WO2019054445A1 - Transgenic plant, method for producing transgenic plant, polynucleotide, polynucleotide cluster, vector, and kit - Google Patents

Abstract

Provided is a transgenic plant that comprises a vanC gene coding a vanC protein, and includes an exogenous gene having an expression target near the following polynucleotide. Also provided is a polynucleotide having a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO. 11), agtattac (SEQ ID NO. 12), agtactat (SEQ ID NO. 13), and agtactac (SEQ ID NO. 14).

Description

Transgenic plant, method of producing transgenic plant, polynucleotide, cluster of polynucleotides, vector, and kit

The present invention relates to transgenic plants, methods of producing transgenic plants, polynucleotides, clusters of polynucleotides, vectors and kits.
Priority is claimed on Japanese Patent Application No. 2017-176009, filed September 13, 2017, the content of which is incorporated herein by reference.

Foreign sequences introduced into the genome of plants and animals often undergo DNA methylation, resulting in transcriptional repression. Particularly in the production of genetically modified (GM) plants, this phenomenon of transcriptional repression may be a hindrance to stable expression of the transgene.
As a method of releasing the transcriptional repression accompanied by DNA methylation, a method of inhibiting the function of factors necessary for DNA methylation such as DNA methylation enzyme is known. For example, there is a method of treating an organism with an inhibitor of the factor, or introducing a mutation into the factor inherent in the organism.
For example, Patent Document 1 describes inhibition of DNA methylation using Zebularine and its related compounds.

JP, 2010-248223, A

However, in the method of treating with an inhibitor of a factor necessary for DNA methylation, or the method of introducing a mutation into a factor necessary for DNA methylation, deregulation of methylation occurs in many regions of the genome, There are concerns of many side effects, such as unhealthy conditions and the occurrence of lethality.
The present invention has been made in view of the above-mentioned circumstances, and for a desired region, a transgenic plant, a method of producing a transgenic plant, a polynucleotide, and a polynucleotide capable of removing repression of transcriptional repression associated with DNA methylation. The task is to provide clusters, vectors, and kits.

As a result of intensive studies to solve the above problems, the present inventors have found that by using a VANC protein and a polynucleotide sequence that is a target of the VANC protein, transcriptional repression associated with DNA methylation is desired for a desired region. The inventors have found that deinhibition can be achieved and completed the present invention.

That is, the present invention provides a transgenic plant, a method of producing a transgenic plant, a polynucleotide, a cluster of polynucleotides, a vector, and a kit, which have the following characteristics.
(1) A transgenic plant having a VANS gene encoding a VANC protein and containing a foreign gene to be expressed in the vicinity of the polynucleotide described below.
Polynucleotide: A polynucleotide consisting of a nucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide A polynucleotide consisting of a polynucleotide sequence wherein 1 to several nucleotides are added to the sequence (2) The transgenic plant according to the above (1), wherein the polynucleotide is foreign.
(3) The transgenic plant according to (1) or (2), wherein the VANC gene is foreign.
(4) The polynucleotide according to the above, wherein two or more of the polynucleotides are assembled on a chromosome, and the number of the polynucleotides per number of base pairs (bp) on the chromosome is 2/300 bp to 30/300 bp. The transgenic plant according to any one of the above (1) to (3), which has a cluster.
(5) The transgenic plant according to any one of (1) to (4) above, wherein the VANS protein is a protein of any of the following (I) to (III):
(I) Protein Having the Amino Acid Sequence Represented by SEQ ID NO: 1 (II) The amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence in which 1 to several amino acids have been deleted, substituted or added A protein having a demethylating activity (III) a protein having a demethylating activity (6) that has an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 The transgenic plant according to any one of the above (1) to (5), which is
(7) A method for producing a transgenic plant, which comprises introducing a gene to be expressed in the vicinity of the polynucleotide described below of a plant having the VANC gene encoding the VANC protein and the polynucleotide described below.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide Polynucleotide comprising a polynucleotide sequence having 1 to several nucleotides added to the sequence (8) Including the following polynucleotide and a gene to be expressed in a plant having a VANC gene encoding a VANC protein The method for producing a transgenic plant, wherein the gene to be expressed is located in the vicinity of the polynucleotide.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide It comprises introducing into a polynucleotide (9) plant consisting of a polynucleotide sequence having 1 to several nucleotides added to the sequence, the VANC gene encoding the VANC protein, the polynucleotide described below, and the gene to be expressed. The method for producing a transgenic plant, wherein the gene to be expressed is located in the vicinity of the polynucleotide.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide Two or more of the polynucleotides are assembled on a polynucleotide (10) chromosome consisting of a polynucleotide sequence having one to several nucleotides added to the sequence, and the number of base pairs (bp) on the chromosome of the polynucleotide is The method for producing a transgenic plant according to any one of the above (7) to (9), which has a cluster of the polynucleotide, wherein the number of present is 2/300 bp to 30/300 bp.
(11) The method for producing a transgenic plant according to any one of (7) to (10) above, wherein the VANS protein is a protein of any one of the following (I) to (III):
(I) Protein Having the Amino Acid Sequence Represented by SEQ ID NO: 1 (II) The amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence in which 1 to several amino acids have been deleted, substituted or added A protein having a demethylating activity (III) a protein having an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 and a protein having a demethylating activity (12) The method for producing a transgenic plant according to any one of the above (7) to (11), which is a plant of the family Brassicaceae.
(13) A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide A polynucleotide consisting of a polynucleotide sequence wherein one to several nucleotides are added to the sequence.
(14) The poly, wherein two or more of the following polynucleotides are assembled on a chromosome, and the number of the polynucleotides per chromosome (bp) is 2/300 bp to 30/300 bp: Cluster of nucleotides.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide A polynucleotide comprising a polynucleotide sequence wherein 1 to several nucleotides are added to the sequence (15) The polynucleotide according to (13) or the cluster of polynucleotides according to (14) And a vector used for introducing the gene of the gene and the polynucleotide into a plant.
(16) The vector according to (15) above, which further comprises a VANS gene encoding a VANC protein.
(17) A kit comprising a vector containing a VANS gene encoding a VANC protein and used for introducing the VANC gene into a plant, and the vector according to (15).

