WO1994023043A2 - Control of plant abscission and pod dehiscence - Google Patents
Control of plant abscission and pod dehiscence Download PDFInfo
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- WO1994023043A2 WO1994023043A2 PCT/GB1994/000689 GB9400689W WO9423043A2 WO 1994023043 A2 WO1994023043 A2 WO 1994023043A2 GB 9400689 W GB9400689 W GB 9400689W WO 9423043 A2 WO9423043 A2 WO 9423043A2
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
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- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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- 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)
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- 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
Definitions
- This invention relates generally to the control of plant abscission and pod dehiscence or shatter.
- Abscission is the process that causes the shedding of a range of plant parts, including leaves, flowers and fruit. The process occurs at precise sites and involves coordinated ceil wall breakdown. Associated with cell separation is an increase in the activity of several hydrolytic enzymes including ?-1 ,4-glucanase (cellulase, EC 3.1.2.4) and polygalacturonase
- This invention takes a completely different approach to solve the problem of dehiscence and the related problem of adequately controlling plant abscission: it involves the use of recombinant DNA technology.
- Roberts and Taylor speculated: By regulating cell separation at abscission sites, it may be possible...to also influence related processes such as pod dehiscence. [Proceedings of the Symposium on the Physiology of Fruit Drop, Ripening, Storage and Post-Harvest Processing of Fruits, Turin, 3-4 October 1988, pp 24-33).
- this exhortation did little to enable the art to address the problem at the cell or genetic level.
- the invention relates to the exploitation of such genes and related DNA sequences (including regulatory sequences) in the manipulation of plant abscission in general and the reduction or prevention of pod dehiscence in particular.
- nucleic acid sequence which:
- (b) contains a promoter or other regulatory sequence which naturally controls expression of a gene involved in plant abscission or dehiscence;
- the recombinant or isolated nucleic acid will generally be DNA, but RNA is not excluded from the scope of the invention.
- the invention is applicable generally to plant abscission or dehiscence, that is to say to the organised shedding of a part of a plant by means of an absciss layer or dehiscence zone.
- Parts of plants that may from time to time be involved in abscission include leaves, petals, pods, seeds and fruit.
- the invention may also have application in regulating the abscission of pollen from anthers, which may be useful in generating artificially male sterile plants, which are useful for hybrid seed production (as, for example, discussed in WO-A-921 1 379).
- the invention has application to all crops that lose seed pre-harvest because of cell separation events.
- An economically important crop to which the invention applies is Brassica napus.
- nucleic acid sequences which satisfy criterion (a) given above are illustrated by the nucleic acid sequence of Figures 3 and 8, which encode the amino acid sequences shown in those figures. All other nucleic acid sequences which, by virtue of the degeneracy of the genetic code, also code for the given amino acid sequences are also preferred embodiments of the invention. Nucleic acid sequences which are substantially homologous to nucleic acid sequences encoding the amino acid sequences shown in Figures 3 and 8 also constitute preferred embodiments of the invention. "Substantial homology" may be assessed either at the nucleic acid level or at the amino acid level.
- sequences having substantial homology may be regarded as those which hybridise to the nucleic acid sequences shown in Figures 3 and 8 under stringent conditions (for example at 35 to 65 °C in a salt solution of about 0.9M).
- a protein sequence may be regarded as substantially homologous to another protein sequence if a significant number of the constituent amino acids exhibit homology. At least 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 99%, in increasing order of preference, of the amino acids may be homologous.
- nucleic acids satisfying the criterion of (a) are nucleic acids encoding the DC2.1 5 and pZRP3 sequences shown in Figure 5.
- Nucleic acids encoding plant abscission or dehiscence proteins having one or more amino acids of pSAC51 not shared with at least one of DC2.1 5 and pZRP3 are, however, within the scope of the invention.
- the most preferred embodiments of the invention satisfying criterion (a) are nucleic acids encoding proteins specifically or at least preferentially expressed in the dehiscence zone of pericarp tissue, particularly in Brassica nap us.
- nucleic acid satisfying criterion (b) given above constitutes a powerful and flexible means of achieving the benefits of the invention.
- Such nucleic acid contains a promoter or other regulatory sequence which naturally controls expression of a gene involved in plant abscission or dehiscence.
- the promoter is that region of a gene which regulates its expression, for example by specifying the time or location of expression. Promoters can be separated from the coding region of a gene and used to drive a different coding region, thus allowing the expression of a different product.
- the promoter may drive a gene which disrupts cellular development.
- the promoter may drive DNA coding a lytic enzyme.
