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WO1999004003A1 - Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede - Google Patents

Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede Download PDF

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Publication number
WO1999004003A1
WO1999004003A1 PCT/NL1997/000424 NL9700424W WO9904003A1 WO 1999004003 A1 WO1999004003 A1 WO 1999004003A1 NL 9700424 W NL9700424 W NL 9700424W WO 9904003 A1 WO9904003 A1 WO 9904003A1
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Prior art keywords
gene
dna
plants
inhibited
flowering
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PCT/NL1997/000424
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English (en)
Inventor
Gerco C. Angenent
Marco Busscher
John Franken
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Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo)
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Priority to PCT/NL1997/000424 priority Critical patent/WO1999004003A1/fr
Priority to EP97930888A priority patent/EP0985041A1/fr
Priority to IL13408397A priority patent/IL134083A0/xx
Priority to PL97338241A priority patent/PL338241A1/xx
Priority to NZ502374A priority patent/NZ502374A/en
Priority to BR9714986-1A priority patent/BR9714986A/pt
Priority to AU34655/97A priority patent/AU742459B2/en
Priority to CA002296761A priority patent/CA2296761A1/fr
Publication of WO1999004003A1 publication Critical patent/WO1999004003A1/fr

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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
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • 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

Definitions

  • the present invention is related to recombinant DNA, particularly to recombinant DNA in relation to genetic modification of plants.
  • the modification relates to altering the flowering process of plants and specifically to inhibiting flowering in genetically engineered plants.
  • the present invention also relates to DNA sequences which code for MADS box transcription factors, which, upon integration into a plant genome, modify flowering.
  • Plant development is characterised by a series of phase changes in which meristems are capable of generating new meristems of different identity.
  • Vegetative meristems produce roots and leaves and may give rise to generative meristems upon floral induction (evocation) .
  • floral induction evocation
  • the switch from vegetative to generative phase is induced in response to many environmental conditions such as temperature, light conditions, and day-length.
  • the process of flower induction is influenced by internal factors such as the age of the plant, hormones and gene products (Bernier, 1988) . Almost nothing is known, however, about the molecular and genetic controls that induce a plant to flower.
  • the new flower meristems form flowers, whereas vegetative meristems produce leaves.
  • the new flower meristems are formed in the axils of small leaf-like organs which are called bracts. These bracts are distinguishable from vegetative leaves with respect to their shape and position in the plant.
  • the next developmental step is the determination of the identity of the floral organs, sepals, petals, stamens and carpels, which develop as distinct primordia from the floral apex. In the stamens and carpels, the reproductive cells are produced and upon fertilisation seeds are formed giving rise to the next generation. Mutants affected in the flowering process have been characterised from many species, in particular, from Arabidopsis thaliana .
  • a number of Arabidopsis mutants are known displaying an early or late flowering phenotype (see for review, eigel, 1995) . Although the transition from vegetative to reproductive phase can be delayed dramatically in some of these late flowering mutants, eventually they start to flower, indicating that the affected genes are not essential for flowering. Many of these early- and late- flowering genes are thought to modify the transduction of the external factors which are involved in flower induction.
  • the late- flowering gene CONSTANS (CO) which encodes a Zinc finger transcription factor (Putterill et al, 1995 ) is involved in light perception or transduction and, upon mutation, results in a late flowering phenotype specifically under short-day conditions.
  • Another Arabidopsis gene that affects flowering time and have been cloned is LUMINIDEPENDENS (Lee et al . , 1994) .
  • ID INDETERMINATE
  • This ID gene encodes a Zinc finger transcription factor and disturbance of this gene by insertion of a DS transposable element resulted in the inhibition of flowering in maize plants.
  • the so-called 'meristem identity' genes that control the identity of the flower meristem can also be used. These genes are LEAFY (LFY, Weigel et al . , 1992 and APETALA1 (API , Mandel et al., 1992J from Arabidopsis and their ho ologues from Antirrhinum FLORICAULA (FLO, Coen et al., 1990) and SQUAMOSA (SQUA, Huijser et al . , 1992), respectively.
