WO1992017596A1 - Dna, dna constructs, cells and plants derived therefrom - Google Patents

Abstract

DNA constructs comprise a DNA sequence homologous to some or all of the gene encoded by the clone pTOM75. The constructs may further comprise a transcriptional initiation region operative in plants for transcribing this DNA sequence, optionally in the antisense direction to produce mRNA complementary to the pTOM75 gene. From such constructs may be derived transformed plant cells and plants in which expression of the pTOM75 gene is inhibited: fruit from the plants (such as tomatoes) can show modified ripening properties, such as increased acid content.

Description

DNA, DNA CONSTRUCTS, CELLS AND PLANTS DERIVED THEREFROM

This application relates to novel DNA constructs, plant cells containing them and plants derived therefrom. In particular it involves the control of gene expression in plants .

As is well known, a cell manufactures protein by transcribing the DNA of the gene for that protein to produce messenger RNA (mRNA), which is then processed (eg by the removal of introns) and finally translated by ribosomes into protein. This process may be inhibited by the presence in the cell of "antisense RNA". By this term is meant an RNA sequence which is complementary to a sequence of bases in the mRNA in question: 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. It is believed that this inhibition takes place by formation of a complex between the two complementary strands of RNA, preventing the formation of protein. How this works is uncertain: the complex may interfere with further transcription, processing, transport or translation, or degrade the mRNA, or have more than one of these effects. Such antisense RNA may be produced in the cell by transformation with an appropriate DNA construct arranged to transcribe backwards part of the coding strand (as opposed to the template strand) of the relevant gene (or of a DNA sequence showing substantial homology therewith). The use of this technology to downregulate the expression of specific plant genes has been described, for example in European Patent publication no 271988 to ICI (corresponding to US serial 119614). Reduction of gene expression has led to a change in the phenotype of the plant: either at the level of gross visible phenotypic difference e.g. lack of anthocyanin production in flower petals of petunia leading to colourless instead of coloured petals (van der Krol et al, Nature, 333, 866-869, 1988); or at a more subtle biochemical level e.g. change in the amount of polygalacturonase and reduction in depoly erisation of pectins during tomato fruit ripening (Smith et al, Nature, 334, 724-726, 1988; Smith et al, Plant Mol. Biol. 14, 369-380, 1990). Thus antisense RNA has been proven to be useful in achieving downregulation of gene expression in plants.

In work leading to the present invention we have identified a gene which expresses a protein during the ripening of tomatoes. This gene is also expressed in roots and senescing leaves of tomato. This gene has been cloned and characterised. We postulate that it will be of use in modifying the ripening of tomatoes and other fruit. The gene in question is encoded (almost completely) in the clone pTOM75, the sequence of which has not previously been disclosed.

According to the present invention we provide DNA constructs comprising a DNA sequence homologous to some or all of the gene encoded by the clone pTOM75. The homologous DNA sequence may be preceded by a transcriptional initiation region operative in plants, so that the construct can generate mRNA in plant cells. In a further aspect, the present invention provides DNA constructs comprising a transcriptional initiation region operative in plants positioned for transcription of a DNA sequence encoding RNA complementary to a substantial run of bases showing substantial homology to an mRNA encoding the protein produced by the gene in pTOM75. The invention also includes plant cells containing such constructs; plants derived therefrom showing modified ripening characteristics; and fruit and seeds of such plants.

The constructs of the invention may be inserted into plants to regulate the production of enzymes encoded by genes homologous to pTOM75. Depending on the nature of the construct, the production of the enzymes may be increased, or reduced (downregulated) , either throughout or at particular stages in the life of the plant. Generally, as would be expected, production of the enzyme is enhanced only by constructs which express RNA homologous to the substantially complete endogenous pTOM75 mRNA. What is more surprising is that constructs containing an incomplete DNA sequence substantially shorter than that corresponding to the complete gene generally inhibit the expression of the gene and production of the enzymes, whether they are arranged to express sense or antisense RNA.

