JP2007202561A - A method for reducing pollen fertility using a pollen-specific zinc finger transcription factor gene - Google Patents

Abstract

Provided is a genetic engineering technique using a pollen-specific gene that is useful for modifying plant traits represented by male sterility.
A method for producing a plant having a modified trait, which comprises a DNA having a specific nucleotide sequence and exhibiting specific promoter activity in a microspore and optionally a cleaved tissue of a cocoon. And a plant expression cassette comprising a heterologous gene operably linked to the promoter. Furthermore, a plant expression cassette useful for imparting male sterility to a plant comprising the promoter and the promoter and a heterologous gene.
[Selection figure] None

Description

  The present invention relates to a gene specifically expressed in pollen-forming tissues of stamens and use thereof. In particular, the present invention relates to genes of a petunia-derived zinc finger type transcription factor group (ZPT2-5, ZPT3-1 and ZPT4-1) that are specifically expressed in microspores, and use thereof.

  Pollen fertility becomes a problem in various aspects of agriculture and horticulture. For example, when crossing in crossbreeding, in order to avoid self-pollination, the work of male removal (removal of stamens), which requires much labor, is performed. In the seed and seedling industry, a trait without pollen fertility is required from the standpoint of commercially protecting excellent varieties obtained by cross breeding. From such a request, a technique for controlling the fertility of pollen (pollination control) has been strongly demanded. Traditionally, in certain crops, cytoplasmic male sterility lines have been used for crossbreeding with some success. However, cytoplasmic sterile traits often have undesirable side effects such as reduced disease resistance, and further have problems such as unstable traits and difficulty in mass production of seeds. A method of reducing fertility by chemical treatment has also been studied, but safety evaluation and action mechanism elucidation have been delayed, and it has not been put into practical use. Therefore, an excellent male sterilization technique using genetic engineering is required.

  Pollen is the male gametophyte in seed plants. The development of pollen that progresses in a state surrounded by cocoons as the support tissue is the four-molecule phase immediately after meiosis of the microspore-forming cells (pollen mother cells), the release phase of microspores from the four molecules, the pollen cell It is divided into the following stages: a mononuclear phase characterized by expansion and vacuolation, a mitotic cell division in which mitosis causes differentiation into vegetative and germ cells, and a subsequent binuclear phase. Through these steps, the wrinkles are finally cleaved and mature pollen grains are released. Therefore, microspores can be said to be one of the target tissues most suitable for artificial control to inhibit pollen development and lose pollen fertility.

  As described above, great expectations are placed on the male sterilization technology by genetic engineering. In particular, if a gene that is specifically expressed in the direct precursors of pollen cells such as microspores can be used, it is highly likely that male sterility can be achieved without imparting undesirable traits to the plant. It is done. Several examples of promoters specific to various tissues of stamens and a gene construct for male sterility containing the same have been reported (Non-patent Document 1). However, there is a continuing need for new genes that are highly tissue-specific and time-specific in expression and useful for controlling pollen fertility.

The present inventors recently include PEThyZPT2-5, PEThyZPT3-1, and PEThyZPT4-1 (hereinafter abbreviated as ZPT2-5, ZPT3-1, and ZPT4-1, respectively) as novel transcription factors derived from petunia. Seven zinc finger (ZF) type transcription factor cDNA sequences were identified. From Northern blot analysis, it was reported that each transcription factor showed transient expression at different stages of wrinkle development in a wrinkle-specific manner (Non-patent Document 2). However, the physiological functions and actions of these transcription factors in plants, and the precise expression site of the gene encoding the transcription factor and its expression regulatory mechanism were unknown.
Shivanna and Sawhney, Pollen biotechnology for crop production and improvement (Cambridge University Press) 1997, pp. 237-257 Kobayashi et al., Plant J. et al. 1998, 13: 571.

  An object of the present invention is to provide a genetic engineering technique using a gene specific for pollen that is useful for modification of plant traits represented by male sterility.

  The present inventors reintroduced into petunia the genes encoding a group of transcription factors (ZPT2-5, ZPT3-1 and ZPT4-1) previously isolated from petunia. As a result, it was found that the normal development of pollen was inhibited and the fertility of the pollen was significantly reduced (ZPT2-5 and ZPT4-1) or almost completely lost (ZPT3-1). Furthermore, the present inventors isolated upstream regions from the genomic genes of ZPT3-1 and ZPT4-1, respectively, and examined the tissue specificity of their promoter activity. As a result, the present inventors have found that promoter activity is expressed in a tissue and time-specific manner in microspores from the first to the second nuclear phase. The present invention has been completed based on these findings.

  The present invention, in its first aspect, relates to a method for producing a male sterile plant comprising the following steps: (i) from position 1 of the base sequence represented by SEQ ID NO: 1 DNA having a sequence up to position 777, (ii) DNA encoding a transcription factor that hybridizes with a DNA having the base sequence of (i) under stringent conditions and controls the development of pollen, or (iii) Providing a plant expression cassette comprising a nucleic acid that is either DNA that is a fragment of (i) or (ii) and a promoter operably linked to the nucleic acid; endogenous controlling pollen development Wherein a gene encoding an endogenous transcription factor hybridizes with the nucleic acid under stringent conditions. A step of introducing an expression cassette into a plant cell; a step of regenerating a plant cell into which the expression cassette has been introduced into a plant body; and a regenerated plant body, wherein the nucleic acid is expressed, A step of selecting a plant in which expression of an endogenous transcription factor is suppressed.

  In its second aspect, the present invention relates to a method for producing a male sterile plant comprising the following steps: (i ′) the first position of the base sequence represented by SEQ ID NO: 3 To a DNA having a sequence from position 1 to position 1640, a DNA encoding a transcription factor that hybridizes with a DNA having a base sequence of (ii ′) (i ′) under stringent conditions and controls the development of pollen, or (Iii ′) providing a plant expression cassette comprising a nucleic acid that is either a DNA that is a fragment of (i ′) or (ii ′) and a promoter operably linked to the nucleic acid; Providing a plant cell with an endogenous transcription factor that controls development, wherein the gene encoding the endogenous transcription factor is under stringent conditions with the nucleic acid. A step of introducing the expression cassette into the plant cell; a step of regenerating the plant cell into which the expression cassette has been introduced into the plant body; and a regenerated plant body, wherein the nucleic acid is expressed. A step of selecting a plant body in which expression of an endogenous transcription factor is suppressed.