  • LEAFY LEAFY, Weigel et al . , 1992
  • API Mandel et al., 1992J from Arabidopsis and their ho ologues from Antirrhinum FLORICAULA (FLO, Coen et al., 1990)
  • SQUAMOSA SQUA, Huijser et al . , 1992
  • API and SQUA are both members of the MADS box gene family coding for transcription factors. These MADS box transcription factors share a highly conserved domain which is called the 'MADS box' and facilitates the DNA binding (Schwarz-Sommer et al., 1990) .
  • the identity of the floral organs, sepals, petals, stamens, carpels, and within the carpels, the ovules is determined by four classes of homeotic genes. These homeotic genes are acting alone or in combination to determine floral organ identity. A model describing the action of these genes has been proposed first by Coen and Meyerowitz (1991) and later modified for genes controlling ovule identity by Colombo et al. (1995). Most of these organ identity genes belong to the MADS box gene family. The function of these genes has been determined in a number of dicotyledon and monocotyledon species by gene inactivation and ectopic expression in transgenic plants. These studies have demonstrated that these homeotic genes are well conserved in the plant kingdom and play similar roles with respect to flower development in many species.
  • the reproductive phase is characterised by the production of flowers which is often preceded by the formation of an inflorescence.
  • Inflorescence An inflorescence is characterised by a structure bearing the flowers which arises from the axils of leaf-like organs called 'bracts'.
  • Antisense construct or gene a gene or a nucleotide sequence derived thereof, having a homology of more than 70%, preferably more than 90% to a target gene and which is linked to a promoter in the inverse 3 ' to 5 ' orientation with respect to the target gene.
  • Cosuppression construct or gene a gene or a nucleotide sequence derived thereof, having a homology of more than 70%, preferably more than 90% to the target gene and which is linked to a promoter in the 5' to 3 ' orientation or which expression is not driven by an exogenous promoter.
  • MADS box gene a gene coding for a transcription factor having a region of 56 a ino acids which is homologous to a similar region in the Arabidopsis AGAMOUS protein and Antirrhinum DEFICIENS protein. This region is called the 'MADS box' . At least 50% of the amino acids in this region should be identical to the amino acid composition in the MADS boxes of AGAMOUS and DEFICIENS.
  • Inhibiting flower formation can be advantageous in plants which multiply in predominantly vegetative manner. Flowers are strong sink tissues and therefore, prevention of flowering may lead to an increased deposition of stored substances in storage organs and increased growth of vegetative organs such as leaves, stems and roots. It therefore appears desirable to provide a method to inhibit flowering independent from external factors and without exogenous application of substances .
  • the present invention provides a method to produce genetically engineered plants in which the flowering process is altered. More specifically, the inventors have found a method for reducing flowering or completely abolishing the formation of inflorescences and flowers.
  • inhibiting flowering means that the transformed plants do not produce inflorescences, neither inflorescences bearing flowers nor inflorescences without flowers, develop fewer inflorescences or short inflorescences with a single or a few flowers.
  • this invention provides an isolated DNA sequence which encodes a MADS box protein indicated FBP10 and having the amino acid sequence given in SEQ ID NO:2 of the sequence listing hereinafter or a functionally homologous protein or part thereof, wherein inactivation of gene function of said DNA sequence, if present as an endogenous gene in a plant, results in inhibition of flowering.
  • the DNA sequence of the invention can also be characterized in that it comprises the FBP10 gene or corresponding cDNA having the nucleotide sequence given in SEQ ID N0:1 or a functionally homologous gene or an essentially identical nucleotide sequence or part thereof or derivatives thereof which are derived from said sequence by insertion, deletion or substitution of one or more nucleotides.
  • the invention provides an RNA sequence encoded by any of the above defined DNA sequences .
  • the invention provides a protein encoded by any of the above defined DNA sequences . Further the invention provides processes of producing transgenic plants in which flowering is inhibited, comprising inhibiting the expression of endogenous FBP10 gene or a homologous gene.
  • the invention provides recombinant double- stranded DNA molecules comprising an expression cassette to be used in the above process .
  • the invention provides transgenic plants showing inhibited flowering, and also plant cells, seeds, tissue culture, plant parts or progeny plants derived from said transgenic plants.
  • Figure 1 is the cDNA sequence of the petunia FBP10 gene and the deduced amino acid sequence.
  • the MADS box region is underlined.
