WO2004070036A1 - Proteine et gene participant a la floraison perpetuelle d'un angiosperme - Google Patents

Proteine et gene participant a la floraison perpetuelle d'un angiosperme Download PDF

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Publication number
WO2004070036A1
WO2004070036A1 PCT/JP2004/001064 JP2004001064W WO2004070036A1 WO 2004070036 A1 WO2004070036 A1 WO 2004070036A1 JP 2004001064 W JP2004001064 W JP 2004001064W WO 2004070036 A1 WO2004070036 A1 WO 2004070036A1
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Prior art keywords
gene
nucleotide sequence
angiosperm
dna
seq
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PCT/JP2004/001064
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Japanese (ja)
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Hikaru Iwata
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Wakunaga Pharmaceutical Co., Ltd.
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Publication of WO2004070036A1 publication Critical patent/WO2004070036A1/fr

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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)
    • C12N15/8241—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]

Definitions

  • the present invention relates to genes and DNA fragments involved in the seasonal blooming of angiosperms,
  • the seasonal varieties produced by cross breeding are accidental products.
  • the transmission of genetic traits by crossing is particularly complex, because many rose cultivars are higher polyploids, such as tetraploids. Therefore, it is difficult to ensure that certain varieties are bloomed in four seasons and that certain traits are introduced into four seasonally bloomed varieties. For this reason, some varieties that are of value in appreciation cannot actually be commercialized because they do not have seasonal blooming properties.
  • most other flowers have few seasonal varieties due to lack of suitable breeding materials.
  • the present inventor succeeded in identifying a protein and its gene involved in the seasonal blooming of angiosperms, and a DNA that suppresses the expression of the gene. It has been found that the formation of flower buds at the top is promoted, and the plant becomes seasonal blooming.
  • the present invention is based on this finding.
  • an object of the present invention is to provide a protein involved in the seasonal blooming, a gene thereof, and a DNA capable of suppressing the expression of the gene.
  • the protein according to the present invention is represented by the following (a) or (b):
  • one or more amino acids include an amino acid sequence in which substitution, deletion, addition, or insertion has been performed, and can suppress flower bud formation at the shoot apex of an angiosperm plant protein.
  • gene according to the present invention encodes the protein according to the present invention.
  • DNA according to the present invention is a DNA represented by the following (a) or (b):
  • a DNA which comprises a nucleotide sequence in which one or more nucleotides have been deleted, substituted, added or inserted in the nucleotide sequence represented by SEQ ID NO: 3, and which can function as a transposon in angiosperms.
  • ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide an angiosperm with the seasonal blooming property, Furthermore, it suppresses the expression of the target protein, target gene, nucleic acid molecule and said gene which are useful in generating an angiosperm with the seasonal blooming. DNA is provided.
  • FIG. 1 shows a partially determined nucleotide sequence of the KSN gene (SEQ ID NO: 16).
  • FIG. 2 shows a partially determined nucleotide sequence of the KSN gene (SEQ ID NO: 17).
  • FIG. 3 shows a partially determined nucleotide sequence of the KSN gene (SEQ ID NO: 18).
  • FIG. 4 shows the nucleotide sequence of the coding region of the KSN gene (SEQ ID NO: 19).
  • FIG. 5 is a view showing the mRNA expression levels of KSN at various sites of rose (R es) axillary buds.
  • the term “four season blooming” refers to a property of a plant that continuously blooms in all seasons.
  • angiosperm refers to a plant having an ovule in the ovary, and most of the plants for enjoying flowers are included in angiosperms.
  • angiosperms include a wide variety of plants, which are not particularly limited in the present invention, but are preferably plants belonging to the order Rosale, more preferably plants belonging to the family Rosaceae, More preferably, it is rose (Rosa).
  • nucleic acid molecule is used to include DNA, RNA, and PNA (peptide nucleic acid).
  • ⁇ specific '' used for a nucleic acid molecule means that the nucleotide sequence of the nucleic acid molecule is present only in the gene of interest, and not in other genes in angiosperms.
