WO1994021803A2 - Fruit ripening-related tomato dna, dna constructs, cells and plants derived therefrom - Google Patents

Abstract

DNA constructs comprising DNA sequences encoding a TOM92 fruit-ripening-related proteins may be transformed into plants to modify plant characteristics (particularly fruit quality). A new DNA sequence is disclosed; a cDNA clone has been deposited; new ripening-related promoter sequences may also be obtained. Sense and antisense constructs for plant transformation are described. Genetically modified plants may be used to produce improved fruit and may also be used in breeding programmes to produce hybrid seed.

Description

FRUIT RIPENING-RELATED TOMATO DNA, DNA CONSTRUCTS, CELLS AND PLANTS DERIVED

THEREFROM

This application relates to novel DNA

constructs, plant cells containing the constructs

and plants derived therefrom. In particular it

involves the use of antisense or sense RNA

technology to control gene expression in plants.

The modification of plant gene expression has been achieved by several methods. The molecular

biologist can choose from a range of known methods to decrease or increase gene expression or to alter the spatial or temporal expression of a particular gene. For example, the expression of either

specific antisense RNA or partial sense RNA has

been utilised to reduce the expression of various

target genes in plants (as reviewed by Bird and

Ray, 1991, Biotechnology and Genetic Engineering

Reviews 9:207-227). These techniques involve the

incorporation into the genome of the plant of a

synthetic gene designed to express either antisense or sense RNA. They have been successfully used to down-regulate the expression of a range of

individual genes involved in the development and

ripening of tomato fruit (Gray et al, 1992, Plant

Molecular Biology, 19:69-87). Methods to increase the expression of a target gene have also been

developed. For example, additional genes designed to express RNA containing the complete coding

region of the target gene may be incorporated into the genome of the plant to "over-express" the gene product. Various other methods to modify gene

expression are known; for example, the use of

alternative regulatory sequences. In work leading to the present invention, we have identified a gene which encodes an enzyme involved in ripening-related processes. A DNA sequence encoding this enzyme has been cloned and characterised. The DNA sequence may be used to modify plant characteristics, particularly the ripening characteristics of fruit, including tomatoes. The sequence in question is encoded (almost completely) in the clone herein referred to as TOM92.

According to the present invention we provide a DNA construct comprising a DNA sequence as encoded by a TOM92 clone or as obtainable by the use of said clone as a hybridization probe. The DNA sequence may be derived from cDNA, from genomic DNA or synthetic polynucleotides (synthesised ab initio).

TOM92 was obtained from a cDNA library

produced from ripe wild-type tomato (Lycopersicon esculentum L cv Ailsa Craig) fruit which was differentially screened against un-ripe green fruit to identify ripening-related clones. TOM92 is a previously-unidentified clone. It has been shown that the mRNA for which TOM92 codes is expressed in ripening tomato fruit. TOM92 mRNA increases at the breaker stage and reaches maximal levels of expression three days post breaker. The levels then decline. In the ripening inhibitor (rin) mutant fruit the expression of TOM92 was not detectable. TOM92 mRNA levels are also not seen in leaves or wounded leaves of tomato. In low ethylene tomatoes (eg low EFE tomatoes) levels of TOM92 mRNA at seven days post breaker are similar to levels found in unmodified tomato fruit. TOM92 is described by Picton et al in Plant Molecular Biology, 1993, 23:627-631.

A cDNA clone encoding the TOM92 sequence was deposited at The National Collections of Industrial and Marine Bacteria (23 St Machar Drive, Aberdeen, Scotland, AB2 1RY) under the terms of the Budapest Treaty on 18 March 1993 under the accession number NCIMB 40552. The TOM92 cDNA has been inserted into a plasmid for replication purposes (designated pTOM92) within an E coli host. The base sequence of TOM92 is set out in SEQ ID NO 1. cDNA clones encoding the TOM92 enzyme may also be obtained from the mRNA of tomatoes or other plants by known screening methods similar to that described by Slater et al (1985, Plant Molecular Biology, 5:137-147) using suitable probes derived from the sequence shown as SEQ ID NO 1. Sequences coding for the whole, or substantially the whole of the mRNA produced by the TOM92 gene or genes may thus be isolated.

DNA sequencing of the TOM92 cDNA revealed an open reading frame with homology to pyridoxal

5'-phosphate histidine decarboxylases.

