WO1992006206A1

WO1992006206A1 - Dna, dna constructs, cells and plants derived therefrom

Info

Publication number: WO1992006206A1
Application number: PCT/GB1991/001721
Authority: WO
Inventors: Colin Roger Bird; Donald Grierson; John Anthony Ray; Wolfgang Walter Schuch
Original assignee: Imperial Chemical Industries Plc
Priority date: 1990-10-09
Filing date: 1991-10-04
Publication date: 1992-04-16
Also published as: GB9021923D0; AU8641391A; EP0552207A1

Abstract

DNA constructs comprising a DNA sequence homologous to some or all of a fruit-ripening gene encoded by the clone pTOM99 preceded by a transcriptional initiation region operative in plants, so that the construct can generate RNA in plant cells. The sequence may be inverted, so as to produce antisense RNA. The invention also includes plant cells containing such constructs; plant (e.g. tomatoes) derived therefrom showing modified ripening characteristics; and fruit and seeds of such plants.

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 (e.g. 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 colour petals (van der Krol et al, Nature, 333, 866-869, 1988); at a more subtle biochemical level e.g. change in the amount of polygalacturonase and reduction in depolymerisation 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 antisen 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 an enzyme involved in ripening of tomatoes. This gene has been cloned and characterised. We postulate that it will be of use in modifying the ripening characteristics of tomatoes and other fruit. The gene in question is encoded (almost completely) in the clone pTOM99, which has 100% homology with the clone E8 disclosed by Diekman and Fischer (The EMBO Journal, 7, 3315, 1988).

According to the present invention we provide DNA constructs comprising a DNA sequence homologous to some all of the gene encoded by the clone pTOM99, preceded by transcriptional initiation region operative in plants, s 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 an mRNA encoding the enzyme produced by the gene for the pTOM99 cDNA. The invention also includes 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 enzymes encoded by genes homologous to pTOM99. Depending on the nature of the construct, the production of the enzymes 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 pTOM99 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 f uit-bearing plants, in particular tomato. In this way, plants can be generated which have modified expression levels of the pTOM99 gene and 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 the tomato flavour. Sweeter tomatoes due tc decrease in the accumulation of acids (e.g. citric or malic acid) thereby allowing the flavour of the sugars to dominate.

Modified colour due to inhibition of the pathways of pigment biosynthesis (e.g. lycopene, β-carotene).

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.

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.

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 clone pTOM99. The required antisense DNA can be obtained in several ways: by cutting with restriction enzymes an appropriate sequence of such DNA; by ς _

synthesismg 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 that the cut DNA sequence is inverted with respect to its orientation in the strand from which i 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 pTOM99 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 pTOM99. The base sequence of pTOM99 is set out in Figure 1. Searches in DNA data bases have revealed 100% homology to a known ethylene-responsive fruit ripening gene from tomato known as E8 (Lincoln et al, Proceedings of the National Academy of Sciences USA, 84, 2793, 1987; Diekman and Fischer ,1988, cited above). The function of the E8 gene is not known.

pTOM99 has been deposited on 14 September 1990 with the National Collections of Industrial and Marine Bacteria, Aberdeen, under Accession No. NCIB 40317. Alternatively, cDNA clones similar to pTOM99 may be obtained from the mRN of ripening tomatoes by the method described by Slater et al, Plant Molecular Biology 5, 137- 147, 1985. In this wa may be obtained sequences coding for the whole, or substantially the whole, of the mRNA produced by pTOM99. Suitable lengths of the cDNA so obtained may be cut out for use by-- means of restriction enzymes.

As previously stated, the preferred source of RNA for use in the present invention is DNA showing homology to the gene encoded by the clone pTOM99. pTOM99 was derived from a cDNA library isolated from ripe tomato. RNA (Slater et al Plant Molecular Biology 5, 137-147, 1985). Seven other clones (pTOM9 , 29, 44, 60, 61, 93 and 103) from the same library cross-hybridise to pTOM99 and probably contain related sequences. DNA sequence analysis has demonstrated that the cDNA insert of pTOM99 is 1166 bases long.

It has been shown that the mRNA for which pTOM99 codes is expressed in ripening tomato fruit. No expre^ssion of pTOM99 could be detected in green fruit (Maunders et al, Plant Cell and Environment, 10, 177, 1987). pTOM99 is expressed most strongly at the full orange stage of ripening. The levels of mRNA then declines in line with the general decline in biosynthetic capacity of the ripening fruit. The expression of pTOM99 is reduced in the Ripening inhibitor (rin) tomato fruit ripening mutants which mature very slowly ( Knapp et al , Plant Molecular Biology, 12, 105, 1989).

Although a considerable body of information on the structure and expression of the pTOM99 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 pTOM99 protein. This gene may differ from the cDNA of, e.g. pTOM99 in that intronε 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 guide.

