
Ectopic expression of pMA:DS3 in transgenic petunia phenocopies the petunia blind mutant. Plant Cell 5, 843-853.
Verwoerd T.C., Dekker, B.M.M, and Hoekema A. (1989}. A
small-scale procedure fox 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) Sequences:
SEQ ID N0:1 amino acid sequence NEC1 SUBSTITUTE SHEET (RULE 26) SEQ ID No: 2 amino acid sequence of FBP15 Met G1y Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu val Ala Leu Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ala Asn Asn Ser Val Lys Ala Thr Ile Asp Arg Tyr Lys Lys Ala Sex Ser Asp Ser Ser Asn Thr Gly 65 70 ?5 80 Ser Thr Ser Glu Ala Asn Thr Gln Phe Tyr Gln Gin Glu Ala Ala Lys Leu Arg Val Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg Asn Met Leu Gly Glu Ser Leu Ser Ser Leu Thr Ala Lys Asp Leu Lys Gly Leu Glu Thr Lys Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys Lys Asn Glu Leu Leu Phe Ala Glu Ile G1u Tyr Met Arg Lys Arg Glu Ile Asp Leu His Asn Asn Asn Gln Met Leu Arg Ala Lys Ile Ala Glu Ser Glu Arg Asn Val Asn Met Met Gly Gly Glu Phe Glu Leu Met Gln Ser His Pro Tyr Asp Pro Arg Asp Phe Phe Gln Val Asn Gly Leu Gln His Asn His Gln Tyr Pro Arg Gln Asp Asn Met Ala Leu Gln Leu Val 210 27.5 220 SUBSTITUTE SHEET (RULE 26) SEQ ID N0:3 amino acid sequence CUSP
MFLPILFTISLLFSSSH.A
SUBSTITUTE SHEET (RULE 26) SEQ ID N0:4 Nucleotide sequence NEC1

4 1001 TTACCTTATAATT.AGCTTGTAGCATAGCCTTCCCACTAATAATTCTGCTT

5 SUBSTtTUTE SHEET (RULE 26~

6 PCT/NL99/00453 SEQ ID N0:5 Nucleotide sequence FBP15 151 GAAGATGTCAGACT(~GCCTCAGAGGAAGATGGGAAGAGGAAAGATTGAGA
l0 i5 301 TGAAGTTGCTCTCATCGTTTTCTCAAGCCGTGGCCGCCTCTATGAATATG

SUBSTITUTE SHEET (RULE 261 SEQ ID N0:6 Nucleotide sequence CVSP
ATGTTTCTTCCAATTCTCTTCACCATTTCCCTCCTCTTCTCCTCCTCCCATGCT
SUBSTITUTE SHEET (RULE 2~j SEQ TD N0:7 Nucleotide sequence NECI promoter 1 CCTAGGAGAAATCAAGCCTACTCTTAAGATGGATGACTCACTTGCCCCGA .

