JP2006158402A - Method for reducing pollen fertility by using gene of tapetal layer specific zinc finger transcription factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene engineering method useful for transforming a plant represented by male sterile by utilizing a specific gene in the tissue of an anther. <P>SOLUTION: This method for producing the transformed plant is provided ty utilizing a plant expression cassette containing a promoter containing any one of the (a) a DNA having a specific base sequence (e.g. base sequence encoding ZPT3-2 derived from petunia) or (b) a DNA having a part of the sequence of (a) and exhibiting the the specific promoter activity of the tapetal layer of an anther, and a xenogenic gene bound to the promoter operably. According to the method, the plant including the promoter, and the promoter and the xenogenic gene imparts male sterility to the plant. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a gene specifically expressed in stamen cocoons and use thereof. In particular, the present invention relates to a petunia-derived zinc finger transcription factor (ZPT3-2) gene that is specifically expressed in the tapetum layer of cocoon 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, pollina control technology (pollina)
(ion 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.

The tapetum layer is a tissue that is located on the innermost side of the fold wall and surrounds spore-forming cells (pollen cells). The role of the tapet layer is important for pollen development, not only as a support for pollen cells, but also for the supply of nutrients necessary for pollen development and for the callose layer that wraps the tetrad. There are a wide variety of factors such as degradation and supply of compounds that constitute the exine layer on the surface of pollen cells. The development of wrinkles is a quasi-phase, characterized by the quaternary phase immediately after meiosis of the spore-forming cells, the release of microspores from the quaternary molecules, the expansion of pollen cells and the formation of vacuoles, It is divided into each stage of the mitotic cell division stage where differentiation into germ cells occurs and the subsequent binuclear stage. Through these steps, the wrinkles are finally cleaved and mature pollen grains are released. In the above development process, if even part of the function of the tapetum layer is impaired, in many cases, it is known that normal pollen development is inhibited and pollen fertility is lost.

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 a small region that is not exposed to the outside, such as a tapete layer, can be used, without imparting undesirable traits to the plant,
It is likely that male sterility can be achieved. Several examples of promoters specific for the tapetum layer and gene constructs for male sterility containing the same have been reported (Shivanna and Sawney, edited by Pollen).
biotechnology for crop production a
nd improvement (Cambridge University
Press) pp. 237-257, 1997). However, there is still a need for new genes that are highly tissue-specific and time-specific in expression and useful for the control of pollen fertility.

The present inventors have recently proposed PEThyZ as a novel transcription factor derived from petunia.
7 zinc fingers (ZF) including PT3-2 (hereinafter abbreviated as ZPT3-2)
) The cDNA sequence of type transcription factor was identified. And from Northern blot analysis,
It has been reported that each transcription factor is transiently expressed at different stages of epilepsy development (Kobayashi et al., Plant J., 13:57).
1, 1998). 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.

An object of the present invention is to provide a genetic engineering technique using a gene specific to a cocoon tissue, which is useful for modifying plant traits represented by male sterility.

We have identified one of the sputum-specific transcription factors previously isolated from petunia (
The gene encoding ZPT3-2) was reintroduced into petunia. As a result, it was found that when the transgene suppresses the expression of the endogenous gene, the normal development of the pollen is inhibited and the fertility of the pollen is significantly reduced. Furthermore, the inventors have
The upstream region was isolated from the T3-2 genomic gene, and the tissue specificity of the promoter activity was examined. As a result, in the tapet layer from the 4th molecular phase to the 1st nuclear phase,
It was found that promoter activity is expressed in a tissue and time specific manner. 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 1886, (ii) DNA encoding a transcription factor that hybridizes with 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 In which a gene encoding an endogenous transcription factor is hybridized with the nucleic acid under stringent conditions. Step of the expression cassette into a plant cell to step;
A step of regenerating a plant cell into which an expression cassette has been introduced into a plant body; and a regenerated plant body, wherein the expression of an endogenous transcription factor is suppressed by the expression of the nucleic acid. The process of selecting. The above method of the present invention can be used as a method for imparting male sterility to a plant.

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

In one embodiment, the nucleic acid can be ligated in the reverse direction to the promoter and transcribed in the antisense direction within the plant cell.

In one embodiment, the plant is a dicotyledonous plant. The dicotyledonous plant is preferably a solanaceous plant, more preferably a petunia plant.

In one embodiment, the expression cassette is incorporated into a plant expression vector.

