WO2004106530A1

WO2004106530A1 - Novel promoters

Info

Publication number: WO2004106530A1
Application number: PCT/GB2004/002334
Authority: WO
Inventors: Simon Paul Vaughan; Andrea Juliet Massiah; David John James
Original assignee: East Malling Research Limited
Priority date: 2003-05-30
Filing date: 2004-06-01
Publication date: 2004-12-09
Also published as: GB0312449D0

Abstract

A promoter sequence associated with the tonoplast intrinsic protein FavRB7 gene, or a variant or derivative thereof, allows tissue specific expression in the Rosaceae and constitutive levels of expression in other plants at levels similar to those observed with CaMV35S.

Description

NOVEL PROMOTERS

The present invention relates to promoters derived from Fragaria spp., and uses therefor.

The field of plant transformation remained largely unexplored until the late 1980's, but this changed with the discovery of the unique abilities of ^' Agrobacterium tumefaciens. Large numbers of plant species have now been transformed in order to incorporate suitable characteristics or traits, varying from pest resistance to hormone production. An example of the former is the production of Bt toxin, while an example of the latter is heightened expression of ethylene in tomatoes.

Despite the success in being able to express virtually any desired protein in a plant cell, relatively few promoters have been employed. Indeed, the standard promoter is the cauliflower mosaic virus 35S RNA (CaMV35S) promoter, as this leads to high levels of constitutive expression, both aspects of which can be valuable properties.

However, it has been established that CaMV35S has a recombination "hotspot", leading to worries that unforeseen recombination events, especially with viruses, could lead to spread of the associated gene, even to humans. In addition, constitutive expression throughout the plant is no longer necessarily desirable, as the gene associated with the promoter will necessarily be expressed in the part of the plant to be consumed, when the plant is a food crop. The constitutive nature of CaMV35S promoter is such that high levels of the associated gene are expressed in all tissues of the transformed plant.

Tissue-specific transgene expression is, therefore, a desirable target. However, the problem lies with the reliability and predictability of tissue-specific promoters from one species being similarly tissue-specific in another species.

For example, haemoglobin genes are preferentially expressed in the roots of several plant species (Appleby, CA. et al. (1988), Plant Cell Environ. 11, 359-367). These genes have also been isolated from non-nodulating species, in addition to the nodulating legumes in which they are most commonly found. However, use of the haemoglobin par promoter in a heterologous host (tobacco) met with limited success. Although the promoter was intended to lead to expression only in the roots, expression was additionally seen in the leaves of 46 out of 140 transgenic lines (Van de Hoven et al. (1994), Transgenic Res.3, 159-165).

Similar findings exist for other root-specific genes, and none has yet been reported that remains identically tissue-specific across all species.

The cultivated strawberry is a complex plant. The modern cultivated strawberry, Fragaria ananassa, is a hybrid octaploid resulting from a cross of two New World octaploid species (E. chiloensis and E. virginiana). Breeding programs for the cultivated strawberry have concentrated primarily on improvements to plant vigour, fruiting habit, fruit quality, winter hardiness, frost tolerance and, to some extent, developing pest and disease resistance. However, the majority of genes in the modern strawberry are derived from only seven nuclear, and ten cytoplasmic, sources (Hancock, J. F., et al. (2001), Hort. Science 36, 221-225). This has resulted in a narrow germplasm base, and sources of resistance are highly restricted, owing both to the narrow germplasm base and to the alternative requirement for interploid crosses.

The strawberry is susceptible to a wide range of pests and diseases, including both insect and fungal diseases. The primary insect disease is that of vine weevil infection, the larvae of which feed on both the crowns and the roots of the strawberry, often leading to plant death. The roots are also subject to infection by a number of soil borne fungi, such as Verticillium dahliae and Phytophthora fragariae, which generally cause the plant to die.

Methyl bromide has been used on a large scale in order to kill the vine weevil, but has been identified as a significant ozone-depleting substance, and is generally being withdrawn from commercial use. Broad-spectrum insecticides and fungicides have limited efficacy, and can also encourage the development of resistant strains of pests.

WO98/31812 identifies promoters from strawberries which are tissue-specific to the receptacle.

US-A-6043010 also discloses tissue-specificity in receptacles of strawberries, while WO 01/51637 discloses the isolation of a promoter from the wild strawberry which is specific, once again, for the receptacle, WO 02/34894 relating to similar subject matter. Thus, tissue-specific promoters so far identified in relation to strawberries are specific for the fruit. This is not a problem if the gene to be promoted is to enhance flavour, for example, but genes controlled by promoters specific for the receptacle will only be expressed in the fruit, and will be unable to affect the plant as a whole, especially outside of the fruiting season. In addition, genetically engineered expression in the fruit is less likely to meet with public acceptance.

Thus, there remains a requirement for a constitutive promoter for plants that has similar activity to that of CaMV35S, and which preferably is not associated with a virus.

There is also a desire for tissue-specific promoters in food crop plants.

