WO1993013212A1

WO1993013212A1 - Dna, dna constructs, cells and plants derived therefrom

Info

Publication number: WO1993013212A1
Application number: PCT/GB1992/002355
Authority: WO
Inventors: Colin Roger Bird; Donald Grierson; Lisa Naomi Hall
Original assignee: Zeneca Limited
Priority date: 1991-12-20
Filing date: 1992-12-18
Publication date: 1993-07-08
Also published as: AU3166693A; GB9127098D0; US5659121A; EP0637338A1

Abstract

DNA constructs comprise a DNA sequence homologous to some or all of the pectinesterase gene encoded by the clone pB8 (Sequence ID No. 1), under control of a transcriptional initiation region operative in plants for transcribing this DNA sequence, optionally in the antisense direction to produce RNA complementary to the gene mRNA. From such constructs may be derived transformed plant cells and plants in which expression of pectinesterase genes is inhibited: fruit from the plants (such as tomatoes) can show modified ripening properties.

Description

DNA, DNA CONSTRUCTS, CELLS AND PLANTS DERIVED THEREFROM

This invention 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.

During tomato fruit ripening, the components of the cell walls undergo a series of modifications. The pectin component is subject to substantial modification and degradation. The major changes to the pectin include: solubilisation, depolymerisation and demethylation. The changes in texture of the fruit that occur during ripening occur in parallel with the changes to the pectin component. It has been demonstrated that many of these changes are the result of the action of enzymes in the cell wall.

The cell wall enzymes polygalacturonase and pectin esterase have a major role in modifying pectin during fruit ripening. Individual cDNA clones that corresponded to mRNAs encoding these two enzymes have previously been identified and characterised. DNA sequences from these clones have been used to demonstrate sense and antisense inhibition of these enzymes (EPA 271,988, ICI). For antisense inhibition of, say, pectin esterase, a fragment of DNA coding for the mRNA that produces pectinesterase is inserted into the plant genome under control of a plant promoter, but in reverse orientation. In this way the plant produces RNA sequences complementary to the mRNA that is translated into pectin esterase: and this interferes in some way with the translation of the mRNA into protein.

For sense inhibition of, say, polygalacturonase, a fragment of DNA coding for the mRNA that produces polygalacturonase is inserted into the plant genome under control of a plant promoter, in normal orientation. The plant produces RNA - 1 - sequences homologous with part of the full-length mRNA that is translated into polygalacturonase. Again, this interferes with and can substantially inhibit the translation of polygalacturonase mRNA into protein. How this happens is not clear.

In this invention, we provide clones comprising further novel DNA coding sequences from a new pectin esterase gene. These can be used to make DNA constructs that will control expression of pectinesterase, either by sense or antisense inhibition.

In work leading to the invention, we have identified a novel cDNA (pB8) which encodes a tomato pectin esterase isoenzyme. The cDNA was identified in a clone from a library constructed using mRNA isolated from tomatoes at an early ripening stage. The cDNA in the clone was approximately 2 kb long. Characterisation of the sequence of the clone indicated in addition to sequences similar to those found in the previously characterised cDNA sequence (pPEl), there was also a 358 bp extension at the 5' end. This extended sequence was not predicted since the size of the previous cDNA (pPEl - 1.655 kb) corresponded to the determined size of the mRNA (1.6 kb). More recent, unpublished determinations indicate that the true size of the PE mRNA in tomato fruit is approximately 2.0 kb (and see Handa, 1992).

According to the present invention, we further provide recombinant DNA comprising an upstream promoter base sequence, a base sequence for transcription into mRNA under control of said upstream promoter base sequence, and a downstream transcription terminator base sequence, characterised in that the base sequence for transcription comprises a sequence of bases complementary to a substantial run of bases in the novel PE cDNA sequence. The sequence for transcription may be oriented in the normal or reverse sense, so as to produce either sense or antisense RNA in plants. The invention also includes plant cells containing such constructs; plants derived therefrom showing modified ripening characteristics; seeds of such plants; and novel recombinant DNA from clones B8 and B16.