51 TGGTAAGGT(3AAGGATCTGTTGATTAGAGTTGGGAAGTTCATGTTCTCTG

301 TGTGTCAGA(~GAGAACATGAAAGTAAACCGAAAGAGGTGTTTGAGCGGAA

451 AAATGCTGGA.AGTGAGCTTAAAGGTGTTGTCGTACTACGACGTTAACTAA

501 GGCGCTTGTCGGGAGGCAACCCTAGCTTTGTATGTAAATGTAAA.AGTAAA

7a1 ACTTTCGGAAGGTGAGGTAATTTCAAGGCATCGCGGTGTGTATTGCAGCG

801 GAGAGGCTTTCAACCTGTTGCGACACGTGAAA.AATTAAGAGCCAGATCTG

901 GAATTCAAACCAAA.ATCAGAAACGCCACAAGAGATGTGTCGCACACTGCA

SUBSTtTUTE SHEET (RULE 26) - 4fi 1451 GCTTCAAA,AGTTTAAATTATTAATATGATAAGTCATCCATAGTCAAACAA

SUBSTITUTE SHEET (RULE 26) SEQ ID N0:8 Nucleotide sequence FBP15 promoter SU9ST~TUTE SHEET (RULE 26) WO 00104176 PCTIlVL99/00453 SE(ZUENCE LISTING
<110> CPRO-DLO
<120> Process to collect metabolites from modified nectar by insects <130> 159782 <190> pct/n199/00953 <141> 1999-07-15 <160> 10 <170> PatentIn Vex. 2.7.
<210> 1 <211> 265 <212> PRT
<213> Petunia x hybrids .. <220>
<223> strain: W115 <220>
<223> tissue type: nectar gland <220>
<223> NEC1 amino acid sequence <900> 1 Met Ala Gln Leu Arg Ala Asp Asp Leu Ser Phe Ile Phe Gly Leu Leu Gly Asn Ile Val Ser Phe Met Val Phe Leu Ala Pro Val Pro Thr Phe Tyr Lys Ile Tyr Lys Arg Lys Ser Ser Glu Gly Tyr Gln Ala Ile Pro Tyr Met Val Ala Leu Phe Ser Ala Gly Leu Leu Leu Tyr Tyr Ala Tyr Leu Arg Lys Asn Ala Tyr Leu Ile Val Sex Ile Asn Gly Phe Gly Cys Ala Tle Glu Leu Thr Tyr Ile Ser Leu Phe Leu Phe Tyr Ala Pro Arg WO 00/04176 . PCT/NL99/00453 Lys Ser Lys Ile Phe Thr Gly Trp Leu Met Leu Leu Glu Leu Gly Ala Leu Gly Met Val Met Pro Ile Thr Tyr Leu Leu Ala Glu Gly Ser fiis Arg Val Met Ile Val Gly Trp Ile Cys Ala Ala Ile Asn Val Ala Val Phe Ala Ala Pro Leu Ser Ile Met Arg Gln Val Ile Lys Thr Lys Ser Val Glu Phe Met Pro Phe Thr Leu Ser Leu Phe Leu Thr Leu Cys Ala Thr Met Trp Phe Phe Tyr Gly Phe Phe Lys Lys Asp Phe Tyr Ile Ala Phe Pro Asn Ile Leu GIy Phe Leu Phe Gly Ile Val Gln Met Leu Leu Tyr Phe Val Tyr Lys Asp Ser Lys Arg Ile Asp Asp Glu Lys Ser Asp Pro Val Arg Glu Ala Thr Lys Ser Lys Glu Gly Val Glu Ile Ile Ile Asn Ile Glu Asp Asp Asn Ser Asp Asn Ala Leu Gln Ser Met Glu Lys Asp Phe Ser Arg Leu Arg Thr Sex Lys <210> 2 <211> 221 <212> PRT
<213> Petunia x hybrida <220>
<223> strain: W115 <220>
<223> tissue type: nectar gland, secretory cell <220>