  In its third aspect, the present invention relates to a method for producing a male-sterile plant comprising the following steps: (i ″) position 1 of the base sequence represented by SEQ ID NO: 5 DNA having a sequence from position 1 to position 1948, DNA encoding a transcription factor that hybridizes with DNA having a base sequence of (ii ″) (i ″) under stringent conditions and controls the development of pollen, or (Iii) providing a plant expression cassette comprising a nucleic acid that is either DNA that is a fragment of (i ") or (ii") and a promoter operably linked to the nucleic acid; Providing a plant cell with an endogenous transcription factor that controls development, wherein the gene encoding the endogenous transcription factor is under stringent conditions with the nucleic acid. A step of introducing the expression cassette into the plant cell; a step of regenerating the plant cell into which the expression cassette has been introduced into the plant body; and a regenerated plant body, wherein the nucleic acid is expressed. A step of selecting a plant body in which expression of an endogenous transcription factor is suppressed.

  However, the DNAs of (ii), (ii ′) and (ii ″) above are stringent with DNA having the sequence from position 1 to position 1886 of the base sequence represented by (iv) SEQ ID NO: 13, respectively. DNA that encodes a transcription factor that hybridizes under mild conditions and controls the development of pollen is not included The base sequence represented by SEQ ID NO: 13 encodes another transcription factor ZPT3-2 isolated from petunia cDNA sequence (Kobayashi et al., supra).

  The method in the first to third aspects of the present invention can be used as a method for imparting male sterility to a plant.

  In one embodiment of the first to third aspects, the nucleic acid is linked to the promoter in the forward direction, and can be transcribed in the sense direction within the plant cell.

  In one embodiment of the first to third aspects, the nucleic acid is linked in the reverse direction to the promoter, and can be transcribed in the antisense direction in the plant cell.

  In one embodiment of the first to third aspects, the plant is a dicotyledonous plant. The dicotyledonous plant is preferably a solanaceous plant, more preferably a petunia plant.

  In one embodiment of the first to third aspects, the expression cassette is incorporated into a plant expression vector.

  According to the first to third aspects of the present invention, a male sterile plant produced by any one of the methods described above is also provided.

  In its fourth aspect, the present invention relates to a method for producing a plant having a modified trait, comprising the following steps: (a ′) position 1 of the nucleotide sequence represented by SEQ ID NO: 7 A promoter comprising either a DNA having a sequence from position 1 to position 2624, or a DNA having a partial sequence of (b ′) (a ′) and exhibiting promoter activity specific to microspores, Providing a plant expression cassette comprising a heterologous gene operably linked to the plant; introducing the expression cassette into the plant cell; and regenerating the plant cell into which the expression cassette has been introduced into the plant body.

  In its fifth aspect, the present invention relates to a method for producing a plant having a modified trait, comprising the following steps: (a ″) position 1 of the nucleotide sequence represented by SEQ ID NO: 8 DNA having a sequence from position 1 to position 3631, or a DNA having a partial sequence of (b ″) (a ″) and exhibiting specific promoter activity in microspores and optionally in cleaved tissues of sputum A plant expression cassette comprising a promoter comprising any of the above and a heterologous gene operably linked to the promoter; introducing an expression cassette into a plant cell; and a plant into which the expression cassette has been introduced A step of regenerating cells into plants.

  In one embodiment of the fourth and fifth aspects, the trait is fertile and the plant whose trait has been modified is a male sterile plant. Therefore, the above method of the present invention can be used as a method for imparting male sterility to a plant.

  In one embodiment of the fourth and fifth aspects, the trait is compatible, and the plant whose trait has been modified is a self-incompatible plant. Therefore, the said method of this invention can be utilized also as a method of providing self-incompatibility to a plant.

  In one embodiment of the fourth and fifth aspects, the plant is a dicotyledonous plant. The dicotyledonous plant is preferably a solanaceous plant, more preferably a petunia plant.

  In one embodiment of the fourth and fifth aspects, the expression cassette is incorporated into a plant expression vector.

  According to the fourth and fifth aspects of the present invention, there is also provided a plant having a modified trait produced by any one of the methods described above.

  The present invention, in its sixth aspect, relates to a promoter comprising the following DNA (I ′) or (II ′): (I ′) From the first position to the 2624th position of the base sequence represented by SEQ ID NO: 7 Or a DNA having a partial sequence of (II ′) (I ′) and exhibiting promoter activity specific to microspores.

  In its seventh aspect, the present invention relates to a promoter comprising the following DNA (I ″) or (II ″): (I ″) from the first position to the 3631 position of the base sequence represented by SEQ ID NO: 8 Or a DNA having a partial sequence of (II ″) (I ″) and exhibiting promoter activity specific to microspores and optionally to cleaved tissue.

  The present invention, in its eighth aspect, is useful for conferring male sterility to a plant comprising any of the above-mentioned microspore-specific promoters and a heterologous gene operably linked to the promoter. A plant expression cassette.

The present invention will be described in more detail below.
(Transcription factors derived from ZPT3-2, ZPT3-1 and ZPT4-1 genes)
The nucleic acid useful in the method for producing a male sterile plant, which is the first to third aspects of the present invention, is any of the following:
(I) DNA having a sequence from position 1 to position 777 of the base sequence represented by SEQ ID NO: 1,
(I ′) DNA having a sequence from position 1 to position 1640 of the base sequence represented by SEQ ID NO: 3;
(I ″) DNA having a sequence from position 1 to position 1948 of the base sequence represented by SEQ ID NO: 5, or
DNA encoding a transcription factor that hybridizes with a DNA having any one of the nucleotide sequences (i) to (i ″) under stringent conditions and controls the development of pollen (ie, (ii), (ii ′) ) Or (ii ")), or
DNA which is a fragment of any of the above DNAs (ie, (iii), (iii ′) or (iii ″)).

  The nucleic acid in the present invention is preferably DNA of (i), (i ′) or (i ″), that is, DNA encoding ZPT2-5, ZPT3-1 or ZPT4-1, or a fragment thereof, More preferred is DNA of (i), (i ′) or (i ″).

  As used herein, the term “transcription factor” refers to a protein that binds to DNA in the regulatory region of a gene to control mRNA synthesis. Certain transcription factors are known to have highly conserved amino acid sequences called zinc finger (ZF) motifs in their DNA binding domains. ZPT2-5 is a Cys2 / His2 type (EPF family) zinc finger (ZF) protein, which contains two ZF motifs within the full-length amino acid sequence consisting of 176 amino acids, and is further referred to as a DLNL sequence. A transcription factor containing a region. Similarly, ZPT3-1 is a ZF protein of the EPF family, which is a transcription factor containing three ZF motifs in the full-length amino acid sequence consisting of 437 amino acids and further containing a DLNL sequence. Similarly, ZPT4-1 is a ZF protein of the EPF family, which is a transcription factor containing four ZF motifs in the full-length amino acid sequence consisting of 474 amino acids and further containing a DLNL sequence. In both cases, see Kobayashi et al. (Supra). CDNA sequences encoding ZPT2-5, ZPT3-1 and ZPT4-1 (SEQ ID NOs: 1, 3 and 5), respectively, together with the corresponding deduced amino acid sequences (SEQ ID NOs: 2, 4 and 6), are shown in FIGS. Shown in 2A-2C and Figures 3A-3D.