DNA constructs according to the invention comprise a base sequence for transcription at least 10 bases in length. For antisense transcription at least, 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. For sense transcription, it may be found that sequences approaching the full length of the corresponding mRNA may lead to over-production of the enzyme rather than inhibition. Where this happens shorter sequences should be used, unless over-production is desired. The preparation of suitable constructs is described in more detail below.

The preferred DNA for use in the present invention is DNA derived from the clone B8. The required antisense DNA can be obtained in several ways: by cutting with restriction enzymes an appropriate sequence of such DNA; by synthesising a DNA fragment using synthetic oligonucleotides which are annealed and then ligated together in such a way as to give suitable restriction sites at each end; by using synthetic oligonucleotides in a polymerase chain reaction (PCR) to generate the required fragment with suitable restriction sites at each end. The DNA is then cloned into a vector containing upstream promoter and downstream terminator sequences, the cloning being carried out so that the cut DNA sequence is inverted with respect to its orientation in the strand from which it was cut. In new vectors expressing antisense RNA, the strand that was formerly the template strand becomes the coding strand, and vice versa. The new vector will thus encode RNA in a base sequence which is complementary to the sequence of pB8 mRNA. Thus the two RNA strands are complementary not only in their base sequence but also in their orientations 5^r to 3').

As source of the DNA base sequence for transcription, it is convenient to use cDNA clones such as pB8. The base sequences of pB8 are set out in Table 1. pB8 has been deposited on 06 December 1991 with the National Collections of Industrial and Marine Bacteria, Aberdeen, under Accession No. NCIB 40463. Alternatively, cDNA clones similar to pB8 may be obtained from the mRNA of ripening tomatoes by the methods similar to that described by Slater et al, Plant Molecular Biology 5, 137-147, 1985. In this way may be obtained sequences coding for the whole, or substantially the whole, of the mRNA produced by pB8. Suitable lengths of the cDNA so obtained may be cut out for use by means of restriction enzymes.

As previously stated, DNA suitable for use in the present invention is DNA showing homology to the gene encoded by the clone pB8. pB8 was derived from a cDNA library isolated from early ripening tomato RNA.

An alternative source of DNA for the base sequence for transcription is a suitable gene encoding the pectinesterase mRNA represented in pB8. Such a gene may be isolated, for example, by probing tomato DNA with cDNA from clone pB8. This gene may differ from the cDNA of, e.g. pB8 in that introns may be present. The introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out). When using such a gene as the source of the base sequence for transcription it is possible to use either intron or exon regions.

A further way of obtaining a suitable DNA base sequence for transcription is to synthesise it ab initio from the appropriate bases, for example using Table 1 as a guide.

Recombinant DNA and vectors according to the present invention may be made as follows. A suitable vector containing the desired base sequence for transcription (for example pB8) 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 ( for example cauliflower mosaic virus 35S RNA promoter or the tomato polygalacturonase gene promoter sequence - UK Patent Application 9024323.9) and the desired terminator sequence (for example the 3' of the Agrobacterium tumefaciens nopaline synthase gene, the nos 3' end).

According to the invention we propose to use both constitutive promoters (such as cauliflower mosaic virus 35S RNA) and inducible or developmentally regulated promoters (such as the ripe-fruit-specific polygalacturonase promoter) as circumstances require. Use of a constitutive promoter will tend to affect functions in all parts of the plant: while by using a tissue-specific promoter, functions may be controlled more selectively. Thus in applying the invention, e.g. to tomatoes, it may be found convenient to use the promoter of the PG gene (UK Patent Application 9024323.9, filed 8 November 1990). Use of this promoter, at least in tomatoes, has the advantage that the production of antisense RNA is under the control of a ripening-specific promoter. Thus the antisense RNA is only produced in the organ in which its action is required.

Other ripening-specific promoters that could be used include the E8 promoter (Diekman & Fischer, 1988 cited above). It may be advantageous to use a ripening-specific promoter that switches on in unripe fruit, so as to leinhibit the formation of pectinesterase from then on.

Vectors according to the invention may be used to transform plants as desired, to make plants according to the invention. Dicotyledonous plants, such as tomato and melon, may be transformed by Agrobacterium Ti plasmid technology, for example as described in EP 271,988 (ICI). Such transformed plants may be reproduced sexually, or by cell or tissue culture.