It is also possible to target the expression of the gene to a particular compartment of the cell, such as the tonoplast, the vacuole, the chloroplasts and chromoplasts or the cell wall. The plants to which the present invention can be applied include commercially important fruit-bearing plants, in particular the tomato. In this way, plants can be generated which may have one or more of the following characteristics: Novel flavour and aroma due to changes in the concentrations and ratios of the many aromatic compounds that contribute to fruit flavour;

Fruit (e.g. tomatoes) of changed or intensified flavour (e.g. sweeter or sharper, or both) due to changes in the accumulation of acids (e.g. citric or malic acid) or of sugars, or both;

Longer shelf life and better storage characteristics due to changed turgor of the tomato fruit cells;

Improved processing characteristics due to changed composition of the fruit leading to altered viscosity, solids, pH, elasticity and sugar content;

Modified fruit shape thus improving packing and storage characteristics;

Extended leaf biosynthetic activity due to inhibition of enzymes responsible for the degradative processes involved in senescence (in particular, leaf senescence); thus improving plant productivity;

Improved stress tolerance, particularly drought tolerance and/ or improved turgor maintenance;

Increased shelf life of fruit and flowers. Delayed abscission due to changes in turgor in cells of the abscission layer.

DNA constructs for downregulating gene expression according to the invention preferably comprise a homologous base sequence at least 20, usually at least 50 bases in length. 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.

The preferred source of RNA for use in the present invention is DNA derived from the clone pTOM75. The required DNA can be obtained in several ways, including: 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 (if so desired) being carried out so that the DNA sequence is inverted with respect to its orientation to the promoter in the strand from which it was cut. If the new .vector is intended to produce antisense RNA, the strand that was formerly the template strand becomes the coding strand, and vice versa. Such a new vector will encode RNA in a base sequence which is complementary to the sequence of pTOM75 mRNA. The two RNA strands are complementary not only in their base sequence but also in their orientations (5' to 3' ) .

As source of the DNA base sequence for transcription, it is convenient to use a cDNA clone such as pTOM75. The base sequence of pTOM75 is set out in Figure

1.

The pTOM75 clone has been deposited at the National

Collections of Industrial and Marine Bacteria, PO Box 31, of 23 St. Machar Drive (formerly of 135 Abbey Road) , Aberdeen AB2 1RY, Scotland, as a plasmid in E. coli , under the reference NCIB 40397, on 25 March 1991.

Alternatively, a cDNA clone similar to pTOM75 may be obtained from the mRNA of ripening tomatoes by the method 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 pTOM75. Suitable lengths of the cDNA so obtained may be cut out for use by means of restriction enzymes.

An alternative source of DNA for the base sequence for transcription is a suitable gene encoding the pTOM75 protein. Such a gene may differ from the cDNA of pTOM75 in that introns may be present. The introns are not transcribed into mRNA (or, if so transcribed, are subsequently cut out). When using part of such a gene as the source of the base sequence for transcription (with the object of downregulating gene expression) 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 a_b initio from the appropriate bases, for example using Figure 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 pTOM75) 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 - Bird et al. , Plant Molecular Biology, 11, 651-662, 1988) and the desired terminator sequence (for example the 3' sequence of the

Agrobacterium tumefaciens nopaline synthase gene, with the nos 3' end, or the 'long' PG promoter, with the PG gene 3' end, as described in ICI's UK patent application 9024323.9, filed 11 November 1990). 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

(Bird et al , 1988, cited above; or the 'long' PG promoter as described in ICI's UK patent application 9024323. 9, filed 11 November 1990). Use of these promoters, 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, EMBO Journal 7, 3315-3320, 1988) and the promoters from the pTOM36 genes.

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, may be transformed by Agrobacterium Ti plasmid technology, for example as described by Bevan (1984) Nucleic Acid Research, 12, 8711-8721. Such transformed plants may be reproduced sexually, or by cell or tissue culture.

The degree of production of 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. Examples of genetically modified plants according to the present invention include, as well as tomatoes, fruits such as mangoes, peaches, apples, pears, strawberries, bananas and melons; and carnations and other ornamental flowers.

As previously stated, the preferred means of producing RNA for use in the present invention is DNA showing homology to the gene encoded by the clone pTOM75. pTOM75 was derived from a cDNA library isolated from ripe tomato RNA (Slater et al Plant Molecular Biology _5, 137-147, 1985). pTOM75 has been characterised by hybrid select translation. it may not contain the full length coding sequence for the gene. Slater et al (Plant

Molecular Biology 5, 137-147, 1985) reported a product of 28 kDa. DNA sequence analysis has demonstrated that the clone is 889 bases long. The longest open reading frame of this clone encodes a protein of 180 amino acids. The putative protein has a predicted molecular weight of 19.8 kDa, rather than the 28 kDa estimated by hybrid-select translation. The extreme hydrophobic nature and low charge of the protein may have led to overestimation in the size previously determined.

