WO2007105967A1 - Novel plant genes and uses thereof - Google Patents
Novel plant genes and uses thereof Download PDFInfo
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- WO2007105967A1 WO2007105967A1 PCT/NZ2007/000047 NZ2007000047W WO2007105967A1 WO 2007105967 A1 WO2007105967 A1 WO 2007105967A1 NZ 2007000047 W NZ2007000047 W NZ 2007000047W WO 2007105967 A1 WO2007105967 A1 WO 2007105967A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- the present invention relates to novel plant genes and uses thereof.
- the present invention relates to novel plant genes involved in plant development and cell differentiation.
- the invention relates to genes and polypeptides capable of: affecting the growth and shape of lateral shoot organs in plants or plant cells; or altering the arrest of meristemoid cell division during organ development.
- Some of the economic value derived from variation in plant organ size or shape is obvious. Larger fruit or seed may represent a greater harvest index, more grain or fruit per plant or per crop area.
- the value of plant ornamentals is often because of unusual leaf, flower or seed pod shapes. Horticulturalists have been collecting "odd" plants because of their commercial value for centuries. Other economic impacts of variability in organ size and shape may not be so obvious.
- leaf blade is the main site of photosynthesis and respiration in higher plants and leaf size and shape are key factors influencing these processes.
- Plants with large leaves grown in hot dry climates lose a lot of water by transpiration.
- Narrow curled leaves maybe a means of avoiding excessive transpiration (i.e. less water use).
- Larger leaves on short stems can result in a higher grain yield, due to more carbon etc being made available for seed development instead of vegetative growth.
- All plant shoot lateral organs have a number of planes of growth.
- One of these planes is the adaxial (upper or adjacent to the shoot meristem)-abaxial (lower or away from the shoot meristem) axis. Generally this plane determines the thickness of a leaf.
- Another is the proximal (base)-distal (tip) axis. This axis contributes to the length of the organ.
- the blade or lamina is most obvious in a leaf but the same laminal plane exists in flower sepals, petals, anthers and seed pods.
- leaves and seed pods that have the longest duration of growth and expand most after the initial shape is determined by development of the organ primordia.
- the leaves and seed pods also have the greatest flexibility in size of the lateral shoot organs. Under optimal growth conditions the leaves and fruit increase most in size whereas flowers are less flexible in size.
- the present invention is derived from the discovery of a novel Arabidopsis thaliana type of gene, named PEAPOD (PPD) that controls the arrest of meristemoid cell division during , organ development. Alterations in the number of copies of this gene or the level of PPD transcription, controls the timing of meristemoid cell division arrest. Fewer copies of PPD or less PPD transcription prolongs meristemoid cell division resulting in a larger organ blade. In the case of a complete loss of PPD function blade growth exceeds the expansion capacity of the margin and the organ develops a bell shape (referred to as positive Gaussian curvature) rather than the normal flat plane (zero Gaussian curvature).
- PEAPOD a novel Arabidopsis thaliana type of gene
- the PPD gene encodes a novel protein. Homologous genes are present in a wide range of eudicot plants and analysis of the primary amino acid structure of these proteins indicates the presence of a highly conserved novel plant specific domain present only in the PEAPOD-like proteins.
- AINTEGUMENTA ANT
- ANT AP2-like transcription factor
- Ectopic expression of the ANT gene (expression in most tissues rather than the restricted tissue expression normally found in wild type plants), enlarges embryonic and all shoot organs without altering superficial morphology by increasing cell number. However, cells expressing ANTm mature organs exhibit neoplastic growth producing calli and adventitious shoots and roots. The ANT gene therefore acts to promote cell division. (Mizukami & Fischer, 2000. PNAS 97: 942-947).
- the JAGGED (JAG) gene of Arabidopsis appears to have a similar effect. Loss-of-function mutations of the JAG gene of Arabidopsis result in abnormal lateral organs including small serrated leaves, narrow floral organs, and petals with fewer larger cells.