The degree of production of sense or antisense RNA in the plant cells can be controlled by suitable choice of promoter sequences, or by selecting the number of copies, or the site of integration, of the DNA sequences according to the invention that are introduced into the plant genome. In this way it may be possible to modify ripening or senescence to a greater or lesser extent.

The constructs of our invention may be used to transform cells of both monocotyledonous and dicotyledonous plants 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. Such plants have modified fruit ripening properties. Examples of genetically modified plants according to the present invention include, as well as tomatoes, fruits of such plants as mangoes, peaches, apples, pears, strawberries, bananas and melons. Tomatoes with reduced pectin esterase activity give improved characteristics in paste made from the processed tomatoes: in particular increased viscosity of the serum from the paste and decreased granularity of the paste, which eventually gives a smoother and more glossy paste.

The invention will now be described further with reference to Table 1, which shows the full base sequence of the clone pB8 (ID sequence No 1) and, for comparison, the base sequence of the clone pB16 (ID Sequence No 2), homologous to the pectinesterase gene disclosed in EPA 271,988.

The following Examples illustrate aspects of the invention.

EXAMPLE 1

Identification and characterisation of novel PE cDNA

A cDNA library in lambda zapll (Stratagene) which had been prepared from mRNA extracted from early ripening tomato fruit was screened with a radiolabelled oligonucleotide probe corresponding to a sequence from the 5' end of pPEl (Ray et al 1988). Several hybridising clones were identified and plasmid DNA was excised ill vivo according to the instructions supplied by Stratagene. The sizes of the cDNA inserts in the clones were determined by digestion with BamHl and Kpnl followed by agarose gel electrophoresis. One clone (pB8) had an insert size of approximately 2 kb. This clone was selected for further analysis since the insert was approximately 300 bases longer than that of the previously identified PE cDNA (pPEl). The nucleotide sequence of the 5' 530 bases of this clone were determined. The 5' end of pPEl was located at base 359 of the new clone. EXAMPLE 2

Construction of a constitutively expressed PE antisense vector

A plant transformation vector is constructed using the sequences corresponding to bases 1 to 338 of pB8 (Fig 1). This fragment is synthesised by polymerase chain reaction using synthetic primers. The fragment is cloned into the vector pJRl which is previously cut with Smal. pJRl (Smith et al Nature 334, 724-726, 1988) is a vector hased on Binl9 (Bevan, Nucleic Acids Research, 12, 8711-8721, 1984), which permits the expression of the antisense RNA under the control of the CaMV 35S promoter. This vector includes a nopaline synthase (nos) 3 ^r end termination sequence.

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

EXAMPLE 3

Construction of a developmentally regulated PE antisense vector

The fragment of the pB8 cDNA that was described in example 2 is also cloned into the vector pJR3.

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 pB8 sequences are identified by DNA sequence analysis.

EXAMPLE 4

Construction of a constitutively expressed PE sense vector

The fragment of pB8 cDNA that was described in example 2 is also cloned into the vector pJRl in the sense orientation.

After synthesis, the vectors with the sense orientation of pB8 sequence are identified by DNA sequence analysis.

EXAMPLE 5

Construction of a developmentally regulated PE sense vector

The fragment of pB8 cDNA that was described in example 2 is also cloned into the vector pJR3 in the sense orientation.

After synthesis, the vectors with the sense orientation of pB8 sequence are identified by DNA sequence analysis. EXAMPLE 6

Production of genetically modified tomato plants

Vectors are transferred to Agrobacteriu tumefaciens

LBA4404 (a micro-organism widely available to plant biotechnologists) and are used to transform tomato plants. Transformation of tomato stem segments follow standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988 - leaf or cotyledon segments may also be used). 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 reduced PE activity and improved fruit quality characteristics.