<223> FBP15 amino acid sequence <900> 2 Met Gly Arg Gly Lys Ile Glu I1e Lys Arg Ile Glu Asn Thr Thr Asn 1 5 10. 15 Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr Glu Leu Ser Vai Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ala Asn Asn Ser Val Lys Ala Thr Ile Asp Arg Tyr Lys Lys Ala Ser Ser Asp Ser Ser Asn Thr Gly Ser Thr Ser Glu Ala Asn Thr Gln Phe Tyr Gln Gln Glu Ala Ala Lys Leu Arg Val Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg Asn Met Leu Gly Glu Ser Leu Ser Sex Leu Thr Ala Lys Asp Leu Lys Gly Leu Glu Thr Lys Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys Lys Asn Glu Leu Leu Phe Ala Glu Iie Glu Tyr Met Arg Lys Arg Glu Ile Asp Leu His Asn Asn Asn Gln Met Leu Arg Ala Lys Ile Ala Glu Ser Glu Arg Asn Val Asn Met Met Gly Gly Glu Phe Glu Leu Met Gln Ser His Pro Tyr Asp Pro Arg Asp Phe Phe Gln Val Asn Gly Leu Gln His Asn His Gln Tyr Pro Arg Gln Asp Asn Met Ala Leu Gln Leu Val <220> 3 <211> 18.
<212> PRT
<213> Calluna vulgaris <220>
<223> tissue type: flower <220>
<223> Calluna vulgaris signal peptide <A00> 3 Met Phe Leu Pro Ile Leu Phe Thr Ile Ser Leu Leu Phe Ser Ser Ser 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 <220>
<223> NEC1 <400> 9 tcgagcggcc gcccgggcag gtattcaaca agagtattca ccacttgaac tcaaaagggg 60 cttcactaaa aaaaaatc atg gcg caa tta egt get gat gac ttg tct ttc 111 Met Ala Gln Leu Arg Ala Asp Asp Leu Ser Phe 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 ccc gtg cca aca ttt tac; aaa ata tat aaa agg aaa tca tca gaa gga 207 Pro Val Pro Thr Phe Tyr Lys Ile Tyr Lys Arg Lys Ser Ser Glu Gly tat caa gca ata cca tat. atg gta gca ctg ttc agc gcc gga cta ttg 255 Tyr Gln Ala Ile Pro Tyr Met Val Ala Leu Phe Ser Ala Gly Leu Leu eta tat tat get tat etc agg aag aat gee tat ett atc gtc age att 303 Leu Tyr Tyr Ala Tyr Leu Arg Lys Asn Ala Tyr Leu Ile Val Ser Ile aat ggc ttt gga tgt gcc att gaa tta aca tat atc tct ctg ttt ctc 351 Asn Gly Phe Gly Cys Ala Ile Glu Leu Thr Tyr Ile Ser Leu Phe Leu 80 85 ~ 90 ttt tac gcg ccc aga aag tct aag att ttc aca ggg tgg ctg atg ctc 399 Phe Tyr Ala Pro Arg Lys Ser Lys Ile Phe Thr Gly Trp Leu Met Leu tta gaa ttg gga gcc cta gga atg gtg atg cca att act tat tta tta 447 Leu Glu Leu Gly Ala Leu Gly Met Val Met Pro Ile Thr Tyr Leu Leu gca gaa ggc tca cat aga gtg atg ata gtg gga tgg att tgt gca get 495 Ala Glu Gly Ser His Arg Val Met Ile Val Gly Trp Ile Cys Ala Ala ate aat gtt get gtc ttt get get cct tta agc ate atg agg caa gta 543 Ile Asn Val Ala Val Phe Ala Ala Pro Leu Ser Ile Met Arg Gln VaI

