JP5374785B2 - Method for producing fertility-controlling Asteraceae - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To stably provide a fertility-inhibited Asteraceae plant. <P>SOLUTION: The RNA expression cassette for inhibiting male fertility or female fertility of the Asteraceae plant includes a DNA encoding an RNA containing an antisense sequence complemental to a base sequence having continuous 21 or more bases in a homologous recombination gene DMC1 at the meiotic stage. The recombinant vector includes the cassette. The method for inhibiting the fertility of the Asteraceae plant includes introducing them to the Asteraceae plant. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method for producing a fertility suppressing asteraceae plant using RNA silencing.

  In recent years, research and cultivation of genetically modified crops have expanded dramatically, and there is concern about the impact on biodiversity caused by unintended gene diffusion. In particular, the cultivated chrysanthemum is an insect-bearing flower and is a cross-breeding species, and since hybrids of cultivated and wild chrysanthemum have been found so far, the development of effective means for preventing the spread of transgenes has been developed for the practical application of transgenic chrysanthemum. It is essential.

  Germ cell sterilization is effective in preventing gene spread. So far, for example, a method has been used to suppress pollen development and induce male sterility by introducing a melon-derived modified ethylene receptor gene (CmETR1 / H69A gene) into plants such as tobacco and tomato. (Patent Document 1). It has also been reported that the fertility of plants has been reduced by suppressing the expression of meiotic homologous recombination gene (DMC1) (Non-Patent Documents 1 to 4).

  In chrysanthemum, male sterility has been attempted by introducing a male sterility gene. However, in chrysanthemum, when a modified ethylene receptor gene is introduced, the senescence of tapete cells is delayed at a flowering temperature of 20 ° C or higher. Then, the expression level of the gene was lowered and a phenomenon of forming fertile pollen was observed (Non-patent Document 5). That is, the conventional method based on the introduction of the modified ethylene receptor gene has a problem in the temperature stability of the sterilization effect. Since chrysanthemum has a wide flowering temperature range of 10 to 35 ° C., in order to more reliably sterilize pollen, development of a method that can stably sterilize chrysanthemum has been desired. On the other hand, since the sterilization when the modified ethylene receptor gene is introduced is limited to quantitative sterilization by suppressing pollen development (maturation), in order to realize more stable sterilization, A technique for achieving qualitative sterility that prevents the formation of monovalent chromosomes (pollen nuclei) caused by meiosis has also been desired. In addition, from the viewpoint of more effective gene diffusion prevention measures, not only male sterilization but also development of a female sterilization method has been demanded. So-called constitutive promoters are widely used for overexpression of transgenes, but such promoters are not always active in the germline, and can induce gene expression in chrysanthemum pollen and egg cells. It was difficult to sterilize chrysanthemum based on the expression of the transgene in pollen and egg cells.

  On the other hand, with the development of plant molecular biology, antisense nucleic acid or double-stranded RNA (dsRNA: see RNA interference (RNAi) described later) for a specific gene is expressed in plant cells and is endogenous to the plant. It has become possible to suppress gene expression (knockdown). For example, a long-lasting tomato (Non-patent Document 6) in which a polygalacturonase gene involved in ripening of tomato fruit is introduced in the antisense direction has been reported. Such suppression of gene expression via RNA is called RNA silencing. RNA interference (RNAi) is a phenomenon in which dsRNA having a base sequence homologous to a gene specifically suppresses the expression of the target gene by promoting the specific degradation of the mRNA of that gene. It is widely used as a highly gene knockdown technique. The proposed RNAi mechanism is as follows. Using mRNA transcribed from an endogenous locus as a template, dsRNA produced by RNA-dependent RNA polymerase [of small RNA (small RNA), the origin of small interfering RNA (siRNA)], Alternatively, dsRNA derived from an endogenous or genomic hairpin dsRNA expression gene (the origin of micro-RNA (miRNA among small RNAs)) is recognized by an enzyme called Dicer belonging to the ribonuclease III family, It is degraded into short 3 'overhanging double-stranded siRNA and miRNA of 21-23 nucleotides. These small RNAs (siRNA and miRNA) are then incorporated into an RNAi target complex called the RNA-induced silencing complex (RISC). This RISC recognizes a target mRNA having a sequence complementary to the small RNA incorporated therein, and cleaves the target mRNA at the center of the complementary sequence. Thereafter, the target mRNA is rapidly degraded, and the expression level of the encoded protein decreases (Non-patent Document 7).

  In the expression suppression technology using RNAi, a technique was first used in which RNA molecules (RNAi molecules) such as dsRNA and siRNA that cause RNAi were directly introduced into cells. However, in recent years, expression vectors capable of biosynthesizing RNAi molecules in an optimal form have been developed and are becoming widely used. In general, shRNA (short hairpin RNA, which can form dsRNA in vivo) is expressed in cells into which this expression vector has been introduced by linking RNAs having mutually complementary sequences with various spacers. SiRNA is generated. Plants have also produced transformants in which target gene expression is suppressed by introducing an expression vector that generates RNAi molecules (Non-Patent Documents 1 to 4).

  In reality, however, the mechanism of RNAi has not yet been fully elucidated. For example, stably achieving gene expression suppression in a wide range of temperature environments is still a challenge with current RNAi technology.

JP 2003-180178 A

Deng, Z. Y. and Wang, T., Plant Mol. Biol., 65 (1-2): 31-42 (2007) Siaud, N. et al., EMBO J., 23 (6): 1392-1401 (2004) Stevens, R. et al., Plant Cell, 16 (1): 99-113 (2004) Couteau, A. et al., Plant Cell, 11 (9): 1623-1634 (1999) Fukui Prefectural Agricultural Experiment Station, 2006 Biotechnology Test Report P5-6 Smith C.J., et al., Nature, 334 (6184): 724-726 (1988) Vaucheret, H., Genes and Dev., 20 (7): 759-771 (2006)

  An object of the present invention is to provide a asteraceae plant that causes stable fertility suppression.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that a meiotic homologous recombination gene (DMC1 gene; also referred to as a meiosis specific homologous recombination gene) is RNA in a asteraceae plant. By suppressing the expression using silencing, the inventors succeeded in stably suppressing the inertia and completed the present invention.

That is, the present invention includes the following.
[1] Male fertility and female fertility of Asteraceae plants containing DNA encoding an RNA containing an antisense sequence complementary to a continuous base sequence of 21 or more bases in the meiotic homologous recombination gene DMC1 RNA expression cassette to suppress sex.

  In a more preferred embodiment of this RNA expression cassette, the RNA comprises a sense sequence consisting of 21 or more consecutive nucleotide sequences in the meiotic homologous recombination gene DMC1, and an antisense complementary to the nucleotide sequence. It preferably contains a sense sequence.

In one embodiment of this RNA expression cassette, it is preferable to use any of the following genes (a) to (e) as the meiotic homologous recombinant gene DMC1:
(a) a gene consisting of the base sequence represented by SEQ ID NO: 1,
(b) a gene comprising a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having recombinase activity;
(c) a gene encoding a protein consisting of a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 1 and having recombinase activity
(d) a gene encoding the amino acid sequence represented by SEQ ID NO: 2,
(e) A gene encoding a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having recombinase activity.

  In this RNA expression cassette, in one embodiment, the continuous base sequence of 21 bases or more in the meiotic homologous recombination gene DMC1 preferably has a length of 500 to 700 bases.

  In this RNA expression cassette, the continuous base sequence of 21 bases or more in the meiotic homologous recombination gene DMC1 includes a region in the gene corresponding to positions 234 to 815 of SEQ ID NO: 1. preferable.

  In this RNA expression cassette, the sense sequence and the antisense sequence may be linked via a spacer sequence.

  An RNA expression cassette according to a particularly preferred embodiment of the present invention comprises a DNA encoding the RNA under the control of a mannopine synthase gene bidirectional mas1′-2 ′ promoter.

  In a preferred embodiment of the RNA expression cassette according to the present invention, the continuous base sequence of 21 bases or more in the meiotic homologous recombination gene DMC1 comprises the base sequence represented by SEQ ID NO: 11. .

[2] A recombinant vector comprising the RNA expression cassette according to [1] above.
[3] By introducing the RNA expression cassette according to [1] above or the recombinant vector according to [2] above into a asteraceae plant cell, a transformed asteraceae having male fertility and female fertility suppression traits A method for suppressing fertility of a asteraceae plant, comprising producing a plant.
Here, it is preferable that the male fertility and female fertility suppressing traits have a property that male fertility and female fertility are suppressed under a temperature condition of 10 to 35 ° C.

[4] A transformed cell into which the RNA expression cassette according to [1] above or the recombinant vector according to [2] above is introduced.
A preferred example of this transformed cell is a transformed Asteraceae plant cell.

[5] A transformed asteraceae plant having male fertility and female fertility suppression traits obtained by regenerating a plant from the transformed asteraceae plant cell according to [4].
[6] crossing the transformed Asteraceae plant according to [5] above as a seed parent and crossing a non-transformed Asteraceae plant as a pollen parent to obtain a progeny plant containing the RNA expression cassette, A method for producing a transformed asteraceae plant having male fertility and female fertility suppression traits.

  By the method of the present invention, a transformed plant exhibiting stable fertility suppression can be produced in a asteraceae plant.

FIG. 1 shows the nucleotide sequence (upper row: SEQ ID NO: 1) and encoded amino acid sequence (lower row: SEQ ID NO: 2) of chrysanthemum-derived meiotic homologous recombination gene (CmDMC1). The underlined part is the sequence used as a trigger for RNAi molecules in the examples. FIG. 2 shows the structure of the binary vector pBIK102DBs. The total length of the pBIK102DBs base sequence is 16,416 bp. The total length of the T-DNA region from RB to LB is 7,850 bp. The numerical value attached to the restriction enzyme name indicates the number of the 5′-most base of each restriction enzyme site when the 5′-end base of T-DNA is the first. FIGS. 3A to 3H show the structure and base sequence of T-DNA in the expression vector pBIK102DBs, and FIG. 3A shows the 5 ′ most sequence. 3 (A) to (H) show the structure and base sequence of T-DNA in the expression vector pBIK102DBs, and FIG. 3 (B) shows the sequence following FIG. 3 (A). 3 (A) to (H) show the structure and base sequence of T-DNA in the expression vector pBIK102DBs, and FIG. 3 (C) shows the sequence following FIG. 3 (B). 3 (A) to (H) show the structure and base sequence of T-DNA in the expression vector pBIK102DBs, and FIG. 3 (D) shows the sequence following FIG. 3 (C). 3 (A) to (H) show the structure and base sequence of T-DNA in the expression vector pBIK102DBs, and FIG. 3 (E) shows the sequence following FIG. 3 (D). 3A to 3H show the structure and base sequence of T-DNA in the expression vector pBIK102DBs, and FIG. 3F shows the sequence following FIG. 3E. 3 (A) to (H) show the structure and base sequence of T-DNA in the expression vector pBIK102DBs, and FIG. 3 (G) shows the sequence following FIG. 3 (F). 3 (A) to (H) show the structure and base sequence of T-DNA in the expression vector pBIK102DBs, and FIG. 3 (H) shows the sequence following FIG. 3 (G). FIG. 4 is a photograph of a cross-sectional image of a cocoon showing the development state of the cocoon according to the flowering temperature. The scale bar in the figure indicates 200 μm. The red part indicates mature tissue and the blue part indicates immature tissue. FIG. 5 is a photograph of the pods and pollen showing the pollen formation status in the pods according to the flowering season temperature. The scale bar in the figure is 1 mm for cocoons and 50 μm for pollen. The red part indicates mature tissue and the blue part indicates immature tissue.

