JP2017018100A - Sterilized plant, method for producing sterile plant, and vector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sterilized plant, a method for producing the sterile plant, and a vector.SOLUTION: The present invention provides a sterile plant in which the function of the FAR gene is defected or suppressed and the function of the PLE gene is defected or suppressed. Preferably, the FAR gene and/or a promoter thereof has a modified site that results in defection or suppression of the function of the FAR gene, and the PLE gene and/or a promoter thereof has a modified site that results in defection or suppression of the function of the PLE gene.SELECTED DRAWING: None

Description

  The present invention relates to a sterile plant, a method for producing a sterile plant, and a vector.

  In order to control the cultivation of newly produced varieties and the spread of genetic information, plant sterility is desired. As an example of the sterilization method, for example, a method of sterilizing a plant by controlling formation of stamens that produce pollen or pistil that pollinates pollen has been attempted.

Torenia is a useful plant that is widely cultivated around the world for viewing.
Regarding the control of the formation of flower organs in Torenia, Non-Patent Document 1 reports that TfFARINELLI (TfFAR) and TfPLENA1 (TfPLE1) genes, which are class C genes of Torenia, have been isolated.
In Non-Patent Document 2, a transposon was inserted into the expression region of the TfFAR gene to obtain a plant with reduced expression of the TfFAR gene. In this plant, a plurality of stamens were converted into petals. Has been disclosed.

Narumi, T. et al. “Chimeric AGAMOUS repressor induces serrated petal phenotype in Torenia fournieri similar to that induced by cytokinin application” Plant Biotechnology, 2008, 25, p.45-53 Takaaki Nishijima, Tomoya Niki, “A new mutant“ Yae Saki ”that appeared in the progeny of Torenia's easily denatured strain, Sparrow”, 2013, Horticultural studies, 12 (1), p.183

Non-Patent Document 1 describes that a plant body in which the function of the TfFAR gene was suppressed was produced, but this plant body was only cut into the edge of the petal. Similarly, the function of the TfPLE gene was Although a suppressed plant was produced, there is a description that this plant was only cut at the edge of the petal. Therefore, a completely sterile plant has not been obtained.
Further, in Non-Patent Document 2, a transposon is inserted into the expression region of the TfFAR gene to obtain a plant body in which the expression of the TfFAR gene is reduced, but there is a description that this plant body can be crossed, Completely sterile plants have not been obtained.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a sterile plant, a method for producing a sterile plant, and a vector by controlling the morphogenesis of a flower organ.

  The present invention is as follows.

[1] The function of the FAR gene of any of the following (A) to (C) is deficient or suppressed,
A sterilized plant, wherein the function of the PLE gene of any one of the following (D) to (F) is deleted or suppressed.
(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function Gene (D) encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (E) In the amino acid sequence represented by SEQ ID NO: 2, one to several amino acids have been deleted, substituted and / or added The gene (F) consisting of an amino acid sequence and encoding a protein having a transcription control function is identical to the amino acid sequence represented by SEQ ID NO: 2 A gene encoding a protein having a transcription control function of [2] the function of the FAR gene is deleted or suppressed by having a modified site in the FAR gene and / or its promoter Has been
The sterilized plant according to the above [1], wherein the function of the PLE gene is deleted or suppressed by having a modification site in the PLE gene and / or its promoter.
[3] The function of the FAR gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the FAR gene.
The sterilized plant according to [1], wherein the function of the PLE gene is deficient or suppressed by introducing a nucleic acid that deficient or suppresses the expression of the PLE gene.
[4] The nucleic acid that suppresses the expression of the FAR gene is any of the following nucleic acids (g) to (i):
The sterilized plant according to [3] above, wherein the nucleic acid that suppresses the expression of the PLE gene is any one of the following (j) to (l).
(G) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(H) In the base sequence represented by SEQ ID NO: 7, a sense strand consisting of a base sequence in which one to several bases are deleted, substituted and / or added, or part thereof, and the sense strand or part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene,
(I) a sense strand consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene,
(J) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(K) In the base sequence represented by SEQ ID NO: 8, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the PLE gene,
(L) a sense strand consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof The sterilized plant according to any one of the above [1] to [4], which is a nucleic acid [5] plant of the family Aperaceae that can suppress the expression of the PLE gene.
[6] In the plant,
The function of the FAR gene of any of the following (A) to (C) is deleted or suppressed,
A method for producing a sterile plant, wherein the function of the PLE gene of any one of (D) to (F) below is deleted or suppressed.
(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function Gene (D) encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (E) In the amino acid sequence represented by SEQ ID NO: 2, one to several amino acids have been deleted, substituted and / or added The gene (F) consisting of an amino acid sequence and encoding a protein having a transcription control function is identical to the amino acid sequence represented by SEQ ID NO: 2 There consists of 80% or more amino acid sequences, and the gene encoding a protein having a transcriptional control function [7] the function of the FAR gene by modifying the FAR gene and / or its promoter, deficient or suppressed,
The method for producing a sterile plant according to the above [6], wherein the function of the PLE gene is deleted or suppressed by modifying the PLE gene and / or its promoter.
[8] The function of the FAR gene is deleted or suppressed by introducing into the plant a nucleic acid that deletes or suppresses the expression of the FAR gene.
The method for producing a sterilized plant according to [6] above, wherein the function of the PLE gene is deficient or suppressed by introducing into the plant a nucleic acid that lacks or suppresses the expression of the PLE gene.
[9] The nucleic acid that suppresses the expression of the FAR gene is any of the following nucleic acids (g) to (i):
The method for producing a sterile plant according to [8] above, wherein the nucleic acid that suppresses the expression of the PLE gene is any one of the following nucleic acids (j) to (l).
(G) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(H) In the base sequence represented by SEQ ID NO: 7, a sense strand consisting of a base sequence in which one to several bases are deleted, substituted and / or added, or part thereof, and the sense strand or part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene,
(I) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene,
(J) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(K) In the base sequence represented by SEQ ID NO: 8, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the PLE gene,
(L) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof And a method for producing a sterilized plant according to any one of [6] to [9], which is a nucleic acid [10] plant belonging to the family Aperaceae that can suppress the expression of the PLE gene.
[11] A nucleic acid that lacks or suppresses the function of the FAR gene of any of the following (A) to (C) and the function of any of the PLE genes of any of the following (D) to (F) A vector capable of expressing a nucleic acid.
(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function Gene (D) encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (E) In the amino acid sequence represented by SEQ ID NO: 2, one to several amino acids have been deleted, substituted and / or added The gene (F) consisting of an amino acid sequence and encoding a protein having a transcription control function is identical to the amino acid sequence represented by SEQ ID NO: 2 A gene encoding a protein having an amino acid sequence of 80% or more and having a transcriptional control function [12] a nucleic acid that lacks or suppresses the function of the FAR gene suppresses the expression of the FAR gene (g ) To (i) any of the nucleic acids,
The vector according to [11] above, wherein the nucleic acid that lacks or suppresses the function of the PLE gene is any one of the following nucleic acids (j) to (l) that suppresses the expression of the PLE gene.
(G) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(H) In the base sequence represented by SEQ ID NO: 7, a sense strand consisting of a base sequence in which one to several bases are deleted, substituted and / or added, or part thereof, and the sense strand or part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene,
(I) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene,
(J) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(K) In the base sequence represented by SEQ ID NO: 8, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the PLE gene,
(L) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof And a nucleic acid capable of suppressing the expression of the PLE gene [13] a nucleic acid that lacks or suppresses the function of the FAR gene to modify the FAR gene and / or its promoter (s) A nucleic acid of
The vector according to [11] above, wherein the nucleic acid lacking or suppressing the function of the PLE gene is the following nucleic acid (t) for modifying the PLE gene and / or its promoter.
(S) A guide RNA comprising a base sequence selected from the sequences of the FAR gene and / or its promoter
(T) Guide RNA containing a base sequence selected from the sequence of the PLE gene and / or its promoter

  According to the present invention, a plant excellent in sterility can be obtained.

