Method for producing fruiting plants with improved fruit flavour

FIELD OF THE INVENTION
This invention relates to DNA sequences encoding tomato alcohol dehydrogenase II (ADH2) enzyme, and hybrid DNA molecules incorporating such sequences. These hybrid DNA molecules can be used in a method for producing fruiting plants, particularly tomato plants, which produce fruit with improved flavour.
BACKGROUND TO THE INVENTION
In the soft fruit industry (e.g. strawberries, peaches, plums, bananas and tomatoes), substantial losses are incurred during transport, storage and marketing because of the susceptibility of softer fruit to mechanical damage and invasion by microorganisms. To limit such losses, plant breeders have selected lines which are less soft, and for many fruits firm cultivars now dominate national and international trade. Unfortunately, while marketing losses may be lower when fruit is less soft, the firmer lines may have reduced market appeal and also tend to be deficient in flavour.
The flavour of ripe fruit is a function of a number of components including sweetness (sugars), sourness (organic acids) and volatile compounds. A large number of volatiles have been identified as contributing to flavour and aroma (1-8). In ripening tomato fruit, approximately 400 volatile compounds have been found in the ripening fruit (5) but of these only a small number have been identified as important components of flavour and aroma. These include Z-3-hexenal, Z-3-hexenol, 2-E-hexenal, hexanal, 3-methylbutanal, 3-methylbutanol, .beta.-ionone, 1-penten-3-one, 2-isobutylthiazole, 6-methyl-5-hepten-2-one, methyl salicylate, geranylacetone, E-2-heptenal, isobutyl cyanide and 2-phenylethanol (9,10,3), Flavour volatiles are formed by several different pathways such as the deamination and decarboxylation of amino acids (3-methylbutanal (11)) and lipid oxidation of unsaturated fatty acids (hexanal and the hexenals (12)) (FIG. 1).
The tomato alcohol dehydrogenase II enzyme (ADH 2; alcohol:NAD.sup.+ oxidoreductase; EC 1.1.1.1) has been implicated in the interconversion of the aldehyde and alcohol forms of these volatiles (13,14). The enzyme has also been shown to accumulate in the fruit during ripening and to have appropriate substrate specificities in vitro (14-16). Further, the activity of ADH2 in ripening tomato fruit appears to be a function of the softness of the fruit. For example, in the typically firm tomato fruit of commercial cultivars, ADH2 activity is low and this may contribute to the perception of poor flavour in these fruit.
The present inventors have sought to improve tomato fruit flavour by increasing the ADH2 activity in ripening tomato fruit through the introduction of an additional ADH2-encoding DNA sequence(s) into the parent plants. This involved, firstly, isolating a cDNA (see FIG. 1A) encoding ADH2 and then transforming tomato plant explants. The present inventors have now produced a number of tomato plants so transformed and have demonstrated significant changes to the ratios of 2-carbon aldehydes to alcohols. Whether directly or indirectly, these changes have altered the aroma and/or flavour of the fruit produced by transformed plants. Thus, transformed fruit plants with altered ADH2 activity may have direct commercial value and provide valuable stocks for breeding programs.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a method for producing a transformed fruit plant which produces ripened fruit with improved flavour as compared to ripened fruit produced by its parental plant, said method comprising;
stably transforming explants of said parental plant with at least one DNA molecule comprising a nucleotide sequence encoding alcohol dehydrogenase II (ADH2) or a functional portion thereof operably linked to a suitable promoter sequence, such that transformed fruit plants grown from said explants express said DNA molecule(s) in fruit tissue, and
selecting a transformed fruit plant which produces ripened fruit with improved flavour as compared to ripened fruit produced by said parental plant.
By "improved flavour" we refer to any change in the organoleptic qualities of the ripened fruit which is perceived by a statistically significant number of persons within a randomly selected test population as an improvement in fruit flavour.
By "functional portion" we refer to any portion of ADH2 which possesses activity which, directly or indirectly, effects interconversion of the 5-carbon and/or 6-carbon alcohols and aldehydes. Such functional portions, which may be identified by routing deletion analysis/mutagenesis methods, may possess lesser or greater activity than the full-length ADH2.
The transformed plants produced according to the method of the invention show altered ADH2 activity and, consequently, demonstrate altered ratios of aldehyde/alcohol volatiles. It is most preferred that the transformed plants show increased ADH2 activity in ripening fruit resulting in increases in the ratios of alcohols/aldehydes in ripened fruit as compared to the ratios of alcohols/aldehydes of ripened fruit produced by the parental plant (which may show low or substantially nil ADH2 activity).
From taste tests conducted on fruit from transformed tomato plants (described hereinafter), the present inventors have determined that increases in ADH2 activity leading to an increase in the amounts of the volatile alcohols hexanol and Z-3-hexenol in ripened fruit, results in improved fruit flavours. Furthermore, the present inventors have recognised that 1 to 5 (approximate) fold increases in the amounts of these volatiles over that typical of control fruit, favourably influences the flavour of the ripened fruit.
Thus, the step of selecting a transformed fruit plant which produces ripened fruit with improved flavour, preferably involves selecting a transformed fruit plant which produces fruit which, when ripened, show a 1 to 10 fold increase in the amounts of the volatile alcohols hexanol and Z-3-hexenol as compared to ripened fruit of said parental plant.
Preferably, the ripened fruit of the selected transformed fruit plant show a 1 to 5 fold increase in the amounts of the volatile alcohols hexanol and Z-3-hexenol. More preferably, the ripened fruit of the selected transformed fruit plants who a 1.0 to 5.0 fold increase in the amount of hexanol and a 1.5 to 4.0 fold increase in the amount of Z-3-hexenol.
The DNA molecule mentioned in the first aspect may comprise an ADH2 nucleotide sequence corresponding to, or derived from the ADH2 nucleotide sequence of the parental plant species/strain. However, more preferably, the DNA molecule comprises an ADH2 nucleotide sequence corresponding to, or derived from the ADH2 nucleotide sequence of a tomato plant species/strain. Most preferably, the DNA molecule comprises an ADH2 nucleotide sequence which substantially corresponds to the cDNA or genomic sequence shown in FIGS. 1a and 1b.
Suitable promoter sequences for use in the present invention include inducible promoters such as the tomato ADH2 promoter (described in International (PCT) patent application No. PCT/AU93/00654 (WO 94/13797)), or endopolygalacturonase (PG), 1-aminocyclopropane-1-carboxylic acid oxidate or E8 promoters. It is, however, preferred to use a constitutive promoter such as the CaMV 35S promoter or one of the family of SCSV (Subterranean Clover Stunt Virus) promoters contained in the pPLEX vectors (International (PCT) patent application No. PCT/AU95/00552 (WO96/06932)).
Further, the DNA molecule encoding the ADH2 or a functional portion thereof may include enhancer elements (e.g. from the octapine synthase gene of Agrobacterium tumefaciens--Ellis, J. G. et al., 1987 EMBO J. 6:11-16) or multiple copies of an anaerobic response element (ARE) (Olive, M. R. et al., 1990 Plant Mol. Biol. 15:593-604).
The plants may be transformed such that they include an additional copy of the ADH2 gene or, alternatively, the native ADH2 gene may be replaced by homologous recombination with a recombinant ADH2 gene which is expressed at modified levels (e.g. through the use of a stronger promoter sequence). In some applications, it may be preferred to transform the parental plant with multiple copies of the said DNA molecule.
Preferably the DNA molecule(s) is stably inserted into the plant genome and will be transferred, via the seed or by clonal propagation, to subsequent generations. Transformation may be via, for example, Agrobacterium tumefaciens-mediated transfer or the well known DNA particle gun method.
The pathways of synthesis of volatiles such as the 6-carbon aldehydes and alcohols, some 5-carbon aldehydes and alcohols (for example 3-methylbutanal/ol), and simple aldehydes and alcohols such as ethananl/ol, appear to be generally associated with flavour production in ripening fruit (cf References 1-15 and general reference Biogeneration of Aroma (1986), Parliment T H and Croteau R (eds), ACS Symposium Series 317, American Chemical Society, Washington D.C.). In all these pathways, ADH plays a role in interconversion of the aldehydes and alcohols, the balance of which is important to perceived aroma and flavour. It is therefore anticipated that the method of the invention is generally applicable to fruit plants. It is, however, preferable that the fruit plant is a soft fruit plant such as a strawberry plant, peach tree or plum tree. However, most preferably, the fruit plant is a tomato plant.