- the lytic enzyme may cause lysis of one or more biologically important molecules, such as macromolecules including nucleic acid, protein (or gl ⁇ coprotein), carbohydrate and in some circumstances lipid.
- Ribonuclease such as RNase T1
- barnase are examples of enzymes which cause lysis of RNA.
- enzymes which lyse DNA include exonucleases and endonucleases, whether site specific (such as EcoRI) or non-site-specific.
- Glucanase is an example of an enzyme which causes lysis of a carbohydrate.
- Lipases whose corresponding nucleic acids may be useful in the invention include phospholipase A 2 .
- Actinidin is an example of a protease, DNA coding for which may be useful in the invention; other examples include papain zymogen and papain active protein. Such "killer" enzymes as these do not have to be lytic enzymes.
- DNA coding for which may be useful in the invention catalyse the synthesis of phytohormones, such as isopentyl transferase, which is involved in cytokinin synthesis, and one or more of the enzymes involved in the synthesis of auxin.
- promoters useful in feature (b) of the invention may drive DNA encoding an enzyme, they may alternatively drive DNA whose transcription product is itself deleterious. Examples of such transcription products include antisense RNA and ribozymes.
- RNA transcribed from antisense DNA is capable of binding to, and destroying the function of, a sense RNA of the sequence normally found in the cell, thereby disrupting function.
- antisense DNAs examples are the antisense DNAs of the sequences shown in Figures 3 and 8. Since these genes are normally expressed in the dehiscence zone, antisense to them may be expected to disrupt normal dehiscence.
- Ribozymes are RNA "enzymes" capable of highly specific cleavage against a given target sequence (Haseloff and Gerlach, Nature 334 585-591 ( 1 988)).
- Promoters useful in feature (b) of the invention may be located in cDNA or genomic libraries using, for example, probe sequences taken from the nucleic acid sequences of Figures 3 and 8.
- a third category of nucleic acid useful in the invention is identified under (c) above, as that which, when introduced into a plant, prevents or otherwise interferes with normal plant abscission of dehiscence.
- dehiscence- or abscission-specific promoters may be useful in this feature of the invention.
- antisense DNA or ribozyme-encoding DNA specific for abscission- or dehiscence-specific genes need not be driven by abscission- or dehiscence-specific promoters. Instead, they could be driven by constitutive or other promoters (such as for example the CaMV 35S, rubisco or plastocyanin promoter).
- Antisense technology and ribozyme technologies have already found application in other areas of plant molecular biology. For example, antisense technology has been used to control tomato fruit ripening. Ribozyme technology has been used to control viral infection of melons.
- DNA or RNA in accordance with this feature of the invention generally interferes with the proper expression of a gene or genes, in preferred embodiments expression is substantially prevented.
- nucleic acids useful in the invention is specified under feature (d); this includes nucleic acids which hybridise under stringent conditions to nucleic acids satisfying the criterion of one or more of features (a), (b) or (c).
- nucleic acid fragments are useful for probing for similar genes involved in abscission or dehiscence.
- an Arabidopsis or other gene library may be probed. Fragments of at least 1 0, 20, 30, 40 or 50 more nucleotides may be used. Many useful probes are from 1 5 to 20 nucleotides in length.
- 3'- transcription regulation signals including a polyadenylation signal
- Preferred 3'-transcription regulation signals may be derived from the cauliflower mosaic virus 35S gene. It should be recognised that other 3'-transcription regulation signals could also be used.
- Recombinant DNA in accordance with the invention may be in the form of a vector.
- the vector may for example be a plasmid, cosmid or phage.
- Vectors will frequently include one or more selectable markers to enable selection of cells transfected (or transformed: the terms are used interchangeably in this specification) with them and, preferably, to enable selection of cells harbouring vectors incorporating heterologous DNA.
- Cloning vectors can be introduced into E. coli or another suitable host which facilitate their manipulation. According to another aspect of the invention, there is therefore provided a host cell transfected or transformed with DNA as described above.
- DNA in accordance with the invention can be prepared by any convenient method involving coupling together successive nucleotides, and/or ligating oiigo- and/or poly-nucleotides, including in vitro processes, but recombinant DNA technology forms the method of choice.
- DNA in accordance with the invention will where appropriate be introduced into plant cells, by any suitable means.
- a plant cell including DNA in accordance with the invention as described above.
- DNA is transformed into plant cells using a disarmed Ti- plasmid vector and carried by Agrobacterium by procedures known in the art, for example as described in EP-A-01 16718 and EP-A-0270822.