Hereinafter, the present invention will be described in detail.
The present invention relates to a double-stranded RNA (dsRNA) comprising a base sequence (antisense sequence) complementary to at least a part of the base sequence of the meiotic homologous recombination gene DMC1 (hereinafter also referred to as DMC1 gene). The present invention relates to a method for stably suppressing fertility of asteraceae plants by RNA silencing based on production.

  In the method of the present invention, an RNA comprising at least the antisense sequence derived from the meiotic homologous recombinant gene DMC1 is expressed from a DNA construct (expression cassette or recombinant vector) introduced into an Asteraceae plant cell, By inhibiting the function of the mRNA transcribed from the plant's endogenous meiotic homologous recombination gene DMC1, it suppresses germ cell or gametogenesis and stabilizes male fertility and female fertility traits in the plant Can be granted. The inhibition of the mRNA function of the DMC1 gene is mainly performed by converting the RNA containing the above-described antisense sequence (preferably a short antisense RNA (eg, 21-25 bp, preferably 21-23 bp siRNA)) into the DMC1 gene mRNA. Intramolecular RNA that contains at least a part of the base sequence (sense sequence) of the DMC1 gene and a complementary base sequence (antisense sequence) to form a double-stranded RNA (dsRNA) by associating Based on the formation of double-stranded RNA (dsRNA) by association, the DMC1 gene is inhibited from translation of mRNA and / or promotes degradation of the mRNA (RNAi). In the method of the present invention, since both male fertility and female fertility can be suppressed, the fertility of Asteraceae can be stably suppressed. Furthermore, the method of the present invention can also achieve temperature-stable fertility suppression that is not easily affected by the width of the flowering period temperature.

  In the present invention, when the base sequence of RNA is specified with reference to the sequence number described in the sequence listing, “thymine (t)” in the base sequence represented by the sequence number is replaced with “uracil (u)”. It is assumed that it is specified by the read base sequence.

  Preparation of mRNA used in the present invention, preparation of cDNA (RT-PCR), PCR, preparation of cDNA library, ligation of DNA fragment into vector, cell transformation, DNA sequencing, primer synthesis, sudden Molecular biological and biochemical experimental methods such as mutagenesis and protein extraction / purification can be carried out according to the description in ordinary experimental documents. Examples of such experiments include Sambrook et al., Molecular Cloning, A laboratory manual, 2001, Eds., Sambrook, J. & Russell, DW. Cold Spring Harbor Laboratory Press.

1. RNA expression cassette for suppressing male fertility and female fertility of asteraceae and recombinant vector comprising the same In the method of the present invention, in order to stably suppress fertility of asteraceae, meiosis An RNA expression cassette containing a DNA encoding an RNA containing an antisense sequence complementary to a continuous base sequence of 21 bases or more in the homologous recombination gene DMC1 can be used.

  In the present invention, “RNA expression cassette” refers to a DNA construct capable of expressing a predetermined RNA molecule. Specifically, an RNA expression cassette is usually a DNA sequence (RNA coding) that encodes the RNA under the control of a transcription control sequence (generally at least a promoter, typically a promoter and a terminator). A DNA fragment having no self-replicating ability, for example, a DNA fragment in which a promoter and a terminator are linked to a DNA sequence encoding the RNA. In the present invention, when a plurality of nucleic acids (sequences) are “linked”, the case where a restriction enzyme site or a short non-coding sequence is inserted between the linked nucleic acids is also included.

  In the present invention, the meiotic homologous recombination gene DMC1 (or DMC1 gene) is a gene encoding a recombinase protein that is expressed during meiosis and has a function of controlling homologous recombination in meiosis. The DMC1 gene is known to exist regardless of animals and plants, and has been isolated in various organisms (for example, Arabidopsis-derived DMC1 gene (AtDMC1), rice-derived DMC1 gene (OsDMC1), budding yeast-derived DMC1 gene (ScDMC1 / ISC), mouse-derived DMC1 gene (MmDMC1), human-derived DMC1 gene (HsDMC1), etc.).

  The DMC1 gene used in the present invention is not limited, and may be isolated from any Asteraceae plant, and in particular, one isolated from an Asteraceae plant that is a target for inhibiting fertility (ie, , Derived from the Asteraceae plant). The DMC1 gene is not limited, but is more preferably derived from chrysanthemum. A particularly suitable example of the chrysanthemum-derived DMC1 gene is a gene consisting of the base sequence represented by SEQ ID NO: 1.

  Specifically, the DMC1 gene used in the present invention hybridizes under stringent conditions with a chrysanthemum-derived gene consisting of the base sequence represented by SEQ ID NO: 1 and a polynucleotide comprising the base sequence represented by SEQ ID NO: 1. It may be a gene consisting of a polynucleotide encoding a protein that is soybean and has recombinase activity. Here, “stringent conditions” refers to conditions under which a specific nucleic acid hybrid is formed. Specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, The reaction conditions are such that the temperature is 25 to 70 ° C., more preferably 55 to 65 ° C., and the formamide concentration is 0 to 50%, more preferably 35 to 45%. Under stringent conditions, the washing conditions of the filter after hybridization are further set such that the sodium salt concentration is 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and the temperature is 50 to 70 ° C., preferably 55 to It is suitable to set it as 70 degreeC, More preferably, it is 60-65 degreeC.

  Further, the DMC1 gene used in the present invention is 90% or more (preferably 95% or more, more preferably 96% or more, 97% or more, or 98% or more, more preferably 99% with respect to the nucleotide sequence represented by SEQ ID NO: 1. % Or more) and a gene encoding a protein having recombinase activity. The identity of the nucleotide sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 87: 2264, 1990, Karlin, S and Altschul. SF, Proc. Natl. Acad. Sci. USA, 90: 5873.). A program called BLANSTN based on the BLAST algorithm has been developed (Altschul, SF et al., J. Mol. Boil., 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and word length = 12. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known and can be used from, for example, http // www.ncbi.nlm.nih.gov /.

  Furthermore, the DMC1 gene used in the present invention may be a gene encoding the amino acid sequence represented by SEQ ID NO: 2, which is a DMC1 protein isolated from chrysanthemum, or one or several (2) in the amino acid sequence represented by SEQ ID NO: 2. (9, preferably 2 to 5) amino acids are deleted, substituted and / or added amino acid sequences, and may be a gene encoding a protein having recombinase activity.

  The “DMC1 gene” of the present invention encodes a protein having a function of controlling recombinase activity, particularly homologous recombination in meiosis in germ cells. The recombinase activity of this protein can be confirmed by methods known to those skilled in the art. For example, when the gene is genetically deleted in a plant having the gene encoding the protein, Based on the fact that pollen cells whose growth has stopped at the tetramolecular stage are observed, it is shown that the protein has a “function to control homologous recombination in meiosis” in germ cells. it can.

  The “gene” in the present invention means a gene consisting of a base sequence of an open reading frame (ORF). In the present invention, the “gene” may be DNA or RNA. DNA includes at least genomic DNA and cDNA, and RNA includes mRNA and the like.

The DMC1 gene of the present invention can be isolated by a conventional method.For example, mRNA is extracted from animals and plants including Asteraceae plants, and reversely transcribed by a conventional method to synthesize cDNA, A general PCR technique (Saiki, RK. Et al. Science (1985) using a specific PCR primer or probe designed on the basis of the known DMC1 gene or the nucleotide sequence of the DMC1 gene derived from chrysanthemum represented by SEQ ID NO: 1 ) 230, 1350, Saiki RK. Et al., Science, (1985) 239, 487) or Southern blotting. The isolated DMC1 gene can be modified in base sequence by site-directed mutagenesis techniques (Kramer, W. and Fritz, HJ, Methods Enzymol, 154: 350, (1987)). For example, a known method such as the Kunkel method, the Gapped duplex method, or the like is adopted, and a site-specific mutagenesis kit such as Mutan ^(R) -Super Express Kit (TAKARA BIO INC.) Or LA PCR ^™ in A mutant DMC1 gene can be obtained by introducing a mutation into the isolated DMC1 gene using an in vitro Mutagenesis series kit (TAKARA BIO INC.) or the like. The nucleotide sequence of the obtained DMC1 gene can be determined by a conventional method. For example, the base sequence can be determined by a known method such as the Maxam-Gilbert chemical modification method or the dideoxynucleotide chain termination method. Usually, an automatic base sequencer (for example, a commercially available DNA sequencer manufactured by ABI) is used. You can use it.

  In the present invention, “DNA encoding an RNA comprising an antisense sequence complementary to a continuous base sequence of 21 bases or more in the meiotic homologous recombination gene DMC1” means the entire base sequence of the DMC1 gene. Or, a base sequence (antisense sequence) that is at least complementary to a part (21 or more consecutive base sequences) of the base sequence (here, “thymine (t)” in the base sequence used for its identification is DNA which preferably encodes an RNA molecule which is preferably non-translatable (not translated into protein).

  When RNA encoded by this DNA is expressed, the function of mRNA of meiotic homologous recombination gene DMC1 is inhibited. This RNA may be an RNA molecule consisting of a base sequence complementary to a continuous base sequence of 21 bases or more in the DMC1 gene, and another base sequence is added to the 5 ′ or 3 ′ end. May be good.

  In a more preferred embodiment, this RNA according to the present invention comprises a sense sequence comprising a base sequence of 21 or more bases in the DMC1 gene and an antisense sequence complementary to the base sequence. Since this RNA contains an inverted repeat sequence, a long or short dsRNA having a hairpin structure is formed by intramolecular association to induce RNAi.

  In a preferred embodiment, the RNA expressed from the RNA expression cassette has a spacer sequence comprising a sense sequence consisting of a base sequence of 21 bases or more in the DMC1 gene and an antisense sequence complementary to the base sequence. It may be an RNA molecule comprising a sequence linked via Such RNA molecules form dsRNA having a stem-loop structure (hairpin structure). In such RNA, the sense sequence and the antisense sequence may be arranged such that either is located 5 ′ or 3 ′ with respect to the spacer sequence. For example, the sense sequence in such RNA may be linked to the 3 ′ end of the spacer sequence, and the antisense sequence may be linked to the 5 ′ end of the spacer sequence.

  The spacer sequence is preferably capable of constituting the loop structure portion of the stem loop without inhibiting double strand formation by intramolecular association. The spacer sequence is not particularly limited, and for example, a partial sequence of ORF of gusA gene (β-D-glucuronidase gene) (in a preferred example, a sequence consisting of the base sequence represented by SEQ ID NO: 16) can be used. Examples of spacer sequences that can be used include fragments containing intron sequences of various genomic genes. The “spacer sequence” can be RNA or DNA encoding the RNA. The base sequence of RNA as a spacer sequence can be specified as a sequence in which “thymine (t)” in the DNA sequence encoding it is replaced with “uracil (u)”.