In an Example, it is a figure which shows typically the area | region used for the silencing vector among TfFAR cDNA and TfPLE cDNA. It is a figure which shows typically the expression cassette produced in the Example. It is the map figure of the vector used in the Example. It is the map figure of the vector used in the Example. It is a photograph which shows the expression analysis result of TfFAR / TfPLE gene in an Example. It is the photograph which compared the form of the flower of the TfFAR / TfPLE-IR recombinant torenia and the control plant produced in the Example. It is the photograph which compared the form of the flower of the TfFAR / TfPLE-IR recombinant torenia and the control plant produced in the Example. It is a figure which shows typically the expression cassette produced in the Example. It is a figure which shows typically the expression cassette produced in the Example. It is the map figure of the vector used in the Example. It is the photograph which compared the morphology of the flower of a TfFAR target 1 & 2 / TfPLE target 1 & 2 genome editing torenia and a control plant produced in the Example. It is a photograph which shows the expression analysis result of TfFAR / TfPLE gene in an Example.

≪Sterile plants≫
The sterile plant of the present invention is
The function of the FAR gene of any of the following (A) to (C) is deficient or suppressed,
The function of the PLE gene in any of the following (D) to (F) is deficient or suppressed.

(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function gene

(D) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (E) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 2 A gene comprising a sequence and encoding a protein having a transcription control function (F) encoding a protein having an amino acid sequence of 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having a transcription control function gene

<FAR gene, PLE gene>
The ABC model is known as a model for explaining the flowering of angiosperms. In the ABC model, the development of flower organs is controlled by the expression pattern of class A gene, class B gene, and class C gene. In the expression region of the class A gene alone, wrinkles are formed, and in the expression region of the class A gene and the class B gene, petals are formed. A stamen is formed in the expression region of the class B gene and the class C gene, and a heart skin is formed in the expression region of the class C gene alone. As a class C gene, AGAMOUS (AG) of Arabidopsis thaliana is known.
However, the above model has been clarified in a study on some plant species such as Arabidopsis and snapdragons, and the above model can be similarly applied to all other plant species, and which gene is Whether it functions as a gene of each class is unknown.

  Also in Torenia, it has been reported that TfFAR gene and TfPLE gene, which are considered to be one of class C genes, have been isolated, and a plant body in which the function of each of these genes is suppressed has been produced. However, the observed flower morphological abnormalities were very slight.

  The inventors cloned the FAR gene and the PLE gene from Torenia fournieri as shown in the Examples described later. Furthermore, the inventors have introduced a stamen and a pistil in a plant into which a nucleic acid that suppresses the expression of the Torenia FAR gene and a nucleic acid that suppresses the expression of the Torenia PLE gene are petalized, and are completely sterile. It was found that a transformed plant body was obtained.

  The FAR gene according to the present invention is any one of the genes (A) to (C).

Generally, it is known that a protein having some physiological function can be deleted, substituted and / or added with one to several amino acids without impairing the original function.
In the protein (B), the number of amino acids from which 1 to several amino acids may be deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 is preferably 1 to 30, 1-20 are more preferable, 1-10 are more preferable, and 1-5 are especially preferable.

In the protein (C), the identity with the amino acid sequence represented by SEQ ID NO: 1 is 80% or more and less than 100%, preferably 90% or more and less than 100%, preferably 95% or more and less than 100% It is more preferable that it is 98% or more and less than 100%.
The identity between amino acid sequences can be determined using various known homology search programs. As the identity between amino acid sequences in the present invention, a value obtained by known homology search software BLASTP can be adopted. As the identity between amino acid sequences in the present invention, a value obtained by a commercially available genetic information processing software GENETYX can be adopted as shown in Examples described later.

  The PLE gene according to the present invention is any one of the genes (D) to (F).

Generally, it is known that a protein having some physiological function can be deleted, substituted and / or added with one to several amino acids without impairing the original function.
In the protein of (E), the number of amino acids from which 1 to several amino acids may be deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 2 is preferably 1 to 30, 1-20 are more preferable, 1-10 are more preferable, and 1-5 are more preferable.

In the protein (F), the identity with the amino acid sequence represented by SEQ ID NO: 2 is 80% or more and less than 100%, preferably 90% or more and less than 100%, preferably 95% or more and less than 100%. It is more preferable that it is 98% or more and less than 100%.
The identity between amino acid sequences can be determined using various known homology search programs. As the identity between amino acid sequences in the present invention, a value obtained by known homology search software BLASTP can be adopted. As the identity between amino acid sequences in the present invention, a value obtained by a commercially available genetic information processing software GENETYX can be adopted as shown in Examples described later.

The proteins (A) to (F) have a transcription control function. The transcription control function is a function of a transcription factor. Whether or not a protein has a transcription control function can be examined by a technique using a known transcription factor measurement reagent.
The proteins (B) and (C) preferably have the same transcriptional control function as the protein (A), and the proteins (E) and (F) are equivalent to the protein (D). It is preferable to have a transcription control function.

The protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in (A) may be a protein consisting only of the amino acid sequence, and any sequence may be included in the protein consisting of the amino acid sequence as long as it has a transcriptional control function. May be added.
The protein consisting of the amino acid sequence represented by SEQ ID NO: 2 in (D) may be a protein consisting only of the amino acid sequence, and any sequence may be included in the protein consisting of the amino acid sequence as long as it has a transcription control function. May be added.
Examples of the arbitrary sequence include a linker sequence, a tag sequence, a modified sequence for protein stabilization, and the like.

  The protein consisting of the amino acid sequence represented by SEQ ID NO: 1 can be produced by a known method. Moreover, the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 can be produced by a known technique. For example, a polynucleotide consisting of a base sequence encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 may be synthesized by incorporating it into an expression vector and expressing it. Or you may synthesize | combine the protein which consists of an amino acid sequence represented by sequence number 1 with a polypeptide synthesizer.

The FAR gene according to the present invention encodes the FAR protein according to the present invention, and is any one of the following polynucleotides (a) to (c). Here, the gene is a concept including an intron and an exon. The FAR gene has a promoter region in the second intron located 3 ′ of the MADS box domain, which is a DNA binding domain.
(A) a polynucleotide comprising a base sequence encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) one to several amino acids in the amino acid sequence represented by SEQ ID NO: 1 are deleted, substituted and / or Or a polynucleotide consisting of an added amino acid sequence and a nucleotide sequence encoding a protein having a transcription control function (c) a transcription control function consisting of an amino acid sequence of 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 A polynucleotide comprising a base sequence encoding a protein having

The PLE gene according to the present invention encodes the PLE protein according to the present invention, and is a polynucleotide of any one of the following (d) to (f). Here, the gene is a concept including an intron and an exon. The PLE gene is said to have a promoter region in the second intron located 3 ′ of the MADS box domain, which is a DNA binding domain.
(D) a polynucleotide comprising a nucleotide sequence encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (e) deletion, substitution and / or deletion of one to several amino acids in the amino acid sequence represented by SEQ ID NO: 2 Or a polynucleotide consisting of an added amino acid sequence and a nucleotide sequence encoding a protein having a transcription control function (f) an amino acid sequence having an identity of 80% or more of the amino acid sequence represented by SEQ ID NO: 2 and a transcription control function A polynucleotide comprising a base sequence encoding a protein having

  The proteins in the polynucleotides shown in (a) to (f) are the same as the proteins described in the genes shown in (A) to (F).

The base sequence encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in (a) may be the base sequence represented by SEQ ID NO: 3, or the base sequence represented by SEQ ID NO: 40 There may be.
The base sequence encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 in (d) above may be the base sequence represented by SEQ ID NO: 4, or the base sequence represented by SEQ ID NO: 41 There may be.

  The polynucleotide according to the present invention can be produced by a known method. The DNA encoding the FAR protein according to the present invention can be obtained, for example, by PCR using the obtained cDNA library as a template and a primer designed based on the nucleotide sequence of SEQ ID NO: 3, etc. It can be amplified and obtained by such as.