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

In its second aspect, the present invention relates to a method for producing a male-sterile plant comprising the following steps: (a) from the first position of the base sequence represented by SEQ ID NO: 3 DNA having a sequence up to position 1991, or (b) (a
DN having a partial sequence of) and exhibiting promoter activity specific to the tapet layer of cocoon
Providing a plant expression cassette comprising a promoter comprising any of A and a heterologous gene operably linked to the promoter; introducing the expression cassette into a plant cell; and introducing the expression cassette A step of regenerating plant cells into plants. The above method of the present invention can be used as a method for imparting male sterility to a plant.

In one embodiment, the plant is a dicotyledonous plant. The dicotyledonous plant is preferably a solanaceous plant, more preferably a petunia plant.

In one embodiment, the expression cassette is incorporated into a plant expression vector.

According to the second aspect of the present invention, there is also provided a male sterile plant produced by any one of the methods described above.

The present invention, in its third aspect, relates to a promoter comprising the following DNA (I) or (II): (I) the first of the nucleotide sequence represented by SEQ ID NO: 3
DNA having a sequence from position 1 to position 1991; or DNA having a partial sequence of (II) (I) and exhibiting a promoter activity specific to the cocoon tapetum layer.

In its fourth aspect, the present invention relates to a plant expression cassette useful for imparting male sterility to a plant, comprising the promoter and a heterologous gene operably linked to the promoter.

DNA encoding a transcription factor derived from the ZPT3-2 gene, and ZPT3-2
The method of the present invention using a gene-derived promoter is useful as a technique for selectively modifying plant traits by genetic engineering, particularly as a technique for imparting male sterile traits.

The present invention will be described in more detail below.
(Transcription factor derived from ZPT3-2 gene)
A nucleic acid useful in the method for producing a male sterile plant, which is the first aspect of the present invention, has (i) a sequence from position 1 to position 1886 of the base sequence represented by SEQ ID NO: 1. DNA, (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) (i) or (ii) It is any of DNA that is a fragment. The nucleic acid in the present invention is preferably the DNA of (i), that is, the DNA encoding ZPT3-2, or a fragment thereof, more preferably the DNA of (i).

In the present specification, the “transcription factor” refers to m that binds to DNA in the regulatory region of a gene.
A protein that regulates RNA synthesis. Certain transcription factors are known to have highly conserved amino acid sequences called zinc finger motifs in their DNA binding domains. ZPT3-2 is a zinc finger protein of Cys2 / His2 type (EPF family), which contains three zinc finger motifs in the full-length amino acid sequence consisting of 444 amino acids, and further DLNL
It is a transcription factor containing a hydrophobic region called a sequence (Kobayashi et al., Supra). A cDNA sequence (SEQ ID NO: 1) encoding ZPT3-2 is shown in FIGS. 1A-1D along with the corresponding deduced amino acid sequence (SEQ ID NO: 2).

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 the DNA of (i) or (ii) other than the region encoding the zinc finger motif, and is at least about 40 base pairs or more, preferably about 50 base pairs or more, more preferably about 70 base pairs. More preferably, the length is about 100 base pairs or more.

In the present specification, “stringent conditions” for hybridization are a specific base sequence (for example, DNA encoding ZPT3-2 derived from petunia) and another base sequence having high homology with this (base sequence). For example, conditions sufficient to form an oligonucleotide duplex with a DNA encoding a homologue of ZPT3-2 present in plants other than petunia are contemplated. Typical examples of stringent conditions applied to the present invention include the following conditions: 1M NaC
l, 1% SDS, 10% dextran sulfate, ³² P-labeled probe DNA (1 × 10
⁷ cpm) and 50 μg / ml salmon sperm DNA in solution at 60 ° C.
After hybridization for 2 hours, washing at 60 ° C. for 30 minutes with 2 × SSC / 1% SDS is repeated twice.

In order to isolate DNA encoding ZPT3-2 and a DNA encoding a transcription factor that hybridizes under stringent conditions and controls the development of pollen in the present invention, a known transcription factor can be isolated. A degenerate primer pair corresponding to the conserved region of the amino acid sequence encoded by the gene can be used. Using this primer pair, a plant cDNA or genomic DNA as a template for PC
R is performed, and then the obtained amplified DNA fragment can be used as a probe to screen a cDNA library or a genomic library of the same plant. As an example of such a primer pair, 5'- CARGCNYTNG
GNGGNCAY-3 ′ (SEQ ID NO: 4) and 3′-RTGNCCNCCNARN
A combination with GCYTG-5 ′ (SEQ ID NO: 5), where N is inosine, R is G or A, and Y is C or T. Each of the above primer sequences corresponds to the amino acid sequence QALGGGH contained in the zinc finger motif of ZPT3-2.