TABLE 1

SEQ ID NO : 1

SEQUENCE TYPE: Nucleotide

SEQUENCE LENGTH: 1989

STRANDEDNESS: Single

TOPOLOGY: Linear

MOLECULE TYPE: DNA

ORIGINAL SOURCE ORGANISM: Tomato var Ailsa Craig

IMMEDIATE EXPERIMENTAL SOURCE: cDNA clone B8

FEATURES:

From 16 to 1654 Open reading frame encoding Pectin Esterase

From 16 to 703 Coding region for N-terminal extension

PROPERTIES: cDNA for tomato fruit pectin esterase

TTGCAAACTT CTAAAATGGC TAATCCTCAA CAACCTTTGT TAATAAAAAC ACACAAACAA 60

AATCCAATAA TCAGCTTCAA GATCCTCAGT TTTGTTATAA CTTTGTTTGT TGCTCTCTTC 120

TTAGTTGCTC CATATCAAGT TGAGATTAAA CATTCTAATC TATGTAAAAC TGCACAAGAT 180

TCCCAACTCT GTCTCAGTTA TGTCTCTGAT TTGATATCCA ATGAAATTGT CACAACAGAA 240

T€AGATGGAC ATAGTATTCT GATGAAATTT TTAGTTAACT ATGTCCATCA AATGAACAAT 300

GCAATTCCAG TGGTTCGCAA AATGAAGAAT CAGATCAATG ACATTCGTCA ACACGGGGCT 360

TTAACTGATT GTCTTGAGCT TCTTGATCAG TCAGTTGATT TCGCATCTGA TTCAATTGCA 420

GCAATTGATA AAAGAAGTCG CTCGGAGCAT GCCAATGCGC AAAGTTGGCT AAGTGGTGTG 480

CTTACTAACC ACGTTACGTG CTTGGATGAG CTTGATTCCT TTACTAAAGC TATGATAAAT 540

GGAACGAATC TTGAAGAGTT GATCTCGAGA GCTAAGGTAG CATTAGCGAT GCTTGCGTCT 600

TTGACAACTC AGGATGAGGA TGTTTTCATG ACGGTTTTAG GAAAAATGCC ATCTTGGGTG 660

AGTTCGATGG ATAGGAAGCT GATGGAGAGT TCGGGTAAGG ACATTATAGC GAATGCAGTG 720

GTGGCACAAG ATGGAACGGG GGATTATCAA ACACTTGCTG AAGCAGTTGC TGCAGCACCA 780

GATAAGAGTA AGACGCGTTA TGTAATTTAT GTAAAGAGGG GAACTTATAA AGAGAATGTT 840

GAGGTGGCTA GCAATAAAAT GAACTTGATG ATTGTTGGTG ATGGAATGTA TGCTACGACC 900

ATTACTGGTA GCCTTAATGT TGTCGATGGA TCAACAACCT TCCGCTCTGC CACTCTTGCT 960 GCAGTCGGCC AAGGATTTAT ACTACAGGAC ATATGTATAC AGAACACAGC AGGGCCAGCG 1020 AAAGACCAAG CAGTGGCACT TCGAGTTGGA GCTGATATGT CTGTCATAAA TCGTTGTCGT 1080