In the FAR gene and PLE gene according to the present invention, “the gene function is deficient or suppressed” means that the activity of the gene product is deficient or suppressed by controlling the gene or its expression. Alternatively, it means that the expression of the gene is deficient or suppressed.
For example, a plant body in which the generation of a functional gene product is deficient or suppressed may be obtained by artificially modifying or mutating the genome of a target gene. For example, there is a method obtained by screening a plant body in which the function of a target gene is deficient or suppressed by treatment with a mutagenic agent. In addition, for example, a plant whose production of a functional gene product is deficient or suppressed by artificially modifying or mutating the genome of a target gene by gene targeting method, genome editing technology, transgenic, etc. You may get The target genes are the FAR gene and the PLE gene according to the present invention.
Examples of genome editing techniques include the CRISPR-Cas system, zinc finger nuclease, TALEN, and other methods that can modify the base sequence of DNA.
Alternatively, a plant body in which generation of a gene product is deficient or suppressed may be obtained by artificially introducing a nucleic acid that deficiently or suppresses the expression of a target gene.
In the sterilized plant of the present invention, for each of the FAR gene and the PLE gene of the present invention, the degree of deficiency or suppression of the activity of the gene product, or the degree of deficiency or suppression of expression of the gene, It is sufficient that the sterilization is achieved, and it is preferable that the complete sterilization of the plant body is achieved.
In the sterile plant of the present invention, for each of the FAR gene and the PLE gene according to the present invention, a site where loss or suppression of the activity of the gene product, or a site where loss or suppression of expression of the gene occurs, It is sufficient that the sterility of the plant body is achieved, and it is preferable that the sterility of the plant body is achieved. For example, plant sterility or complete sterility may be achieved by specifically deleting or suppressing the activity of a gene product in a flower.

In the present invention and the present specification, “sterility” means that some or all of the stamens and / or pistil fertility are lost among the floral organs of a plant individual. Female sterility is the loss of fertility of some or all of the pistil among the floral organs of a plant individual. Male sterility is the loss of fertility of some or all of the stamens among the floral organs of a plant individual. Complete sterility is the loss of all stamens and pistil fertility of the floral organs of a plant individual.
The stamens are sterilized because the stamens are not formed normally and no pollen is produced, the stamens are replaced by other organs and no pollen is produced, and pollen is produced but fertile. The thing by not having, etc. are mentioned. Similarly, the sterility of the pistil is due to the fact that the pistil is not formed normally, due to the replacement of the pistil with other organs, due to the formation of the pistil but no fertility. And the like.
The stamen's sterility may be examined by morphological observation, such as stamens being replaced by other organs such as petals, and it is easy to determine whether seeds are formed by crossing with normal individuals' pistil. You can investigate. The sterility of the pistil may be examined by morphological observation, such as the replacement of pistil by other organs such as petals, and it is easy to determine whether seeds are formed by crossing with normal male stamens. You can investigate.

  In the present invention, the sterilized plant is used to include cells as long as the plant is not sterilized when the plant is produced. The cell may be in a tissue or organ state. In the present invention, the sterile plant may be a progeny or clonal organism or cell thereof. For example, as the plant body of the clone, a plant body obtained by cutting or cutting buds may be mentioned.

In the sterilized plant of the present invention, the function of the FAR gene is preferably one in which the FAR gene and / or its promoter is altered or suppressed.
In the sterilized plant of the present invention, the function of the PLE gene is preferably one in which the PLE gene and / or its promoter is altered or suppressed by modification.

The sterile plant of the present invention is
The function of the FAR gene is deleted or suppressed by having a modification site in the FAR gene and / or its promoter,
It is preferable that the function of the PLE gene is deficient or suppressed by having a modification site in the PLE gene and / or its promoter.

One embodiment of a nucleic acid for modifying a gene and / or its promoter is a nucleic acid used in the CRISPR-Cas system.
Examples of the nucleic acid for modifying the FAR gene and / or its promoter include the following nucleic acid (s).
(S) A guide RNA comprising a base sequence selected from the sequences of the FAR gene and / or its promoter
Examples of the nucleic acid for modifying the PLE gene and / or its promoter include the following nucleic acid (t).
(T) Guide RNA containing a base sequence selected from the sequence of the PLE gene and / or its promoter

  Guide RNA is guide RNA in the CRISPR-Cas system. The sequence to be linked as a part of the guide RNA can be designed in consideration of the presence of the PAM sequence, CRISPR-Cas nucleic acid cleavage activity, and the like.

  In the nucleic acid of (s), the sequence of the FAR gene and / or its promoter linked as part of the guide RNA is, for example, one or more sequences as appropriate based on the sequence of the FAR gene and / or its promoter. It is possible to design, and one or two or more arrangements can be used as appropriate. When two or more sequences are used, they can be used in appropriate combination. The sequence may be, for example, a sequence of 17 to 24 bases in the sequence of the FAR gene represented by SEQ ID NO: 40, and the base sequence represented by SEQ ID NO: 26 and / or SEQ ID NO: 29 is preferable. It can be illustrated as a thing. As long as the FAR gene and / or its promoter can be modified, 1 to several bases in the base sequence represented by SEQ ID NO: 26 or 29 instead of the base sequence represented by SEQ ID NO: 26 or 29 May consist of a base sequence deleted, substituted and / or added.

  In the nucleic acid of (t), the sequence of the PLE gene and / or its promoter linked as part of the guide RNA is appropriately selected from one or more sequences based on the sequence of the PLE gene and / or its promoter, for example. It is possible to design, and one or two or more arrangements can be used as appropriate. When two or more sequences are used, they can be used in appropriate combination. The sequence may be, for example, a sequence of 17 to 24 bases in the sequence of the PLE gene represented by SEQ ID NO: 41, and the base sequence represented by SEQ ID NO: 32 and / or SEQ ID NO: 35 is preferable. It can be illustrated as a thing. As long as the PLE gene and / or its promoter can be modified, 1 to several bases in the base sequence represented by SEQ ID NO: 32 or 35 instead of the base sequence represented by SEQ ID NO: 32 or 35 May consist of a base sequence deleted, substituted and / or added.

Here, two or more sequences that can be appropriately designed as a part of the guide RNA may be 2 to 4 per gene and its promoter, and two are preferable.
In the base sequences represented by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, and SEQ ID NO: 35, the 1 to several may be 1 to 3, or 1 to 2, One may be sufficient.

The sterile plant of the present invention is preferably one in which the function loss or suppression of the FAR gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the FAR gene. .
In the sterilized plant of the present invention, it is preferable that the PLE gene function deficiency or suppression is deficient or suppressed by introducing a nucleic acid that deficient or suppresses the expression of the PLE gene. .

The sterile plant of the present invention is
The function of the FAR gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the FAR gene,
It is preferable that the function of the PLE gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the PLE gene.

  Examples of nucleic acids that suppress gene expression include RNAi-inducible nucleic acids, antisense nucleic acids, and ribozymes. Examples of the RNAi-inducible nucleic acid include siRNA, miRNA, shRNA, and nucleic acids serving as precursors thereof.

  In the present invention, the term “nucleic acid” is used in a sense that encompasses not only RNA and DNA, but also nucleic acid analogs such as LNA, peptide nucleic acids (PNA), and hybrids thereof.

Examples of the antisense nucleic acid include a nucleic acid having a base sequence complementary to a sense sequence that is an mRNA sequence of a gene to be suppressed.
The base sequence represented by SEQ ID NO: 5 is the base sequence of the cDNA of the FAR gene according to the present invention. In the base sequence represented by SEQ ID NO: 5, the 759th to 1021st regions are 3′UTR regions.
An antisense nucleic acid that suppresses the expression of the FAR gene according to the present invention can be designed by those skilled in the art based on the base sequence represented by SEQ ID NO: 5.
The base sequence represented by SEQ ID NO: 6 is the base sequence of cDNA of the PLE gene according to the present invention. In the base sequence represented by SEQ ID NO: 6, the 784-999th region is a 3 ′ UTR region.
An antisense nucleic acid that suppresses the expression of the PLE gene according to the present invention can be designed by those skilled in the art based on the base sequence represented by SEQ ID NO: 6.

Ribozymes are RNAs that have catalytic activity. Ribozymes have an active site that reacts with the target nucleic acid and a substrate binding site that binds to the target nucleic acid. Examples of the ribozyme include ribozymes having a sequence complementary to the mRNA sequence of the gene to be suppressed as a substrate binding site.
A ribozyme that suppresses the expression of the FAR gene according to the present invention can be designed by those skilled in the art based on the base sequence represented by SEQ ID NO: 5. The ribozyme that suppresses the expression of the PLE gene according to the present invention can be designed by those skilled in the art based on the base sequence represented by SEQ ID NO: 6.

  Examples of RNAi-inducible nucleic acids include shRNA, siRNA, miRNA, and precursors thereof. For example, double-stranded RNA having the same base sequence or partial sequence as the mRNA sequence of the gene to be suppressed Can be given. As long as the expression of the gene to be suppressed can be suppressed, the RNAi-inducible nucleic acid may be a double-stranded RNA having the same base sequence as the mRNA sequence of the gene to be suppressed or a partial sequence thereof. Good.