The method of the invention thus permits suitable "slow ripening" or "slow softening" fruit cultivars to be transformed with a DNA molecule to enable the production of the aroma/flavour enhancing ADH2 or functional portion thereof. Where a constitutive promoter is used, production of the ADH2 or functional portion thereof would be enhanced in all tissues of the plant including ripening fruit. Where an inducible promoter is used, production of the ADH2 or functional portion thereof can be controllably regulated by, for example, selected temperature or gaseous treatments applied at a late stage in the fruit distribution and marketing chain.
In a specific case, benefits can be envisaged in modifying a slow-ripening tomato cultivar by incorporation of a DNA molecule comprising the ADH2 promoter from tomato, suitable enhances, and the tomato ADH2-encoding nucleotide sequence. During the early stages of distribution after harvesting advantage could be taken of the cultivar's inherent resistance to physical damage, but the promoter could be activated, at a point close to sale, to bring out a flavour akin to that of a fast ripening cultivar.
Northern analysis of ripening fruit of two tomato cultivars has shown that the mRNA for ADH2 is present in low abundance in mature green fruit and increases in abundance through ripening and, particularly, late in ripening, In fruit pericarp tissue exposed to atmospheres with 3% (v/v) oxygen, the ADH2mRNA level increases to a maximum within 8-16 hours, and returns to the basal level within 16 hours of return to air. The mRNA level was sensitive to the oxygen level in the atmosphere, increasing 20 fold in 12% (v/v) oxygen and 100 fold in 3% oxygen. These oxygen levels may reflect appropriate levels for the induction of some inducible promoters in commercial amounts of fruit harvested from transformed plants. Alternative methods for induction may include UV light, low temperatures (e.g. 0.degree.-10.degree. C.) and exposure to some organic acids or gaseous CO.sub.2.
In a second aspect, the present invention provides a fruit plant transformed with at least one DNA molecule comprising a nucleotide sequence encoding alcohol dehydrogenase II (ADH2) or a functional portion thereof operably linked to a promoter sequence suitable for expression of the said DNA molecule(s) in fruit tissue, such that the transformed fruiting plant produces ripened fruit with improved fruit flavour as compared to ripened fruit produced by said parental plant.
In a third aspect, the present invention provides reproductive material derived or produced from a fruit plant according to the third aspect.
Such reproductive material may include seed, cuttings or any other part or product of the plant from which another plant can be produced.
In a fourth aspect, the present invention provides fruit produced by a fruit plant according to the second aspect.
The term "substantially corresponds" as used herein in relation to the nucleotide sequence encoding tomato ADH2, is intended to encompass minor variations in the DNA sequence which due to degeneracy in the DNA code do not result in a significant change in the encoded protein. Further this term is intended to encompass other minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded protein.
The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated component or feature with or without the inclusion of a further component or feature or group of components or features.
The invention will now be further described by way of the following, non-limiting examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
FIG. 1a provides the nucleotide sequence of the tomato ADH2 cDNA, pTADH2 (SEQ ID NO:1).
FIG. 1b provides the nucleotide sequence of the tomato ADH2 gene (SEQ ID NO:3).
FIG. 1c is a digram of the genomic clone lambda 2A3 indicating the location of exons 1 to 9 of the ADH2 gene, the relative positions of the EcoR1 fragments and the 5' region comprising the promoter. The region shown dashed has not been sequenced.
FIGS. 2a-c provide diagrammatic representations of constructs PJR-ADH and PRD-ADH.
Construct PJR-ADH: The tomato ADH2 cDNA was inserted between a CaMV 35S-promoter and a Nos 3' termination sequence (B) ligated into the T-DNA region of the binary vector PRJ (A) described by Smith et al., (21) and derived from Bin 19 (22,23).
Construct PRD-ADH: The tomato ADH2 cDNA was inserted between the promoter and 3' regions of the tomato polygalacturonase gene (C) ligated into the T-DNA region (see also A) in the binary vector PRD. The PRD vector is described by Nicholass et al. (24) and, as with the PJR vector, was derived from Bin 19.
FIG. 3 provides graphical results of ADH specific activities in fruit from transformed plants. ADH activities were determined in pericarp from breaker, 2 days post breaker (Breaker+2) or 7 days post breaker (Breaker+7) fruit from transformed tomato plants. The top two histograms show results for plants transformed with the PJR-ADH construct containing the tomato ADH2 cDNA coupled with the constitutive CaMV 35S-promoter. The bottom two histograms show results from plants transformed with the PRD-ADH construct containing the tomato ADH2 cDNA coupled with the tomato fruit ripening specific PG-promoter. Results are arranged in order of increasing activity in Breaker+7 fruit (right hand histograms), and are indicated in the same order in the left hand histograms. Three fruit were averaged for each data point. Ac in each histogram is a control mean.+-.S.D. obtained from 3 fruit (each control point) from separate, untransformed plants.
FIG. 4 provides graphical results of ADH specific activity in ripening fruit from transformed plants. ADH activities in pericarp tissue are shown for ripening fruit from three transformed plants. Plant C13 was transformed with the ADH2 cDNA coupled with the fruit ripening specific PG-promoter. Plants C20 and C23 were transformed with constructs containing the CaMV 35S-promoter. Control, untransformed fruit data are shown as open histograms. Data from transformed plants are shown as closed histograms. Three fruit were averaged for each data point.
FIG. 5 shows the spectrum of volatiles isolated from headspace above macerated whole fruit tissue. Typical spectrum of compounds detected by GC analysis of headspace volatiles.
FIG. 6 provides graphical results of the aldehyde:alcohol ratios for a number of headspace volatiles from fruit of three ADH-modified plants, compared with ratios for control plants. Ratios of aldehydes to alcohols were determined for individual fruit from each plant and means.+-.S.D. were calculated. The number of fruit sampled in each case is shown in Table 1. Control fruit ratios were determined from pooled ratios of all the control fruit, AC#1, AC#2 and AC#4 (total n=10). Hexenal/ol (1) is the ratio of Z-3-hexenal:Z-3-hexenol. Hexenal/ol (2) is the ratio of Z-3-hexenal+E-2-hexenal:Z-3-hexenol. For statistical analysis the plants were grouped into the four genotypes: C,20, C13, C23 and untransformed controls. The data were non-normal and non-orthogonal. Analysis was therefore carried out on log transformed data to give approximately normality. The fixed effects of the genotypes were then measured using restricted maximum likelihood (REML--from the GENSTAT package). The test statistic for the REML analysis is a .chi..sup.2 on 3 degrees of freedom. The critical values of .chi..sup.2 are 7.815 for P=0.05 and 11.345 for P=0.01. The probabilities of differences between all four groups being significant are as follows: Hexanal/ol (.chi..sup.2 =23.1) P<0.001; Z-3-hexenal/ol (.chi..sup.2 =11.02) P<0.01; Hexenal(3Z+2E)/Hexenol 3Z(.chi..sup.2 =10.94) P<0.01; Z-3-hexenal/E-2-hexenol (.chi..sup.2 =5.87) ns; Octenal/ol (.chi..sup.2 =8.39) P<0.05; Citral/Geraniol (.chi..sup.2 =0.98) ns.

DETAILED DESCRIPTION OF THE INVENTION
Example 1
Isolation of a cDNA Encoding Tomato ADH2
Poly A.sup.+ RNA was isolated from pericarp tissue of tomato (cv. de Ruiter 83G38) fruit, 9 days after the first appearance of colour (Breaker+9). A cDNA library was constructed from the poly A.sup.+ RNA and was cloned into lambda Gem 11 using the protocol or Promega Ltd. The library was screened by hybridisation with a .sup.32 P-labelled fragment of tomato genomic DNA with sequence homology to ADH genes of pear (Llewellyn et al. J. Mol. Biol. 195:115-127, 1987) potato (Matton et al., Plant Mol. Biol. 14: 775-783, 1990) and maize (Dennis et al. Nucleic Acids Res. 13: 727-743, 1985). Four positive colonies were isolated, phage DNA was purified and cDNA insets were transferred from the phage vector to a plasmid vector pGem 11 (Promega) for sequencing, Sequencing was carried out by the dideoxy-method of Sanger et al. PNAS, USA 784: 5463-5467 (1977) on double stranded DNA using pUC universal and reverse primers and oligonucleotide primers to extend known sequence. The four cDNAs had identical 5' sequences and identical open reading frames encoding a protein of 379 amino acids.