Decarboxylation of histidine results in the

formation of histamine, which is associated with allergic reactions and smooth muscle contraction in mammals. Enzyme activity is induced by various psychological and physical stresses resulting in changes in capillary dilation and permeability. There is no known reported role for either the enzyme or its product, histamine, in plants

although the action of an elderberry fruit

histidine decarboxylase has been implicated in the production of a histamine contaminant in wine

(Porgorzelski, 1992, J Sci Food Agric, 60:239-244).

DNA sequencing of TOM92 (SEQ ID NO 1) revealed an open reading frame encoding a polypeptide with high homology to several bacterial pyridoxal phosphate requiring histine decarboxylases (Picton et al, 1993, Plant Molecular Biology, 23:627-631). Other members of this group include the mammalian histidine decarboxylases (Taguchi et al, 1984, J Biol Chem, 259). The TOM92-encoded protein (TOM92 protein) has 35% identity and 53.8% similarity over the region of amino acid residues 8-386 with a pyridoxal phosphate-dependant histidine

decarboxylase sequence from Morganella morgani

(Vaaler et al, 1986, J Biol Chem, 261:11010-11014). This homology spans the entire length of the bacterial sequence. There is also homology with the same enzyme from Klebsiella planticola and

Enterobacter aerogenes (Kamath et al, 1991, J Biol Chem, 266:9432-9437). The TOM92 derived sequence and all three of the bacterial amino acid sequences contain the conserved lysine residue which is known to bind the pyridoxal phosphate coenzyme, and a serine residue which binds α-fluoromethylhistidine, and inhibitor of enzyme activity (Hayashi et al, 1986, J Biol CHem, 261:11003-11009). The derived TOM92 peptide sequence also contains additional stretches of 7 amino acids at the N-terminus and 27 amino acids at the C-terminus which are not present in the bacterial histidine decarboxylases. There are two stop codons, upstream of the first

methionine, at positions -28 and -37. As compared to the bacterial sequences, the TOM92 peptide has 44-57% more leucine residues, whilst glucine residues are particularly well conserved, with 20 out of 23 residues common to all sequences

compared.

An alternative source of the TOM92 DNA

sequence is a suitable gene encoding the TOM92 protein/enzyme. This gene may differ from the cDNA in that introns may be present. The introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out). Oligonucleotide probes or the cDNA clone may be used to isolate the actual TOM92 gene(s) by screening genomic DNA libraries. Such genomic DNA sequences may also be used as sources of gene promoters ( transcriptional

initiation sequences). The genomic clones may include control sequences operating in the plant genome. Thus it is also possible to isolate promoter sequences which may be used to drive expression of the TOM92 protein or any other protein. These promoters may be particularly responsive to ripening-related events and

conditions. A TOM92-gene promoter may be used to drive expression of any target gene.

A TOM92 gene has been mapped to chromosome 8 of tomato (chromosome 8 is described by Kinzer et al, 1990, Theor Appl Genet, 79:489-496). Southern analysis of tomato DNA cut with EcoRI and Hindlll showed 3 and 4 major hybridising bands respectively indicating a small number of TOM92 genes are present in the tomato genome. Our studies have shown that TOM92 gene expression is not directly regulated by ethylene, which is known to affect the expression of some but not all ripening-related genes (Picton et al, 1993, Plant Molecular Biology, 23:627-631). TOM92 mRNA was not detected in wild-type immature and mature green tomato fruit and began to accumulate at the first sign of colour change (the breaker stage). TOM92 mRNA levels peaked three days later, when fruit approach the fully red stage, and declined as the fruit aged. From gel blot analysis of fruit RNA, the transcript size of TOM92 was estimated to be 1.85 Kb which indicates that the sequenced cDNA (SEQ ID NO 1) is not full length.

A further way of obtaining a TOM92 DNA

sequence is to synthesise it ab initio from the appropriate bases, for example using SEQ ID NO 1 as a guide.

DNA sequences encoding the TOM92

ripening-related protein or enzyme may be isolated not only from tomato but from any suitable plant species. Alternative sources of suitable genes may include bacteria, yeast, lower and higher

eukaryotes.

The TOM92 sequences may be incorporated into DNA constructs suitable for plant transformation. These DNA constructs may then be used to modify TOM92 gene expression in plants. "Antisense" or "partial sense" or other techniques may be used to reduce the expression of the TOM92 protein(s) in developing and ripening fruit. The levels of the TOM92 proteins(s) may also be increased; for example, by incorporation of additional TOM92 sequence(s). The additional sequence(s) may be designed to give either the same or different spatial and temporal patterns of expression in the fruit. The overall level of TOM92 gene activity and the relative activities of the various

ripening-related proteins/enzymes affect plant (notably fruit) development and thus determine certain characteristics of the plant/fruit.