  • Figure 2 is a comparison of the petunia FBP10 protein sequence and known homologous protein sequences from potato (P0TM1) , tomato (TDR4) , and Arabidopsis (AGL8) .
  • P0TM1 petunia FBP10 protein sequence
  • TDR4 tomato
  • AGL8 Arabidopsis
  • Figure 3 shows the expression of FBP10 in wild type petunia plants (line W115) and in FBP10 plants as determined by Northern blot analysis.
  • the blot contains total RNA from the tissues as indicated: leaf, stem, bract, sepal, petal, stamen, style/stigma, ovary and leaf, stem, shoot and flower from the cosuppression plant.
  • the Smal/Hindlll DNA fragment (nucleotide 325-885) of FBP10 cDNA was used as probe.
  • Figure 4 shows the expression of FBP10 by in si tu hybridisations using a digoxygenin labeled antisense RNA probe ( Figures 4B, 4D, and 4F) derived from the Smal/Hindlll fragment (nucleotide 325-885) of FBP10 cDNA.
  • Sense control probe has been used for figures 4A, 4C, and 4E.
  • Figures A and B show longitudinal sections through an apical meristem, C and D show an axillary vegetative meristem and in E and F floral buds are depicted.
  • a apical mersitem
  • am axillary meristem
  • l leaf
  • b bract
  • f floral meristem
  • l sepal
  • 2 petal
  • 3 stamen.
  • Figure 5 depicts a schematic presentation of the T-DNA region between the borders of the binary vector containing the FBPIO sense construct.
  • This binary vector designated pFBP113, was used to generate transgenic petunia plants in which FBPI O expression was inhibited.
  • Figure 6 shows the phenotype of a wild type petunia plant (a left) and representative plants in which the expression of FBPI O was inhibited (a right and b) .
  • the DNA sequence of the invention encodes a MADS box protein indicated FBPIO or a fucntionally homologous protein or part thereof.
  • the cDNA sequence of the FBP10 gene and the deduced amino acid sequence are given in Fig. 1 and in SEQ ID No:l and 2.
  • the term "functionally homologous" in the present description and claims should be understood to mean proteins and genes belonging to the MADS box family and being functionally equivalent to FBP10 protein and FBP10 gene, respectively. Inhibition of the endogenous FBP10 gene in a plant results in inhibition of flowering.
  • Homologous sequences are, for example, sequences derived from other organisms than petunia. The percentage of sequence similarity of FBP10 and a homologous gene may vary.
  • the functionally homologous gene from a species related to petunia may have more than 90% sequence identity, whereas the functional homologue from a non-related species may have only 65% or less sequence identity.
  • the invention also relates to derivatives of the FBP10 gene or a homologous gene, which can be derived from the parent sequence by insertion, deletion or substitution of one or more nucleotides. This includes naturally occurring variations or variations introduced through targeted mutagenesis or recombination.
  • the expression of endogenous FBPI O gene or a homologous gene is inhibited by the use of anti-sense RNA.
  • the process comprises the following steps: a) a DNA, or a DNA fragment having at least 15 base pairs, which is complementary to a degree of at least 70% to the FBP10 gene or homologous gene is introduced in a plant cell, b) said introduced DNA is transcribed into anti- sense RNA, said DNA being expressed constitutively or tissue- specific, or being induced by promoter elements controlling the expression of the introduced DNA, c) the expression of endogenous FBP10 gene or homologous gene is inhibited because of the anti-sense effect, d) plants are regenerated from the transgenic cells, and e) plants exhibiting inhibited flowering are selected.
  • step a) instead of the complete FBP10 gene or homologus gene sequence, partial sequences thereof can be used to obtain antisense inhibition. Sequences up to a minimum length of 15 base pairs can be used. It is also possible to use DNA sequences which have a high degree of similarity to the FBP10 gene or homologous gene sequence. The minimum similarity should be 70%, preferably more than 90%.
  • the expression of endogenous FBP10 or a homologous gene is inhibited through the use of sense/cosuppression.