A novel tonoplast intrinsic protein (TIP) gene homologue has now been identified in strawberry, which is expressed primarily in root tissue, and the promoter of which is useful in the expression of foreign genes.

Thus, in a first aspect, the present invention provides a promoter sequence associated with the tonoplast intrinsic protein gene homologue, FavRB7, from strawberry, or a variant or derivative thereof, especially as shown in SEQ. ID NO. 1.

In an alternative aspect, there is provided a promoter sequence as shown in SEQ. ID NO. 1, or a variant or derivative thereof.

The preferred promoters are associated with the tonoplast intrinsic protein gene homologue FavRB7 found in the strawberry, by which is meant that this gene has been found to be controlled by this promoter, and that variants and derivatives of this promoter sequence associated with FavRB7, preferably as found in nature, are included in the invention.

The preferred promoter of the present invention is designated SEQ ED NO. 1 in the accompanying Sequence Listing. The same sequence is illustrated in Figure 1, but has the ATG initiation codon.

It is preferred that the promoter is operably linked to an initiation codon, by which is meant that the promoter promotes transcription of a translatable sequence containing the codon as a translation initiator. The promoters of the present invention are constitutive or tissue-specific, depending on the plant species in which they are used and/or the nature of the derivative of the promoter. In particular, when used in conjunction with Rosaceae spp., and especially Fragaria spp., the promoters of the invention are largely root-specific while, in other species, such as tobacco, the promoters of the invention tend to be constitutive.

Whether the promoters of the invention are constitutive or tissue-specific, they have advantages over the prior art. Where the promoters are constitutive, they are of plant origin and are not associated in any way with viruses, so that any threats associated specifically with viral promoters are avoided. Although such threats may not be as substantial as public opinion would suggest, the effect on sales can be very substantial. Moreover, the FavRB7 gene promoter confers activity similar to that of the CaMV35S promoter when used in a heterologous environment, so that it may readily be used in the place of the CaMV35S promoter, where such is currently used or considered for use, for example.

Especially in the species Rosaceae, promoters of the present invention are generally root-specific. In fact, expression is confined mainly to the root, but some expression has been noted in the petiole. Virtually no expression is observed in the leaves, floral organs or fruit, so that any gene associated with the promoters of the present invention will not be expressed in the fruit, thereby minimising or obviating the risk of the consumer eating the expression product.

It will be appreciated that the term "tissue-specific", and associated terms, indicate that the promoter is active primarily in a given tissue, or range of tissues, and that it will be substantially dormant in at least one tissue type. For the purposes of the present invention, the term "dormant" is taken to mean that activity of the promoter, such as by detection of associated marker activity, is substantially undetectable in the tissue in which the promoter is dormant. For example, if the promoter is associated with the gusA marker, then the tissue in which the promoter is dormant will not appear to have any blue colour when subject to histological analysis.

The term "variants" includes all forms of the promoter found in nature within the genus Rosaceae and, particularly, within the Fragariae. Although this may encompass various deletions, insertions, inversions and replacements, variants are generally of identical, or very nearly identical, length, and any difference is generally limited to replacement.

Variants preferably have an identity of at least 80% with SEQ ID NO 1. More preferably there is at least 90% identity, such as at least 95% or at least 98% identity.

"Identity" is a measure of the identity of nucleotide sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognised meaning and can be calculated using published techniques. See: e.g. : (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed.: Academic Press, Now York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

While there exists a number of methods to measure identity between two polynucleotide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAMJ Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Marlin J. Bishop, ed., Academic Press, San Diego, 1 994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073.

The present invention also extends to "derivatives" of the promoter of the FavRB7 gene, and these include any modified version of the promoter, provided that the activity of the promoter is retained to the extent that it remains essentially root-specific in Fragaria spp. Derivatives include mutations such as deletions, insertions, inversions, replacements and truncations.

Truncated forms of the promoter should preferably be no less than 600 base pairs in length, and preferably between 1400 and 2500 base pairs. Where convenient, if there is no requirement for amending the promoter sequence, then it is generally preferred to use the full sequence of SEQ JJD NO. 1, or preferably at least 50% of the 3' end, together with the ATG codon, and more preferably 70% and more preferably 80% of the 3' end. Amendments may include incorporating splice sequences, for example.

The FavRB7 gene promoter sequence was subject to a search for -acting regulatory elements using the PlantCARE database (www.oberon.rug.ac.be). This search identified only one regulatory motif associated with root-specific expression, an asl motif at position - 2271. However, several regulatory motifs linked to hormone response were identified; auxin response, an AuxRR-core element at - 215 and TGA elements at - 1975 and - 2271 ; an abscisic acid response, CE1 element, at - 198; ethylene response, ERE, elements at - 542, - 935, 1184 and - 2113 and gibberellin response, P-box and TATC box, elements at - 785, - 1868, - 1904 and - 503.

