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United States Patent Application	20010016955
Kind Code	A1
Woodhead, Mary Rose ;   et al.	August 23, 2001

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Foreign Application Data

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Date	Code	Application Number
Nov 3, 1995	GB	9522558.7

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Claims

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1. A process for isolating a promoter capable of driving fruit-specific expression of DNA sequences in transgenic blackcurrant and other non-climacteric fruit comprising a) isolating mRNA from ripening blackcurrant fruit b) preparing a cDNA library from the isolated mRNA c) differentially screening the library from b) to identify genes expressed during the ripening period and d) screening a genomic library with probes prepared from cDNA identified according to c) to isolate the corresponding gene and its promoter region.

2. A promoter capable of driving fruit-specific expression of DNA sequences in transgenic blackcurrant and other non-climacteric fruit obtainable by the process of claim 1.

3. A promoter according to claim 2 which comprises the sequence of nucleic acid bases in FIG. 9 or IDSEQ 11 (the RIB1 gene promoter) or IDSEQ 14 (the RIB 7 gene promoter)..

4. Promoter DNA sequences which hybridise to the DNA of claim 3 under conditions of high stringency.

5. cDNA for genes which exhibit differential expression in fruit during the ripening period of fruit development selected from pRIB1 (IDSEQ 1), pRIB3 (IDSEQ 3), pRIB5 (IDSEQ 5), pRIB6 (IDSEQ 7) and pRIB7 (IDSEQ 9).

6. DNA encoding the RIBI or RIB 7 gene.

7. A vector comprising the DNA as claimed in any one of claims 2 to 6.

8. Use of a promoter according to claim 2, 3 or 4 to control the expression of one or more genes in climacteric or non-climacteric fruit.

9. Use according to claim 8 wherein the non-climacteric fruit is blackcurrant.

10. Use of a promoter according to claim 2, 3 or 4 in the transformation of plant cells.

11. Plant cells and plants transformed using a promoter according to claims 2, 3 or 4 or a vector according to claim 7.

12. Plants comprising cells according to claim 11 and descendants thereof.

13. Plants and seeds according to claim 12 which are blackcurrants and products prepared therefrom.

14. A process according to claim 1 wherein the method for extracting nucleic acid from blackcurrant fruit comprises homogenising by pulping blackcurrant fruit in a buffer containing insoluble polyvinylpolypyrrolidone.

15. Proteins encoded by the DNA sequences of claims 5 or 6.

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Description

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[0001] The present invention relates to transgenic plant production and the expression of gene sequences introduced by genetic transformation procedures. In particular the present invention relates to blackcurrant (Ribes nigrum L.) fruit-specific gene promoters and their use in the expression of nucleic acid sequences in transgenic fruit.

[0002] Studies on the molecular basis of fruit ripening have concentrated on species whose fruit exhibit a climacteric pattern of ripening, for example tomato, avocado, apple, kiwifruit, peach and mango. Ripening in the fruit from these species is accompanied by a burst in the rate of respiration and a generally large increase in the rate of biosynthesis of the plant growth regulator, ethylene.

[0003] Non-climacteric fruit have a considerably different ripening mechanism. Examples of non-climacteric fruit are blueberry, cucumber, grape, orange and strawberry.

[0004] Fruit ripening is an important area of scientific research with particular attention being paid to high value fruits such as tomato, kiwifruit and avocado. In the tomato some of the genes involved in the ripening process have been isolated and characterised, for example the gene for polygalacturonase an enzyme which acts on cell wall pectin. The level of expression of the polygalacturonase gene has been down-regulated in transgenic tomato fruit resulting in increased fruit firmness and consequently extended storage life (Schuch et al, 1991).

[0005] In contrast, less is known about the molecular basis of fruit ripening in non-climacteric fruit. In the work leading to the present invention we have found from measurements of respiration rate that blackcurrant fruit do not exhibit a respiratory climacteric during ripening and that ripe fruit produce very low levels of ethylene, hence blackcurrant can be classed as a non-climacteric fruit.

[0006] The blackcurrant is the most widely grown bush fruit in Europe, valued particularly for its high content of ascorbic acid and anthocyanin pigments. Areas for potential improvement in blackcurrants include enhancing pigment levels, aroma, flavour, texture, nutritional values (e.g. vitamin content), storage life, weather resistance, pest or pesticide resistance and manipulating sugar , soluble solids or acid levels in the fruit.

[0007] Plants with novel/improved characteristics can be produced by introducing genes or DNA sequences from the same or a different organism. Many examples are now in the literature of plant DNA sequences which have been used to drive the expression of foreign genes in plants. In most instances the regions adjacent to the 5' terminus of the coding regions of genes have been used in gene constructs. These regions are referred to as promoter sequences. In order to produce novel phenotypes it is necessary to have active expression of the introduced DNA sequence by cloning the sequence downstream of a promoter sequence active in plant tissue. These promoters may be derived from plant DNA or from other sources e.g. viruses. In most cases sequences up to 500-1000 bases are sufficient to allow for the regulated expression of foreign genes. However sequences longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants. Examples of fruit-specific promoters isolated from climacteric fruit such as tomato include the 2All promoter, and the polygalacturonase gene promoter.

[0008] Of considerable importance to the development of genetically improved blackcurrants is the finding in the work of the present invention that blackcurrant is in fact a non-climacteric fruit.