Modification of TOM92 enzyme activity can therefore be used to modify various aspects of plant or fruit quality when compared to similar unmodified plants or fruit at a corresponding development stage.

The invention further provides a DNA construct comprising a DNA sequence as encoded by a TOM92 clone or as obtainable by the use of said clone as a hybridization probe, in which said DNA sequence is under the control of a transcriptional

initiation region operative in plants, so that the construct can generate RNA in plant cells. Such a DNA construct may be an "antisense" construct generating "antisense" RNA or a "sense" construct (encoding at least part of the functional TOM92 protein) generating "sense" RNA. "Antisense RNA" is an RNA sequence which is complementary to a sequence of bases in the corresponding mRNA:

complementary in the sense that each base (or the majority of bases) in the antisense sequence (read in the 3' to 5' sense) is capable of pairing with the corresponding base (G with C, A with U) in the mRNA sequence read in the 5' to 3' sense. Such antisense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged to generate a transcript with at least part of its sequence complementary to at least part of the coding strand of the relevant gene (or of a DNA sequence showing substantial homology

therewith). "Sense RNA" is an RNA sequence which is substantially homologous to at least part of the corresponding mRNA sequence. Such sense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged in the normal orientation so as to generate a transcript with a sequence identical to at least part of the coding strand of the relevant gene (or of a DNA sequence showing substantial homology therewith). Suitable sense constructs may be used to inhibit gene expression (as described in International Patent Publication WO91/08299) or to over-express the enzyme.

The transcriptional initiation region may be derived from any plant-operative promoter. The transcriptional initiation region may be positioned for transcription of a DNA sequence encoding RNA which is complementary to a substantial run of bases in a mRNA encoding an enzyme produced by a TOM92 gene (making the DNA construct a full or partial antisense construct).

The characteristics of plant parts,

particularly fruit, may be modified by

transformation with a DNA construct according to the invention. The invention also provides plant cells containing such constructs; plants derived therefrom showing modified ripening

characteristics; and seeds of such plants.

The constructs of the invention may be inserted into plants to regulate the production of proteins encoded by genes homologous to the ripening-related TOM92 clone. The constructs may be transformed into any dicotyledonous or

monocotyledonous plant. Depending on the nature of the construct, the production of the protein may be increased, or reduced, either throughout or at particular stages in the life of the plant.

Generally, as would be expected, production of the protein is enhanced only by constructs which express RNA homologous to the substantially complete endogenous TOM92 mRNA. Constructs containing an incomplete DNA sequence shorter than that corresponding to the complete gene generally inhibit the expression of the gene and production of the proteins, whether they are arranged to express sense or antisense RNA. Full-length antisense constructs also inhibit gene expression.

The plants to which the present invention can be applied include commercially important

fruit-bearing plants, in particular tomato. In this way, plants can be generated which, amongst other phenotypic modifications, may have one or more of the following fruit characteristics:

improved resistance to damage during harvest, packaging and transportation due to slowing of the ripening and over-ripening processes;

longer shelf life and better storage

characteristics due to reduced activity of

degradative pathways (e.g. cell wall hydrolysis); improved processing characteristics due to changed activity of enzymes contributing to factors such as: viscosity, solids, pH, elasticity;

improved flavour and aroma at the point of sale due to modification of the sugar/acid balance and other flavour and aroma components responsible for characteristics of the ripe fruit;

modified colour due to changes in activity of enzymes involved in the pathways of pigment biosynthesis (e.g. lycopene, b-carotene, chalcones and anthocyanins);

increased resistance to post-harvest pathogens such as fungi.

As the TOM92 mRNA specifically accumulates during tomato ripening, is undetected in the extreme ripening mutant rin, and its encoded peptide has high homology with bacterial histidine decarboxylases, it may be that a histidine

decarboxylase-like protein, or its product

histamine, may play a role in the normal fruit ripening process. Modification of TOM92 gene expression thus allows modification of

fruit-ripening and other characteristics. For example, modification of TOM92 gene expression may affect the content of flavour-giving compounds and/or the aroma-volatile components found in the fruit. It may also affect the level of any

allergenic compounds found in the fruit.