  As used herein, a “fragment” of nucleic acid or DNA refers to a fragment that can suppress the expression of an endogenous transcription factor of the plant when introduced into a plant body and expressed in an appropriate manner. This fragment is selected from a region other than the region encoding the zinc finger motif in the DNA of (i), (i ′), (i ″), (ii), (ii ′) or (ii ″), and at least about It has a length of 40 base pairs or more, preferably about 50 base pairs or more, more preferably about 70 base pairs or more, and further preferably about 100 base pairs or more.

In the present specification, “stringent conditions” for hybridization refer to a specific base sequence (for example, DNA encoding ZPT2-5, ZPT3-1 or ZPT4-1 derived from petunia) and homologous thereto. Sufficient to form an oligonucleotide duplex with other highly likely nucleotide sequences (for example, DNA encoding a homologue of ZPT2-5, ZPT3-1 or ZPT4-1 present in plants other than petunia) Intended conditions. Typical examples of stringent conditions applied to the present invention include the following conditions: 1M NaCl, 1% SDS, 10% dextran sulfate, ³² P-labeled probe DNA (1 × 10 ⁷ cpm) and 50 μg / ml. After hybridization at 60 ° C. for 16 hours in a solution containing salmon sperm DNA, washing with 2 × SSC / 1% SDS at 60 ° C. for 30 minutes is repeated twice.

  In order to isolate DNAs encoding ZPT2-5, ZPT3-1 and ZPT4-1, and DNAs encoding a transcription factor which hybridizes with these under stringent conditions and controls the development of pollen in the present invention. A degenerate primer pair corresponding to a conserved region of an amino acid sequence encoded by a gene of a known transcription factor can be used. Using this primer pair, PCR can be performed using plant cDNA or genomic DNA as a template, and then the obtained amplified DNA fragment can be used as a probe to screen a cDNA library or genomic library of the same plant. . An example of such a primer pair is a combination of 5'-CARGCNYTNGGGNGCAY-3 '(SEQ ID NO: 9) and 3'-RTGNCCNCNARGNGCYTG-5' (SEQ ID NO: 10), where N is inosine. , R is G or A, and Y is C or T). Each of the primer sequences corresponds to the amino acid sequence QALGHH contained in the zinc finger motif of the ZPT transcription factor group.

  Therefore, stringent hybridization conditions applied to the present invention can also be defined as PCR conditions, and in representative examples, the above degenerate primers (SEQ ID NOs: 9 and 10) can be used. PCR reaction conditions were as follows: denaturation at 94 ° C for 5 minutes, 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 1 minute, and finally incubation at 72 ° C for 7 minutes It may be a condition to perform.

  PCR can be performed based on the guidelines of the manufacturers of commercially available kits and devices, or can be performed by techniques well known to those skilled in the art. Methods for preparing gene libraries and gene cloning methods are also well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory, 1989). The base sequence of the obtained gene can be determined using a nucleotide sequence analysis method known in the art or a commercially available automatic sequencer.

  In the present specification, a transcription factor “controls the development of pollen” typically means that a significant change in the morphology or function of pollen is observed when the expression of the transcription factor is inhibited. Say. Typically, the transcription factor in the present invention is preferably about 75% or more, more preferably about 90% or more, more preferably about 95% or more pollen by inhibiting the expression of the gene encoding it. Cells are killed before maturation. Here, the expression of the transcription factor being inhibited means that the amount of mRNA is about 1/10 or less as compared with the wild type control plant as measured by Northern blotting.

  Transcription factors (ie, ZPT2-5, ZPT3-1 and ZPT4-1, and homologs thereof) encoded by the genes isolated and identified by screening as described above control pollen development. It can be confirmed by creating a transformed plant according to the disclosure of the specification and observing its pollen characteristics.

  In the present invention, by using a DNA encoding a transcription factor that controls the development of pollen, the expression of an endogenous gene containing a base sequence identical or homologous to the DNA can be suppressed in plant cells. The target endogenous gene is also a transcription factor that controls pollen development. According to the method of the present invention, preferably, by selectively suppressing only the expression of the endogenous transcription factor without substantially suppressing the expression other than the endogenous transcription factor that controls the development of pollen, Is given male sterility.

  That is, the plant cell to which the expression suppression technique in the present invention is applied is a plant cell having an endogenous transcription factor that controls the development of pollen, and the gene encoding this endogenous transcription factor is the above-mentioned ZPT2 -5, defined as hybridizing under stringent conditions with ZPT3-1 or ZPT4-1, or their homologous DNA. Here, the definition of “stringent conditions” is the same as the conditions described above in connection with the identification of homologs of ZPT2-5, ZPT3-1, and ZPT4-1. Although not intended to limit the scope of the present invention, plants capable of conferring male sterility by the above method are petunia from which the ZPT gene group has been isolated, or genes encoding the ZPT homologs have been isolated. It is desirable that the plant is a plant closely related to evolutionary phylogeny. Here, evolutionary phylogenetic related is typically a plant classified in the same eye, preferably classified in the same family, more preferably classified in the same genus, and more preferably classified in the same species. is there. However, considering the fact that pollen development is essential for the reproduction of seed plants, transcription factors that perform the same or similar functions as ZPT2-5, ZPT3-1, and ZPT4-1 are widely present in other plants. Getting is easily understood.

  As a technique for suppressing the expression of an endogenous gene, typically, co-suppression and antisense techniques can be used. Cosuppression means that when a recombinant gene is introduced into a plant cell, expression of the gene itself and an endogenous gene containing a sequence homologous to a part of the gene are suppressed. When cosuppression is used, the expression cassette in the present invention contains DNA encoding a transcription factor or a fragment thereof in a form linked in a forward direction to a promoter. A DNA encoding a transcription factor or a fragment thereof can be transcribed in the sense direction under the control of a promoter after being introduced into a plant cell as an expression cassette. By the action of the introduced DNA, it becomes possible to suppress the desired gene expression. Cosuppression is observed in some individuals of transformed plants and often does not occur well in other individuals. For this reason, individuals whose gene expression is repressed as intended are usually selected according to routine procedures.

  In antisense, when a recombinant gene is introduced into a plant cell, the transgene transcript (mRNA) forms a hybrid with a complementary sequence of the endogenous gene transcript (mRNA), and the endogenous gene encodes. It means that the translation of the protein is inhibited. When antisense is used, the expression cassette in the present invention contains DNA encoding a transcription factor or a fragment thereof in a form linked in the reverse direction to the promoter. A DNA encoding a transcription factor or a fragment thereof can be introduced into a plant cell as an expression cassette and then transcribed in the antisense direction under the control of a promoter. By the action of this antisense transcript, it becomes possible to suppress the desired gene expression.