Several regulatory motifs associated with environmental stresses were also identified; drought inducibility, MBS, elements were found at - 68, - 2069 and - 2185; a low temperature response, LTR, element at - 1557; heat responsive elements, HSE at - 274, - 893, - 1009, - 1107, -1145 and - 2213; wound response, WUN, motifs were found at - 38, - 544, - 815, - 1186, - 1343, - 1657, - 1734, - 2115, - 2281 and - 2617 and an anoxic specific inducible, GC-motif was found at - 126.

It will be appreciated that motifs may be added to the sequence, where they are not already present, or where it is desired to supplement the existing motif, or motifs may be deleted. Any one of the motifs listed above, or a combination thereof, may be deleted from the promoter sequence, in order to disable variable expression. For example, deleting one or more of the motifs corresponding to hormone responses can stabilise resulting expression at consistent levels, unaffected by growing conditions.

Several regions of repetitive sequence were identified in the FavRB7 promoter (Figure 1), the longest of which is a direct repeat of 22/23 bases occurring at positions - 1798 / - 1819. A direct repeat of 13 bp occurs at - 718 / - 772 and four 11 bp direct repeats were also identified. A 15 bp dyad repeat was identified at - 1883 / - 1416 and a 12 bp dyad repeat at - 1350 / - 942. Inverted repeats of 12 and 11 bp were found at - 2033 / - 537 and- 2429 / - 1817.

Figure 1 shows the nucleotide sequence of the FavRB7 gene promoter. Nucleotide positions are given distal to the proposed ATG FavRB '7 gene initiation codon. Putative regulatory motifs are indicated in bold:- Asl- root specificity, AuxRR - auxin response, TGA - auxin response, CE1 - abscisic acid response, ERE - ethylene response, P-box and TATC boxes - gibberellin response, MBS - drought inducibility, LTR - low temperature response, HSE - heat responsive elements, WUN- wound response and GC-motif - anoxic inducible. Regions of repetitive sequence identified are underlined and highlighted in grey, the type of repeat is indicated and letters indicate pairs of repetitive sequence.

There is no particular restriction on the nature of the gene or genes that may be operably associated with the promoter of the present invention, and neither is there any restriction on the number of occasions the promoter of the invention may be used in any one genome. For example, the promoter may be associated with a Bt toxin gene, together with any other appropriate sequences, such as a termination sequence, and the resulting operon may be incorporated once or a number of times into the genome of a desired plant. The promoter may also be associated with another gene, such as a gene involved in the synthesis of ethylene, and this may also be incorporated once or a number of times into the genome of the same or another plant. The operon usefully further comprises a termination sequence and any other expression sequences that may be desired.

It will be appreciated that the promoter and any associated sequence may be incorporated at random, on a vector, or by targeted transformation techniques, all of which are well known in the art.

Exemplary genes for use with the present invention in order to confer resistance against some of the insect pests of strawberry, for example, include the proteinase inhibitors, Bacillus thuringiensis (Bt) toxins and plant lectins. Proteinase inhibitors generally form complexes with proteinases, thereby inhibiting enzyme activity and, thus, disrupting digestion in insects that consume these compounds. Bt toxin gene products are δ-protoxins that form an active toxin during digestion by insects that consume the δ-protoxin. Plant lectins act by binding to carbohydrates and also have a disruptive effect on insect metabolism.

Suitable genes for conferring resistance against fungal pathogens of strawberry, for example, are the endochitinases, which hydrolyse the chitin component of fungal cell walls; various antimicrobial peptides which have exhibited antimicrobial activity against bacteria and fungi when expressed in transgenic plants (Park, et al. (2000), Plant Mol. Biol. 44, 187-197; DeGray, et al., (2001), Plant Physiol. 127, 852-862); the beta-l-3-glucanase protein, which has been shown to confer a degree of resistance to several fungal pathogens in tobacco and wheat; the ribosome-inactivating proteins (REPs), which have displayed varying antifungal and antiviral activities (Bieri, S., et al., (2000), Theor. Appl. Genet. 100, 755-763; Nielsen and Boston, (2001) Annu. Rev. Plant Physiol. Plant Molec. Biol. 52, 785-816; Park et al., (2002) Planta 216, 227-234; Parkash et al., (2002) Peptides 23, 1019-1024). These last often serve as plant defence proteins, and specifically inhibit protein synthesis through the depurination of the conserved alpha-sarcin loop of large ribosomal RNAs (Park et al., supra).

Sequence homology analysis between the FavRB7 and TobRB7 (EMBL: S45406) promoters revealed 23% overall homology between the two sequences. Repetitive sequence elements designated B and C boxes in the TobRB7 promoter were not present in the FavRB7 promoter. However, the sequence motif, CGAGCTCGATA (SEQ ID NO. 10), which forms the consensus of the TobRB7 promoter A box, was present several times in the FavRB7 promoter (Figure 2).

Figure 2 shows sequence positions distal to the FavRB7 gene initiation codon for sequence motifs identified in the FavRB7 promoter homologous to the A box motifs identified in the TobRB7 promoter. Regions of homology are indicated by black or grey shading; regions of divergence are highlighted in white. The TobRB7 promoter A box consensus sequence is presented below the FavRB7 sequence motifs in bold and underlined.