- the JAG gene encodes a protein with a single C2H2 zinc-finger domain (transcription factor). Misexpression of JAG results in leaf fusion and the development of ectopic leaf-like outgrowth from both leaf and floral tissue. (Ohno et al., 2004. Development 131 : 1111- 1122).
- ARGOS is another Arabidopsis gene that is involved in organ size control. Over expression of ARGOS in Arabidopsis increases cell proliferation resulting in larger organs, reduced expression reduces growth and results in smaller organs. It is proposed that ARGOS regulates growth through ANT during development. (Hu et al., 2003. Plant Cell 15: 1951- 1961 ).
- CIN In Antirrhinum (snapdragon), two members of the TCP family of transcription factors, encoded by Cl NCI NNATA (CIN) and CYCLOIDEA (CYC), appear to affect organ shape by promoting cell differentiation. CIN controls growth of the leaf blade and is required to produce flat leaves. Mutations in the CIN gene result in excessive growth at the periphery of the leaf blade, resulting in wavy leaves (negative Gaussian curvature). CIN appears to prevent excessive growth by sensitising peripheral cells to the cell cycle arrest front that moves from the tip to the base. (Nath et al., 2003. Science 299: 14011407). CYC suppresses growth of dorsal floral structures producing asymmetric flowers and also appears to regulate growth by altering the cell cycle. (Luo et al., 1996. Nature 383:794- 799).
- PPD is distinct in its predicted protein sequence, mode of action, loss-of-function phenotype, and elevated expression phenotype, from any gene previously described.
- the protein appears to act by restricting meristemoid cell division during growth of shoot organ laminal tissue. The result of this action is to co-ordinate growth of the organ blade and margin so that a "normal" curvature is maintained.
- the level of PPD transcription (or gene dosage) modulates the timing of meristemoid cell cycle arrest (this could be either by promoting differentiation or inhibiting cell division) the PPD gene appears to be a key regulator of plant shoot organ size flexibility.
- an isolated nucleic acid molecule having a nucleotide sequence comprising:
- an isolated polypeptide having an amino acid sequence comprising:
- an isolated nucleic acid molecule encoding a domain having conserved amino acids:
- an isolated polypeptide encoding a domain having a conserved amino acid sequence: SXLXKPLXXLTXXDISQXTREDCRXXLKXKGMRXPSWNKSQAIQQVXXXKXLXE
- nucleic acid molecule encoding a polypeptide substantially as described herein, comprising an amino acid sequence substantially as set forth in the sequence listing; or a functional fragment or variant thereof; or a homolog or ortholog thereof.
- nucleic acid molecule substantially as described above to alter a plant or plant cell.
- nucleic acid molecule substantially as described above to alter the growth and/or shape of lateral shoot organs in plants or plant cells.
- nucleic acid molecule substantially as described above wherein the plants or plant cells are eudicots.
- nucleic acid molecule substantially as described above wherein the plants or plant cells are Trifolium repens.
- nucleic acid molecule substantially as described above wherein the plants or plant cells are Arabidopsis.
- nucleic acid molecule substantially as described above wherein the plants or plant cells are cotton According to another aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above wherein the plants or plant cells are soya bean.
- nucleic acid molecule substantially as described above wherein the plants or plant cells are Nicotiana.
- a polypeptide substantially as described above wherein the plant cells are Nicotiana.
- a cell which has been altered from the wild type to include a nucleic acid molecule substantially described herein.
- a probe comprising at least 20 or more contiguous nucleotides selected from a sequence substantially as set forth in the sequence listing; or a functional fragment or variant thereof; or a homolog or ortholog thereof.
- a plant which has been altered from the wild type to include a nucleic acid molecule substantially as described above.
- nucleic acid molecule substantially as described herein to control the arrest of meristemoid cell division during organ development.
- a polypeptide substantially as described herein to control the arrest of meristemoid cell division during organ development.
- a primer for the conserved PEAPOD domain which has the following nucleotide sequence:
- the primer may have the sequence:
- a primer for the conserved PEAPOD domain which has the following nucleotide sequence:
- the found primer may have the sequence:
- RNAi gene silencing - refer Stoutjesdijk, P.A., Singh, S.P., Liu, Q., Hurlston, CJ. ,
- nucleic acid molecule as used herein may be an RNA, cRNA, genomic DNA or cDNA molecule, and may be single- or doublestranded.