ATCGATGCTT ATCAAGACAC CCTTTATGCA CATTCTCAAA GGCAATTCTA TCGAGACTCC 1140

TACGTGACAG GTACTGTTGA TTTCATATTT GGTAATGCAG CAGTTGTATT CCAGAAATGC 1200

CAGCTCGTAG CTAGAAAACC GGGTAAATAC CAGCAAAACA TGGTGACTGC ACAAGGCAGG 1260

ACGGACCCAA ATCAGGCCAC GGGGACATCA ATTCAGTTCT GTAACATAAT AGCAAGTTCG 1320

GACCTAGAAC CAGTCCTGAA AGAATTCCCA ACATATCTTG GTAGGCCATG GAAAGAATAT 1380

TCAAGAACTG TAGTGATGGA ATCATACTTA GGTGGTCTCA TTAATCCAGC GGGTTGGGCT 1440

GAGTGGGACG GAGATTTTGC GTTGAAGACA TTGTATTATG GTGAATTTAT GAACAATGGA 1500

CCTGGTGCTG GTACTAGTAA GCGTGTCAAG TGGCCTGGTT ATCATGTCAT TACTGATCCC 1560

GCTAAAGCTA TGCCGTTCAC TGTGGCTAAG CTGATTCAGG GCGGATCATG GTTGAGGTCT 1620

ACTGGCGTGG CGTATGTGGA TGGATTATAT GATTAGAGTA TATATATGAT GTGCCACATG 1680

AGCAGGGCAG AGCAAGCATA ACACACAACT CTAGTGTGAC AAGCATTTAC ATGGCTCATT 1740

CGTTACTACT AAGTTGTCAA TAAGTTCTGT TTAGGGGTTC ATAAGTTTAT ATACGTATAT 1800

ACATTTACAT TGGTGATGAA GCTGAAACTG ATGATGCTTT AATGTAATTA TAGTTTTCTG 1860

AAAAAGGATA TGAGTAATAT TAGTTTTTCC CAGATGTGTA TGGTTGTGGA ACTGTTTATG 1920

CTTAAATTGG CAAGGGGTAT TGAATAAAAA TCTATTGTGT TAAAAAAAAA AAAAAAAAAA 1980

AAAAAAAAAA 1989

TABLE

SEQ ID NO: 2 SEQUENCE TYPE: Nucleotide SEQUENCE LENGTH: 2059

STRANDEDNESS: Single TOPOLOGY: Linear MOLECULE TYPE: DNA

ORIGINAL SOURCE ORGANISM: Tomato var Ailsa Craig IMMEDIATE EXPERIMENTAL SOURCE: cDNA clone B16

FEATURES:

From 13 to 1672 Open reading frame encoding Pectin Esterase

From 13 to 711 Coding region for N-terminal extension

Base 368 Start of cDNA pPEl published by Ray et al (1988)

PROPERTIES: cDNA for tomato fruit pectin esterase

CGAACTTCTA AAATGGCTAC TCCTCAACAA CCTTTGTTAA CAAAAACACA CAAACAAAAT 60

TCCATAATCA GCTTCAAGAT CCTCACTTTT GTTGTAACTT TGTTTGTTGC TCTCTTCTTA 120

GTTGTGTTTC TTGTTGCTCC ATATCAATTT GAGATTAAAC ATTCTAATCT GTGTAAAACT 180

GCACAAGATT CCCAACTCTG TCTCAGTTAT GTTTCTGATT TAATATCCAA TGAAATTGTC 240

ACATCTGATT CAGATGGACT AAGTATTCTG AAGAAATTTT TAGTTTACTC TGTTCATCAA 300

ATGAACAATG CAATTCCAGT GGTTCGCAAA ATCAAGAATC AGATCAATGA CATTCGTGAA 360

CAAGGGGCTT TAACTGATTG TCTTGAGCTT CTTGATCTGT CAGTTGATTT AGTATGTGAT 420

TCAATTGCAG CAATTGATAA GAGAAGTCGT TCGGAGCATG CCAATGCGCA AAGTTGGCTA 480

AGTGGTGTGC TTACTAACCA CGTTACGTGC TTGGATGAGC TTGATTCCTT TACTAAAGCT 540

ATGATAAATG GAACGAATCT TGATGAGTTG ATCTCGAGAG CTAAGGTAGC ATTGGCGATG 600

CTTGCGTCTG TGACAACTCC AAATGATGAA GTTTTGAGGC CGGGTTTAGG AAAAATGCCA 660

TCTTGGGTGA GTTCGAGGGA TAGGAAGCTG ATGGAGAGTT CGGGTAAGGA CATTGGAGCG 720

AATGCAGTGG TGGCAAAAGA TGGAACAGGG AAATATCGAA CACTTGCTGA AGCTGTTGCT 780 GCAGCACCAG ATAAGAGTAA GACGCGTTAT GTAATTTATG TAAAGAGGGG AACTTATAAA 840