ata aaa aca aag agt gta gag ttc atg ccc ttc act tta tct ttg ttc 591 Ile Lys Thr Lys Ser Val Glu Phe Met Pro Phe Thr Leu Ser Leu Phe ctc act ctc tgt gcc act atg tgg ttt ttc tat ggg ttt ttc aag aag 639 Leu Thr Leu Cys Ala Thr Met Trp Phe Phe Tyr Gly Phe Phe Lys Lys gac ttt tac att gcg ttt cca aat ata ctg ggc ttt cta ttc gga atc 687 Asp Phe Tyr Ile Ala Phe Pro Asn Ile Leu Gly Phe Leu Phe Gly Ile gtt caa atg cta tta tat ttt gtt tac aag gat tca aag aga ata gat 735 Val Gln Met Leu Leu Tyr Phe Val Tyr Lys Asp Ser Lys Arg Ile Asp WO 00!04176 PCT/NL99100453 gat gaa aaa tct gat cct: gtt cga gaa get aca aaa tca aaa gaa ggt 783 Asp Glu Lys Ser Asp Pra Val Arg Glu Ala Thr Lys Ser Lys Glu Gly gta gaa atc att atc aac: att gaa gat gat aat tct gat aac gca ttg 831 Val Glu Ile Ile Ile Asn Ile Glu Asp Asp Asn Ser Asp Asn Ala Leu cag tcc atg gag aag gat ttt tcc aga ctg cgg aca tca aaa g73 Gln Ser Met Glu Lys Asp Phe Ser Arg Leu Arg Thr Ser Lys taagcaagaa gatgatcaaa aaatgacaaa gctaaggagt ttgaagtaag gcaaggaact 933 tgacactgaa tatctaagct aattagcaag actttagcag cttgtaatat ttagtgtttg 993 tgaggtgtta ccttataatt agcttgtagc atagccttcc cactaataat tctgcttagc 1053 gaatcttata tatgggaaat acttacacta gtatgcatct tctatataca tgtttggcac 1113 ttgactatac atagaaaaat taacaagcat ttctcacctc aatttgtcac ttacttataa 1173 gtagctgaat aatataatgc aattttcacc cc 1205 <210> 5 <211> 265 <2I2> PRT
<213> Petunia x hybrida <223> NEC1 <400> 5 Met Ala Gln Leu Arg Ala Asp Asp Leu Ser Phe Ile Phe Gly Leu Leu Gly Asn Ile Val Ser Phe Met Val Phe Leu Ala Pro Val Pro Thr Phe Tyr Lys Ile Tyr Lys Arg Lys Ser Ser Glu Gly Tyr Gln Ala Ile Pro Tyr Met Val Ala Leu Phe Ser Ala Gly Leu Leu Leu Tyr Tyr Ala Tyr Leu Arg Lys Asn Ala Tyr Leu Ile Val Ser Ile Asn Gly E'he Gly Cys Ala Ile Glu Leu Thr Tyr Ile Ser Leu Phe Leu Phe Tyr Ala Pro Arg 85 90 g5 Lys Sex Lys Ile Phe Thr Gly Trp Leu Met Leu Leu Glu Leu Gly Ala Leu Gly Met Val Met Pro Ile Thr Tyr Leu Leu Ala Glu Gly Ser His Arg Val Met Ile Val Gly Trp Ile Cys Ala Ala Ile Asn Val Ala Val Phe Ala Ala Pro Leu Ser Ile Met Arg Gln Val Ile Lys Thr Lys Ser 195 , i50 155 160 Val Glu Phe Met Pro Phe Thr Leu Ser Leu Phe Leu Thr Leu Cys Ala Thr Met Trp Phe Phe Tyr Gly Phe Phe Lys Lys Asp Phe Tyr Ile Ala Phe Pro Asn Ile Leu Gly Phe Leu Phe Gly Iie Val Gln Met Leu Leu Tyr Phe Val Tyr Lys Asp 5er Lys Arg Iie Asp Asp Glu Lys Ser Asp zlo 215 220 Pro Val Arg Glu Ala Thr Lys Ser Lys Glu Gly Val Glu Ile Ile Ile 225 230 235 2qp Asn Ile Glu Asp Asp Asn Ser Asp Asn Ala Leu Gln Ser Met Glu Lys Asp Phe Ser Arg Leu Arg Thr Ser Lys <zlo> 6 <211> 1157 <212> DNA
<213> Petunia x hybrida <220>
<221> CDS
<222> (I79} " (841) <220>
<223> strain: WlIS