- the foreign DNA could be introduced directly into plant cells using an electrical discharge apparatus. This method is preferred where
- Agrobacterium is ineffective, for example where the recipient plant is monocotyledonous. Any other method that provides for the stable incorporation of the DNA within the nuclear DNA of any plant cell of any species would also be suitable. This includes species of plant which are not currently capable of genetic transformation.
- DNA in accordance with the invention also contains a second chimeric gene (a "marker" gene) that enables a transformed plant containing the foreign DNA to be easily distinguished from other plants that do not contain the foreign DNA.
- a marker gene examples include antibiotic resistance (Herrera-Estrella et al. , EMBO J. 2(6) 987-95 (1983) and Herrera-Estrella er a/. , Nature 303 209-13 (1983)), herbicide resistance (EP-A-0242246) and glucuronidase (GUS) expression (EP-A- 0344029).
- Expression of the marker gene is preferably controlled by a second promoter which allows expression in cells other than the tapetum, thus allowing selection of ceils or tissue containing the marker at any stage of regeneration of the plant.
- the preferred second promoter is derived from the gene which encodes the 35S subunit of Cauliflower Mosaic Virus (CaMV) coat protein.
- CaMV Cauliflower Mosaic Virus
- any other suitable second promoter could be used.
- a whole plant can be regenerated from a single transformed plant cell, and the invention therefore provides transgenic plants (or parts of them, such as propagating material) including DNA in accordance with the invention as described above. The regeneration can proceed by known methods.
- a further aspect of the invention is constituted by novel proteins which are preferentially or exclusively expressed in dehiscence zones or abscission layers.
- novel proteins are those whose amino acid sequences are given in Figures 3 and 8.
- FIGURE 1 relates to Example 1 and is a diagrammatic representation of a transverse section through an oilseed rape pod showing the distinction between 'zone' and 'non-zone' pericarp tissue used for protein extraction.
- FIGURE 2 also relates to Example 1 and is a Northern blot analysis of total RNA ( 10 ⁇ g).
- the RNA was hybridised to radiolabelled pSAC51 cDNA insert.
- FIGURE 3 also relates to Example 1 and shows the nudeotide and deduced amino acid sequence of pSAC51 cDNA. The initiation and termination codons are indicated by a single asterisk. Amino acid domains of interest are double underlined and a possible glycosylation site is underlined. A putative polyadenyiation signal has asterisks above it.
- FIGURE 4 also relates to Example 1 and shows the hydropathy profile of the cDNA clone pSAC51 deduced amino acid sequence.
- the profile was computer generated according to Kyte and Doolittle
- FIGURE 5 also relates to Example 1 and shows the sequence alignment of amino acids deduced from nudeotide sequences of the following cDNAs: pSAC51 - Oilseed rape pods DC2.1 5 - Carrot embryos, Aleith and Richter Planta 183, 1 7-24 ( 1 990) pZRP3 - Maize roots, John et al, Plant Mol. Biol. 20 821 -831 ( 1 992)
- FIGURE 6 also relates to Example 1 and shows a genomic Southern blot analysis of B. napus DNA probed with the pSAC51 cDNA. 10 ⁇ g DNA was digested using the following restriction enzymes:
- FIGURE 8 also refers to Example 2 and shows the pSAC40 nudeotide and deduced amino acid sequences.
- FIGURE 9 also refers to Example 2 and shows a hydropathy profile of the cDNA clone pSAC25 deduced amino acid sequence.
- the profile was computer generated according to Kyte and Doolittle, J. Mol. Biol. 157 105-1 32 ( 1 982).
- FIGURE 1 A shows a restriction map of the A. thaliana genomic clone gSAC25. The position of the SAC25 coding region is shown as a filled box and the extent of the insert in pDH30 is indicated.
- FIGURE 1 1 B shows a restriction map of the A. thaliana genomic clone gSAC51 .
- the position of the SAC51 coding region is shown as a filled box and the extent of the insert in pDH31 is indicated.
- FIGURE 1 2 shows the construction of a chimeric gene that expresses SAC25 anti-sense RNA from the CaMV double 35S promoter in transgenic plants.
- Plant Material Seeds of B. napus cv Rafal were grown as described by Meakin and
- the plant material was ground to a powder in liquid N 2 and then in 10 volumes of extraction buffer (200mM Tris-acetate [pH
- RNA precipitated Poly(A) + RNA was isolated from total RNA extracted, from both the zone and non-zone tissue of 40, 45 and 50 DAA pods, using a Poly(A) QUIK” mRNA purification kit (Stratagene, Cambridge, UK) following the manufacturers instructions, and then bulked together. Total RNA was also extracted from leaves, seeds and pods using a method described by Dean et al, EMBO J. 4 3055-3061 (1985) for use in Northern analyses.