One aspect of the nucleic acid that suppresses the expression of the FAR gene is any of the following nucleic acids (g) to (i).
(G) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(H) In the base sequence represented by SEQ ID NO: 7, a sense strand consisting of a base sequence in which one to several bases are deleted, substituted and / or added, or part thereof, and the sense strand or part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene,
(I) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene

One embodiment of the nucleic acid that suppresses the expression of the PLE gene includes any of the following nucleic acids (j) to (l).
(J) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(K) In the base sequence represented by SEQ ID NO: 8, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the PLE gene,
(L) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the PLE gene

Even when the antisense strand corresponding to the nucleotide sequence represented by SEQ ID NO: 7 is expressed, it becomes an antisense nucleic acid, and the expression of the FAR gene can be suppressed. By expressing a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof and an antisense strand capable of forming a duplex with the sense strand or a part thereof, two strands are expressed. Strand RNA is formed, and silencing is efficiently caused in the plant body by the formation of siRNA and the like.
Similarly, even when an antisense strand corresponding to the base sequence represented by SEQ ID NO: 8 is expressed, it becomes an antisense nucleic acid and can suppress the expression of the PLE gene. By expressing a nucleic acid containing a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof and an antisense strand capable of forming a duplex with the sense strand or a part thereof, two strands are expressed. Strand RNA is formed, and silencing is efficiently caused in the plant body by the formation of siRNA and the like.

Generally, it is known that a nucleic acid having some physiological function can be deleted, substituted and / or added with one to several base sequences without impairing the original function.
In the nucleic acid of (h) above, in the base sequence represented by SEQ ID NO: 7, the number of bases from which 1 to several bases may be deleted, substituted and / or added is preferably 1 to 30 1 to 20 is more preferable, 1 to 10 is more preferable, and 1 to 5 is more preferable.

In the base (i), the identity with the base sequence represented by SEQ ID NO: 7 is 80% or more and less than 100%, preferably 90% or more and less than 100%, preferably 95% or more and less than 100%. It is more preferable that it is 98% or more and less than 100%.
The identity between base sequences can be determined using various known homology search programs. As the identity between base sequences in the present invention, a value obtained by known homology search software BLAST can be adopted. As the identity between base sequences in the present invention, a value obtained by commercially available genetic information processing software GENETYX can be adopted as shown in the examples described later.

Generally, it is known that a nucleic acid having some physiological function can be deleted, substituted and / or added with one to several base sequences without impairing the original function.
In the nucleic acid of (k), in the base sequence represented by SEQ ID NO: 8, the number of bases from which 1 to several bases may be deleted, substituted and / or added is preferably 1 to 30 1 to 20 is more preferable, 1 to 10 is more preferable, and 1 to 5 is more preferable.

In the base (1), the identity with the base sequence represented by SEQ ID NO: 8 is 80% or more and less than 100%, preferably 90% or more and less than 100%, preferably 95% or more and less than 100%. It is more preferable that it is 98% or more and less than 100%.
The identity between base sequences can be determined using various known homology search programs. As the identity between base sequences in the present invention, a value obtained by known homology search software BLAST can be adopted. As the identity between base sequences in the present invention, a value obtained by commercially available genetic information processing software GENETYX can be adopted as shown in the examples described later.

  In the nucleic acids shown in the above (g) to (l), the antisense strand capable of forming a duplex with the sense strand or part thereof is composed of a sequence that is completely complementary to the sense strand or part thereof. Preferably, it consists of a sequence that is partially complementary to the sense strand as long as it can form a double strand with the sense strand or a part thereof in the plant and can suppress the expression of the gene to be suppressed. It may be.

In the nucleic acids shown in the above (g) to (i), a part of the sense strand consisting of the base sequence represented by SEQ ID NO: 7 refers to 19 or more consecutive strands in the base sequence represented by SEQ ID NO: 7. The thing which consists of bases can be illustrated.
In the nucleic acids shown in the above (g) to (i), the length of 19 or more consecutive bases in the base sequence represented by SEQ ID NO: 7 as a part of the sense strand is appropriately determined depending on the gene to be suppressed. For example, it may be about 19 to 493 bases, about 100 to 493 bases, about 300 to 493 bases, about 19 to 400 bases, about 20 to 100 bases, or about 21 to 50 bases.
In the nucleic acids shown in (g) to (i) above, the 19 or more consecutive bases in the base sequence represented by SEQ ID NO: 7 that is part of the sense strand are the base sequence represented by SEQ ID NO: 7. It is preferable to include a part (19 or more consecutive bases) or all of the region consisting of the 366th to 494th base sequences. The region consisting of the 366th to 494th base sequences in the base sequence represented by SEQ ID NO: 7 corresponds to the 3′UTR region of the base sequence represented by SEQ ID NO: 5.

In the nucleic acids shown in the above (j) to (l), a part of the sense strand consisting of the base sequence represented by SEQ ID NO: 8 refers to 19 or more consecutive nucleotides in the base sequence represented by SEQ ID NO: 8. The thing which consists of bases can be illustrated.
In the nucleic acids shown in the above (j) to (l), the length of 19 or more consecutive bases in the base sequence represented by SEQ ID NO: 8 that is a part of the sense sequence is appropriately determined depending on the gene to be suppressed. For example, it may be about 19 to 506 bases, about 100 to 506 bases, about 300 to 506 bases, about 19 to 400 bases, about 20 to 100 bases, or about 21 to 50 bases.
In the nucleic acids shown in the above (j) to (l), the 19 or more consecutive bases in the base sequence represented by SEQ ID NO: 8 which is a part of the sense strand are the base sequence represented by SEQ ID NO: 8. It is preferable to include a part (continuous 19 or more bases) or all of the region consisting of the 450th to 507th base sequences. The region consisting of the 450th to 507th base sequences in the base sequence represented by SEQ ID NO: 8 corresponds to the 3′UTR region of the base sequence represented by SEQ ID NO: 6.

  The nucleic acids (g) to (i) have an activity of suppressing the expression of the FAR gene. Whether or not the nucleic acids (g) to (i) have the activity of suppressing the expression of the FAR gene can be examined by a conventional method. For example, as illustrated in the examples described below, a plant into which any of the nucleic acids (g) to (i) is introduced, and a plant into which any of the nucleic acids (g) to (i) is not introduced When the expression level of the FAR gene is significantly reduced in a plant into which any of the nucleic acids of (g) to (i) is introduced, the nucleic acid of any of (g) to (i) However, it can be judged that it has the activity which suppresses expression of a FAR gene.

  The nucleic acids (j) to (l) have an activity of suppressing the expression of the PLE gene. Whether or not the nucleic acids (j) to (l) have the activity of suppressing the expression of the PLE gene can be examined by a conventional method. For example, as exemplified in the examples described below, a plant into which any of the nucleic acids (j) to (l) is introduced, and a plant into which any of the nucleic acids (j) to (l) is not introduced And the nucleic acid of any one of (j) to (l) when the expression level of the PLE gene is significantly reduced in a plant into which any of the nucleic acids of (j) to (l) has been introduced However, it can be judged that it has the activity which suppresses expression of a PLE gene.

As long as the nucleic acids shown in (g) to (i) can suppress the expression of the FAR gene, in addition to each of the sense strands or a part thereof and each of the antisense strands or a part thereof, Arbitrary nucleic acid may be added.
As long as the nucleic acids shown in (j) to (l) can suppress the expression of the PLE gene, in addition to each of the sense strands or a part thereof and each of the antisense strands or a part thereof, Arbitrary nucleic acid may be added.

The expression of the FAR gene and the PLE gene may be deleted or suppressed by introducing a nucleic acid capable of suppressing the expression of the FAR gene and the PLE gene.
One embodiment of the nucleic acid capable of suppressing the expression of the FAR gene and the PLE gene includes any of the following nucleic acids (m) to (o).
(M) a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof;
A nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand or a part thereof capable of forming a duplex with the sense strand,
(N) In the base sequence represented by SEQ ID NO: 7, a sense strand or a part thereof consisting of a base sequence in which one to several bases are deleted, substituted and / or added, and the sense strand or a part thereof An antisense strand capable of forming a duplex with
In the base sequence represented by SEQ ID NO: 8, a sense strand or a part thereof consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, and the sense strand or a part thereof and a duplex An antisense strand capable of forming a strand, and a nucleic acid capable of suppressing the expression of the FAR gene and the PLE gene,
(O) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof When,
A sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof. A nucleic acid capable of suppressing the expression of the FAR gene and the PLE gene,
Is mentioned.