  • the process comprises the following steps: a) a DNA which is the FBP10 gene or homologous gene or a sequence having at least 70% sequence similarity to said
  • FBP10 or homologous gene, or a fragment thereof having at least 15 base pairs is introduced in a plant cell, b) said DNA being expressed constitutively or tissue- specific or being induced by promoter elements controlling the expression of the introduced DNA in such a way that transcription produces sense RNA, or being introduced without the use of a promoter, c) the expression of endogenous FBPI O gene or homologous gene and the introduced gene are inhibited by the cosuppression effect, d) plants are regenerated from the transgenic cells, and e) plants exhibiting inhibited flowering are selected.
  • partial sequences of at least 15 base pairs, and sequences having a similarity of at least 70%, preferably at least 90%, can be used.
  • the expression of endogenous FBP10 or a homologous gene is inhibited by the use of dominant- negative mutations.
  • the process comprises the following steps: a) a DNA which encodes a modified FBP10 protein or homologous protein or an essential part thereof, whereby said modified protein or part thereof is suitable for inhibiting the function of the endogenous FBP10 protein or homologous protein, is introduced in a plant cell, b) said introduced DNA is transcribed into RNA and translated into a polypeptide, said DNA being expressed constitutively or tissue- specific, or being induced by promoter elements controlling the expression of the introduced DNA, c) the function of the endogenous FBP10 protein or homologous protein is inhibited by the dominant-negative mutation effect, d) plants are regenerated from the transgenic cells, and e) plants exhibiting inhibited flowering are selected.
  • the transgenic plants produced by this process express an altered FBP10 protein or homologue thereof. As a result said plants are changed in their flowering behaviour.
  • This strategy has been successful for the inhibition of the Arabidopsis MADS box protein AGAMOUS by expressing a c-terminal truncated AGAMOUS protein (Ma et al, 1996) .
  • the invention also relates to recombinant double-stranded DNA molecules for use in the above processes for producing transgenic plants.
  • the invention provides a recombinant double-stranded DNA molecule for use in the anti-sense method, comprising an expression cassette comprising the following constituents: i) a promoter functional in plants, ii) a DNA sequence which is FBP10 gene or a homologous gene as defined above, which is fused to the promoter in anti-sense orientation so that the non- coding strand is transcribed, and if necessary iii) a signal fucntional in plants for the transcription termination and polyadenylation of an RNA molecule.
  • the invention provides a recombinant double-stranded DNA molecule for use in the sense/cosuppression method, comprising an expression cassette comprising the following constituents: i) a DNA sequence which is FBP10 gene or a homologous gene as defined above, ii) optionally a promoter functional in plants, which is fused to the DNA sequence in sense orientation, and if necessary iii) a signal functional in plants for the transcription termination and polyadenylation of an RNA molecule.
  • the invention provides a recombinant double-stranded DNA-molecule for use in the dominant-negative mutation method, comprising an expression cassette comprising the following constituents: i) a promoter functional in plants, ii) a DNA sequence which is a modified FBP10 gene or homologous gene as defined above, which is fused to the promoter in sense orientation, and if necessary iii) a signal functional in plants for the transcription termination and polyadenylation of an RNA molecule.
  • a preferred promoter to be used in any of the above processes or recombinant double-stranded DNA molecules for expressing the said recombinant polynucleotide that is active in the shoot apical meristems comprises the cauliflower mosaic virus (CaMV) 35S promoter.
  • the promoter is an inducible promoter active in the shoot meristem or a tissue-specific promoter active in the shoot meristem.
  • an inducible promoter which can be activated or suppressed in response to external stimuli. Activation or suppression of the promoter results in plants displaying characteristics according to the present invention.
  • the present invention provides plants without inflorescences or flowers. Because flowers and seeds and fruits produced thereof are high energy demanding tissues
  • the present invention provides a method to increase the total biomass of vegetative tissues, such as roots, tubers, stems and leaves.
  • the present invention can be used in Gymnosperms and
  • Angiosperms The present invention is especially useful for plant species for which vegetative propagation is possible.
  • parent plants carrying transgenes related to the DNA sequence shown in figure 1 (SEQ ID NO: 1
  • the parent plants have a wild-type phenotype.
  • the present invention is especially useful for plant species for which the vegetative part of the plant is used as the economical product such as vegetables (e.g. lettuce, spinach, chicory, etc.), sugar beet, potato, trees for wood production (e.g. Eucalyptus, oak, willow, etc), tobacco, grasses, plants for nitrogen fixation, ornamental plants for production of cuttings.