The invention provides an operon comprising at least a promoter as defined, in operable association with a gene, preferably a gene having an expression product.

h general, it has been established that there is no particular advantage to incorporating the operon any more than once into any given genome and, indeed, there may be disadvantages associated with multiple occurrences where the primary insertion event is random.

It is generally preferred to associate the promoter of the present invention with a marker therefor, in order to be able to establish whether a particular cell line has been transformed, and to follow the efficiency of transformation. Suitable markers may include resistance markers, such as kanamycin, gentamycin, tetracycline, streptomycin and spectinomycin, although there is popular antipathy to such markers, owing to the possibility of spreading resistance to other species. A particularly useful marker is the gusA marker referred to above. The product of this gene is β-glucuronidase which, when incubated with the substrate 5-bromo-4-chloro-3-indolyl-l-glucuronide, produces a blue stain visible to the naked eye. The lack of detectable levels of β-glucuronidase in plants and the wide availability of spectrophotometric substrates for the enzyme, makes the gusA system particularly suitable as a marker.

Other selectable markers, such as other antibiotic resistance markers, non- antibiotitc resistance markers, including phosphomannose/xylose isomerase, and other scorable markers, such as green fluorescent protein (GFP), and luciferase may also be used.

The present invention also extends to vectors, such as plasmids, comprising a promoter of the present invention, especially where the promoter is associated with a gene to be expressed in a plant.

The present invention further extends to methods for incorporating promoters of the present invention into plants, especially where the promoter is associated with a gene to be expressed. Such methods may include microinjection, electroporation, PEG- transformation, transformation with a vector, and biolistics, such as exemplified by Sanford, et al (Method Enzymol. (1993), 217, 483-509).

The present invention envisages plants, or reproductive material thereof, comprising a promoter as defined above in expressible form. Thus, it is preferred that the promoter be in operable connection with at least one gene. The promoter is preferably incorporated in to the genome, although it may be present in a vector.

It will be appreciated that reference to a nucleotide sequence of the invention can generally be taken to include reference to a sequence comprising at least a promoter of the invention. Preferred sequences are DNA.

The present invention also envisages a plant expressing at least one transgene in at least one tissue thereof at at least one stage in the lifecycle thereof, wherein the transgene is under the control of a promoter as defined above. In this respect, a transgene may be either homologous or heterologous, although the latter is preferred, and is a gene not normally expressed in association with a promoter of the invention. For example, a gene normally expressed only in the fruit may be expressed in the root, but it is generally preferred that a heterologous gene be expressed constitutively, or in a tissue specific manner, in the plant.

The present invention will now be illustrated by way of the following, non- limiting Example.

EXAMPLE 1

Construction of the binary vector, pSCVFavRB7

The binary vector pSCVl .6 (Figure 3) harbours the gusA reporter gene driven by the CaMV 35S promoter next to the left T-DNA border and the neomycin phosphotransferase (nptll) gene also driven by the CaMV 35S promoter, next to the right T-DNA border. This vector was used to confer constitutive gusA expression, to serve as a comparison to the FavRB7 gene promoter in these investigations.

To create a vector comparable to pSCVl .6, but with the gusA gene under control of the FavRB7 promoter, the CaMV 35S RNA promoter controlling the gusA gene in pSCVl .6 was removed and replaced with the 2843 bp FavRB7 gene promoter isolated from strawberry, creating the vector pSCVFavRB7.

Primers, FavRB7SS and FavRB7N (SEQ ID NO's 8 and 9, respectively), were designed to incorporate Sph I and Sma I sites at the 5' end of the promoter and anNco I site across the initiation codon of the FavRB7 gene to facilitate cloning. PCR was carried out using the high fidelity proofreading enzyme Pfx and pFavRB72.8 as template. A 2860 bp amplification product incorporating the FavRB7 promoter sequence and FavRB7 gene 5' untranslated region up to the ATG initiation codon was gel isolated and transformed into the vector pCR-Blunt-TOPOII. Plasmid DΝA was prepared from transformed colonies and sequenced to confirm the correct addition of restriction sites. The authenticated plasmid was named pFavRB7P. Figure 3 shows the binary vector pSCVl.6. Left (LB) and right (RB) T-DNA borders are indicated. OD indicates an overdrive T-DNA transfer enhancer. Arrows indicate the orientation of promoters and genes. Key restriction endonuclease sites are indicated and their positions noted.

The plasmid pFavRB7P was digested with Sph I and Nco I and the resulting 2856 bp restriction fragment ligated into Sph I and Nco I cut, dephosphorylated pGUS358-S. The plasmid pGUS358-S (EMBL: U02441) harbours the gusA gene with an Nco I site across the initiation codon. Correct linkage of the FavRB7 gene promoter to the gusA gene in pGUS358-S was confirmed by sequencing and the authenticated plasmid named pGFavRB7P.