Computer analysis and data base searching has revealed that the derived amino acid sequence shows homology with bovine lens fibre major intrinsic protein (MIP), soybean nodulin-26, the glycerol facilitator protein of E. coli, the Drosophila big brain gene, a turgor- responsive gene expressed in wilted pea shoots and a seed-specific tonoplast intrinsic protein amongst others. MIP is believed to be involved in the formation of aqueous channels in lens tissue and has been shown to allow the passive transport of ions and small molecules when inserted into artificial membranes. Its function may be to allow transport of metabolites within the avascular tissue of the lens to regulate the volume of the extracellular space. The glycerol transporter from E. coli forms a passive channel allowing bi-directional transport of neutral molecules less than 0.4 nm in diameter across the inner cytoplasmic membrane. Antisense RNA could be used to clarify whether these rather tentative sequence relationships imply a functional homology or whether they are not significant.

We have demonstrated that the mRNA for which pTOM75 codes is expressed in ripening tomato fruit, in roots and in senescing leaves of tomatoes. Almost no expression could be detected in mature green fruit. pTOM75 is expressed most strongly at the full orange stage of ripening. The level of mRNA then declines in line with the general decline in biosynthetic capacity of the ripening fruit. Expression of pTOM75 is also detected at high levels in immature green fruit. pTOM75 can also be induced by exposing mature green fruit to exogenous ethylene. The expression of pTOM75 is apparently increased in the ripening inhibitor (rin) tomato fruit ripening mutant which mature very slowly ( Knapp et al , Plant Mol . Biol 12, 105-118, 1989). The relative expression levels of pTOM75 in fruit is low compared with pTOM6 (polygalacturonase ) or pTOMδ (prephytoene synthase ) . Although a considerable body of information on the structure and expression of the pTOM75 gene or genes is known, the biochemical function of this clone has not hitherto been elucidated.

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

Figure 1 shows the base sequence of the clone pTOM75; Figure 2 shows the construction of plant transformation antisense RNA vectors according to the invention. Figure 2 also shows the construction of the pTOM75 expression vectors according to the invention.

The following Examples illustrate aspects of the invention.

EXAMPLE 1

Identification of base sequence of pTOM75

The base sequence of pTOM75 has not previously been determined. The sequence was determined by standard DNA sequencing procedures and is shown in Figure 1. Knowledge of this sequence is essential for determining the orientation of the open reading frame and for the subsequent construction of RNA antisense vectors.

EXAMPLE 2A

Construction of pTOM75 antisense RNA vectors with the CaMV 35S promoter (vector containing the 695 base pair Pstl-Dral fragment) A vector pBDH75A was constructed using the base pair Pstl-Dral fragment derived from pTOM75 cDNA by digestion of pTOM75 with Pstl and Dral , followed by isolation of the 695 base pair fragment after electrophoresis. The fragment was then cloned into the vector pDH51 or pjRl which had previously been cut with Pstl and Smal . Recombinant plasmids were isolated and characterised. Representative vectors of this series were called pDH75A (derived from pDH51 - see Figure 2) and pJR75A (derived from pJRl ). pDH51 is a pUC based cloning vector containing a CaMV35S promoter and terminator fragment. pJRl (Smith et al Nature 334, 724-726, 1988) is a Binl9 ( Bevan, Nucleic Acids Research, 12, 8711-8721, 1984) based vector, which permits the expression of the antisense RNA under the control of the CaMV 35S promoter. This vector includes a nopaline synthase (noε) 3' end termination sequence.

After synthesis of the vector pDH75A, the expression cassette was transferred to Binl9 (Bevan, Nucleic Acids Research, 12, 8711-8721, 1984) to yield pBDH75A. After synthesis of the vector, the structure and orientation of the pTOM75 sequence it contained were confirmed by DNA sequence analysis.

EXAMPLE 2B

Construction of pTOM75 antisense RNA vectors with the CaMV 35S promoter (vector containing the complete pTOM75 fragment)

Vectors pJR75CA and pJR75CS are prepared as follows: the complete cDNA pTOM75 insert (889 bases) is inserted into pJRl as a Pstl fragment. This results in clones having the pTOM75 fragment either in the antisense or sense orientation inserted into the cloning vector pJR1. The antisense vector is called pJR75CA. The sense vector is called pJR75CS.

EXAMPLE 3A

Construction of pTOM75 antisense RNA vector with the polygalacturonase promoter (vector containing the 695 base pair Pstl-Dral fragment).

The fragment produced in Example 2A by cleavage of pTOM75 with Dral and Pstl is cloned into the vector pJR2 to give the clone pJR275A. pJR2 is a Binl9-based vector, which permits the expression of the antisense RNA under the control of the tomato polygalacturonase promoter. This vector includes a nopaline synthase (nos) 3' end termination sequence.