The cDNA hybridised strongly to a 1.8 kb RNA species in RNA from ripe but not from unripe tomato fruit. Similarly, the cDNA hybridised to RNA from tomato seedling roots kept anaerobic overnight, but not to RNA from aerobic roots. In contrast, a BGl11 fragment of tomato genomic of cDNA, which contains regions encoding the tomato ADH1 isozyme and hybridises strongly to ADH1 mRNA but weakly to ADH2 mRNA (Wisman et al. Mol. & Gen. Genet. 226:120-128, 1991), did not hybridise to RNA from either ripe fruit or anaerobic root tissue. Thus it was concluded that the cDNAs isolated from the ripening fruit library, encode a tomato ADH2 enzyme.
The nucleotide sequence for the cDNA encoding the ADH2 mRNA is provided at FIG. 1a.
The 5' 843 nucleotides of the cDNA including sequence encoding 250 amino acids of the ripening associated ADH2 enzyme provides a particularly useful probe by virtue of its location adjacent to the promoter in the genomic sequence.
Example 2
Isolation of Tomato ADH2 Genomic DNA
Isolation of Tomato Leaf DNA
Young leaves of tomato plants (cv. de Ruiter 83G38) were harvested and snap-frozen in liquid nitrogen. The leaves were ground to a fine powder under liquid nitrogen and DNA was isolated by the method of Thomas et al. Theor. Appl. Genetic 86: 173-180 (1993).
Construction of Genomic Library
Aliquots of high molecular weight tomato genomic DNA were digested with varying amounts of Mbol restriction endonuclease for 1 hour at 37.degree. C. and digestion was stopped by heating the samples at 75.degree. C. for 10 minutes. The samples were fractionated on a 0.5% agarose gel and the mean size and spread of the digested DNA was determined. 20 .mu.g of genomic DNA as digested with Mbol enzyme, under conditions designed to give a fragment size range of between 10 kb and 20 kb, and the reaction was stopped as before. The digested fragments were partially end-filled with dATP and dGTP and were ligated into Lambda GEM-11 Xhol Half-site arms (Promega Corp.), according to the Promega protocols. Three ligations were carried out with arms:fragment ratios of: 0.5 .mu.g:0.25 .mu.g; 0.5 .mu.g:0.5 .mu.g; 0.5 .mu.g:0.75 .mu.g.
The ligated DNA samples were packaged according to Hohn, Methods in Enzymology 68:299-309 (1979) and titred, giving average titres of 1.8.times.10.sup.5 pfu/.mu.g arms. The three libraries were pooled.
Library Screening
E. coli, strain KW251 cells, were infected with 2.times.10.sup.5 phage, were spread on six, 10 cm.times.10 cm Luria agar plates (33,000 pfu/plate) and were incubated at 37.degree. C. for 6 hours until small phage plaques had developed. Lifts were taken off the plates onto Biotrace NT nitrocellulose membranes (Gelman Sciences), and the membranes were prehybridised, hybridised and washed according to the Gelman protocols. Dried membranes were exposed to X-ray film (Fuji, RX) with an intensifying screen (Du Pont--Cronex, Lightning-Plus) at -70.degree. C. Hybridisation probes were labelled with .sup.32 p dATP by oligopriming (Feinberg and Voglestein, Anal. Biochem 132: 6-13, 1983).
Subcloning and Sequencing
The genomic DNA insert of lambda clone 2A3 was digested with EcoR1 restriction enzyme and the resulting fragments subcloned by ligation into the plasmid vector pUC18 (Yanisch-Peron et al., Gene 33: 103-119, 1985) by standard methods.
Double stranded sequencing was carried out by the enzymatic chain-termination method of Sanger et al. Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977), using universal and reverse M13 primers based on determined sequence.
PCR amplification of the 5' region of the ADH2 gene was carried out on a tomato genomic DNA template using oligonucleotide primers to regions of the ADH2 cDNA as follows:
5' Primer #1078 5' CCACTGCCTCAACTGAG 3' (SEQ ID NO:22)
3' Primer #1057 3' CGACCATTCGGTAATCA 5' (SEQ ID NO:23)
PCR reactions were carried out in 50 .mu.l reactions containing 16.6 mM (NH).sub.4 SO.sub.4, 67 mM Tris-HCl pH 8.8, 6.7 mM EDTA, 2 mM MgCl.sub.2, 0.15% Triton X-100, 200 .mu.M dNTPs and 200 .mu.g/.mu.l gelatin, with primers added at 22 pmol/.mu.l, 100 ng tomato genomic DNA and 2.5 units of Taq polymerase. Reactions were carried out as follows:
1st cycle
94.degree. C. 5 mins.
45.degree. C. 1 min.
72.degree. C. 1 min.
Followed by 38 cycles as follows:
94.degree. C. 1 min.
45.degree. C. 1 min.
72.degree. C. 1.5 mins.
The PCR product was ligated into pBluescript KS.sup.+ vector (Stratagene) linearised at the EcoRV site and T-tailed according to the procedure of Marchuk et al. Nucleic Acids Research 19.1154 (1990).
Results
Duplicate lifts were taken of six plates containing a total 2.times.10.sup.5 pfu of a phage library of tomato genomic DNA. The lifts were screened by hybridisation with two probes. The first probe, pADHCR1 was designed to be specific to the 5' end of the tomato ADH2 gene which is expressed in ripening fruit. pADHCR1 was generated by PCR amplification of the genomic sequence bounded by primers #1078 and #1057 defined by the sequence of the ADH2 cDNA and spans exon 1, intron 1 and 34 nucleotides of exon 2 of the ADH2 gene. The second probe, pADH2-3' constitutes the entire ADH2 cDNA 3' of the EcoR1 site at nucleotide 290 on the cDNA (nucleotide 382 on the genomic DNA--Table 1).
Screening with the 5' specific probe, pADHCR1, gave 28 positives. Screening with the 3' specific probe, pADH2-3' gave 20 positives. Only one lambda phage plaque, 2A3, hybridised with both probes, and this plaque was isolated and purified for further characterisation.
EcoR1 digestion of DNA from the lambda genomic clone, 2A3, generated a number of fragments including the left and right arms of the lambda vector and three fragments of insert DNA with sizes of 7.8 kb, 1.4 kb and 1.2 kb. Subsequent subcloning revealed a further insert fragment of 63 bp in size. The EcoR1 fragments were subcloned into pUC18 and were sequenced, allowing alignment of the fragments to be determined (FIG. 1). The 1.4 kb subclone, pADH2-1.4 was found to contain the 5' end of the ADH2 gene, from the EcoR1 site at nucleotide 290 in the cDNA and including introns 1 and 2. The clone includes the transcription start site and extends a further 800 bp upstream encompassing the gene promoter.
The sequence of the clone 2A3 including the ADH2 promoter and the protein-encoding region (including introns) is provided at FIG. 1b. The ADH2 gene has an overall length of 2334 bp from transcription site to poly A addition site.
Example 3
Production of Transformed Tomato Plants Expressing ADH2
Construction of ADH transgenes
The ADH cDNA used for the construction of the ADH transgene, was modified by PCR using the tomato ADH2 cDNA pTADH2 as template (20). The 1.5 Kb ADH2 cDNA includes a 5' untranslated region of 91 bp upstream of the ATG start codon and a 320 bp untranslated region 3' of the TAA stop codon. The integrity of the sequences was confirmed by sequence analysis after modification. Two ADH2 transgenes were constructed. The first construct, PJR-ADH (FIG. 2), consists of the ADH2 cDNA ligated into the binary vector PJR1. The PJR1 vector, described in Smith et al. (21), is a derivative of Bin 19 (22,23) and contains the constitutive CaMV 35S-promoter and the Nos 3' terminator sequence. The second construct, PRD-ADH (FIG. 2), consists of the ADH2 cDNA ligated into the binary vector PRD. The PRD binary vector was derived from Bin 19 and contains the tomato fruit specific PG-promoter (4.8 Kb) and PG-terminator (1.8 kb), (24). The ADH2 cDNA fragment was inserted into the Bsa1 and Spe1 sites of the PRD binary vector.
Tomato Transformation
The constructs PJR-ADH and PRD-ADH were transferred from E. coli DH5.alpha. to Agrobacterium tumefaciens LBA4404 by triparental mating via E. coli HB101 and pRK2013. Tomato (Lycopersicum Esculentum Mill cv. Ailsa Craig) transformations were carried out according to Bird et al. (25). Southern analysis of DNA from young leaf tissue was used to estimate the number of NptII and ADH2 inserts integrated into each plant and plants were allowed to grow to maturity and set fruit. Fruit were harvested at first colour change (Breaker, Br) or two days post Breaker (Br+2) and 7 days post breaker (Br+7) and ADH activity in the pericarp tissue was determined.