According to the present invention, a transgenic plant, a method for producing a transgenic plant, a polynucleotide, a cluster of polynucleotides, a vector, and a kit capable of derepressing transcriptional repression associated with DNA methylation in a desired region are provided. it can.

It is a figure which shows the mapping result of the chromatin immunoprecipitation sequence (ChIP-seq) by the anti-VANC antibody obtained in the Example. It is the graph which displayed distribution of the RPM (Reads per million) value per copy about the read-out of IP product by anti-VANC antibody. It is a graph which shows the result of having analyzed accumulation of VANC protein, and the hypomethylation state of DNA by VANC gene introduction. It is an AGTAYTAY sequence found in the region where VANC protein is accumulated. It is a graph which shows the appearance frequency in each transposon of AGTAYTAY sequence | arrangement found in the area | region which VANC protein has accumulated. It is the figure which integrated and showed the presence region of AGTATTAY sequence, and the result of ChIP-seq in a VANC gene transfer line as a result of Bisulfite-seq. FIG. 1 shows the construction of a vector containing the VANDAL 21 sequence. After seeding the seeds of 4 lines into which the vector shown in FIG. 7 has been introduced in kanamycin medium and storing them at 4 ° C. for 1 day, they are cultivated for 12 days under long conditions of 22 ° C. is there.

<< Polynucleotide >>
The polynucleotide of the present invention consists of the polynucleotide sequence represented by any one of SEQ ID NOs: 11 to 14 shown in Table 1 below. The polynucleotide sequence is a sequence commonly found in the region where VANC proteins are accumulated, which has been revealed by ChIP-Seq, and is considered to be a region to which VANC proteins specifically bind. The polynucleotide consisting of the polynucleotide sequence represented by any one of SEQ ID NOs: 11 to 14 is the target of the binding of the VANC protein, and hence the polynucleotide consisting of any one of SEQ ID NOs: 11 to 14 below A polynucleotide may be referred to as a "target polynucleotide".

In addition, the polynucleotide of the present invention may be a polynucleotide consisting of a polynucleotide sequence in which 1 to several nucleotides are added to the polynucleotide sequence represented by any one of SEQ ID NOs: 11 to 14. Here, as the number of nucleotides which may be added, 1 to 10 can be mentioned. The number of nucleotides which may be added is preferably 1 to 5, more preferably 1 to 3, still more preferably 1 to 2, and particularly preferably 1.
The addition of nucleotides may be either 5 'or 3'. A nucleotide may be added to the 5 'side of the polynucleotide sequence represented by any one of SEQ ID NOs: 11 to 14, or a nucleotide may be added to the 3' side, and the 5 'side and the 3' side Nucleotides may be added to both.
The type of nucleotide added to the polynucleotide sequence represented by any one of SEQ ID NOs: 11 to 14 is not particularly limited, and may be any nucleotide selected from adenine, cytosine, guanine and thymine. In addition, when a nucleotide sequence consisting of a plurality of nucleotides is added, the nucleotide sequence may also be arbitrary.
The present inventors have found that thymine or cytosine is often adjacent to the 5 'side of the polynucleotide sequences represented by SEQ ID NOs: 11 to 14 in the region to which VANC protein specifically binds. Therefore, as a preferable example of “a polynucleotide consisting of a polynucleotide sequence in which 1 to several nucleotides are added to the polynucleotide sequence represented by any one of SEQ ID NOs: 11 to 14”, “SEQ ID NO: 11 to The polynucleotide consisting of the polynucleotide sequence to which thymine or cytosine is added to 5 'side of the polynucleotide sequence represented by any of 14 is mentioned. The polynucleotide sequences are shown in SEQ ID NOs: 15-22 of Table 2 below. Hereinafter, the “target polynucleotide” also includes “a polynucleotide consisting of a polynucleotide sequence in which 1 to several nucleotides are added to the polynucleotide sequence represented by any of SEQ ID NOs: 11 to 14”. There is.