(Promoter derived from ZPT3-1 and ZPT4-1 genes)
Promoters useful in the fourth and fifth aspects of the present invention for producing a plant with altered traits are (a ′) from position 1 to position 2624 of the nucleotide sequence represented by SEQ ID NO: 7. (A ″) DNA having the sequence from position 1 to position 3631 of the base sequence represented by SEQ ID NO: 8, or (a ′) or a partial sequence of (a ″) It is a promoter containing any one of DNAs having promoter activity specific to microspores. The promoter in the present invention is preferably the promoter (a ′) or (a ″), that is, the promoter of the ZPT3-1 or ZPT4-1 gene.

  A sequence having promoter activity specific to microspores obtained by removing a sequence not essential for tissue-specific expression activity in the promoter region of ZPT3-1 and ZPT4-1 gene is within the scope of the present invention. is there. Such a sequence can be obtained by conducting a promoter deletion experiment according to a conventional method. Briefly, various deletion mutants of the promoter region of the ZPT3-1 or ZPT4-1 gene (for example, the promoter region of the ZPT3-1 or ZPT4-1 gene is deleted to various lengths from the 5 ′ upstream side. The region essential for the activity is determined by measuring the tissue-specific promoter activity of the deletion mutant using a plasmid fused with an appropriate reporter gene (for example, GUS gene). It can be specified.

  Once a region essential for promoter activity is identified, it is possible to further modify the sequence in the region or adjacent sequences to increase the level of expression activity of the promoter. The variants thus obtained are also within the scope of the present invention as long as they exhibit promoter activity specific to microspores.

  In the present invention, “showing a promoter activity specific to microspores” means that a promoter is introduced into a plant in a natural plant or as an expression cassette linked to an arbitrary structural gene. It means to specifically show in microspores the ability to direct gene expression by initiating transcription. The term “specific” as used herein includes all other tissues (tapeto layer, flower thread, style, flower head, petal, wrinkles, etc.) of flowers of the same plant; ) Is higher than the expression activity of the promoter. The specific promoter preferably has higher promoter expression activity than all other tissues and non-flower parts (roots, leaves, stems, etc.) of flowers of the same plant, more preferably the same plant. It exhibits virtually no activity in all other tissues and non-flower parts of the body. “Showing a promoter activity specific to the cleavage tissue of cocoon” is also defined in the same manner as described above. The degree of expression activity can be assessed by comparing the expression level of the promoter in the microspore with the expression level of the same promoter in the other tissues of the flower according to conventional methods. The expression level of a promoter is usually determined by the amount of gene product expressed under the control of that promoter.

  The methods of the present invention that utilize the specific promoters described above are intended to modify traits associated with plant reproduction. Here, “modified” means that at least a part of the reproductive organs in the transformed plant loses the function that existed in the plant before transformation (wild type or horticultural variety), or the plant before transformation It refers to acquiring a function that did not exist, or enhancing or reducing the degree of a specific function compared to the plant before transformation. Such a modification of the trait is that any heterologous gene operably linked to the promoter in the present invention is specifically expressed in microspores under the control of the promoter in a transformed plant into which this gene has been introduced. Can result. In many tissue specific promoters, it is well known that the tissue specificity is conserved across species, so it is readily understood that the promoters of the present invention can also be applied to a wide range of plant species. The degree of trait modification can be assessed by comparing the plant trait after transformation with the plant trait prior to transformation. Preferred traits to be modified include, but are not limited to, female sterility and self-incompatibility.

  The promoter of the present invention can be obtained, for example, by screening a plant genomic library using a known cDNA as a probe and isolating the upstream sequence of the coding region from the corresponding genomic clone. Examples of the cDNA include ZPT3-1 and ZPT4-1 cDNAs that are the above-mentioned transcription factors derived from petunia.

  The promoter in the present invention is not limited to those isolated from nature, and may include synthetic polynucleotides. Synthetic polynucleotides can be obtained, for example, by synthesizing or modifying the promoter sequence sequenced as described above or the active region thereof by techniques well known to those skilled in the art.

(Construction of expression cassette and expression vector)
The DNA encoding the transcription factor of the present invention is introduced into a plant cell using a known gene recombination technique as an expression cassette operably linked to an appropriate promoter using a method well known to those skilled in the art. obtain. Similarly, the microspore-specific promoter in the present invention can be introduced into a plant cell as an expression cassette operably linked to a desired heterologous gene.

  The “promoter” that can be linked to the transcription factor means any promoter expressed in a plant, and can include any of a constitutive promoter, a tissue-specific promoter, and an inducible promoter.

  “Constitutive promoter” refers to a promoter that allows a structural gene to be expressed at a certain level regardless of stimulation inside or outside a plant cell. If a heterologous gene is expressed in other tissues or organs of the plant but does not give the plant an undesirable trait, the use of a constitutive promoter is convenient and preferred. Examples of constitutive promoters include, but are not limited to, cauliflower mosaic virus (CaMV) 35S promoter (P35S) and nopaline synthase promoter (Tnos).

  In the present invention, “tissue-specific promoter” refers to a promoter that specifically expresses a structural gene in at least microspores. Such tissue-specific promoters include, in addition to promoters derived from the ZPT3-1 and ZPT4-1 genes in the present invention, other known promoters that exhibit expression activity specifically. Thus, the natural ZPT3-1 and ZPT4-1 gene expression cassettes themselves, including microspore-specific promoters and sequences encoding transcription factors, may be used in combination with other regulatory elements as necessary. It is included in the scope of the present invention.

  The “inducible promoter” refers to a promoter that causes a structural gene to be expressed when a specific stimulus such as a chemical agent or physical stress is applied and does not exhibit an expression activity in the absence of the stimulus. Examples of inducible promoters include the auxin-inducible glutathione S-transferase (GST) promoter (van der Kop, DA, et al., Plant Mol. Biol., 39: 979, 1999), It is not limited to this.

  In the present specification, the term “expression cassette” or “plant expression cassette” is linked to DNA encoding the transcription factor of the present invention operably (that is, so that expression of the DNA can be controlled). And a nucleic acid sequence comprising a microspore-specific promoter in the present invention and a heterologous gene operably linked thereto (ie, in frame).

  The “heterologous gene” that can be linked to the above-mentioned microspore-specific promoter is an endogenous gene in Petunia other than the ZPT3-1 and ZPT4-1 genes, or an endogenous gene in a plant other than Petunia, or foreign to the plant Genes (eg, genes derived from animals, insects, bacteria, and fungi), wherein expression of the gene product is desired in microspores. A preferred example of the heterologous gene in the present invention is a gene whose encoding gene product exhibits cytotoxicity and inhibits pollen development by its expression. Specific examples of such genes include, but are not limited to, the barnase gene (Beals, TP and Goldberg, RB, Plant Cell, 9: 1527, 1997).