  • vegetables e.g. lettuce, spinach, chicory, etc.
  • sugar beet e.g.
  • trees for wood production e.g. Eucalyptus, oak, willow, etc
  • tobacco, grasses e.g. Eucalyptus, oak, willow, etc
  • ornamental plants for production of cuttings e.g. Eucalyptus, oak, willow, etc
  • the use of the present invention is of particular interest to sugar beet, since "bolting" can be prevented by inhibition of inflorescence formation.
  • This flower induction process is induced by vernalization (cold treatment) and can be circumvented by planting relatively late in the year (April/May) . Inhibition of inflorescence formation by applying the present invention will allow an earlier planting of the sugar beet resulting in an increase in yield.
  • the use of the present invention is of particular interest for grasses to improve its quality as feed for cattle. Inflorescences of grasses contain relatively low quantities of carbohydrates and large quantitives of lignin, which cannot be digested by cattle.
  • Petunia MADS box cDNA clones were isolated from a cDNA library made from young petunia pistils (Angenent et al . , 1993)
  • the cDNA library was constructed using the lambda ZAP cloning vector (Stratagene)
  • the library was screened under low stringency hybridization conditions with a mixed probe comprising the MADS box regions of Floral binding protein gene 1 ⁇ FBP1 ) and FBP2 (Angenent et al . , 1993).
  • the hybridizing phage plaques were further purified using standard techniques. Using the in vivo excision method, E.
  • coli clones which contain a double-stranded Bluescript SK-plasmid with the cDNA insertion between the EcoRl and Xhol cleavage site of the polylinker were generated.
  • Cross-hybridization of the purified clones revealed 10 independent clones designated FBP2, FBP6- 14.
  • FBP10 The nucleotide sequence of a full length clone (clone CIO) was determined by the dideoxynucleotide-mediated chain termination method and is depicted in Figure 1 (SEQ ID NO:l).
  • the FBPI O cDNA clone has a length of 1131 nucleotides and encodes for a polypeptide of 246 amino acid residues.
  • FBPIO a N-terminal located MADS box region which shows a high degree of similarity with other MADS box proteins, and a K-box in the middle of the protein with an alpha helical structure.
  • the alignment of FBPIO and homologues from Arabidopsis, potato and tobacco is shown in Figure 2.
  • FBPIO is most similar to the potato MADS box protein POTM1 (Kang and Hannapel, 1995) .
  • the functions of POTM1, TDR4, and AG18 have not been determined yet.
  • FBPI O The expression of FBPI O was determined by standard Northern blot hybridization experiments according to Angenent et al. (1992). A Smal/Hindll DNA fragment containing nucleotides 325 to 885 of FBPIO cDNA was used as a probe. Using 10 ⁇ g of total RNA from various petunia tissues, expression of FBP10 was detectable in young leaves, stems, bracts, and all floral organs except anthers. No expression was detectable in roots and old leaves ( Figure 3) . The expression in the apical meristem was determined by in situ hybridization using a DIG labeled antisense RNA probe corresponding to the Smal/Hindlll fragment of the FBP10 cDNA. ( Figure 4) .
  • RNA transcripts were made using T7 RNA polymerase.
  • a standard protocol for in situ hybridization was used as described by Canas et al . , 1994. Strong hybridizing signals were observed in the vegetative apical meristem, floral meristem and weaker signals in the developing leaves .
  • the full length FBP10 cDNA was subcloned into the binary vector pFBP20 (Angenent et al . , 1993).
  • This binary vector contains the CaMV 35S promoter, the adh intron, a multiple cloning site for insertion of the cDNA and the nos terminator.
  • the full length cDNA clone (CIO) present in the bluescript SK- vector (Strategene lambda ZAP excision vector) was cut with BamHl and Xhol and reinserted in the binary vector pFBP20 yielding plasmid pFBP113 ( Figure 5) .
  • the binary vector containing the FBPI O cDNA in the sense orientation behind the CaMV 35S promoter was transferred to Agrobacterium tumefaciens strain LBA4404 by triparental mating.
  • the plasmid was transferred from E.coli HB101 to LBA4404 using a strain containing plasmid pRK2013.
