CA2106091A1 - Expression of genes in transgenic plants - Google Patents
Expression of genes in transgenic plantsInfo
- Publication number
- CA2106091A1 CA2106091A1 CA 2106091 CA2106091A CA2106091A1 CA 2106091 A1 CA2106091 A1 CA 2106091A1 CA 2106091 CA2106091 CA 2106091 CA 2106091 A CA2106091 A CA 2106091A CA 2106091 A1 CA2106091 A1 CA 2106091A1
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- dna construct
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8237—Externally regulated expression systems
- C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8235—Fruit-specific
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- General Chemical & Material Sciences (AREA)
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Abstract
DNA construct for use in transforming plant cells which comprises an exogenous coding sequence with upstream promoter and downstream terminator sequences, the promoter being a DNA sequence homologous to the DNA control sequence found upstream of a gene involved in carotenoid biosynthesis, for example the gene encoding phytoene synthase. The invention also includes plant cells containing such constructs and plants derived therefrom. Plants according to the invention may be stimulated to express the exogenous coding sequences by application of ethylene.
Description
2 i O ~ 3 ~1 PCT/GB92/00~2 EXPRESSION OF GENES I~ TRANSGENIC PLANTS
The present invention relates to the expression of genes in transgenic plants. In particular it is concerned with the isolation and use of DNA sequences which control the expression of foreign genes in ripening fruits and in - response to ethylene.
The ability to isolate and manipulate plant genes has opened the way to gain understanding about the mechanisms involved in the regulation of plant gene expression. This knowledge is important for the exploitation of genetic engineering techniques to applied problems such as the expression of genes in genetically manipulated crop plants exhibiting improved quality and production characteristics.
Many examples are now in the literature of plant DNA
sequences which have been used to drive the expression of l; foceign genes in plants. In most instances the regions immediately 5' to the coding regions of genes have been used in gene constructs. These regions are referred to as promoter sequences. They may be derived from plant DNA; or Crom other sources, eq, viruses. It has been demonstrated 'O that sequences up to 500-1000 bases in most instances are sufficient to allow for the regulated expression of foreign genes. This regulation has involved tissue-specificity, regulation by external factors such as light, heat treatment, chemicals, hormones, and developmental ~; regulation. However, it has also been shown that sequences much longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants.
These experiments have been carried out using gene fusions between the promoter sequences and foreign genes such as bacterial promoter genes, etc. This has led to the identification of useful promoter sequences. In work leading to the present invention we have identified a gene which expresses an enzyme involved in the ripenina of ~` .
..
-t ; , , ~106091 W O 92/16635 P(~r/GB92/00442 tomatoes. We have now shown that it is involved in carotenoid synthesis. The gene in question is encoded (almost completely) in the clone pTOM5, disclosed by Ray et al (Nucleic Acids Research, 1;, 10587, 19~9). Hereinafter this gene is referred to as the phytoene synthase (or PS~
gene; the enzyme for which the pTOM5 gene codes is the ; pTOM5 gene product. We have shown that the pTOM5 gene is involved in the step or steps of the pathway between geranylgeranyl pyrophosphate and phytoene, and that the pTOM5 gene product is the enzyme known as phytoene synthase. Among the products produced by this branch of the pathway are carotenes, lutein, xanthophylls, and pigments such as lycopene, as well as plant growth regulators such as IBA. We have now isolated a part of the chromosomes of tomato in which the pTOM5 gene is localised.
We now disclose the structure of this gene and its transcriptional control sequences, in particular its promoter.
Evidence for the involvement of the pTOMS gene product in carotenoid synthesis has come from experiments in which the expression of the pTOM5 gene has been inhibited using antisense RNA (see PCT patent application 90/01924). The resulting plants have fruit which are yellow and Lack lycopene t indicating that lycopene synthesis has been inhibited. Biochemical precursor feeding experiments have shown that geranylgeranyl pyrophosphate accumulates in extracts of these fruit, indicating that phytoene synthase is inhibited.
Further evidence for the function of the pTOM5 gene in the carotenoid pathway is the significant degree of homology (27~ identity; 17~ similari~y) between the polypeptide predicted from the translation of the open sequence in the clone pTOMS and the protein encoded by the crtB gene from Rhodobacter capsulatus, a gram-negative purple non-sulphur bacterium. The crtB gene product catalyses the tail-to-tail dimerisation of geranylgeranyl ~092/1~63~ 210 ~ O ~1 PCT/GB92/00442 diphosphate to form prephytoene diphosphate. This enzyme (phytoene synthase) is the point of divergence of carotenoid biosynthesis from other isoprenoid metabolism.
Further, an enzyme has been isolated from Capsicum annuum - fruit chromoplasts which is believed to catalyse both the synthesis of prephytoene diphosphate and its subsequent conversion to phytoene. This enzyme has a molecular weight of 47,500, in close agreement with the predicted size of the pTOM5 gene product (48,000). The ~inal conclusion comes from complementation experiments in which pTOM5 cDNA
has been used to complement an Erwinia mutant which is deficient in phytoene synthase.
We have shown that phytoene synthase mRNA is expressed in ripening tomato fruit. No expression could be l- detected in green fruit. The phytoene synthase gene 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 phytoene synthase mRNA could also be ~0 induced by exposing mature green fruit to exogenous ethylene. The expression of the phytoene synthase gene is reduced in the Ripening Inhibitor (rin) and Neverripe (Nr) tomato fruit ripening mutants, which mature very slowly and never achieve the full red c~lour of ordinary tomato fruit.