The VANS protein according to the present invention is one of the proteins encoded by the gene possessed by the transposon belonging to the VANDAL family. A VANS protein according to the invention is capable of binding to said target polynucleotide. Whether or not a protein can bind to a target polynucleotide can be determined by known methods. For example, as shown in the examples described below, ChIP-Seq can be used to confirm the binding of a protein to a target polynucleotide. The VANC protein and target polynucleotide may be directly linked or indirectly linked via other factors.

The VANS protein according to the present invention is preferably capable of binding to a target polynucleotide and having a function of suppressing methylation of the polynucleotide. The function of suppressing polynucleotide methylation can also be called "demethylation action". The VANC protein is capable of suppressing the methylation of nearby polynucleotides of a target polynucleotide in DNA containing the target polynucleotide.
Here, the term "vicinity" refers to the extent to which the demethylation action of the VANC protein affects, and may include the target polynucleotide.
It is believed that the VANS protein according to the present invention binds to a target polynucleotide and suppresses the methylation of nearby polynucleotides. Therefore, the extent of demethylation by the VANC protein may be examined with respect to the region around the DNA of the target polynucleotide. The range can be confirmed by a known method. For example, as shown in the examples described later, by performing bisulfite sequencing, it is possible to confirm the state of DNA methylation in the region around the DNA of the target polynucleotide. For example, the plant A in which the expression of the VANC protein is enhanced is compared with the plant B in which the expression of the VANC protein is lower than that of the plant A. Then, with respect to the region around the DNA of the target polynucleotide, the range in which the degree of DNA methylation was reduced in plant A rather than in plant B is the range covered by the demethylation action by the VANC protein and can do.
According to the results shown in the following examples, it has been suggested that the VANC protein can suppress the methylation of DNA in the range of several kilobps surrounding it starting from the position of the end of the target polynucleotide . Therefore, as a specific example of the range in which the VANC protein can suppress the methylation of the polynucleotide, for example, it may be in the range of 1 bp to 5000 bp starting from the end position of the target polynucleotide, 1 bp to It may be in the range of 3000 bp, in the range of 1 bp to 1000 bp, or in the range of 1 bp to 500 bp.

The VANS proteins according to the present invention may be those mentioned above, but may be, for example, proteins having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. The protein having the amino acid sequence represented by SEQ ID NO: 1 is a protein encoded by the VANS gene (At2g23480) contained in VANDAL21 of Arabidopsis thaliana.
Arabidopsis thaliana VANDAL 21 is known to function as a transposon, and contains three genes, VANA, VANB and VANC. The VANA protein encoded by the VANA gene (At2g23500) is considered to function as a MURA-type transposase. The function of the VANB gene (At2g23490) is unknown. The VANC protein encoded by the VANC gene (At2g23480) has a DNA demethylating action.

Examples of the protein encoded by the VANC gene (At2g23480) or a homolog, variant or variant of the VANC gene include the following proteins (I), (II) and (III).
(I) a protein having the amino acid sequence represented by SEQ ID NO: 1, (II) an amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 A protein having a demethylating activity, (III) a protein having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having a demethylation activity.

Here, in the amino acid sequence of (II) above, the number of amino acids which may be deleted, substituted or added is preferably 1 to 30, preferably 1 to 20, and more preferably 1 to 10. , 1 to 7 are more preferable, 1 to 5 is more preferable, 1 to 3 is particularly preferable, and 1 to 2 is most preferable.
Here, the identity with the amino acid sequence of the above (III) is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more. . The identity of amino acid sequences can be determined, for example, by BLAST (Reference URL: blast. Ncbi. Nlm. Nih. Gov / Blast. Cgi). The parameters are, for example, score = 100 and wordlength = 12. Further, when analyzing an amino acid sequence by BLASTX based on BLAST, parameters are set, for example, as score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program.

The above-mentioned proteins (I), (II) and (III) can bind to a target polynucleotide and can suppress the methylation of a target polynucleotide in the vicinity of the polynucleotide.

The target polynucleotide may form a cluster of the polynucleotide in which two or more of the polynucleotides are assembled.
The cluster may be present in a number of 2/1000 bp to 30/1000 bp, or 2/500 bp to 20/500 bp per base pair [bp] of the region on the chromosome of the target polynucleotide. It may be 2/500 bp to 4/500 bp. The target polynucleotide may be on the positive strand (5 'to 3') or on the complementary strand (3 'to 5').

The target polynucleotides form clusters of the above-mentioned polynucleotides at the above ratio, whereby the methylation suppression effect by the VANC protein is more effectively exerted.