7 WO 00/04176 PCTlNL99/00453 <220>
<223> tissue type: nectar gland <220>
<223> cDNA library of nectaries from Petunia hybrida flowers <220>
<223> FBP15 <400> 6 tctgaataca agctgtgtgt gtagagagat ttcataaaga cagcaaacat cccttctttt 60 tgttctgttt t~aaagttcc cttcttcaac cagctctttt cctcatcagg gtaagttgca 120 aataaagggg atgttccaga atcaagaaga gaagatgtca gactcgcctc agaggaag 178 atg gga aga gga aag att gag att aag agg att gaa aat aca aca aat 226 Met Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn cgt caa gtc act ttc tgt aag aga aga aat ggg ttg ctt aaa aaa get 274 Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala tat gaa ctt tct gtt ctt tgt gat get gaa gtt get ctc atc gtt ttc 322 Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe tca agc cgt ggc egc ctc tat gaa tat get aac aac agt gtg aag gca 370 Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ala Asn Asn Ser Val Lys Ala aca att gat aga tat aag aaa gca tcc tca gat tcc tcc aac act gga 418 Thr Ile Asp Arg Tyr Lys Lys Ala Ser Ser Asp Ser Ser Asn Thr Gly tct act tet gaa get aac act cag ttt tat caa caa gaa get gec aaa 466 Ser Thr Ser Glu Ala Asn Thr Gln Phe Tyr Gln Gln Glu Ala Ala Lys ctc cga gtt cag att ggt aac tta cag aac tca aac agg aac atg cta 519 Leu Arg Val Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg Asn Met Leu ggc gag tct cta agt tct ctg act gca aaa gat ctg aaa ggc ctg gag 562 Gly Glu Ser Leu Ser Ser Leu Thr Ala Lys Asp Leu Lys Gly Leu Glu