; The genomic locations in the tomato of sequences komologous to the pTOM5 clone have been identified using RFLP mapping: two ioci, on chromosome 2 and chromosome 3 respectively, carry sequences homologous to the pTOM5 clone. It has also been shown by Southern blotting that the pTOM5 gene may be present as a small multigene family.
The present invention proposes to use the promoters of the phytoene synthase and similar genes to control the expression of novel and exogenous proteins and genes in somato fruit.
According to the present invention we provide a DNA
-onstruct for use in transforming plant cells which W092/1663~ 21~ 1 PCT/GB92/00442 comprises an exogenous coding sequence under the controi c-upstream promoter and downstream terminator sequences, characterised in that the upstream promoter has homology ~c a promoter of a gene of the carotenoid biosynthesis 5 pathway. We further provide novel plant cells, and plants, particularly tomatoes, transformed with constructs according to the present invention.
We further provide a process for stimulating the expression of exogenous coding sequences in plants by applying ethylene to plants transformed with constructs according to the invention.
Promoters for use in the invention may be derived from genes such as phytoene desaturase, cyclase and epoxydase. Such promoters may be isolated from genomic 1; libraries by the use of cDNA probes, as has been done in the case of pTOM5. We particularly prefer to use the promoter of the phytoene synthase gene.
The downstream (3') terminator sequences can also be derived from the phytoene synthase gene: or they can be 0 derived from other genes such as the polygalacturonase gene (see UK Patent Application 9025323.9 filed ~3 November 1390). Many other possibilities are available from the literature.
8y the term 'exogenous coding sequence' we indicate a sequence of DNA, other than that which follows the promoter region in the natural pTOMS gene, that is adapted to be transcribed into functional RNA under the action of plant cell enzymes such as RNA polymerase. Functional RNA is RNA
which affects the biochemistry of the cell: it may for example be mRNA which is translated into protein by ribosomes; or antisense RNA which inAibits the translation of mRNA complementary (or otherwise related) to it into protein. In principle all kinds of exogenous coding sequences are useful in the present in~ention.
Where the exogenous coding sequence codes for mRNA
for a protein, this protein may be of bacterial origin W092/16635 210 ~ O 91 PCT/GB92/00~2 (sucn as enzymes involved in polysaccharide metabolism and cell wall metabolism), of eukaryotic origin (such as pharmaceutically active polypeptides) or of plant origin (such as the product of the phytoene synthase gene itself, enzymes involved in respiration, ethylene synthesis, sugar metabolism, aroma and flavour production and cell wall metabolism), or genes or parts thereof in sense and antisense orientation. Of particular interest is the ability of the phytoene synthase gene promoter to respond to exogenously supplied ethylene.
A wide variety of exogenous coding sequences is known from the literature, and the present invention is applicable tO these as well as many others yet to be reported. As well as functional mRNA, the exogenous gene may code for RNA that interferes with the function of any kind of mRNA produced by the plant cell: for example, antisense RNA complementary to mRNA for fruit ripening genes such as polygalacturonase, pectinesterase, ~-1,4-glucanase, pTOM13 etc.
'0 The construction of these vectors and constructs is described in more detail in the Examples below. For convenience it will be generally found suitable to use promoter sequences (upstream - i.e. S' - of the coding sequence of the gene) of between 100 and 2000 bases in length.
Plant cells according to the invention may be transf~rmed with constructs of the invention according to a variety of known methods (Aarobacterium Ti plasmids, electroporation, microinjection, microprojectile gun, etc).
The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated in~o the genome. ~oth transformed monocot and dicot plants may be obtained in this way, although the latter are usually more easy to regenerate.
Examples of genetically modified plants according to WO92/16635 ~ 1 0 6 ~ ~1 PCT/GB92/~0~42 the present invention include, as well as tomatoes, fruits such as mangoes, peaches, apples, pears, strawberries, bananas and melons; and field crops such as maize (corn), sunflowers, sugarbeet, canola, and smallgrain cereals sucn ; as wheat, barley and rice.
Plants produced by the process of the invention may contain more than one recombinant construct. As well as one or more constructs containing the phytoene synthase promoter, they may contain a wide variety of other recombinant constructs, for example constructs having different effects on fruit ripening. In particular where the invention is applied to tomatoes, these may be of enhanced colour (as a result of inserting extra gene copies of the PS gene and thereby overexpressing phytoene i5 synthase) and may also contain constructs inhibiting the production of enzymes such as polygalacturonase and pectinesterase, or interfering with ethylene production (eg from pTOMl3, see PCT Application 90~01072 filed 12 July l990). Such tomatoes can have higher solids contents than conventional tomatoes and produce more tomato paste per unit of fruit weight. The extra lycopene production in such tomatoes is desirable to prevent any lightening of colour that might otherwise be observed in such pastes.
Tomatoes containing rlo,re than one type of recombinant construct may be made either by successive transformations, or by successively crossing varieties that each contain one of the constructs, and selecting among the progeny for those that contain all the desired constructs.