Plasmid pGFavRB7P was digested with Sma I and Sna Bl to release a 3228 bp fragment representing the FavRB7 promoter and the first 385 bp of the gusA gene. This fragment was then ligated into Sma I and Sna Bl digested, dephosphorylated pSCVl.6 replacing the CaMV 35S promoter and the first 385 bp of the gusA gene. Correct linkage of the components was confirmed by sequencing and the authenticated binary vector containing the FavRB 7 promoter linked to the gusA gene named pSCVFavRB7 (Figure 4).

Both pSCVl.6 and pSCVFavRB7 were transformed into electro-competent cells of Agrobacterium tumefaciens strain EHAIOI prior to use in nuclear transformation experiments.

FavRB 7 promoter activity in strawberry and tobacco transgenics

a) Agrobacterium-mediated nuclear transformation

Strawberry, cv. Calypso, and tobacco cvs Ottawa and Petit Havana were transformed using the binary vectors pSCVl .6 and pSCVFavRB7 in Agrobacterium tumefaciens strain EHAIOI. Shoots regenerating from transformation experiments for both strawberry and tobacco were maintained on selective medium for a minimum of 12 weeks prior to weaning. Figure 4 shows the binary vector pSCVFavRB7. The FavRB7 gene promoter replaces the CaMV 35S promoter driving gusA in pSCVl .6 to create ρSCVFavRB7. Left (LB) and right (RB) borders are indicated. OD indicates an overdrive T-DNA transfer enhancer. Arrows indicate the orientation of promoters and genes. Key restriction endonuclease sites are indicated and their positions noted.

Two replicates of each putative transgenic strawberry line were weaned and transferred to a controlled environment cabinet. After six weeks under standard growth conditions the plants were subject to a 14 d chilling treatment of 10°C day and night to induce flowering. After a further six weeks under standard growth conditions the plants were transferred to the glasshouse under natural heat and light. All strawberry lines produced displayed the phenotype typical of Calypso. Runners generated from the RO lines were rooted and then detached to produce Rl lines. All Rl lines produced displayed the typical phenotype.

Tobacco plants were weaned and transferred to the glasshouse. All of the tobacco lines produced displayed the typical phenotype for Nicotiana tabacum.

Primers FavPGUSl and FavPGUS2 (SEQ ID NO' s 2 and 3, respectively) were designed to anneal in the FavRB 7 gene promoter and the gusA gene respectively, and were used in PCR to confirm the presence of this region in pSCVFavRB7 transgenic lines of both strawberry and tobacco. Primers GUS27 and GUS392 (SEQ ID NO's 6 and 7, respectively) were used to confirm the presence of the gusA gene in pSCVl.6 transgenic lines.

Thirty-two strawberry lines containing pSCVFavRB7 and one line containing pSCVl.6 T-DNA were generated. In tobacco, eight Ottawa lines containing pSCVFavRB7 and two lines containing pSCVl .6 T-DNA were generated. Eleven Petit Havana lines containing pSCVFavRB7 and two lines containing pSCV1.6 were also generated. PCR was used to detect the FavRB7 gene promoter and gusA gene in these lines. Strawberry lines 50, 59a, 59b and 60, and tobacco lines 35 and 68 transformed with pSCVFavRB7 were additionally generated, however the FavRB 7 gene promoter: gusA region was not detected in these lines. Southern analysis of transgenic strawberry

Southern analysis was carried out on 36 putative pSCVFavRB7 lines and one putative pSCVl .6 line of strawberry. The transgenic tobacco lines generated were not subject to Southern analysis due to time constraints.

To establish site integration number of the gusA and nptll genes and to confirm the presence of the FavRB7 gene promoter:gw^ gene region in transgenic strawberry lines, genomic DNA was digested with Kpn I and Sma I. Digoxigenin-II-dUTP (DIG) labelled nptll and gusA probes were synthesised using the primers NPTJIA and NPTIIB and GUS27 and GU392, respectively, using pSCVl.6 plasmid DNA as template in PCR. These probes were then used to evaluate the number of transgene integration sites.

Southern analysis of Kpn I digested genomic DNA revealed the number of integration sites for the nptll gene, which was confirmed through Southern using Sma I digested genomic DNA. Southern analysis of Sma I digested genomic DNA revealed the number of integration sites for the gusA gene.

The presence of a second Kpn I site within the T-DNA (Figure 4) meant that a 4.6 kb restriction fragment representing the FavRB 7 gene promoter :gusA gene region should be detected in all pSCVFavRB7 transgenic lines using the gusA probe. Identification of fragment greater than or less then 4.6 kb would imply that some manner of deletion or rearrangement has occurred within this region to remove a Kpn I site at either the 5' of 3' end of this region. In both cases it is important to note that the integrity of the promoter driving gusA cannot be guaranteed. Evidence of such alterations were observed in lines 41, 53a, 53b, 61, 67, 72 and 78.