  The RNA expressed from the RNA expression cassette according to the present invention comprises a combination of a sense sequence consisting of a continuous base sequence of 21 bases or more in the DMC1 gene and an antisense sequence complementary to the base sequence. A set may be included, or two or more sets may be included. When two or more sets are included, it is preferable that the sense sequences of each set and the antisense sequences complementary thereto are arranged adjacent to each other or linked via a spacer sequence.

  With respect to the RNA expression cassette according to the present invention, the “base sequence of 21 or more bases in the DMC1 gene” is a sequence of 21 to 49 bases in the DMC1 gene, preferably 21 to The base sequence may be 25 bases, more preferably 21-23 bases. On the other hand, when forming a long dsRNA, the “base sequence of 21 or more bases in the DMC1 gene” is 50 or more bases in the DMC1 gene, 1032 bases or less, more preferably 100 bases or more, particularly The base sequence is preferably 500 bases or more, typically 800 bases or less, preferably 500 to 700 bases, more preferably 500 to 600 bases.

  In a particularly preferred embodiment, in the above RNA according to the present invention, a continuous base sequence of 21 bases or more in the meiotic homologous recombination gene DMC1 is a sequence in the gene corresponding to positions 234 to 815 of SEQ ID NO: 1. It is preferable to include a region. In the present invention, the “region in the DMC1 gene corresponding to positions 234 to 815 of SEQ ID NO: 1” refers to the nucleotide sequence represented by SEQ ID NO: 1 which is the sequence of the DMC1 gene derived from chrysanthemum and any origin (preferably When the base sequence “X” of the DMC1 gene of the Asteraceae plant is aligned, it starts from the base that is aligned with the base A (alanine) at position 234 of SEQ ID NO: 1, and the base at position 815 of SEQ ID NO: 1. It means a region on the base sequence “X” that ends with a base aligned with A (alanine). The alignment of the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence “X” of the DMC1 gene of any origin can be performed manually, for example, the Clustal W multiple alignment program (Thompson, JD et al. (1994) Nucleic Acids Res. 22 (22): 4673-4680) with default settings. In a more preferred embodiment, the continuous base sequence of 21 bases or more in the DMC1 gene in the RNA according to the present invention is a sequence of 582 bases or more including positions 234 to 815 in the base sequence represented by SEQ ID NO: 1. As a more preferable example, a base sequence of 582 bases (SEQ ID NO: 11) including positions 234 to 815 in the base sequence represented by SEQ ID NO: 1 may be used.

  The RNA according to the present invention may have a total length of 21 to 5000 bases, preferably 500 to 3000 bases, more preferably 2000 to 2500 bases in length.

  In the RNA expression cassette according to the present invention, the DNA encoding the RNA is ligated so as to be arranged under the control of a transcription control sequence. Examples of transcription control sequences include promoters, terminators, TATA boxes, initiators, etc. In general, it is preferable that at least a promoter is included in an RNA expression cassette. The RNA expression cassette according to the present invention more preferably includes a promoter and a terminator, and preferably includes a DNA encoding the RNA under their control. The promoter that can be used is not limited to the following, but is preferably a plant promoter (promoter capable of functioning in plant cells), and is a promoter capable of inducing high expression in asteraceae plant cells (ie, exhibiting high promoter activity). In particular, a promoter exhibiting promoter activity in germ cells such as pollen and egg cells or gametes is even more preferable. The promoter may be an inducible promoter or a constitutive promoter. Suitable promoters include, for example, a mannopine synthase gene bidirectional promoter, preferably derived from Rhizobium (Agrobacterium) T-DNA, an EF1α promoter derived from tobacco (Aida, R., et al., JARQ, 39: 269 (2005)), DMC1 promoter (for example, but not limited to AtDMC1 promoter (Klimyuk, VI and Jones, JD Plant J. 11 (1): 1-14 (1997)), etc. Mannopine synthase gene Bidirectional promoters include mannopine synthase gene bidirectional mas1'-2 'promoter (Pmas102; also referred to as mas102 promoter) and mannopine synthase gene bidirectional mas2'-1' promoter (Pmas201; Any of these may be used.Mannopine synthase gene bidirectional mas1'-2 'promoter and mannopine synthase The gene bi-directional mas2'-1 'promoter is a naturally derived sequence in which two gene promoters, mas1' and mas2 ', are linked at the 5' ends. Pmas102 is, for example, the vector in FIG. Referring to the map and the T-DNA sequence in FIG. 3, mas1 ′ is located on the 5 ′ side, and mas2 ′ is located on the 3 ′ side, whereas Pmas201, on the other hand, has mas2 ′, 3 on the 5 ′ side. It has a structure in which 'mas1' is arranged on the side.Mannopine synthase gene bidirectional promoter, particularly preferably the mannopine synthase gene bidirectional mas1'-2 'promoter Pmas102 (typically (Consisting of the base sequence represented by SEQ ID NO: 20) is a plant promoter useful in that it stably exhibits high promoter activity in pollen and egg cells, and is very effective in causing temperature-stable fertility suppression. Mannopine synthase gene bi-directional When using lomotor Pmas102, an RNA expression cassette can also be constructed by linking a terminator to the 5 ′ end of the DNA encoding the RNA and a promoter Pmas102 to the 3 ′ end. Although not particularly limited, it is preferably a plant terminator (terminator that can function in plant cells), and more preferably a terminator that can function in asteraceae plant cells. Examples of suitable terminators include, but are not limited to, a terminator derived from cauliflower mosaic virus (CaMV 35S terminator) or a terminator derived from a nopaline synthase gene (nos terminator). The RNA expression cassette according to the present invention may further contain other components (for example, restriction enzyme sites) in addition to the DNA fragment encoding RNA and the transcription control sequence.

  In the method of the present invention, a recombinant vector containing the above RNA expression cassette can also be used in order to introduce the above RNA expression cassette into an Asteraceae plant. The present invention also provides a recombinant vector comprising the above RNA expression cassette. The vector into which the RNA expression cassette according to the present invention is inserted is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA and phage DNA. For example, plasmid DNA includes plasmids derived from E. coli (eg, pET22b (+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and plasmids derived from yeast. (For example, YEp13, YCp50, pPICZαA, etc.) and the like, and examples of phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, λZAPII, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used as vectors. In order to incorporate the RNA expression cassette according to the present invention into a vector, for example, each DNA fragment constituting the RNA expression cassette is cleaved with an appropriate restriction enzyme and ligated to a restriction enzyme site of a vector DNA or a multicloning site. What is necessary is just to confirm and select what was inserted in an appropriate order.

  For the purpose of expressing an RNA expression cassette in a host Asteraceae plant cell, the vector containing the RNA expression cassette according to the present invention is preferably a vector capable of replicating in a plant cell. Furthermore, for the purpose of integrating the RNA expression cassette into the genome of the host Asteraceae plant cell, the vector containing the RNA expression cassette according to the present invention is derived from Rhizobium (former name: Agrobacterium) that can be suitably used for the Agrobacterium method. These plasmid vectors (for example, binary vectors such as pBI101, pBI102, pBI120, pBI121) or their series vectors are preferably used. Specifically, for example, a pBIK102 vector that can be prepared according to the Examples can be mentioned as a suitable example.

  When the Agrobacterium method is applied to the recombinant vector containing the RNA expression cassette according to the present invention, the RNA expression cassette according to the present invention is inserted between the right boundary sequence (RB) and the left boundary sequence (LB). It is preferable to include within the region (T-DNA region). In the Agrobacterium method using such a recombinant vector, the region sandwiched between RB and LB in the recombinant vector according to the present invention introduced into the plant cell is introduced into the genome of the plant cell as T-DNA. Thus, the RNA expression cassette according to the present invention is introduced into the plant.

  The recombinant vector according to the present invention may further contain a selection marker gene and the like. Selectable marker genes include, for example, neomycin phosphotransferase gene (neomycin phosphotransferase II gene etc.) conferring resistance to antibiotics kanamycin or G418, modified cry1Ab gene, hygromycin phosphotransferase conferring resistance to hygromycin Examples thereof include genes and acetyltransferase genes that confer resistance to the herbicide phosphinothricin.

  The recombinant vector containing the RNA expression cassette according to the present invention may further contain other components. For example, it may contain a transcription control sequence such as a promoter and terminator for inducing the expression of a selectable marker gene, or may contain an additional restriction enzyme site, but is not limited thereto. When additional components such as an expression cassette containing a selectable marker gene and its transcription control sequence are additionally contained in the vector containing the T-DNA region, the additional component may be contained in the T-DNA region, It may be included in the other area inside.

  The RNA expression cassette according to the present invention and the recombinant vector containing the same can be prepared by methods known to those skilled in the art. The DNA fragment encoding the “sense sequence consisting of a continuous base sequence of 21 bases or more in the DMC1 gene”, which is a component of the RNA expression cassette according to the present invention, may be any organism (preferably as described above). Is an mRNA, cDNA, cDNA library, or genomic DNA library prepared from an Asteraceae plant subject to fertility suppression) or a DMC1 gene isolated therefrom as a template, and 21 or more consecutive bases The sequence can be obtained by amplifying by a nucleic acid amplification technique such as PCR using a specific primer designed based on the base sequence of the DMC1 gene, or an appropriate DNA fragment containing the isolated DMC1 gene can be obtained. It can also be obtained by cutting with a restriction enzyme. On the other hand, as a DNA fragment encoding an “antisense sequence complementary to a continuous nucleotide sequence of 21 bases or more in the DMC1 gene”, a DNA fragment encoding the sense sequence thus obtained (for example, What is arranged in the antisense direction (reverse direction) may be used in the RNA expression cassette. In the case of a spacer sequence derived from a gusA gene, for example, an mRNA, cDNA, cDNA library, or genomic DNA library prepared from a living organism (eg, E. coli) by a conventional method. Alternatively, a region to be used as a spacer sequence using an isolated gusA gene or the like as a template can be obtained by nucleic acid amplification by a technique known to those skilled in the art such as PCR, and includes an isolated gusA gene. It can also be obtained by excising from a DNA fragment with an appropriate restriction enzyme. Alternatively, these DNA fragments can be prepared by other conventional nucleic acid synthesis methods such as chemical synthesis. The DNA fragments prepared as described above may be ligated to each other in the intended order by a ligation reaction, for example, in an RNA expression cassette. In one embodiment, a DNA fragment encoding such an antisense sequence, and if necessary, a DNA fragment encoding a sense sequence and / or a DNA fragment containing a spacer sequence, etc. are contained in a vector such as an RNAi expression vector. A recombinant vector containing the RNA expression cassette according to the present invention can be prepared by inserting it into a restriction enzyme site typically between a promoter and a terminator under the control of a transcription control sequence. If necessary, the RNA expression cassette can be isolated by excising the RNA expression cassette from this recombinant vector by appropriate restriction enzyme treatment.

  In the present invention, male fertility and female fertility of an Asteraceae plant can be suppressed by introducing the above RNA expression cassette or a recombinant vector containing the RNA expression cassette into the Asteraceae plant. That is, the RNA expression cassette according to the present invention and the recombinant vector containing the same are useful as reagents for imparting male fertility and female fertility suppressing traits to asteraceae plants. The RNA expression cassette according to the present invention or the recombinant vector containing the same is particularly preferably one that can suppress male and female fertility in a temperature-independent manner.

