EP1196582A1 - Modulation genetique de la floraison au moyen du gene fwa - Google Patents

Modulation genetique de la floraison au moyen du gene fwa

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Publication number
EP1196582A1
EP1196582A1 EP00946522A EP00946522A EP1196582A1 EP 1196582 A1 EP1196582 A1 EP 1196582A1 EP 00946522 A EP00946522 A EP 00946522A EP 00946522 A EP00946522 A EP 00946522A EP 1196582 A1 EP1196582 A1 EP 1196582A1
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Prior art keywords
plant
acid sequence
nucleic acid
fwa
gene
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EP00946522A
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German (de)
English (en)
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Wilhelmus Jakobus Johannes Soppe
Antonius Johan Maria Peeters
Maarten Koornneef
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Wageningen Universiteit
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Wageningen Universiteit
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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
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation

Definitions

  • This invention relates to the determination, cloning and expression of the flowering time gene FWA and the use of this gene to delay or accelerate flowering in a large variety of plant species.
  • the gene that was determined is that of Arabidopsis thaliana.
  • Table 1 Morphological markers, used for the genetic mapping of the FWA locus.
  • the plant selected as example for such manipulation is the Arabidopsis thaliana. This is a facultative long day plant, which means that its flowering is promoted by long day photoperiods. Also many strains of this species respond strongly to vernalisation, the cold treatment given at the young plant stage.
  • the fwa gene, located on the long arm of chromosome 4 of Arabidopsis thaliana has now been cloned and isolated.
  • the nucleic acid sequence of the gene is presented in SEQ ID No. 1 and is explained below.
  • the cDNA encoding the FWA protein has been sequenced and its nucleic acid sequence is presented in SEQ ID No. 2.
  • the amino acid sequence of the expression product FWA is presented in SEQ ID No. 3.
  • the invention firstly relates to a nucleic acid sequence encoding FWA having the amino acid sequence of SEQ ID No 3, or a functional equivalent or part of FWA having flowering time associated activity in the vernalisation pathway. This includes homologous flowering time associated proteins in other plants than Arabidopsis thaliana.
  • a protein is considered homologous with FWA if its amino acids sequence of 686 amino acids shows at least 60% identity with the amino acid sequence of SEQ ID No 3.
  • the amino acid identity is at least 70%, more preferably at least 80%, most preferably at least 90% with the amino acid sequence of SEQ ID No. 3.
  • Percentage amino acid identity is determined using conventional methods, such as the BLAST algorithm.
  • a functional equivalent contains a stretch of at least 80 amino acids having at least 80% or even at least 90% homology with the corresponding part of the amino acid sequence of SEQ ID No. 3.
  • the functional equivalent or part comprises at least 4, preferably at least 6, and more preferably at least 8 or even at least 12 consecutive amino acids from the amino acid sequence of SEQ ID No 3.
  • a nucleic acid sequence according to the invention is preferably also capable of hybridising under stringent conditions with the nucleic acid sequence of SEQ ID No 1 or 2, wherein the hydridisation conditions are described below.
  • the invention also relates to expression regulating sequences derived from the 5' non-coding region of the nucleic acid sequence of SEQ ID No. 1.
  • the FWA locus had been identified in Arabidopsis thaliana by its mutant phenotype. In comparison to the wild type, the fwa mutant was delayed in the transition from the vegetative to the generative phase. There were two mutant alleles available; fwa-1 was obtained by EMS mutagenesis and fwa-2 by ⁇ -irradiation. The FWA locus had already been mapped to chromosome 4. However, nothing was known about the molecular function of FWA. To understand this function and the role of the FWA gene in the flowering process an attempt was made to clone this gene. There are several strategies to clone a gene, depending on the available molecular information (Gibson and Somerville, 1993).
  • the gene can be cloned by subtractive hybridisation.
  • subtractive hybridisation in Arabidopsis is tricky and has only been proven successful for two loci (Silverstone et al, 1998; Sun et al, 1992).