  “Plant expression vector” refers to a nucleic acid sequence to which various regulatory elements are operably linked in a host plant cell in addition to an expression cassette. Suitably, a terminator, a drug resistance gene, and an enhancer may be included. It is well known to those skilled in the art that the type of plant expression vector and the type of regulatory element used can vary depending on the host cell. The plant expression vector used in the present invention may further have a T-DNA region. The T-DNA region increases the efficiency of gene introduction, particularly when a plant is transformed with Agrobacterium.

  A “terminator” is a sequence that is located downstream of a region encoding a protein of a gene and is involved in termination of transcription when DNA is transcribed into mRNA, and addition of a poly A sequence. Terminators are known to contribute to mRNA stability and affect gene expression levels. Examples of terminators include, but are not limited to, nopaline synthase gene terminator (Tnos) and cauliflower mosaic virus (CaMV) 35S terminator.

  The “drug resistance gene” is preferably one that facilitates selection of transformed plants. A neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, a hygromycin phosphotransferase gene for conferring hygromycin resistance, and the like can be preferably used, but are not limited thereto.

  The plant expression vector in the present invention can be prepared using gene recombination techniques well known to those skilled in the art. For the construction of a plant expression vector, for example, a pBI-type vector or a pUC-type vector is preferably used, but is not limited thereto.

(Creation of transformed plants)
The expression cassette constructed as described above or an expression vector containing the expression cassette can be introduced into a desired plant cell using a known gene recombination technique. The introduced expression cassette is incorporated into DNA in the plant cell. The DNA in plant cells includes not only chromosomes but also DNAs contained in various organelles (for example, mitochondria and chloroplasts) contained in plant cells.

  As used herein, the term “plant” includes both monocotyledonous and dicotyledonous plants. A preferred plant is a dicotyledonous plant. Dicotyledonous plants include both petal flower subclasses and joint flower subclasses. A preferred subclass is the joint flower subclass. The joint flower subclass includes any of Gentian, Eggplant, Perilla, Abagoceae, Psyllium, Oleander, Pleurotus, Akane, Nematode, and Chrysanthemum. Preferred eyes are eggplant eyes. The solanidae includes any of the solanaceae, sericidaceae, red-breasted, lanterns, and convolvulaceae. A preferred family is the solanaceous family. The solanaceae includes Petunia, Datura, Tobacco, Eggplant, Tomato, Pepper, Physalis, and Culicidae. Preferred genera are Petunia, Datura, and Tobacco, more preferably Petunia. Petunia is P. hybrida sp. Axillaris sp. inflata species, and including violacea species. Preferred species are P. pylori. It is a hybrida species. “Plant” means a plant having a flower and a seed obtained from the plant unless otherwise indicated.

  Examples of “plant cells” include cells of each tissue, callus, and suspension culture cells in plant organs such as flowers, leaves, and roots.

  For introduction of a plant expression vector into a plant cell, methods well known to those skilled in the art, for example, an Agrobacterium-mediated method and a method of direct introduction into a cell can be used. As a method using Agrobacterium, for example, the method of Nagel et al. (FEMS Microbiol. Lett., 67: 325 (1990)) can be used. In this method, Agrobacterium is first transformed with a plant expression vector (for example, by electroporation), and then transformed Agrobacterium is introduced into plant cells by a well-known method such as the leaf disk method. Is the method. Examples of methods for directly introducing a plant expression vector into cells include an electroporation method, a particle gun, a calcium phosphate method, and a polyethylene glycol method. These methods are well known in the art, and a method suitable for the plant to be transformed can be appropriately selected by those skilled in the art.

  A cell into which a plant expression vector has been introduced is selected based on drug resistance such as kanamycin resistance. The selected cells can be regenerated into a plant body by a conventional method.

  In a regenerated plant body, it can be confirmed that the introduced plant expression vector is functional using a technique well known to those skilled in the art. For example, when the purpose is to suppress the expression of an endogenous gene, this confirmation can be performed by measuring a transcription level using Northern blot analysis. In this manner, a desired transformed plant body in which the expression of an endogenous transcription factor is suppressed can be selected. Even when the purpose is to express a heterologous gene using a tissue-specific promoter, the expression of the heterologous gene can usually be confirmed using Northern blot analysis using RNA extracted from the target tissue as a sample. The procedure of this analysis method is well known to those skilled in the art.

  The expression of the endogenous transcription factor is suppressed according to the method of the present invention, and the fertility of the pollen is reduced. For example, the pollen of a plant obtained by transformation with an expression vector containing DNA encoding the transcription factor The morphology can be confirmed by microscopic observation after performing tissue chemical staining as necessary.

  In addition, the specific expression of the promoter in the microspores according to the method of the present invention means that, for example, in a plant obtained by transformation with an expression vector in which a promoter and a GUS gene are operably linked, The distribution of GUS activity in the included flower tissue can be confirmed by tissue chemical staining according to a conventional method.

  Hereinafter, the present invention will be described based on examples. The scope of the present invention is not limited to only the examples. Restriction enzymes, plasmids, etc. used in the examples are available from commercial sources.

(Example 1: Construction of a plant expression vector containing a polynucleotide encoding a group of ZPT transcription factors)
Among the previously reported sputum-specific ZF genes (Kobayashi et al., Supra), the PEThyZPT2-5 (ZPT2-5), PEThyZPT3-1 (ZPT3-1), and PEThyZPT4-1 (ZPT4-1) cDNAs, In each case, a plant expression vector was prepared by connecting to the downstream of the 35S promoter of cauliflower mosaic virus. Specifically, it is as follows.

(Example 1-1)
A DNA fragment (HindIII-XbaI fragment) containing a cauliflower mosaic virus 35S promoter in a plasmid pBI221 (purchased from CLONTECH Laboratories Inc.) and a DNA fragment (SacI-EcoRI fragment) containing a NOS terminator were sequentially added to the plasmid pUCAP (van Engelen, F. et al. A. et al., Transgenic Res., 4: 288, 1995) was inserted into the multicloning site to prepare pUCAP35S. On the other hand, the pBluescript vector containing the ZPT2-5 cDNA was cleaved at the KpnI site and the SacI site (both sites in the vector) and inserted between the KpnI and SacI sites of the above pUCAP35S. The recombinant plasmid was further digested with EcoRI and HindIII, and a DNA fragment encoding ZPT2-5 was introduced into the EcoRI and HindIII sites of the binary vector pBINPLUS (van Engelen, FA, et al., Supra). As is clear from FIG. 4 (a), the constructed ZPT2-5 gene is a 35S promoter region (P35S; 0.9 kb) of cauliflower mosaic virus (CaMV), a polynucleotide encoding ZPT2-5 of the present invention. (ZPT2-5; about 0.8 kb) and a terminator region of nopaline synthase (Tnos; 0.3 kb). In FIG. 4, Pnos represents the promoter region of nopaline synthase, and NPTII represents the neomycin phosphotransferase II gene.