  In the nucleic acids shown in the above (m) to (o), the sense strand, a part of the sense strand, and the antisense strand are the same as those described in the nucleic acids shown in the above (g) to (l). .

  As long as the nucleic acids shown in (m) to (o) can suppress the expression of the FAR gene and the PLE gene, in addition to each of the sense strands or a part thereof and each of the antisense strands, any nucleic acid may be used. The nucleic acid may be added.

As an example of the nucleic acid capable of suppressing the expression of the FAR gene and the PLE gene, any one of the following nucleic acids (p) to (r) can be mentioned.
(P) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 9 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(Q) In the base sequence represented by SEQ ID NO: 9, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene and the PLE gene,
(R) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 9 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene and the PLE gene

  Since the base sequence represented by SEQ ID NO: 9 is a concatenation of the base sequence represented by SEQ ID NO: 7 and the base sequence represented by SEQ ID NO: 8, the nucleic acids represented by the above (p) to (r) The sense strand, a part of the sense strand, and the antisense strand are the same as those described for the nucleic acids shown in the above (g) to (l).

  As long as the nucleic acids shown in (p) to (r) can suppress the expression of the FAR gene and the PLE gene, in addition to each of the sense strands or a part thereof and each of the antisense strands, any nucleic acid can be used. The nucleic acid may be added.

Examples of the nucleic acid (p) include a polynucleotide comprising the base sequence represented by SEQ ID NO: 10.
In the base sequence represented by SEQ ID NO: 10, the portion consisting of the 1st to 1001st bases is the portion of the antisense strand, the portion consisting of the 1002nd to 1920th bases is the spacer portion, The portion consisting of the base at position 2921 is the sense strand portion.
As will be described later in <Method for producing sterile plants> and Examples, by introducing a polynucleotide consisting of the base sequence represented by SEQ ID NO: 10 into a plant, expression of the FAR gene and PLE gene is performed. It can be suppressed.

  The sterilized plant of the present invention is preferably an angiosperm, and more preferably a plant of the Linderniaceae family. As a plant of the azepule family, plants classified into the genus Torenia and Torenia are preferable, and Torenia fournieri is preferable.

The sterile plant of the present invention is
The function of the FAR gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the FAR gene,
When the function of the PLE gene is one that is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the PLE gene, it is preferable that these nucleic acids are expressed at least in flowers. .
When the nucleic acid is expressed in a flower, the nucleic acid may be expressed by a transcriptional regulatory region that specifically expresses the nucleic acid in the flower, and a transcriptional regulatory region such as a constant promoter that expresses the nucleic acid in the entire plant including the flower The nucleic acid may be expressed by
An example of a constitutive promoter is the CaMV35S (cauliflower mosaic virus 35S) promoter.

≪How to make sterile plants≫
The method for producing the sterilized plant of the present invention comprises:
In the plant,
The function of the FAR gene of any of the following (A) to (C) is deleted or suppressed,
The function of the PLE gene in any of the following (D) to (F) is lost or suppressed.
(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function Gene (D) encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (E) In the amino acid sequence represented by SEQ ID NO: 2, one to several amino acids have been deleted, substituted and / or added The gene (F) consisting of an amino acid sequence and encoding a protein having a transcription control function is identical to the amino acid sequence represented by SEQ ID NO: 2 Gene but which consists of 80% or more amino acid sequences, and encodes a protein having a transcriptional control function

  Since the FAR gene and the PLE gene according to the present invention are the same as those described in the above << Sterile plants >>, description thereof is omitted.

As a method for deleting or suppressing the function of a gene, for example, by artificially modifying or mutating the genome of a target gene, a plant body in which the generation of a functional gene product is deleted or suppressed is obtained. Also good. For example, the method of screening the plant body by which the function of the target gene was deleted or suppressed by the process of a mutagen is mentioned. In addition, for example, a plant whose production of a functional gene product is deficient or suppressed by artificially modifying or mutating the genome of a target gene by gene targeting method, genome editing technology, transgenic, etc. You may get The target genes are the FAR gene and the PLE gene according to the present invention.
Or you may obtain the plant body in which the production | generation of the gene product was deleted or suppressed by introduce | transducing into the plant the nucleic acid which loses or suppresses the expression of the target gene.
In the sterilized plant of the present invention, for each of the FAR gene and the PLE gene of the present invention, the degree of deficiency or suppression of the activity of the gene product, or the degree of deficiency or suppression of expression of the gene, It is sufficient that the sterilization is achieved, and it is preferable that the complete sterilization of the plant body is achieved.

In the method for producing a sterilized plant of the present invention, the function of the FAR gene is preferably deleted or suppressed by modifying the FAR gene and / or its promoter.
In the method for producing a sterilized plant of the present invention, the function of the PLE gene is preferably deleted or suppressed by modifying the PLE gene and / or its promoter.

In the method for producing a sterile plant of the present invention, the function of the FAR gene is deleted or suppressed by modifying the FAR gene and / or its promoter,
It is preferable that the function of the PLE gene be deleted or suppressed by modifying the PLE gene and / or its promoter.

The method for producing an sterilized plant of the present invention may be a deletion or suppression of the function of the FAR gene into a plant by introducing a nucleic acid for modifying the FAR gene and / or its promoter. preferable.
The method for producing an sterilized plant of the present invention may be a method of deleting or suppressing the function of the PLE gene into a plant by introducing a nucleic acid for modifying the PLE gene and / or its promoter. preferable.

The method for producing an sterilized plant of the present invention comprises deleting or suppressing the function of the FAR gene into a plant by introducing a nucleic acid for modifying the FAR gene and / or its promoter,
The function of the PLE gene is preferably deleted or suppressed by introducing a nucleic acid for modifying the PLE gene and / or its promoter.

Since the nucleic acid for modifying the FAR gene and / or its promoter is the same as that described in the above << Sterile plant >>, description thereof is omitted.
Since the nucleic acid for modifying the PLE gene and / or its promoter is the same as that described in the above << Sterile Plant >>, description thereof is omitted.

The method for producing a sterilized plant of the present invention is preferably one in which the function of the FAR gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the FAR gene into the plant.
The method for producing a sterilized plant of the present invention is preferably one in which the function of the PLE gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the PLE gene into the plant.

The method for producing a sterilized plant of the present invention comprises deficient or suppressed the function of the FAR gene by introducing a nucleic acid that deletes or suppresses the expression of the FAR gene into the plant,
It is preferable that the function of the PLE gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the PLE gene.

The method for producing a sterilized plant of the present invention is a method for deleting or suppressing the functions of the FAR gene and the PLE gene by introducing into the plant a nucleic acid that deletes or suppresses the expression of the FAR gene and the PLE gene. It is preferable that

The nucleic acid that suppresses the expression of the FAR gene is the same as that described in the above-mentioned << sterile plants >>, and thus the description thereof is omitted.
The nucleic acid that suppresses the expression of the PLE gene is the same as that described in the above-mentioned << sterile plants >>, and thus the description thereof is omitted.
Since the nucleic acid that suppresses the expression of the FAR gene and the PLE gene is the same as that described in the above-mentioned <sterile plants>, description thereof is omitted.

  The method for producing the sterilized plant of the present invention can also be carried out by a known genetic recombination technique, and introduces the FAR gene according to the present invention and the nucleic acid capable of suppressing the expression of the PLE gene according to the present invention into the plant Examples thereof include a method of introducing into a plant a vector capable of expressing in the plant a nucleic acid capable of suppressing the expression of the FAR gene according to the present invention and the PLE gene according to the present invention. As another example, a vector capable of expressing in a plant a nucleic acid capable of suppressing the expression of the FAR gene according to the present invention, and a vector capable of expressing in a plant a nucleic acid capable of suppressing the expression of the PLE gene according to the present invention. The method of introduce | transducing into a plant is mentioned. The nucleic acid that suppresses the expression of the introduced gene may or may not be introduced on the genome, but is preferably introduced on the genome.

  Examples of plants to which a nucleic acid that suppresses gene expression is introduced include plant bodies, plant cells, plant culture cells, and callus.