  • map-based cloning In the instant case of FWA the gene product and function were unknown and no tagged alleles were available, so the first methods mentioned above were excluded as cloning strategy.
  • the first step in map-based cloning is to locate genetically the locus of interest as accurately as possible with the help of linked markers, either morphological or molecular.
  • linked markers either morphological or molecular.
  • the most closely linked molecular markers can be used to isolate clones that contain the region of the genome covering the locus.
  • the gene can be identified by complementation of the mutant phenotype in transgenic plants containing the candidate gene.
  • DNA sequencing of the wild type and mutant alleles can reveal the nature of the mutation and a comparison with sequences in the databases can indicate the putative molecular function of the gene.
  • a set of overlapping cosmid clones isolated from a cosmid library was thus made by the inventors from the mutant allele fwa-1.
  • Agrobacterium-mediated transformation of two overlapping cosmids conferred lateness to transformed early wild type plants, similar to the lateness of the ⁇ va mutants.
  • This DNA sequence is hereafter called the FWA gene.
  • RTPCR reverse transcriptase PCR
  • Northern blot analysis it was shown that the FWA gene is not expressed in the wild-type but is expressed in the mutant.
  • the lack of expression in the wild type could be attributed to the methylation of a part of the promoter of the gene.
  • the promoter contains two blocks of repeated sequences in which the C residues of the DNA are methylated.
  • sequences can be used to modify flowering time in those other species by overexpression or by co-suppression.
  • the expression regulation of the genes can be amended to the direction required i.e. earlier or later flowering as desired.
  • the other FWA genes can be manipulated in other plants analogously to the methodology illustrated herein for Arabidopsis. The same can of course be carried out obviously for Arabidopsis itself as is illustrated herein.
  • the Arabidopsis or any other plant foreign FWA gene can also be introduced into any desired plant.
  • the alteration in other plants can also for example occur by use of the FWA gene of Arabidopsis or of the plant in question to repress the expression of the plants own FWA genes via the anti-sense or co-suppression technology.
  • This method will be effective as is apparent from our illustration that some transformed late ⁇ va mutant plants, which express FWA, become early after transformation with a mutant copy of the FWA encoding sequence. Silencing of the ⁇ va gene in the ⁇ va mutant due to gene-silencing induced by the transgene is the most likely explanation for this observation.
  • the nucleic acid sequence encoding FWA can also be placed under the control of externally inducible gene promoters, which may allow the exact timing of the crop plant.
  • externally inducible gene promoters which may allow the exact timing of the crop plant.
  • This example describes the map-based cloning of the FWA gene. First a segregating population that was constructed for the fine mapping of FWA is described. Thereafter the YAC and cosmid contigs that were constructed and finally the plant transformation experiments that showed complementation are described
  • mapping population For the genetic mapping of FWA both morphological and molecular markers were employed. Morphological markers are based on differences in phenotype while molecular markers detect polymorphisms at the DNA level. The latter implies that a mapping population should be derived from a cross between two plants that do not only differ for the locus of interest but that also differ in their DNA sequence. Such DNA polymorphisms can be found between ecotypes in Arabidopsis. For the mapping of FWA the ecotypes Landsberg erecta (Ler) and Columbia (Col) were used. A complication of the use of ecotypes is that they may differ in loci affecting the trait of interest, in this case flowering time.
  • a mapping population was constructed with a more uniform genetic background ( Figure 1).
  • Table 2 Genetic distances between the morphological markers.
  • the FWA genotype of the F2 plants was determined by analysing the flowering time of the F3 lines, derived from recombinant plants. These lines were only grown from the 120 recombinants between GA5 and EMB35. F3 lines from homozygous ⁇ va mutant F2 plants were completely late flowering; F3 lines from heterozygous FWA/ ⁇ va F2 plants segregated flowering time while F3 lines from homozygous wild type FWA F2 plants were early flowering. Out of these 120 recombinants, only two were recombinant between GAS and FWA and 118 had undergone recombination between FWA and EMB35. This means that FWA maps only 0.1 cM from GA5.