(Example 1-2)
The pBluescript vector containing the ZPT3-1 cDNA was cleaved at the KpnI site and the SacI site (both sites in the vector) and inserted between the KpnI and SacI sites of pUCAP35S. This recombinant plasmid was further digested with EcoRI and HindIII, and a DNA fragment encoding ZPT3-1 was introduced into the EcoRI and HindIII sites of the binary vector pBINPLUS. As is clear from FIG. 4 (b), the constructed ZPT3-1 gene is a 35S promoter region (P35S; 0.9 kb) of cauliflower mosaic virus (CaMV), a polynucleotide encoding ZPT3-1 of the present invention ( ZPT3-1; about 1.7 kb) and a terminator region of nopaline synthase (Tnos; 0.3 kb).

(Example 1-3)
The pBluescript vector containing the cDNA of ZPT4-1 was cleaved at the KpnI site and the SacI site (both sites in the vector), and inserted between the KpnI and SacI sites of the above pUCAP35S. This recombinant plasmid was further digested with EcoRI and HindIII, and a DNA fragment encoding ZPT4-1 was introduced into the EcoRI and HindIII sites of the binary vector pBINPLUS. As is clear from FIG. 4 (c), the constructed ZPT4-1 gene was a 35S promoter region (P35S; 0.9 kb) of cauliflower mosaic virus (CaMV), a polynucleotide encoding ZPT4-1 of the present invention ( ZPT4-1; about 2.0 kb), and a terminator region of nopaline synthase (Tnos; 0.3 kb).

(Example 2: Isolation of ZPT3-1 and ZPT4-1 promoter regions and ligation with GUS reporter gene)
Using the cDNAs of ZPT3-1 and ZPT4-1 as probes, the corresponding genomic clones were isolated from a Petunia genomic DNA library, and a DNA fragment (promoter region; about 2.7 kb and about 3. 6 kb) was subcloned. Each DNA fragment was linked upstream of the GUS reporter gene and cloned into a binary vector. Specifically, it is as follows.

(Example 2-1)
ZPT3-1 cDNA is labeled with [α32P] dCTP using a normal random prime method (Sambrook et al., Supra) to produce a radiolabeled probe, which is used to create an EMBL3 vector (Stratagene). A petunia (Petunia hybrida var. Mitchell) genomic library was screened. From the obtained clone, a genomic DNA fragment (PstI-SacI) of about 2.7 kb containing the gene upstream region was subcloned into the PstI-SacI site of the pBluescriptSK vector (pBS-ZPT3-1-PS), and the nucleotide sequence was determined ( 5A-5B). Next, using this plasmid as a template, PCR was performed using a primer containing a SalI recognition sequence (3′-TATGGAGCTCCGTCGACATGTTGATGTTCATTTTTCTGGCTATTTGTC-5 ′; SEQ ID NO: 11) and a commercially available M13-20 primer, and the ZPT3-1 protein translation start point A SalI site was introduced immediately downstream (base number 2661). Then, a DNA fragment obtained by cutting with PstI and SalI was inserted upstream of the GUS coding region of pUCCAPUSNT (pUCAP-ZPT3-1-GUSNT). As a result, the region near the N-terminal of the ZPT3-1 gene coding region was connected to the GUS coding region in frame. Further, a DNA fragment (including ZPT3-1 promoter, GUS coding region and NOS terminator) obtained by cleaving pUCAP-ZPT3-1-GUSNT with AscI and PacI is inserted into the pBINPLUS vector, and pBIN-ZPT3-1-GUS is inserted. Obtained (FIG. 7A).

(Example 2-2)
Similarly, genomic DNA was isolated for ZPT4-1, and an approximately 3.6 kb DNA fragment (EcoRI-EcoRI) containing the upstream region of the ZPT4-1 gene was subcloned into the EcoRI-EcoRI site of the pBluescriptSK vector (pBS-ZPT4-1). -EE), the base sequence was determined (FIGS. 6A-6B). Using this plasmid as a template, PCR was performed using a primer containing a BamHI recognition sequence (3′-CATGGATATAGGATCCCTATC-5 ′; SEQ ID NO: 12) and an M13-20 primer, and immediately downstream (base number) of the ZPT4-1 protein translation start point. 3641) was introduced a BamHI site. Then, a DNA fragment obtained by digestion with EcoRI and BamHI was inserted upstream of the GUS coding region of pUCAPPGUSNT (pUCAP-ZPT4-1-GUSNT). As a result, the region near the N-terminal of the ZPT4-1 gene coding region was connected to the GUS coding region in frame. Further, a DNA fragment (AscI-PacI) was inserted into the pBINPLUS vector in the same manner as described above to prepare pBIN-ZPT4-1-GUS (FIG. 7 (b)).

(Example 3: Introduction of each fusion gene into petunia cells)
Each of the above expression vectors was introduced into Petunia (Petunia hybrida var. Mitchell) via Agrobacterium according to the following procedure.

  (1) Agrobacterium tumefaciens LBA4404 strain (purchased from CLONTECH Laboratories Inc.) was cultured at 28 ° C. in L medium containing 250 mg / ml streptomycin and 50 mg / ml rifampicin. A cell suspension was prepared according to the method of Nagel et al. (1990) (supra), and the plasmid vector constructed in Examples 1 and 2 was introduced into the strain by electroporation.

  (2) Polynucleotides encoding each fusion gene were introduced into petunia cells by the following method: Agrobacterium tumefaciens LBA4404 obtained in (1) above was transformed into YEB medium (DNA Cloning No. 2 Volume, p. 78, Glover DM, edited by IRL Press, 1985), and cultured with shaking (28 ° C., 200 rpm). The obtained culture broth was diluted 20-fold with sterilized water and co-cultured with petunia (Petunia hybrida var. Mitchell) leaf pieces. Two to three days later, the bacteria were removed with a medium containing antibiotics, subcultured with a selective medium every two weeks, and introduced with the five types of fusion genes, and kanamycin resistance due to expression of the NPTII gene derived from pBINPLUS. Transformed petunia cells were selected based on the presence or absence. The selected cells were induced to callus by a conventional method and then redifferentiated into plants (Jorgensen RA, et al., Plant Mol. Biol., 31: 957, 1996).