- a cDNA library was constructed using 5 ⁇ g poly(A) + RNA extracted from the dehiscence zone of pods prior to and during dehiscence.
- the library was constructed using the ⁇ ZAP-cDNA synthesis kit according to the manufacturers' instructions (Stratagene). This resulted in the production of a library containing 1 .2 X 10 6 recombinants.
- Several plaques were picked at random and in vivo excised (Short et al, Nucl. Acids Res. 16 7583-7600 ( 1 988)) .
- the average insert size was 1 Kb.
- Differential screening was performed using single-stranded cDNA probes synthesised from poly(A) + RNA isolated from dehiscence zone or non-zone pod material.
- the probes were synthesised using the method of Picton et al, Plant Mol. Biol in press ( 1993) and used to screen 40,000 recombinant plaques by in situ plaque hybridisation.
- Duplicate plaque lifts were obtained using HYBOND" N + membranes (Amersham, Aylesbury, UK) and were then treated and hybridised according to manufacturers instructions but were washed at 65 °C in 0.1 X sodium chloride, sodium phosphate, EDTA (SSPE), 0.1 % SDS. Any plaques hybridising preferentially to zone probes were re-screened at densities of 50-100 plaques/plate. Chosen plaques were cored out of the plate and plasmids isolated using the in vivo excision procedure (Short et al, Nucl. Acids Res. 16 7583-7600
- Inserts were amplified by polymerase chain reaction (PCR) using the T3, T7 bacteriophage promoters and subsequently used for probes. Isolated plasmid was also used as a template for sequencing.
- RNA samples 10 ⁇ g total RNA isolated from various parts of the oilseed rape plant were separated on a 1 X TBE, 1 % agarose/6% formaldehyde denaturing gel. The RNA was transferred onto a nylon membrane (GeneScreen, NEN-Du Pont, UK) using capillary transfer. The gel, RNA samples, blot and hybridisation were performed in accordance with the membrane manufacturers instructions.
- a radio-labelled probe was generated using 100ng of insert from the plasmid pSAC51 , using [ 32 P] dCTP (1 10 TBq nmole "1 , Amersham) and a nick translation kit (Boehringer Mannheim, Lewes, UK).
- Unincorporated label was removed from the probe by passing it through a SEPHADEXTM G-50 column and eluting the probe with TE (pH 8). The blot was washed at 65°C in 0.1 X SSPE, 0.1 % SDS and exposed to KODAK X-AR5 film with intensifying screens at -70°C.
- the resulting pellet was resuspended in 300 ⁇ l TE, 10 ⁇ l of RNaseA (10 mg ml '1 ) added, and then incubated at 37°C for 1 5 mins before 300 ⁇ l CTAB buffer (0.2M Tris.HCI pH 7.5, 0.05M EDTA, 2M NaCl and 2% w/v CTAB) was added before a further incubation at 60°C for 1 5 mins. Following re-extraction with an equal volume of chloroform the DNA was precipitated with an equal volume of isopropanol at -20°C.
- CTAB buffer 0.2M Tris.HCI pH 7.5, 0.05M EDTA, 2M NaCl and 2% w/v CTAB
- Plasmid DNA was isolated by the alkaline-lysis method (Sambrook et al,
- pSAC51 When screened with the insert from the clone designated pSAC51 , 1 9 other clones were shown to have homology (data not shown), indicating that this cDNA may encode an abundant mRNA.
- the insert from pSAC51 was approximately 700 bp in length by comparison with DNA standards on an ethidium bromide stained agarose gel.
- pSAC51 mRNA expression by Northern analysis Pods were harvested at 20, 40, 45, 50, 60 DAA and dehiscence zone (Z) and flanking non-zone (NZ) tissue isolated (see Figure 1 ). Total RNA was extracted from these excised parts and from the seed, leaves and roots. Northern analysis revealed that the 700 bp insert from pSAC51 hybridised to a mRNA of about 700 nucleotides (See Figure 2). At 20 DAA hybridisation was apparent in both Z and NZ, but subsequently disappeared in NZ tissues and preferentially accumulated in the dehiscence zone tissue with maximum signal occurring at 60 DAA. The process of dehiscence is visible to the naked eye at 50 DAA. The transcript could not be detected in the leaves, seeds and roots. The cDNA pSAC51 was deemed to be near full-length because the mRNA transcript size was similar to that of the cDNA insert size.
- the hydropathy plot ( Figure 4) of the peptide indicates that the protein has several distinct domains.