[0009] Promoters can vary in the level of expression and in the tissue-specific or developmental stage-specific pattern of expression that they drive. Some promoters are expressed in a tissue-specific or developmental stage-specific manner whereas others are expressed in each and every cell and are called constitutive promoters.

[0010] The most widely used constitutive promoters are the Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthetase (nos) and the octopine synthetase (ocs) promoters. Due to the different molecular mechanisms of ripening between climacteric and non-climacteric fruit it is hardly appropriate to use fruit-specific promoters isolated from climacteric fruit such as tomato (e.g. the 2All promoter or the polygalacturonase gene) in non-climacteric fruit.

[0011] Climacteric fruit-specific promoters therefore may not be suitable for many potential biotechnological applications for the improvement of non-climacteric fruit such as the blackcurrant which ideally require high levels of fruit-specific expression. In the case of the commonly used constitutive promoters, they have the disadvantage that they drive expression at high levels in all or nearly all cell types and throughout the development of the plant. Expression of the introduced gene or DNA sequence driven by a constitutive promoter can have a deleterious effect on normal plant development. Additionally, the commonly used constitutive promoters are derived from plant infectious agents such as plant viruses or Agrobacterium, a soil-borne infectious bacteria. The source of these promoters is a cause for concern in risk assessment of transgenic plant production.

[0012] Accordingly, the present invention provides promoters and a process for obtaining promoters capable of driving fruit-specific expression of DNA sequences in transgenic blackcurrant and other non-climacteric fruit. The process is as defined in claim 1 and the promoters as defined in claim 2. Preferably the promoter comprises the sequence of nucleic acid bases in FIG. 9 or IDSEQ 11 herein designated the RIBI promoter or in IDSEQ 14 herein designated the RIB 7 promoter. No previous promoters have been reported to be suitable to drive fruit-specific expression in blackcurrant and other non-climacteric fruit.

[0013] One advantage of the present invention is that because of the developmental stage specificity of the expression ie. it offers high level expression in fruit and only very low levels in other tissues, there is a reduced chance that the introduced DNA sequences will have an adverse effect on normal plant development.

[0014] The promoters of the present invention also have the advantage over some constitutive promoters in that they are naturally occurring plant gene sequences derived from blackcurrants, ie. a plant that is consumed by humans and not from plant pests or other infectious agents; this overcomes objections to the use of such sequences due to potential recombination.

[0015] The isolation and characterisation of blackcurrant fruit-specific gene promoters and how they can be used to drive the expression of genes of interest in plants is given below and in the following examples. This description is purely for the purpose of illustrating the invention. It should be noted that the gene promoter may function in a similar (that is, fruit-specific) manner in other related species of non-climacteric fruit, in particular other Ribes species.

[0016] Promoters for use in the invention may be isolated from genomic libraries by the use of cDNA probes. The cDNA clones of genes highly expressed specifically in ripe blackcurrant fruit were obtained by differentially screening a cDNA library constructed from mRNA isolated from ripening blackcurrant fruit.

[0017] In a further aspect of the invention there is also provided cDNA for genes which exhibit differential expression in fruit during the ripening period of fruit development. In particular the cDNA is identified herein as pRIB1, pRIB3, pRIB5, pRIB6 and pRIB7.

[0018] The promoters of the present invention can be used to control the expression of one or more genes in non-climacteric and/or climacteric fruit. Preferably the non-clinacteric fruit is the blackcurrant. Suitably the genes are novel/exogenous.

[0019] According to the present invention we also provide the use of promoters of the present invention in the transformation of plant cells to control the expression of one or more genes in non-climacteric/climact- eric fruit.

[0020] In a further aspect of the invention there are provided novel plant cells and plants transformed using the promoter according to the present invention. Preferably the plants or seeds are blackcurrants.

[0021] According to the present invention, plant cells may be transformed using promoters of the invention using a variety of known transformation methods such as Agrobacterium-mediated or other vector-mediated transformation methods or physical transformation methods such as biolistics, chemical or electrical transfection or micro-injection.

[0022] In particular the RIB1 or RIB 7 promoter regions are suitable for incorporation into plasmid vectors designed for general use in construct production in E. coli, and for use in stable, Agrobacterium-mediated transformation (Bevan, 1984) and in transient transformation (Fromm et al., 1985) or stable, physical transformation methods (Klein et al., 1987). DNA sequences which one wishes to have expressed only in the fruit of transgenic blackcurrants and possibly other non-climacteric soft fruit can be inserted downstream of the promoter region of the blackcurrant RIB1 or RIB 7 gene, prior to introduction into plant cells or production of transgenic plants.

[0023] The transformed cells may then, in suitable cases, be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome.

[0024] Examples of genetically modified plants according to the invention include as well as blackcurrants, fruits such as blueberry, cucumber, grape, orange and strawberry. Plants produced by the process of the invention may contain more than one recombinant gene. In order to prepare RNA suitable for a cDNA library construction, an improved method for the RNA extraction was developed as the available methods were found not to be applicable to blackcurrent fruit. The problems in working with blackcurrant tissue include the combination of the high levels of phenolic compounds and polysaccharides and the high acidity of berry extracts.

[0025] Accordingly in a further aspect of the present invention there is provided a method of extracting nucleic acid in particular RNA from blackcurrant fruit. One known method for grape berries (Tesniere & Vayda, 1991) was found to be unable to yield large quantities of good quality RNA from blackcurrant fruit which was not contaminated with coloured substances. This method was the basis for the modified method for the extraction of RNA from blackcurrant fruit.