Southern analysis established the number of integration sites for both nptll and gusA genes in the transgenic lines generated (Table 1). In the majority of the transgenic lines integration of both the nptll and gusA genes was found to have occurred at a single site, 20/34 and 27/34, respectively. Six lines (41, 53a, 53b, 67, 78 and 102) were found to contain multiple insertions of both genes. In four lines (41, 53a, 53b and 67) the integrity of the FαvRB7 gene promoter:gw.s7l gene region could not be confirmed at all integration sites. Lines 58a and 58b were found to be clonal, having arisen from a single transformation event, and so only line 58a was subject to further analysis. Lines 50, 59a, 59b and 60 were found to contain neither the nptll or gusA gene, which supported the PCR observations for these lines. These lines were not subject to further analysis.

Table 1 Number of integration sites observed for gusA and nptll in pSCVFavRB7 strawberry lines

The number of integration sites of each transgene in each pSCVFavRB7 strawberry line is indicated. Non-transformed control (-) and pSCVl.6 (+) lines are included. An asterisk denotes those lines highlighted in Figure 4.8, which may contain deletions or rearrangements within the FavRB7 gene promoteπgztf gene region. Histochemical analysis of transgenic strawberry

A β-glucuronidase (X-Gluc) histochemical assay was carried out to assess expression of the gusA gene in a range of tissue types in the pSCVFavRB7 transgenic strawberry lines. Leaf, petiole and root samples were collected from all glasshouse grown lines. Not all lines flowered within the study period, however where possible floral organs (whole buds and open flowers) were also collected. All samples were stored at -80°C prior to analysis.

Histochemical analysis was carried out. Blue staining was observed in the roots of all pSCVFavRB7 transgenic lines, at similar levels to that observed in the CaMV 35S line. No blue staining was observed for the FavRB 7 gene promoter lines in the leaves or floral organs, whereas intense blue staining was observed in the CaMV 35S line. Light blue staining was observed in the petioles of several of the FavRB 7 gene promoter lines, although not to the intensity of the CaMV 35 S line, suggesting that gusA is also expressed to a lesser degree in the petioles of some FavRB7 gene promoter lines. This suggests that the FavRB7 gene promoter confers expression of gusA preferentially in the roots of strawberry.

Further samples of each tissue type were taken from non-transformed control, the CaMV 35S line and pSCVFavRB7 lines 37 and 53b and subject to histochemical assay. These samples were then fixed in 10% formalin prior to histological study. To obtain detailed visualisation of gusA activity within the various tissue types, 10 μm sections were taken from the various tissues and photographed under magnification using the Zeiss axiophot microscope and a Nikon coolpix digital camera.

Intense staining as a result of gusA activity was observed in all tissues of the CaMV 35S line and was non-detectable in the non-transformed control line. Intense staining was only observed in the root tissues of the pSCVFavRB7 lines and no staining was observed in the leaf, floral organs or fruit of the pSCVFavRB7 lines.

However, slight staining was observed in the petioles. Microscopy revealed that gusA activity in the roots of the pSCVFavRB7 lines was highest in the central vascular region of the root and in the tip of the lateral root cap. Intense staining was often observed in the root tips, however it was thought that this may be a result of older root tissues being more highly suberised and less permeable to the β-glucuronidase substrate used in the histochemical assay rather than lack of activity in the older tissues.

To establish if the expression pattern conferred by the FavRB7 gene promoter was maintained through vegative propagation in strawberry histochemical analysis was also carried out on a population of eight Rl lines, propagated from stolons of RO pSCVFavRB7 lines. In all of the lines examined, the spatial expression pattern observed in the RO lines of strong blue staining in the roots, weak staining in some petioles and no staining in the floral organs or leaves, was maintained in the Rl lines. This suggests that the transgene expression pattern conferred by the FavRB 7 gene promoter is maintained through vegetative propagation.

The ρSCVFavRB7 transgenic tobacco lines were also subject to β-glucuronidase (X-Gluc) histochemical assay to assess the activity of the FavRB 7 gene promoter in a heterologous system. Tobacco tissue samples were collected from the leaf at the fourth internode, the petiole of this leaf, stem tissue above the fourth internode, young root tissue and floral organs. Comparable levels of blue staining were observed in all tissues for lines transformed with pSCVl.6 and pSCVFavRB7. Although not quantitative, the histochemical assay showed that gusA expression levels were uniformly high in all tissues and not preferentially expressed in the roots of tobacco. This result clearly shows that FavRB 7 gene promoter activity differs significantly in tobacco compared to strawberry, behaving constitutively in the heterologous system.

GUS fluorometric analysis of transgenic strawberry

Fluorometric analysis was carried out to quantitatively assess the expression pattern of the gusA gene under control of the FavRB 7 gene promoter in strawberry. Leaf, petiole and root samples were collected from one pSCV1.6 and 32 pSCVFavRB7 lines of glasshouse grown transgenic strawberry. Not all clones flowered during the study period and no mature fruit was available for fluorometric analysis. However, floral organs, whole buds and flowers were collected from those clones producing flowers over a period of several weeks and stored at - 80°C. To account for variation within the lines, samples were taken from 2 replicates of each line and a minimum of 6 replicates of each tissue type collected. For each line, leaves were excised using a 1 cm cork borer and petiole and root tissues were excised in sections of -0.25 cm and tissues were flash frozen in liquid nitrogen. Floral organs stored at - 80°C were pooled prior to analysis. An aliquot of approximately 100 mg of each tissue type was taken for fluorometric assay.