- the nucleic acid molecule may also optionally comprise one or more synthetic, non-natural or altered nucleotide bases, or combinations thereof.
- isolated means substantially separated or purified away from contaminating sequences in the cell or organism in which the nucleic acid naturally occurs and includes nucleic acids purified by standard purification techniques as well as nucleic acids prepared by recombinant technology, including PCR technology, and those chemically synthesised.
- variant' refers to a nucleic acid molecule or polypeptide wherein the nucleotide or amino acid sequence exhibits substantially 70, 80, 95, or 99% homology with the nucleotide or amino acid sequence as set forth in the sequence listing - as assessed by GAP or BESTFIT (nucleotides and peptides), or BLASTP (peptides), or BLASTX (nucleotides). It should be appreciated that the variant may result from a modification of the native nucleotide or amino acid sequences, or by modifications including insertion, substitution or deletion of one or more nucleotides or amino acids.
- the nucleotide sequence of the native DNA may be altered appropriately for example by synthesis of the DNA de novo, or by modification of the native DNA, for example by site-specific or cassette mutagenesis.
- site-specific primer directed mutagenesis is employed using techniques standard in the art.
- a variant may be naturally occurring.
- the term variant also encompasses homologous sequences which hybridise under stringent conditions to the sequences of the invention.
- variant' also encompasses "conservative substitutions" wherein the alteration of the nucleotide or amino acid sequences, as set out in the sequence listing of this specification, results in the substitution of a functionally similar amino acid residue (Creighton T. E. 'Proteins Structure and Molecular Properties.' WH Freeman and Co. 1984).
- fragment nucleic acid molecule' refers to a nucleic acid molecule which represents a portion of the nucleic acid molecule of the present invention and is therefore less than full length and comprises at least a minimum sequence capable of hybridising stringently with a nucleic acid molecule of the present invention (or a sequence complementary thereto).
- a "fragment" of a polypeptide of the present invention is a portion of the polypeptide that is less than full length.
- the polypeptide fragment has at least approximately 60% identity to a polypeptide of the present invention, more preferably at least approximately an 80% identity, and most preferably at least approximately a 90% identity.
- the fragment has size of at least 10 amino acids, more preferably at least 15 amino acids, and most preferably at least 20 amino acids.
- a "substantially identical" amino acid sequence is an amino acid sequence which differs only by conservative amino acid substitutions. For example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine and so forth) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein.
- a sequence is at least 85%, more preferably 90% and more preferably 95% identical to amino acid level to the sequence of the protein or peptide to which it is being compared.
- a "substantially identical" nucleotide sequence codes for a substantially identical amino acid sequence as defined above.
- the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
- ortholog refers to a functionally equivalent yet distinct corresponding nucleotide or amino acid sequence that may be derived from another plant.
- an ortholog may have a substantially identical nucleotide or amino acid sequence to the sequences of the present invention as set forth in the sequence listing.
- construct refers to an artificially assembled or isolated nucleic acid molecule which includes the gene or nucleic acid molecule of interest.
- a construct may include the gene or genes of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used.
- construct includes vectors but should not be seen as being limited thereto.
- vector encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources.
- the cloning vector selected may depend on the host and host cell as used. In general the vector may:
- Suitable vectors may include:
- nucleic acid molecules of the present invention may be expressed via a control sequences such as a promoter operably linked thereto.
- control sequences such as a promoter operably linked thereto.
- Other control sequences may also be employed and may include origins of replication, enhancer and transcriptional terminator sequences.
- clone refers to a population of cells derived from a single cell.
- stable clone refers to a host cell which incorporates and expresses the exogenous nucleic acid or polypeptide molecule introduced via a vector or construct.
- transformed cell refers to a cell into which (or into an ancestor of which) there has been introduced, by means of recombinant DNA techniques, a nucleic acid molecule of interest.