[0026] Two key modifications were the method of tissue homogenisation and the inclusion of 8.5% (w/v) insoluble polyvinylpolypyrrolidone (PVPP) in the homogenisation buffer. The use of PVPP resulted in the removal of pigment from the fruit pulp at the start of the extraction procedure producing a clear final RNA pellet. Pulping fruit in the homogenisation buffer rather than grinding frozen fruit in a fine powder in liquid nitrogen and then adding the buffer was a less harsh method of tissue maceration and resulted in less disruption of cells and a reduction in the amount of gelatinous material. Pulping also reduced the problem of extracting large amounts of seed as well as fruit RNA which otherwise occurred during grinding in liquid nitrogen. Each fruit can frequently contain over twenty seeds and these are impossible to manually extract quickly enough to prevent the expression and subsequent isolation of wound-induced mRNA's from the fruit. In ripe fruit the problem can be solved using a juicerator (Acme). This macerates the fruit tissue to a pulp which can be collected and retains the seed and large pieces of skin material. Unripe fruit (i.e. green or green/red) were too hard to be pulped using this method so a coffee grinder was used instead.

[0027] The average yield of total RNA using this method is 15-20 .mu.g RNA per g fresh weight of fruit, for each stage of ripening investigated. The ratio of A.sub.260/A.sub.280 nm was between 1.8-2.0. The yield was the same whether RNA was extracted from the pulp on the day of fruit harvest or whether the pulp was stored at -80.degree. C., defrosted and subsequently used in an extraction. This implies that the RNA remains stable in the pulp. The yields are similar to those obtained from other fruit tissues e.g. apples (13 .mu.g RNA per g fresh weight Lay-Yee et al., 1990) and peaches (12-15 .mu.g RNA per g fresh weight, Callahan et al, 1989).

[0028] Denaturing agarose gel electrophoresis revealed that two ribosomal RNA bands were clearly visible suggesting that the RNA extracted using this new procedure was undegraded. In addition the RNA isolated from the fruit was capable of directing the synthesis of polypeptides as demonstrated by in vitro translation using a wheat germ lysate system. Polypeptides of up to approximately 80 kD were synthesised and the incorporation of .sup.35S--methionine into TCA precipitable products was about 30 times higher than background values when 20 .mu.g of total RNA were used compared with the minus RNA control.

[0029] The new extraction method described below in Example 2 allowed for the first time the extraction of RNA from blackcurrant fruit. This RNA has been shown to be biologically active, as demonstrated by in vitro translation results. In addition this RNA has been used to construct a cDNA library from an early ripening stage (Example 4 below). The cDNA library contained approx. 6.6.times.10.sup.6 primary clones with an average insert size of 900 base pairs. Differential screening of 10,000 clones has resulted in the isolation of 5 clones which show an increase in expression during ripening.

[0030] The invention will be described further with reference to the following figures, in which;

[0031] FIG. 1 shows the results of an RNA blot analysis of total RNA isolated from blackcurrant (cv Ben Alder);

[0032] FIG. 2 shows the results of a DNA blot analysis;

[0033] FIG. 3 shows the nucleotide sequence of the pRIB1 cDNA clone (IDSEQ 1);

[0034] FIG. 4 shows the deduced amino acid sequence encoded by pRIB1 (IDSEQ 2);

[0035] FIG. 5 shows the nucleotide and predicted amino acid sequence of pRIB3 (IDSEQ 3 and 4 respectively);

[0036] FIG. 6 shows the nucleotide and predicted amino acid sequence of pRIB 5 (IDSEQ 5 and 6 respectively);

[0037] FIG. 7 shows the nucleotide and predicted amino acid sequence of pRIB 6 (IDSEQ 7 and 8 respectively);

[0038] FIG. 8 shows the nucleotide and predicted amino acid sequence of pRIB 7 (IDSEQ 9 and 10 respectively);

[0039] FIG. 9 shows the nucleotide sequence of the RIB1 promoter up to the transcription start site (IDSEQ 11), and

[0040] FIG. 10 shows the RIBI gene sequence (IDSEQ 12) and the deduced amino acid sequence (IDSEQ 13). The transcription start site was located by primer extension analysis and this C residue in position 1797 is indicated in bold type and underlined in the figure.

EXAMPLES

[0041] Unless indicated otherwise the methods and standard techniques used below are as given in Sambrook et al (1989).

Example 1

Pigmnent and Respiratory Analysis

[0042] 1.1 Plant material

[0043] Fruit, leaves and stems were harvested from blackcurrant (Ribes nigrum L. cv, Ben Alder) plants grown in experimental field plots at the Scottish Crop Research Institute, Invergowrie, Dundee, UK. Blackcurrant tissues were harvested and frozen immediately in liquid nitrogen. Thereafter, tissues were stored at -80.degree. C. prior to analysis. Roots, leaves and stems were harvested from either one year old plants that had not yet borne fruit or from two-year-old plants that were producing fruit. Fruits were harvested at five stages of ripening as determined by fruit colour (designated green, green/red, red/green, red and black).

[0044] 1.2 Determination of fruit anthocyanin content

[0045] Blackcurrant fruit (0.5 g FWt) was ground to fine powder in liquid nitrogen and extracted with 1 ml of methanol containing 1% (v/v) trifluroacetic acid. After centrifugation (16000 g, 10 min) the pellet was re-extracted with a further 1 ml of methanol/trifluroacetic acid. The absorbance of the combined extracts at 518 nm was determined spectrophotometrically. Anthocyanin concentration in the extracts was estimated by comparison with a standard curve produced using the artificial pigment, amaranth (trisodium 3-hydroxy4(4-sulphonato-1 -naphthylazo)naphthalene-2, 7-disulphonate).