  • FWA FWA fine mapping
  • molecular markers 16 different restriction fragment length polymorphism (RFLP) markers and one codominant cleaved amplified polymorphism (CAPS) marker were used (Table 3).
  • RFLP restriction fragment length polymorphism
  • CAS codominant cleaved amplified polymorphism
  • Table 3 DNA probes used to detect molecular polymorphisms.
  • AtDB A. thaliana database (http://genome-www.stanford.edu/Arabidopsis/) DNA isolated from F3 lines derived from the 120 F2 plants, showing recombination between GA5 and EMB35, were analysed for polymorphic molecular markers. From this analysis the location of the FWA locus could be limited to a region of 0.7 cM, between the morphological marker ga5, and the molecular marker pcr23 ( Figure 3). For ga5 also a molecular marker was available (Xu et al, 1995), which was used as an extra check for the scoring of this morphological marker, indicating that for one recombinant the ga5 phenotype was misscored.
  • a YAC contig was constructed in order to locate FWA within a YAC.
  • nine YAC's were selected from the published YAC contig of chromosome 4 (Schmidt et al, 1995).
  • the relative positions of these YAC'S were refined by hybridising them with all the molecular markers in this region that were used for the mapping.
  • the relative position of a YAC was deduced, according to whether a marker hybridised completely, partially or not at all with the YAC.
  • the YAC contig made in this way is shown in Figure 3.
  • the ratio of physical to genetic distance in this region of chromosome 4 is about 300 Kb/cM.
  • the average ratio for this chromosome is 175 Kb/cM, varying from 30 Kb/cM to more than 550 Kb/cM (Schmidt et al, 1995). Therefore the ratio in the FWA region is higher than average, which is not favourable for map-based cloning. If this ratio was lower it would have been possible to further reduce the physical distance where FWA is located, using the same number of recombinants.
  • The/vv ⁇ -mutant is semi-dominant and probably a gain of function mutation. This raises the possibility that complementation of a mutant plant with the wild type gene might not be possible. Therefore the complementation experiment should be done by transforming a wild type plant with the ⁇ va mutation. In this case a complementing transformant should confer later flowering to wild type plants.
  • a genomic library was made from ⁇ va- 1 mutant DNA.
  • This library was constructed in a cosmid binary vector because of the relatively large insert size and the advantage of being able to use the clones directly for plant transformation.
  • the resulting cosmid library consists of 27.264 clones with an average insert size of 16 Kb.
  • the library should contain four genome equivalents.
  • the library probably contains between two and four genome equivalents. From the contig a YAC, covering the genomic region that contains FWA, was selected that could be used for the screening of this library. This was the case for YAC EG1F12, which contains both markers, CC128 and pcr28, flanking the FWA locus.
  • the library was screened by hybridisation with this YAC and 21 hybridising cosmids were obtained. Four pairs of these clones were identical, which means that the screen yielded in total 17 different cosmids.
  • the overlaps between the different cosmids were at least five Kb, apart from the overlap between cosmids 2/5 and 120, which was only a few Kb.
  • Ten of the cosmids covered the region between the markers CC128 and pcr28.
  • cosmids were selected for the plant transformation experiment. These cosmids span the complete region where FWA is located, ranging from the left end of CC128 to the right end of pcr28. All these cosmids were introduced in wild type Ler plants. The number of Tl transformants from every cosmid that was checked for flowering time and the flowering time behaviour of these transformants are shown in Table 4.
  • cosmids 20, 28 and 31 were also transformed to ⁇ va mutant plants. With cosmids 20 and 28 several early flowering Tl transformants were obtained which is possibly caused by cosuppression of the ⁇ va mutant gene.
  • the sequence of the FWA genomic region was obtained from the thus generated database, together with open reading frames (ORF's). In the overlap of cosmids 20 and 28 only one complete ORF was found.
  • This ORF has homology to homeodomain genes and highest homology with ANL, which is a homeobox gene, involved in the accumulation of anthocyanin.