The activity of the TOM92 protein may be either increased or reduced depending on the characteristics desired for the modified plant part (fruit, leaf, flower, etc). The levels of TOM92 protein may be increased; for example, by

incorporation of additional TOM92 genes. The additional genes may be designed to give either the same or different spatial and temporal patterns of expression in the fruit. "Antisense" or "partial sense" or other techniques may be used to reduce the expression of TOM92 protein.

The activity of the TOM92 protein or enzyme may be modified either individually or in

combination with modification of the activity of one or more other proteins/enzymes. In addition, the activities of the TOM92 enzyme may be modified in combination with modification of the activity of other enzymes involved in fruit ripening or related processes.

DNA constructs according to the invention may comprise a base sequence at least 10 bases

(preferably at least 35 bases) in length for transcription into RNA. There is no theoretical upper limit to the base sequence - it may be as long as the relevant mRNA produced by the cell - but for convenience it will generally be found suitable to use sequences between 100 and 1000 bases in length. The preparation of such

constructs is described in more detail below.

As a source of the DNA base sequence for transcription, a suitable cDNA or genomic DNA or synthetic polynucleotide may be used. The

isolation of suitable TOM92 sequences is described above; it is convenient to use DNA sequences derived from the TOM92 clone deposited at NCIMB in Aberdeen. Sequences coding for the whole, or substantially the whole, of the TOM92 protein may thus be obtained. Suitable lengths of this DNA sequence may be cut out for use by means of restriction enzymes. When using genomic DNA as the source of a base sequence for transcription it is possible to use either intron or exon regions or a combination of both.

To obtain constructs suitable for expression of the TOM92-related sequence in plant cells, the cDNA sequence as found in the pTOM92 plasmid or the related gene sequence as found in the chromosome of the plant may be used. Recombinant DNA constructs may be made using standard techniques. For example, the DNA sequence for transcription may be obtained by treating a vector containing said sequence with restriction enzymes to cut out the appropriate segment. The DNA sequence for

transcription may also be generated by annealing and ligating synthetic oligonucleotides or by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to give suitable restriction sites at each end. The DNA sequence is then cloned into a vector containing upstream promoter and

downstream terminator sequences. If antisense DNA is required, the cloning is carried out so that the cut DNA sequence is inverted with respect to its orientation in the strand from which it was cut.

In a construct expressing antisense RNA, the strand that was formerly the template strand becomes the coding strand, and vice versa. The construct will thus encode RNA in a base sequence which is complementary to part or all of the sequence of the TOM92-protein-encoding mRNA. Thus the two RNA strands are complementary not only in their base sequence but also in their orientations (5' to 3').

In a construct expressing sense RNA, the template and coding strands retain the assignments and orientations of the original plant gene.

Constructs expressing sense RNA encode RNA with a base sequence which is homologous to part or all of the sequence of the mRNA. In constructs which express the functional TOM92 protein/enzyme, the whole of the coding region of the gene is linked to transcriptional control sequences capable of expression in plants. For example, constructs according to the present invention may be made as follows. A suitable vector containing the desired base

sequence for transcription (such as pTOM92) is treated with restriction enzymes to cut the

sequence out. The DNA strand so obtained is cloned (if desired, in reverse orientation) into a second vector containing the desired promoter sequence and the desired terminator sequence. Suitable

promoters include the 35S cauliflower mosaic virus promoter and the tomato polygalacturonase gene promoter sequence (Bird et al, 1988 , Plant

Molecular Biology , 11 : 651-662 ) or other

developmetally regulated fruit promoters. Suitable terminator sequences include that of the

Agrobacterium tumefaciens nopaline synthase gene (the nos 3' end).

The transcriptional initiation region (or promoter) operative in plants may be a constitutive promoter (such as the 35S cauliflower mosaic virus promoter) or an inducible or developmentally regulated promoter (such as fruit-specific

promoters), as circumstances require. For example, it may be desirable to modify TOM92 protein

activity only during fruit development and/or ripening. Use of a constitutive promoter will tend to affect TOM92 protein levels and functions in all parts of the plant, while use of a tissue specific promoter allows more selective control of gene expression and affected functions. Thus in

applying the invention (for example, to tomatoes) it may be found convenient to use a promoter that will give expression during fruit development and/or ripening. Thus the antisense or sense RNA is produced only in the organ in which its action is required and/or only at the time required.

