WO1993007275A1 - Dna, dna constructs, cells and plants derived therefrom - Google Patents

Abstract

DNA constructs comprise a DNA sequence homologous to some or all of a gene encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase, 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 the glyceraldehyde 3-phosphate dehydrogenase gene is inhibited: fruit from the plants (such as tomatoes) can show modified ripening properties, such as inhibition of respiration.

Description

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.

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, in for example 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 Molecular Biology, 13, 303-311, 1990). Thus antisense RNA has been proven to be useful in achieving downregulation of gene expression in plants.

In this invention we postulate that antisense RNA can be used to downregulate the expression of genes encoding enzymes involved in respiration during ripening of tomato and other fruit. The effect of this downr.gula ion in gene expression will be to reduce the respirative capacity of the fruit. The consequent retardation of the metabolism will slow the ripening and subsequent deterioration of the fruit.

In work leading to the present invention we have identified tomato genes which encode the enzyme glyceraldehyde 3-phosphate dehydrogenase which is involved in respiration during the ripening of tomatoes. Clones encoding parts of these genes have been identified and characterised. We postulate that they will be of use in modifying the ripening characteristics of tomatoes and other fruit. The genes in question are encoded (in part) in the clones pT0M38, M13T38S3.1 and M13T38S4.1 not previously disclosed.

According to the present invention we provide DNA constructs comprising a DNA sequence homologous to some or all of a gene encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase, under control of a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells. Such DNA constructs may comprise a DNA sequence homologous to some or all of a gene partially encoded by any of the clones pT0M38, M13T38S3.1 and M13T38S4.1 preceded by a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells.

In a further aspect, the 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 mRNA encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase. The invention also includes plant cells containing such constructs; plants derived therefrom showing modified ripening characteristics; and seeds of such plants. Suitable DNA may be obtained from the genes which are partially encoded by the clones pT0M38, M13T38S3.1 and M13T38S4.1 or from cDNA of such genes.

The constructs of the invention may be inserted into plants to regulate the production of the enzyme glyceraldehyde 3-phosphate dehydrogenase. Depending on the nature of the construct, the production of the enzyme 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 enzyme is enhanced only by constructs which express RNA homologous to the substantially complete endogenous 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.

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 have modified expression levels of genes encoding respiration enzymes and which may have one or more of the following characteristics:

Improved resistance to damage during harvest, packaging and transportation due to slowing of the ripening and overripening 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 the ability of fruit ripened on the plant to withstand handling and transportation.

Increased sweetness of the fruit due to prolonged accumulation of sugars whilst ripening on the plant and decreased degradation of sugars due to retarded overripening.

Modified colour due to inhibition of the pathways of pigment biosynthesis (e.g. lycopene, β-carotene). DNA constructs according to the invention preferably comprise a base sequence at least 10 bases in length for transcription into antisense 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.

The preferred DNA for use in the present invention is DNA derived from the clones pT0M38, M13T38S3.1 or M13T38S4.1. 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 pT0M38, M13T38S3.1 or M13T38S4.1 mRNA. Thus 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 cDNA clones such as pTOM38, M13T38S3.1 or M13T38S4.1. The base sequences of pT0M38, M13T38S3.1 and M13T38S4.1 are set out in Figure 1.

Searches in DNA data bases indicate that these clones show significant homology to clones for the glycolysis enzyme, cytosolic glyceraldehyde 3-phosphate dehydrogenase, from mustard, maize and tobacco. pT0M38 has been deposited on 1

September 1989 with the National Collections of Industrial and Marine Bacteria, Aberdeen, under Accession No.

NCIB 40193. M13T38S3.1 and M13T38S4.1 have been similarly deposited on 26 September 1991 under Accession Nos. NCIB 40448 and 40449, respectively. Alternatively, cDNA clones similar to pT0M38 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 that produces the cytosolic glyceraldehyde 3-phosphate dehydrogenase enzyme.

Suitable lengths of the cDNA so obtained may be cut out for use by means of restriction enzymes.

As previously stated, a preferred source of RNA for use in the present invention is DNA showing homology to the gene encoded by the clone pT0M38. pT0M38 was derived from a cDNA library isolated from ripe tomato RNA (Slater et al Plant Molecular Biology 5, 137- 147, 1985). DNA sequence analysis has demonstrated that the cDNA insert of pT0M38 is 848 bases long. M13T38S3.1 and M13T38S4.1 were derived from a cDNA library obtained from Clontech Laboratories Incorporated. The 5' ends of the cDNA inserts of two clones (lambdaT38S3.1 and lambdaT38S4.1) from this library which hybridised to pT0M38 were amplified by PCR and subcloned into plasmid vectors to give M13T38S3.1 and M13T38S4.1 respectively. These two clones contain sequence 5' of the cDNA sequence in pT0M38. It has been shown that the mRNA for which pT0M38 codes is expressed in ripening tomato fruit. pT0M38 mRNA was detected at reduced levels in green fruit, and in ripening fruit of the rin mutant (Knapp PhD Thesis, University of Nottingham 1988). Although information on the structure and expression of the pT0M38 gene family is known, the biochemical function of the products of these genes has not hitherto been fully elucidated.