  The present invention also provides a transformed cell in which the RNA expression cassette as described above or a recombinant vector containing the same is introduced into any host cell using transformation techniques known to those skilled in the art. This transformed cell is not limited to those using asteraceae plant cells as host cells, but also bacteria such as E. coli, Rhizobium and Bacillus subtilis, yeast cells, insect cells, animal cells (for example, mammalian cells), plant cells. Any of these may be used. For example, Rhizobium spp. (Such as Rhizobium radiobacter) into which a recombinant vector has been introduced are very useful for introducing the recombinant vector into Asteraceae plant cells by the Agrobacterium method in the method of the present invention. It is. Known transformation techniques include, for example, the calcium phosphate method, electroporation method, lipofection method, particle gun method, PEG method and the like.

2. Production of transformed Asteraceae and fertility suppression In the method of the present invention, in order to suppress the fertility, preferably male fertility and female fertility of the Asteraceae, according to the present invention produced as described above. An RNA expression cassette or a recombinant vector containing it is introduced into a asteraceae plant. Introduction of an RNA expression cassette or a recombinant vector according to the present invention into an Asteraceae plant can be performed by methods known to those skilled in the art.

  The asteraceae plant into which the RNA expression cassette or recombinant vector according to the present invention is introduced by the fertility suppression method of the present invention is a plant that is a target for suppressing male fertility and female fertility, and preferably its RNA expression It is a asteraceae plant having a DMC1 gene in the genome having a base sequence complementary to the antisense sequence in the cassette or a base sequence of the sense sequence. As a suitable Asteraceae plant as a target of fertility suppression by the method of the present invention, for example, Chrysanthemum, Zion (Aster), Achillea, Ageratum, and Ammobium , Burdock (Arctium), Argyranthemum, Artemisia, daisies (Belis), calendula, callistephus, carthamus, centaurea, Cichorium, Cirsium, Coreopsis, Cosmos, Cynara, Dahlia, Dimorphotheca, Echinops , Farfugium, Felicia, Gaillardia, Gazania, Gerbera ), Genus Glebionis, genus Gynnaster, genula (Gynura), sunflower (Helianthus), wheat genus (Helichrysum), genus Rhodanse (Helipterum), genus Lactuca, Leucantia French genus Leucanthemum, Liatris, Matricaria, Nipponanthemum, Petasites, Pyrethrum, Rudbeckia, Senecio, Strokesia Examples include the genus (Stokesia), the genus Tagetes, the genus Tanacetum, the dandelion (Taraxacum), and the genus Zinnia. As for the Asteraceae plants, a plant belonging to the genus Chrysanthemum is more preferable, but is not limited thereto. Chrysanthemum arcticum, Chrysanthemum rupestre, Chrysanthemum yoshinaganthum, Chrysanthemum indicum, Chrysanthemum japonense, Chrysanthemum pumense, Chrysanthemum pistol (Chrysanthemum shiwogiku), Pireogiku (Chrysanthemum weyrichii), Oshimashimajikiku (Chrysanthemum crassum), Kahamagiku (Chrysanthemum yezoense), Sea anemone (Chrysanthemum pacificum) and the like. As chrysanthemum (Chrysanthemum morifolium), various cultivars can be used, but not limited to, for example, “Shiyoshi no Aji”, “Yamate Shiro”, “Happiness Bird”, “Hiroshima Beni”, “ "Summer Yellow", "Seiun", "Seimarin", "Shinma", "Seiko no Makoto", "Iwa no White Fan", "Floral", "Tsubasa", "Akiyoshi", "Kinhide", "Oki no Otome" ”,“ Sakura ”,“ Saiplin ”,“ Maifu ”,“ Remidas ”,“ Seielsa ”, and the like.

  The asteraceae plant cell used when introducing the RNA expression cassette or the recombinant vector according to the present invention is not particularly limited as long as it is a reproducible form into a plant body. For example, cells such as scutellum in leaves, roots, stems, flowers and seeds, callus, suspension culture cells and the like can be mentioned.

  As a method for introducing the RNA expression cassette or the recombinant vector according to the present invention into Asteraceae plant cells, a method generally used for plant transformation, for example, but not limited to, Agrobacterium method, particle Physical and chemical methods such as a cancer method, an electroporation method, a polyethylene glycol (PEG) method, a microinjection method, a protoplast fusion method, and a liposome fusion method can be used. For details on these plant transformation methods, refer to general textbooks such as “Isao Shimamoto, Kiyotaka Okada“ Experimental Protocols from Model Plants to Genetic Analysis ”(2001) Shujunsha) References such as Potrykus, I., Annu. Rev. Plant Physiol. Plant Mol. Biol., 42: 205-225, 1991 may be referred to.

  When the Agrobacterium method is used, for example, a recombinant vector prepared as described above and incorporating the RNA expression cassette according to the present invention in a vector suitable for the Agrobacterium method is used as an appropriate Rhizobium, for example, Rhizobium radiobactor (R. radiobactor); former name: Agrobacterium tumefaciens (for example, R. radiobacter EHA105 strain), and the resulting recombinant strain is the leaves of Asteraceae After inoculating and infecting a tissue-derived explant (preferably sterilized or aseptic plant), and then preferably sterilizing, the transformed cells are further selected with a selective medium or the like. Together with the induction of callus, the recombinant vector is introduced into the asteraceae cells, and as a result, the RNA expression cassette is introduced into its genome. Can. A more preferable method is, for example, the Agrobacterium method according to the method of Shinoyama et al. (Plant Biotechnol. 19: 335 (2002)). According to this method, the recombinant vector is transformed into R. radiobacter bacteria, and the transformed R. radiobacter is then introduced into plant cells by a known method such as the leaf disk method.

  When using a particle gun method, an electroporation method, or the like, the RNA expression cassette according to the present invention may be directly introduced into a cell. Alternatively, the recombinant vector according to the present invention may be introduced into cells by a particle gun method, an electroporation method, or the like. As a sample used for introduction of an RNA expression cassette or a recombinant vector, a plant body, a plant organ, a plant tissue itself may be used, a section thereof may be used, or a protoplast may be prepared and used. Good (Christou P., et al., Biotechnology 9: 957 (1991)). For example, in the particle gun method, an RNA expression cassette or a recombinant vector is applied to such a sample using a gene transfer device (for example, PDS-1000 (BIO-RAD), etc.) according to the manufacturer's instructions. By implanting metal particles into a sample, it can be introduced into the cells to obtain transformed asteraceae cells. The operating conditions are usually a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

  For example, in the case of using a selection marker gene, selection of a transformed asteraceae plant cell into which an RNA expression cassette or a recombinant vector has been introduced is carried out according to a conventionally known plant tissue culture method or the cell. Can be carried out by placing the explants / callus containing the seeds on a selective medium and culturing. The selected transformed asteraceae plant cells or callus derived therefrom are further cultured in a regeneration medium (containing appropriate concentrations of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.)). By doing so, the plant body can also be regenerated (Shinoyama, H. et al., Plant Biotechnol., 19: 335 (2002)). Moreover, you may obtain the transformed Asteraceae plant as a plant body, without using such a regeneration technique by introduce | transducing a transformed Asteraceae plant cell into the shoot tip.

  The “transformed asteraceae plant” in the present invention includes a transformation obtained directly by regeneration or the like from a asteraceae plant cell into which an RNA expression cassette or a recombinant vector has been introduced or a callus derived therefrom as described above. It includes not only Asteraceae plants but also Asteraceae plants that are progeny cells thereof and retain the RNA expression cassette or recombinant vector heterozygously or homozygously in the chromosomal genome or in the extrachromosomal cytoplasm. In the present invention, the term “transformed asteraceae plant” refers to a plant body comprising at least a part of a asteraceae plant cell that retains an RNA expression cassette or a recombinant vector introduced into a cell in or outside the chromosome genome. Or a part thereof is also meant. That is, a transformed Asteraceae plant is an Asteraceae plant produced by introduction of an RNA expression cassette or a recombinant vector, or a plant body (plant individual) of a progeny plant holding the RNA expression cassette or the recombinant vector, or a plant thereof Organs (eg leaves, petals, stems, roots, seeds, pollen, buds, pistils, stamens, etc.), plant tissues (eg epidermis, phloem, soft tissues, xylem, vascular bundles, palisade tissues, spongy tissues, etc.) Or a cultured cell (for example, callus) is included.

  The transformed Asteraceae plant cell and the transformed Asteraceae plant produced as described above hold a RNA expression cassette or a recombinant vector according to the present invention, whereby a post-transcription-inhibiting RNA molecule against the DMC1 gene (preferably Can stably express RNAi molecules). As a result, the transformed Asteraceae plant according to the present invention has male fertility and female fertility suppression traits.

  In the present invention, male fertility and female fertility-inhibiting traits refer to properties in which the plant individual or the plant body of the plant exhibits sterility or reduced fertility of male gametes and female gametes. In the present invention, “fertility” refers to male fertility or female fertility, and “suppresses fertility” refers to sterilization or significantly reducing fertility.

  In a preferred embodiment of the method according to the present invention, fertility (male fertility and female fertility) is made temperature-independent in a transformed Asteraceae plant by introducing an RNA expression cassette or a recombinant vector according to the present invention. Can be suppressed. Specifically, in a preferred example, in the transformed Asteraceae plant according to the present invention, the flowering temperature that affects the fertility temperature condition, particularly fertility is, for example, 10 ° C to 35 ° C, more preferably 15 ° C to 30 ° C. Even if it fluctuates under a relatively wide range of conditions such as ℃, male fertility and female fertility are stably suppressed (sterility or decreased fertility) without depending on the specific flowering temperature. In addition, fertility suppression does not disappear at a predetermined flowering temperature (for example, less than 20 ° C.). In providing this temperature-independent male fertility and female fertility suppression trait, the mannopine synthase gene bidirectional promoter Pmas102, which exhibits high promoter activity in germ cells and gametes, is present in the present invention. It is very useful to use in such RNA expression cassette.

  In a more preferred embodiment of the method according to the present invention, the introduction of the RNA expression cassette or the recombinant vector according to the present invention may bring about a significant decrease in male sterility and female fertility in the transformed Asteraceae plants. it can. That is, the transformed asteraceae plant according to the present invention preferably has such male sterility and female fertility-reducing traits. Such transformed asteraceae plants according to the present invention are preferably male sterilized at a relatively wide range of temperatures (eg, stably at a flowering temperature of 10 to 35 ° C.), and female fertility is remarkable. Shows a trait that decreases. In the present invention, the “flowering temperature” means the time until flowering including the meiosis period, particularly the ambient temperature during the meiosis period. For example, the meiosis period of the chrysanthemum variety “Yamate Shiro” has a cocoon size. It can be judged as an index that the diameter has reached 6 mm.