  • the nucleic acid sequences according to the invention exhibit a higher degree of homology and identity with the sequences of Sequence id no 1-4 than with ANL. They also illustrate a higher degree of homolgy with a fwa gene encoding sequence than with the ANL sequence.
  • Seeds were sown in plastic Petri dishes on a filter paper soaked with water and incubated in a cold room (4°C) for three days. After this they were transferred to a climate room (25°C, 16 hours light per day) and incubated for two days. Germinated seeds were planted on potting compost in individual clay pots and grown in a greenhouse with long daylight conditions (at least 14 hours daylight).
  • DNA was isolated from plants grown in the greenhouse, following basically the protocol of Bernatzky and Tanksley (1986). Approximately 4 g of fresh leaf material was ground in a mortar filled with liquid nitrogen. The powder was transferred to a tube containing 20 ml extraction buffer (0.1 M Tris pH7.5, 0.35 M Sorbitol, 5 mM EDTA). After centrifuging at 4000rpm for 30 min the supernatant was discarded and 1.25 ml extraction buffer, 1.75 ml nuclei lysis buffer (0.2 M Tris pH7.5, 50 mM EDTA, 2M NaCl, 2% CTAB) and 300 ⁇ l 10% sarkosyl were added, mixed with the pellet and incubated at 65°C for 30 min.
  • RNAase A was added to an end concentration of 10 ⁇ g/ml and the tube was incubated at 37°C for 30 min. The solution was extracted twice, first with phenol/chloroform isoamylalcohol (25:24:1) and then with chloroform/isoamylalcohol.
  • DNA concentrations were measured with a TKO 100 fluorimeter (Hoefer Scientific Instruments, San Francisco, CA, USA). Plasmid and cosmid DNA was isolated, following the "small-scale preparations of plasmid DNA” protocol of Sambrook et al (1989). When the DNA was used as a probe it was purified with Qiagen-tip 20 columns (Qiagen, Chatsworth, CA, USA) following the manufacturers instructions. Phage DNA was isolated, following the "rapid analysis of bacteriophage ⁇ isolates, plate lysate method" protocol of Sambrook et al (1989).
  • Total genomic YAC DNA was isolated from a 5 ml culture of yeast, which was grown in YPD medium (10 g yeast extract, 20 g peptone and 20 g dextrose per liter) at 30°C. After centrifuging the culture at 4K for 5 min, the pellet was washed in 5 ml of 50 mM EDTA, then washed in 20 mM EDTA, 1 M sorbitol; after this it was resuspended in 150 ⁇ l of 20mM EDTA, 1M sorbitol. Hereafter 35 ⁇ l lyticase (5U/ ⁇ l) and 11.5 ⁇ l ⁇ - mercaptoethanol was added and the solution was incubated for 2 hours at 37°C.
  • YPD medium 10 g yeast extract, 20 g peptone and 20 g dextrose per liter
  • the pellet was dissolved in 0.5 ml of 0.1 M EDTA, 0.15 M NaCl, then 25 ⁇ l of 20% SDS was added and the solution was incubated at 65°C for 20 min. Next, 200 ⁇ l of 5 M KAc was added and the tube was left on ice for 30 min after which it was centrifuged for 3 min. The supernatant was poured in a 1.5 ml Eppendorf tube that' was filled with 96% ethanol and then centrifuged for 10 min at RT. The pellet was resuspended in 250 ⁇ l of mQ, after which an equal volume of 4.4 M LiCl was added and the tube was left on ice for 30 min.
  • the plugs were transferred to a small volume of LET (0.5 M EDTA, 10 mM Tris pH8.0) with 7.5 ⁇ l ⁇ -mercaptoethanol and 0.1 mg/ml RNAaseA and incubated overnight at 37°C. Hereafter they were washed three times in NDS buffer (0.5 M EDTA, 10 mM Tris pH8.0, 1% sodium N-Lauroylsarcosine) for 15 min. Then they were transferred to NDS with 2 mg/ml proteinase K and incubated overnight at 50°C. Finally they were washed in 50 mM EDTA pH8.0 for 15 min, left overnight in fresh 50 mM EDTA and washed again.