Fruit development and/or ripening-specific

promoters that could be used include the

ripening-enhanced polygacturonase promoter

(International Patent Publication Number

WO92/08798), the E8 promoter (Diekman & Fischer, 1988, EMBO, 7:3315-3320), the fruit specific 2A11 promoter (Pear et al, 1989, Plant Molecular

Biology, 13:639-651), the histidine decarboxylase promoter (HDC, Sibia) and the phytoene synthase promoter.

TOM92 protein or enzyme activity (and hence ripeing-related processes and fruit ripening characteristics) may be modified to a greater or lesser extent by controlling the degree of the TOM92 protein's sense or antisense mRNA production in the plant cells. This may be done by suitable choice of promoter sequences, or by selecting the number of copies or the site of integration of the DNA sequences that are introduced into the plant genome. For example, the DNA construct may include more than one DNA sequence encoding the TOM92 protein or more than one recombinant construct may be transformed into each plant cell.

The activity of the TOM92 protein may be separately modified by transformation with a suitable DNA construct comprising a TOM92 sequence. In addition, the activity of two or more proteins may be simultaneously modified by transforming a cell with two or more separate constructs: the first comprising a TOM92 sequence and the second (or further) comprising a second sequence encoding a related or unrelated protein. Alternatively, a plant cell may be transformed with a single DNA construct comprising both a first TOM92 sequence and a second protein sequence.

It is thus possible to modify the activity of the TOM92 protein while also modifying the activity of one or more other enzymes. The other enzymes may be involved in cell metabolism or in fruit development and ripening. Cell wall metabolising enzymes that may be modified in combination with a TOM92 protein include but are not limited to:

pectin esterase, polygalacturonase, β-galactanase, β-glucanase. Other enzymes involved in fruit development and ripening that may be modified in combination with a TOM92 protein include but are not limited to: ethylene biosynthetic enzymes, carotenoid biosynthetic enzymes including phytoene synthase, carbohydrate metabolism enzymes including invertase.

Several methods are available for modification of the activity of the TOM92 protein(s) in

combination with other enzymes. For example, a first plant may be individually transformed with a TOM92 construct and then crossed with a second plant which has been individually transformed with a construct encoding another enzyme. As a further example, plants may be either consecutively or co- transformed with TOM92 constructs and with

appropriate constructs for modification of the activity of the other enzyme(s). An alternative example is plant transformation with an TOM92 construct which itself contains an additional gene for modification of the activity of the other enzyme(s). The TOM92 construct may contain sequences of DNA for regulation of the expression of the other enzyme(s) located adjacent to the TOM92 sequences. These additional sequences may be in either sense or antisense orientation as described in International patent application publication number W093/23551 (single construct having distinct DNA regions homologous to different target genes). By using such methods, the benefits of modifying the activity of the TOM92 protein may be combined with the benefits of modifying the activity of other enzymes.

A DNA construct of the invention is

transformed into a target plant cell. The target plant cell may be part of a whole plant or may be an isolated cell or part of a tissue which may be regenerated into a whole plant. The target plant cell may be selected from any monocotyledonous or dicotyledonous plant species. Suitable plants include any fruit-bearing plant (such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, melons). For any particular plant cell, the TOM92 sequence used in the transformation construct may be derived from the same plant species, or may be derived from any other plant species (as there will be sufficient sequence similarity to allow modification of related

isoenzyme gene expression).

Constructs according to the invention may be used to transform any plant using any suitable transformation technique to make plants according to the invention. Both monocotyledonous and dicotyledonous plant cells may be transformed in various ways known to the art. In many cases such plant cells (particularly when they are cells of dicotyledonous plants) may be cultured to

regenerate whole plants which subsequently reproduce to give successive generations of genetically modified plants. Any suitable method of plant transformation may be used. For example, dicotyledonous plants such as tomato and melon may be transformed by Agrobacterium Ti plasmid

technology, such as described by Bevan (1984, Nucleic Acid Research, 12:8711-8721) or Fillatti et al (Biotechnology, July 1987, 5:726-730). Such transformed plants may be reproduced sexually, or by cell or tissue culture.

Transgenic plants and their progeny may be used in standard breeding programmes, resulting in improved plant lines having the desired

characteristics. For example, fruit-bearing plants expressing a TOM92 construct according to the invention may be incorporated into a breeding programme to alter fruit-ripening characteristics and/or fruit quality. Such altered fruit may be easily derived from elite lines which already possess a range of advantageous traits after a substantial breeding programme: these elite lines may be further improved by modifying the expression of a single targeted TOM92 protein/enzyme to give the fruit a specific desired property.