  The male fertility and female fertility of the transformed Asteraceae plant according to the present invention can be confirmed by those skilled in the art by a well-known cross test or the like. Chrysanthemum is self-incompatible, so self-propagating seeds cannot be obtained. However, since it is possible to cross between related species, the transformed Asteraceae plant according to the present invention is used as a seed parent or a pollen parent to cross with the Asteraceae related species, and the results are suppressed in fertility. Compared to the result of crossing non-transformed asteraceae with related species, the fertility is lost (sterile), reduced, or maintained (or promoted) You can find out. In addition, by using the Alexander staining method (Alexander, MP, Stain Technology, 44 (3): 117, 1969), the pollen produced by transformed Asteraceae is distinguished from mature pollen and immature pollen. It is also possible to test. Specifically, the degree of male fertility of the transformed Asteraceae plant according to the present invention is determined by crossing the transformed Asteraceae plant (pollen parent) with the Asteraceae related species (seed parent), for example, maturation. We investigated pollen formation rate, pollen solidity, pollen germination ability, pollen solidity, pollen germination ability, etc. This can be determined by comparing the result of crossing with the relative species (control). On the other hand, the degree of female fertility of the transformed asteraceae plant according to the present invention is, for example, formed by crossing the transformed asteraceae plant (seed parent) with the asteraceae related species (pollen parent). It can be determined by examining the rate of formation of seeds (fruiting rate), etc., and comparing the results with the results of crossing non-transformed Asteraceae plants that are not fertilely suppressed and related species of Asteraceae (control) . In the present invention, the value indicating male fertility or female fertility (for example, the above-mentioned mature pollen formation rate and seedling rate of other seeds), when the value obtained in the control experiment is 100%, When the value is 60% or less, preferably 50% or less, more preferably 20% or less, further preferably 15% or less, still more preferably 10% or less, and particularly preferably 5% or less, inertia is suitable. It is determined that it has been suppressed. In addition, when the value indicating male fertility or female fertility does not show fertility, that is, for example, when the above-mentioned mature pollen formation rate or seedling rate of other breeding seeds is 0%, it is sterilized. It is determined that

  Alternatively, the amount of mRNA of the DMC1 gene or the protein encoded by the DMC1 gene (SEQ ID NO: 2), whose expression is suppressed by the expression of the above RNA (ie, post-transcription inhibitory RNA molecule) from the introduced RNA expression cassette, is intracellular. By measuring with (preferably germ cells or gametes such as pollen and egg cells), it is also possible to confirm whether or not the transformed Asteraceae plant has male fertility and female fertility suppression traits.

  The temperature-independence of the male fertility and female fertility-inhibiting effects is determined by measuring male fertility and female fertility, preferably in the flowering period, for transformed Asteraceae plants treated under various temperature conditions. be able to.

  As described above, a fermented and suppressed Asteraceae plant into which the RNA expression cassette or recombinant vector according to the present invention has been introduced is obtained, and the male fertile or female anther of the transformed Asteraceae plant is obtained. If at least one of the sexes is not completely sterile, the progeny can be obtained by crossing the transformed asteraceae plant with a non-transformed asteraceae plant (which is not fertilely suppressed) according to a conventional method. As a hybrid, a transformed Asteraceae plant having male fertility and female fertility suppression traits can be obtained. For example, when the obtained transformed Asteraceae plant exhibits male sterility and female fertility traits, the transformed Asteraceae plant according to the present invention is a seed parent, and a non-transformed Asteraceae plant is a pollen parent. You only have to cross.

  The above-mentioned non-transformed Asteraceae plants that are not fertilely suppressed are those in which the RNA expression cassette of the present invention has not been introduced and fertility is not suppressed (that is, preferably wild-type fertile traits) Asteraceae). The non-transformed Asteraceae plant used to cross with the transformed Asteraceae plant here is a related Asteraceae plant belonging to the same genus but different varieties or species from the transformed Asteraceae plant. Is preferred. In the present invention, the “related species” includes not only different asteraceae plant species but also different cultivars belonging to the same species. For example, when using the chrysanthemum (Chrysanthemum morifolium) cultivar "Yamate Shiro" as a transformed Asteraceae plant, the non-transformed Asteraceae plant that is not fertilely suppressed as a cross partner is not limited. However, for example, other chrysanthemum cultivars such as Hiroshima Beni and other chrysanthemum genus plants such as wakasa hamagiku, nojigiku and isogiku can be preferably used.

  The transformed asteraceae plant according to the present invention in which an RNA expression cassette is introduced into a chromosome by the Agrobacterium method is usually a heterozygote having the RNA expression cassette only on one chromosome. Therefore, a hybrid F1 plant obtained by crossing such a transformed Asteraceae plant and a non-transformed Asteraceae plant according to the present invention includes a hybrid F1 plant having an introduced RNA expression cassette and a hybrid F1 plant not having it. And can be included. Therefore, in the method according to the present invention, in order to isolate hybrid F1 plant individuals having male fertility and female fertility suppression traits from hybrid F1 plants, for example, the presence or absence of an RNA expression cassette in the genome of each individual is detected. It is also preferable to select and obtain a hybrid F1 plant individual containing the RNA expression cassette. The present invention also provides these hybrid plants, which are fertility-suppressed transformed asteraceae plants and methods for producing the same.

  According to the present invention, by introducing the RNA expression cassette or the recombinant vector according to the present invention into an Asteraceae plant, male fertility and female fertility can be stably and significantly suppressed in the transformed Compositae plant. In addition, even when gametes that can be formed slightly from the transformed Asteraceae are crossed with non-transformed Asteraceae plants, the male fertility and female fertility of the hybrid F1 plant are stable and prominent ( For example, at most 10% or less of control inertia can be suppressed. In particular, when the transformed Asteraceae plant according to the present invention exhibits male sterility and female fertility traits, it is useful because it can prevent gene diffusion due to pollen scattering with high certainty.

In addition, each sequence number quoted in this specification respond | corresponds to the following arrangement | sequences.
SEQ ID NO: 1: nucleotide sequence of chrysanthemum meiosis homologous recombination gene (CmDMC1) SEQ ID NO: 2: amino acid sequence encoded by chrysanthemum meiosis homologous recombination gene (CmDMC1) SEQ ID NO: 3: highly conserved region amplification of AtDMC1 Forward primer SEQ ID NO: 4: Reverse primer for amplification of AtDMC1 highly conserved region SEQ ID NO: 5: Internal primer sequence (forward)
SEQ ID NO: 6: internal primer sequence (reverse)
Sequence number 7: Primer GSP1
Sequence number 8: Primer NGSP1
Sequence number 9: Primer GSP2
Sequence number 10: Primer NGSP2
SEQ ID NO: 11: CmDMC1 trigger fragment SEQ ID NO: 12: EcoRV site added primer SEQ ID NO: 13: XbaI-HindIII site added primer SEQ ID NO: 14: SmaI site added primer SEQ ID NO: 15: SpeI-HindIII site added primer SEQ ID NO: 16: spacer derived from gusA SEQ ID NO: 17: antisense sequence complementary to the base sequence of the CmDMC1 trigger fragment SEQ ID NO: 18: DNA construct DMC1-RNAi
Sequence number 19: CaMV 35S terminator (T35S)
Sequence number 20: mas102 promoter (Pmas102)
SEQ ID NO: 21: Base sequence of entire T-DNA in pBIK102DBs SEQ ID NO: 22: Primer for detection of Pmas102-mcbt (forward)
SEQ ID NO: 23: Primer for detecting Pmas102-mcbt (reverse)
SEQ ID NO: 24: Primer for detecting CmCMC1 trigger fragment (forward)
SEQ ID NO: 25: Primer for detecting CmCMC1 trigger fragment (reverse)
SEQ ID NO: 26: primer for detecting spacer sequence derived from gusA (forward)
SEQ ID NO: 27: primer for detecting spacer sequence derived from gusA (reverse)
SEQ ID NO: 28: primer for detection of w-nptII (forward)
SEQ ID NO: 29: primer for detection of w-nptII (reverse)

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[Example 1] Preparation of expression vector encoding RNAi molecule Chrysanthemum camellia-derived total RNA was prepared using RNeasy Plant mini kit (Qiagen). First, a 6mm-diameter medium-flowered cultivar "Chrysanthemum morifolium Ramat. Cv. Syuho-no-chikara" was frozen in liquid nitrogen and powdered with a cooled mortar and pestle. It was crushed until. RNA was extracted according to the protocol of the above kit and dissolved in 50 μl of RNase-free sterilized water.

  Using the mRNA in the prepared total RNA as a template, cDNA was synthesized using the Ready-To-Go T-Primed First-Strand Kit (Amersham Bioscience) according to the protocol of the kit.

  In addition, based on the base sequence of the meiotic homologous recombination gene (AtDMC1) derived from Arabidopsis (GenBank accession number U76670), a region that is highly conserved among organisms (corresponding to positions from base 200 to 850) Primers 5′-TGCAATGGTCTCATGATGCATA-3 ′ (SEQ ID NO: 3) and 5′-TGGGTCTGATATGAACATTCC-3 ′ (SEQ ID NO: 4) for amplification were designed. In addition to these primers, 1 μl of the 20-fold diluted DNA solution prepared above was used as a template, dissociation (denaturation) at 94 ° C. for 30 seconds, annealing at 45 ° C. for 30 seconds, 72 ° C., 1 Fast PCR was performed under reaction conditions of 30 cycles of extension in minutes.

  Using the obtained DNA solution (first PCR product) diluted 100-fold as a template, the primers 5'-TCTGAGGCCAAAGTTGACAA-3 '(SEQ ID NO: 5), 5'-CTGGGTCAGCTATGACTTGG designed inside the first PCR product -3 ′ (SEQ ID NO: 6) was subjected to second PCR under reaction conditions of 30 cycles of dissociation at 94 ° C. for 30 seconds, annealing at 45 ° C. for 30 seconds, and extension at 72 ° C. for 1 minute. .

  A DNA fragment of about 600 bp amplified by second PCR was introduced into a T-vector prepared from plasmid pUC19 cleaved with SmaI, and the nucleotide sequence of 10 clones was determined by a conventional method and sequence analysis was performed. As a result, the DNA fragment of # 4 clone having the highest homology with the nucleotide sequence of AtDMC1 gene was identified as a partially amplified fragment of chrysanthemum-derived meiotic homologous recombinant gene DMC1 (CmDMC1).

Subsequently, using SMART ^™ RACE cDNA Amplification Kit (Clontech), the full-length sequence of chrysanthemum meiotic homologous recombination gene CmDMC1 (SEQ ID NO: 1) was determined as follows.

  First, the 5 'upstream base sequence was determined. First, a primer (GSP1) 5'-GTGAAGTCAACTCGGAACAGAGC-3 '(SEQ ID NO: 7) for amplifying a 5' unknown sequence was designed based on the base sequence of the CmDMC1 partial fragment isolated above. In addition, as described above, cDNA was synthesized using 1 ng of RNA extracted from “Hideyoshi's power” as a template, and using the 5′RACE primer (5′CDS primer) and SD SMART II A Oligo attached to the kit. . Dissociation (denaturation) at 94 ° C. for 30 seconds using DNA solution (2.5 μl) diluted 6-fold as a template, using primer GSP1 and 10 × Universal primer A mix attached to the above kit, Fast PCR was performed under the reaction conditions of 25 cycles of annealing at 68 ° C. for 30 seconds and extension at 72 ° C. for 4 minutes.

  A primer (NGSP1) 5′-GTTCTTCAGCCATCTTTGCTGCC-3 ′ (SEQ ID NO: 8) designed above the first PCR product was used as a template with a solution (5 μl) obtained by diluting the obtained DNA solution (first PCR product) 50-fold. Reaction conditions for 20 cycles of 94 ° C, 30 seconds of dissociation (denaturation), 68 ° C, 30 seconds of annealing, 72 ° C, 4 minutes of extension using Nested Universal Primer A (10 µM) attached to the kit Second PCR was performed.