  • LET 0.5 M EDTA, 10 mM Tris pH8.0
  • the plugs were stored at 4°C in 50 mM EDTA pH 8.0. To separate complete YAC's, the plugs were cast in a 1% agarose gel, which was run by pulsed field gel electrophoresis in a CHEF-DRTMII (Bio-Rad, Hercules, CA, USA) apparatus.
  • CHEF-DRTMII Bio-Rad, Hercules, CA, USA
  • Electrophoresis lasted 45 minutes at 80V after which the DNA was pipetted out of the salt trap (two times 175 ⁇ l).
  • the DNA was first extracted with phenol/chloroform/IAA (25:24:1), then with chloroform/IAA (24:1) and finally precipitated with 2.5 volumes of absolute ethanol overnight at -20°C. The precipitate was washed with 70% ethanol and dissolved in mQ water.
  • the blot was soaked in 2 x SSC for 1 min, UV irradiated in an ultraviolet crosslinker (Ultra Lum, Paramount, CA, USA) with 120,000 ⁇ J/cm 2 and baked at 80°C for 2 hours. Hybridisations were performed in a Hybaid oven (Hybaid, Teddington, UK). A blot was prehybridised with 10 ml of hybridisation solution (5 x SSC, 5 x Denhardt's solution and 0.5% SDS) for 4 hours at 65°C. [ 32 P] Random prime labelled DNA fragments were used as probe for hybridisation overnight.
  • Blots were washed at 65°C in 0.1% SDS and respectively 5 x SSC, 3 x SSC and 1 x SSC (every wash step took half an hour). The activity of a blot was visualised with a phosphor imager.
  • the T-DNA cosmid vector 04541 was used to prepare the genomic library. This vector was derived from SLJ1711 (Jones et al, 1992) by the insertion of a fragment containing a cos site between the Bglll sites. SLJ1711 was derived from pRK290 (Ditta et al, 1980). The vector contains the kanamycine resistance gene (NPTII), a cos site and a polylinker, with blue/white selection, between T-DNA borders. Furthermore it carries a SURETM bacterial tetracycline resistance gene.
  • NPTII kanamycine resistance gene
  • genomic DNA of the w ⁇ -1 mutant was partially digested with the restriction enzyme Sau3AI, treated with calf intestinal phosphatase and size fractionated over a sucrose gradient to obtain fragments in between 15 and 25 Kb. These fragments were ligated into the BamHI site of the cosmid vector.
  • the DNA was packaged with Gigapack II packaging extract (Stratagene, La Jolla, CA, USA), mixed with SURETM cells (Stratagene, La Jolla, CA, USA) and plated out on LB (10 g peptone, 5 g yeast extract and 5 g NaCl per liter) plates with tetracycline (lO ⁇ g/ml), 0.004% Xgal and 0.2 mM IPTG for blue/white selection.
  • Gigapack II packaging extract (Stratagene, La Jolla, CA, USA)
  • SURETM cells Stratagene, La Jolla, CA, USA
  • LB 10 g peptone, 5 g yeast extract and 5 g NaCl per liter
  • Hybond-N filters (Amersham Pharmacia, Uppsala, Sweden) were placed on the plates with colonies for 1 minute, denatured and neutralised in trays containing these solutions and baked at 80°C for 2 hours. Hybridisation of the filters was similar as mentioned above (southerns, blotting and hybridisation), but the filters were hybridised in trays instead of bottles. Electroporation of Agrobacterium tumefaciens
  • Cosmids that were selected for plant transformation were transferred from Escherichia coli cells (SURETM) to Agrobacterium tumefaciens (AGLO strain; Lazo et al, 1991) by electroporation.