The isolated fragment is made flush-ended with T4 poly erase and then cloned into the Hindi site of pJR2. After synthesis, vectors are identified which have the pTOM75 insert fragment in the antisense (pJR275A) and sense (pJR275S) orientation.

EXAMPLE 4

Generation of transformed plants

The vector pBDH75A of Example 2A was transferred to

Agrobacterium tumefaciens LBA4404 (a micro-organism widely available to plant biotechnologists) and used to transform tomato plants ( Lycopersicon esculentum, var. Ailsa Craig) . Transformation of tomato stem segments and cotyledons followed standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988). Transformed plants were identified by their ability to grow on media containing the antibiotic kanamycin. Plants were regenerated and grown to maturity. Ripening fruit were analysed biochemically and the presence of the antisense pTOM75 gene construct was verified by Southern and PCR analysis .

Similar plants may be produced using the vectors PJR275A and pJR275S from Example 3A in place of pBDH75A. These are also expected to show inhibition of expression of the pTOM75 gene.

EXAMPLE 5

Drought stress testing,

Drought stress tests were carried out on leaves of a transformed plant produced in Example 4, found to contain the pBDH75A construct. Comparisons were made with the leaves of similar wild-type tomatoes. In each case total leaf RNA was probed with pTOM75 sense and antisense transcript, before or after 24 hours drought stress. Samples were hybridised with nick-translated pTOM75 insert 5xSSPE at 65°C, with a final wash at 65°C in 0.2x55PE. The wild-type plant showed no pTOM75 mRNA before drought-stress. After drought-stress the wild-type plant showed substantial amounts of pTOM75 mRNA, while pBDH75A showed only traces.

EXAMPLE 6

Malic acid fruit content

A plant from Example 4 transformed with the pBDH75A construct was grown to maturity and produced fruit. individual fruit were sampled for malic acid content, using a commercially available food analysis kit (Boehringer Mannheim) . Results are shown in Table 1 below, compared with similar results for wild-type fruit. The mean malic acid level in transformed fruit was over 25% above the mean level in wild type fruit. Application of Student's T test suggests that the result was statistically significant at the 2% level.

This result suggests that inhibition of the pTOM75 antisense gene can be used to produce fruit of greater acidity, which will be preferred by certain tastes.

TABLE 1

PLANT TYPE Fruit # μq MALIC ACID,g ^λ MEAN

212 242 232.7

244

158 200

156 178.4

198

180

Claims

We claim :

1. DNA constructs comprising a DNA sequence homologous to some or all of the gene encoded by the clone pTOM75.

_

2. DNA constructs as claimed in claim 1 in which the homologous DNA sequence is preceded by a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.

10

3. DNA constructs as claimed in claim 2 comprising a transcriptional initiation region operative in plants positioned for transcription of a DNA sequence encoding RNA complementary to a substantial run of bases showing

15 substantial homology to pTOM75 mRNA.

4. DNA constructs as claimed in any of claims 1 to 3 in which the homologous DNA derives from pTOM75.

20 5. DNA constructs as claimed in any of claims 1 to 4 which contain DNA homologous to the sequence set forth in Figure 1.

6. DNA constructs as claimed in claim 2 in which the

25 transcriptional initiation region operative in plants is a constitutive promoter.

7. DNA constructs as claimed in claim 6 in which the constitutive promoter is CaMV 35S.

30

8. DNA constructs as claimed in claim 2 in which the transcriptional initiation region operative in plants is an inducible or developmentally regulated promoter.

35 9. DNA constructs as claimed in claim 8 in which the promoter is that for the polygalacturonase gene.

10. Plant cells transformed with a construct claimed in any of claims 1 to 9.

11. Plant cells claimed in claim 10 which are cells of tomato.

12. Plant cells claimed in claim 10 which are cells of mangoes, peaches, apples, pears, bananas, melons or strawberries, or of carnations or other ornamental flowers,

13. Plants containing cells claimed in any of claims 10 to 12.

14. Plants claimed in claim 13 which bear climacteric fruit.

15. Fruit or seeds of plants claimed in either of claims

13 or 14.

16. Tomato seeds as claimed in claim 15 containing a construct adapted to express mRNA antisense to pTOM75.

17. Genetically-modified fruit having a higher malic acid content as compared with corresponding wild-type fruit.

18. Tomatoes as claimed in claim 17