(Example 4: Phenotype of transformed petunia introduced with ZPT gene group)
By using the transformant obtained by introducing the vector of Example 1 and observing the morphological changes of pollen accompanying the regulation of the expression of ZPT2-5, ZPT3-1 and ZPT4-1, The effect on plants by introduction of cDNA was examined. Specifically, it is as follows.

(Example 4-1)
From the transformants (14 individuals) into which the cDNA of ZPT2-5 was introduced under the control of the 35S promoter, individuals (3 individuals) in which suppression of gene expression due to cosuppression occurred were selected by Northern blot analysis. (In addition, overexpression of the introduced ZPT2-5 gene was observed in the other 4 individuals out of 14 individuals.) The conditions for Northern blot analysis were as follows: 7% SDS, 50% formamide, 5 × SSC, 2 % Blocking reagent (Boehringer Mannheim), 50 mM sodium phosphate buffer (pH 7.0), 0.1% lauryl sarcosine sodium, 50 μg / ml yeast tRNA, and ³² P-labeled probe DNA (1 × 10 ⁷ cpm) Hybridize at 68 ° C. for 16 hours, then wash with 2 × SSC / 0.1% SDS at 68 ° C. for 30 minutes.

  The following phenotypes were observed in the above three cosuppression transformants (FIG. 8).

  In the process of meiosis that occurs just before the tetramolecular phase, in normal (wild-type) petunias, the formation of a filamentous structure by the condensation of chromatin (prophase I fibril phase), homologous chromosomes pair ( The joining yarn period of the first period I). Next, when metaphase I is entered, the quadrant is aligned with the cell equatorial plane, and then homologous chromosomes are evenly separated into the cell poles by the spindle apparatus. On the other hand, in the transformant in which the co-suppression of the ZPT2-5 gene occurred, the chromosomal segregation proceeded in the middle stage I without correctly arranging the quadrants on the cell equatorial plane. At this time, the distribution of chromosomes to both extremes was extremely uneven.

  In the normal meiosis process, after the first chromosome segregation, a second chromosomal segregation forms four haploid groups, followed by cytoplasmic segregation. On the other hand, in the transformant in which the above co-suppression occurred, cytoplasm separation and cell division occurred immediately after the first chromosome separation. In addition, this unequal cell division was not limited to once, but was repeated at least twice more to form up to 8 microspore cells. Due to unequal chromosome segregation, the number of chromosomes contained in these microspore cells was heterogeneous, and with it, the size of the cells was also significantly uneven. As a result, at the time of the tetramolecular phase in normal petunia, these transformants formed more microspores (up to 8) than normal (FIG. 8 (f); at a bud size of 6 mm). A photograph of pollen cells of the ZPT2-5 cosuppression transformant (see also FIG. 9B; a photograph of pollen cells of the transformant in the four-molecule phase).

  In co-suppression transformants, some of the microspores (10-20%) continued to develop, but most microspore cells ruptured before the callose layer surrounding them was degraded. The microspores that survived without rupture at this stage showed an abnormal morphology close to the hexahedron, clearly different from the tetrahedral morphology of normal microspores. The abnormally shaped microspores then became binucleated by apparently normal mitosis to form pollen grains. However, these pollen grains almost lost their fertility. That is, when pollen grains of these transformants are applied to a normal petunia pistil, there is no seed formation or only slight seed formation in the pollen from three lines showing cosuppression. (At most 10%, that is, the number of seeds produced per petunia strain was 10% of the control as an average of about 10 flowers). In the pollen from the three transformants showing no cosuppression, normal seed formation was confirmed as in the case of the wild type control plant.

  The above cosuppression transformants also showed abnormalities in female gametophyte formation, with female fertility reduced to 25-35% of normal individuals. In other words, the development of ovules (female gametophytes) was normal in appearance, but when pollinated with wild-type pollen, the majority of ovules could not be fertilized, and those that were fertilized also showed abnormalities in subsequent development, and many died (Abort). Also in this case, normal female gametophyte formation was observed in the transformant showing no cosuppression, as in the case of the wild type control plant.

(Example 4-2)
When the ZPT3-1 cDNA was introduced under the control of the 35S promoter, the same phenotypic changes as in the case of introducing the ZPT2-5 gene appeared in 3 of 15 individuals (FIG. 9). That is, in these transformants, the same abnormality as in the case of ZPT2-5 was observed during the meiosis. The number of cells that developed until the microspore stage was very small, and the remaining microspores showed morphological abnormalities (hexahedral). In addition, mature pollen grains lost their fertility. However, unlike ZPT2-5, these individuals had no effect on female fertility.

  As a result of gene expression analysis by Northern blotting under the same conditions as in Example 4-1, suppression of expression of both ZPT3-1 and ZPT4-1 genes was observed in the individuals into which the ZPT3-1 gene was introduced. . Both genes are highly structurally similar. That is, the homology of the base sequence of the entire coding region is 37%, the second ZF region of ZPT3-1 and the third ZF region of ZPT4-1, and the third ZF region of ZPT3-1 and ZPT4- When the 4th ZF region of 1 is compared at the base sequence level including the neighboring sequences so that the homology value is maximized, the average value of the homology is 86% (sequence comparison). (According to Clustal V program). Therefore, there is a high possibility that the above-mentioned expression suppression event is caused by the suppression of expression (cosuppression) of two genes by introduction of one gene. This suggests that the functions of these two genes are duplicated, and is consistent with the common phenotypic change observed with the introduction of both genes.

(Example 4-3)
When the cDNA of ZPT4-1 was introduced under the control of the 35S promoter, the same phenotypic changes as when the ZPT2-5 gene was introduced appeared in 2 of 13 individuals. That is, in these transformants, the same abnormality as in the case of ZPT2-5 was observed during the meiosis. The number of cells that developed until the microspore stage was very small, and the remaining microspores showed morphological abnormalities (hexahedral). Moreover, mature pollen grains almost lost their fertility. However, as with ZPT3-1, these individuals had no effect on female fertility. For the reasons described above, also in this example, there is a high possibility that expression suppression (cosuppression) of both genes of ZPT3-1 and ZPT4-1 has occurred.

  As described above, introduction of a gene encoding ZPT2-5, ZPT3-1, or ZPT4-1 can inhibit pollen development and lose fertility with extremely high efficiency (99 for ZPT3-1). % Or more, 90% or more for ZPT2-5 and ZPT4-1). In addition, the introduction of these genes is a selective trait in that the effect is specific to pollen (pollen and female gametophytes in the case of ZPT2-5) and does not affect other traits of the plant. It can be useful as a modification technique.

(Example 5: Tissue specificity of ZPT3-1 and ZPT4-1 promoter activity)
Using the transformant obtained by introducing the vector of Example 2, tissue-specific promoter activity of the DNA fragment was detected by performing histochemical staining with GUS activity. Specifically, it is as follows.