  • Means The numerical values of homology described herein may be numerical values calculated using a homology search program known to those skilled in the art, unless otherwise specified, but are preferably BLAST CJ. Mol. .
  • Isseki parameters of default (initial setting) in e.g., Protein -Based BLAST Search (FiHer: 0N 5 Scoring Matr ix: BL0SUM62, Word Size: 3, E value: 10, Gap costs: II, I, Aliguments: 50)).
  • the protein according to the present invention comprises the amino acid sequence represented by SEQ ID NO: 2, and the protein can suppress flower bud formation at the shoot apex of an angiosperm.
  • the protein according to the present invention is not limited to the above, and as long as the formation of flower buds at the shoot apex of an angiosperm can be suppressed, one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 May be a mutant comprising a substituted, deleted, added or inserted amino acid sequence.
  • the number of amino acids to be substituted, deleted, added or inserted is not particularly limited, but is preferably 1 to 9, more preferably 1 to 5, and still more preferably 1 to 3.
  • the amino acid sequence of the above mutant preferably has at least 83%, more preferably at least 90%, even more preferably at least 95% homology with the amino acid sequence represented by SEQ ID NO: 2. It is assumed to have.
  • the gene according to the present invention encodes the protein according to the present invention.
  • the nucleotide sequence is not particularly limited, but is preferably the nucleotide sequence represented by SEQ ID NO: 1.
  • the nucleotide sequence represented by SEQ ID NO: 1 is a sequence from the translation initiation codon to the translation termination codon in the cDNA for mRNA, and encodes the amino acid sequence represented by SEQ ID NO: 2.
  • the gene according to the present invention may not only include a sequence of cDNA but also a sequence of genomic DNA.
  • Such a gene comprising the genomic DNA sequence is a sequence comprising an intron sequence in addition to the exon sequence encoding the protein of the present invention, and is preferably SEQ ID NO: 1.
  • the gene according to the present invention has one or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 1.
  • the tide comprises a substituted, deleted, added or inserted nucleotide sequence.
  • the number of nucleotides to be substituted, deleted, added or inserted is not particularly limited, but is preferably 1 to 9, more preferably 1 to 5, and even more preferably 1 to 3.
  • such a nucleotide sequence preferably has at least 81%, more preferably at least 85%, and still more preferably at least 90% homology to the nucleotide sequence represented by SEQ ID NO: 1. It is assumed that.
  • genes and proteins according to the present invention can be obtained according to methods known to those skilled in the art.
  • a method for obtaining the gene according to the present invention for example, a method of chemically synthesizing by the phosphoramidite method or the like, a nucleic acid sample from an angiosperm plant such as Rosa kinensis spontanea (Rosa c hinensis spontanea), Examples include a nucleic acid amplification method using a primer designed based on the nucleotide sequence of the target gene.
  • Methods for obtaining the protein according to the present invention include, for example, a method of purifying the angiosperm as a natural product from the shoot apex, and a method of producing the protein by incorporating the gene of the present invention into an expression system using Escherichia coli, yeast, or the like as a host. ⁇
  • the protein according to the present invention suppresses flower bud formation at the shoot apex of an angiosperm, and the gene according to the present invention encodes the protein. Therefore, when the function of the protein or gene according to the present invention in the angiosperm is inhibited, the suppression of flower bud formation at the shoot apex is released, so that flower bud formation is promoted, and the angiosperm becomes seasonally blooming. Therefore, according to the present invention, there is provided a method for imparting seasonal blooming to an angiosperm plant, comprising inhibiting the function of the protein or gene according to the present invention. The method can be performed according to methods known to those skilled in the art, but preferably comprises suppressing the expression of the gene according to the present invention.
  • RNAZDNA technology Bioscience and Industry, 50, 322 (1992), Chemistry, 46, 681 (1991), Biotechnology, 9, 358 (1992), Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152 (1992), Cell Engineering, 16, 1463 (1997)] Triple 'Helix Technology
  • RNA interference [(Chua Natl. Acad. Sci. USA 97, 4985, 2000; Elbashir, SM et al., Nature 411, 494-498, 2001].