The polynucleotide of the present invention can bind to a VANC protein, and the action of the VANC protein can cause demethylation of DNA in the vicinity of the polynucleotide of the present invention. It is prevented that transcription is suppressed.
Conventionally, when demethylation of DNA is attempted by a method of treating with an inhibitor of a factor necessary for DNA methylation, or a method of introducing a mutation into a factor necessary for DNA methylation, methyl in many regions of the genome There is concern about side effects such as the unhealthy condition of the organism and the developmental lethality, etc., because
On the other hand, according to the polynucleotide of the present invention, demethylation of DNA can be specifically caused in a region near the polynucleotide. Therefore, the above-mentioned side effects are avoided, and the target gene can be stably expressed while keeping the organism healthy.

«Transgenic plants»
The transgenic plant of the present invention has a VANC gene encoding a VANC protein, and contains a foreign gene to be expressed in the vicinity of the polynucleotide described below.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence represented by any of SEQ ID NOs: 11 to 14, or a polynucleotide consisting of a polynucleotide sequence in which one to several nucleotides are added to the polynucleotide sequence

Here, as the “VNC protein”, the “polynucleotide”, and “vicinity” of the polynucleotide, those described in the above-mentioned “polynucleotide” can be mentioned.

The plant in the transgenic plant is not particularly limited, and is preferably a land plant, more preferably a seed plant, still more preferably an angiosperm. As angiosperms, for example, rice (Oryza sativa), wheat (Triticum aestivum), rice (Poaceae) plants such as corn (Zea mays); Arabidopsis thaliana (Arabidopsis thaliana), Brassica napus (Brassica)
Brassicaceae plants such as napus); Solanaceous plants such as potato (Solanum tuberosum), tomatoes (Solanum lycopersicum) etc .; Leguminous plants such as soybean (Glycine max) etc. (Fabaseae); onion (Allium cepa) And allegaceae (Alliaceae) plants, and among the above, Brassicaceae plants are preferable, and Arabidopsis thaliana is more preferable.
Transgenic plants include primary transgenic plants and their offspring or clones in which the introduction of foreign genes has been performed.

The VANC gene, the target polynucleotide, and the foreign gene are preferably present on the genome of a transgenic plant. Also, as used herein, the term "genome" is used to include all DNAs contained in the chromosome of transgenic plants.
The foreign gene to be expressed is not particularly limited, and any gene can be adopted.
Whether the gene located in the vicinity of the target polynucleotide is a foreign gene or not is determined by introducing a new gene, as compared with the sequence information of the control plant DNA such as wild type, which has not been introduced into the transgenic plant. It can be determined by examining the

In the transgenic plant of the present invention, the target polynucleotide may be an endogenous polynucleotide inherent to a plant, but is preferably a foreign polynucleotide introduced into a plant.
In the part where the target polynucleotide is newly introduced, the polynucleotide can be newly demethylated in the vicinity of the target polynucleotide by the action of the VANC protein.
Whether the target polynucleotide is a foreign polynucleotide or not is determined whether the target polynucleotide has been newly introduced by comparison with the sequence information of the control plant DNA such as wild-type which has not been introduced into the transgenic plant. It can be judged by examining.

In the transgenic plant of the present invention, the VANC gene may be an endogenous VANC gene originally possessed by a plant, but is preferably a foreign VANC gene introduced into a plant.
Whether the VANC gene is a foreign VANC gene or not is to determine whether the VANC gene has been newly introduced by comparison with the sequence information of the control plant DNA, such as wild type, which has not been introduced into the transgenic plant. It can be judged by

In wild type Arabidopsis thaliana, the target polynucleotide is present as part of a transposon belonging to the VANDAL family. On the other hand, for example, when the target polynucleotide is a foreign polynucleotide, the target polynucleotide may not be introduced as part of a transposon belonging to the VANDAL family. Similarly, when the VANS gene is a foreign VANS gene, the VANC gene may not be introduced as part of a transposon belonging to the VANDAL family.
Specifically, it is preferable not to have the VANA gene and / or the VANB gene in the vicinity of the foreign target polynucleotide, and more preferable not to have the VANA gene in the vicinity of the foreign target polynucleotide. Since the VANA gene is presumed to function as a MURA-type transposase, the absence of the VANA gene in the vicinity of the foreign target polynucleotide can prevent the transposon from being activated unintentionally.

The transgenic plant of the present invention preferably has a VANC gene encoding the VANC protein and expresses the VANC protein.
When the VANC gene is a foreign VANC gene, the introduction position of the VANC gene on the genome is not particularly limited, and it is preferable that the VANC protein is introduced so as to be expressible.

The transgenic plant of the present invention preferably has enhanced expression of the VANC gene or VANC protein. The enhanced expression of the VANC gene or the VANC protein means that the expression of the VANC gene or the VANC protein is increased as compared to a non-transgenic control plant such as a wild type of a transgenic plant. is there.