- the nucleic acid of interest will typically encode a peptide or protein.
- the transformed cell may express the sequence of interest or may be used only to propagate the sequence.
- transformed may be used herein to embrace any method of introducing exogenous nucleic acids including, but not limited to, transformation, transfection, electroporation, microinjection, viral-mediated transfection, and the like.
- probe refers to a single-stranded nucleic acid molecules with a known nucleotide sequence which are labelled in some way (for example, radioactively, fluorescently or immunologically), or are otherwise detectable, and which are used to find and mark a target DNA or RNA sequence by hybridizing to it.
- protein refers to a protein encoded by the nucleic acid molecules of the invention, including fragments, mutations and homologs, orthologs, or analogs having the same biological activity.
- the protein or polypeptide or peptide of the invention can be isolated from a natural source, produced by the expression of a recombinant nucleic acid molecule, or can be chemically synthesized.
- plant' refers to the plant in it's entirety or a part thereof including selected portions of the plant during the plant life cycle such as the plant seeds, shoots, leaves, bark, pods, roots, flowers, stems and the like, or parts thereof.
- plant cell refers to any plant cell(s) from any stage of the plant life cycle, including plant seed cells, shoot cells, leave cells, bark cells, root cells, flower cells, pod cells, stem cells and the like.
- PEAPOD gene sequence of the present invention
- PEAPOD gene from Arabidopsis or the target crop
- Adding 1 or more copies of the PEAPOD gene to a crop or ornamental plant by transformation to reduce leaf and/or seed pod size.
- the intact gene with its own promoter may be added.
- the present invention may be useful in achieving genetically directed increases or decreases in the level of PEAPOD gene expression (this extends to orthologous genes where ever they are found in plants) to either increase or decrease the size of shoot organs.
- Altering the expression includes changes to the number of PEAPOD genes in the genome, gene silencing, mutating the PEAPOD gene, modification of the level of peapod gene transcription, steady state levels of mRNA or activity of the PEAPOD protein by alterations to the "PEAPOD Domain" conserved amino acid sequence.
- the invention may be used to genetically direct increases or decreases in the level of PEAPOD gene expression to alter the shape of plant shoot organs.
- PEAPOD genes can be reasonably predicted to be capable of manipulating; leaves, seed pods, fruits and flower petals.
- Figure 1 Leaves of peapod mutant plants are enlarged and have a bell-shaped curvature rather than the flat blade of wild type plants.
- the seed pods of peapod plants have a wide and flattened shape rather than the long tubular shape of wild type plants;
- Figure 2 Replica SEM of the leaf surface of wild type and peapod mutant leaves 14 days after germination
- Figure 7 Multiple alignment of PPD-like proteins.
- Figure 8 Phenotypes of Arabidopsis peapod mutant plants are complemented by transformation with TrPPD cloned cDNA.
- Figure 8 Mature leaves of peapod mutant (left) and three different complemented transgenic plants (right).
- TrPPD cDNA transgenic plants (right).
- the plant having the mutant peapod gene was identified during a screen for morphological mutants of ca 3,500 M2 seedlings grown from a commercially supplied Arabidopsis thaliana Landsberg erecta ecotype population obtained from M1 plants grown from seed treated by fast neutron mutagenesis. Fast neutrons typically cause mutations by deletion of segments of the genome.
- a single plant with the peapod mutant phenotype (large bell shaped leaves, short wide pea pod-like siliques, and reduced trichome branching) was identified. All M3 and M4 seed (self) collected and germinated from this plant retained the mutant phenotype.
- Arabidopsis plants were germinated after stratification at 4C for 5 days in 0.1 % agarose and grown in seedling soil mix either in an Environ cabinet, 2OC, 16 hour day length, or in a heated greenhouse with supplemented artificial light as required to give a 16 day length.
- peapod X wild type F1 selfed each time with selection of peapod homozygotes from the F2 before repeating the backcross to wild type i.e. a total of 10 generations).