  • the suppression of the expression of the gene according to the present invention is carried out using a single-stranded nucleic acid molecule comprising all or a part of the same nucleotide sequence as the antisense strand of the gene according to the present invention. Done.
  • a single-stranded nucleic acid molecule comprising all or a part of the same nucleotide sequence as the antisense strand of the gene according to the present invention.
  • the antisense method has a sequence complementary to the gene whose expression is to be suppressed: Expression of a high level of RNA suppresses the expression of the target gene.
  • a single-stranded RNA comprising the entire nucleotide sequence identical to the antisense strand of the gene according to the present invention can be used.
  • a single-stranded RNA comprising a part of the same nucleotide sequence as the antisense strand of the gene according to the present invention can also be used.
  • a partial single-stranded RNA may be any as long as it can suppress the expression of the gene according to the present invention, and can be appropriately designed by those skilled in the art, but is preferably specific to the gene according to the present invention.
  • the chain length is preferably 5 to 100 nucleotides, more preferably 5 to 50 nucleotides, and still more preferably 10 to 20 nucleotides.
  • the expression of the gene of the present invention is suppressed by using a single-stranded nucleic acid molecule comprising all or a part of the same nucleotide sequence as the sense strand of the gene of the present invention. It is done. That is, this sense single-stranded nucleic acid can be used for suppressing the expression of the gene according to the present invention, like the above-mentioned antisense single-stranded nucleic acid.
  • a single-stranded RNA comprising the entire nucleotide sequence identical to the sense strand of the gene according to the present invention can be used.
  • a single-stranded RNA comprising a part of the same nucleotide sequence as the sense strand of the gene of the present invention can also be used.
  • Such a partial single-stranded RNA may be any as long as it can suppress the expression of the gene of the present invention, and can be appropriately designed by those skilled in the art, but is preferably specific to the gene of the present invention.
  • the chain length is preferably 5 to 100 nucleotides, more preferably 5 to 50 nucleotides, and still more preferably 10 to 20 nucleotides.
  • the suppression of the expression of the gene according to the invention is carried out by means of a two or more nucleotide sequence comprising the same nucleotide sequence as the gene according to the invention.
  • This is performed using a single-stranded nucleic acid molecule.
  • an antisense or sense single-stranded nucleic acid of the gene according to the present invention can be expressed in an angiosperm plant.
  • the double-stranded nucleic acid molecule according to the present invention is preferably DNA, and its chain length and specific nucleotide sequence correspond to the chain length and nucleotide sequence of the target single-stranded nucleic acid molecule.
  • the double-stranded nucleic acid molecule according to the present invention when the antisense single-stranded nucleic acid is expressed, includes the antisense strand of the gene according to the present invention as a coding strand.
  • the double-stranded nucleic acid molecule of the present invention when expressing the sense single-stranded nucleic acid, includes the sense strand of the gene of the present invention as a code chain.
  • the double-stranded nucleic acid molecule according to the present invention can be expressed in angiosperms using methods known to those skilled in the art.
  • an expression construct comprising a promoter, a double-stranded nucleic acid molecule according to the present invention, a transcript, a mineral, and the like is introduced into a target angiosperm plant, and the obtained plant is cultivated.
  • the double-stranded nucleic acid molecule according to the present invention can be expressed.
  • Introduction of the expression construct into a plant can be performed by a method known to those skilled in the art, for example, an agrobacterium method, a binary vector method, an electroporation method, a PEG method, a particle gun method, or the like.
  • rose transformation for gene transfer can be performed by the method described in US Pat. No. 5,480,789.
  • Examples of the method for suppressing gene expression using the nucleic acid molecule according to the present invention include a transformation method using an antisense strand (Proc. Natl. Acad. Sci. USA 90: 6160-6164, 1993).
  • an antisense double-stranded DNA having the entire nucleotide sequence of the gene according to the present invention is introduced into a plant of interest.