Even when the wild-type Arabidopsis thaliana has a VANC gene, such as Arabidopsis thaliana, the amount of expression of the VANC protein can be improved by newly having a foreign VANC gene. In addition, a plant that originally does not have a VANC gene can newly have the function of a VANC protein by newly having a foreign VANC gene.

The foreign gene, the target polynucleotide, and the VANC gene may be derived from a different species of organism from the transgenic plant, or may be artificially produced. Moreover, although it originates in the wild type plant of a transgenic plant, it may be newly introduce | transduced in the position different from the original position on a chromosome.

The target polynucleotide may form a cluster of the polynucleotide in which two or more of the polynucleotides are assembled. As the said cluster, what was demonstrated by said <polynucleotide> is mentioned.
The target polynucleotides form clusters of the above-mentioned polynucleotides at the above ratio, whereby the methylation suppression effect by the VANC protein is more effectively exerted.

As described above, the transgenic plant has the VANC gene encoding the VANC protein, and includes the foreign gene to be expressed in the vicinity of the target polynucleotide, thereby causing the vicinity of the polynucleotide of the present invention by the action of the VANC protein. It is prevented that the DNA of the foreign gene is methylated. Therefore, it is possible to prevent the foreign gene in the vicinity of the target polynucleotide from being transcriptionally repressed due to methylation, and the foreign gene can be stably expressed. In addition, since demethylation occurs specifically in the region near the target polynucleotide, foreign genes can be stably expressed while maintaining the transgenic plant in a healthy state.

«Method for producing transgenic plants»
In one embodiment, the method for producing a transgenic plant of the present invention comprises introducing a gene to be expressed in the vicinity of the polynucleotide below of a plant having the VANC gene encoding the VANC protein and the polynucleotide below including.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence represented by any of SEQ ID NOs: 11 to 14, or a polynucleotide consisting of a polynucleotide sequence in which one to several nucleotides are added to the polynucleotide sequence

In one embodiment, the method for producing a transgenic plant of the present invention comprises introducing the following polynucleotide and a gene to be expressed into a plant having a VANC gene encoding a VANC protein, wherein the gene to be expressed is A method of production located in the vicinity of said polynucleotide.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence represented by any of SEQ ID NOs: 11 to 14, or a polynucleotide consisting of a polynucleotide sequence in which one to several nucleotides are added to the polynucleotide sequence

The target polynucleotide and the gene to be expressed may be introduced as a construct comprising the target polynucleotide and the gene to be expressed. In the construct, the gene to be expressed is preferably located in the vicinity of the target polynucleotide.

In one embodiment, the method for producing a transgenic plant of the present invention comprises introducing into a plant a VANS gene encoding a VANC protein, the following polynucleotide, and a gene to be expressed, said gene to be expressed being: A method of production located in the vicinity of said polynucleotide.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence represented by any of SEQ ID NOs: 11 to 14, or a polynucleotide consisting of a polynucleotide sequence in which one to several nucleotides are added to the polynucleotide sequence

The VANC gene, the target polynucleotide and the gene to be expressed may be introduced as a construct comprising the VANC gene, the target polynucleotide and a gene to be expressed. In the construct, the gene to be expressed is preferably located in the vicinity of the target polynucleotide.

Here, as the “plant” in the transgenic plant, the “VANC protein”, the target polynucleotide, and “vicinity” of the target polynucleotide, those described in the aforementioned “polynucleotide” can be mentioned.
The gene to be expressed is not particularly limited, and includes the foreign gene described in the above-mentioned «Transgenic plant».

The target polynucleotide may form a cluster of the polynucleotide in which two or more of the polynucleotides are assembled. As the said cluster, what was demonstrated by said <polynucleotide> is mentioned.
The target polynucleotides form clusters of the above-mentioned polynucleotides at the above ratio, whereby the methylation suppression effect by the VANC protein is more effectively exerted.

A known method can be used as a method for introducing the above-mentioned VANC gene, target polynucleotide, and gene to be expressed into a plant. For example, the Agrobacterium method, particle gun method, electroporation method, PEG (polyethylene glycol) method and the like can be mentioned. It is preferable that the above-mentioned VANC gene, target polynucleotide and gene to be expressed are introduced into genomic DNA of plant cells.

Preferably, the transgenic plant produced expresses the VANC protein.
When the above-mentioned VANC gene is introduced into a plant, the introduction position of the VANC gene on the genome is not particularly limited, and it is preferable to introduce the VANC protein in an expressible manner.

The transgenic plants to be produced preferably have enhanced expression of the VANC gene or VANC protein. The enhanced expression of the VANC gene or the VANC protein means that the expression of the VANC gene or the VANC protein is increased as compared to a non-transgenic control plant such as a wild type of a transgenic plant. is there.
As a method of increasing the expression of the VANC gene or the VANC protein, for example, a method of linking the constitutive expression promoter to the VANC gene and expressing the VANC gene under the control of the promoter can be mentioned.