Fluorescence was quantified using a FLUOstar Galaxy multi well plate reader with the excitation filter set at 360 nm and the emission filter set at 460 nm. Each fluorescence reading was taken a minimum of four times and the average value calculated. Specific activity of gusA was quantified in terms of pmol 4-MU produced per min per mg total protein. Total protein was determined using the BioRad assay using the FLUOstar Galaxy multi well plate reader with the absorbance filter set at 595 nm. Each protein assay was read a minimum of four times and the average value calculated.

The fluorometric data generated from the 32 pSCVFavRB7 lines analysed revealed an average specific activity fox gusA of 36,780 pmol 4-MU/min/mg in the roots and a comparable value of 40,482 pmol 4-MU/min/mg in the roots of the CaMV 35S line. By contrast, average gusA specific activities in the petioles, leaves and floral organs in the pSCVFavRB7 lines were found to be greatly reduced compared to the gusA expression in the roots. Average gusA specific activities for petiole, leaf and floral organs were found to be 7,417, 858 and 419 pmol 4-MU/min/mg respectively. For the same tissues CaMV 35S values were 44,908, 132,387 and 75,953 respectively. In pSCVFavRB7 lines the median gusA specific activity ratio between root: petiole was found to be 1: 0.3, root: leaf 1: 0.04 and root: floral organ 1: 0.009. These data corroborate the observations made using the histochemical assay that the FavRB7 gene promoter confers expression of the gusA gene in a near root-specific manner.

Further examination of the fluorometric data revealed that the majority (25/32) of gusA specific activities in roots were over 10000 pmol 4-MU/min/mg for the pSCVFavRB7 lines. The lowest and highest values observed in root tissue were 3,518 and 154,500 pmol 4-MU/min/mg, respectively. By contrast the majority (25/32) of gusA specific activities in leaves were beneath 900 pmol 4-MU/min/mg. The lowest value observed for leaf tissue was 100 pmol 4-MU/min/mg. The majority (23/32) of gusA specific activities observed in petioles were beneath 10,000 pmol 4-MU/min/mg. However, lines 1 and 55 both showed specific activities of over 30,000 pmol 4-MU/min/mg, which although lower than the root specific activities observed in these lines, is comparable with root specific activities for gusA observed in other lines. Only 1 lines contributed to the data generated for gusA specific activity in pSCVFavRB7 floral organs, of these lines the majority (13/17) exhibited specific activities beneath 160 pmol 4-MU/min/mg.

No significant correlation could be made between the number of copies of the gusA gene present in each line and the expression levels observed, however the majority (4/6) of those lines containing two or more copies of the gusA gene ranked in the bottom 30 % for gusA root expression levels. By contrast the line shown to have the second highest level of gusA expression in the roots (53a) contains four copies of the gusA gene and was implicated as containing possible rearrangements in the FavRB 7 gene promoter: gusA gene region. Anomalous levels of gusA gene expression were noted in several of the other lines suspected to contain such rearrangements. Line 53b consistently displayed very low gusA expression levels in all tissue types other than roots, and lines 61 and 78 displayed the highest levels of gusA expression observed in floral organs and leaf tissues, respectively.

Reverse transcriptase PCR analysis of transgenic strawberry

Expression of the nptll and gusA genes were studied at the molecular level in both pSCVFavRB7 and pSCVl .6 lines of transgenic strawberry using reverse transcriptase PCR (RT-PCR). Messenger RNA extraction, cDNA synthesis and PCR were carried out. Primers NptllA, NptllB (SEQ ID NO's 4 and 5, respectively), GUS27 and GUS392 were used in a multiplexed RT-PCR reaction to detect expression of nptll and gusA transgenes in the root, leaf, petiole, floral organs and fruit. Lines 6, 14, 37, 46 and 55 were selected from the pSCVFavRB7 strawberry transgenics because fluorometric analysis had shown these lines to represent a range of gusA expression levels and samples were available for all of the tissue types examined in the fluorometric study. Lines 6, 46 and 55 also produced fruit during the study period. cDNA was synthesised from fruit tissues for these lines as well as the CaMV 35S constitutive control line.

Amplification fragments corresponding to the nptll and gusA genes were generated from all tissue types for the CaMV 35S line using RT-PCR. This confirmed that the assay was sensitive enough to detect the transgenes being expressed under control of the CaMV 35S promoter. Expression of the nptll gene under control of the CaMV 35S promoter was detected in all of the pSCVFavRB7 transgenic lines. The amplification fragment generated for the gusA gene under control of the FavRB7 promoter was noticeably less intense than the nptll fragment generated in the ρSCVFavRB7 lines. Only in the root tissues of line 55 was agusA amplification fragment generated at similar levels to that of nptll. Although not quantitative this assay confirmed gusA activity conferred by the FavRB 7 promoter at the molecular level as was previously revealed through histochemical and fluorometric assay.