Recombinant DNA and vectors according to the presen invention may be made as follows. A suitable vector containing the desired base sequence for transcription (fo example pTOM99) is treated with restriction enzymes to cut the sequence out. The DNA strand so obtained is cloned (i desired, in reverse orientation) into a second vector containing the desired promoter sequence ( for example cauliflower mosaic virus 35s RNA promoter or the tomato oolygalacturonase gene promoter sequence - Bird et al., Plant Molecular Biology, 11, 651-662, 1988) and the desire 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 i 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). Use of this promoter, at least in tomatoes, has the advantage that the production o 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).

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 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 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, melons and citrus fruit.

The invention will now be described further with reference to the accompanying drawings, in which: Figure 1 shows the base sequence cf the clones pTOM99;

Figure 2 shows the method of construction of pJRl99A.

The following Examples illustrate aspects of the invention.

EXAMPLE 1

Construction of pTOM99 antisense RNA vectors with th CaMV 35S promoter

The vector pJRl99A was constructed using the sequences corresponding to bases 1 to 776 of pTOM99 (Fig 2) . This fragment was synthesised by polymerase chain reaction using synthetic primers. The fragment was cloned into the vector pJRI which had previously been cut with Smal. pJRI (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 pTOM99 sequences were confirmed by DNA sequence analysis.

EXAMPLE 2

Construction of pTOM99 antisense RNA vectors with th polygalacturonase promoter. The fragment of the pTOM99 cDNA that was described in Example 2 is also cloned into the vector pJR2 to give pJR299A.

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.

After synthesis, vectors with the correct orientation ύf pTOM99 sequences are identified by DNA sequence analysis.

EXAMPLE 3

Construction of pTOM99 sense RNA vectors with the CaMV 35S promoter

The fragment of pTOM99 cDNA that was described in Example 2 was cloned into the vector pJRI in the sense orientation to give pJRl99S.

After synthesis, the vectors with the sense orientation of pTOM99 sequence were identified by DNA sequence analysis.

EXAMPLE 4

Generation of transformed plants

Vectors from Example 1 were transferred to Agrobacterium tumefacienε LBA4404 (a micro-organism widely available to plant biotechnologists ) and were used to transform Ailsa Craig tomato plants. Transformation of tomato stem segments followed standard protocols (e.g. Bird et al Plant Molecular Biology 11, 6.51-662, 1988). Transformed plants were identified by their ability to grow on media containing the antibiotic kanamycin. 36 transformed plants were grown to maturity. Northern blot analysis of ripe fruit mRNA from 9 of these transformants showed that in 8 of the plants the abundance of the 1.45kb pTOM99 gene transcript is greatly reduced. Ripening fruit from the transformed plants were observed for modifications to their ripening characteristics. Some of them exhibited slow fruit coloration. This may be related specifically to reduced pigment accumulation during fruit ripening, or it may be a symptom of a more general delay in the fruit ripening process. Some plants also showed retarded seed development or abnormal leaf growth - it is not clear whether these effects are due to the antisense mRNA or to tissue-culture-induced mutations.

Selfed progeny from 5 of the primary transformant identified were germinated. Genomic Southern analysis of the progeny from one such primary transformant has confirmed that the pTOM99 antisense gene is inherited. The selfed progeny are being grown to maturity so that their fruit can be observed.

Claims

CLAIMS :

1. DNA constructs comprising a DNA sequence showing homology to at least some of the gene for the gene product encoded by the clone pTOM99, preceded by a transcriptional initiation region operative in plants, so that the construct can generate mRNA 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 base sequence showing substantial homology to an mRNA encoding the enzyme produced by the gene of pTOM99.

3. DNA constructs as claimed in claim 1 comprising a transcriptional initiation region operative in plants positioned for transcription of a DNA sequence to give mRNA which can be translated to give the enzyme produced by the gene of pTOM99.

4. DNA constructs as claimed in claim 1 comprising a transcriptional initiation region operative in plants positioned for transcription of a DNA sequence encoding RNA showing substantial homology to part only of the mRNA encoding the enzyme produced by the gene of pTOM99.

5. DNA constructs as claimed in any of claims 1 to 4 in which the transcriptional initiation region is a constitutive promoter such as CaMV 35S. 13 -

6. DNA constructs as claimed in any of claims 1 to 4 in which the transcriptional initiation region is an inducible or developmentally-regulated or tissue- specific promoter.

7. DNA constructs as claimed in claim 6 in which the promoter is the polygalacturonase promoter.

8. Plant cells transformed with constructs claimed in any of claims 1-7, and descendants thereof.

9. Plants comprising cells claimed in claim 8; and seeds thereof.

10. Plants and seeds as claimed in claim 9 which are fruit, eg tomatoes, mangoes, peaches, apples, pears, s-trawberries , bananas, melons or citrus fruit.