  • Plasmid DNA from the Agrobacterium conjugates were isolated and the structure of the binary vector was verified by restriction analysis.
  • Agrobacterium conjugants were used to transform Petunia hybrida leaf disks as described by Horsch et al. (1985) .
  • Leaf disks were prepared from top leaves of young Petunia hybrida variety W115 plants. After shoot and root induction on Kanamycin selection media, plants were planted in soil and transferred to the greenhouse.
  • Wild-type petunia plants have indetermined inflorescences of the raceme type ( Figure 6a) .
  • Figure 6a In the axils of two bracts, which are positioned opposite to each other, a flower is formed and the inflorescence continues. Under normal greenhouse conditions, this inflorescence maintains an inflorescence identity and never reverses to the vegetative phase. Vegetative leaves are not positioned opposite to each other like bracts, but are arranged in a spiral phylotaxy. Two plants were selected which showed abberations in inflorescence structure. Occasionally, part of these inflorescences reverted to short vegetative shoots.
  • T30.009 and T30.012 were self pollinated and the offspring were analyzed.
  • the offspring of T30.009 could be divided into three classes: 8 plants with a wild-type phenotype, 12 plants with a phenotype resembling the primary transformant, and 5 plants with severe alterations in inflorescence development. Plants from the latter class are affected in the switch from vegetative to inflorescence phase, in that vegetative shoots are produced instead of indetermined inflorescences ( Figures 6a and 6b) .
  • Vernalization is a well known inducer of flowering for many species. Therefore, we analysed the stability of the non- flowering phenotype of the FBP10 mutant by exposure of the plants to cold conditions (4 °C) for 4 and 8 weeks. Wild type plants (line W115) were treated simultaneously. The plants (4 plants of each) were transferred to the cold about two weeks before they start to flower under normal greenhouse conditions (20 °C) . The cold treatments did not affect the timing of flowering, neither for the wild type plants nor for the FBPIO mutants. About two weeks after the treatment wild type plants started to flower, while the FBPIO mutants remained vegetative just like the untreated FBPIO mutant plants.
  • Floricaula a homeotic gene required for flower development in Antirrhinum majus. Cell, 63, 1311-1322.
  • Bracteomania an inflorescence anomaly, is caused by the loss of function of the MADS box gene sguamosa in Antirrhinum majus .
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • AAAA ATG GGA AGA GGA AGA GTG CAG ATG AAG AGA ATT GAG AAT AAA ATT 109 Met Gly Arg Gly Arg Val Gin Met Lys Arg lie Glu Asn Lys lie 1 5 10 15
  • GCA AAA CTT AAG GCC AGA ATT GAG GTT GTG CAG AGA AAC CAA AGG CAT 397 Ala Lys Leu Lys Ala Arg lie Glu Val Val Gin Arg Asn Gin Arg His 100 105 110
  • GAG AAA GAG TTG GCT CAA CAA ACT CAA TGG GAG CAG CAG AAT AAT CAT 637 Glu Lys Glu Leu Ala Gin Gin Thr Gin Trp Glu Gin Gin Asn Asn His 180 185 190
  • GATCTTATTT ACTGTATCAG CAGCCTTGCC TTGAATAACT TAAATATTCT GAATGATCT 1131

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Abstract

L'invention porte sur une séquence d'ADN de pétunia codant pour une protéine de MADS box dite FBP10 présentant la séquence d'acide aminé donnée sous SEQ ID NO:2, ou une protéine à fonctionnalité homologue. Ladite séquence d'ADN contient le gène FBP10 présentant la séquence nucléotidique d'ADNc donnée sous SEQ ID NO:2 ou une protéine à fonctionnalité homologue. L'inactivation de la fonction de ladite séquence d'ADN, si elle se trouve dans un gène endogène de plante, provoque l'inhibition de la floraison. L'invention porte également sur le procédé d'obtention d'une plante transgénique dont la floraison est inhibée, consistant à empêcher l'expression du gène endogène FBP10 ou d'un gène homologue.