  A DNA fragment of about 700 bp amplified by second PCR was introduced into a T-vector prepared from plasmid pUC19 cleaved with SmaI, and the nucleotide sequence of 5 clones was determined by a conventional method and sequence analysis was performed. As a result, the DNA fragment of clone # 9 having the highest homology with the nucleotide sequence of AtDMC1 gene was identified as a partial fragment 5 ′ upstream of chrysanthemum-derived meiotic homologous recombination gene DMC1 (CmDMC1).

  Next, the 3 ′ downstream base sequence was determined. Based on the base sequence of the CmDMC1 partial fragment isolated above, a primer (GSP2) 5′-GCTGGCTCATACTCTTTGCGTTTC-3 ′ (SEQ ID NO: 9) for amplifying the 3 ′ unknown sequence was designed. As described above, cDNA was synthesized using 1 ng of RNA extracted from “Hideyoshi's power” as a template and using the 3 ′ RACE primer (3 ′ CDS primer A) attached to the kit. Dissociation (denaturation) at 94 ° C. for 30 seconds using the DNA solution (2.5 μl) diluted 6-fold as a template, using primer GSP2 and 10 × Universal primer A mix attached to the kit, Fast PCR was performed under the reaction conditions of 25 cycles of annealing at 68 ° C. for 30 seconds and extension at 72 ° C. for 4 minutes.

  A primer (NGSP2) 5′-GAAAGGTGGAAATGGGAAGGTTGC-3 ′ (SEQ ID NO: 10) designed above the first PCR product was used as a template with a solution (5 μl) obtained by diluting the obtained DNA solution (first PCR product) 50-fold. Reaction conditions for 20 cycles of 94 ° C, 30 seconds of dissociation (denaturation), 68 ° C, 30 seconds of annealing, 72 ° C, 4 minutes of extension using Nested Universal Primer A (10µM) attached to the kit Second PCR was performed.

  The DNA fragment of about 850 bp amplified by the second PCR was introduced into a T-vector prepared from plasmid pUC19 cleaved with SmaI, and the base sequence of 10 clones was determined by a conventional method, and the sequence analysis was performed. As a result, the DNA fragment of # 16 clone having the highest homology with the nucleotide sequence of AtDMC1 gene was identified as a partial fragment 3 'upstream of chrysanthemum-derived meiotic homologous recombination gene DMC1 (CmDMC1).

  As described above, the base sequence of the full length CmDMC1 gene was determined (FIG. 1). The base sequence of the entire ORF of the CmDMC1 gene is shown in SEQ ID NO: 1. The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2. By sequence analysis, it was confirmed that the DNA fragment of the identified # 4 clone corresponded to a part of the ORF sequence (SEQ ID NO: 1) of the CmDMC1 gene.

  Subsequently, the full-length sequence of the obtained CmDMC1 gene was examined, and a DNA construct encoding an RNAi molecule that caused suppression of CmDMC1 gene expression was designed. Here, a 582 bp CmDMC1 partial fragment (base positions 234 to 815 of SEQ ID NO: 1; SEQ ID NO: 11) containing a highly conserved region between organisms is used as a trigger that constitutes a double-stranded siRNA that triggers the RNAi reaction It was decided to use it as a fragment. This 582 bp CmDMC1 partial fragment is also referred to herein as a CmDMC1 trigger fragment.

  Therefore, using the above plasmid # 4 clone as a template, using the forward primer 5′-CCGATATCGGAATCTGTGAAGCTGCT-3 ′ (SEQ ID NO: 12) and reverse primer 5′-GCTCTAGAGCCCAAGCTTGGGTTTGTCATATACAC-3 ′ (SEQ ID NO: 13), the above CmDMC1 trigger fragment A DNA fragment (EcoRV-CmDMC1 trigger fragment-HindIII-XbaI) in which a restriction enzyme EcoRV cleavage site was added on the 5 ′ side and XbaI and HindIII cleavage sites were added on the 3 ′ side was resynthesized by PCR amplification. Similarly, using the plasmid of # 4 clone as a template, using forward primer 5′-TCCCCCGGGGGAAATCTGTGAAGCTGCT-3 ′ (SEQ ID NO: 14) and reverse primer 5′-GACTAGTCCCAAGCTTGGGTTTGTCATATACACTGC-3 ′ (SEQ ID NO: 15), the above CmDMC1 trigger fragment A DNA fragment (SmaI-CmDMC1 trigger fragment-HindIII-SpeI) added with restriction enzyme SmaI cleavage site on the 5 ′ side and SpeI and HindIII cleavage site on the 3 ′ side was resynthesized by PCR amplification.

  The RNAi construction vector pBS-RNAi- containing these two DNA fragments as a spacer sequence from the base position 790 to 1729 (931 bp; SEQ ID NO: 16) of the ORF of the E. coli gusA gene (β-D-glucuronidase gene) GUS (Fumihiko Sato, “Regulation of gene expression via small RNA in plants: present status and applications of siRNA and miRNA research”, Plant growth regulation, Society of Plant Chemistry Regulation, 42 (2): 130-138); Nagoya University The CmDMC1 trigger fragment was placed in the opposite direction across the spacer sequence (Fig. 2). This spacer sequence forms a loop structure in the expressed RNA.

  Further, the recombinant vector thus obtained was treated with the restriction enzyme HindIII to cut out a DNA construct containing the CmDMC1 trigger fragment as an inverted repeat sequence with a spacer sequence in between.

  Next, an expression vector in which the DNA construct was cloned was prepared. The vector pBIK102mcbt (Shinoyama, H et al., Breeding Sci., 53: 359 (2003)) containing the mannopine synthase gene bidirectional promoter Pmas102 and the modified cry1Ab gene was used as the vector backbone. This pBIK102mcbt was cleaved with BamHI to remove the wild-type nptII gene fragment, then treated with HindIII, and the DNA construct was inserted into the cleaved site to obtain the vector pBIK102dmc1RNAi. Further, a wild-type nptII gene (Beck et al., Gene) is expressed in a high expression promoter (EF1α promoter) of tobacco-derived elongation factor gene (EF1α promoter) (Aida, R. et al., JARQ, 39: 269 (2005); JP-A-2004-65096). 1982, 19 (3): 327-336) was ligated to the XhoI cleavage site of pBIK102dmc1RNAi to obtain the expression vector pBIK102DBs (FIG. 2). In FIG. 2, DMC1-RNAi is the above DNA construct containing a CmDMC1 trigger fragment (base positions 234 to 815 of SEQ ID NO: 1) as an inverted repeat sequence. The DNA construct DMC1-RNAi comprises an antisense sequence (SEQ ID NO: 17) complementary to the base sequence of the CmDMC1 trigger fragment (SEQ ID NO: 11), a spacer sequence (gusA loop fragment), and a CmDMC1 trigger fragment sequence from 5 ′ to 3 'Include in this order in the direction. The nucleotide sequence of the DNA construct DMC1-RNAi is shown in SEQ ID NO: 18.

The abbreviations in FIG. 2 are as follows. RB: right border sequence of T-DNA region (25bp), T35S: cauliflower mosaic virus-derived 35S (CaMV 35S) terminator, w-nptII: wild-type neomycin phosphotransferase II gene, P _EF1α : tobacco-derived elongation factor gene High expression promoter, DMC1-RNAi: DNA construct containing RNAi sequence derived from chrysanthemum meiosis homologous recombination gene (CmDMC1), Pmas102: Mannopine synthase (MAS) gene bidirectional mas1 from Rhizobium radiobacter AtC1 strain (MAFF301276) '-2' promoter, mcbt: modified cry1Ab gene, Tnos: terminator derived from Rhizobium radiobacter nopaline synthase gene, LB: T-DNA region left border sequence (26bp), RK2ori (oriV): broad host plasmid RK2 Replication origin, nptIII: wild-type neomycin phosphotransferase I from Streptococcus faecalis II gene.

  RB is a sequence that functions as a starting point of transmission when T-DNA is transferred from Rhizobium to the plant genome. LB is a sequence that functions as an end point of transmission when T-DNA is transferred from Rhizobium to the plant genome. PEF1α, w-nptII, and T35S constitute a w-nptII gene expression cassette. Pmas102, DMC1-RNAi, and T35S constitute an RNA expression cassette. Pmas102, mcbt, and Tnos constitute the mcbt gene expression cassette. The modified cry1Ab gene is an artificially synthesized gene prepared by modifying the Lepidoptera pest resistance gene cry1Ab gene isolated from Bacillus thuringiensis var. Kurstaki HD-1. The base sequence of T35S used here is shown in SEQ ID NO: 19, and the base sequence of Pmas102 is shown in SEQ ID NO: 20.

  When the T-DNA (FIGS. 3 (A) to (H), SEQ ID NO: 21) in the expression vector pBIK102DBs produced in this way is integrated into the plant genome by the Agrobacterium method, the promoter Pmas102 in the RNA expression cassette Thus, the expression of an RNAi molecule capable of suppressing the expression of the CmDMC1 gene is induced.

[Example 2] Transformation of chrysanthemum In this example, in order to introduce the expression vector pBIK102DBs prepared in Example 1 into chrysanthemum by the Agrobacterium method, first, pBIK102DBs was electrolyzed by Rhizobium radiobacter ( Rhizobium radiobactor (old name: Agrobacterium tumefaciens) EHA105 strain (sold by Zeneca Morgen). Then, R. radiobacter introduced with pBIK102DBs was selected and cultured in YEP medium (composition: 1% peptone, 1% yeast extract, 0.5% sodium chloride, pH 7.2) at 28 ° C. for 5 hours. R. Radiobacter suspension was prepared.

Chrysanthemum transformation was performed by the following procedure. A leaf disc was prepared by punching the leaves of a sterile plant produced from the small sterility cultivar "Yamate Shiro" (Chrysanthemum morifolium Ramat. Cv. Yamate-shiro) with a 6 mm diameter cork borer. did. After the explants were immersed in the R. radiobacter suspension prepared above for about 15 minutes, the excess bacterial solution was removed with filter paper, and the coexisting medium (Murashige-Skoog (MS) basic medium, 3% sucrose, Add 0.3% gellan gum, 1mg / l naphthalene acetic acid, 0.5mg / l benzyladenine, and 1g / l casamino acid, pH 5.8), place the leaves face up, and co-culture for 3 days at 25 ° C in the dark did. At the end of co-cultivation, explants were sterilized (added 3% sucrose, 0.3% gellan gum, 1 mg / l naphthalene acetic acid, 0.5 mg / l benzyladenine, and 250 mg / l cefotaxime sodium salt to the MS basic medium, The sample was placed at pH 5.8) and sterilized at low illuminance (photosynthesis effective photon flux density (PPFD): 60 μmol / m ² / s) for 7 days. The explants were then added to selective medium A (MS basic medium, 3% sucrose, 0.3% gellan gum, 1 mg / l naphthalene acetic acid, 0.5 mg / l benzyladenine, 250 mg / l cefotaxime sodium salt, and 20 mg / l G418. Added, pH 5.8), and cultured at 25 ° C. and medium illuminance (PPFD: 60 μmol / m ² / s) for 2 weeks. Two weeks later, the cells were transferred to a new selection medium A, and two weeks later, the cells were transferred to a new selection medium A. Then, after 2 weeks, selective medium B (3% sucrose, 0.3% gellan gum, 1 mg / l naphthalene acetic acid, 0.5 mg / l benzyladenine, 100 mg / l cefotaxime sodium salt, and 20 mg / l G418 was added to MS basic medium. PH 5.8), and further two weeks later, the cells were transferred to a new selection medium B to select transformed cells and promote callus induction from the transformed cells. Then transfer to redifferentiation medium (MS basic medium, 3% sucrose, 0.3% gellan gum, 0.5 mg / l benzyladenine, 0.2 mg / l gibberellin A ₃ and 100 mg / l cefotaxime sodium salt, pH 5.8) The plant body was regenerated from callus. The regeneration medium was renewed every 3 weeks, and when the shoots became 2 to 3 mm long, the shoots were scraped and transferred to a test tube containing hormone-free MS basic medium to promote rooting. For the shoots causing vitrification (vitrification: vitrification), air permeability was ensured by using a breathable membrane milliseal (Millipore) to eliminate the vitrification.