  The method for producing a transformant using a vector is not particularly limited, and can be carried out by a method commonly used in this technical field. Examples of the method include an Agrobacterium method, a particle gun method, and an electroporation method, and the Agrobacterium method is preferable.

  In the present invention, the sterilized plant is not only a plant body, but if the plant body is sterilized when the plant body is produced from the cell, it is used in the sense of including the cell from which the plant is derived. Yes. The cell may be in a tissue or organ state. In the present invention, the sterile plant may be a progeny or clonal organism or cell thereof. For example, as the plant body of the clone, a plant body obtained by cutting or cutting buds may be mentioned.

Introducing a nucleic acid that suppresses the expression of the FAR gene into a plant so that the function of the FAR gene is deficient or suppressed, and the function of the PLE gene is introduced by introducing a nucleic acid that suppresses the expression of the PLE gene. These nucleic acids are preferably expressed in at least flowers.
When the nucleic acid is expressed in a flower, the nucleic acid may be expressed by a transcriptional regulatory region that specifically expresses the nucleic acid in the flower, and a transcriptional regulatory region such as a constant promoter that expresses the nucleic acid in the entire plant including the flower The nucleic acid may be expressed by
An example of a constitutive promoter is the CaMV35S (cauliflower mosaic virus 35S) promoter.
A method for expressing a nucleic acid by a transcriptional regulatory region is known, and for example, the transcriptional regulatory region is operably linked to a polynucleotide comprising a base sequence encoding the nucleic acid to be expressed.
“Operably linked” means that the transcriptional regulatory region is capable of regulating nucleic acid expression.
The “transcription regulatory region” is a sequence capable of controlling the initiation of transcription and transcription efficiency, and includes a promoter, an enhancer, and a response element.
The “transcriptional regulatory region capable of regulating transcription in a flower” means that transcription of a nucleic acid can be regulated in a cell constituting the flower. More specifically, a transcriptional regulatory region that can be transcriptionally regulated in a flower is a transcriptional regulatory region that can be transcriptionally regulated so that the nucleic acid can act on the formation of stamens and / or pistils. Are preferred, and more preferred are transcriptional regulatory regions capable of specifically producing nucleic acid expression in stamens and pistil.

  The plant produced by the method for producing an sterilized plant of the present invention is preferably an angiosperm, and more preferably a plant of the Linderniaceae family. As a plant of the azepule family, plants classified into the genus Torenia and Torenia are preferable, and Torenia fournieri is preferable.

≪Vector≫
The vector of the present invention lacks the function of a nucleic acid that deletes or suppresses the function of any of the following FAR genes (A) to (C) and the function of any of the following PLE genes of (D) to (F): Alternatively, the nucleic acid to be suppressed can be expressed.
(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function Gene (D) encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (E) In the amino acid sequence represented by SEQ ID NO: 2, one to several amino acids have been deleted, substituted and / or added The gene (F) consisting of an amino acid sequence and encoding a protein having a transcription control function is identical to the amino acid sequence represented by SEQ ID NO: 2 Gene but which consists of 80% or more amino acid sequences, and encodes a protein having a transcriptional control function

Since the nucleic acid that lacks or suppresses the function of the FAR gene is the same as that described in the above << Sterile plant >>, description thereof is omitted.
The nucleic acid that lacks or suppresses the function of the PLE gene is the same as that described in the above-mentioned <sterile plants>, and thus the description thereof is omitted.

  The vector of the present invention is preferably capable of expressing the FAR gene and / or promoter thereof and the PLE gene and / or nucleic acid for modifying the promoter.

Since the nucleic acid for modifying the FAR gene and / or its promoter is the same as that described in the above << Sterile plant >>, description thereof is omitted.
Since the nucleic acid for modifying the PLE gene and / or its promoter is the same as that described in the above << Sterile Plant >>, description thereof is omitted.

  The vector of the present invention is preferably capable of expressing a nucleic acid that suppresses the expression of the FAR gene and the PLE gene.

The nucleic acid that is deficient or suppresses the expression of the FAR gene is the same as that described in the above-mentioned <sterile plants>, and thus the description thereof is omitted.
The nucleic acid that is deficient or suppresses the expression of the PLE gene is the same as that described in the above-mentioned <sterile plants>, and thus the description thereof is omitted.
Since the nucleic acid that deletes or suppresses the expression of the FAR gene and the PLE gene is the same as that described in the above << Sterile plants >>, description thereof is omitted.

In the vector of the present invention,
The nucleic acid that suppresses the expression of the FAR gene is any one of the following nucleic acids (g) to (i):
The nucleic acid that suppresses the expression of the PLE gene is preferably any of the following nucleic acids (j) to (l).
(G) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(H) In the base sequence represented by SEQ ID NO: 7, a sense strand consisting of a base sequence in which one to several bases are deleted, substituted and / or added, or part thereof, and the sense strand or part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene,
(I) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene,
(J) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(K) In the base sequence represented by SEQ ID NO: 8, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the PLE gene,
(L) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the PLE gene

In the vector of the present invention,
The nucleic acid deficient or suppresses the function of the FAR gene is the following nucleic acid (s) for modifying the FAR gene and / or its promoter:
The nucleic acid deficient or suppressed in the function of the PLE gene is preferably the following nucleic acid (t) for modifying the PLE gene and / or its promoter.
(S) A guide RNA comprising a base sequence selected from the sequences of the FAR gene and / or its promoter
(T) Guide RNA containing a base sequence selected from the sequence of the PLE gene and / or its promoter

  Examples of the vector include DNA having a promoter sequence, DNA that suppresses expression of the FAR gene, DNA that suppresses expression of the PLE gene, and a vector that includes DNA having a terminator sequence. A vector containing the DNA is preferred. The vector of the present invention has a sequence such as a drug resistance sequence in addition to the nucleic acid that lacks or suppresses the expression of the FAR gene according to the present invention and the nucleic acid that lacks or suppresses the expression of the PLE gene according to the present invention. May be.

  The vector of the present invention incorporates a nucleic acid that deletes or suppresses the expression of the FAR gene according to the present invention and a nucleic acid that deletes or suppresses the expression of the PLE gene according to the present invention into the vector using a known genetic recombination technique. Can be manufactured. When incorporating the nucleic acid, a commercially available expression vector preparation kit may be used.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

1. Isolation of genes First, cDNA was obtained by RT-PCR from total RNA extracted from male and female pupae of Torenia fournieri.
Using the primer: 5′-ATGGAGATTCAAAGCGATCAATC-3 ′ (SEQ ID NO: 11) and the primer: 5′-TATAATATAGCAGCAATCGAAGG-3 ′ (SEQ ID NO: 12), the sequence is amplified from the cDNA, and the base represented by SEQ ID NO: 5 A polynucleotide containing the sequence was obtained.
Using the primer: 5′-ATGGATTTTCCTAATGATGAATCCG-3 ′ (SEQ ID NO: 13) and the primer: 5′-GTTTTCTTCTAGACACTTAATAACG-3 ′ (SEQ ID NO: 14), the sequence was amplified from the cDNA, and the base represented by SEQ ID NO: 6 Obtained polynucleotide containing sequence

  An open reading frame was estimated from the obtained sequence containing the nucleotide sequence represented by SEQ ID NO: 5, and these were converted into an amino acid sequence to obtain the amino acid sequence represented by SEQ ID NO: 1. The gene encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is the FAR gene according to the present invention.

  An open reading frame was estimated from the obtained sequence containing the base sequence represented by SEQ ID NO: 6, and these were converted into an amino acid sequence to obtain the amino acid sequence represented by SEQ ID NO: 2. The gene encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is the PLE gene according to the present invention.

  When identity was calculated using GENETYX (Ver. 8), the identity between the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2 was 63%. In addition, in the nucleotide sequence, the identity was calculated using GENETYX (Ver. 8). As a result, the homology in the sequence portion used for silencing was found to be TfFAR (the nucleotide sequence represented by SEQ ID NO: 7) and TfPLE. The homology with (base sequence represented by SEQ ID NO: 8) was 66%. Including the whole not used for silencing, the homology between TfFAR (base sequence represented by SEQ ID NO: 3) and TfPLE (base sequence represented by SEQ ID NO: 4) was 69%.