(Example 5-1)
Using a flower of a transformant obtained by introducing a fusion gene between the upstream region of the ZPT3-1 gene and GUS, the distribution of GUS activity was examined using X-GUS as a substrate (Edited by Gallagher, SR). , GUS protocols: using the GUS gene as a reporter of gene express, Academic Press, Inc., pp. 103-114, 1992). As a result, GUS activity was specifically detected in mononuclear microspores (FIGS. 10A to 10C).

(Example 5-2)
The distribution of GUS activity was examined in the same manner as described above using a flower of a transformant obtained by introducing a fusion gene between the upstream region of the ZPT4-1 gene and GUS. As a result, GUS activity was specifically observed in the microspore and the cleaved tissue from the first to second nuclei (FIGS. 10 (d) to (f); FIG. 10 (e) ) And (f) with arrows).

  As described above, the promoters of the ZPT3-1 and ZPT4-1 genes are specific for mononuclear microspores (ZPT3-1) and mononuclear to binuclear microspores (ZPT4-1), respectively. Shows activity. The promoter of the ZPT4-1 gene exhibits specific activity also in the cleaved tissue of the wing from the first to the second nuclear phase.

  Microspores are progenitor cells that mature later to produce pollen grains. Therefore, these promoters are useful as tools for detailed studies related to pollen development. In addition, these promoters or active fragments thereof can be used to directly and efficiently improve pollen development by specifically expressing cytotoxic genes, etc. in microspores, killing pollen cells or losing function. Can be controlled.

  The method of the present invention using DNAs encoding transcription factors derived from ZPT2-5, ZPT3-1 and ZPT4-1 genes, and promoters derived from ZPT3-1 and ZPT4-1 genes, It is useful as a technique for selectively modifying, particularly a technique for imparting male sterility.

It is a figure which shows the cDNA sequence of the gene which codes ZPT2-5 (this specification is also only called "ZPT2-5 gene"), and a corresponding amino acid sequence. Two zinc finger motifs and the DLNL sequence (amino acids 145-155) are underlined. It is a continuation of FIG. 1A. It is a figure which shows the cDNA sequence of the gene which codes ZPT3-1 (this specification is also only called "ZPT3-1 gene"), and a corresponding amino acid sequence. Three zinc finger motifs and the DLNL sequence (amino acids 408-417) are underlined. It is a continuation of FIG. 2A. It is a continuation of FIG. 2B. It is a figure which shows the cDNA sequence of the gene which codes ZPT4-1 (this specification is also only called "ZPT4-1 gene"), and a corresponding amino acid sequence. Four zinc finger motifs and the DLNL sequence (amino acids 438-449) are underlined. It is a continuation of FIG. 3A. It is a continuation of FIG. 3B. It is a continuation of FIG. 3C. Construction of plant expression vectors (pBIN-35S-ZPT2-5, pBIN-35S-ZPT3-1 and pBIN-35S-ZPT4-1) for expressing the cDNA sequences of ZPT2-5, ZPT3-1 and ZPT4-1 FIG. It is a figure which shows the upstream sequence of the coding region of ZPT3-1 gene. The transcription start point (position 2567) is indicated by a bold arrow, and the translation start codon (ATG) is indicated by bold underline. It is a continuation of FIG. 5A. It is a figure which shows the upstream sequence of the coding region of ZPT4-1 gene. The transcription start point (position 3503) is indicated by a bold arrow, and the translation start codon (ATG) is indicated by underlined bold. FIG. 6B is a continuation of FIG. 6A. It is the schematic which shows the structure of the plant expression vector (pBIN-ZPT3-1-GUS and pBIN-ZPT3-1-GUS) for analyzing the promoter of ZPT3-1 and ZPT4-1 gene. It is the photograph which shows the form of the living body which image | photographed the pollen of a wild type petunia, and the pollen of the petunia (transformant which raised cosuppression) which introduce | transduced pBIN-35S-ZPT2-5 (magnification is 400 times). . FIGS. 8 (a)-(d) show the pollen at different growth stages of the buds for wild-type petunia and FIGS. 8 (e)-(h) for cosuppression transformed petunias, respectively. All pollen was dye | stained by DAPI (4 ', 6-diamidino-2-phenylindylene chloride n-hydrate) by a conventional method. It is the photograph which shows the form of the living body which image | photographed the pollen of a wild type petunia, and the petunia which introduce | transduced pBIN-35S-ZPT3-1 (magnification is 700 times). FIGS. 9 (a) and (c) show the pollen at the tetramolecular and microspore stages for wild-type petunia and FIGS. 9 (b) and 9 (d), respectively, for the transformed petunia. Tetramolecular pollen was DAPI, and microspore pollen was safranin, which was stained by conventional methods. In addition, in petunia pollen into which pBIN-35S-ZPT4-1 was introduced, the same form as in FIGS. 9B and 9D was observed. It is the photograph which shows the form of the organism which image | photographed the organ of the flower which carried out the GUS dyeing | staining of the petunia which introduce | transduced pBIN-ZPT3-1-GUS and pBIN-ZPT4-1-GUS. In both cases, flowers (buds) in which the buds were in the nucleus stage were taken as subjects. 10 (a) and 10 (d) show the appearance of the bud in full scale, and FIGS. 10 (b) and 10 (e) show a cross-sectional view of the heel at a low magnification (40 times), FIG. 6 (c) and FIG. (F) shows a cross-sectional view at a high magnification of microspores (FIG. 6 (c); magnification is 700 times) or the vicinity of the cleaved tissue (FIG. 6 (f); magnification is 200 times).

Claims (1)

A method of creating a male sterile plant:
(I) DNA consisting of the sequence from position 1 to position 777 of the base sequence represented by SEQ ID NO: 1, (ii) hybridizing with DNA consisting of the base sequence of (i) under stringent conditions, and pollen A nucleic acid that is either a DNA encoding a transcription factor that controls the development of DNA, or a DNA that is a fragment of (iii) (i) or (ii), and a promoter operably linked to the nucleic acid, Providing a plant expression cassette;
Providing a plant cell having an endogenous transcription factor that controls pollen development, wherein the gene encoding the endogenous transcription factor hybridizes under stringent conditions with the nucleic acid. The process,
Introducing the expression cassette into the plant cell;
A step of regenerating a plant cell into which the expression cassette has been introduced into a plant, and the regenerated plant, wherein the expression of the endogenous transcription factor is suppressed by the expression of the nucleic acid. Selecting a body,
Including
However, the DNA of (ii) above (iv) hybridizes under stringent conditions with the DNA consisting of the sequence from the 1st position to the 1886th position of the base sequence represented by SEQ ID NO: 13, and promotes the development of pollen. Does not contain DNA encoding the transcription factor to be controlled,
Method.

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