  • SURETM Escherichia coli cells
  • Agrobacterium tumefaciens Agrobacterium tumefaciens
  • To prepare competent cells a 50 ml liquid culture of LB with selective antibiotics was inoculated with A. tumefaciens and grown overnight at 28°C. The next day a 500 ml liquid culture (LB without salts) was inoculated with 25 ml of the overnight culture. Cells were harvested at OD600 by centrifugation (5K, 5min, 4°C) and gently resuspended in 250 ml of ice-cold mQ water.
  • the cells were harvested at an OD600 of 0.8 by centrifugation (5K, 15min, RT) after which the pellet was gently resuspended in 0.5 liter of infiltration medium, pH 5.8 (0.5 X Murashige & Skoog salts, 5% sucrose, 0.05% MES, 0.02% Silwet L-77 (Lehle seeds, Round Rock, TX, USA).
  • the infiltration medium with A. tumefaciens was put in two jars on top of which the pots with Arabidopsis were placed upside down with the flowering shoots completely submerged in the medium. Thereafter the jars with pots were placed in vacuum for five minutes. Finally, the pots with Arabidopsis were transferred to the greenhouse.
  • the seeds that were harvested from these plants were sterilised for 15 minutes with 20% bleach in absolute ethanol solution, then they were rinsed two times in absolute ethanol and dried overnight in a flow cabinet. Seeds were sown on plates with selective medium (1 X Murashige & Skoog salts, 1% sucrose, 40 ⁇ g/ml kanamycine, 0.8%o agar, pH 5.8). The plates were kept in the cold room (4°C) for 4 days and then transferred to the growth room (16 hours light, 25°C). After 10 days, transformed seedlings were visible as green plants with several green leaves and a root, whereas untransformed seedlings were yellow and did not develop further than the cotyledons.
  • DNA was isolated from a few leaves of a transformant plant and amplified through 35 cycles (10 sec 94°C, 30 sec 54°C and 2 min 72°C) in standard PCR conditions. Presence of the cos20 insert in the plant was confirmed by appearance of a 1.1 Kb band after amplification with the T3 primer, 5'-AATTAACCCTCACTAAAGGG-3' (SEQ ID No 5) and the primer 5'-GCTTCGGAACTAAGGAACCCAAGC-3' (SEQ ID No 6). For cos28 a 0.8 band was amplified, using the T3 primer and the primer 5'-GAGTCTTGCTTTATGCCAAGCCGC-3' (SEQ ID No 7).
  • Sequencing of the FWA region in wild type and mutants revealed that the lack of expression in the wild type (in contrast to the suppressors) is not due to a mutation within the FWA region.
  • the phenotype of the suppressor mutants further confirmed that FWA is not essential for normal development of the plant and that its expression results in lateness, which property we expect to be transferred to other plants too.
  • a third proof that the ⁇ va sequence confers lateness is that sequencing of three intra- genic suppressors (1R1, 1R2 and 1R3) showed mutations that either lead to an early stop codon (1R1 and 1R3) or to an amino acid change in a part of the gene that is probably for the function (table 6 and gDNA sequence, Sequence id no 1).
  • Figure 1 Five weeks old plants grown under long daylight conditions: left: Ler wild type; right: ⁇ va mutant.
  • Figure 2 Leaf number (as a measurement for flowering time) of wild-type Ler, heterozygous and homozygous ⁇ va plants, grown under long daylight conditions.
  • Figure 3 Position of the ⁇ va locus on chromosome 4. The upper part of the figure (Fig 3(1)) shows the whole chromosome with some morphological markers. Below this the ⁇ va region is shown with morphological and molecular markers that were used for the mapping of the ⁇ va locus. The middle of the figure (Fig 3(2)) shows the YAC contig from a small part of this region, together with the probes that were used to construct this contig. The number of recombinants that were left between these probes is indicated.
  • FIG. 3(3) The cosmid contig that was generated after screening of the fwa-1 cosmid library with YAC EG1F12 is shown in the bottom of the figure (Fig 3(3)). Cosmids in white were used for plant transformation experiments.