- the protein has a hydrophobic amino terminus, characteristic of a membrane spanning cleavable signal sequence (von Heijne, Nucl. Acids Res. 144683-4690 (1986)), extending to position 30. It then has a hydrophilic region extending to position 70, followed by a further hydrophobic region extending to the carboxy terminus.
- the 756 bp insert of pSAC51 was used as a probe for hybridisation to Southern blots of B. napus genomic DNA digested with EcoRI, Hind ⁇ and Bam ⁇ ( Figure 6).
- the probe hybridised to several fragments ranging in size from 5 kb to 1 kb.
- the cDNA has an internal restriction site for Hind ⁇ at nudeotide 32 and this may account for extra fragments.
- Pod dehiscence is a hard phenotype to measure accurately and therefore the precise start of the process is not known.
- Cellulase activity increases in the dehiscence zone from 40 DAA and precedes the first visible signs of cell wall breakdown by 1 5-20 days (Meakin and Roberts, J. Exp. Bot. 41 1003-101 1 (1990)). Therefore mRNA extracted from different developmental stages (40, 45 and 50 DAA) were bulked in order to increase the chances of obtaining mRNAs that are present prior to and during the process of dehiscence.
- This example relates to the isolation of a cDNA clone that corresponds to a mRNA that preferentially accumulates in the dehiscence zone of the developing pod ( Figure 2).
- the pSAC51 mRNA is present at the early stages of pod development in both the zone and non-zone tissue. The presence of signal in the non-zone RNA at 20 DAA cannot be fully explained.
- the deduced protein sequence of pSAC51 has several features that are worth mentioning (Figure 3).
- the protein is rich in the amino acid, proline, whose arrangement is in the form of a repeated motif "Pro-X". It also has another arrangement of amino acids into a "DALK" motif.
- Other characteristics include defined hydrophobic and hydrophilic domains ( Figure 4) and a membrane spanning cleavable signal peptide.
- the pSAC51 deduced amino acid sequence has an unknown function but has significant homology to other characterised protein whose functions are also unknown (Figure 5). These proteins are from different plant species and from different plant organs; carrot embryos ( Aleith and Richter, Planta 183 1 7-24 ( 1 990)) and young maize roots (John et al, Plant Mol. Biol. 20 821 -831 (1 992)).
- the pSAC51 protein also has significant homology ( > 40%) with a protein of unknown function from immature tomato fruit
- pSAC51 This may give some insight into the role of the protein encoded by pSAC51 in that it may be connected with the developing seed. Given that the pSAC51 protein is likely to be transported and that the seed attachments to the pod occur in the region of the dehiscence zone then it may have a role in seed development, although no pSAC51 mRNA was detected by Northern analysis.
- the processes of abscission and dehiscence involve the breakdown of ceil walls.
- the cell walls of a plant are composed of cellulose, hemicellulose, pectic compounds, proteins, suberin, lignin and water (Cassab and Verner, Ann. Rev. Plant Physiol. Plant Mol. Biol. 39 321 -353 ( 1 988)), but can be grouped into three main types: hydroxyproline-rich glycoproteins (HRGPs) (Chen and Varner, Proc. Natl. Acad. Sci. 82 4399-4403 (1985), Zheng-Hua and Varner, Plant Cell.
- HRGPs hydroxyproline-rich glycoproteins
- GRPs glycine-rich proteins
- PRPs proline- (or hydroxyproline)-rich proteins
- all these proteins are characterised by basic repeat motifs that are different for each type: Ser-(Hyp) 4 for HRGPs; (Gly-X) n for GRPs; and Pro-Pro-Val-X-Y for PRPs.
- proline-rich protein encoded by pSAC51 shows no significant identity to any of the aforementioned groups described so far (Cassab and Varner, Ann. Rev. Plant Physiol. Plant Mol. Biol. 39 321 -353 (1988)).
- the proline-rich proteins that do show homology to pSAC51 have ail been characterised since that review was written and, given the different proline repeat motif "Pro-X", they may be a new sub-group of proline-rich proteins.
- proline-rich proteins that do show homology to pSAC51 have ail been characterised since that review was written and, given the different proline repeat motif "Pro-X", they may be a new sub-group of proline-rich proteins.
- Pro-X proline repeat motif
- the pSAC51 deduced amino acid sequence has one glycosylation site as does the maize protein (John et al, Plant Mol. Biol. 20 821 -831 (1 992)) but the tomato (Salts et al, Plant Mol. Biol. 17 149- 1 50 ( 1 991 )) and carrot (Aleith and Richter, Planta 183 1 7-24 ( 1 990)) proteins do not.