2-1. Production of Torenia transformant As shown in FIG. 1, it is represented by SEQ ID NO: 7 which is a partial sequence of SEQ ID NO: 5 based on SEQ ID NO: 5 (TfFAR cDNA sequence) obtained above so as not to include MADS domain. The base sequence of 494b was selected.
As shown in FIG. 1, based on SEQ ID NO: 6 (TfPLE cDNA sequence) obtained above, the base sequence of 507b represented by SEQ ID NO: 8, which is a partial sequence of SEQ ID NO: 6, so as not to include the MADS domain Was elected.

  Downstream of the 35S promoter, DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 8 (partial sequence of the antisense strand of TfPLE) and a base complementary to the base sequence represented by SEQ ID NO: 7 DNA comprising the sequence (partial sequence of the antisense strand of TfFAR), DNA comprising the base sequence represented by SEQ ID NO: 15 as a spacer (GUS gene sequence), and DNA comprising the base sequence represented by SEQ ID NO: 7 (Partial sequence of TfFAR sense strand), DNA consisting of the base sequence represented by SEQ ID NO: 8 (partial sequence of TfPLE sense strand) and HSPter are linked in this order, and represented by SEQ ID NO: 10 DNA consisting of the base sequence was obtained (FIG. 2).

  The expression cassette schematically shown in FIG. 2 is replaced with the position of the SmaI / SalI site (FIG. 3) on the vector (P35SSRDXG-HSPter), and a binary vector introduced into the pBCKK vector (FIG. 4) by LR reaction is used. Then, it was introduced into Agrobacterium, and then Agrobacterium was used to introduce it into Torenia by the Agrobacterium method to produce a transformant (TfFAR / TfPLE-IR recombinant Torenia).

2-2. Expression analysis Total RNA extracted from leaves, petals, stamens and pistil of wild-type Torenia, total extracted from leaves, petals, stamens (petalization) and pistil (petalization) of TfFAR / TfPLE-IR recombinant torenia RT-PCR was performed on each RNA. The following expression analysis of TfFAR gene and TfPLE gene [TfFAR; primer: 5′-ATGGAGATTCAAAGCGATCAATC-3 ′ (SEQ ID NO: 16), primer: 5′-GACAAGGTGAAGAGATGTCTGG-3 ′ (SEQ ID NO: 17), and TfPLE ; Primer: 5′-ATGGATTTTCCTAATGATGAATC-3 ′ (SEQ ID NO: 18) and primer: 5′-CACGAGCCGAAGGGGTGTTT-3 ′ (SEQ ID NO: 19)]. GUS gene expression analysis was performed using the following primers [5′-GTCGTCATGAAGATGCGGAC-3 ′ (SEQ ID NO: 20) and 5′-CCCTGCTGCGGTTTTTCACC-3 ′ (SEQ ID NO: 21)]. Expression analysis (positive control) of the TfACT3 gene was performed using the following primers [5′-AAATACAGTGTTTGGATCGGAGGTTC-3 ′ (SEQ ID NO: 22) and 5′-GAATAGCACACAGAGAATAGCAAACC-3 ′ (SEQ ID NO: 23)].
FIG. 5 shows the results of RT-PCR. In confirming the transgene (spacer part), GUS expression was confirmed, and the introduction of the silencing vector in the TfFAR / TfPLE-IR recombinant torenia was confirmed. In the TfFAR / TfPLE-IR recombinant torenia, it was confirmed that the expression of the TfFAR gene and the TfPLE gene was almost silenced.

2-3. FIG. 6 shows the flower morphology of the obtained transformant (TfFAR / TfPLE-IR recombinant torenia). In the transformant (TfFAR / TfPLE-IR recombinant torenia), the pistil was completely petalized and the stamen was completely petaled compared to the control torenia (wild type) flower. As a result, the transformant (TfFAR / TfPLE-IR recombinant torenia) was completely sterilized.
As described above, the torenia according to the present invention is a very useful plant having both high appreciability and complete sterility of the figure of flowers unique to Torenia (FIG. 7).

3-1. Production of Torenia transformant Based on SEQ ID NO: 5 (TfFAR cDNA sequence), primer: 5′-attgCAAGAGAAATCTCGCCGCAG-3 ′ (SEQ ID NO: 24) and primer: 5′-aaacCTGCGGCGAGATTTCTCTTG-3 ′ (SEQ ID NO: 25) After annealing, an oligonucleotide represented by TfFAR target1 (CAAGAGAAATCTCGCCGCAG, SEQ ID NO: 26) was prepared and selected. Similarly, based on SEQ ID NO: 5 (sequence of TfFAR cDNA), primer: 5′-attgCGAGAACACGACGAATCGAC-3 ′ (SEQ ID NO: 27) and primer: 5′-aaacGTCGATTCGTCGTGTTCTCG-3 ′ (SEQ ID NO: 28) are annealed, and TfFAR An oligonucleotide represented by target2 (CGAGAACACGACGAATCGAC, SEQ ID NO: 29) was prepared and selected.

  Based on SEQ ID NO: 6 (sequence of TfPLE cDNA), primer: 5′-attgCCTGAAGGAACTCAAGAACA-3 ′ (SEQ ID NO: 30) and primer: 5′-aaacTGTTCTTGAGTTCCTTCAGG-3 ′ (SEQ ID NO: 31) are annealed, and TfPLE target1 ( An oligonucleotide represented by CCTGAAGGAACTCAAGAACA, SEQ ID NO: 32) was prepared and selected. Similarly, based on SEQ ID NO: 6 (sequence of TfPLE cDNA), primer: 5′-attgGCCAGGACCAAACACCCCTT-3 ′ (SEQ ID NO: 33) and primer: 5′-aaacAAGGGGTGTTTGGTCCTGGC-3 ′ (SEQ ID NO: 34) are annealed. An oligonucleotide represented by target2 (GCCAGGACCAAACACCCCTT, SEQ ID NO: 35) was prepared and selected.

  TfFAR target1 (SEQ ID NO: 26), which is an oligonucleotide sequence based on SEQ ID NO: 5 (TfFAR cDNA sequence), is introduced at the position of the BbsI site on the pMR217_pDONR_AtU6gRNA vector (FIG. 8) to newly create a pMR217_pDONR_AtU6gRNA-TfFAR_target1 vector (FIG. 9). The base sequence represented by TfFAR target2 (SEQ ID NO: 29) was introduced at the position of the BbsI site on the pMR219_pDONR_AtU6gRNA vector (FIG. 8) to newly produce a pMR219_pDONR_AtU6gRNA-TfFAR_target2 vector (FIG. 9). Thereby, downstream of AtU6promoter, the base sequence represented by TfFAR target1 (SEQ ID NO: 26) and the sgRNA scaffold sequence were linked in this order, and the base sequence represented by TfFAR target2 (SEQ ID NO: 29) The sgRNA scaffold sequence was ligated in this order (FIG. 9).

  TfPLE target1 (SEQ ID NO: 32), which is an oligonucleotide sequence based on SEQ ID NO: 6 (TfPLE cDNA sequence), is introduced into the BbsI site on the pMR204_pDONR_AtU6gRNA vector (FIG. 8) to newly create a pMR204_pDONR_AtU6gRNA-TfPLE_target1 vector. did. The base sequence represented by TfPLE target2 (SEQ ID NO: 35) was introduced at the position of the BbsI site on the pMR205_pDONR_AtU6gRNA vector (FIG. 8) to newly produce a pMR205_pDONR_AtU6gRNA-TfPLE_target2 vector. Thereby, downstream of AtU6promoter, the base sequence represented by TfPLE target1 (SEQ ID NO: 32) and the sgRNA scaffold sequence are linked in this order, and the base sequence represented by TfPLE target2 (SEQ ID NO: 35) The sgRNA scaffold sequence was ligated in this order (FIG. 9).

  The expression cassette schematically shown in FIG. 9 was transformed into a vector in which the selection marker of pDeCas9_Kan vector (pDeCas9; Fauser et al. 2014, was changed from hygromycin to kanamycin by LR reaction. Fauser F, Schiml S, Puchta H ( 2014) Both CRISPR / Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79: 348-359) (Fig. 10) and the pDeCas9-TfFAR_target-1 & 2 / TfPLE_target-1 & 2 binary vector Produced. pDeCas9-TfFAR_target-1 & 2 / TfPLE_target-1 & 2 binary vector is introduced into Agrobacterium, then Agrobacterium is used to introduce it into Torenia by Agrobacterium method, and transformant (TfFAR target 1 & 2 / TfPLE target 1 & 2 genome) Editorial Torenia). In the transformant, it was confirmed that the base sequence of the TfFAR gene genome was shortened by about 60 bases, and the base sequence of the TfPLE gene genome was shortened by about 460 bases.