A further aspect of the present invention is a process of activating exogenous coding sequences in plants under the control of the phytoene synthase promoter which comprises the application of exogenous ethylene. This may find particular use when fruit is stored in the absence of ethylene, and ethylene is then used to switch on the production of a given useful character providing extra value to the fruit at the point of sale. This may lead tc 2~06091 , increase in sweetness of the fruit, or the production of special flavours or aromas, or the production of special polypeptides desired by the consumer. This will enable more flexibility in control of the fruit ripening process, 3 particularly at the point of sale.
We now describe the isolation of genomic clones from a tomato library encoding the phytoene synthase gene and related sequences. Genomic clones representing two individual genes have been isolated and characterised by DNA sequence analysis. The clone gTOM5 represents part of a gene with exon sequence identical to the clone pToM5.
Clone F contains a sequence similar but not identical to pTOM5. Details of these clones are given below. Sequence and expression data suggest that Clone F encodes an untranscribed pseudogene. The genomic clones described in the Examples cover most of the coding region and the complete transcriptional initiation region of the phytoene synthase gene. The clone gTOM5 has been deposited at the National Collections of Industrial and Marine Bacteria (NCIa), now at 23 St. Machar Drive, Aberdeen AB2 lRY, Scotland, on 11 March 1991 under the reference ~CIB Number 40382 while pTOM5 has been deposited at NC~B as a plasmid in E.coli, under the reference NCIB 40191, on 1 September 1989.
'; The invention will be further described with reference to the following drawings, in which:
Figures 1 and lA show the nucleotide sequence of the 3.5 kb EcoRI - SalI fragment of gTOM5 (SEQ ID: 1) and the 3' region of the phytoene synthase gene (SEQ ID:
2);
Fi~ure 2 is a diagram of the structure of the phytoene synthase gene;
.
.
WO92/16635 %1~ ~ 0 91 P~T/GB92/00442 Figure 3 outlines a scheme for polymerase cAain reaction amplification of the phytoene synthase aene promoter fragment;
- Figure 4 outlines a scheme for construction of the plant transformation vector p5TA~.
1.1 Isolation of pToM5 related genes A library was constructed from tomato (Lycopersicon esculentum var. Ailsa Craig) genomic DNA which was ~ partially digested with Sau3A and cloned into lambda EMLL3 i (~ird et al (1988) Plant Molecular Biology 11, 651-662).
The library was screened with the pTOM5 CDNA insert (Ray et al (1987) Nucleic Acids Research 15, 10587) and positive phages were purified by four successive cycles of plaque purification. Five positive clones were isolated.
Restriction fragment mapping and DNA sequence '0 analysis of these clones indicated that all ; clones were overlapping and related. The clones did not have 100%
sequence homology to pTOM5 in the regions that probably represented exons. This indicated that these clones represented a gene (designated clone F) that was not the '; pTOM5 gene.
In order to isolate the phytoene synthase gene, synthetic oligonucleotides were designed that hybridised speciFically to either pTOM5 or the clone F. The sequences of oligonucleotides CL100 and CL99 represented a region where the pTOM5 sequence is only 54~ homologous to the sequence of clone F:
CL100 ~ CATCTGTTCCGATGTCATCGTCCG-3~ pTOM5 specific CL99 - 5~-TTTTTTTTCTGATGACACAGCCAT-3~ clone ~ specific WO92/1~35 210 ~ 0 91 PCT/GB92/00442 CL100 was used to screen the same genomic library. After four rounds of purification, one phage (designated GTOM5) was isolated which hybridised to CL100 and pTOM5 but not to CL99.
~ 5 `~ 1.2 Characterisatlon of the phytoene synthase gene promoter sequence :, A 3.5 kb EcoRI - SalI fragment was isolated from GTOM5 and the complete nucleotide sequence of fragment has been determined (Fig 1). This sequence contained exon regions that were 100% homologous to pTOM5 but did not contain the 3' end of the cDNA ( Fig 2~. The fragment contained 1.1 kb of sequence extending 5' of the end of the CDNA. This sequence represents the pTOM5 gene promoter.
1.3 Isolation and characterisation of the 3' region of the phytoene synthase gene Synthetic oligonucleotides were designed for use as primers for polymerase chain reaction (PCR) amplification of a specific fragment containing the 3' region of the pTOM5 gene with BamHI restriction sites at each end. The oligonucleotides (designated 5GENE-5 and 5GENE-3) contain sequences from base 3405 to 3442 of SEQ ID:l and 1604 to 1630 of the pTOM5 cDN~.
After PCR followed by BamHI digestion, two fragments (approximately 800 and 570 bp) were identified by agarose gel electrophoresis. These fragments were isolated, restricted with 8amHI and cloned into M13mpl8. Clones containing each fragment were identified and the nucleotide sequence was determined (Fig 1).
1.4 Isolation of a phytoene synthase gene promoter fragment 211)1j0~1 WO9~/16635 - I PCT/GB92/00442 Synthetic oligonucleotides were designed for use as primers for polymerase chain reaction (PCR) amplification of a specific fragment containing the phytoene synthase gene promoter with restriction sites at each end (5~- HindIII :
5 3~- BamHI). The oligonucleotides ~designated 5PRo-5 and 5PRO-3) contain sequences from base 1 to 30 and 115; to 1105 of the phytoene synthase gene:
.
aindIII
BamHI
These primers were used in a PCR with tomato genomic DNA (Lycopersicon esculentum var. Ailsa Craig) to amplify a 1171 bp fragment that contained the phytoene synthase gene promoter sequence and 52 bp of the 5' untranslated region of pTOM5 ~Fig 3). This fragment was digested with HindIII and BamHI and cloned into M13mpl8. The nucleotide sequence of one clone (pSpRo~ was found to be identical to that of the same region of GTOM5.