  • Figure 4 Detection of FWA and ANL2 mRNA by RT-PCR; RNA was isolated from whole plants before flowering. The first three lanes show RT-PCR results of late flowering ddm ⁇ lines, the fourth of the ⁇ va mutant, the fifth of the late flowering comutant and the sixth of wild-type Ler. The lane on the right shows the result of PCR with the smae primers on genomic DNA.
  • Figure 5 Methylation differences between the wild type and ⁇ va. DNA was digested with Mspl or with the methylation sensitive isoschizomer Hpall. Cosmid 31 was used as a probe.
  • FIG. 6 Schematic representation of the ⁇ va gene. Open boxes represent exons. The start codon (ATG), stop codon (TAA) and the position and nature of the mutations in the three revertants are indicated. The arrows above the 5' region mark the two direct repeat sequences, while arrows within the first exon show the position of the direct repeat in the untranslated leader of the mRNA.
  • Figure 7 shows the segregation of flowering time in this mapping population.
  • the plants were grown under long day light conditions in a greenhouse and in these conditions Col flowered between 27 and 31 days, whereas the progeny of the parental ⁇ va mutant plant that was selected for the cross flowered between 42 and 51 days.
  • the overall shape of the flowering time frequency distribution with two major peaks of different size can be explained because approximately 2/3 of these plants will be heterozygous fox ⁇ va (the heterozygous FWAIfwa plant flowers earlier than the homozygous fwa fwa plant).
  • the flowering time of most of the plants of the mapping population is between the values of the parental lines, although a very small fraction of transgressive phenotypes might be present due to the segregation of some other flowering loci of minor effect differing between Ler and Col.
  • the Figure shows all recombinants that were obtained between the different morphological markers classified according to their flowering time. From this mapping population the recombinants between ga5 and emb35 were selected for the fine mapping of FWA, using molecular markers.
  • ORGANISM Arabidopsis thaliana Arabidopsis thaliana.
  • SEQ ID No. 1 presents the nucleic acid sequence of the ⁇ va gene.
  • SEQ ID No. 2 presents the nucleic acid sequence of the experimentally found cDNA corresponding to the mRNA encoding the FWA protein.
  • SEQ ID No. 3 presents the amino acid sequence of FWA as encoded by the cDNA of
  • SEQ ID No. 2 presents the amino acid sequence of FWA as encoded by a predicted cDNA.
  • Cys Asn lie Cys Gly Lys Ala Thr Asn Cys Gly Asp Thr Glu Tyr Glu 130 135 140
  • Ser Thr His Lys Val lie Ser Thr Gly Ser Gly Gly Thr Lys Ser Gly 305 310 315 320
  • Gly lie Gly Leu Gly Ala Lys Arg Trp Leu Ala Thr Leu Gin Arg His 420 425 430
  • Trp Asp lie Leu Thr Asn Asp Thr Thr Met Glu Glu Thr lie Arg lie 545 550 555 560

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Abstract

Cette invention concerne la détermination, le clonage et l'expression du gène FWA de la durée de la floraison, et l'utilisation dudit gène pour retarder ou accélérer la floraison dans de multiples espèces de plantes. Plus précisément, on a déterminé le gène de Arabidopsis thaliana, mais il va de soi que cette invention englobe d'autres végétaux.
EP00946522A 1999-07-02 2000-07-03 Modulation genetique de la floraison au moyen du gene fwa Withdrawn EP1196582A1 (fr)

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WOPCT/NL99/00414 1999-07-02
PCT/NL1999/000414 WO2001002572A1 (fr) 1999-07-02 1999-07-02 Regulation genique de la floraison au moyen du gene fwa
PCT/NL2000/000465 WO2001002573A1 (fr) 1999-07-02 2000-07-03 Modulation genetique de la floraison au moyen du gene fwa

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KR100510959B1 (ko) 2001-08-22 2005-08-30 제노마인(주) 식물의 개화시기를 조절하는 유전자 및 이를 이용한식물의 개화시기 조절방법
CN111402964B (zh) * 2020-03-19 2023-07-25 西南医科大学 一种基于混合烟花算法的分子构象搜索方法

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