Therefore, stringent hybridization conditions applied to the present invention can also be defined as PCR conditions, and in a typical example, the above degenerate primers (SEQ ID NOs: 4 and 5) can be used. The PCR reaction conditions were as follows: denaturation at 94 ° C. for 5 minutes, 94 ° C. for 30 seconds, 50 ° C. for 30 seconds,
Then, the conditions may be such that 30 minutes are repeated at 72 ° C. for 1 minute, and finally, incubation is performed at 72 ° C. for 7 minutes.

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. For example, Sambrook et al., Molec
ultra Cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory, 1989)
checking ... 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, the transcription factor "controls the development of pollen" typically means
When expression of this transcription factor is inhibited, it means that a significant change is observed in pollen morphology or function. Typically, the transcription factor in the present invention is preferably about 60% or more, more preferably about 75% or more, and further preferably about 90% 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 encoded by genes isolated and identified by screening as described above (ie, ZPT3-2 and ZPT3-2 homologs) regulate pollen development according to the disclosure herein. Can be confirmed by observing the characteristics of the pollen.

In the present invention, DNA encoding a transcription factor that controls the development of pollen or a fragment thereof can be used to suppress the expression of an endogenous gene containing a base sequence identical or homologous to the DNA in plant cells. . The target endogenous gene is also
It is a transcription factor that controls the development of pollen. 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 technology 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 ZPT3. -2 or ZPT3-2 homolog DN
It is defined as hybridizing with A under stringent conditions. Here, the definition of “stringent conditions” is the same as the conditions described above in connection with the identification of the ZPT3-2 homolog. Although not intended to limit the scope of the present invention, a plant capable of conferring male sterility by the above method is obtained by isolating a petunia from which a ZPT3-2 gene has been isolated or a gene encoding a ZPT3-2 homolog. It is desirable to be 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, since it is a fact common to most seed plants that the tapet layer exists in the innermost layer of the cocoon and plays an essential function for the development of pollen, considering this point, it is the same as ZPT3-2 or It is readily understood that transcription factors that perform similar functions can be widely present in other plants.

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 utilized, the expression cassette in the present invention is a D encoding a transcription factor.
NA or a fragment thereof is included in a form linked in the forward direction to the 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. This introduction D
The desired gene expression can be suppressed by the action of NA. Cosuppression is observed in some individuals of transformed plants and often does not occur well in other individuals. Therefore, normally, individuals whose gene expression is suppressed as intended are
Selected according to conventional 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 ZPT3-2 gene)
A promoter useful in the method for producing a plant having a modified trait, which is the second aspect of the present invention, has (a) a sequence from the first position to the 1991 position of the base sequence represented by SEQ ID NO: 3. It is a promoter comprising either DNA or DNA having a partial sequence of (b) and (a) and exhibiting promoter activity specific to the cocoon tapetum layer. The promoter in the present invention is preferably the promoter (a), that is, the promoter of the ZPT3-2 gene.

A sequence having promoter activity specific to the tapetum layer obtained by removing a sequence not essential for tissue-specific expression activity in the promoter region of the ZPT3-2 gene is within the scope of the present invention. Such a sequence can be obtained by conducting a promoter deletion experiment according to a conventional method. Briefly, ZPT3
-Deletion mutants of the promoter region of the -2 gene (for example, mutants obtained by deleting the promoter region of the ZPT3-2 gene to various lengths from the 5 'upstream side),
By measuring the tissue-specific promoter activity of the deletion mutant using a plasmid fused with an appropriate reporter gene (for example, GUS gene), a region essential for the activity can be identified.

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 the tapetum layer.