- peapod Ler procipient
- wild type Columbia ecotype polylen donor
- F1 plants where selected by the dominant CoI phenotype and confirmed using a molecular CAPS marker that distinguishes Ler X CoI heterozygotes.
- F2 (self) progeny where screened to identify peapod homozygote segregants (those with all the original characteristics). Inheritance of peapod was determined in BC5 self progeny by scoring leaf curvature, trichome branching, and measuring silique width.
- Leaf dimension measurements of first and fourth leaves collected at the floral bolt stage where made by flattening the leaves between two microscope slides, scanning to produce a computer image together with an internal mm rule, and then calculating length, width, area and perimeter using a public domain image analysis programme (ImageJ).
- Mature silique dimensions where determined by measuring at least 30 siliques from each genotype using a digital micrometer.
- a cyc1At::GUS reporter gene was introgressed into wild type Ler and peapod Ler genetic backgrounds. Detection of GUS activity was carried out as described by Donnelly et al., 1999, Developmental
- DNA extracted from individual F2 peapod homozygous segregants was bulked into a single sample and analysed using a set of CAPS molecular markers suitable for detecting heterozygote loci (and map homozygote recombination positions) in a CoI X Ler population.
- the set of CAPS markers used are spaced throughout the Arabidopsis genome such that they can be used to distinguish the general chromosome arm position of a homozygous mutant locus. (Note: At the mutant locus all sequences flanking peapod are Ler. Linked markers have a higher than 50% chance of being Ler homozygotes. In bulk segregant analysis linkage is indicated by little or no apparent heterozygosity of a marker).
- Linkage was then confirmed by testing individual DNA samples with the CAPS marker identified by bulk segregant analysis. To fine map the position of the peapod locus DNA from individuals of a larger population of homozygous mutant F2 segregants was analysed with a series of CAPS, PCR or InDeI markers spanning regular intervals along the mapped chromosome arm. Once linkage distance and direction of the peapod locus from markers was established it became apparent that a known trichome branching gene was possibly collocated. PCR analysis using primers specific for genes located adjacent to the trichome branching gene At4g 14750 established the site and extent of chromosomal deletion in the peapod mutant.
- Plant lines containing T-DNA insertions in At4g14713 or At4g14720 in the Columbia (CoI) ecotype background were identified by searching the TAIR Arabidopsis web site. Insertion lines for At4g14713 (SALKJ 49924, SALK_057237) and At4g14720 (SALKJ 4698) were obtained from the Arabidopsis Biological Resource Center, Ohio State University, Columbus, OH, USA and verified by PCR analysis with T-DNA left border and gene specific primers, together with sequence analysis of the amplified fragment. Identification of homozygous T-DNA insertion plants was confirmed by co-segregation of the T-DNA and mutant phenotype.
- the At4g14713 and At4g14720 genes were cloned from wild type CoI ecotype genomic DNA by Hi Fidelity PCR using gene specific primers for sequences immediately at the end of the 3' UTR and about 1.5 Kb upstream of the beginning of the 5' UTR.
- the At4g14713 and At4g14720 gene PCR fragments were each cloned first into pGEMT and confirmed by PCR and sequence analysis.
- the gene specific primers each incorporated a terminal Not 1 restriction enzyme site. Clones were excised from pGEMT by Not 1 digestion and subcloned into the unique Not 1 site of pHZbar, a binary Agrobacterium transformation vector.
- Plasmids pHZbAt4g14713 and pHZbAt4g14720 were transferred to Agrobacterium tumefaciens strain AGL1. Mutant peapod plants were transformed by using an /t ⁇ rojbactera/m-mediated floral dip infiltration method. (Clough & Bent. 1998. Plant J 16: 735-743). Soil grown transgenic T1 plants were selected by repeated (3X) spraying with a solution of Buster (phosphothicin herbicide, 375 ug/L). Transgenic plants were confirmed by PCR analysis; with a combination of transgene specific and T-DNA primers, the absence of PCR products for other genes in the deleted region and the mutant unbranched trichome phenotype.