[0046] 1.3 Ethylene and CO.sub.2 determinations

[0047] The rate of ethylene and CO.sub.2 evolution from harvested blackcurrant fruit was determined using a Hewlett Packard 5890A gas chromatograph. Blackcurrant fuit were placed in gas-tight jars and incubated at 15.degree. C. for up to 24 h. Sampling was carried out using a gas-tight syringe. For CO.sub.2 determinations, the gas chromatograph was fitted with a thermal conductivity detector and a Porapak Q column (2 mm internal diameter, 1.85 M length) maintained at 50.degree. C. A flow rate of 20 cm.sup.3 min.sup.-1, was set for the carrier gas (helium) and the peaks were integrated on a Spectra-Physics integrator (San Jose, Calif., USA). The chromatograph was calibrated with injections of 1 ml samples of 1% CO.sub.2 (Phase Separations Ltd, Clwyd, Wales, UK). For ethylene measurements, the gas chromatograph was fitted with a flame ionization detector and a Porapak R column (2 mm internal diameter, 1.85 M length) maintained at 80.degree. C. The flow rate of carrier gas (helium) was 50 cm.sup.3 min.sup.-1 and the system was calibrated by injecting 1 ml samples of ethylene gas at a concentration of 91 ppm (Phase Separations Ltd, Clwyd, Wales, UK). All peaks were integrated using a Hewlett-Packard 3390A integrator.

[0048] Results

[0049] Rate of ethylene and carbon dioxide production by blackcurrant fruit

[0050] Very low levels of ethylene were produced by fruit from all stages of ripening (the level of ethylene from green, green/red and red/green fruit was below the detection limit of the gas chromatograph (approximately 0.1 ppm)). As an indication of the rate of respiration of the ripening fruit, the rate of CO.sub.2 production was determined. There was no burst in respiration rate as the fruit ripened. In fact, the highest rate of CO.sub.2 production was produced by green fruit. In the later ripening stages, the level was approximately 20% lower than in the green fruit and remained constant as the fruit ripened from the green/red to the black stage.

Example 2

RNA Extraction

[0051] RNA was extracted from Ben Alder fruit at five ripening stages, and from leaf, root and stem material from fruited and non-fruited Ben Alder plants. Glassware was baked at 180.degree. C. for 12 h and plasticware and Miracloth (Calbiochem) were autoclaved prior to use. Solutions were prepared from stocks by dilution in sterile DEPC-treated (diethyl pyrocarbonate) distilled water before autoclaving. Unless otherwise stated, the procedures were carried out at 4.degree. C. Freshly harvested berries were weighed into 50 g portions and stored on ice. Leaf, root and stem material was harvested, rapidly frozen in liquid nitrogen and stored at -80.degree. C. until required. Fruit (50 g) was pulped with 100 ml of homogenisation buffer (200 mM Tris.HCl pH 8.5, 300 mM LiCl, 10 mM Na.sub.2EDTA, 1% (w/v) sodium deoxycholate, 1.5% (w/v) sodium dodecyl sulphate, 8.5% (w/v) insoluble polyvinylpolypyrrolidone (PVPP), 1% (v/v) Nonidet P-40, 1 mM aurintricarboxylic acid, 5 mM thiourea, and 10 mM dithiothreitol (the last three components were added as solids after autoclaving)) in a domestic coffee grinder for 45 s. Leaves, roots and stems were ground to a fine powder in a sterile pestle and mortar, with a little sand (previously baked at 180.degree. C. for 12 h) in liquid nitrogen and 5 vol of homogenisation buffer (containlng 4% PVPP instead of 8.5%) was added per gramme of tissue. The viscous material was poured into sterile 50 ml tubes. If not required for immediate use, the fruit pulp was frozen in liquid nitrogen and stored at -80.degree. C.

[0052] Frozen fruit pulp was defrosted rapidly in a microwave oven prior to use in the extraction. To proceed with the extraction, the homogenate was diluted 1:1 with sterile water and mixed well. 20 ml of diluted homogenate was placed in a 50 ml Oak Ridge-type centrifuge tube containing 15 ml homogenisation buffer and shaken. The tubes were placed in a waterbath at 65.degree. C. for 10 min, with occasional mixing, and then centrifuged at 12,000 x g for 30 min at 4.degree. C. The supernatant was filtered through two layers of Miracloth and collected in an Oak Ridge-type centrifuge tube and solid CsCl was dissolved in the supernatant to a final concentration of 0.2 g CsCl per ml of filtered extract. The extract was gently layered onto a 10 ml cushion of 5.7 M CsCl containing 10 mM Tris.HCl pH 7.5 and 10 mM Na.sub.2EDTA, in a Beckman 50 ml ultracentrifuge tube and centrifuged at 100,000 x g for 20 h at 20.degree. C. After centrifugation, the supernatant was carefully removed with a syringe and discarded. The RNA pellet remained at the bottom of the tube.

[0053] The pellet was washed with 5 ml of ice-cold 70% ethanol, centrifuged at 10,000 x g for 10 min at 4.degree. C. and the tubes inverted to allow the pellet to dry. The RNA was resuspended in a total of 1 ml of sterile distilled water and transferred to a sterile microfuge tube. 200 .mu.l of 3 M LiCl (0.5 M final concentration) and 2.5 ml of 95% ethanol was added to precipitate the RNA (overnight at -20.degree. C.).