PCT/NL1997/000424 1997-07-18 1997-07-18 Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede WO1999004003A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
PCT/NL1997/000424 WO1999004003A1 (fr) 1997-07-18 1997-07-18 Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede
EP97930888A EP0985041A1 (fr) 1997-07-18 1997-07-18 Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede
IL13408397A IL134083A0 (en) 1997-07-18 1997-07-18 Process of producing transgenic plants in which flowering is inhibited, and dna sequences used in said process
PL97338241A PL338241A1 (en) 1997-07-18 1997-07-18 Method of obtaining transgenic plants of inhibited efflorescence
NZ502374A NZ502374A (en) 1997-07-18 1997-07-18 Production of transgenic plants in which flowering is inhibited by using the FBP10 gene
BR9714986-1A BR9714986A (pt) 1997-07-18 1997-07-18 Processo de produção de plantas transgênicasnas quais a floração é inibida e sequências de dnausadas no referido processo
AU34655/97A AU742459B2 (en) 1997-07-18 1997-07-18 Process of producing transgenic plants in which flowering is inhibited, and DNA sequences used in said process
CA002296761A CA2296761A1 (fr) 1997-07-18 1997-07-18 Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede

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WO2001004315A3 (fr) * 1999-07-12 2001-07-12 Texas A & M Univ Sys Compositions de genes rin et leurs procedes d'utilisation
WO2002036776A1 (fr) * 2000-10-30 2002-05-10 National Institute Of Agrobiological Sciences Amelioration apportee a un gene a mads box ciblant un type de fleur
EP1231835A4 (fr) * 1999-10-12 2005-03-16 Mendel Biotechnology Inc Modification du temps de floraison
US8022274B2 (en) 1998-09-22 2011-09-20 Mendel Biotechnology, Inc. Plant tolerance to low water, low nitrogen and cold
EP2272962B1 (fr) * 2002-09-18 2017-01-18 Mendel Biotechnology, Inc. Polynucléotides et polypeptides dans les plantes
CN108949773A (zh) * 2017-05-18 2018-12-07 萧郁芸 兰花胚珠发育调控基因及其方法
US11124801B2 (en) 2018-04-18 2021-09-21 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
US12371702B2 (en) 2018-04-18 2025-07-29 Pioneer Hi-Bred International, Inc. Improving agronomic characteristics in maize by modification of endogenous mads box transcription factors

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US8022274B2 (en) 1998-09-22 2011-09-20 Mendel Biotechnology, Inc. Plant tolerance to low water, low nitrogen and cold
EP1055729A1 (fr) * 1999-05-18 2000-11-29 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Plantes tansgeniques presentant un debut de floraison modifie
WO2000070053A1 (fr) * 1999-05-18 2000-11-23 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Plantes transgeniques a temps de floraison modifie
WO2001004315A3 (fr) * 1999-07-12 2001-07-12 Texas A & M Univ Sys Compositions de genes rin et leurs procedes d'utilisation
EP1231835A4 (fr) * 1999-10-12 2005-03-16 Mendel Biotechnology Inc Modification du temps de floraison
WO2002036776A1 (fr) * 2000-10-30 2002-05-10 National Institute Of Agrobiological Sciences Amelioration apportee a un gene a mads box ciblant un type de fleur
US7282622B2 (en) 2000-10-30 2007-10-16 National Institute Of Agrobiological Sciences Flower morphology of plants by targeting mads-box gene
EP2272962B1 (fr) * 2002-09-18 2017-01-18 Mendel Biotechnology, Inc. Polynucléotides et polypeptides dans les plantes
CN108949773A (zh) * 2017-05-18 2018-12-07 萧郁芸 兰花胚珠发育调控基因及其方法
CN108949773B (zh) * 2017-05-18 2023-12-26 萧郁芸 产生转基因植物的方法
US11124801B2 (en) 2018-04-18 2021-09-21 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
US11421242B2 (en) 2018-04-18 2022-08-23 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
US12234470B2 (en) 2018-04-18 2025-02-25 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
US12371702B2 (en) 2018-04-18 2025-07-29 Pioneer Hi-Bred International, Inc. Improving agronomic characteristics in maize by modification of endogenous mads box transcription factors

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IL134083A0 (en) 2001-04-30
EP0985041A1 (fr) 2000-03-15
AU742459B2 (en) 2002-01-03
AU3465597A (en) 1999-02-10
CA2296761A1 (fr) 1999-01-28

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