  • the antisense single-stranded RNA according to the present invention is expressed as an RNA complementary to the mRNA of the gene according to the present invention, and the expression of the gene is extremely suppressed by the complementary RNA.
  • Another example of the method for suppressing gene expression using the nucleic acid molecule according to the present invention includes a co-suppression method.
  • a sense double-stranded DNA having the entire nucleotide sequence of the gene according to the present invention is introduced into a target plant.
  • the sense single-stranded RNA according to the present invention is expressed, and the expression of the gene is Is extremely suppressed (Plant Cell 9: 1357-1368, 1997).
  • RNAi RNA-induced silencing complex
  • siRNA nucleic acid molecule comprising a part of the same nucleotide sequence as the gene of the present invention is used, whereby the expression of the gene of the present invention is specifically suppressed.
  • siRNA nucleic acid molecule means not only the siRNA itself, but also a longer double-stranded RNA molecule capable of introducing the siRNA into a target cell.
  • the siRNA typically comprises a 19-21 base pair nucleotide sequence homologous to the sequence specific for the mRNA of the target gene.
  • the double-stranded RNA molecule described above typically comprises a longer nucleotide sequence homologous to the sequence specific for the mRNA of the target gene.
  • the siRNA nucleic acid molecule can be expressed by an appropriate vector delivered into a cell. Therefore, for suppressing the expression of the gene according to the present invention, a vector that expresses a siRNA nucleic acid molecule that specifically suppresses the expression of the gene according to the present invention can be used.
  • Such vectors can be readily constructed by standard procedures well known in the art (Bass, BL, Cell 101, 235-238, 2000; Tavernarakis, N. et al. 24, 180-183, 2000; Malagon 5 F. et al., Mol. Gen. 6e net. 259, 639-644, 1998; Parrish, S. et al. Mol. Cell 6 1077-. 1087, 2000).
  • the suppression of gene expression according to the present invention can also be achieved by utilizing a network originally existing in a plant.
  • TFL 1 protein the induction of flower bud formation at the shoot apex of Arabidopsis is suppressed by the TFL 1 protein, but the regulation of flower bud formation by TFL 1 requires additional LFY (LEAFY) protein and API (APE T ALA 1). Knowing that proteins are involved Have been. That is, TFL 1 and LFY suppress the expression of each other, ! ⁇ 1 also suppresses each other's expression, while L FY and AP 1 promote each other's expression.
  • TFL1 when TFL1 is constitutively expressed, expression of L FY and AP1 is suppressed, and when LFY or AP1 is constitutively expressed, expression of TFL1 is suppressed (Mechanisms in Plant Development Otto line Leyser, P 208-210, Stephen Day: Blackwell Pub; ISBN: 0865427429; 2003). Since the gene and protein according to the present invention have the same function as TFL1, expression of the gene according to the present invention can be suppressed by constitutively expressing AP1 or L FY in plants.
  • the DNA according to the present invention comprises the nucleotide sequence represented by SEQ ID NO: 3, and the DNA can function as a transposon in angiosperms.
  • residues 1 to 856 are LTRs
  • residues 857 to 6105 are ORFs
  • residues 8069 to 8925 are LTRs.
  • the DNA according to the present invention is not limited to the above, and as long as it can function as a transposon in an angiosperm, one or more nucleotides in the nucleotide sequence represented by SEQ ID NO: 3 are deleted, replaced, or substituted. It may comprise an added or inserted nucleotide sequence.
  • the number of nucleotides to be substituted, deleted, added or inserted is not particularly limited, but is preferably 1 to 9, more preferably 1 to 5, and still more preferably 1 to 3.
  • Such a nucleotide sequence preferably has at least 70%, more preferably at least 80%, and even more preferably at least 90% homology to the nucleotide sequence represented by SEQ ID NO: 3. It is said.
  • the DNA according to the present invention is prepared by a method known to those skilled in the art, for example, a method of chemically synthesizing by a phosphoramidite method or the like, a nucleic acid sample from a seasonally blooming rose such as old blush, and the like.