In the present invention, “showing the promoter activity specific to the tapetum layer” means that the promoter is introduced into a plant in a natural plant or as an expression cassette linked to an arbitrary structural gene. It means that the ability to direct the expression of a gene by initiating transcription is specifically shown in the tapetum layer.
Here, “specific” means that the promoter has more expression activity than all other tissues (including pollen, flower thread, style, flower head, petal, bud, etc.) of the flower of the same plant. It's expensive. 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. The degree of expression activity can be assessed by comparing the expression level of the promoter in the tapetum layer 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 provision of male sterility to a plant using the above specific promoter is achieved by controlling the promoter in a transformed plant into which any heterologous gene operably linked to the promoter in the present invention has been introduced. This can occur as a result of being specifically expressed in the tapetum layer below. 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 promoter of the present invention, for example, using a known cDNA as a probe,
It can be obtained by screening a genomic library of plants and isolating the upstream sequence of the coding region from the corresponding genomic clone. cD
As an example of NA, cPT of ZPT3-2 which is a transcription factor derived from petunia described above
NA.

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 tapetum layer-specific promoter in the present invention can be introduced into plant cells 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 the cauliflower mosaic virus (CaMV) 35S promoter (P35S),
And nopaline synthase promoter (Tnos), but is not limited thereto.

In the present invention, the “tissue-specific promoter” refers to a promoter that specifically expresses a structural gene in anther tissue containing at least the tapetum layer.
Such tissue-specific promoters include, in addition to the promoter derived from the ZPT3-2 gene in the present invention, other known promoters that exhibit expression activity specifically. Therefore, it is also within the scope of the present invention to use the natural ZPT3-2 gene expression cassette itself, including a tapete layer specific promoter and a sequence encoding a transcription factor, optionally in combination with other regulatory elements. include.

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 d
er Kop, D.M. A. Et al., Plant Mol. Biol. 39: 979
, 1999), but is not limited thereto.

As used herein, the term “expression cassette” or “plant expression cassette”
DNA encoding the transcription factor in the present invention and operably thereto (ie,
Nucleic acid sequence comprising a plant expression promoter linked so that expression of the DNA can be controlled, as well as a tapete layer specific promoter in the present invention, operably linked to it (ie in frame) A nucleic acid sequence containing a heterologous gene.

A “heterologous gene” that can be linked to the above-mentioned tapetum layer-specific promoter is Z
Endogenous genes in Petunia other than the PT3-2 gene, or endogenous genes in plants other than Petunia, or genes that are foreign to the plant (for example,
A gene derived from animals, insects, bacteria, and fungi), wherein expression of the gene product is desired in the tapetum layer. 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. As a specific example of such a gene, b
arnase gene (Beals, TP and Goldberg, RB
, Plant Cell, 9: 1527, 1997), but is not limited thereto.

“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
Examples 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 venture flower rope is
Includes Gentian eyes, Eggplant eyes, Perilla eyes, Abagoceae, Psyllium eyes, Oxaloptera eyes, Ganoderma eyes, Acne eyes, Tricholoma eyes, and Chrysanthemum eyes. 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
Species, P. 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 involving Agrobacterium, for example, the method of Nagel et al. (F
EMS Microbiol. Lett. 67: 325 (1990)). 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 the regenerated plant body, the introduced plant expression vector is functional.
This can be confirmed using techniques 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 the plant obtained by transforming with an expression vector containing DNA encoding the transcription factor is used. 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 tapetum layer according to the method of the present invention means that, for example, in the 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 ZPT3-2)
Among previously reported sputum-specific ZF genes (Kobayashi et al., Supra),
PEThyZPT3-2 (ZPT3-2) cDNA was connected downstream of the 35S promoter of cauliflower mosaic virus to prepare a plant expression vector.

Specifically, the plasmid pBI221 (CLONTECH Laborato
ries Inc. The DNA fragment containing the cauliflower mosaic virus 35S promoter (HindIII-XbaI fragment) and the DNA fragment containing the NOS terminator (SacI-EcoRI fragment) in sequence from plasmid pU
CAP (van Engelen, FA, et al., Transgenic Res
. , 4: 288, 1995) and inserted into the pUCAP.
35S was produced. On the other hand, pBluescript containing ZPT3-2 cDNA
The t vector 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 ZPT3-2 was converted into a binary vector pBINP
EcoRI and Hi of LUS (van Engelen, FA et al., Supra)
It introduced into the ndIII site. As is clear from FIG. 2, the constructed ZPT3-2 gene is a 35S promoter region (P35S; 0.9 kb) of cauliflower mosaic virus (CaMV), a polynucleotide encoding ZPT3-2 in the present invention (ZPT3-2). About 1.9 kb), and the terminator region of nopaline synthase (Tnos; 0.3 kb). Pno in Figure 2
s represents the promoter region of nopaline synthase, and NPTII represents the neomycin phosphotransferase II gene.
(Example 2: Isolation of ZPT3-2 promoter region and ligation with GUS reporter gene)
Using the ZPT3-2 cDNA as a probe, the corresponding genomic clone was isolated from the Petunia genomic DNA library, and the DNA upstream of the transcription start site
The fragment (promoter region; about 2.0 kb) was subcloned. This DNA
The fragment was linked upstream of the GUS reporter gene and cloned into a binary vector.