[0054] RNA was recovered by centrifugation at 16,000 x g for 30 min at 4.degree. C., and the pellet was washed three times with 0.5 ml 2.5 M sodium acetate (pH 5.5). Following centrifugation at 16,000 x g for 15 min at 4.degree. C. and removal of the supernatant, the pellet was resuspended in 100 .mu.l of sterile distilled water. Ethanol (95%) was slowly added to a final concentration of 30% (v/v) of the total and the tube vortexed briefly. After centrifugation at 16,000 x g for 2 min at 4.degree. C. the supernatant containing the RNA was transferred to a fresh microfuge tube and precipitated by the addition of 0.1 vol sodium acetate pH 5.2 and 3 vol ethanol and incubation at -20.degree. C. overnight. The RNA was recovered by centrifugation at 16,000 x g for 30 min at 4.degree. C., the pellet washed in 0.5 ml 70% ethanol and allowed to dry before it was suspended in sterile water.

Example 3

RNA analysis

[0055] Total RNA was extracted from blackcurrant tissues as described above in Example 2. Steady-state transcript levels were determined by RNA blot analysis. Total RNA (15 .mu.g/track) was separated electrophoretically under denaturing conditions and transferred by capillary action onto Hybond-N membranes (Amersham) as recommended by the manufacturer. Blots were probed with .sup.32P labelled cDNA inserts isolated from cDNA clones following restriction endonuclease digestion. Inserts were separated by electrophoresis through agarose gels and purified by electroelution. After hybridisation for 16-24 h at 42.degree. C. in 50% formamide, filters were washed sequentially in 2 x SSC, 0.5% SDS followed by 2 x SSC, 0.1% SDS and then 0.1% x SSC, 0.1% SDS for 20 min per wash at 52.degree. C. prior to exposure to X-ray film at -70.degree. C. for between 24 and 96 h. Transcript size was determined by comparison of electrophoretic mobility with RNA markers of known sizes (Life Technologies). The intensity of the hybridisation signal was determined by densitometry using a Millipore Bio-Imager (Millipore, Mich., USA).

[0056] FIG. 1 shows the results of one RNA blot analysis. Total RNA was isolated from blackcurrant (cv. Ben Alder) leaves (L), sterns (S) and roots (R) from plants that had borne fruit and from those that had not, and from fruit at five ripening stages (G green; GR=green/red; R/G=red/green; R=red; B=black). Total RNA (20 .mu.g per lane) was analysed by electrophoresis through a 1.2% denaturing agarose gel, blotted onto nylon membrane and hybridised with a labelled probe prepared to pRIB1, using standard techniques.

Example 4

cDNA Clone Isolation and Analysis

[0057] A cDNA library was constructed from polyadenylated RNA (7 .mu.g) extracted from green/red blackcurrant fruit. Polyadenylated RNA was prepared by affinity chromatography using oligo d(T) cellulose (Life Technologies). Double stranded cDNA was synthesised and directionally ligated into EcoRI/XhoI digested lambda Zap arms using a Uni-Zap XR vector kit (Stratagene). The library was packaged using an in vitro kit (Stratagene) and plated on the XL1-Blue strain of E.coli (Stratagene).

[0058] Differential gene expression during ripening

[0059] The cDNA library was screened with .sup.32P labelled cDNA from green fruit and green/red fruit. By differentially screening a total of 10,000 plaques, five were found to be differentially expressed between these stages. The in vivo excision protocol of Stratagene with the R408 helper phage was used to rescue putative ripening-related cDNAs in pBluescript SK (-) plasmids. The plasmids were purified using Qiagen columns (Qiagen Ltd., Dorking, UK). Steady-state expression levels of the corresponding genes (designated RIB1, RIB3, RIB5, RIB6 and RIB7) were determined by RNA blot analysis. The intensities of the hybridisation signals were determined by densitometry. For all clones, very low or negligible levels of expression could be detected in the green fruit and the highest levels of expression were detected in black, fully ripe fruit. In the quantitative densitometric analysis therefore, steady-state transcript levels are expressed relative to the level in black fruit. In order to demonstrate equal loading and transfer of RNA during this analysis, filters were stripped and hybridised with a potato 25S ribosomal RNA probe. An equivalent hybridisation signal was detected for RNA extracted from tissue at all stages (data not shown).

[0060] Expression in other blackcurrant tissues

[0061] Steady-state expression levels of the RIB genes were also determined in leaves, stems and roots of blackcurrant plants that had borne fruit and from those that had not. A variety of expression patterns were observed. For example, the expression of RIB1 and RIB7 was confined largely to fruit. RIB3, RIB5 and RIB 6 expression however was less specific to fruit and relatively high expression levels could be detected in some of the other plant tissues that were tested. The expression level of some of the clones was different depending on whether the blackcurrant plants had produced fruit or not. For example, the expression level of RIB5 was higher in plants that had never produced fruit compared with tissues from plants that had.