The FavRB 7 gene promoter isolated from strawberry was shown to act in a strongly root preferential manner in strawberry. Histochemical, fluorometric and molecular analyses revealed that its activity, as measured by activity of the expressed product of the gusA gene linked to the promoter, was low in tissues other than the root. In root however, FavRB7 promoter activity was high and comparable to that of the CaMV 35S promoter.

Histochemical analysis showed that the FavRB7 gene promoter imparts a very different spatial gene expression pattern in tobacco to that observed in strawberry. In tobacco the FavRB7 gene promoter was found to act in a strong constitutive manner and accordingly may provide a useful alternative the CaMV 35S promoter in heterologous systems.

Claims

Claims:

1. A nucleic acid sequence comprising a promoter sequence as shown in SEQ. JD NO. 1, or a variant or derivative thereof.

2. A nucleic acid sequence according to claim 1, wherein the promoter is as found in association with a tonoplast intrinsic protein gene in the strawberry.

3. A nucleic acid sequence according to claim 1 or 2, wherein the promoter is operably linked to an ATG initiation codon.

4. A nucleic acid sequence according to any preceding claim, wherein the promoter is operably linked with a marker therefor.

5. A nucleic acid sequence according to claim 4, wherein the marker is gusA.

6. A nucleic acid sequence according to claim 4, wherein the marker is selected from: antibiotic resistance markers, non-antibiotitc resistance markers, and scorable markers.

7. A nucleic acid sequence according to any preceding claim, wherein the the promoter is a variant having an identity of at least 80% with SEQ ID NO 1.

8. A nucleic acid sequence according to claim 7, wherein the identity is at least 90%.

9. A nucleic acid sequence according to claim 8, wherein the identity is at least 95%.

10. A nucleic acid sequence according to claim 9, wherein the identity is at least 98%.

11. A nucleic acid sequence according to any preceding claim, wherein the promoter is a truncated form of SEQ ED NO. 1.

12. A nucleic acid sequence according to claim 11 , wherein the truncated promoter is no less than 600 base pairs in length.

13. A nucleic acid sequence according to claim 11 or 12, wherein the length of the truncated promoter is between 1400 and 2500 nucleotides.

14. A nucleic acid sequence according to any preceding claim, wherein the promoter comprises at least 50% of the 3' end of SEQ JD NO. 1.

15. A nucleic acid sequence according to claim 14, wherein the promoter comprises at least 70% of the 3' end of SEQ ED NO. 1.

16. A nucleic acid sequence according to claim 14, wherein the promoter comprises at least 80% of the 3' end of SEQ ID NO. 1.

17. A nucleic acid sequence according to any preceding claim, wherein the promoter comprises the full sequence of SEQ JD NO. 1.

18. A nucleic acid sequence according to any preceding claim, further comprising one or more response motifs located within the promoter sequence.

19. A nucleic acid sequence according to any preceding claim, wherein one or more response motifs occurring within SEQ ID NO. 1 are deleted.

20. A nucleic acid sequence according to any preceding claim, wherein the promoter is operably linked with at least one gene encoding an expression product.

21. A nucleic acid sequence according to claim 20, wherein the gene encodes pest resistance.

22. A nucleic acid sequence according to claim 21, wherein the resistance is to a pathogen.

23. A nucleic acid sequence according to claim 21 or 22, wherein the resistance is to a fungus.

24. A nucleic acid sequence according to claim 20 or 21, wherein the expression product is selected from: proteinase inhibitors, Bacillus thuringiensis (Bt) toxins and plant lectins, and combinations thereof.

25. A nucleic acid sequence according to claim 20 or 21, wherein the expression product is selected from: endochitinases; antimicrobial peptides; beta-l-3-glucanase protein; ribosome-inactivating proteins, and combinations thereof.

26. A sequence according to any preceding claim, which is DNA.

27. A plant, or reproductive material thereof, comprising a nucleic acid sequence according to any preceding claim in expressible form.

28. A plant, or reproductive material thereof, according to claim 27, wherein the promoter is constitutive.

29. A plant, or reproductive material thereof, according to claim 27, wherein the promoter is tissue-specific.

30. A plant, or reproductive material thereof, according to any of claims 27 to 29, which is Rosaceae spp.

31. A plant, or reproductive material thereof, according to claim 30, which is Fragaήa spp.

32. A plant, or reproductive material thereof, according to any of claims 27 to 31 , wherein the promoter is root-specific.

33. A plant, or reproductive material thereof, according to any of claims 27 to 29, that is tobacco.

34. A vector comprising a nucleic acid sequence according to any of claims 1 to 26.

35. A vector according to claim 34 that is an expression vector.

36. A plant expressing at least one transgene in at least one tissue thereof at at least one stage in the lifecycle thereof, wherein the transgene is under the control of a promoter as defined in any of claims 1 to 26.