By transforming plants with DNA constructs according to the invention, it is possible to produce plants having an altered (increased or reduced) level of expression of one or more TOM92 proteins, resulting from the presence in the plant genome of DNA capable of generating sense or antisense RNA homologous or complementary to the RNA that generates such protein(s). For

fruit-bearing plants, fruit may be obtained by growing and cropping using conventional methods. Seeds may be obtained from such fruit by

conventional methods (for example, tomato seeds are separated from the pulp of the ripe fruit and dried, following which they may be stored for one or more seasons). Fertile seed derived from the genetically modified fruit may be grown to produce further similar modified plants and fruit.

The fruit derived from genetically modified plants and their progeny may be sold for immediate consumption, raw or cooked, or processed by canning or conversion to soup, sauce or paste. Equally, they may be used to provide seeds according to the invention.

The genetically modified plants (transformed plants and their progeny) may be heterozygous for the TOM92 DNA construct. The seeds obtained from self fertilisation of such plants are a population in which the DNA constructs behave like single Mendelian genes and are distributed according to Mendelian principles: eg, where such a plant contains only one copy of the construct, 25% of the seeds contain two copies of the construct, 50% contain one copy and 25% contain no copy at all. Thus not all the offspring of selfed plants produce fruit and seeds according to the present invention, and those which do may themselves

be either heterozygous or homozygous for the defining trait. It is convenient to maintain a stock of seed which is homozygous for the TOM92 DNA construct. All crosses of such seed stock will contain at least one copy of the construct, and self-fertilized progeny will contain two copies, i.e. be homozygous in respect of the character. Such homozygous seed stock may be conventionally used as one parent in F1 crosses to produce

heterozygous seed for marketing. Such seed, and fruit derived from it, form further aspects of our invention. We further provide a method of

producing F1 hybrid plants expressing a TOM92 DNA sequence which comprises crossing two parent lines, at least one of which is homozygous for a TOM92 DNA construct. A process of producing F1 hybrid seed comprises producing a plant capable of bearing genetically modified fruit homozygous for a TOM92 DNA construct, crossing such a plant with a second homozygous variety, and recovering F1 hybrid seed. It is possible according to our invention to transform two or more plants with different TOM92 DNA constructs and to cross the progeny of the resulting lines, so as to obtain seed of plants which contain two or more constructs leading to reduced expression of two or more fruit-ripening -related TOM92 proteins.

The invention will now be described further with reference to the drawings in which:

Figure 1 is a diagram showing the construction of a TOM92 antisense construct.

Figure 2 is a diagram showing the construction of a TOM92 sense construct.

The invention will also be described with reference to the SEQUENCE LISTING in which:

SEQ ID NO 1 shows the base sequence of the cDNA clone pTOM92. The sequence has been placed on the EMBL database (accession number X719000).

The following Examples illustrate aspects of the invention. EXAMPLE 1

Construction of antisense RNA vectors with the CaMV 35S promoter.

A vector is constructed using sequences corresponding to a restriction fragment obtained from pTOM92 and is cloned into the vectors GA643 (An et al, 1988, Plant Molecular Biology Manual A3: 1-19) or pDH51 (Pietrzak et al, 1986, Nucleic Acids Research, 14:5875-5869) which has previously been cut with a compatible restriction enzyme(s). A restriction fragment from the pTOM92/pDH51 clone containing the promoter, the pTOM92 fragment and other pDH51 sequence is cloned into SLJ44026B or SLJ44024B (Jones et al, 1990, Transgenic Research, 1) or a Bin19 (Bevan, 1984, Nucleic Acids Research, 12:8711-8721) which permits the expression of the antisense RNA under control of the CaMV 35S

promoter.

After synthesis of the vector, the structure and orientation of the sequences are confirmed by DNA sequence analysis.

EXAMPLE 2

Construction of antisense RNA vectors with the polygalacturonase promoter.

The fragment of the pTOM92 cDNA described in Example 1 is also cloned into the vector pJR3 to give pJR392A. pJR3 is a Binl9 based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase

promoter. This vector includes approximately 5 kb of promoter sequence and 1.8 kb of 3' sequence from the PG promoter separated by a multiple cloning site.

After synthesis, vectors with the correct orientation of pTOM92 sequences are identified by DNA sequence analysis.