8 acc aaa ctt gag aaa gga att agt aga att agg tcc aaa aag aat gaa 610 Thr Lys Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys Lys Asn Glu i30 135 140 ctc ctg ttt get gag att gag tat atg cga aaa agg gaa att gat ttg 658 Leu Leu Phe Ala Glu Ile Glu Tyr Met Arg Lys Arg Glu Ile Asp Leu cac aac aac aat cag atg ctt cgg gca aag ata get gag agt gaa aga 706 His Asn Asn Asn Gln Met Leu Arg Ala Lys Ile Ala Glu Ser Glu Arg aat gtg aac atg atg.gga gga gaa ttt gag ctg atg caa tct cat ccg 754 Asn Val Asn Met Met Gly Gly Glu Phe Glu Leu Met Gln Ser His Pro tac gat cca aga gac ttc ttc caa gtg aac ggc tta cag cat aat cat 802 Tyr Asp Pro Arg Asp Phe Phe Gln Val Asn Gly Leu Gln His Asn His caa tat cca cgc caa gac aac atg get ctt caa tta gta taagtttata 851 Gln Tyr Pro Arg Gln Asp Asn Met Ala Leu Gln Leu Val 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 <21i> 221 <212> PRT
<213> Petunia x hybrida <223> FBP15 <900> 7 Met Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Asn

9 Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ala Asn Asn Ser Val Lys Ala Thr Ile Asp Arg Tyr Lys Lys Ala Sex Ser Asp Ser Ser Asn Thr Gly Ser Thr Ser Glu Ala Asn Thr Gln Phe Tyr Gln Gln Glu Ala Ala Lys Leu Arg Val Gln Ile Gly Asn Leu Gln Asn Ser Asn Arg Asn Met Leu Gly Glu 5er Leu Ser Ser Leu Thr Ala Lys Asp Leu Lys Gly Leu Glu Thr Lys Leu Glu Lys Gly Ile Ser Arg Ile Arg Ser Lys Lys Asn Glu Leu Leu Phe Ala Glu Ile Glu Tyr Met Arg Lys Arg Glu Ile Asp Leu His Asn Asn Asn Gln Met Leu Arg Ala Lys Ile Ala Glu Ser Glu Arg Asn Val Asn Met Met Gly Gly Glu Phe Glu Leu Met Gln Ser His Pro Tyr Asp Pro Arg Asp Phe Phe Gln Val Asn Gly Leu Gln His Asn His Gln Tyr Pro Arg Gln Asp Asn Met Ala Leu Gln Leu Val <210> 8 <211> 59 <212> DNA
<213> Calluna vulgaris <220>
to <221> CDS
<222> (1)..(54) <220>
<221> sig_peptide <222> (1)..(54) <400> 8 atg ttt ctt cca att ctc ttc acc att tcc ctc ctc ttc tcc tcc tcc 48 Met Phe Leu Pro Ile Leu Phe Thr Ile Ser Leu Leu Phe Ser Ser Ser cat get 54 His Ala <210> 9 <211> 18 <212> PRT
<213> Calluna vulgaris <400> 9 Met Phe Leu Pro Ile Leu Phe Thr Ile Ser Leu Leu Phe Ser Ser Sex His Ala <210> 10 <211> 2141 <212> DNA
<213> Petunia x hybrida <220>
<223> strain: W115 <220>
<223> NEC1 promoter <400> 10 cctaggagaa atcaagccta ctcttaagat ggatgactca cttgccccga tggtaaggtg 60 aaggatctgt tgattagagt tgggaagttc atgttctctg ctgattttat tattctagac 120 tatgaagagg accaagaagc tccaataatt ttgggaagag cattcttaat cacatcgatg 180 gcaattattg acatggaact tggggagatg actgtgagag cgcatggaga aaaggttact 240 ttcaaggttt ataataaaaa ggatcatatg gctaagtttg aagagtgttc tttgatagaa 300 tgtgtcagac gagaacatga aagtaaaccg aaagaggtgt ttgagcggaa tgtagaacaa 360 agtgaccacg gcacaataat_tgacaagttg aaggaaaatt cacctaaagg aaggaagaag 420 acaaaagttc gtcgtaacaa gaggagacgt aaatgctgga agtgagctta aaggtgttgt 480 cgtactacga cgttaactaa ggcgcttgtc gggaggcaac cctagctttg tatgtaaatg 540 taaaagtaaa aaatatatat atagaaaaag gaaaatacaa aaagagtcgt gccgcgacgt 600 taaatcaagc gcttgttgga aggcaaccca atttttattg ttttagttgt tttacttatt 660 tagtattacg tagtttcttg ttgtttttgt agggctcggg actttcggaa ggtgaggtaa 720 tttcaaggca tcgcggtgtg tattgcagcg aggtaagtgt aagagttgag ttggaagcgt 780 ttggccaagt gttgcaccgt gagaggcttt caacctgttg cgacacgtga aaaattaaga 840 gccagatctg ctacattagc actgaagcat cgcttggcca atagcttgga atggaagcaa 900 gaattcaaac caaaatcaga a.acgccacaa gagatgtgtc gcacactgca aagctttgtg 960 caaactagtg aacgcagaaa tagaaatgct acagcccatg cgtcgcttgg cttatggcag 1020 gcagcaaaaa ttcagcagca aaacagaaac gctgcgagaa acgcgtcgca tacgccatag 1080 ctttgtgtca aacagaacgt ccagaaattg aaaagctata agcctgcgtc gcttggctca 1190 tggcgtgcag actagaaaag ctctagcaga tgcgtcgcgt attgtatagc ttggtgtgaa 1200 acagaaagtt cgaaacttgg aaaacgataa cccagcgtcg cctcttcaac cgcgtccagg 1260 taagttcaag attcttacgg g~ttgacccat taacccattg atcggctgat tataaacaat 1320 aaaacatcac cttcaactat cacatgattt cataagtttg acctaggata ttttatatat 1380 atatatatat atatacacac acacaccatt tccagcgatc ttacctcatt tttattcaaa 1440 ccatttttct gcttcaaaag tttaaattat taatatgata agtcatccat agtcaaacaa 1500 gattttctat actattttgt cccttgtaat tttaaaaaaa aaatgagcga tggtaagata 1560 aacattgttt gcaagtgtac aattttagta tatgcaaacc aacgcttctt cttccaacta 1620 tcacctaaaa ctacatcatt tatggcgggc ggactagacg tagccaaata taaaaacgca 16$0 atggccattc agttcatgtc atttttatat ccttcatcca ataatattac tcaaaattga 1790 tgtacagttt ggtctctgat gtgcacttta ctatacgtaa tacggaattt acattataat 1800 taaagagaac tgttccacta aattttaatg atttaattaa tttaactcgg ttacttgtat 1860 tattattatt gctgtatttg tttgtcattt gaatttggca ccgcagattt ttgtatgcaa 1920 ttaaccctca tatatctttt ggccaaataa agaaaaagtc tgcatatttc ttgccaaaca 1980 tttatcatac tttaccgaat tcttgttttt tgtttctctg ttgttgttct ccactataaa 2040 taacatttgc agtgagtaaa gtttcttcag gtctcttttg tagattcaac aagagtattc 2100 agcacttgaa ctcaaaaggg gcttcactaa aaaaaatcat g 2141