  • the DNA according to the present invention functions as a transposon in angiosperms and suppresses the expression of the gene by being inserted into the gene according to the present invention. This releases the suppression of flower bud formation at the shoot apex and promotes flower bud formation The angiosperms become seasonally blooming. Therefore, according to the present invention, there is provided a transposon comprising the DNA according to the present invention, and further comprising suppressing the expression of the gene according to the present invention by using the DNA according to the present invention or the transposon comprising the same.
  • a method for imparting seasonal blooming properties to an angiosperm plant comprising:
  • a DNA or transposon according to the invention is introduced into an angiosperm. This is appropriately performed by those skilled in the art so that the expression of the gene according to the present invention is suppressed, but the DNA or transposon according to the present invention is preferably introduced into or near the gene according to the present invention, more preferably. Is introduced into the second intron of the gene according to the present invention, more preferably at the same location as the seasonal rose.
  • DNA into a plant can be carried out by a method known to those skilled in the art, for example, an agrobacterium method, a binary vector method, an electroporation method, a PEG method, a particle gun method and the like.
  • rose transformation for gene transfer can be performed by the method described in US Pat. No. 5,480,789.
  • Example 1 Identification of factors involved in seasonal blooming
  • the present inventor has found knowledge on the wild-type (infinite inflorescence) and abruptly variant (finite inflorescence) of Arabidopsis, as well as seasonal blooming in modern four-season roses. Based on the fact that there are many mutations to sex roses, the following hypothesis was established and proved.
  • Hypothesis 2 Seasonal roses are caused by mutation of transposons inserted into the orthologous gene of TFL1 gene, and seasonal vine roses are caused by reversion of deletion of the transposons.
  • the nucleotide sequence of the rose ortholog of the TFL 1 ⁇ gene was clarified as follows, and the transposon was inserted into all of the genes in the seasonal blooming type, while the gene without the transposon was inserted in the seasonal blooming type. Prove that at least one exists.
  • the TFL1 gene belongs to a family of genes called phosphatidylethanolamine-binding proteins (PEBP).
  • PEBP phosphatidylethanolamine-binding proteins
  • Arabidopsis six PEBP genes are known. Based on the nucleotide sequence of known PEBP genes such as Arabidopsis, rice, Kingfisher, petunia, and tomato, the following primers were designed by selecting particularly conserved portions: ⁇
  • the DNA of Rosa chinensis spontanea was used as a type III DNA and PCR was performed to obtain a plurality of amplified products.
  • the nucleotide sequences of these amplification products were determined, and four types of PEBP family genes were selected from the nucleotide sequences, and these were named RPEBP1, RPEBP2, RPEBP3, and KSN.
  • RPEBP1, RPEBP2, RPEBP3, and KSN the nucleotide sequence of the product of more than 400 bp of KSN is shown in FIG.
  • This nucleotide sequence is the exokine of the Arabidopsis TFL1 gene. It had a high homology with the nucleotide sequence from steps 2 to 3.
  • the nucleotide sequence of the entire KSN gene was determined as follows. That is, it was determined by the Inverse PCR method using the Rcs DNA as a ⁇ type.
  • Rcs DNA is digested with the restriction enzyme Cac81 from NEB (DNA171, enzyme 1-1, 10-fold buffer 2 zl is reacted at 37 ° C for 4 hours), and the enzyme is inactivated by heating. After activation (65 ° C for 20 minutes), it was cyclized using NEB's T4-DNA-Ligase (enzyme-digested DNA 20 ⁇ 1, ligase 11 and 10-fold buffer 12.5 ⁇ 1,. And purified water 91.5 51 at 8 ° C all day and night). This circular DNA was designated as type I and PCR was performed using the following two primers:
  • KSN-IR1 CTGTAAGTTATACCAGTGCAGGTGC (SEQ ID NO: 6);
  • KSN-IF 2 GGAAGTACATATTATGGCATAATC (SEQ ID NO: 7).