GAGAATGTTG AGGTGAGTAG CAGGAAAATG AATTTGATGA TTATTGGTGA TGGCATGTAT 900

GCTACCATCA TTACTGGGAG CCTTAATGTT GTCGATGGAT CAACAACCTT CCACTCTGCC 960

ACTCTTGCTG CAGTTGGCAA AGGATTTATA CTACAGGACA TATGTATACA GAACACAGCA 1020

GGACCAGCTA AACACCAAGC TGTTGCACTT CGAGTTGGAG CTGATAAGTC TGTCATAAAT 1080

CGTTGTCGTA TCGATGCTTA TCAAGACACC CTTTATGCAC ATTCTCAAAG GCAATTCTAT 1140

CGAGACTCCT ACGTGACAGG GACTATTGAT TTCATATTCG GTAATGCAGC AGTTGTATTC 1200

CAGAAATGCC AGCTCGTAGC TAGAAAACCG GGTAAATACC AGCAAAACAT GGTGACTGCA 1260

CAAGGCAGGA CGGACCCAAA TCAGGCCACG GGGACATCAA TTCAGTTTTG TGATATAATA 1320

GCAAGTCCTG ACCTAAAACC AGTCGTGAAA GAATTCCCAA CATATCTTGG TAGGCCATGG 1380

AAAAAATATT CAAGAACTGT AGTGATGGAA TCATCATTGG GTGGTCTCAT TGATCCATCG 1440

GGTTGGGCTG AGTGGCACGG AGATTTTGCG TTAAAGACAT TGTATTATGG TGAATTTATG 1500

AATAATGGAC CTGGTGCTGG TACTAGTAAG CGTGTCAAGT GGCCTGGCTA TCATGTCATT 1560

ACTGACCCCG CTGAAGCTAT GTCATTCACT GTGGCTAAGC TGATTCAGGG CGGATCATGG 1620

TTGAGGTCTA CTGACGTGGC GTATGTGGAT GGATTATATG ATTAGAGTGA TATAAAATTA 1680

CTCTTTGTTT ATGTAACAAG ACATCTTTAA AAAGTTCAAA GTAAGTAGTA GTAATATATC 1740

CATATGAAGT GCCACATGAG CAGGGCAGAG CCGGGATTAA GTGTCTAAAG CATAACACAC 1800

AACTCTAGTG TGACAAGCAT TTACATGGCT CATTCCTTAC TACTAAGTCG TCAATAAGTT 1860

CAGTTAAGGG GTTCATAAGT TAATATACGT ATATATATTT ATGTTGGCGA TAAAGCTGAA 1920

ACTGATGATG CTTTAATGTA ATTATAGTTT TCTGAAAAAG GATATGTGTA ATATTAGGTT 1980

TTCCCTGATG TTTATGGTTG TGGGGTGGTG GTTATGATAA AAATATGCAA GATGAAAGTC 2040

AAAAAAAAAA AAAAAAAAA 2059

Claims

We claim:

1. DNA constructs comprising a DNA sequence homologous to some or all of the gene encoded by the clone pB8 (Sequence ID No 1), under control of a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.

2. DNA constructs as claimed in claim 1 comprising a transcriptional initiation region operative in plants positioned for transcription of a DNA sequence encoding RNA complementary to a sequence of bases showing homology to mRNA of the gene encoded by the clone pB8 (Sequence ID No

1).

3. DNA constructs as claimed in claim 1 in which the transcriptional initiation region operative in plants is a constitutive promoter.

4. DNA constructs as claimed in claim 3 in which the constitutive promoter is CaMV 35S.

5. DNA constructs as claimed in claim 1 in which the transcriptional initiation region operative in plants is an inducible or developmentally regulated promoter.

6. DNA constructs as claimed in claim 5 in which the promoter is that for the polygalacturonase gene.

7. Plant cells transformed with a construct claimed in claim 1.

8. Plant cells claimed in claim 7 which are cells of tomato.

9. Plant cells claimed in claim 8 which are cells of mangoes, peaches, apples, pears, bananas, melons or strawberries.

10. Plants containing cells claimed in claim 7.

11. Plants claimed in claim 10 which bear climacteric fruit.

12. Fruit or seeds of plants claimed in claim 11.

13. Tomato seeds as claimed in claim 12 containing a construct adapted to express RNA antisense to mRNA expressed by the gene encoded by the clone pB8 (Sequence ID No 1).

14. Recombinant DNA comprising a base sequence substantially as shown in Sequence ID No 1.

15. Recombinant DNA comprising a base sequence at least 16 bases in length that is homologous to the sequence from base 16 to base 1654 shown in Sequence ID No 1 or to the sequence from base 13 to base 367 shown in Sequence ID No 2.