- Many proline-rich proteins that have been characterised so far have been isolated from tissue capable of growth and cell expansion and they may have a role in cell wall formation and structure (Cassab and Varner, Ann. Rev. Plant Physiol. Plant Mol. Biol. 39 321 -353 ( 1 988)).
- pSAC25 which has a different pattern of gene expression from that of pSAC51 .
- pSAC40 is one of several cDNAs with sequences that match pSAC25: the insert is slightly larger than pSAC25.
- Pods were harvested at 20, 30, 35, 40, 45, 50 and 60 DAA, and dehiscence zone and flanking non-zone tissue isolated, as described in Example 1 .
- Northern analysis of extracted RNA reveals that expression was taking place in zone tissue from 30 DAA, with only slight expression in non-zone tissue as late as 50 DAA ( Figure 7). The transcript could not be detected in leaves, seeds or roots.
- the cDNA sequence of pSAC40 was sequenced and the result, with the deduced amino acid sequence, is shown in Figure 8.
- the hydropathy plot ( Figure 9) of the deduced amino acid sequence shows distinct hydrophobic and hydrophilic regions.
- the insert of pSAC25 was used as a probe for hybridisation to Southern blots of B. napus genomic DNA digested with EcoRI, HindlW and Bam ⁇ .
- the probe hybridised to several fragments ranging in size from 7 or 8 kb to 1 kb.
- EXAMPLE 3 Cloning of gSAC51 and gSAC51 a) Isolation and characterisation of the SAC 25 gene.
- a 10 kb A. thaliana genomic clone [gSAC25] was isolated that hybridised to the pSAC25 cDNA ( Figure 1 1 A).
- a 3.5 kb EcoRI/H/ ⁇ dlll fragment of this DNA that hybridised to the pSAC25 cDNA was subcloned into EcoR ⁇ /Hind ⁇ cut pBluescript KS + , forming pDH30. Nudeotide sequencing shows that this fragment contains an ORF that is highly homologous to the pSAC25 ORF. This sequence also determines the position of the SAC25 promoter region relative to the SAC25 ORF, as it is that region upstream of (ie 5' to) the ORF.
- a 10 kb A. thaliana genomic clone (gSAC51 ] was isolated that hybridised to the pSAC51 cDNA ( Figure 1 1 B).
- a 4.0 kb EcoRI fragment of this DNA that hybridised to the pSAC51 cDNA was subdoned into EcoRI cut pBluescript KS + , forming pDH31.
- Nudeotide sequencing shows that this fragment contains an ORF that is highly homologous to the pSAC51 ORF. This sequence also determines the position of the SAC51 promoter region relative to the SAC51 ORF, as it is that region upstream of (ie 5' to) the ORF.
- transcriptional fusions of the promoters can be made to the E. coli gene encoding /?-glucuronidase (GUS). Fragments of the clones [gSAC25] or [gSAC51 ) containing the putative promoter region are subdoned into pBII OI (Jefferson et al, EMBO J. 6 3901 (1987)). The GUS constructs are then transformed into A. thaliana, B. napus or N. tabacum, using standard transformation techniques.
- Either pod shatter zone-specific or constitutive promoters can be used to drive expression of sense or anti-sense RNA corresponding to shatter zone-specific transcripts in transgenic plants, thus potentially creating pod mutations and shatter-resistance (indehiscence).
- the same pod shatter zone-specific promoters can be used to drive the pod shatter zone expression of genes encoding proteins or enzymes detrimental to shatter- zone function thereby creating shatter-resistance (indehiscence).
- pJIT 60 is identical to pJIT30 (Guerineau et al, Plant Mol. Biol. 15 1 27-1 36 ( 1 990)) except that the CaMV 35S promoter is replaced by a double CaMV 35S promoter.
- p35S-antiSAC25 a portion of the SAC25 cDNA is cloned in an antisense orientation between a double 35S promoter and a CaMV polyadenylation signal. This chimeric gene was then cloned into pBin 1 9 (Bevan et al, Nucl. Acids Res. 22 871 1 -8721 ( 1 984)) as a Ssn, Xho ⁇ fragment forming p35S-antiSAC25.
- B. napus plants transformed with the 35S-antiSAC25 chimeric gene are resistant to pod-shatter (indehiscent).
- Double CaMV 35S promoter linked to the coding region of the SAC25 cDNA or gene such that sense SAC25 RNA is produced ii) Double CaMV 35S promoter linked to the coding region of the SAC51 cDNA or gene such that sense or anti-sense SAC51 RNA is produced;
- SAC25 promoter linked to the coding region of the SAC25 cDNA or gene, such that sense or anti-sense SAC25 RNA is produced;
- plasmids could also be transformed into other members of the Brassicaceae causing shatter-resistance in the transgenic plants.