EXAMPLE 3

Construction of truncated sense RNA vectors with the CaMV 35S promoter.

The fragment of pTOM92 cDNA described in

Example 1 is also cloned into the vectors described in Example 1 in the sense orientation.

After synthesis, the vectors with the sense orientation of pTOM92 sequence are identified by DNA sequence analysis.

EXAMPLE 4

Construction of truncated sense RNA vectors with the polygalacturonase promoter.

The fragment of pTOM92 cDNA that was described in Example 1 is also cloned into the vector pJR3 in the sense orientation.

After synthesis, the vectors with the sense orientation of pTOM92 sequence are identified by DNA sequence analysis. EXAMPLE 5

Construction of a pTOM92 overexpression vector using the CaMV35S promoter.

The complete sequence of the pTOM92 cDNA clone is inserted into pJRl.

EXAMPLE 6

Construction of a pTOM92 overexpression vector using the polygalacturonase promoter.

The complete sequence of the pTOM92 cDNA clone is inserted into pJR3.

EXAMPLE 7

Constructs made for plant transformations

Figure 1 is a diagram showing the construction of a TOM92 antisense construct. Digestion of pTOM92 with Spel and Rsal yielded a 580 base pair fragment which was cloned into pDH51 cut with Xbal and Smal. The EcoRI/SacI fragment released from pDH51 was ligated into EcoRI/SacI-cut pSLJ44026B.

Figure 2 is a diagram showing the construction of a TOM92 sense construct. Digestion of pTOM92 with Nsil and Kpnl yielded a 230 base pair sense fragment which was cloned into Pstl/KpnI-cut pDH51. PvuII and Sad were used to cut a fragment from pDH51. pSLJ44026B was digested initially with EcoRI and the cohesive ends blunted using Klenow enzyme. It was then cut with Sad and the

PvuII/Sacl fragment from pDH51 ligated into it.

EXAMPLE 8

Generation of transformed plants

Vectors are transferred to Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and are used to transform tomato plants.

Transformation of tomato cotyledons follows standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988). Transformed plants are identified by their ability to grow on media containing the antibiotic kanamycin. Plants are regenerated and grown to maturity.

Ripening fruit are analysed for modifications to their ripening characteristics.

EXAMPLE 9

Transgenic plants

Table 1 summarises the numbers of plants which have been transformed using the various constructs described in Example 7. These plants and their fruit are currently being analysed. Progeny will also be developed and analysed. Further plants are being transformed.

SEQUENCE LISTING

1: GENERAL INFORMATION :

( i) APPLICANT: SENECA, Limited

(ii) TITLE OF INVENTION: DNA, DNA CONSTRUCTS, CELLS AND PLANTS

DERIVED THEREFROM

(iii) NUMBER OF SEOUENCES: 1

(iv) CORRESPONDENCE ADDRESS :

(A) ADDRESSEE: ICI GROUP PATENTS SERVICES DEPARTMENT

(B) STREET: PO BOX 6, SHIRE PARK, BESSEMER ROAD

(C) CITY: WELWYN GARDEN CITY

(D) STATE: HERTFORDSHIRE

(E) COUNTRY: UNITED KINGDOM

(F) ZIP: AL7 1HD

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floopy disk

(B) COMPUTER: IBM PC comoatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9305861.8

(B) FILING DATE: 22-MAR-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: ROBERTS, TIMOTHY W

(B) REGISTRATION NUMBER: (GEN AUTHN) 31435

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (+44) 0707 323400

(B) TELEFAX: (+44) 0707 337454

(C) TELEX: 94028500 ICIC G

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1412 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: smαie

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE:

(A) ORGANISM: PTOM92

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TTTAATTTTA TTTATTTATT TTTATAACAT AGGAGTTTGA TTTAACGATA GTTCCAACAG 60