Specifically, the cDNA of ZPT3-2 was synthesized by the usual random prime method (Sam
Brook et al., supra) was used to produce a radiolabeled probe by labeling with [α32P] dCTP, which was used to make a petunia (Petunia hybrida var. Mitc) produced in the EMBL3 vector (Stratagene).
hell) genomic library was screened. About 2.0 kb genomic DNA fragment containing the upstream region of the gene was obtained from the obtained clone.
Subcloning into the EcoRI site of the iptSK vector (pBS-ZPT
3-2-E), the base sequence was determined (FIG. 3). Next, a linker DNA containing a SalI recognition sequence was inserted into the BglII site (base number 2006) in the upstream sequence of the ZPT3-2 gene to introduce the SalI site (base number 2016). And
The DNA fragment obtained by digestion with EcoRI and SalI was pUCCAPGUSN.
Inserted upstream of the GUS coding region of T (pUCAP-ZPT3-2-GUSN
T). As a result, in the region near the N-terminus of the ZPT3-2 gene coding region,
The frame is connected to the GUS code area. Furthermore, pUCAP-ZPT3-2
-A DNA fragment (including ZPT3-2 promoter, GUS coding region and NOS terminator) obtained by cutting GUSNT with EcoRI and HindIII was inserted into the pBINPLUS vector, and pBIN-ZPT3-2-GUS
Was obtained (FIG. 4).
(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 (CLON
TECH Laboratories Inc. Purchased from 250mg / ml)
In L medium containing 1 mg streptomycin and 50 mg / ml rifampicin,
Incubated at 0 ° C. A cell suspension was prepared according to the method of Nagel et al. (Supra), and the plasmid vector constructed in Examples 1 and 2 was introduced into the strain by electroporation.

(2) A polynucleotide encoding each fusion gene is obtained by the following method.
Introduced into petunia cells: Agrobacterium tumefaciens LBA4404 strain obtained in (1) above was transferred to YEB medium (DNA Cloning Vol. 2, p. 78, Glover DM, IRL Press, 1985). Shake culture (28 ° C., 200 rpm) was performed. The obtained culture broth was diluted 20-fold with sterilized water, and petunia (Petania hybrida var. Mitche).
ll) co-cultured with 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 two types of fusion genes, and kanamycin resistance by expression of the NPTII gene derived from pBINPLUS Transformed petunia cells were selected based on the presence or absence of. The selected cells were induced to callus by a conventional method and then re-differentiated into a plant body (Jorgensen).
R. A. Et al., Plant Mol. Biol. , 31: 957, 1996).
(Example 4: Phenotype of transformed petunia introduced with ZPT3-2 gene)
By using the transformant obtained by introducing the vector of Example 1 and measuring the expression level of ZPT3-2 and observing the form of pollen, the cDNA of ZPT3-2 was obtained.
The effects and effects on the plant by introduction were examined.

Specifically, individuals (4 individuals) in which suppression of gene expression due to co-suppression occurred were selected from transformants (15 individuals) into which ZPT3-2 cDNA was introduced under the control of the 35S promoter by Northern blot analysis. did. The conditions for Northern blot analysis are as follows: 7% SDS, 50% formamide, 5 × SSC
2% blocking reagent (Boehringer Mannheim), 50
mM sodium phosphate buffer (pH 7.0), 0.1% sodium lauryl sarcosine, 50 μg / ml yeast tRNA, and ³² P-labeled probe DNA
Hybridization in a solution containing (1 × 10 ⁷ cpm) at 68 ° C. for 16 hours, followed by washing with 2 × SSC / 0.1% SDS at 68 ° C. for 30 minutes.

In four cosuppression transformants, about 90% of pollen cells
It was observed to die before maturation. As a result of flavonol staining by a conventional method,
In the ruptured cells of these transformants, almost no flavonols that should have been contained in the exin layer on the pollen cell surface were detected (FIG. 5; irregular shape that appears pale in the photograph showing the pollen of the transformed plant). Particles are ruptured cells).