Genomic Southern Blot Analyses
Genomic DNA was extracted from young leaves by the method of Thomas et al. (26). 7 .mu.g of Hind III digested genomic DNA was fractionated by electrophoresis on 0.7% agarose-TBE gels. The DNA was transferred onto nylon membrane (Zetaprobe, BioRad) as described by the manufacturer. The number of transgene(s) inserted was determined by hybridisation using 32p labelled probes corresponding to the NptII and ADH2 cDNA regions of the transgene. Hybridisation was according to the procedure recommended for Zetaprobe, at 65.degree. C. for 16 h. The filters were washed twice with 2.times.SSC, 0.1% SDS at 65.degree. C. for 15 min each, followed by two washes of 10 min with 0.1.times.SSC, 0.1% SDS at 65.degree. C. The membranes were blotted dry and autoradiographed.
Extraction and Assay of ADH
Tomato pericarp tissues, and whole fruit tissues were extracted and assayed according to the method of Longhurst et al. (15). Protein concentration was measured using a protein quantification Kit II (BioRad).
Volatile Analyses
Fruit were harvested 7 days post breaker. 10 g of pericarp tissue was taken from freshly harvested fruit, sliced and then briefly macerated using a Polytron PT2000 homogenizer (Kinematica AG, Switzerland). The slurry was allowed to stand at room temperature for exactly 3 min after which 3.3 g solid CaCl.sub.2 were added to inhibit further enzyme activity. 1 .mu.l of uniformly labeled, deuterated Hexanol (80 nmoles/.mu.l) were added as an internal standard. 6.6 g of the mixture was transferred to a 20 ml headspace vial which was sealed with a silicon/teflon septum. The vial was incubated at 40.degree. C. for 30 min. Sampling of the headspace was carried out by insertion of a Solid Phase MicroExtraction (SPME, Supelco) fiber (65 .mu.m Carbowax-Divinylbenzene), for 30 min while continuing incubation of the vial at 40.degree. C. The absorbed sample was analysed by gas chromatography on an HP-GC series 6809 fitted with a capillary DB-wax column, 30 m.times.0.25 mm ID.times.0.25 .mu.m) and individual peaks were identified by mass spectrometry. Peak areas were measured by integration and were normalized against the internal deuterated hexanol standard.
Tasting Trial
Tomatoes from each treatment were harvested at between 7 and 9 days into ripening (Breaker+7-9, where Breaker is first colour change). The tomatoes were matched on the basis of size. Each tomato was quartered and the locular tissue, containing the seeds, removed. Each quarter was used for each of the four attributes (ripe flavour, green flavour, sweetness and acidity) by a single panelist.
Four sets of samples were presented to each panelist, each in a different random order. Each set was used to rank only one of the attributes. The order of assessment of the attributes was the same for each panelist (ripe flavour, green flavour, sweetness and acidity).
Panelists were asked to rank the samples on each of the four attributes. Samples with the greatest intensity of the attribute were given a rank of 1, whilst the least intense was ranked 4.
An orthogonal latin square design was used to balance out carry-over effects. This design required 12 assessments. However, due to insufficient samples, only 11 were made in this trial.
Panelists
Seven of the eleven panelists were trained oenologists with extensive experience in wine flavour and acid balance assessment (mean experience=10 years). One panelist whilst not formally trained had three years regular experience in assessing these attributes in wine. Three panelists were CSIRO employees. Whilst all panelists knew the general purpose of the tasting, is a comparison of flavour modified tomatoes, none were aware of the exact nature of the samples.
Results
Transformation with ADH2 constructs and initial screening
Tomato explants were transformed with constructs containing the tomato ADH2 cDNA as described in FIG. 2, and 30 transformed plants from each experiment were selected.
Two types of transformation construct were used. Both contained the tomato ADH2 cDNA in a sense orientation relative to the construct promoter. In one set of experiments the CaMV 35S-promoter was used to provide constitutive expression of the cDNA. In the other the tomato polygalacturonase gene promoter (25,26) (PG-promoter) was used to provide fruit ripening-specific expression of the cDNA.
Constitutive expression of the introduced cDNA(s) resulted in both enhanced and inhibited accumulation of the ADH2 enzyme in the ripening fruit (FIG. 3). Enhanced levels of accumulation were most evident in Breaker (Br; first colour change) fruit but a number of fruit continued to show significantly (P<0.05) enhanced levels at Br+7 (Breaker+7 days). About 40% of the plants analysed had fruit in which ADH2 expression was completely inhibited, presumably by co-suppression.
In the second set of experiments, in which the introduced cDNA(s) were expressed in a fruit ripening-specific manner, ADH activities at Br+2 varied from approximately that in control fruit to a 6-7 times control levels, while activities at Br+7 varied from approximately control fruit levels to 2-3 times control levels (FIG. 3).
ADH activities during ripening
ADH activity was measured in pericarp, locular and whole fruit tissues. Activity was found to vary in the different tissues but was highest in pericarp tissue (data not shown). Because of this, and because of the uniform composition of the tissue, only pericarp activities are presented here.
A small number of plants showing significant modifications in pericarp ADH activities were taken for more extensive analysis during fruit ripening and for analysis of fruit flavour volatiles. Plants C20 and C23 contained constructs with constitutive expression of the inserted cDNA(s). Fruit from these plants showed respectively either enhanced ADH activity or minimal activity in fruit throughout ripening relative to levels in fruit from untransformed control plants (FIG. 4). Plant C13 contained ADH2 cDNAs regulated by the fruit ripening-specific PG-promoter. Fruit from the C13 plant showed significantly enhanced levels of ADH activity which continued to increase up to 7 days post breaker then declined. In a separate series of experiments, ADH levels and volatiles were determined for breaker+7 fruit from these plants and a number of other transformed plants. These data are shown in Table 1.
Analysis of volatiles
A number of the transformed plants were selected for analysis of volatiles produced in their fruit. Solid Phase Micro Extraction (SPME) was used as a quick and convenient method for measuring headspace volatiles from individual fruit. This method revealed some 15 major peaks of volatiles with many minor peaks (FIG. 5). A number of the volatiles identified in FIG. 5 are amongst those considered to be important to flavour and aroma development. Of particular interest were the hexanal/ol and hexenal/ol compounds, peak areas of which are listed in Table 1, together with areas of some other aldehydes and alcohols detected.
Comparison of areas of individual peaks between tissue from control, high ADH and low ADH fruit showed, in general, little variation. Peak areas of the aldehydes Z-3-hexenal and E-2-hexenal did not vary greatly between the various plants. Similarly the areas of the aldehyde hexanal varied little between plants except in the case of the C13 plant where it was lower, but not significantly different to controls (Table 1). In contrast, levels of the alcohols, hexanol and Z-3-hexenol, did show significant variation between plants with differing ADH levels (Table 1), while E-2-hexenol was not detected in any of the fruit.