AAGGTGAAAT TGATGCACCA TCATCGCCAA GGAAGAATTT ATGTCTCAGT GTGATGGAAT 120

CTGATATTAA AAATGAAACG TCTTTTCAAG AACTCGACAT GATTTTGACT CAATATTTAG 180

AGACATTGTC CGAGCGAAAA AAGTATCATA TAGGTTATCC AATTAACATG TGTTACGAAC 240

ATCATGCCAC TTTAGCCCCA CTTTTGCAAT TTCATTTGAA CAATTGTGGA GATCCCTTTA 300

CTCAGCACCC TACAGATTTC CATTCAAAAG ATTTTGAAGT GGCTGTTTTA GATTGGTTTG 360

CACAACTCTG GGAAATAGAG AAAGATGAAT ATTGGGGATA CATTACTAGT GGTGGCACTG 420

AGGGCAATCT CCATGGCCTT TTGGTTGGGC AGAAGAGAGC TACTTCCTAA TGGATATTAT 480

ATGCATCAAA AGATTCACAT TACTCGATTT TCAAAGCAGC AAGAATGTAT CGAATGGAGC 540

TACAAACTAT CAACACTTTA GTTAATGGGG AAATTGATTA TGAAGATTTA CAATCAAAGT 600

TACTTGTCAA CAAGAACAAA CCAGCTATCA TCAATATCAA TATTGGAACA ACCTTCAAAG 660

GAGCTATTGA TGACCTCGAT TTCGTCATAC AAACACTTGA AAATTGTGGT TATTCAAATG 720

ACAATTATTA TATCCATTGC GATCGAGCAT TATGTGGGCT AATTCTCCCA TTTATCAAAC 780

ATGCAAAAAA AATTACCTTC AAGAAACCAA TTGGAAGTAT TTCAATTTCA GGGCACAAAT 840

TCTTGGGATG TCCAATGTCT TGTGGCGTTC AGATAACAAG GAGAAGTTAC GTTAGCACCC 900

TCTCAAAAAT TGAGTATATT AATTCCGCAG ATGCTACAAT TTCTGGTAGT CGAAATGGAT 960

TTACACCAAT ATTCTTATGG TACTGTTTAA GCAAGAAAGG ACATGCTAGA TTGCAACAAG 1020

ATTCCATAAC ATGCATTGAA AATGCTCGGT ATTTGAAAGA TCGACTTCTT GAAGCAGGAA 1080

TTAGTGTTAT GCTGAATGAT TTTAGTATTA CTGTTGTTTT TGAACGACCT TGTGACCATA 1140

AATTCATTCG TCGTTGGAAC TTGTGTTGCT TAAGAGGCAT GGCACATGTT GTAATTATGC 1200

CAGGTATTAC AAGAGAAACT ATAGATAGTT TCTTCAAAGA TCTAATGCAA GAGAGGAACT 1260

ATAAGTGGTA CCAAGATGTA AAAGCTCTGC CTCCTTGCCT AGCTGATGAT TTGGCTCTAA 1320

ATTGTATGTG CTCCAATAAA AAGATGCATA ACTAAATTAT ATCAAGAGTT TTCAAATAAA 1380

TTTTCCATAT ATAAAAAAAA AAAAAAAAAA AA 1412

Claims

1. A DNA construct comprising a DNA sequence as encoded by a TOM92 clone or as obtainable by the use of said clone as a hybridization probe.

2. A DNA construct as claimed in claim 1 in which the DNA sequence is under the control of a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.

3. A DNA construct as claimed in claim 1 or claim 2 in which the DNA sequence is derived from cDNA.

4. A DNA construct as claimed in claim 1 or claim 2 in which the DNA sequence is derived from genomic DNA.

5. A DNA construct as claimed in claim 1 or claim 2 in which the DNA sequence is synthetic DNA.

6. A DNA construct as claimed in claim 1 which is a cDNA clone deposited at The National

Collections of Industrial and Marine Bacteria under the accession number NCIMB 40552.

7. A transcriptional initiation sequence derived from a genomic DNA sequence encoding a TOM92 protein.

8. A method to produce a plant having a modified characteristic when compared to a similar unmodified plant at a corresponding

development stage comprising transformation of a plant with a DNA construct as claimed in claim 2.

9. A method as claimed in claim 8 comprising

transformation of a fruit-bearing plant with a DNA construct as claimed in claim 2 to modify fruit-ripening characteristics.

10. A plant cell comprising a DNA construct as claimed in claim 2.

11. A plant derived from a plant cell as claimed in claim 10.

12. A fruit comprising a DNA construct as claimed in claim 2.

13. A seed comprising a DNA construct as claimed in claim 2.

14. A method of producing an F1 hybrid plant

comprising crossing two parent lines, at least one of which is a genetically modified plant homozygous for a DNA construct as claimed in claim 2.

15. A process of producing F1 hybrid seed

comprising producing a genetically modified plant which is homozygous for a DNA construct as claimed in claim 2, crossing such a plant with a second homozygous variety, and

recovering F1 hybrid seed.