  • FIG. 2 shows the nucleotide sequence of the obtained amplification product. As apparent from FIG. 2, the nucleotide sequence of 416 bp upstream of Utfl-FI was determined.
  • zg of Rcs DNA was digested with NEB's restriction enzyme Sau96I (reacting DNA 171-1, enzyme 1-1, 10-fold buffer 1-2-1 at 37 ° C for 4 hours) and heating. After inactivating the enzyme (80 ° C for 20 minutes), it was cyclized using NEB's T4-DNA-Ligase (enzyme-digested DNA 201, ligase 1/1, 10-fold buffer 12. 5 j and purified water 9 1.5 1 at 8 ° C all day and night). This circular DNA was designated as type I and PCR was performed using the above primer KSN-IR1 and the following primer KSN-IF1:
  • KSN-IF1 AGGAGCTAACATTTTTGCCTTG (SEQ ID NO: 8).
  • FIG. 3 shows the nucleotide sequence of the obtained amplification product. As is clear from FIG. 3, the nucleotide sequence of 401 bp downstream of UtflRl was determined.
  • FIG. 4 shows the nucleotide sequence of KSN that summarizes the sequences determined as described above.
  • portions that are considered to be exons compared with the nucleotide sequence of the Arabidopsis TFL1 gene are boxed.
  • the nucleotide sequence of the coding region of KSN is shown in SEQ ID NO: 1, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 2.
  • the nucleotide sequences of RPEBP1, RPEBP2, and RPEBP3 were revealed (sequences not shown).
  • primers were designed that specifically amplify almost the entire coding region of these genes.
  • the primer for KSN amplification had the following sequence:
  • KSNF1 ATTAGGAGTACAATCCTTCCTTCC (SEQ ID NO: 9);
  • KSNR2 ACCTGCCTCCTGCTAGCTGC (SEQ ID NO: 10).
  • the four seasonal old brush (OBB) and its mutant, one-year old cold plus climbing (OBC 1) DNA are designated as type I, and the above primers for coding region amplification are used.
  • the PCR was performed under the conditions to obtain an amplified product of about 1 Kbp (using PCR Master Mix manufactured by Promega according to the attached protocol).
  • an amplification product of the expected size was not observed, so the PCR conditions were such that an amplification product of about 10 Kbp could be obtained (using One Shot LAPCR Mix manufactured by Yukara according to the attached protocol). )
  • re-performed the CRC As a result, an amplification product of about 10 Kbp was obtained with OBB, and an amplification product of about 2 Kbp was obtained with 0BC1.
  • KSNIF3 CATATTATGGCATAGGGTGTGGC (SEQ ID NO: 11);
  • KSNR5 CAAATGTAACATCTGTGGTGCCTG (SEQ ID NO: 12).
  • the transformed Escherichia coli was previously purified according to the attached protocol using 2% 5-bromo-14-chloro-1-3-indolyl-D-galactoside (Xgal) 50 / zl and 0.1 M isopropyl-D- Spread the cells onto L-agar medium (containing 50 g / m1 of ampicillin) coated with 10 ⁇ 1 of thiogalactobilanoside (IPTG), and grow white colonies from the resulting colony.
  • L-agar medium containing 50 g / m1 of ampicillin coated with 10 ⁇ 1 of thiogalactobilanoside (IPTG)
  • clone 6 was presumed to contain the full-length PCR amplification product, and its nucleotide sequence was determined as follows.
  • the vector was digested with HindII I or EcoRI, a restriction enzyme having a break at the closing site, and religated using DNA Ligation Kit Ver.2 (Yukara) to circularize.
  • a competent cell strain JM109 Yukara
  • Clones 6-16 having a large DNA fragment using EcoRI were further cut at their corresponding sites using Spel, Nolel, Xbal, and Pstl, and clones were obtained in the same manner.
  • clones 6—16—27, clones 6—16—32, clones 6—16—34, clones 6—16—40, and clones 6—16—43 It was presumed to contain different DNA fragments.