2;
1.5 Construction of plant transformation vector - pSTAK
The 1151 bp HindIII/BamHI phytoene synthase gene promoter fragment from the M13mpl8 clone (pSPRO) is exciseà
from replicative form DNA and cloned into HindIII and BamH;
cut pTAKl (described in EP 271988 A). Plasmids with the correct orientation of the PS gene promoter are identified by restriction analysis and DNA sequencing. One such clone is designated p5TAK (Fig 4).
WO92/16~35 21~ ~ 0 91 PCT/~B92/00~42 EXAMPLE
Generation of transformed plants The vector p5TAK (from Example 1.5) is transferred to Aqrobacterium tume~aciens LBA4404 (a micro-organism widely 5 available to plant biotechnologists) and is used to -transform tomato plants. Transformation of tomato stem segments follows standard protocols (eg. Bird et al Plant Molecular Biology _, 651-662, 1988). Transformed plants are identified by their ability to grow on media containing iO the antibiotic kanamycin. Plants are regenerated and grown to maturity.
The ripening-specific eXprecsion of the ~-glucuronidase (GUS) gene as determined by the phytoene synthase gene promoter is demonstrated by analysis of mature green, breaker and ripening fruit for GUS enzyme activity. The response of the gene to exogenous ethylene is demonstrated by incubation of breaker stage fruit in an atmosphere containing additional ethylene followed by 0 analysis of GUS enzyme activity.
The present invention relates to the expression of genes in transgenic plants. In particular it is concerned with the isolation and use of DNA sequences which control the expression of foreign genes in ripening fruits and in - response to ethylene.
The ability to isolate and manipulate plant genes has opened the way to gain understanding about the mechanisms involved in the regulation of plant gene expression. This knowledge is important for the exploitation of genetic engineering techniques to applied problems such as the expression of genes in genetically manipulated crop plants exhibiting improved quality and production characteristics.
Many examples are now in the literature of plant DNA
sequences which have been used to drive the expression of l; foceign genes in plants. In most instances the regions immediately 5' to the coding regions of genes have been used in gene constructs. These regions are referred to as promoter sequences. They may be derived from plant DNA; or Crom other sources, eq, viruses. It has been demonstrated 'O that sequences up to 500-1000 bases in most instances are sufficient to allow for the regulated expression of foreign genes. This regulation has involved tissue-specificity, regulation by external factors such as light, heat treatment, chemicals, hormones, and developmental ~; regulation. However, it has also been shown that sequences much longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants.
These experiments have been carried out using gene fusions between the promoter sequences and foreign genes such as bacterial promoter genes, etc. This has led to the identification of useful promoter sequences. In work leading to the present invention we have identified a gene which expresses an enzyme involved in the ripenina of ~` .
..
-t ; , , ~106091 W O 92/16635 P(~r/GB92/00442 tomatoes. We have now shown that it is involved in carotenoid synthesis. The gene in question is encoded (almost completely) in the clone pTOM5, disclosed by Ray et al (Nucleic Acids Research, 1;, 10587, 19~9). Hereinafter this gene is referred to as the phytoene synthase (or PS~
gene; the enzyme for which the pTOM5 gene codes is the ; pTOM5 gene product. We have shown that the pTOM5 gene is involved in the step or steps of the pathway between geranylgeranyl pyrophosphate and phytoene, and that the pTOM5 gene product is the enzyme known as phytoene synthase. Among the products produced by this branch of the pathway are carotenes, lutein, xanthophylls, and pigments such as lycopene, as well as plant growth regulators such as IBA. We have now isolated a part of the chromosomes of tomato in which the pTOM5 gene is localised.
We now disclose the structure of this gene and its transcriptional control sequences, in particular its promoter.
Evidence for the involvement of the pTOMS gene product in carotenoid synthesis has come from experiments in which the expression of the pTOM5 gene has been inhibited using antisense RNA (see PCT patent application 90/01924). The resulting plants have fruit which are yellow and Lack lycopene t indicating that lycopene synthesis has been inhibited. Biochemical precursor feeding experiments have shown that geranylgeranyl pyrophosphate accumulates in extracts of these fruit, indicating that phytoene synthase is inhibited.
Further evidence for the function of the pTOM5 gene in the carotenoid pathway is the significant degree of homology (27~ identity; 17~ similari~y) between the polypeptide predicted from the translation of the open sequence in the clone pTOMS and the protein encoded by the crtB gene from Rhodobacter capsulatus, a gram-negative purple non-sulphur bacterium. The crtB gene product catalyses the tail-to-tail dimerisation of geranylgeranyl ~092/1~63~ 210 ~ O ~1 PCT/GB92/00442 diphosphate to form prephytoene diphosphate. This enzyme (phytoene synthase) is the point of divergence of carotenoid biosynthesis from other isoprenoid metabolism.