TABLE 1__________________________________________________________________________Number of introduced genes, ADH specific activites and relative abundances of some volatiles in fruit from transformed and control plants. Constitutive promoter PG-prom. Controls Constitutive promoter High-ADH High-ADH untransformed Low-ADH C7 C14 C20 C13 AC#1, 2 & 3 C4 C11 C23__________________________________________________________________________Estimated 1/1 1/1 2/3 7/7-8 -- 3/4 2-3/3 5/6 number of genes inserted (Adh2/NptII) Number of fruit n = 4 n = 3 n = 5 n = 3 n = 10 n = 2 n = 4 n = 3 sampled ADH specific 0.67 .+-. 0.16* 0.56 .+-. 0.04.sup.ns 0.76 .+-. 0.15** 1.26 .+-. 0.31*** 0.375 .+-. 0.15 0.015 .+-. 0.02.sup.ns 0.006 .+-. 0.01** 0.011 .+-. 0.02* activity (U/mg protein) Hexanal 8.48 .+-. 1.60.sup.ns 9.13 .+-. 0.31.sup.ns 9.94 .+-. 1.98.sup.ns 6.95 .+-. 0.89.sup.ns 9.43 .+-. 1.44 9.65 .+-. 0.88.sup.ns 8.45 .+-. 0.44.sup.ns 10.09 .+-. 2.27.sup.ns 3-hexenal Z 8.42 .+-. 1.07.sup.ns 7.28 .+-. 0.40.sup.ns 7.73 .+-. 1.28.sup.ns 7.67 .+-. 1.42.sup.ns 7.76 .+-. 1.57 9.44 .+-. 1.51.sup.ns 7.57 .+-. 0.72.sup.ns 8.71 .+-. 2.46.sup.ns 2-hexenal E 14.38 .+-. 3.24.sup.ns 13.57 .+-. 1.76.sup.ns 15.99 .+-. 1.72.sup.ns 12.92 .+-. 2.78.sup.ns 12.37 .+-. 2.03 13.17 .+-. 0.49.sup.ns 15.88 .+-. 1.42.sup.ns 10.82 .+-. 3.23.sup.ns Hexanol 0.25 .+-. 0.09.sup.ns 0.40 .+-. 0.05.sup.ns 0.52 .+-. 0.17*** 0.29 .+-. 0.08.sup.ns 0.22 .+-. 0.07 0.029 .+-. 0.04.sup.ns 0.04 .+-. 0.02* 0.02 .+-. 0.002* 3-hexen-1-ol Z 3.08 .+-. 0.28.sup.ns 4.12 .+-. 0.79.sup.ns 6.02 .+-. 1.58*** 4.48 .+-. 1.27* 2.51 .+-. 0.46 0.25 .+-. 0.15* 0.065 .+-. 0.08*** 0.057 .+-. 0.05** Geraniol 0.72 .+-. 0.34 0.71 .+-. 0.16 0.62 .+-. 0.14 0.49 .+-. 0.10 0.89 .+-. 0.25 0.88 .+-. 0.09 0.6 .+-. 0.09 0.92 .+-. 0.15 2-octenal E 0.43 .+-. 0.01 0.50 .+-. 0.09 0.57 .+-. 0.06 0.45 .+-. 0.03 0.61 .+-. 0.09 0.51 .+-. 0.07 0.52 .+-. 0.05 0.54 .+-. 0.20 1-octen-3-ol 0.09 .+-. 0.01 0.07 .+-. 0.02 0.11 .+-. 0.02 0.08 .+-. 0.01 0.10 .+-. 0.03 0.11 .+-. 0.03 0.10 .+-. 0.02 0.09 .+-. 0.03 Citral 0.30 .+-. 0.10 0.90 .+-. 0.14 0.69 .+-. 0.28 0.38 .+-. 0.10 0.70 .+-. 0.22 0.6 .+-. 0.01 0.35 .+-. 0.07 0.44 .+-. 0.15__________________________________________________________________________ Number of inserted genes was estimated by quantitating the hybridisation to genomic DNA of probes for Nptil and for Adh2 cDNA. Headspace volatiles were collected by SPME absorption, and analysed by GC/MS. Peal areas were determined by integration and were normalized against the area of a deuterated hexanol standard introduced during maceration of the tissue. Numbers of fruit sampled are indicated (n =). Averaged ADH activites from pericarp tissue and volatile abundances are derived from the same set #o fruits, and were derived separately from the data presented in FIG. 3. Statistical comparisons, where shown, were obtained using TukeyKramer Comparisons Test (GraphPad InStat package) to compare data in the same row. . . Data from individual transformed plants were compared with poole data from the control plants: ns -- not significantly different to fruit from control plants (P > 0.05); * -- significantly different to fruit fro control plants at P < 0.05; ** -- P < 0.01; #*** -- P < 0.001.
Ratios of the fruit aldehydes to alcohols have been calculated for the plants C20, C13 and C23 and are shown plotted as histograms in comparison with ratios in control fruit (FIG. 6). A statistical comparison of the ratios was also carried out, with the probability of each ratio differing between the four classes of plant, C20, C13, C23 and control, being given in the legend to FIG. 6. As Z-3-hexanal isomerises to E-2-hexenal, the ratio of hexenal (3Z+2E):hexenol (3Z) has been included. Hexanal; hexanol, Z-3-hexenal:Z-3-hexanol and the combined hexenal (3Z+2E):hexenol (3Z) ratios are significantly higher in fruit from the low ADH plant C23 than in fruit from the control plants. In some fruit from C23, no Z-3-hexenol was detectable, resulting in aldehyde to alcohol ratios of infinity. In fruit from the high-ADH plants, C20 and C13, the ratios of aldehyde:alcohol are significantly lower than those of the control fruit.
Included in FIG. 6 are ratios of Octenal:Octenol and Citral:Geraniol, (trans-3,7-dimethyl-2,6-octadien-1-al:trans-3,7-dimethyl-2,6-octadien-1-ol). Ratios of those aldehydes and their alcohols varied from plant to plant but did not show any clear trend related to ADH activity.
Taste characteristics
Results of the taste trial are provided at Table 2. Of the eleven (11) panellists involved in the trial, ten (10) selected the fruit from the constitutive high-ADH plant C20 as having a greater intensity of ripened fruit flavour than the other fruit sampled. This result is statistically significant (0.2% probability of error). The fruit from the constitutive low-ADH plant C23 was generally considered to be bland in flavour, whilst the fruit from the fruit-specific high ADH plant C13 was generally considered as having enhanced aroma but not flavour.
TABLE 2__________________________________________________________________________ C23 C13 fruit C20 Control constitutive specific high- constitutive untransformed low-ADH ADH high-ADH P LSD 5%__________________________________________________________________________Ripe tomato 28.sup.b 34.sup.b 34.sup.b 14.sup.n 0.02 10 flavour Green tomato 28.sup.d 25.sup.u 23.sup.a 34.sup.n 0.288 10 leaf character Sweetness .sup. 24.sup.ab 32.sup.b 36.sup.b 16.sup.h 0.014 10 Acidity 30.sup.a 26.sup.a 29.sup.a 25.sup.d 0.819 10__________________________________________________________________________ Samples with greatest intensity of attribute were given a ranking of 1, while those with least were ranked 4. Low rank sums imply a high intensity of that attribute. The lowest rank sum for Ripe Tomato Flavour consisted of ten "1" rankings and a single "4" ranking.
Discussion
By introducing tomato ADH2 cDNA constructs, coupled to either a constitutive promoter or a fruit ripening specific promoter, the present inventors have produced a number of transgenic tomato plants with modified levels of ADH2 activity in their ripening fruit. The introduction of the ADH2 cDNA under the control of the constitutive promoter resulted in a spectrum of transformed plants including those with enhanced levels of ADH2 activity in the ripening fruit (and in other tissues, unpublished result) and plants with barely detectable levels of ADH2 activity in the ripening fruit. Transformed plants containing constructs with the tomato PG-promoter, produced fruit showing enhanced levels of the ADH2 activity. In fruit from these plants, ADH2 activity increased as the fruit ripened, consistent with the fruit/ripening specificity of the PG-promoter. No plants containing this construct were found with suppressed ADH2 activity in the ripening fruit, suggesting that pre-existing threshold levels of mRNA must be present before cosuppression of endogenous plus exogenous genes occurs.
Constitutive promoter--high-ADH plant C20
Associated with the increased ADH activity in fruit from plant C20 were increases in the alcohol forms of the hexanol/ol and hexenal/ol volatiles (Table 1) giving rise to reduced ratios of aldehyde to alcohol as seen in FIG. 6. These are consistent with an increased conversion of the C-6 aldehydes to their alcohols by the increased level of ADH in the transformed fruit and is the first direct evidence that the tomato ADH2 enzyme mediates interconversion of hexanal/ol and the Z-3- form of hexenal/ol in the ripening fruit.
Fruit specific promoter--high-ADH plant C13
In fruit from the C13 plant, ADH activity was marginally higher during development, relative to control fruit, but increased to approximately 3 times the level in control fruit, during ripening (FIG. 4). As with the constitutive high-ADH plant C20, an increase in Z-3-hexenol was found in fruit from this plant, resulting in a decrease in hexenal/ol ratios (FIG. 6). However, no increase in hexanol was observed while a decrease in hexanal was evident (Table 1). While this results in the same reduction in aldehyde to alcohol ratio evident in the C20 plant, the mechanism clearly differs, differing also from that mediating the balance between the hexenals and hexenols in both the C13 and C20 plants. Assuming that ADH is involved, the high ADH activity attained in the fruit, or the specific timing of its increase in the tissues may be affecting the mechanism.
Constitutive promoter--low-ADH plant C23
ADH activity was reduced to barely detectable levels in fruit of plant C23. Also barely detectable in these fruit was hexanol and hexenol (Table 1) with the resulting aldehyde to alcohol ratios for C23 fruit differing grossly from those of control fruit (FIG. 6). Reduction of ADH2 activity has therefore resulted in inhibition of the conversion of hexanal and hexenal to their alcohols providing further direct evidence of at least one of the roles of ADH2 in the ripening fruit.
All the transformed plants showing marked modifications of phenotype contained more than one introduced ADH2 construct. The two plants C7 and C14 contained only single insertions (Table 1) and showed only marginal modifications of ADH activity and aldehyde:alcohol ratios. While this suggested a correlation between the number of genes inserted and the magnitude of their effect, the correlation did not hold in all cases. Gene silencing was only observed in plants transformed with constructs regulated by the constitutive promoter and only in plants containing more than one inserted gene (Table 1). The results indicate that modification of ADH2 activity in the tomato fruit affects the regulation of balance between the 6-carbon aldehydes and alcohols.