- SAC25 and SAC51 promoters could also be harnessed by expressing gene fusions to barnase, or other genes that disrupt cellular development or otherwise interfere in the function of the shatter zone in pod shatter, in transgenic plants.
- barnase gene to cause cell ablation has been described in EP-A-0344029 (Plant Genetic Systems NV) and WO-A-921 1379 (Nickerson International Seed Company Limited), particularly at pages 28 and 29 of the latter document.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP94911257A EP0692030A1 (en) | 1993-03-31 | 1994-03-31 | Control of plant abscission and pod dehiscence |
US08/530,165 US5907081A (en) | 1993-03-31 | 1994-03-31 | Control of plant abscission and pod dehiscence |
CA002159614A CA2159614C (en) | 1993-03-31 | 1994-03-31 | Control of plant abscission and pod dehiscence |
AU63819/94A AU6381994A (en) | 1993-03-31 | 1994-03-31 | Control of plant abscission and pod dehiscence |
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GB939306726A GB9306726D0 (en) | 1993-03-31 | 1993-03-31 | Plant molecular biology |
GB9306726.2 | 1993-03-31 |
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WO1994023043A2 true WO1994023043A2 (en) | 1994-10-13 |
WO1994023043A3 WO1994023043A3 (en) | 1994-11-24 |
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PCT/GB1994/000689 WO1994023043A2 (en) | 1993-03-31 | 1994-03-31 | Control of plant abscission and pod dehiscence |
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US (1) | US5907081A (en) |
EP (1) | EP0692030A1 (en) |
AU (1) | AU6381994A (en) |
CA (1) | CA2159614C (en) |
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Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996030529A1 (en) * | 1995-03-31 | 1996-10-03 | Nickerson Biocem Limited | Control of pod dehiscence |
WO1997013865A1 (en) * | 1995-10-06 | 1997-04-17 | Plant Genetic Systems, N.V. | Seed shattering |
WO1999000502A1 (en) * | 1997-06-27 | 1999-01-07 | The Regents Of The University Of California | Seed plants characterized by delayed seed dispersal |
WO1999015680A1 (en) * | 1997-09-19 | 1999-04-01 | Biogemma Uk Limited | Control of plant abscission and pod dehiscence or shatter |
WO1999015681A1 (en) * | 1997-09-19 | 1999-04-01 | Biogemma Uk Limited | Control pop dehiscence or shatter |
WO1999049046A1 (en) * | 1998-03-20 | 1999-09-30 | Biogemma Uk Limited | Signal transduction protein involved in plant dehiscence |
WO2004057004A2 (en) * | 2002-12-23 | 2004-07-08 | Melinka Butenko | Plant genes and their use in controlling abcission in plants |
US6998517B1 (en) | 1998-06-25 | 2006-02-14 | The Regents Of The University Of California | Control of fruit dehiscence in Arabidopsis by indehiscent1 genes |
US7109395B2 (en) * | 2000-02-11 | 2006-09-19 | Temasek Life Sciences Laboratory Limited | Dehiscence gene and methods for regulating dehiscence |
WO2007105002A2 (en) * | 2006-03-16 | 2007-09-20 | Norwegian University Of Life Sciences | Nucleic acid molecules involved or associated with abscission |
US7528294B2 (en) | 2004-06-18 | 2009-05-05 | The Regents Of The University Of California | Brassica INDEHISCENT1 sequences |
WO2012059497A1 (en) | 2010-11-02 | 2012-05-10 | Bayer Cropscience Ag | N-hetarylmethyl pyrazolylcarboxamides |
WO2012089757A1 (en) | 2010-12-29 | 2012-07-05 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
US8722072B2 (en) | 2010-01-22 | 2014-05-13 | Bayer Intellectual Property Gmbh | Acaricidal and/or insecticidal active ingredient combinations |
US9206137B2 (en) | 2010-11-15 | 2015-12-08 | Bayer Intellectual Property Gmbh | N-Aryl pyrazole(thio)carboxamides |
US9265252B2 (en) | 2011-08-10 | 2016-02-23 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040154053A1 (en) * | 1998-03-20 | 2004-08-05 | Wyatt Paul | Signal transduction protein involved in plant dehiscence |
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- 1994-03-31 US US08/530,165 patent/US5907081A/en not_active Expired - Lifetime