- Quantitative RT-PCR assays for the relative level of At4g14713 gene transcript were performed on a Bio-Rad MyIQ colour Real Time PCR instrument (Bio-Rad) using Ornithine Transfer Carboxylase (OTC) as an internal control.
- One of the gene-specific primers used to detect each gene were designed to span an intron boundary thereby eliminating detection of any contaminating genomic DNA.
- the discovery leading to this invention is based on the identification of an Arabidopsis mutant, peapod, with enlarged and altered curvature of the laminal plane (mediolateral axis or blade) of lateral shoot organs.
- the peapod mutant has enlarged bell shaped instead of flat leaves.
- the seed pods of peapod plants have a wide and flattened shape rather than the long tubular shape of wild type plants ( Figure 1 ).
- Trichomes (hairs) on the mutant have only two branches rather than the wild type 3-4 branches.
- peapod leaves have a greater length, width and lamina area, but the same perimeter as wild type leaves - refer Table 1.
- Table 1 Measurements of mature wild type and peapod leaf laminae. Mean mm (SD) from 14 leaves; Leaf Genotype Length Width Area Perimeter
- Table 2 Measurements of wild type and peapod siliques. Mean mm (SD) from 30 siliques;
- Table 3 Homozygous T-DNA insertions in individual peapod genes produce a semi- dominant peapod phenotype Influence of PEAPOD gene copy number on silique dimensions. Mean mm (SD) from 30 siliques.
- the expression pattern of the PEAPOD gene was established using mRNA in situ localisation in tissue sections. Expression of the peapod gene coincides with the onset of cell differentiation and the arrest of meristemoid cell division in developing leaves and seed pods. Expression is absent from tissues undergoing cell division (not shown).
- At4g14713 and At4g14720 cDNA, genomic, and predicted amino acid sequence
- EST's a sequence of proteins that have the "PEAPOD Domain" sequence runs at the 5' end of cDNA clones.
- these sequences are useful for determining the likely distribution of the PEAPOD-Wke genes in the plant kingdom.
- the Medicago truncatula primer sequences used to amplify the white clover PPD-like gene are:
- TrPPD cDNA expression cassette was subsequently subcloned into the Not1 restriction site of the Agrobacterium binary vector pHZbar and transformed into peapod mutant Arabidopsis plants using the floral dip method (Clough & Bent. 1998. Plant J 16: 735-743)
- RNA interference construct was prepared using a 165 base pair DNA fragment spanning the start codon and predicted PPD Domain. The DNA fragment was amplified from the cloned TrPPD cDNA sequence by PCR using sequence specific primers incorporating EcoR1, Xho1, BamHI, or Xbal restriction enzyme sites.
- the sequence amplified was:
- RNAi vector pRNA 69 Equivalent 5 ' BamH1/Xba1-3' and 5 ⁇ coR1/Xho1 3' fragments were cloned into RNAi vector pRNA 69 to produce a expression cassette composed of CaMV 35S promoter - reverse TrPPD fragment -intron - forward TrPPD fragment - OCS 3' terminator.
- This TrPPD RNAi cassette was subcloned into the Not1 site of Agrobacterium plant transformation vector pHZbar. The contruct was used to transform white clover, cultivar Huia, cotyledons using established tissue culture and clover transformation methods (White & Voisey 1994. Plant Cell Reports 13: 303-308; Voisey, White, Dudas, Appleby, Ealing, Scott 1994. Plant Cell Reports 13:309-314).
- Leaf and petiole length dimension measurements of the fourth fully expanded leaf from the shoot tip, and intemode length between the fourth and fifth observable nodes were obtained using a digital micrometer (measured in mm). At least five samples were measured for each reported dimension.
- TrPPD PEAPOD homolog
- Table 4 Silique sizes of Arabidopsis peapod plants complemented with a white clover cDNA gene construct.
- the white clover PEAPOD-WWe protein coding sequence is therefore functionally equivalent to the Arabidopsis PPD protein coding sequence. Furthermore, experiments silencing the endogenous TrPPD gene in transgenic white clover plants established that the TrPPD gene also acts in white clover to limit leaf lamina size. When a TrPPD RNA interference gene construct was introduced into white clover some of the transgenic plants had abnormally enlarged leaves with positive Gaussian curvature and elongated petioles. An example, comparing some leaf and stem dimensions of wild type and RNAi transgenic plants, is given in Table 5.