  • nucleotide sequence of the DNA inserted into the plasmids of the eight clones above was determined from before and after using the following primers and joined together:
  • M13R18 CAGGAAACAGCTATGACC (SEQ ID NO: 13);
  • MR13F18 TGTAAAACGACGGCCAGT (SEQ ID NO: 14).
  • this DNA fragment (Li) was about 9 Kbp in size, and as a result of a b1 ast homologous search, it was highly homologous to a copier-type retrotransposon.
  • This transposon (SEQ ID NO: 3) was inserted into intron 2 of the KSN gene of 0BB.
  • about 90 Obp of 0BC 1 is It was found to be an approximately 900 bp LTR (long terminal repeat) left in place when the sponges disappeared.
  • RCS, OBB, and OBCI KSNs include wild-type without insertion (w-type, about 1 Kbp), retrotransposon-containing type (Li type, about 10 Kbp), and retrotransposon as LTR. (Si type, about 2Kbp).
  • KSNInsR3 which was used together with KSNIF3 to amplify a part of the retrotransposon, was designed inside the LTR (KSNI nsR3: TGTAATCTGTAGGAGATCCCATGC, SEQ ID NO: 15).
  • KSNIF3 TGTAATCTGTAGGAGATCCCATGC, SEQ ID NO: 15
  • KSNIF3 and KSNR5 primer pairs yielded amplification products.
  • Table 1 mRNA expression level in each sample derived from Rcs

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Abstract

L'invention concerne un gène participant à la floraison perpétuelle d'un angiosperme. Ce gène code une protéine décrite dans (a) ou dans (b) qui suivent : (a) une protéine contenant la séquence d'acides aminés représentée par SEQ ID NO : 2 ; et (b) une protéine contenant une séquence d'acides aminés dérivée de la séquence d'acides aminés représentée par SEQ ID NO : 2, par substitution, par délétion, par ajout, ou par insertion d'un ou de plusieurs acides, et pouvant réguler la formation de bourgeons à l'apex d'une pousse d'un angiosperme. Le fait d'inhiber la fonction de ce gène dans un angiosperme permet d'obtenir la floraison perpétuelle de l'angiosperme.
PCT/JP2004/001064 2003-02-04 2004-02-03 Proteine et gene participant a la floraison perpetuelle d'un angiosperme WO2004070036A1 (fr)

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* Cited by examiner, † Cited by third party
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WO2023192838A1 (fr) * 2022-03-31 2023-10-05 Pairwise Plants Services, Inc. Plantes rosacées à floraison précoce présentant des caractéristiques améliorées

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Publication number Priority date Publication date Assignee Title
WO2008117860A1 (fr) * 2007-03-23 2008-10-02 International Flower Developments Proprietary Limited Procédé pour déterminer la présence d'un croisement avec un plant de rose d'une espèce de jardin dans un plant de rose d'espèce sauvage
EP2128274A1 (fr) * 2007-03-23 2009-12-02 International Flower Developments Proprietary Limited Procédé pour déterminer la présence d'un croisement avec un plant de rose d'une espèce de jardin dans un plant de rose d'espèce sauvage
EP2128274A4 (fr) * 2007-03-23 2010-07-07 Int Flower Dev Pty Ltd Procédé pour déterminer la présence d'un croisement avec un plant de rose d'une espèce de jardin dans un plant de rose d'espèce sauvage
JPWO2008117860A1 (ja) * 2007-03-23 2010-07-15 インターナショナル フラワー ディベロプメンツ プロプライアタリー リミティド 野生種のバラにおける園芸種のバラとの交雑の有無を検定する方法
US8206928B2 (en) 2007-03-23 2012-06-26 Suntory Holdings Limited Method for determination of presence of crossing with cultivated rose in wild rose
AU2008230321B2 (en) * 2007-03-23 2013-04-04 Suntory Holdings Limited Method for determination of presence of crossing with garden-species rose plant in wild-species rose plant
WO2023192838A1 (fr) * 2022-03-31 2023-10-05 Pairwise Plants Services, Inc. Plantes rosacées à floraison précoce présentant des caractéristiques améliorées

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