- 1994-03-31 EP EP94911257A patent/EP0692030A1/en not_active Ceased
- 1994-03-31 CA CA002159614A patent/CA2159614C/en not_active Expired - Fee Related
- 1994-03-31 WO PCT/GB1994/000689 patent/WO1994023043A2/en not_active Application Discontinuation
- 1994-03-31 AU AU63819/94A patent/AU6381994A/en not_active Abandoned
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US6096946A (en) * | 1995-03-31 | 2000-08-01 | Biogemma Uk Limited | Control of pod dehiscence |
WO1996030529A1 (en) * | 1995-03-31 | 1996-10-03 | Nickerson Biocem Limited | Control of pod dehiscence |
AU722501B2 (en) * | 1995-03-31 | 2000-08-03 | Biogemma Uk Limited | Control of pod dehiscence |
WO1997013865A1 (en) * | 1995-10-06 | 1997-04-17 | Plant Genetic Systems, N.V. | Seed shattering |
US6797861B2 (en) | 1995-10-06 | 2004-09-28 | Bayer Bioscience Nv | Seed shattering |
US6420628B1 (en) | 1995-10-06 | 2002-07-16 | Plant Genetic Systems, N.V. | Seed shattering |
AU718082B2 (en) * | 1995-10-06 | 2000-04-06 | Plant Genetic Systems N.V. | Seed shattering |
US6198024B1 (en) | 1997-06-27 | 2001-03-06 | The Regents Of The University Of California | Seed plants characterized by delayed seed dispersal |
US6288305B1 (en) | 1997-06-27 | 2001-09-11 | The Regents Of The University Of California | Seed plants characterized by delayed seed dispersal |
WO1999000502A1 (en) * | 1997-06-27 | 1999-01-07 | The Regents Of The University Of California | Seed plants characterized by delayed seed dispersal |
WO1999015681A1 (en) * | 1997-09-19 | 1999-04-01 | Biogemma Uk Limited | Control pop dehiscence or shatter |
WO1999015680A1 (en) * | 1997-09-19 | 1999-04-01 | Biogemma Uk Limited | Control of plant abscission and pod dehiscence or shatter |
WO1999049046A1 (en) * | 1998-03-20 | 1999-09-30 | Biogemma Uk Limited | Signal transduction protein involved in plant dehiscence |
US6998517B1 (en) | 1998-06-25 | 2006-02-14 | The Regents Of The University Of California | Control of fruit dehiscence in Arabidopsis by indehiscent1 genes |
US7109395B2 (en) * | 2000-02-11 | 2006-09-19 | Temasek Life Sciences Laboratory Limited | Dehiscence gene and methods for regulating dehiscence |
WO2004057004A2 (en) * | 2002-12-23 | 2004-07-08 | Melinka Butenko | Plant genes and their use in controlling abcission in plants |
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US7528294B2 (en) | 2004-06-18 | 2009-05-05 | The Regents Of The University Of California | Brassica INDEHISCENT1 sequences |
US8143481B2 (en) | 2004-06-18 | 2012-03-27 | The Regents Of The University Of California | Brassica indehiscent1 sequences |
US9200294B2 (en) | 2004-06-18 | 2015-12-01 | National Science Foundation | Brassica indehiscent1sequences |
WO2007105002A2 (en) * | 2006-03-16 | 2007-09-20 | Norwegian University Of Life Sciences | Nucleic acid molecules involved or associated with abscission |
WO2007105002A3 (en) * | 2006-03-16 | 2008-03-27 | Norwegian University Of Life S | Nucleic acid molecules involved or associated with abscission |
US8722072B2 (en) | 2010-01-22 | 2014-05-13 | Bayer Intellectual Property Gmbh | Acaricidal and/or insecticidal active ingredient combinations |
WO2012059497A1 (en) | 2010-11-02 | 2012-05-10 | Bayer Cropscience Ag | N-hetarylmethyl pyrazolylcarboxamides |
US9206137B2 (en) | 2010-11-15 | 2015-12-08 | Bayer Intellectual Property Gmbh | N-Aryl pyrazole(thio)carboxamides |
WO2012089757A1 (en) | 2010-12-29 | 2012-07-05 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
US9265252B2 (en) | 2011-08-10 | 2016-02-23 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
Also Published As
Publication number | Publication date |
---|---|
US5907081A (en) | 1999-05-25 |
AU6381994A (en) | 1994-10-24 |
WO1994023043A3 (en) | 1994-11-24 |
CA2159614C (en) | 2007-11-06 |
EP0692030A1 (en) | 1996-01-17 |
GB9306726D0 (en) | 1993-05-26 |
CA2159614A1 (en) | 1994-10-13 |
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