Further, an enzyme has been isolated from Capsicum annuum - fruit chromoplasts which is believed to catalyse both the synthesis of prephytoene diphosphate and its subsequent conversion to phytoene. This enzyme has a molecular weight of 47,500, in close agreement with the predicted size of the pTOM5 gene product (48,000). The ~inal conclusion comes from complementation experiments in which pTOM5 cDNA
has been used to complement an Erwinia mutant which is deficient in phytoene synthase.
We have shown that phytoene synthase mRNA is expressed in ripening tomato fruit. No expression could be l- detected in green fruit. The phytoene synthase gene 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 phytoene synthase mRNA could also be ~0 induced by exposing mature green fruit to exogenous ethylene. The expression of the phytoene synthase gene is reduced in the Ripening Inhibitor (rin) and Neverripe (Nr) tomato fruit ripening mutants, which mature very slowly and never achieve the full red c~lour of ordinary tomato fruit.
; The genomic locations in the tomato of sequences komologous to the pTOM5 clone have been identified using RFLP mapping: two ioci, on chromosome 2 and chromosome 3 respectively, carry sequences homologous to the pTOM5 clone. It has also been shown by Southern blotting that the pTOM5 gene may be present as a small multigene family.
The present invention proposes to use the promoters of the phytoene synthase and similar genes to control the expression of novel and exogenous proteins and genes in somato fruit.
According to the present invention we provide a DNA
-onstruct for use in transforming plant cells which W092/1663~ 21~ 1 PCT/GB92/00442 comprises an exogenous coding sequence under the controi c-upstream promoter and downstream terminator sequences, characterised in that the upstream promoter has homology ~c a promoter of a gene of the carotenoid biosynthesis 5 pathway. We further provide novel plant cells, and plants, particularly tomatoes, transformed with constructs according to the present invention.
We further provide a process for stimulating the expression of exogenous coding sequences in plants by applying ethylene to plants transformed with constructs according to the invention.
Promoters for use in the invention may be derived from genes such as phytoene desaturase, cyclase and epoxydase. Such promoters may be isolated from genomic 1; libraries by the use of cDNA probes, as has been done in the case of pTOM5. We particularly prefer to use the promoter of the phytoene synthase gene.
The downstream (3') terminator sequences can also be derived from the phytoene synthase gene: or they can be 0 derived from other genes such as the polygalacturonase gene (see UK Patent Application 9025323.9 filed ~3 November 1390). Many other possibilities are available from the literature.
8y the term 'exogenous coding sequence' we indicate a sequence of DNA, other than that which follows the promoter region in the natural pTOMS gene, that is adapted to be transcribed into functional RNA under the action of plant cell enzymes such as RNA polymerase. Functional RNA is RNA
which affects the biochemistry of the cell: it may for example be mRNA which is translated into protein by ribosomes; or antisense RNA which inAibits the translation of mRNA complementary (or otherwise related) to it into protein. In principle all kinds of exogenous coding sequences are useful in the present in~ention.
Where the exogenous coding sequence codes for mRNA
for a protein, this protein may be of bacterial origin W092/16635 210 ~ O 91 PCT/GB92/00~2 (sucn as enzymes involved in polysaccharide metabolism and cell wall metabolism), of eukaryotic origin (such as pharmaceutically active polypeptides) or of plant origin (such as the product of the phytoene synthase gene itself, enzymes involved in respiration, ethylene synthesis, sugar metabolism, aroma and flavour production and cell wall metabolism), or genes or parts thereof in sense and antisense orientation. Of particular interest is the ability of the phytoene synthase gene promoter to respond to exogenously supplied ethylene.
A wide variety of exogenous coding sequences is known from the literature, and the present invention is applicable tO these as well as many others yet to be reported. As well as functional mRNA, the exogenous gene may code for RNA that interferes with the function of any kind of mRNA produced by the plant cell: for example, antisense RNA complementary to mRNA for fruit ripening genes such as polygalacturonase, pectinesterase, ~-1,4-glucanase, pTOM13 etc.
'0 The construction of these vectors and constructs is described in more detail in the Examples below. For convenience it will be generally found suitable to use promoter sequences (upstream - i.e. S' - of the coding sequence of the gene) of between 100 and 2000 bases in length.
Plant cells according to the invention may be transf~rmed with constructs of the invention according to a variety of known methods (Aarobacterium Ti plasmids, electroporation, microinjection, microprojectile gun, etc).
The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated in~o the genome. ~oth transformed monocot and dicot plants may be obtained in this way, although the latter are usually more easy to regenerate.
Examples of genetically modified plants according to WO92/16635 ~ 1 0 6 ~ ~1 PCT/GB92/~0~42 the present invention include, as well as tomatoes, fruits such as mangoes, peaches, apples, pears, strawberries, bananas and melons; and field crops such as maize (corn), sunflowers, sugarbeet, canola, and smallgrain cereals sucn ; as wheat, barley and rice.
Plants produced by the process of the invention may contain more than one recombinant construct. As well as one or more constructs containing the phytoene synthase promoter, they may contain a wide variety of other recombinant constructs, for example constructs having different effects on fruit ripening. In particular where the invention is applied to tomatoes, these may be of enhanced colour (as a result of inserting extra gene copies of the PS gene and thereby overexpressing phytoene i5 synthase) and may also contain constructs inhibiting the production of enzymes such as polygalacturonase and pectinesterase, or interfering with ethylene production (eg from pTOMl3, see PCT Application 90~01072 filed 12 July l990). Such tomatoes can have higher solids contents than conventional tomatoes and produce more tomato paste per unit of fruit weight. The extra lycopene production in such tomatoes is desirable to prevent any lightening of colour that might otherwise be observed in such pastes.