  Furthermore, in the same manner, the medium-glyph cultivar "Chrysanthemum morifolium Ramat. Cv. Shuho-no-chikara" and the spray cultivar "Happiness Bird" (Chrysanthemum morifolium Ramat. Cv. Kouhuku-no-tori) Also, a transformed plant body was prepared.

  As a result, regenerated plant bodies of 682 individuals from the chrysanthemum cultivar “Yamate Shiro”, 131 individuals from “Shiho no Aji”, and 102 individuals from “Happiness Bird” were obtained. Therefore, genomic DNA was extracted from each of these individuals, and the introduced sequence was detected based on the PCR method using it as a template. The following introduced sequence-specific primer pairs were used for detection by PCR.

(a) Mannopine synthase gene bidirectional mas1'-2 'promoter (Pmas102) -mcbt detection primer pair:
5'-GAATAGATGATCTGACACGCC-3 '(SEQ ID NO: 22)
5'-AGTGGTGTGGAGTACCTGTAG-3 '(SEQ ID NO: 23)
(b) CmDMC1 trigger fragment detection primer pair:
5'-AATCTGTGAAGCTGCTGAGA-3 '(SEQ ID NO: 24)
5'-TTTGTCATATACACTGCAAGC-3 '(SEQ ID NO: 25)
(c) Primer pair for detecting spacer sequence derived from gusA:
5'-ATCTACCCGCTTCGCGTCGGCATC-3 '(SEQ ID NO: 26)
5'-TCGCGAGTGAAGATCCCCTTCTTG-3 '(SEQ ID NO: 27)
(d) w-nptII detection primer pair:
5'-ATGATTGAACAAGATGGATTGCAC-3 '(SEQ ID NO: 28)
5'-TCAGAAGAACTCGTCAAGAAGGCG-3 '(SEQ ID NO: 29)

  By this detection, in all redifferentiated individuals (regenerated plants), the presence of component mas102 promoter (Pmas102), modified cry1Ab gene (mcbt), DMC1-RNAi, and wild-type nptII gene (w-nptII) in pBIK102DBs A PCR amplification band was confirmed. That is, it was shown that all the obtained regenerated plants were transformed chrysanthemum plants into which pBIK102DBs had been introduced (CmDMC1 gene-derived chrysanthemum plant).

[Example 3] Fertility test of male chrysanthemum grown at different flowering temperatures (male fertility)
The CmDMC1 gene-derived RNAi sequence-introduced chrysanthemum plant (transformed chrysanthemum) obtained in Example 2 was subjected to Southern blot analysis and Northern blot analysis to confirm introduction and expression of CmDMC1 gene-derived RNAi. As transformed chrysanthemum, recombinant DB315, DB569, DB576 individuals were used. Thereafter, CmDMC1 gene-derived RNAi sequence-introduced chrysanthemum plants were acclimated and subjected to low-temperature treatment in a closed greenhouse. For the control experiment, non-transformed chrysanthemum (Phyrus varieties “Yamate white”; strain used as host in transformation) into which pBIK102DBs had not been introduced was also grown and used in the same manner.

  Chrysanthemum individuals (transformed chrysanthemum and non-transformed chrysanthemum) having a cocoon size of 6 mm in diameter (meiotic phase) were placed in artificial weather devices set at different temperatures of 10 to 35 ° C. and allowed to bloom. For cylindrical flowers one day before flowering, Alexander staining (Alexander, MP, Stain Technology, 44 (3): 117, (1969)), acetocarmine staining, pollen medium (Ikeda, H and Numata, S, Acta Hort., 454: 329, (1998)) and subjected to cross test with relatives of Asteraceae, etc. to confirm the degree of male fertility.

  As a result, mature pollen was formed in the non-transformed chrysanthemum at a flowering time treatment temperature of 10 to 25 ° C. On the other hand, in the chrysanthemum, no mature pollen formation was observed at any temperature, and no pollen formation was observed except for a treatment temperature of 15 ° C. Even immature pollen was only observed in transformed chrysanthemum at a treatment temperature of 15 ° C.

  4 and 5 show the results of Alexander staining of buds and pollen at each flowering period treatment temperature condition. In this test, a paraffin-embedded section was prepared after staining with a cylindrical flower, and a red-stained portion showing mature tissue and a blue-stained portion showing immature tissue were observed under a microscope. FIG. 4 shows the development state of the cocoon, while FIG. 5 shows the state of pollen formation in the cocoon. As shown in FIG. 5, in the non-transformed chrysanthemum, individually separated cells (mature pollen) having nuclei (red-stained portions) were observed. In contrast, in the transformed chrysanthemum (DB315 strain), no pollen formation was observed at the flowering temperature of 20 ° C. and 25 ° C., and even at 15 ° C. where pollen was observed, the small molecule ( Pollen that has stopped growing at the tetramolecular stage and pollen stained blue with no nuclei (red dyed part) (unripe pollen; meiosis does not go well, halfway) Only the growth was observed). Furthermore, in the transformed chrysanthemum DB260 and DB194 prepared according to Example 2, only the pollen mass and immature pollen that stopped growing at the tetramolecular stage under the flowering temperature condition of 15 ° C. were observed. .

  Table 1 shows the results of counting the number of mature pollen, the number of immature pollen and the number of four-phase pollen in each cocoon of Alexander-stained tubular flowers (the data shown is the average number of pollen per cocoon ± standard deviation). Pollen stained red by the Alexander staining method was determined as mature pollen, pollen stained blue was immature pollen, and a pollen mass in which small molecules stained red and blue were collected was determined as four-phase pollen.

  Tables 2 and 3 show the fertility and longevity of the pollen formed. Table 2 shows the results of estimation of pollen solidity. First, for the transformed chrysanthemum individual DB315 and the control non-transformed chrysanthemum individual (host “Yamate Shiro”), buds were collected from the cylindrical flower one day before flowering and the pollen was placed on the slide. The preparation on which the pollen was placed was placed in an artificial weather apparatus (continuous light, 60% humidity) set at 10, 15, 20 and 25 ° C. and incubated. Acetocarmine staining solution was dropped onto the pollen incubated for 0, 1, 5, 10, and 24 hours, and cultured in a 25 ° C. artificial weather device (continuous light). The degree of pollen staining was observed 1 hour after acetocarmine staining. The numerical values in the table indicate the percentage (%) of the pollen stained for each elapsed time, and the solidity (maturity) of the pollen was estimated therefrom.

Table 3 shows the test results of germination ability for each elapsed time in the pollen medium. This is because there are some pollens that are full but have no germination ability (that is, they cannot be fertilized). Germination ability was measured for the transformed chrysanthemum individuals DB315, DB569, DB576 and control non-transformed chrysanthemum individuals using a pollen medium. As the pollen medium, the following liquid medium: ethyl acetate extract = 4: 1 was mixed and used.
・ Liquid medium: 100 ppm H ₃ BPO ₄ , 300 ppm CaCl ₂ · 2H ₂ O, 0.01 ppm CoCl ₂ · 6H ₂ O, 30% saccharose • Ethyl acetate extract: 100 ml of chrysanthemum flower (20 g) It is a residual extract obtained by soaking in ethyl acetate, sealing and storing in a refrigerator for 24 hours, and then volatilizing the ethyl acetate.

  For the assay, first, buds were collected from the non-transformed chrysanthemum and the cylindrical flowers one day before the flowering of the transformed chrysanthemum, and the pollen was placed on the slide. The preparation on which the pollen was placed was placed in an artificial weather apparatus (continuous light, 60% humidity) set at 10, 15, 20 and 25 ° C. and incubated. Put 1-2 drops of pollen medium on pollen incubated for 0, 1, 5, 10 and 24 hours, put in a plastic petri dish (diameter 9 cm), seal, and culture in a 25 ° C artificial weather (continuous light) did. Two hours later, when the pollen tube reached the pollen diameter, it was regarded as germination. The numerical value shows the ratio (%) of the pollen germinated for each elapsed time, and the germinating ability of the pollen was estimated therefrom.

  As described above, pollen whose formation was observed at 15 ° C. in the transformed chrysanthemum had a low degree of solidity (Table 2) and did not show germination ability in the pollen medium (Table 3).

  Further, using pollen whose formation was observed in transformed chrysanthemum at 15 ° C., a cross test was conducted with a related species of Asteraceae (seed parent) to examine male fertility. As for the related species of the Asteraceae, there are small varieties “Hiroshimabeni”, edible chrysanthemum varieties “Mottenohoka”, Chrysanthemum wakasaense, Chrysanthemum japonense, Chrysanthemum pacificumster, and konjac (Chrysanthemum pacificumster). ageratoides ssp. avalus).

  In this cross test, no hybrid seed was formed (Table 4), and thus the pollen of the transformed chrysanthemum formed at 15 ° C. was recognized as sterile pollen.

In Table 4, “self” indicates self-breeding (self-crossing).
From the above results, it was shown that the transformed chrysanthemum according to the present invention stably exhibits male sterility in the chrysanthemum flowering temperature zone (10 ° C to 35 ° C).

[Example 4] Fertility test of female chrysanthemum grown at different flowering temperatures (female fertility)
Next, the CmDMC1 gene-derived chrysanthemum plant (transformed chrysanthemum; recombinant DB315, DB569, DB576 individuals) obtained in Example 2 was crossed with the Asteraceae related species (pollen parent). A test was conducted to examine female fertility. As for the Asteraceae related species, “Hiroshima Beni”, “Moretomi Other”, Wakasa Hamagiku, Nojigiku, Isogiiku, and Kongi were used in the same manner as described above. Furthermore, the self-fertilizing fertility of the transformed chrysanthemum was also investigated (“self” in Table 5 below).

  Specifically, the pollen parent was low-temperature treated with an artificial meteor (16-hour day length) set at 10 ° C for 40 days from June 1, 2007. On August 9, the plant height was 5 cm ( Plants of 3 developed true leaves) were inserted and buds proliferated, and seedlings were grown with an artificial meteorometer (16 hours long) set at 25 ° C. On August 25, it was raised in a polypot (diameter 15 cm) containing mixed soil (Taro: vermiculite = 5: 1) and grown in a specific net room with a window open through a screen door.