From the above observation results, in the pollen cells of the transformant in which the suppression of gene expression occurred,
It is presumed that cells that cannot tolerate osmotic pressure have ruptured due to the failure of cell wall development due to abnormal accumulation of the exin layer. There is no difference in the development of pollen cells up to the quadruple phase from normal plants. The time when the anomaly appears in the pollen coincides well with the time when the exin layer accumulates and the time when the ZPT3-2 gene is originally expressed, and supports the above mechanism of action. In a transformant in which gene expression was suppressed,
No changes were observed in traits other than pollen. In the individuals overexpressing the ZPT3-2 gene, no change was observed in either pollen or other traits.

As described above, by introducing a gene encoding ZPT3-2, the development of pollen can be inhibited with high efficiency, and fertility can be lost. This efficiency can be further increased by optimizing the promoter used for gene transfer. For example, instead of the single CaMV35S promoter used in the vector of Example 1, two CaMV35S promoters connected in series to enhance the activity (so-called E2 promoter) can be used. The introduction of the ZPT3-2 gene can be useful as a selective trait modification technique in that the effect is specific to pollen and does not affect other traits of the plant.
(Example 5: Tissue specificity of ZPT3-2 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, the distribution of GUS activity was examined using X-GUS as a substrate using a flower of a transformant obtained by introducing a fusion gene between the upstream region of the ZPT3-2 gene and GUS (Gallagher, S.R., GUS protocols: us
ing the GUS gene as a reporter of ge
ne expressin, Academic Press, Inc. , Pp.
103-114, 1992). As a result, GUS activity was detected specifically in the tapet layer of cocoon (FIG. 6). The time of expression was transient, and the peak of activity almost corresponded to the tetramolecular phase immediately after meiosis in pollen cells. Therefore, it was found that the promoter of the ZPT3-2 gene exhibits a specific activity in the cocoon tapetum layer.

As described above, the promoter of the ZPT3-2 gene exhibits an activity specific to the tapetum layer around the tetramolecular phase. As described above, the tapetum layer is a tissue that plays an essential role in the development of pollen. This promoter is therefore useful as a tool for detailed studies related to pollen development. Furthermore, using this promoter or an active fragment thereof, pollen development can be inhibited by specifically expressing a cytotoxic gene or the like and destroying the tissue of the tapetum layer or losing its function.

It is a figure which shows the cDNA sequence of the gene which codes ZPT3-2 (it is only called "ZPT3-2 gene" in this specification), and a corresponding amino acid sequence. Three zinc finger motifs and the DLNL sequence (amino acids 411-425) are underlined. It is a continuation of FIG. 1A. It is a continuation of FIG. 1B. It is a continuation of FIG. 1C. It is the schematic which shows the structure of the plant expression vector (pBIN-35S-ZPT3-2) for expressing a ZPT3-2 cDNA sequence. It is a figure which shows the upstream sequence of the coding region of ZPT3-2 gene. The transcription start point (position 1721) is indicated by a bold arrow, and the translation start codon (ATG) is indicated by underlined bold. It is the schematic which shows the structure of the plant expression vector (pBIN-ZPT3-2-GUS) for analyzing the promoter of ZPT3-2 gene. It is the photograph which shows the form of the living body which image | photographed the pollen of wild-type petunia and the pollen of petunia which introduce | transduced pBIN-35S-ZPT3-2 (magnification is 240 times). All pollen was flavonol stained. FIGS. 5 (a) to (d) show pollen at different growth stages of wild type petunia buds, and FIGS. 5 (e) to (h) show pollen at different growth stages of transformed petunia buds. Show. 5 (a) and (e) are pollen with a bud size of 5 mm, FIGS. 5 (b) and (f) are pollen with a bud size of 35 mm, and FIGS. 5 (c) and (g) are buds. Pollen at a size of 50 mm and FIGS. 5 (d) and (h) are pollen at the mature stage. It is the photograph which shows the form of the living body which image | photographed the organ of the flower which GUS dye | stained the petunia which introduce | transduced pBIN-ZPT3-2-GUS. A flower (bud) in which the bud is in the tetramolecular period was taken as a subject of photography. FIG. 6A shows the appearance of the bud at a magnification of 2.5 times, FIG. 6B shows a cross-sectional view of the heel at a low magnification (60 times), and FIG. Sectional views at a high magnification (280 times) around the tapet layer are shown.

Claims (1)

A method of creating a male sterile plant:
(I) DNA consisting of the sequence from position 1 to position 1886 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 the method.

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