The tasting trials showed a clear preference by the panelists for fruit from the plant C20. Fruit of this plant showed a statistically significant increase in the alcohols hexanol and 3-Z-hexenol. In order to quantify the increases in these volatiles, simple calculations were made by dividing the hexanol peak area from C20 fruit (Table 1) with that from control fruits. Similar calculations were made using the 3-Z-hexenol peak areas from the two sets of fruit. Such calculations indicated an increase in the amount of hexanol of about 1.2 to 4.6.times.control fruit and an increase in the amount of 3-Z-hexenol of about 1.5 to 3.7.times.control fruit.
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It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 23 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1520 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - AAATATCCAC TGCCTCAACT GAGTAAACAA CCAAAATTTG TGTTCTATAA AA -#AGTTTTCA 60 - - TATTTAGTGA TCACTAAAAA AAAATCAAGA AGATGTCGAC TACTGTAGGC CA -#AGTCATTC 120 - - GTTGCAAAGC TGCTGTGGCA TGGGAGGCTG GTAAGCCATT AGTGATGGAG GA -#AGTGGATG 180 - - TTGCTCCTCC ACAGAAAATG GAAGTTCGTC TTAAGATCCT CTACACTTCA CT -#CTGTCATA 240 - - CTGATGTATA CTTCTGGGAA GCTAAGGGTC AAAATCCAGT CTTTCCTCGA AT -#TCTTGGAC 300 - - ATGAAGCAGC AGGGATTGTG GAGAGTGTTG GAGAGGGAGT AACAGACCTT GC -#ACCAGGAG 360 - - ACCATGTTCT TCCTGTCTTT ACAGGGGAAT GTAAAGATTG CGCTCACTGT AA -#ATCTGAAG 420 - - AAAGCAATAT GTGTAGCCTC TTAAGGATTA ACACTGACAG GGGAGTGATG CT -#TAATGATG 480 - - GAAAATCAAG ATTTTCCATC AATGGAAACC CCATTTACCA TTTTGTTGGG AC -#CTCTACTT 540 - - TTAGTGAGTA CACCGTGGTT CATGTTGGAT GTGTTGCAAA AATTAACCCT CT -#TGCTCCTC 600 - - TTGACAAAGT ATGTGTCCTT AGTTGTGGAA TTTCGACAGG CCTTGGAGCA AG -#TTTGAATG 660 - - TTGCTAAACC AACAAAAGGC TCAAGTGTGG CTATATTTGG ACTAGGAGCT GT -#AGGCCTCG 720 - - CGGCTGCAGA AGGAGCCAGA ATTGCTGGTG CCTCGAGGAT AATTGGTGTT GA -#TTTAAATG 780 - - CTAGTAGATT TGAGCAAGCT AAGAAATTTG GTGTGACAGA GTTTGTGAAC CC -#AAAGGACT 840 - - ATAGTAAACC AGTTCAAGAG GTAATTGCTG AGATGACTGA TGGCGGAGTC GA -#TAGGAGTG 900 - - TGGAATGTAC TGGTCACATT GATGCTATGA TTTCAGCATT TGAATGTGTC CA -#TGATGGCT 960 - - GGGGAGTGGC GGTTCTTGTT GGTGTACCCC ATAAAGAAGC TGTGTTCAAG AC -#ACATCCTC 1020 - - TGAACTTTTT GAATGAACGG ACTCTCAAAG GAACCTTCTT TGGAAACTAC AA -#ACCTCGTT 1080 - - CGGATATTCC TTGTGTTGTT GAGAAATACA TGAACAAAGA ACTTGAATTG GA -#GAAATTCA 1140 - - TCACTCATAC ACTTCCATTT GCTGAAATCA ATAAGGCTTT CGATTTAATG CT -#GAAGGGAG 1200 - - AAGGCCTTCG TTGCATCATC ACCATGGCGG ACTAAACTTT CTGTCCTAGA AA -#AGGAGCTT 1260 - - CTACTGTTTG AGAAAAAAGA CCAATAAATT GTCACTGTCT TATTTTCCCT TT -#CGTGTTTG 1320 - - GTTGAGTTGT AACATTCCAT CCATGTCTCT TCTTTTGTCT TTTGCTTAGA TG -#TTGTGCTT 1380 - - TGCCATATCT CTTTCGATTC TTGTAAAAAA TGCAAATTCT CTCTGTTTTA TC -#TCAAGTAT 1440 - - ATTTACAGAA TTTCAGTGAT TTGATAAATC TAAACTTTAT CATAATATAA TC -#CAAACAGA 1500 - - ATTTCAATTG AAAAAAAAAA - # - # 152 - #0 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 379 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Ser Thr Thr Val Gly Gln Val Ile Arg Cy - #s Lys Ala Ala Val Ala 1 5 - # 10 - # 15 - - Trp Glu Ala Gly Lys Pro Leu Val Met Glu Gl - #u Val Asp Val Ala Pro 20 - # 25 - # 30 - - Pro Gln Lys Met Glu Val Arg Leu Lys Ile Le - #u Tyr Thr Ser Leu Cys 35 - # 40 - # 45 - - His Thr Asp Val Tyr Phe Trp Glu Ala Lys Gl - #y Gln Asn Pro Val Phe 50 - # 55 - # 60 - - Pro Arg Ile Leu Gly His Glu Ala Ala Gly Il - #e Val Glu Ser Val Gly 65 - #70 - #75 - #80 - - Glu Gly Val Thr Asp Leu Ala Pro Gly Asp Hi - #s Val Leu Pro Val Phe 85 - # 90 - # 95 - - Thr Gly Glu Cys Lys Asp Cys Ala His Cys Ly - #s Ser Glu Glu Ser Asn 100 - # 105 - # 110 - - Met Cys Ser Leu Leu Arg Ile Asn Thr Asp Ar - #g Gly Val Met Leu Asn 115 - # 120 - # 125 - - Asp Gly Lys Ser Arg Phe Ser Ile Asn Gly As - #n Pro Ile Tyr His Phe 130 - # 135 - # 140 - - Val Gly Thr Ser Thr Phe Ser Glu Tyr Thr Va - #l Val His Val Gly Cys 145 1 - #50 1 - #55 1 -#60 - - Val Ala Lys Ile Asn Pro Leu Ala Pro Leu As - #p Lys Val Cys ValLeu 165 - # 170 - # 175 - - Ser Cys Gly Ile Ser Thr Gly Leu Gly Ala Se - #r Leu Asn Val Ala Lys 180 - # 185 - # 190 - - Pro Thr Lys Gly Ser Ser Val Ala Ile Phe Gl - #y Leu Gly Ala Val Gly 195 - # 200 - # 205 - - Leu Ala Ala Ala Glu Gly Ala Arg Ile Ala Gl - #y Ala Ser Arg Ile Ile 210 - # 215 - # 220 - - Gly Val Asp Leu Asn Ala Ser Arg Phe Glu Gl - #n Ala Lys Lys Phe Gly 225 2 - #30 2 - #35 2 -#40 - - Val Thr Glu Phe Val Asn Pro Lys Asp Tyr Se - #r Lys Pro Val GlnGlu 245 - # 250 - # 255 - - Val Ile Ala Glu Met Thr Asp Gly Gly Val As - #p Arg Ser Val Glu Cys 260 - # 265 - # 270 - - Thr Gly His Ile Asp Ala Met Ile Ser Ala Ph - #e Glu Cys Val His Asp 275 - # 280 - # 285 - - Gly Trp Gly Val Ala Val Leu Val Gly Val Pr - #o His Lys Glu Ala Val 290 - # 295 - # 300 - - Phe Lys Thr His Pro Leu Asn Phe Leu Asn Gl - #u Arg Thr Leu Lys Gly 305 3 - #10 3 - #15 3 -#20 - - Thr Phe Phe Gly Asn Tyr Lys Arg Ser Asp Il - #e Pro Cys Val ValGlu 325 - # 330 - # 335 - - Lys Tyr Met Asn Lys Glu Leu Glu Leu Glu Ly - #s Phe Ile Thr His Thr 340 - # 345 - # 350 - - Leu Pro Phe Ala Glu Ile Asn Lys Ala Phe As - #p Phe Met Leu Lys Gly 355 - # 360 - # 365 - - Glu Gly Leu Arg Cys Ile Ile Thr Met Ala As - #p 370 - # 375 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3088 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - GATCAAAAGA TGAACTAAGG ATAATATTTT TGATATTTTA CCTTTTTTTT AA -#TTATAACG 60 - - TTGTTAAGAT ATCAAAGGAC CAATTATAAG AAAACCACCC AAAGGTTTCA TG -#TTTTGATT 120 - - GAAAAAACCA TCAAACAACG CAATACAACT GCTCAACTAG AATCAACATA AC -#AAAAAAAT 180 - - ATACTTAATG AGATCATTTA TAACCTAAAT TATAACCCCT CCGTGCACTT TC -#ATTTATCA 240 - - TGTTATATTT TACGCAAGTC AATTTGATTC ATTTTAAAAG TTAAATGAGA TT -#ATATTAAT 300 - - TTAATATTTT AAATAAAATT TTCAGATATT TAAAAATTAT ATGAAAAGTA TC -#ATGAATTG 360 - - TAGTTTTTTT TTGCATATAT GAAAAAATAC ATTATAAATA TTAGTCAATT TT -#TTTATAAT 420 - - TTGACTCTAA ATATGAAAAA AAATGACAAT TAAAAATAGA CGGAGGTTGT AA -#ATTAGCTT 480 - - ATTAATTATT AATTTGATAA ATATCATAAT TAACTGATAA TGACAATTAA AT -#ATTTAGAA 540 - - GACGATAATG ACAGAAATCA ACGTTATTTT AGGTATAATT TTTGTTGTAT TT -#TTGAAAAA 600 - - AATAATCTTT TTTCTGCAAC TGGTTATATT AAGTGAACAA ACAAAAAACA AG -#TAGTATAA 660 - - AAAAATTACA AGTGGACATA AAACAAAATG AGATACAGTA TTTGTGTTTC CA -#TTGGAATA 720 - - TTAGCTTGAC AAAAACTCAA ACGAGCAACA CAAAACAAAC AGCTAAAAAA CC -#TGTTTTGA 780 - - AAAATCCAGT GACCAAAACA TGTAAATGGT TTTACTGTGG CCTATTGTTT TT -#TCACCTTT 840 - - CCCAATTATA AATATCCACT GCCTCAACTG AGTAAACAAC CAAAATTTGT GT -#TCTATAAA 900 - - AAGTTTTCAT ATTTAGTGAT CACTAAAAAA AAATCAAGAA GATGTCGACT AC -#TGTAGGCC 960 - - AAGTCATTCG TTGCAAAGGT ATAATAATTC CATGATTCTG TAATTTCCTC GT -#TTTTTTTT 1020 - - TTAAGTTTGA TAATTTTTGT GGTAATTATA TATTATTTAT AGCTGCTGTG GC -#ATGGGAAG 1080 - - CTGGTAAGCC ATTAGTGATG GAGGAAGTAG ATGTTGCTCC TCCACAGAAA AT -#GGAAGTTC 1140 - - GTCTTAAGAT CCTCTATACT TCACTCTGTC ATACTGATGT ATACTTCTGG GA -#AGCTAAGG 1200 - - TAAACAAAAC TAAATTACGG GACTACGTTG AGTATGTTAG TGTTGTCAGC AA -#ATTTTATA 1260 - - AGGGGATTAT TTCCTTTGAA CTGATTTCAG GGTCAAAATC CAGTCTTTCC TC -#GAATTCTT 1320 - - GGACATGAAG CAGCAGGGTA TGTGTTATCT TGTTTCAATT GATTGATTTG AA -#TTCATCAT 1380 - - TTACTGTTTC TAAAGCTAAA AGGGTACTGA ATTTTGTTGT CTTCTTGATA TT -#TAGGATTG 1440 - - TGGAGAGTGT TGGAGAGGGA GTAACAGACC TTGCACCAGG AGACCATGTT CT -#TCCTGTCT 1500 - - TTACAGGGGA ATGTAAAGAT TGTGCTCACT GTAAATCTGA AGAAAGCAAT AT -#GTGTAGCC 1560 - - TCTTAAGGAT TAACACTGAC AGGGGAGTGA TGCTTAATGA TGGAAAATCA AG -#ATTTTCCA 1620 - - TCAATGGAAA CCCCATTTAC CATTTTGTTG GGACCTCTAC TTTTAGTGAG TA -#CACCGTGG 1680 - - TTCATGTTGG ATGTGTTGCA AAAATTAACC CTCTTGCTCC TCTTGACAAA GT -#ATGTGTCC 1740 - - TTAGTTGTGG AATTTCGACA GGTATAGACG AAGACAACGA TAGATTATGT TA -#CTAGTTTC 1800 - - TTTTTAAGGA GCTGCTCAAT TGTTGATTGA TATGAATACT TTTCCAGGCC TT -#GGAGCAAG 1860 - - TTTGAATGTT GCTAAACCAA CAAAAGGCTC AAGTGTGGCT ATATTTGGAC TA -#GGAGCTGT 1920 - - AGGCCTCGCG GTGAGTATGC TCCGTTGTGT TGTTTTATTG TTTCCCGTAT AT -#GTGTTAGT 1980 - - CTTACAGATG ACTGACTCAT TTGGTCAGGC TGCAGAAGGA GCCAGAATTG CT -#GGTGCCTC 2040 - - GAGGATAATT GGTGTTGATT TAAATGCTAG TAGATTTGAG CAAGGTAATA TA -#AATTTTTC 2100 - - CTTATACATT ATCTTAAAAT TCCTTAGTAA AACAACTAAT TCATCCATTT TA -#CTTGTATT 2160 - - CTACAGCTAA GAAATTTGGT GTGACAGAGT TTGTGAACCC AAAGGACTAT AG -#TAAACCAG 2220 - - TTCAAGAGGT ACTCAAATCA TATTTAATTT ACTTTAATCG AAGAAGAAAA AA -#GACAGGTC 2280 - - TGAGTTAATA GTTGATGTCT TTTCTTGAAT TCTGATTATT TGATCAGGTA AT -#TGCTGAGA 2340 - - TGACTGATGG CGGAGTCGAT AGGAGTGTGG AATGTACGGG TCACATTGAT GC -#TATGATTT 2400 - - CAGCATTTGA ATGTGTCCAT GATGTATGTT TTCTGTAATC AAATTAATTT CC -#TTAGCTGT 2460 - - ATGTTTGCGT TCATCTTAAC GAACATTGTT GTATTAACTT TAGGGCTGGG GA -#GTCGCGGT 2520 - - TCTTGTTGGT GTACCCCATA AAGAAGCTGT GTTCAAGACA CATCCTCTGA AC -#TTTTTGAA 2580 - - TGAACGGACT CTCAAAGGAA CCTTCTTTGG AAACTACAAA CCTCGTTCGG AT -#ATTCCTTG 2640 - - TGTTGTTGAG AAATACATGA ACAAAGAACT TGAATTGGAG AAATTCATCA CT -#CATACACT 2700 - - TCCATTTGCT GAAATCAATA AGGCTTTCGA TTTAATGCTG AAGGGAGAAG GC -#CTTCGTTG 2760 - - CATCATCACC ATGGCGGACT AAACTTTCTG TCCTAGAAAA GGAGCTTCTA CT -#GTTTGAGA 2820 - - AAAAAGACCA ATAAATTGTC ACTGTCTTAT TTTCCCTTTC GTGTTTGGTT GA -#GTTGTAAC 2880 - - ATTCCATCCA TGTCTCTTCT TTTGTCTTTT GCTTAGATGT TGTGCTTTGC CA -#TATCTCTT 2940 - - TCGATTCTTG TAAAAAATGC AAATTCTCTC TGTTTTATCT CAAGTATATT TA -#CAGAATTT 3000 - - CAGTGATTTG ATAAATCTAA ACTTTATCAT AATATAATCC AAACAGAATT TC -#AATTGAAA 3060 - - ATGATGAAGC CCTTACCGTC ATTGTTCC - # - # 3088 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 129 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - AAATATCCAC TGCCTCAACT GAGTAAACAA CCAAAATTTG TGTTCTATAA AA -#AGTTTTCA 60 - - TATTTAGTGA TCACTAAAAA AAAATCAAGA AGATGTCGAC TACTGTAGGC CA -#AGTCATTC 120 - - GTTGCAAAG - # - #- # 129 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - Met Ser Thr Thr Val Gly Gln Val Ile Arg Cy - #s Lys 1 5 - # 10 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 137 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - CTGCTGTGGC ATGGGAAGCT GGTAAGCCAT TAGTGATGGA GGAAGTAGAT GT -#TGCTCCTC 60 - - CACAGAAAAT GGAAGTTCGT CTTAAGATCC TCTATACTTC ACTCTGTCAT AC -#TGATGTAT 120 - - ACTTCTGGGA AGCTAAG - # - # - # 137 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - Ala Ala Val Ala Trp Glu Ala Gly Lys Pro Le - #u Val Met Glu Glu Val 1 5 - # 10 - # 15 - - Asp Val Ala Pro Pro Gln Lys Met Glu Val Ar - #g Leu Lys Ile Leu Tyr 20 - # 25 - # 30 - - Thr Ser Leu Cys His Thr Asp Val Tyr Phe Tr - #p Glu Ala Lys 35 - # 40 - # 45 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - GGTCAAAATC CAGTCTTTCC TCGAATTCTT GGACATGAAG CAGCAGG - # 47 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - Gly Gln Asn Pro Val Phe Pro Arg Ile Leu Gl - #y His Glu Ala Ala Gly 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 326 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: - - GATTGTGGAG AGTGTTGGAG