  The seed parent was acclimatized in October 2006 as a sterile plant in a test tube and subjected to low-temperature treatment with an artificial meteor (16-hour day length) set to 10 ° C for 40 days from June 1, 2007. On August 9, the plant was planted and grown with a plant height of 5 cm (three real leaves unfolded), and seedlings were grown using an artificial meteorometer (16 hours long) set at 25 ° C. On August 25, it was raised in a polypot (diameter 15 cm) containing mixed soil (Taro: vermiculite = 5: 1) and grown in a specific net room with a window open through a screen door. Plants having a cocoon size of 5 mm in diameter were placed in an artificial weather device (8 hours long) set at 10, 15, 20, 25, 30 and 35 ° C. and grown.

  The pollen parent pollen collected from the cylindrical flower one day before flowering is cultivated seed parent, and the seed parent in the cut flower state cut into a length of 50 cm plant length one day before pollination and dipped the cut into tap water The pollen was pollinated with a small brush on the stigma of a cylindrical flower, and an artificial hybridization test was conducted. Thereafter, the non-recombinant chrysanthemum and the recombinant chrysanthemum, which are the seed parents, were grown on an artificial meteorological apparatus set at 10 to 35 ° C. to maturate other seeds. Two months after pollination, the ovary was collected, placed in a petri dish (diameter 9 cm) containing a damp filter paper, placed in a dark at 25 ° C. for 3 days, and examined for germination. The fruiting rate was calculated by dividing the number of germinated seeds by the number of pollinated cylindrical flowers.

  As a result, as shown in Table 5 below, when non-transformed chrysanthemum (host; seed parent) was crossed with Asteraceae related species (pollen parent), in both cultivation state and cut flower state, 15 to Other seeds were formed under each treatment condition of 30 ° C. In contrast, when transformed chrysanthemum (recombinant DB315, DB569, DB576 individuals) was crossed with an Asteraceae related species (pollen parent), the treatment temperature was 20 ° C or less, both in the cultivated state and in the cut flower state. The formation of breeding seeds was not observed, and the formation of other breeding seeds was not observed at 25 ° C or 30 ° C in the cut flower state. In the case of transformed chrysanthemum, other seeds were formed only in the cultivation state under the treatment conditions of 25 ° C to 30 ° C, but the fruiting rate was only one-tenth to one-twentieth that of non-transformed chrysanthemum. There wasn't.

“Self” in the table indicates self-breeding (self-crossing).
Thus, the transformed chrysanthemum according to the present invention stably showed a significant decrease in female fertility in the chrysanthemum flowering temperature range (10 ° C. to 35 ° C.), and showed female sterility particularly at 20 ° C. or lower.

  Therefore, as described above, genomic DNA was extracted from the cross-bred seed (F1 hybrid) obtained by crossing with a chrysanthemum family related species (pollen parent) using the transformed chrysanthemum DB315 strain individual as a seed parent, and using it as a template, The introduced sequence was detected based on the PCR method. For detection by PCR, the primer pair for detecting Pmas102-mcbt, the primer pair for detecting CmDMC1 trigger fragment, and the primer pair for detecting w-nptII described in Example 2 were used. As a result, it was confirmed that many of the F1 hybrids between transformed chrysanthemum DB315 and Asteraceae related species have an introduced sequence containing the DNA construct CmDMC1-RNAi.

[Example 5] Transmission of sterile traits to F1 hybrids Crossbreds were grown by breeding other seeds obtained in the crossing of transformed chrysanthemum (seed parent) and asteraceae related species (pollen parent) in Example 4. F1 lines were obtained, and the pollen formation state was examined by acetocarmine staining and Alexander staining (Table 6). In Table 6, DB315 / H1 ~ 5: transformed chrysanthemum DB315 and Hiroshima red hybrid F1 line, DB315 / M1 ~ 5: other hybrid F1 lines with DB315, DB315 / W1 ~ 5: DB315 and Wakasagamagiku Fukui preservation line No.1 Hybrid F1 line of DB315 / N1 ~ 5: Hybrid F1 line of DB315 and Nojigiku Fukui Conservation Line No. 1, DB315 / I1 ~ 5: Hybrid F1 line of DB315 and seagull.

Specifically, first, non-transformed chrysanthemum having a diameter of 5 mm, transformed chrysanthemum, and hybrid F ₁ strains were placed in an artificial weather apparatus (8 hours long) set at 15 and 20 ° C. . Untransformed chrysanthemum, anthers were collected from transformed chrysanthemum and hybrids F ₁ strains flowering 1 day prior to the tubular flowers were aceto carmine staining and Alexander staining. For acetocarmine staining, the degree of pollen staining was observed 1 hour after staining to determine the degree of pollen fertilization. For Alexander staining, the degree of staining was observed after treatment at 50 ° C. for 24 hours (Table 6). Pollen stained red by the Alexander staining method was determined as mature pollen, and the number was counted as the number of fruit pollen. In addition, pollen stained blue by Alexander staining method is judged as immature pollen, and pollen mass consisting of small molecules dyed in red and blue is judged as tetramolecular pollen, and the total number of immature pollen and tetramolecular pollen as sterile pollen. Was counted as the number of sterile pollen. The numerical values in Table 6 indicate the average number of pollen per cocoon ± standard deviation.

  As can be seen from Table 6, the hybrid lines obtained by crossing the transformed chrysanthemum with related species of the family Asteraceae also showed male sterility that was temperature independent, comparable to that of the transformed chrysanthemum of one parent. Among hybrid F1 lines, fertile pollen was formed only in those in which no introduced sequence (CmDMC1-RNAi etc.) was detected in Example 4. This is because the parental transformed chrysanthemum introduced the pBIK102DBs-derived T-DNA into only one of the chromosomes, resulting in both F1 hybrids with and without the introduced sequence (CmDMC1-RNAi, etc.). Therefore, fruit pollen formation in F1 hybrids that do not have such introduced sequences (CmDMC1-RNAi etc.) can be taken as a suitable control for sterility in F1 hybrids that have the introduced sequences. it can.

  Furthermore, a female fertility test was also carried out by carrying out a cross test with the Asteraceae related species (pollen parent) in the same manner as described above using the hybrid F1 line as a seed parent (Table 7). ). In Table 7, DB315 / H1 ~ 5: transformed chrysanthemum DB315 and hybrid F1 line of Hiroshima red, DB315 / M1 ~ 5: other hybrid F1 lines with DB315, DB315 / W1 ~ 5: DB315 and Wakasamagiku Fukui preservation line No.1 Hybrid F1 line of DB315 / N1 ~ 5: Hybrid F1 line of DB315 and Nojigiku Fukui Conservation Line No. 1, DB315 / I1 ~ 5: Hybrid F1 line of DB315 and seagull.

  As can be seen from Table 7, the hybrid lines obtained by crossing the transformed chrysanthemum and related species of the family Asteraceae also show a marked temperature-independent decline in fertility in female fertility, similar to the single parental chrysanthemum. It was. Among the hybrid F1 lines, the fruiting rate was relatively high only in those in which the introduction sequence (CmDMC1-RNAi etc.) was not detected in Example 4. Such a result of an F1 hybrid that does not have an introduced sequence (CmDMC1-RNAi, etc.) can be taken as a suitable control for a significant reduction in female fertility in an F1 hybrid that has the introduced sequence.

  From the above results, it was shown that male fertility and female fertility can be stably suppressed in the Asteraceae plants by introduction (transformation) of an RNAi molecule encoding CmDMC1 gene. In addition, the mannopine synthase gene bidirectional mas1'-2 'promoter used for inducing the expression of DMC1-RNAi sequence exhibits high promoter activity in pollen and egg cells, and is not affected by a wide range of flowering temperatures, and is not temperature-insensitive. It was also shown that expression can be induced in a dependent manner and is very useful for suppression of male fertility and female fertility by the method of the present invention. Furthermore, the male sterility and low female fertility traits were shown to be inherited by progeny.

  The method of the present invention and the RNA expression cassette used therefor suppress fertility with high probability even in normal temperature conditions during cultivation (especially under flowering temperature conditions) in the production of transgenic Asteraceae plants. And can be used to effectively prevent unintended diffusion of the transgene in the plant.

The sequences of SEQ ID NOs: 3 to 10, 12 to 15, and 22 to 29 are primers.
The sequence of SEQ ID NO: 17 is an antisense sequence complementary to the CmDMC1 trigger.
The sequence of SEQ ID NO: 18 is the DNA construct DMC1-RNAi.
The sequence of SEQ ID NO: 21 is T-DNA.

Claims (14)

Suppresses male fertility and female fertility of Asteraceae plants, including DNA encoding an RNA containing an antisense sequence complementary to a continuous base sequence of 500 or more bases in the meiotic homologous recombination gene DMC1 An RNA expression cassette , wherein the continuous base sequence of 500 bases or more includes a region in the gene DMC1 corresponding to positions 234 to 815 of SEQ ID NO: 1 . 2. The RNA according to claim 1, wherein the RNA comprises a sense sequence consisting of a base sequence of 500 bases or more in the meiotic homologous recombination gene DMC1 and an antisense sequence complementary to the base sequence. The RNA expression cassette as described. The RNA expression cassette according to claim 1 or 2, wherein the meiotic homologous recombination gene DMC1 is any one of the following genes (a) to (e).
(a) a gene consisting of the base sequence represented by SEQ ID NO: 1,
(b) a gene comprising a polynucleotide that hybridizes with a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having recombinase activity;
(c) a gene encoding a protein consisting of a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 1 and having recombinase activity
(d) a gene encoding the amino acid sequence represented by SEQ ID NO: 2,
(e) a gene encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 2 and having recombinase activity The RNA expression cassette according to any one of claims 1 to 3, wherein a continuous base sequence of 500 bases or more in the meiotic homologous recombination gene DMC1 has a length of 500 to 700 bases. The RNA expression cassette according to claim 2 , wherein the sense sequence and the antisense sequence are linked via a spacer sequence. The RNA expression cassette according to any one of claims 1 to 5 , comprising DNA encoding the RNA under the control of a mannopine synthase gene bidirectional mas1'-2 'promoter. The RNA expression cassette according to any one of claims 1 to 6 , wherein a continuous base sequence of 500 bases or more in the meiotic homologous recombination gene DMC1 comprises the base sequence represented by SEQ ID NO: 11. A recombinant vector comprising the RNA expression cassette according to any one of claims 1 to 7 . A transformation having male fertility and female fertility suppression traits by introducing the RNA expression cassette according to any one of claims 1 to 7 or the recombinant vector according to claim 8 into an Asteraceae plant cell. A method for suppressing fertility of a asteraceae plant, comprising producing a asteraceae plant. The method according to claim 9 , wherein the male fertility and female fertility suppressing traits have a property that male fertility and female fertility are suppressed under a temperature condition of 10 to 35 ° C. A transformed cell into which the RNA expression cassette according to any one of claims 1 to 7 or the recombinant vector according to claim 8 has been introduced. The transformed cell according to claim 11 , which is a transformed Asteraceae plant cell. A transformed asteraceae plant having male fertility and female fertility suppression traits obtained by regenerating a plant from the transformed asteraceae plant cell according to claim 12 . Male fertility, comprising obtaining a progeny plant containing the RNA expression cassette by crossing a transformed Asteraceae plant according to claim 13 as a seed parent and a non-transformed Asteraceae plant as a pollen parent. A method for producing a transformed asteraceae plant having a female fertility suppressing trait.

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