An alternative source of DNA for the base sequence for transcription is a suitable gene encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase. This gene may differ from the cDNA of, e.g. pT0M38 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 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 pT0M38) 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 Agrobacteriu tumefaciens nopaline synthase gene, the nos 3' end). According to one aspect of the invention we propose to use inducible or developmentally regulated promoters (such as the ripe-fruit-specific polygalacturonase promoter) since it is often desirable to inhibit respiration only during fruit development and ripening. Use of a constitutive promoter will tend to affect respiration in all parts of the plant which may inhibit growth and development of the whole plant. By using a tissue-specific promoter, respiration 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). 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 ripening- specific E8 promoter (Diekman & Fischer, 1988 EMBO 7, 3315-3320) and the fruit-specific 2A11 promoter (Pear et al, 1989 Plant Molecular Biology 13, 639-651).

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 Agrobacteriu Ti plasmid technology. For example, tomato transformation is described by Fillatti et al. , Biotechnology, Vol. 5, July 1987, pp 726-730. Such transformed plants may be reproduced sexually, or by cell or tissue culture.

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

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

Figure 1 shows the base sequence of the clones pT0M38, M13T38S3.1 and M13T38S4.1.

Figure 2 shows the base sequence of oligonucleotide primers for amplification of a fragment of pT0M38 by PCR.

The following Examples illustrate aspects of the invention.

EXAMPLE 1

Construction of antisense RNA vectors with the CaMV 35S promoter

The vector pJR138A is constructed using the sequences corresponding to the complete insert of M13T38S3.1. This 626bp fragment is synthesised by polymerase chain reaction using synthetic primers (Figure 2). The ends of the fragment are made flush with T4 polymerase and it is cloned into the vector pJRl which has previously been cut with Smal. 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 (nos) 3' end termination sequence.

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 M13T38S3.1 cDNA that was described in Example 1 is also cloned into the vector pJR3 to give pJR338A.

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 pT0M38 sequences are identified by DNA sequence analysis.

EXAMPLE 3 Construction of sense RNA vectors with the CaMV 35S promoter The fragment of M13T38S3.1 cDNA that was described in Example 1 is also cloned into the vector pJRl in the sense orientation to give pJR138S.

After synthesis, the vectors with the sense orientation of pT0M38 sequence are identified by DNA sequence analysis.

EXAMPLE 4

Construction of sense RNA vectors with the polygalacturonase promoter.

The fragment of M13T38S3.1 cDNA that was described in Example 1 is also cloned into the vector pJR3 in the sense orientation to give pJR338S.

After synthesis, the vectors with the sense orientation of pT0M38 sequence are identified by DNA sequence analysis.

EXAMPLE 5

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 stem segments follow 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 kana ycin. Plants are regenerated and grown to maturity. Ripening fruit are analysed for modifications to their ripening characteristics.

Claims

We claim:

1. DNA constructs comprising a DNA sequence homologous to some or all of a gene encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase, 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 a gene encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase.

3. DNA constructs as claimed in claim 1 in which the homologous DNA is homologous to DNA from pT0M38, M13T38S3.1 or M13T38S4.1.

4. DNA constructs as claimed in claim 1 which contain DNA homologous to the sequences set forth in any of Figures 1A, IB or lC.

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

6. DNA constructs as claimed in claim 5 in which the constitutive promoter is CaMV 35S.

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

8. DNA constructs as claimed in claim 7 in which the promoter is that for the polygalacturonase gene.

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

10. Plant cells claimed in claim 9 which are cells of tomato.

11. Plant cells claimed in claim 9 which are cells of mangoes, peaches, apples, pears, bananas, melons or strawberries, or of carnations or other ornamental flowers.

12. Plants containing cells claimed in claim 9.

13. Plants claimed in claim 12 which bear climacteric fruit.

14. Fruit or seeds of plants claimed in claim 13.

15. Tomato seeds as claimed in claim 14 containing a construct adapted to express RNA antisense to mRNA expressed by a gene encoding the plant enzyme glyceraldehyde 3-phosphate dehydrogenase.