When the VANC gene encoding the VANC protein and the target polynucleotide are introduced into a plant, it is preferable not to introduce the VANA gene and / or the VANB gene in the vicinity of the foreign target polynucleotide, and in the vicinity of the foreign target polynucleotide. More preferably, no VANA gene is introduced. Since the VANA gene is presumed to function as a MURA-type transposase, by not introducing the VANA gene in the vicinity of the foreign target polynucleotide, unintentional transposon activation can be avoided.

The plant or plant material to be introduced of these VANC gene, target polynucleotide, and polynucleotide of the gene to be expressed may be any part of a plant, and a root, a stem, a leaf, a seed, Embryos, ovules, ovaries, stem tips, buds, pollen, etc. may be mentioned. Also, the polynucleotide may be introduced into cells such as cultured cells instead of plants. For example, a transgenic plant may be obtained from somatic embryos obtained by forming callus, introducing a polynucleotide, and redifferentiating it, adventitious buds, and the like.

A known method can be used as a method for confirming whether the above-mentioned VANC gene, target polynucleotide, or gene to be expressed has been introduced into a plant. For example, it may be performed by PCR method, Southern hybridization method, Northern hybridization method or the like, or may be performed using a selective marker gene. Examples of selectable marker genes include drug resistance genes and reporter genes.

According to the method of producing a transgenic plant of the present invention, the transgenic plant of the present invention can be produced. In addition, as described above in the section «Transgenic plants», the gene to be expressed can be stably expressed. In addition, since demethylation occurs specifically in the region near the target polynucleotide, the gene to be expressed can be stably expressed while keeping the transgenic plant in a healthy state.

«Vector kit»
The vector of the present invention comprises the following polynucleotides, or clusters of the following polynucleotides described above, and is used for the introduction of genes to be expressed and the polynucleotides into plants.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence represented by any of SEQ ID NOs: 11 to 14, or a polynucleotide consisting of a polynucleotide sequence in which one to several nucleotides are added to the polynucleotide sequence

The vector of the present invention may be an expression vector capable of expressing a gene to be expressed after transformation.
The insertion site of the gene to be expressed is preferably located in the vicinity of the target polynucleotide.
The vector may further comprise a VNC gene encoding a VAN protein.
In the vector of the present invention, the VANC gene is preferably placed in the vicinity of the target polynucleotide.

The vectors of the present invention may be provided as a kit.
The kit of the present invention comprises a VANS gene encoding a VANC protein, and comprises a vector used for introducing the VANC gene into a plant, and the vector of the present invention.

According to the vector of the present invention, since the polynucleotide of the present invention is contained, transcription of the gene to be expressed can be prevented from being suppressed due to methylation, and the gene to be expressed can be stably expressed. . Moreover, since demethylation by the action of the VANC protein is specific, the gene to be expressed can be stably expressed while keeping the transgenic plant in a healthy state.

EXAMPLES The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.

1. ChIP-seq for VANC transgenic lines
The VANC gene (At2g23480) was introduced into wild type Arabidopsis thaliana by a conventional method. A chromatin sample is obtained from a VANC gene-introduced line in which the VANC gene has been confirmed to be introduced, reacted with an anti-VANC antibody and subjected to immunoprecipitation (IP), and then subjected to proteinase K treatment and RNase A treatment to recover DNA. The sample (VANC + IP) was prepared. A control sample (VANC + Input) was prepared in the same manner as the preparation of the sample (VANC + IP) except that the reaction with the anti-VANC antibody was not performed.
Thereafter, sequencing was performed on both samples and the obtained sequences were mapped on the reference genome.

The results are shown in FIG. FIG. 1 shows the results of mapping of ChIP-seq with an anti-VANC antibody for a 500 kb region of Arabidopsis thaliana chromosome 2. The vertical axis of the graph indicates the number of reads of the mapped array. In FIG. 1, two VANDAL 21 regions “AT2TE19615_VANDAL21” and “AT2TE20140_VANDAL21” are shown. From the results of VANC + IP, the lead of the IP product by the anti-VANC antibody was mapped to only the VANC region.
FIG. 2 is a graph showing the distribution of RPMs (Reads per Million) values per copy for reading of IP products with anti-VANC antibodies for each VANDAL family. As shown in FIG. 2, RPM values were high only for the VANDAL 21 family.
From these results, it became clear that the VANS protein specifically accumulates in the partial region of transposon belonging to the VANDAL 21 family.

Next, for each transposon, the accumulation of VANC protein and the state of hypomethylation of DNA by VANC gene transfer were analyzed.
The results are shown in FIGS. 3 (a) to 3 (c). Methylation was compared for each context of CG site (a), CHG site (b) and CHH site (c). H represents a base other than G. The vertical axis shows the RPM value of the IP product by the anti-VANC antibody, and the horizontal axis shows the relative hypomethylation amount of the VANC transgenic line relative to the wild type. Light dots (() indicate transposons of the VANDAL 21 family, and dark dots (●) indicate VANDAL-type transposons other than the VANDAL 21 family. In all the contexts analyzed, it was revealed that hypomethylation of DNA was triggered in correlation with the accumulation of VANC protein.