[0062] The clone pRIB1 hybridised to cDNA probes prepared from mRNA from ripe fruit but not to cDNA probes prepared from green, unripe fruit. Using the cloned pRIB 1 cDNA as a probe, a blackcurrant (cv. Ben Alder) genomic library constructed in .lambda. Fix II (custom synthesised by Stratagene Ltd, Cambridge, UK) was screened using standard techniques (Sambrook et al., 1989). A genomic clone corresponding to the cDNA clone was isolated and the blackcurrant RIB1 genomic clone was plaque purified. DNA was prepared and fragments subcloned into plasmid vectors by standard procedures (Sambrook et al., 1989). The RIBI genomic clone contained an insert of 18 kilobase pairs (kbp) from which the relevant sub-fragments were cloned into plasmid vectors. One subclone contains approximately 3 kbp of gene sequence (two exons and one intron) including approximately 1.8 kbp of 5' flanking sequence which contains the blackcurrant RIB1 promoter region.

[0063] RNA blot analysis (Sambrook et al., 1989) of blackcurrant tissues indicates that the gene is highly expressed in ripe blackcurrant fruit and expressed at negligible levels in other tissues of the blackcurrant plant (FIG. 1). Therefore this promoter region will be suitable to drive the expression of any piece of DNA cloned downstream of it (that is, following the 3' terminus of the promoter region) in ripening fruit but not in unripe fruit. A positive genomic clone corresponding to the RIB 7 CDNA (RIB 7) was isolated from the blackcurrant (Ribes nigrum L., cv. Ben Alder) genomic library and sub-cloned using the same techniques as for RIB 1. Two adjacent sub-clones (as determined by PCR) were sequenced and the RIB7 gene is contained within this sequence.

[0064] DNA sequence analysis

[0065] Plasmid DNA for sequencing was prepared using Qiagen columns. DNA sequence was obtained from both strands of alkaline denatured plasmid by manual dideoxysequencing using Sequenase version 2.0 (United States Biochemical Corporation) or by automated sequencing using an ABl 373 automated sequencer. DNA sequences were compiled and compared using the sequence analysis software and databases available on the SEQNET Computational Molecular Biology facility at SERC Daresbury Laboratory, UK.

[0066] Genomic DNA isolation and Southern analysis

[0067] Genomic DNA was isolated from the leaves of three blackcurrant cultivars (Ben Alder, Ben Sarek and Baldwin), Tayberries (Rubus loganobaccus) and raspberries (Rubus idaeus cv. Glen Moy). Leaves (1 g FWt) were ground to a fine powder in liquid nitrogen. 2.5 ml buffer containing 2% (w/v) CTAB, 100 mM Tris.HCl pH 8.0, 1.4 M NaCl, 20 mM Na.sub.2EDTA, 0.1% (w/v) DTT at 65.degree. C. was added and mixed gently prior to the addition of 0.1 g Polyclar AT (BDH). After a 30 min incubation at 65.degree. C., 7.5 ml of chloroform:isoamyl alcohol (24:1 [v/v]) was added and gently mixed. Following centrifugation (5000 g, 5 min) the aqueous phase was removed and mixed with an equal volume of propan-2-ol. After a 15 min incubation at room temperature, nucleic acids were pelleted by centrifugation (10000 g, 15 min). The air-dried pellet was resuspended in 0.85 ml water before the addition of 50 .mu.l 1 M KAc, pH 5.5, 20 .mu.l of 0.5 M Na.sub.2EDTA, 50 .mu.l Caylase (10 mg/ml [Cayla, Toulouse, France]), 1 .mu.l RNase A (10 mg/ml [Sigma]) and 29 .mu.l water . The mixture was incubated for 14 h at 37.degree. C. 50 .mu.l of 1 M Tris.HCl (pH 8.0) was then added to the solution prior to extraction with one volume of chloroform:IAA (24:1 [v/v]). Genomic DNA was precipitated with three volumes of ethanol, washed with 70% ethanol, air dried and finally resuspended in TE buffer (pH 8.0).

[0068] 5 .mu.g of each DNA sample was digested separately with the restriction endonucleases EcoRI, BamHI and HindIII and resolved by electrophoresis on 0.8% (w/v) agarose gels. DNA was transferred under vacuum to Hybond N membranes (Amersham) and hybridised with the .sup.32P labelled inserts of the pRIB 1 clone, prepared as above. Filters were washed at high stringency (0.1 x SSC, 0.1% SDS at 65.degree. C.) and exposed to X-ray film for 24-72 h at -70.degree. C. with intensifying screens. FIG. 2 shows the results of one DNA blot analysis: Genomic DNA (5 .mu.g per lane) from the blackcurrant cultivars Ben Alder (lane 1), Ben Sarek (lane 2) and Baldwin (lane 3), Tayberry (lane 4) and the raspberry cultivar Glen Moy (lane 5), was digested with either of the restriction endonucleases EcoRI, BamHI or HindIII, and fractionated on an 0.8% (w/v) agarose gel. The DNA was blotted onto nylon membrane hybridised with a labelled probe prepared to pRIB1, using standard techniques (Sambrook et al., 1989).

[0069] Results

[0070] Sequence analysis of the pRIB clones

[0071] pRIB 1

[0072] The size of the insert in pRIB1 is 882 base pairs, similar to that expected from the estimate of transcript size. A potential long open reading frame was identified from nucleotide position 3 to the TAA termination codon at position 489. A translation start codon is not present in this ORF indicating that the 5' portion of the cDNA is absent. A polyadenylation signal was identified in the cDNA sequence. Comparison of the deduced amino acid sequence of this ORF and the nucleotide sequence of the cDNA did not reveal any significant sequence similarity to other sequences in the European Molecular Biology Laboratory (EMBL) database of gene sequences.