Claims (30)

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 N0:4, or b) a nucleotide sequence which hybridises with (a) or with a fragment of (a) under the following conditions:
pre-hybridisation for 1h at about 65 °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 65 °C, followed by washing three times in 0.1 x SSC, 0.1% SDS at about 65 °C for 30 min., or c) a nucleotide sequence that has at least 85% homology to the nucleotide sequence of a).

3. An isolated DNA sequence according to claim 1 or 2, 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.

4. An isolated DNA sequence encoding a protein comprising the amino acid sequence given in SEQ ID N0:1, or a protein having at least 60% homology with the amino acid sequence given in SEQ ID NO:1, 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.

5. An isolated DNA sequence according to claim 4 having:
a) a nucleotide sequence given in SEQ ID N0:4, or b) a nucleotide sequence that hybridises with the nucleotide sequence of (a) or with a fragment of (a) under the hybridisation conditions as defined in claim 2, or c) a nucleotide sequence that has at least 85% homology to the nucleotide sequence of a).

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 occurring variations or variations introduced by targeted mutagenesis 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 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.

9. An isolated DNA sequence according to claim 8, having:
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 a), under the hybridisation conditions as defined in claim 2, or c) a nucleotide sequence that has at least 95% homology to the nucleotide sequence of a).

10. A recombinant double-stranded DNA molecule comprising an expression cassette comprising the following constituents:
i) a promoter functional in plants, ii) a DNA sequence coding for a protein as defined in any of claims 4 to 7 which is fused to the promoter sequence in sense ar antisense orientation, and optionally iii) a signal sequence functional in plants for the transcription detemination and polyadenylation of an RNA
molecule .

11. A recombinant double-stranded DNA molecule comprising an expression cassette comprising the following constituents:
i) a promoter fuzlctional in nectaries of plants, ii) a DNA sequence coding for a protein which is fused to the promoter sequence in sense or antisense orientation, and optionally iii) a signal sequence functional in plants for the transcription termination and polyadenylation of an RNA
molecule.

12. A recombinant double-stranded DNA molecule comprising an lion cassette comprising the following constituents:
i) a promoter funcaional 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.

13. A recombinant double-stranded DNA molecule according to claim 11 or 12 wherein the promoter is as defined in any of claims 1-3.

14. A recombinant double-stranded DNA molecule according to claim 12 or 13 wherein the DNA sequence encoding a signal peptide is as defined in claim 8 or 9.
15. A process for producing a transgenic plant exhibiting excretion of a recombinant protein in its nectar, comprising:
i) introducing in a plant cell a recombinant double-stranded DNA-molecule as defined in any of claims 12 to 14, wherein the recombinant, protein is excreted in nectar, ii) regenerating plants from the transgenic cell, and iii) selecting transgenic plants.

15. A process for producing a transgenic plant exhibiting a modified nectar composition, comprising:
i) introducing in a plant cell a recombinant double-stranded

DNA-molecule as defined in any o~ claims 11 to 14, wherein the recombinant protein interferes with metabolic pathways in the nectaries, ii) regenerating plants from the transgenic cell, and iii) selecting transgenic plants.

17. A process for producing a transgenic plant exhibiting a modified nectar secretion, comprising:
i) introducing in a plant cell a recombinant double-stranded DNA-molecule as defined in any of claims 11 to 14, wherein the recombinant protein interferes with sink strength of nectaries, ii) regenerating plants from the transgenic cell, and iii) selecting transgenic plants.

18. A process for producing a transgenic plant exhibiting a modified nectary development, comprising;
i) introducing in a plant cell a recombinant double-stranded DNA-molecule as defined in claims 11 or 14, wherein the recombinant protein interferes with the development of nectaries, ii) regenerating plants from the transgenic cell, and iii) selecting transgenic plants.

19. A process far producing honey from modified nectar of transgenic plants, comprising:
i) producing a transgenic plant by introducing in a plant cell a recombinant double-stranded DNA molecule as defined in any of claims 11 to 14, regenerating plants from the transgenic cell, and selecting modified plants exhibiting the excretion of nectar with a modified composition, ii) allowing insects, preferably bees, to collect nectar from the transgenic plants and to process the nectar into honey.

20. A process for producing a recombinant gene product from honey, comprising:
i) producing a tramsgenic plant by introducing in a plant cell a recombinant double-stranded DNA molecule as defined in any of claims 12 to 14, regenerating plants from the transgenic cell, and selecting modified plants exhibiting excretion of the recombinant gene product in nectar, ii) allowing insects, preferably bees, to collect nectar from the transgenic plants and to process the nectar into honey, and iii) isolating and purifying the gene product from the honey.

21. A process for producing a metabolite from honey, comprising:
i) producing a plant that excretes this metabolite in nectar and which plant has been produced by current breeding and selection methods, ix) allowing insects, preferably bees, to collect nectar from the selected plants and to process the nectar into honey, and iii) isolating and purifying the metabolite from the honey.

22. Micro organisms containing DNA sequences according to one or more of claims 1 to 9.

23 . Micro organisms containing recombinant DNA molecules according to any of claims 10 to 14.

24. A plant cell or plant cell culture transformed with one or more DNA sequences according to claims 1 to 9

25. A plant cell or plant cell culture transformed with recombinant DNA molecules according to any of 10 to 14.

26. A plant consisting essentially of the plant cells of claims 24 or 25.

27. A transgenic plant obtained by the process of any of claims 15 to 18.

28. Seeds; tissue culture, plant parts or progeny plants derived from a transgenic plant according to claim 27.

29. Honey obtained from nectar from transgenic plants, which nectar has a modified composition.

30. Honey obtained from nectar from transgenic plants, which nectar comprises a recombinant gene product.