The data obtained by ChIP-seq was subjected to peak detection by Model-based Analysis of ChIP-Seq data 2 (MACS2) to identify 61 regions in which VANC proteins were accumulated. From each region identified, the average value of DNA methylation every 50 bp was shown for each context for each of the upper and lower 5 kb.
The results are shown in FIGS. 3 (d) to 3 (f). VANC transgenic lines (VANC TG) and wild type (WT) were compared. For all of the CG site (d), the CHG site (e) and the CHH site (f), a marked decrease in DNA methylation of the VANC gene transfer line in the region where the VANC protein is accumulated was confirmed.

It was analyzed using the DREME (4.11.0) script whether there is a common sequence in 61 regions where the above identified Vanc proteins are accumulated. As a result, AGTAYTAY sequence (Y represents T or C) was confirmed at 60 out of 61 places (FIG. 4).

Since Y bases are present at two places among eight bases, there are four possible sequences of eight bases. Therefore, with respect to four sequences of AGTATTAT (b), AGTACTAT (c), AGTACTAC (d) and AGTATTAC (e), the appearance frequency was analyzed for each VANDAL family and for transposons other than VANDAL (FIG. 5).
As a result, AGTATTAT and AGTATTAC sequences were found to be frequently present only in the VANDAL 21 family (FIG. 5).

Bisulfite-seq was performed in a conventional manner for wild type and the above-mentioned VANC gene-introduced lines. The location of the AGTATTAY sequence and the results of ChIP-seq in the VANC transgenic line were integrated, and the regions of the third chromosome and the second chromosome were displayed in the genome browser (FIG. 6).
For bisulfite-seq, DNA methylation of wild type (WT) and VANC transgenic lines (TG +) per context of CG, CHG, CHH was shown.
For AGTATTAC and AGTATTAT sequences, the locations where the sequences are present are shown, respectively. Positive values indicate that the sequence is present on the positive strand and negative values are present on the complementary strand.
For ChIP-seq, the region to which INPUT DNA reads were mapped and the region to which IP products were mapped with anti-VANC antibody are shown, respectively.
In the region where the AGTATTAC or AGTATTAT sequence indicated by the black arrow in FIG. 6 was present, the VANc protein was accumulated, and further, hypomethylation of DNA was significantly occurred.
From these results, it was suggested that the VANS protein recognizes and binds to the AGTATTAC sequence or the region where the AGTATTAT sequence exists, and causes hypomethylation of nearby DNA.

2. Foreign Gene Transfer Using VANDAL 21 Sequence Four constructs shown in FIG. 7 were prepared as a transfer vector to Arabidopsis thaliana. First, pGreen II-0179 (Hellens, RP, Edwards, EA, Leyland, NR, Bean, S. & Mullineaux, PM Green: a versatile and flexible binary vector for Agrobacterium-mediated plant transformation. Plant Mol. Biol. 42, 819 The VANDAL21 sequence was incorporated into -32 (2000).) To create a vector containing the VANDAL 21 sequence (FIG. 7 Hiun). The nucleotide sequences of the DNA fragment incorporated into pGreenII-0179 and the VANDAL 21 sequence contained in the DNA fragment are shown in SEQ ID NOs: 2 and 4, respectively. The nucleotide sequences of VANA gene, VANB gene and VANC gene contained in the VANDAL 21 sequence are shown in SEQ ID NOs: 5, 6 and 7, respectively.
Then, the sequence of the VANB gene was replaced with the sequence of the kanamycin resistant gene NPTII (NOS promoter-NPTII gene-NOS terminator) (FIG. 7 Hiun_VANB21Δ: NPTII). The base sequence of the DNA fragment after substitution is shown in SEQ ID NO: 3. The nucleotide sequences of NPTII gene and NOS promoter and NOS terminator are shown in SEQ ID NO: 10 and SEQ ID NO: 8 and 9, respectively. When NPTII is expressed in plants, roots grow in kanamycin medium.
Then, a stop codon was introduced into the VANC gene sequence to impair the function of the VANC gene (FIG. 7 Hiun_VANB21Δ: NPTII_VANC21-endo1.1 and Hiun_VANB21Δ: NPTII_VANC21-endo2). In FIG. 7, the configuration represented by the dot pattern indicates the VANS gene, and “*” indicates the stop codon introduced into the VANC gene sequence.

Four types of vectors shown in FIG. 7 were introduced into wild type Arabidopsis thaliana by the floral dip method of infecting flowers with Agrobacterium, respectively, to prepare four lines of transformants. The seeds of the four lines were sown in kanamycin medium and stored at 4 ° C. a day, and then grown for 12 days under long-term conditions of 22 ° C., and the root length was measured. The results are shown in FIG. 8 as box and whisker plots of 10 individual strains.