Tomatoes containing rlo,re than one type of recombinant construct may be made either by successive transformations, or by successively crossing varieties that each contain one of the constructs, and selecting among the progeny for those that contain all the desired constructs.
A further aspect of the present invention is a process of activating exogenous coding sequences in plants under the control of the phytoene synthase promoter which comprises the application of exogenous ethylene. This may find particular use when fruit is stored in the absence of ethylene, and ethylene is then used to switch on the production of a given useful character providing extra value to the fruit at the point of sale. This may lead tc 2~06091 , increase in sweetness of the fruit, or the production of special flavours or aromas, or the production of special polypeptides desired by the consumer. This will enable more flexibility in control of the fruit ripening process, 3 particularly at the point of sale.
We now describe the isolation of genomic clones from a tomato library encoding the phytoene synthase gene and related sequences. Genomic clones representing two individual genes have been isolated and characterised by DNA sequence analysis. The clone gTOM5 represents part of a gene with exon sequence identical to the clone pToM5.
Clone F contains a sequence similar but not identical to pTOM5. Details of these clones are given below. Sequence and expression data suggest that Clone F encodes an untranscribed pseudogene. The genomic clones described in the Examples cover most of the coding region and the complete transcriptional initiation region of the phytoene synthase gene. The clone gTOM5 has been deposited at the National Collections of Industrial and Marine Bacteria (NCIa), now at 23 St. Machar Drive, Aberdeen AB2 lRY, Scotland, on 11 March 1991 under the reference ~CIB Number 40382 while pTOM5 has been deposited at NC~B as a plasmid in E.coli, under the reference NCIB 40191, on 1 September 1989.
'; The invention will be further described with reference to the following drawings, in which:
Figures 1 and lA show the nucleotide sequence of the 3.5 kb EcoRI - SalI fragment of gTOM5 (SEQ ID: 1) and the 3' region of the phytoene synthase gene (SEQ ID:
2);
Fi~ure 2 is a diagram of the structure of the phytoene synthase gene;
.
.
WO92/16635 %1~ ~ 0 91 P~T/GB92/00442 Figure 3 outlines a scheme for polymerase cAain reaction amplification of the phytoene synthase aene promoter fragment;
- Figure 4 outlines a scheme for construction of the plant transformation vector p5TA~.
1.1 Isolation of pToM5 related genes A library was constructed from tomato (Lycopersicon esculentum var. Ailsa Craig) genomic DNA which was ~ partially digested with Sau3A and cloned into lambda EMLL3 i (~ird et al (1988) Plant Molecular Biology 11, 651-662).
The library was screened with the pTOM5 CDNA insert (Ray et al (1987) Nucleic Acids Research 15, 10587) and positive phages were purified by four successive cycles of plaque purification. Five positive clones were isolated.
Restriction fragment mapping and DNA sequence '0 analysis of these clones indicated that all ; clones were overlapping and related. The clones did not have 100%
sequence homology to pTOM5 in the regions that probably represented exons. This indicated that these clones represented a gene (designated clone F) that was not the '; pTOM5 gene.
In order to isolate the phytoene synthase gene, synthetic oligonucleotides were designed that hybridised speciFically to either pTOM5 or the clone F. The sequences of oligonucleotides CL100 and CL99 represented a region where the pTOM5 sequence is only 54~ homologous to the sequence of clone F:
CL100 ~ CATCTGTTCCGATGTCATCGTCCG-3~ pTOM5 specific CL99 - 5~-TTTTTTTTCTGATGACACAGCCAT-3~ clone ~ specific WO92/1~35 210 ~ 0 91 PCT/GB92/00442 CL100 was used to screen the same genomic library. After four rounds of purification, one phage (designated GTOM5) was isolated which hybridised to CL100 and pTOM5 but not to CL99.
~ 5 `~ 1.2 Characterisatlon of the phytoene synthase gene promoter sequence :, A 3.5 kb EcoRI - SalI fragment was isolated from GTOM5 and the complete nucleotide sequence of fragment has been determined (Fig 1). This sequence contained exon regions that were 100% homologous to pTOM5 but did not contain the 3' end of the cDNA ( Fig 2~. The fragment contained 1.1 kb of sequence extending 5' of the end of the CDNA. This sequence represents the pTOM5 gene promoter.
1.3 Isolation and characterisation of the 3' region of the phytoene synthase gene Synthetic oligonucleotides were designed for use as primers for polymerase chain reaction (PCR) amplification of a specific fragment containing the 3' region of the pTOM5 gene with BamHI restriction sites at each end. The oligonucleotides (designated 5GENE-5 and 5GENE-3) contain sequences from base 3405 to 3442 of SEQ ID:l and 1604 to 1630 of the pTOM5 cDN~.
After PCR followed by BamHI digestion, two fragments (approximately 800 and 570 bp) were identified by agarose gel electrophoresis. These fragments were isolated, restricted with 8amHI and cloned into M13mpl8. Clones containing each fragment were identified and the nucleotide sequence was determined (Fig 1).