[0073] When compared with the SwissProt protein database using the `Blitz` programme (MPsrch programme, Biocomputing Research Unit, University of Edinburgh, UK) the putative amino acid sequence shows similarity (% 50.9% similarity, 36.9% identity) to a cDNA encoding a protein isolated from kiwifruit (Ledger and Gardner, 1994). The steady state level of the kiwifruit transcript increases during fruit development, but declines during ripening. This is in contrast to the expression of the RIB1 gene in blackcurrant fruit where the steady state transcript level increases during the ripening period. Importantly, like the blackcurrant transcript, the kiwifruit gene is expressed almost entirely in the fruit.

[0074] pRIB 3

[0075] The ORF present in pRIB3 encodes a polypeptide which shares a high degree of sequence similarity with group one metallothioneins. The most similar metallothionein protein to the blackcurrant deduced sequence was from kiwifruit (79% similarity, 67% identity). Typical of metallothioneins, the putative blackcurrant polypeptide has a low M.sub.r value (M.sub.r 6808) and is acidic (pI 4.56). Metallothioneins also contain characteristic cysteine rich domains and the arrangement of these regions in blackcurrant and in a kiwifruit metallothionein is highly conserved. There are two Cys pairs in the N-terminal domain and three Cys pairs in the C-terminal domain separated by a hydrophobic domain. This organisation has also been observed in putative metallothioneins isolated from rice and Arabidopsis but differs from some plant sequences where there are three Cys pairs in the N-terminal domain.

[0076] pRIB 5

[0077] A long ORF was also identified in the pRIB5 cDNA sequence, extending from the nucleotide in position 3 to the termination codon in position 777. A methionine initiation codon was not present in this ORF indicating that the cDNA was not full length. Searches of the EMBL database with the deduced amino acid sequence of this ORF and also with the nucleotide sequence did not reveal any significant similarities to known sequences. The putative amino acid sequence encoded by pRIB5 does not show significant similarity to other amino acid sequences in the SwissProt database.

[0078] pRIB6

[0079] pRIB6 encodes the C-terminal portion of a polypeptide that shares sequence similarity with the cysteine proteinase family. This group of proteins includes actinidin from kiwifruit, papain from papaya and bromelain from pineapple. The putative protein encoded by pRIB6 shows most similarity to a cysteine proteinase precursor from Arabidopsis thaliana (74% similarity, 60% identity), the expression of which is induced by high salt conditions. Five of the highly conserved residues found in or near the active site of all cysteine proteases are present in the blackcurrant sequence.

[0080] pRIB7.

[0081] pRIB7 contains a long ORF extending from a putative methionine initiation codon at nucleotide 29 to a TAA termination codon at position 860. The ORF encodes a protein of M.sub.r 29,215 and a pl of 7.9. However, a common poly(A).sup.+addition sequence is not present. The pRIB7 ORF was most similar to the yeast mitochondrial protein MRS4, a mitochondrial RNA splicing protein (62% similar and 42% identical at the amino acid level). Hydropathy plots have shown that the MRS4 protein contains potential membrane spanning domains and analysis of the pRIB7 ORF sequence shows that this may also be the case for the blackcurrant polypeptide. The MRS4 protein contains three repeated amino acid sequences of approximately 100 residues and a characteristic highly conserved domain. Such sequence motifs are also seen in a number of mitochondrial carrier proteins.

[0082] RIB 7

[0083] The 5150 nucleotide sequence contains a `TATA box` element at nucleotide 3041 and a putative ATG translational start codon at position 3156. This translational start codon is in the context TTTTCAATGGCG and matches the optimal context consensus sequence (NNANNATGGCT), where N is any nucleotide) proposed by Heidecker and Messing (1986) in all but two positions (these are underlined).

[0084] By comparison with the cDNA sequence, the RIB 7 gene conatins two exons and one intron. The 454 nucleotide intron is located between bases 3927 and 4381. On the basis of the translational start codon being located at position 3156, the putative polypeptide encoded by the RIB 7 gene is composed of 328 amino acids. The deduced amino acid sequnce has been compared with others in the SwissProt database and is most similar to a mitochondrial RNA splicing protein (MRS4:Accession number P32500) from yeast (60.3% similarity and 40.3% identity).

[0085] Southern analysis

[0086] Southern blots of genomic DNA from R. nigrum (cvs Ben Alder, Ben Sarek and Baldwin), R.loganobaccus (Tayberry) and R. idaeus (cv Glen Moy), were hybridised with probes from the RIB genes. Generally, with all these probes, a small number (2 to 4) of hybridising bands were detected by Southern analysis when the genomic DNA was digested with BamHI, EcoRI or HindIII. This indicates that the RIB genes are present in low copy number in the genomes of these diploid species. Blots probed with RIB3 and RIB5 showed that these or similar sequences are not present in the genomes of raspberry and Tayberry as no hybridising bands could be detected on the Southern blots (data not shown). As a control, these blots were stripped and re-probed with a potato .beta.-tubulin probe which gave multiple hybridisation signals with genomic DNA from all the samples that were probed (data not shown).

[0087] Discussion

[0088] On the basis of respiration measurements, blackcurrants do not exhibit a typical climacteric pattern of ripening. Additionally, the large increase in ethylene evolution that commonly accompanies the respiratory climacteric was not detected. Compared with the rate of ethylene production from ripening avocado fruit (internal ethylene levels increase 1 000-fold between the pre-climacteric and climacteric peak) the amount of ethylene produced by blackcurrant fruit was very low. It is not clear which plant growth regulators trigger ripening processes in blackcurrant fruit.