3-2. Characteristics of Torenia transformant The above-described pDeCas9-TfFAR_target-1 & 2 / TfPLE_target-1 & 2 binary vector was introduced into Torenia by the Agrobacterium method. The morphology of the resulting transformant (TfFAR target 1 & 2 / TfPLE target 1 & 2 genome editing Torenia) is shown in FIG. The transformant (TfFAR target 1 & 2 / TfPLE target 1 & 2 genome-edited torenia) has petals in the pistil that do not have the stigma necessary for fertilization, compared to the control torenia (wild-type) flowers. Was confirmed. The stamen disappeared or completely petaled. As a result, the transformant (TfFAR target 1 & 2 / TfPLE target 1 & 2 genome editing torenia) was completely sterilized.
As described above, the torenia according to the present invention was a very useful plant having both high appreciability and complete sterility of the flower-like shape of the torenia (FIG. 11).

3-3. Expression analysis RT-PCR was performed on total RNA extracted from wild-type torenia pistil and total RNA extracted from pistil (petalization) of genome-edited torenia. The following expression analysis of TfFAR gene and TfPLE gene [TfFAR; primer: 5′-TGGAGATTCAAAGCGATCAATCAAG-3 ′ (SEQ ID NO: 36), primer: 5′-aaacCAGCATCACAAAGCACAGAC-3 ′ (SEQ ID NO: 37), and TfPLE ; Primer: 5′-ATGCCCCTGAAGGAACTCAAG-3 ′ (SEQ ID NO: 38) and primer: 5′-TTGTTTGTAGCAAGCTAGTTATATC-3 ′ (SEQ ID NO: 39)]. Expression analysis (positive control) of the TfACT3 gene was performed using the following primers [5′-AAATACAGTGTTTGGATCGGAGGTTC-3 ′ (SEQ ID NO: 22) and 5′-GAATAGCACACAGAGAATAGCAAACC-3 ′ (SEQ ID NO: 23)].
FIG. 12 shows the results of RT-PCR. In the pistilized petals of genome-edited torenia, the expression of TfFAR and TfPLE genes found in wild-type torenia was not observed.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

Claims (13)

The function of the FAR gene of any of the following (A) to (C) is deficient or suppressed,
A sterilized plant, wherein the function of the PLE gene of any one of the following (D) to (F) is deleted or suppressed.
(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function Gene (D) encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (E) In the amino acid sequence represented by SEQ ID NO: 2, one to several amino acids have been deleted, substituted and / or added The gene (F) consisting of an amino acid sequence and encoding a protein having a transcription control function is identical to the amino acid sequence represented by SEQ ID NO: 2 Gene but which consists of 80% or more amino acid sequences, and encodes a protein having a transcriptional control function The function of the FAR gene is deleted or suppressed by having a modification site in the FAR gene and / or its promoter,
The sterilized plant according to claim 1, wherein the function of the PLE gene is deleted or suppressed by having a modified site in the PLE gene and / or its promoter. The function of the FAR gene is deleted or suppressed by introducing a nucleic acid that deletes or suppresses the expression of the FAR gene,
The sterilized plant according to claim 1, wherein the function of the PLE gene is deficient or suppressed by introducing a nucleic acid that deficient or suppresses the expression of the PLE gene. The nucleic acid that suppresses the expression of the FAR gene is any one of the following nucleic acids (g) to (i):
The sterilized plant according to claim 3, wherein the nucleic acid that suppresses the expression of the PLE gene is any one of the following nucleic acids (j) to (l).
(G) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(H) In the base sequence represented by SEQ ID NO: 7, a sense strand consisting of a base sequence in which one to several bases are deleted, substituted and / or added, or part thereof, and the sense strand or part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene,
(I) a sense strand consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene,
(J) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(K) In the base sequence represented by SEQ ID NO: 8, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the PLE gene,
(L) a sense strand consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the PLE gene   The sterilized plant according to any one of claims 1 to 4, which is a plant belonging to the family Zetidae. In the plant,
The function of the FAR gene of any of the following (A) to (C) is deleted or suppressed,
A method for producing a sterile plant, wherein the function of the PLE gene of any one of (D) to (F) below is deleted or suppressed.
(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function Gene (D) encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (E) In the amino acid sequence represented by SEQ ID NO: 2, one to several amino acids have been deleted, substituted and / or added The gene (F) consisting of an amino acid sequence and encoding a protein having a transcription control function is identical to the amino acid sequence represented by SEQ ID NO: 2 Gene but which consists of 80% or more amino acid sequences, and encodes a protein having a transcriptional control function The function of the FAR gene is deleted or suppressed by modifying the FAR gene and / or its promoter,
The method for producing a sterile plant according to claim 6, wherein the function of the PLE gene is deleted or suppressed by modifying the PLE gene and / or its promoter. The function of the FAR gene is deleted or suppressed by introducing into the plant a nucleic acid that deletes or suppresses the expression of the FAR gene.
The method for producing a sterile plant according to claim 6, wherein the function of the PLE gene is deficient or suppressed by introducing into the plant a nucleic acid that lacks or suppresses the expression of the PLE gene. The nucleic acid that suppresses the expression of the FAR gene is any one of the following nucleic acids (g) to (i):
The method for producing a sterile plant according to claim 8, wherein the nucleic acid that suppresses the expression of the PLE gene is any one of the following nucleic acids (j) to (l).
(G) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(H) In the base sequence represented by SEQ ID NO: 7, a sense strand consisting of a base sequence in which one to several bases are deleted, substituted and / or added, or part thereof, and the sense strand or part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene,
(I) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene,
(J) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(K) In the base sequence represented by SEQ ID NO: 8, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the PLE gene,
(L) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the PLE gene   The method for producing a sterilized plant according to any one of claims 6 to 9, wherein the plant is a plant of the family Zetidae. Expression of a nucleic acid that lacks or suppresses the function of any of the following FAR genes (A) to (C) and a nucleic acid that deletes or suppresses the function of any of the following PLE genes (D) to (F) Possible vector.
(A) a gene encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (B) an amino acid in which one to several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A gene encoding a protein comprising a sequence and having a transcriptional control function (C) An amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and encoding a protein having a transcriptional control function Gene (D) encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (E) In the amino acid sequence represented by SEQ ID NO: 2, one to several amino acids have been deleted, substituted and / or added The gene (F) consisting of an amino acid sequence and encoding a protein having a transcription control function is identical to the amino acid sequence represented by SEQ ID NO: 2 Gene but which consists of 80% or more amino acid sequences, and encodes a protein having a transcriptional control function The nucleic acid that lacks or suppresses the function of the FAR gene is any of the following nucleic acids (g) to (i) that suppresses the expression of the FAR gene:
The vector according to claim 11, wherein the nucleic acid that lacks or suppresses the function of the PLE gene is any one of the following nucleic acids (j) to (l) that suppresses the expression of the PLE gene.
(G) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(H) In the base sequence represented by SEQ ID NO: 7, a sense strand consisting of a base sequence in which one to several bases are deleted, substituted and / or added, or part thereof, and the sense strand or part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the FAR gene,
(I) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 7 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the FAR gene,
(J) a nucleic acid comprising a sense strand consisting of the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a duplex with the sense strand or a part thereof,
(K) In the base sequence represented by SEQ ID NO: 8, a sense strand consisting of a base sequence in which 1 to several bases are deleted, substituted and / or added, or a part thereof, and the sense strand or a part thereof And an antisense strand capable of forming a duplex, and a nucleic acid capable of suppressing the expression of the PLE gene,
(L) a sense strand consisting of a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 8 or a part thereof, and an antisense strand capable of forming a double strand with the sense strand or a part thereof A nucleic acid capable of suppressing the expression of the PLE gene The nucleic acid deficient or suppresses the function of the FAR gene is the following nucleic acid (s) for modifying the FAR gene and / or its promoter:
The vector according to claim 11, wherein the nucleic acid deficient or suppresses the function of the PLE gene is the following nucleic acid (t) for modifying the PLE gene and / or its promoter.
(S) A guide RNA comprising a base sequence selected from the sequences of the FAR gene and / or its promoter
(T) Guide RNA containing a base sequence selected from the sequence of the PLE gene and / or its promoter