1.4 Isolation of a phytoene synthase gene promoter fragment 211)1j0~1 WO9~/16635 - I PCT/GB92/00442 Synthetic oligonucleotides were designed for use as primers for polymerase chain reaction (PCR) amplification of a specific fragment containing the phytoene synthase gene promoter with restriction sites at each end (5~- HindIII :
5 3~- BamHI). The oligonucleotides ~designated 5PRo-5 and 5PRO-3) contain sequences from base 1 to 30 and 115; to 1105 of the phytoene synthase gene:
.
aindIII
BamHI
These primers were used in a PCR with tomato genomic DNA (Lycopersicon esculentum var. Ailsa Craig) to amplify a 1171 bp fragment that contained the phytoene synthase gene promoter sequence and 52 bp of the 5' untranslated region of pTOM5 ~Fig 3). This fragment was digested with HindIII and BamHI and cloned into M13mpl8. The nucleotide sequence of one clone (pSpRo~ was found to be identical to that of the same region of GTOM5.
2;
1.5 Construction of plant transformation vector - pSTAK
The 1151 bp HindIII/BamHI phytoene synthase gene promoter fragment from the M13mpl8 clone (pSPRO) is exciseà
from replicative form DNA and cloned into HindIII and BamH;
cut pTAKl (described in EP 271988 A). Plasmids with the correct orientation of the PS gene promoter are identified by restriction analysis and DNA sequencing. One such clone is designated p5TAK (Fig 4).
WO92/16~35 21~ ~ 0 91 PCT/~B92/00~42 EXAMPLE
Generation of transformed plants The vector p5TAK (from Example 1.5) is transferred to Aqrobacterium tume~aciens LBA4404 (a micro-organism widely 5 available to plant biotechnologists) and is used to -transform tomato plants. Transformation of tomato stem segments follows standard protocols (eg. Bird et al Plant Molecular Biology _, 651-662, 1988). Transformed plants are identified by their ability to grow on media containing iO the antibiotic kanamycin. Plants are regenerated and grown to maturity.
The ripening-specific eXprecsion of the ~-glucuronidase (GUS) gene as determined by the phytoene synthase gene promoter is demonstrated by analysis of mature green, breaker and ripening fruit for GUS enzyme activity. The response of the gene to exogenous ethylene is demonstrated by incubation of breaker stage fruit in an atmosphere containing additional ethylene followed by 0 analysis of GUS enzyme activity.
Claims (15)
1. A DNA construct for use in transforming plant cells which comprises an exogenous coding sequence under the control of upstream promoter and downstream terminator sequences, characterised in that the upstream promoter has homology to a promoter of a gene of the carotenoid biosynthesis pathway.
2. A DNA construct as claimed in claim 1 in which the gene of the carotenoid biosynthesis pathway is the phytoene synthase gene.
3. A DNA construct as claimed in claim 2 in which the exogenous coding sequence codes for RNA that inhibits expression of a plant gene.
4. A DNA construct as claimed in claim 3 in which the exogenous coding sequence is antisense to part of the coding strand of a plant gene.
5. A DNA construct as claimed in claim 1 in which the exogenous coding sequence codes for mRNA that is translated into an enzyme functional in plants.
6. A DNA construct claimed in any of claims 2 to 4 in which the upstream promoter is homologous to the sequence shown in Figure 1.
7. A DNA construct claimed in claim 6 in which the upstream promoter is a DNA sequence homologous to at least 100 bases of the sequence shown in Figure 1.
8. Plant cells transformed with DNA constructs claimed in any of claims 1 to 7.
9. Plants comprising cells as claimed in claim 8.
10. Plants as claimed in claim 8 which are tomatoes, mangoes, peaches, apples, pears, strawberries, bananas or melons.
11. A process for stimulating the expression of exogenous coding sequences in plant cells by applying ethylene to plant cells claimed in claim 8.
12. A process as claimed in claim 11 in which the plant cells form part of a growing plant.
13. A process as claimed in claim 11 in which the plant cells form part of harvested material.
14. A process as claimed in any of claims 11-13 in which the exogenous coding sequences express mRNA that is translated into protein functional in the plant cell.