AGGGAGTAAC AGACCTTGCA CCAGGAGACC AT -#GTTCTTCC 60 - - TGTCTTTACA GGGGAATGTA AAGATTGCGC TCACTGTAAA TCTGAAGAAA GC -#AATATGTG 120 - - TAGCCTCTTA AGGATTAACA CTGACAGGGG AGTGATGCTT AATGATGGAA AA -#TCAAGATT 180 - - TTCCATCAAT GGAAACCCCA TTTACCATTT TGTTGGGACC TCTACTTTTA GT -#GAGTACAC 240 - - CGTGGTTCAT GTTGGATGTG TTGCAAAAAT TAACCCTCTT GCTCCTCTTG AC -#AAAGTATG 300 - - TGTCCTTAGT TGTGGAATTT CGACAG - # - # 326 - - - - (2) INFORMATION FOR SEQ ID NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 108 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - Ile Val Glu Ser Val Gly Glu Gly Val Thr As - #p Leu Ala Pro Gly Asp 1 5 - # 10 - # 15 - - His Val Leu Pro Val Phe Thr Gly Glu Cys Ly - #s Asp Cys Ala His Cys 20 - # 25 - # 30 - - Lys Ser Glu Glu Ser Asn Met Cys Ser Leu Le - #u Arg Ile Asn Thr Asp 35 - # 40 - # 45 - - Arg Gly Val Met Leu Asn Asp Gly Lys Ser Ar - #g Phe Gly Ile Asn Gly 50 - # 55 - # 60 - - Asn Pro Ile Tyr His Phe Val Gly Thr Ser Th - #r Phe Ser Glu Tyr Thr 65 - #70 - #75 - #80 - - Val Val His Val Gly Cys Val Ala Lys Ile As - #n Pro Leu Ala Pro Leu 85 - # 90 - # 95 - - Asp Lys Val Cys Val Leu Ser Cys Gly Ile Se - #r Thr 100 - # 105 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 83 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: - - GCCTTGGAGC AAGTTTGAAT GTTGCTAAAC CAACAAAAGG CTCAAGTGTG GC -#TATATTTG 60 - - GACTAGGAGC TGTAGGCCTC GCG - # - # 83 - - - - (2) INFORMATION FOR SEQ ID NO:13: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: - - Gly Leu Gly Ala Ser Leu Asn Val Ala Lys Pr - #o Thr Lys Gly Ser Ser 1 5 - # 10 - # 15 - - Val Ala Ile Phe Gly Leu Gly Ala Val Gly Le - #u Ala 20 - # 25 - - - - (2) INFORMATION FOR SEQ ID NO:14: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 76 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: - - GCTGCAGAAG GAGCCAGAAT TGCTGGTGCC TCGAGGATAA TTGGTGTTGA TT -#TAAATGCT 60 - - AGTAGATTTG AGCAAG - # - # - # 76 - - - - (2) INFORMATION FOR SEQ ID NO:15: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: - - Ala Ala Glu Gly Ala Arg Ile Ala Gly Ala Se - #r Arg Ile Ile Gly Val 1 5 - # 10 - # 15 - - Asp Leu Asn Ala Ser Arg Phe Glu Gln 20 - # 25 - - - - (2) INFORMATION FOR SEQ ID NO:16: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 62 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: - - CTAAGAAATT TGGTGTGACA GAGTTTGTGA ACCCAAAGGA CTATAGTAAA CC -#AGTTCAAG 60 - - AG - # - # - # 62 - - - - (2) INFORMATION FOR SEQ ID NO:17: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: - - Ala Lys Lys Phe Gly Val Thr Glu Phe Val As - #n Pro Lys Asp Tyr Ser 1 5 - # 10 - # 15 - - Lys Pro Val Gln Glu 20 - - - - (2) INFORMATION FOR SEQ ID NO:18: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 97 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: - - GTAATTGCTG AGATGACTGA TGGCGGAGTC GATAGGAGTG TGGAATGTAC TG -#GTCACATT 60 - - GATGCTATGA TTTCAGCATT TGAATGTGTC CATGATG - #- # 97 - - - - (2) INFORMATION FOR SEQ ID NO:19: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: - - Val Ile Ala Glu Met Thr Asp Gly Gly Val As - #p Arg Ser Val Glu Cys 1 5 - # 10 - # 15 - - Thr Gly His Ile Asp Ala Met Ile Ser Ala Ph - #e Glu Cys Val His Asp 20 - # 25 - # 30 - - - - (2) INFORMATION FOR SEQ ID NO:20: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 560 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: - - GCTGGGGAGT GGCGGTTCTT GTTGGTGTAC CCCATAAAGA AGCTGTGTTC AA -#GACACATC 60 - - CTCTGAACTT TTTGAATGAA CGGACTCTCA AAGGAACCTT CTTTGGAAAC TA -#CAAACCTC 120 - - GTTCGGATAT TCCTTGTGTT GTTGAGAAAT ACATGAACAA AGAACTTGAA TT -#GGAGAAAT 180 - - TCATCACTCA TACACTTCCA TTTGCTGAAA TCAATAAGGC TTTCGATTTA AT -#GCTGAAGG 240 - - GAGAAGGCCT TCGTTGCATC ATCACCATGG CGGACTAAAC TTTCTGTCCT AG -#AAAAGGAG 300 - - CTTCTACTGT TTGAGAAAAA AGACCAATAA ATTGTCACTG TCTTATTTTC CC -#TTTCGTGT 360 - - TTGGTTGAGT TGTAACATTC CATCCATGTC TCTTCTTTTG TCTTTTGCTT AG -#ATGTTGTG 420 - - CTTTGCCATA TCTCTTTCGA TTCTTGTAAA AAATGCAAAT TCTCTCTGTT TT -#ATCTCAAG 480 - - TATATTTACA GAATTTCAGT GATTTGATAA ATCTAAACTT TATCATAATA TA -#ATCCAAAC 540 - - AGAATTTCAA TTGAAAAAAA - # - # - #560 - - - - (2) INFORMATION FOR SEQ ID NO:21: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 92 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: - - Gly Trp Gly Val Ala Val Leu Val Gly Val Pr - #o His Lys Glu Ala Val 1 5 - # 10 - # 15 - - Phe Lys Thr His Pro Leu Asn Phe Leu Asn Gl - #u Arg Thr Leu Lys Gly 20 - # 25 - # 30 - - Thr Phe Phe Gly Asn Tyr Lys Pro Arg Ser As - #p Ile Pro Cys Val Val 35 - # 40 - # 45 - - Glu Lys Tyr Met Asn Lys Glu Leu Glu Leu Gl - #u Lys Phe Ile Thr His 50 - # 55 - # 60 - - Thr Leu Pro Phe Ala Glu Ile Asn Lys Ala Ph - #e Asp Leu Met Leu Lys 65 - #70 - #75 - #80 - - Gly Glu Gly Leu Arg Cys Ile Ile Thr Met Al - #a Asp 85 - # 90 - - - - (2) INFORMATION FOR SEQ ID NO:22: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: - - CCACTGCCTC AACTGAG - # - # - # 17 - - - - (2) INFORMATION FOR SEQ ID NO:23: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: - - ACTAATGGCT TACCAGC - # - # - # 17__________________________________________________________________________

Number	Date	Country
PL6349	Dec 1992	AUX
PL9059	May 1993	AUX
PM0712	Aug 1993	AUX
PM0713	Aug 1993	AUX
PO8533	Aug 1997	AUX
PO2662	Sep 1999	AUX

Method for producing fruiting plants with improved fruit flavour

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