[0089] Irrespective of the plant growth regulators that control ripening in blackcurrant fruit, until now, none of the genes that are differentially expressed during fruit ripening have been isolated. A cDNA library constructed from the green/red stage of ripening was differentially screened with probes from this stage and from green fruit, since genes that are differentially expressed as anthocyanin accumulation commences are good candidates for having an important role in this and other ripening processes. In fact the expression of all five genes corresponding to the isolated cDNAs, continued to increase as ripening progresses and reached a maximum steady-state level in fully ripe, black fruit (FIG. 1). The expression of these genes showed varying degrees of fruit specificity. RIB1 and RIB7 were expressed only at very low levels in non-fruit tissues. The promoters driving the expression of these two genes therefore are good candidates for being fruit specific promoters and therefore suitable for use in manipulating ripening processes in transgenic fruit. RIB3, RIB5 and RIB6 were also expressed in roots leaves and stems. RIB3 exhibited a markedly different expression pattern in stems and roots from plants that had not bome fruit (no detectable expression) compared with plants that had (relatively high steady-state transcript levels). It seems likely that the expression of these genes is highly regulated in a tissue-and developmental-stage specific manner.

[0090] In order to determine the copy number and occurrence of the RIB genes in other soft fruit species, Southern blot analyses were performed. Of the five clones isolated from the cDNA library, three of them, pRIB 1, pRIB6 and pRIB 7 hybridised to DNA from three blackcurrant cultivars, Tayberry and red raspberry. These clones may represent genes that occur widely in soft fruit species. Interestingly, in Southern blots probed with pRIB3 and pRIB5, hybridising bands were only present in lanes containing blackcurrant DNA, suggesting these genes and related sequences are absent in other soft fruit species.

[0091] It was possible to identify tentatively three of the blackcurrant sequences based on similarity searches of databases. Sequences similar to pRIB3, encoding a metallothionein-like protein and pRIB6, encoding a cysteine proteinase have been found previously to be expressed in many plant species. A number of highly conserved amino acid residues, essential for protease activity, are present in the putative blackcurrant sequence.

[0092] The pRIB3 ORF has strong sequence similarity to a number of metallothionein-like proteins that have been isolated previously from plants. It is interesting, that of these proteins, the most similar to the pRIB3 sequence, was isolated from the ripening fruit of kiwifruit. Like pRIB3, high steady-state transcript levels of the kiwifruit gene were detected in ripe fruit. In animals, metallothioneins function to maintain metal ion homeostasis and are involved in metal ion detoxification. Additionally they may provide protection against oxidative stress. Although no similar functions have yet been demonstrated for plant metallothioneins, it is possible that they have similar roles. Indeed plant metallothionein-like proteins have been shown to bind cadmium and copper. However it is unclear at the moment, why the steady-state level of the metallothionein-like protein specific transcript increases in ripe fruit. It is interesting that DNA sequences hybridising to the RIB3 probe on the Southern blot were only present in blackcurrant, and not in raspberry or Tayberry.

[0093] pRIB7 was most significantly similar to a gene that has not been previously found to be expressed in plants, the yeast MRS4 gene. This nuclear gene encodes a mitochondrial RNA splicing protein. Although most similar to the MRS4 gene product, the pRIB7 ORF shares some sequence motifs with a number of mitochondrial carrier proteins such as the phosphate carrier protein and the ADP/ATP translocase. The mitochondrial carrier family is characterised by three tandem repeats of a domain of approximately 100 residues, and a highly conserved region within the repeated domain serves as a signature pattern. This consensus pattern (P-Xaa-[D,E]-Xaa [L, V, A, T]-[R, K]-Xaa-[L,R]-[L, I, V, M, F, Y]) is found three times in the pRIB7 ORF although one amino acid residue in the repeat in the --COOH-domain differs from this consensus pattern (Q in place of L or R). The role of the pRIB7 polypeptide therefore is unknown but it may be related to changes in solute transport across the mitochondrial membrane, reflecting changes in metabolism as fruit ripen. The pRIB1 and pRIB5 ORFs did not show any sequence similarity to known sequences in the EMBL database.

[0094] REFERENCES

[0095] Bevan, M. (1984). Nucl. Acid. Res. 12, 8177.

[0096] Callahan, H., Morgens, P and Walton, E (1989). Hortsci. 24, 356-358

[0097] Fromm, M. E., Taylor, L. P. & Walbot, V. (1985). Proc. Natl. Acad. Sci. USA 82, 5824-8.

[0098] Heidecker, G. and Messing, J. (1986). Ann. Review of Plant Physiology, 37, 439-466.

[0099] Klein, T. M., Wolf, E. D., Wu, R. & Sandford, J. C. (1987). Nature 327, 70-73.

[0100] Lay-Yee, M., Dellapenna, D. amd Ross, G. S. (1990). Plant Physiol. 94, 850-853

[0101] Ledger, S. E. and Gardner, R. C. (1994) Plant Mol. Biol. 25, 877-886

[0102] Martin, C., Prescon, A., Mackay, S., Bartlett, J. & Vrijlandt, E. (1991). Plant J. 1, 37-49.

[0103] Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press.

[0104] Schuch, W., Kanczler,J., Robertson, D., Hobson, G, Tucker, G, Grierson, D, Bright, S, Bird, C. Hortsci 26, 1517-1520. (1991).

[0105] Tesniere, C. & Vayda, M. E. (1991). Plant Mol. Biol. Reptr. 9, 242-251.

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