15. A process as claimed in claim 14 in which the protein is a fruit ripening enzyme.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB919105420A GB9105420D0 (en) | 1991-03-14 | 1991-03-14 | Expression of genes in transgenic plants |
| GB9105420.5 | 1991-03-14 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2106091A1 true CA2106091A1 (en) | 1992-09-15 |
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ID=10691573
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2106091 Abandoned CA2106091A1 (en) | 1991-03-14 | 1992-03-12 | Expression of genes in transgenic plants |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0619844A1 (en) |
| JP (1) | JPH06505871A (en) |
| AU (1) | AU1369092A (en) |
| BR (1) | BR9205770A (en) |
| CA (1) | CA2106091A1 (en) |
| GB (1) | GB9105420D0 (en) |
| WO (1) | WO1992016635A1 (en) |
Families Citing this family (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL107239A0 (en) * | 1992-10-15 | 1994-01-25 | Gen Hospital Corp | Crucifer acc synthase polypeptide, methods for the production thereof and uses thereof |
| AU690530B2 (en) * | 1992-12-15 | 1998-04-30 | Commonwealth Scientific And Industrial Research Organisation | DNA molecules encoding inducible plant promoters and tomato ADH2 enzyme |
| CA2158473A1 (en) * | 1993-03-22 | 1994-09-29 | Umi Kalsom Abu-Bakar | Dna, dna constructs, cells and plants derived therefrom |
| GB9314261D0 (en) * | 1993-07-09 | 1993-08-18 | Zeneca Ltd | Dwarf plants |
| AU2805395A (en) * | 1994-07-18 | 1996-02-16 | Zeneca Limited | Dna, constructs, cells and plants derived therefrom |
| AU719671B2 (en) * | 1995-06-07 | 2000-05-18 | Howard Foundation, The | Pharmaceutically active carotenoids |
| US6784351B2 (en) | 2001-06-29 | 2004-08-31 | Ball Horticultural Company | Targetes erecta marigolds with altered carotenoid compositions and ratios |
| US7575766B2 (en) | 2001-06-29 | 2009-08-18 | Ball Horticultural Company | Tagetes erecta with altered carotenoid compositions and ratios |
| US7081478B2 (en) | 2001-06-29 | 2006-07-25 | Chrysantis, Inc. | Mixed zeaxanthin ester concentrate and uses thereof |
| US7456335B2 (en) | 2001-09-03 | 2008-11-25 | Basf Plant Science Gmbh | Nucleic acid sequences and their use in methods for achieving pathogen resistance in plants |
| DE10212892A1 (en) | 2002-03-20 | 2003-10-09 | Basf Plant Science Gmbh | Constructs and methods for regulating gene expression |
| US7223909B2 (en) | 2002-03-21 | 2007-05-29 | Ball Horticultural | 4-ketocarotenoids in flower petals |
| DE10224889A1 (en) | 2002-06-04 | 2003-12-18 | Metanomics Gmbh & Co Kgaa | Process for the stable expression of nucleic acids in transgenic plants |
| WO2004013333A2 (en) | 2002-07-26 | 2004-02-12 | Basf Plant Science Gmbh | Inversion of the negative-selective effect of negative marker proteins using selection methods |
| CA2497635A1 (en) | 2002-09-03 | 2004-04-01 | Sungene Gmbh & Co. Kgaa | Transgenic expression cassettes for expressing nucleic acids in non-reproductive flower tissues of plants |
| US7608751B2 (en) | 2003-08-11 | 2009-10-27 | Kweek-En Researchbedruf Agrico B.V. | Fungus resistant plants and their uses |
| US20070003934A1 (en) | 2003-12-02 | 2007-01-04 | Basf Akiengesellschaft | 2-Methyl-6-solanylbenzoquinone methyltransferase as target for herbicides |
| CA2604807C (en) | 2005-04-19 | 2018-06-12 | Basf Plant Science Gmbh | Improved methods controlling gene expression |
| WO2007039454A1 (en) | 2005-09-20 | 2007-04-12 | Basf Plant Science Gmbh | Methods for controlling gene expression using ta-siran |
| AU2006311089C1 (en) | 2005-11-08 | 2012-11-08 | Basf Plant Science Gmbh | Use of armadillo repeat (ARM1) polynucleotides for obtaining pathogen resistance in plants |
| US8178751B2 (en) | 2006-01-12 | 2012-05-15 | Basf Plant Science Gmbh | Use of stomatin (STM1) polynucleotides for achieving a pathogen resistance in plants |
| DK2059600T3 (en) | 2006-08-30 | 2014-07-07 | Basf Plant Science Gmbh | PROCEDURE FOR INCREASING PATHOGEN RESISTANCE IN TRANSGENE PLANTS |
| AU2007306345B2 (en) | 2006-10-12 | 2013-05-23 | Basf Plant Science Gmbh | Method for increasing pathogen resistance in transgenic plants |
| DK2202314T3 (en) | 2007-01-15 | 2014-06-16 | Basf Plant Science Gmbh | Use of Subtilisin (RNR9) Polynucleotides to Achieve Pathogen Resistance in Plants |
| EP2199399A1 (en) | 2008-12-17 | 2010-06-23 | BASF Plant Science GmbH | Production of ketocarotenoids in plants |
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| GB8626879D0 (en) * | 1986-11-11 | 1986-12-10 | Ici Plc | Dna |
| GB8916213D0 (en) * | 1989-07-14 | 1989-08-31 | Ici Plc | Dna constructs,cells and plants derived therefrom |
-
1991
- 1991-03-14 GB GB919105420A patent/GB9105420D0/en active Pending
-
1992
- 1992-03-12 AU AU13690/92A patent/AU1369092A/en not_active Abandoned
- 1992-03-12 EP EP92906497A patent/EP0619844A1/en not_active Withdrawn
- 1992-03-12 WO PCT/GB1992/000442 patent/WO1992016635A1/en not_active Application Discontinuation
- 1992-03-12 JP JP4505831A patent/JPH06505871A/en active Pending
- 1992-03-12 CA CA 2106091 patent/CA2106091A1/en not_active Abandoned
- 1992-03-12 BR BR9205770A patent/BR9205770A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| GB9105420D0 (en) | 1991-05-01 |
| JPH06505871A (en) | 1994-07-07 |
| WO1992016635A1 (en) | 1992-10-01 |
| EP0619844A1 (en) | 1994-10-19 |
| AU1369092A (en) | 1992-10-21 |
| BR9205770A (en) | 1994-06-07 |
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