As shown in FIG. 8, in the line (Hiun_VANB21Δ: NPTII) in which the VANC gene functions, root elongation was not inhibited at any kanamycin concentration. On the other hand, in the lines where the VANC gene does not function (Hiun_VANB21Δ: NPTII_VANC21-endo1.1 and Hiun_VANB21Δ: NPTII_VANC21-endo2), root elongation was inhibited at kanamycin concentrations of 50 mg / mL or more. This result indicates that the presence of functional VANS protein contributed to the stable expression of NPTII. That is, it is considered that NPTII is stably expressed by accumulation of the VANC protein in the target polynucleotide present in the peripheral region introduced together with NPTII and suppression of the methylation of the NOS promoter.

The configurations and combinations thereof in the embodiments described above are merely examples, and additions, omissions, substitutions, and other modifications of the configurations can be made without departing from the spirit of the present invention. Moreover, this invention is not limited by each embodiment, It limits only by the range of a claim (claim).

Claims (17)

A transgenic plant having a VANC gene encoding a VANC protein and containing a foreign gene to be expressed in the vicinity of the polynucleotide described below.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide Polynucleotide consisting of polynucleotide sequence wherein 1 to several nucleotides are added to the sequence The transgenic plant according to claim 1, wherein the polynucleotide is foreign. The transgenic plant according to claim 1 or 2, wherein the VANC gene is foreign. It has a cluster of the polynucleotide in which two or more of the polynucleotides are assembled on a chromosome, and the number of the polynucleotides per chromosome base pair [bp] is 2/1000 bp to 30/1000 bp. Transgenic plant according to any one of claims 1 to 3. The transgenic plant according to any one of claims 1 to 4, wherein the VANC protein is any of the following proteins (I) to (III):
(I) Protein Having the Amino Acid Sequence Represented by SEQ ID NO: 1 (II) The amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence in which 1 to several amino acids have been deleted, substituted or added And a protein having a demethylating activity (III) having an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 The transgenic plant according to any one of claims 1 to 5, which is a plant of the family Brassicaceae. A method for producing a transgenic plant, which comprises introducing a gene to be expressed in the vicinity of the following polynucleotide of a plant having a VANC gene encoding a VANC protein.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide Polynucleotide consisting of polynucleotide sequence wherein 1 to several nucleotides are added to the sequence A method for producing a transgenic plant, comprising introducing the following polynucleotide and a gene to be expressed into a plant having a VANC gene encoding a VANC protein, wherein the gene to be expressed is located in the vicinity of the polynucleotide: .
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide Polynucleotide consisting of polynucleotide sequence wherein 1 to several nucleotides are added to the sequence A method for producing a transgenic plant, comprising introducing into a plant a VANC gene encoding a VANC protein, the following polynucleotide, and a gene to be expressed, wherein the gene to be expressed is located in the vicinity of the polynucleotide.
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide Polynucleotide consisting of polynucleotide sequence wherein 1 to several nucleotides are added to the sequence It has a cluster of the polynucleotide in which two or more of the polynucleotides are assembled on a chromosome, and the number of the polynucleotides per chromosome base pair (bp) is 2/1000 bp to 30/1000 bp. A method of producing a transgenic plant according to any one of claims 7 to 9. The method for producing a transgenic plant according to any one of claims 7 to 10, wherein the VANC protein is any of the following proteins (I) to (III):
(I) Protein Having the Amino Acid Sequence Represented by SEQ ID NO: 1 (II) The amino acid sequence represented by SEQ ID NO: 1 has an amino acid sequence in which 1 to several amino acids have been deleted, substituted or added And a protein having a demethylating activity (III) having an amino acid sequence having an identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 The method for producing a transgenic plant according to any one of claims 7 to 11, which is a plant of the family Brassicaceae. a polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or A polynucleotide consisting of a polynucleotide sequence to which several nucleotides are added. A cluster of the polynucleotides, wherein two or more of the following polynucleotides are assembled on a chromosome, and the number of the polynucleotides per chromosome base pair (bp) is 2/1000 bp to 30/1000 bp: .
Polynucleotide: A polynucleotide consisting of a polynucleotide sequence selected from the group consisting of agtattat (SEQ ID NO: 11), agtattac (SEQ ID NO: 12), agtactat (SEQ ID NO: 13), and agtactac (SEQ ID NO: 14), or the polynucleotide Polynucleotide consisting of polynucleotide sequence wherein 1 to several nucleotides are added to the sequence A vector comprising the polynucleotide according to claim 13 or the cluster of polynucleotides according to claim 14, and used for introducing a gene to be expressed and the polynucleotide into a plant. The vector according to claim 15, further comprising a VANC gene encoding a VANC protein. A kit comprising a vector containing a VANC gene encoding a VANC protein and used for introducing the VANC gene into a plant, and the vector according to claim 15.

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