- the peapod mutant phenotype is due to the loss-of-function of a key gene controlling the arrest of meristemoid cell division during organ development
- the peapod mutation is due to a deletion of a ca 60 Kilobase segment of the Arabidopsis genome, located between ca 8,420Kb and 84800Kb.
- the peapod mutation is due to the deletion of a duplicated pair of genes, i.e. there are two adjacent peapod genes (At4g14713 & At4g 14720) that have to be inactivated to give the mutant phenotype.
- PEAPOD is a protein unlike any other previously described.
- PEAPOD has a protein domain conserved in a range of plants. • The PEAPOD gene is only found in plants.
- PEAPOD is a key gene regulating flexibility in organ size.
- PEAPOD gene function is conserved in other eudicot plants.
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Cited By (4)
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CN102260679A (en) * | 2010-05-27 | 2011-11-30 | 中国科学院上海生命科学研究院 | Protein for regulating growth of fruits and seeds and use thereof |
WO2016005449A1 (en) | 2014-07-08 | 2016-01-14 | Vib Vzw | Means and methods to increase plant yield |
WO2016071830A2 (en) | 2014-11-04 | 2016-05-12 | Agresearch Limited | Methods for plant improvement |
WO2016071829A1 (en) * | 2014-11-04 | 2016-05-12 | Agresearch Limited | Methods for monocot plant improvement |
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CN102260679A (en) * | 2010-05-27 | 2011-11-30 | 中国科学院上海生命科学研究院 | Protein for regulating growth of fruits and seeds and use thereof |
CN102260679B (en) * | 2010-05-27 | 2013-06-05 | 中国科学院上海生命科学研究院 | Protein for regulating growth of fruits and seeds and use thereof |
WO2016005449A1 (en) | 2014-07-08 | 2016-01-14 | Vib Vzw | Means and methods to increase plant yield |
WO2016071830A2 (en) | 2014-11-04 | 2016-05-12 | Agresearch Limited | Methods for plant improvement |
WO2016071829A1 (en) * | 2014-11-04 | 2016-05-12 | Agresearch Limited | Methods for monocot plant improvement |
WO2016071830A3 (en) * | 2014-11-04 | 2016-07-14 | Agresearch Limited | Methods for plant improvement |
CN107105627A (en) * | 2014-11-04 | 2017-08-29 | 农牧研究公司 | The method improved for monocotyledon |
CN107205355A (en) * | 2014-11-04 | 2017-09-26 | 农牧研究公司 | method for plant improvement |
EP3214921A4 (en) * | 2014-11-04 | 2018-08-08 | Agresearch Limited | Methods for monocot plant improvement |
EP3214922A4 (en) * | 2014-11-04 | 2018-08-22 | Agresearch Limited | Methods for plant improvement |
US10337022B2 (en) | 2014-11-04 | 2019-07-02 | Agresearch Limited | Methods of increasing root biomass in plants |
US10487337B2 (en) | 2014-11-04 | 2019-11-26 | Agresearch Limited | Methods for monocot plant improvement |
RU2727424C2 (en) * | 2014-11-04 | 2020-07-21 | Агрисерч Лимитед | Methods of plants improvement |
RU2727428C2 (en) * | 2014-11-04 | 2020-07-21 | Агрисерч Лимитед | Methods for improvement of monocotyledon plants |
AU2015344482B2 (en) * | 2014-11-04 | 2021-04-01 | Agresearch Limited | Methods for monocot plant improvement |
CN107205355B (en) * | 2014-11-04 | 2021-04-30 | 农牧研究公司 | Method for plant improvement |
Also Published As
Publication number | Publication date |
---|---|
WO2007105967A8 (en) | 2009-07-23 |
NZ545621A (en) | 2008-05-30 |
AU2007225511A1 (en) | 2007-09-20 |
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