JP2006246744A - Gene-transducing method, gene targeting method and method for producing transgenic plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for transducing a gene into a plant, by which gene-silencing can more efficiently be inhibited, and to provide a gene targeting method by which homologous recombination efficiency is improved. <P>SOLUTION: This method for transducing a gene comprises preparing a plant mutant in which the functional expression of one or more genes encoding a factor participating in a nucleosome-forming reaction accompanied by DNA replication is inhibited, and transducing a polynucleotide containing a foreign gene into the mutant to obtain the foreign gene-transduced mutant. The gene-targeting method comprises transducing a polynucleotide containing the target gene and/or a portion homologous to a region adjacent the target gene and a foreign gene into the above-mentioned plant mutant by the method for transducing the gene to obtain the foreign gene-transduced mutant by the homologous recombination. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a gene introduction method, a gene targeting method, and a method for producing a transgenic plant.

  There are roughly two methods for introducing foreign genes into plants. The first method is a direct / physical method in which a hole is physically made in a part of a plant cell, and a gene is introduced into the plant cell from this hole, and the second method is a gene introduction of a microorganism or virus. It is an indirect and biological method that uses the ability.

  Examples of the first method include a particle gun method (see Patent Document 1), a polyethylene glycol method (PEG method), an electroporation method, and the like. The particle gun method is a method in which a foreign gene is attached to minute metal particles, the metal particles are driven into a plant cell with an air gun, and the gene is introduced. Examples of the second method include an Agrobacterium method (see Patent Documents 2 to 6), a virus vector method (see Patent Documents 7 to 8), and the like. The Agrobacterium method is a method for introducing a gene using a mechanism for inserting a portion called a T-DNA region on a plasmid into a plant chromosome when the soil bacterium Agrobacterium tumefaciens infects a plant. The viral vector method is, for example, a method of introducing a foreign gene using a vector prepared by modifying a plant RNA virus such as tobacco mosaic virus (TMV), bromo mosaic virus (BMV), or cucumber mosaic virus (CMV). It is.

  According to these gene introduction techniques, a transgenic plant having a useful trait that is difficult to impart by cross breeding can be efficiently produced. The use of transgenic plant production technology for food plants, feed plants, ornamental plants, environmental plants, experimental plants, resource plants, etc. is important in general industries including agriculture, commerce, and industry. There is significant significance.

  The following three problems are listed as technical problems for the practical application of transgenic plants. The first problem is the low gene transfer efficiency of plants. That is, unlike the case of animal cells, there is a problem that the efficiency of introducing foreign genes into plant cells is low. In order to solve this problem, various physical and biological gene transfer methods have been developed as described above. However, there is an additional second problem in plants. That is, even if a foreign gene can be introduced into the genome of a plant, the expression of the introduced gene is reduced by so-called gene silencing. In order to solve this problem, for example, a method using a special promoter sequence (see Patent Document 9), a method for introducing an exonuclease gene (see Patent Document 10), and the like have been developed.

  The third problem for the practical application of transgenic plants is low homologous recombination efficiency. Particularly in higher plants, there is a problem that homologous recombination efficiency of the introduced foreign gene is low and gene targeting is difficult. Several conventional methods for improving homologous recombination in plants are known. For example, there is a method for improving homologous recombination efficiency by expressing a protein involved in microbial DNA replication / repair / recombination in plant cells. Specifically, RecA gene of Escherichia coli (see Patent Document 11 and Non-patent Document 1), RuvC gene, RecQ gene (see Patent Document 12), Rad52 gene of yeast (see Patent Document 13), HO endonuclease gene, It is disclosed that the homologous recombination in Arabidopsis thaliana can be improved by the expression of the I-SceI endonuclease gene, the red mold UVDE gene (see Patent Document 14) and the like. Moreover, it has been found that homologous recombination in tobacco can be increased by using a PARP inhibitor (see Non-Patent Document 2). Furthermore, a method is also disclosed that makes it possible to easily select whether gene integration is by homologous recombination or non-homologous recombination using a positive selection marker and a negative selection marker (patent). Reference 15).

Japanese Patent Publication No. 5-508316 JP 2003-274777 A Japanese Patent Laid-Open No. 9-252674 JP-A-6-209779 Japanese Unexamined Patent Publication No. 60-70080 Japanese Patent No. 1633546 JP 2005-013164 A JP 2004-065009 A Special Table 2002-533057 JP-T-2004-504839 WO97 / 08331 pamphlet JP 2001-321169 Special Table 2003-526376 Japanese Patent Laid-Open No. 2004-275012 WO2003 / 020940 pamphlet Reiss B et al. (1996) Proc Natl Acad Sci USA 93 (7): 3094-3098 Puchta H et al. (1995) Plant J. 7: 203-210

  However, even in light of these conventional technologies, the transgenic plant production technology is more efficient, and there are new technologies for gene transfer methods that can cancel gene silencing suppression and homologous recombination efficiency. New techniques for improved gene targeting methods are desired.

  Accordingly, the first object of the present invention is to provide a plant gene introduction method that is more efficient and capable of releasing suppression of gene silencing, and secondly to provide a gene targeting method with improved homologous recombination efficiency. The purpose.

  In order to achieve the first object, the gene introduction method of the present invention is a gene introduction method for introducing a foreign gene into a plant, which encodes a factor involved in a nucleosome formation reaction associated with DNA replication. Gene introduction comprising a preparatory step of preparing a plant mutant in which the expression of the above gene is suppressed, and a gene introduction step of obtaining a foreign gene-introduced mutant by introducing a polynucleotide containing the foreign gene into the mutant Is the method.

  In order to achieve the second object, the gene targeting method of the present invention is a gene targeting method for incorporating a foreign gene into a target gene locus of a plant genome by homologous recombination, wherein the target gene and / or Homologous recombination of the polynucleotide to the mutant of the plant by a polynucleotide preparation step for preparing a polynucleotide comprising a portion homologous to a region adjacent to the target gene and a foreign gene, and the gene introduction method of the present invention And a gene introduction step of obtaining an exogenous gene introduction mutant or an exogenous gene introduction plant by introducing by the above.

  According to the gene introduction method of the present invention, for example, a foreign gene-introduced transgenic plant by non-homologous recombination can be efficiently produced by introduction of a foreign gene with improved introduction efficiency by non-homologous recombination. Moreover, according to the gene introduction method of the present invention, gene silencing of a foreign gene randomly inserted into a plant genome is derepressed.

  According to the gene targeting method of the present invention, for example, a foreign gene-introduced transgenic plant can be efficiently produced by introducing a foreign gene with improved introduction efficiency by homologous recombination. In this method of producing a transgenic plant by introduction of a foreign gene by homologous recombination, the foreign gene is inserted into the target gene on the plant genome, and therefore unpredictable risk compared to randomly inserted non-homologous recombination. Can be avoided, and safety to the human body and the natural environment is improved.

  Moreover, since the mutant of the plant used in the method of the present invention is a mutant in which the gene encoding a factor relating to nucleosome formation is suppressed in its function, the transgenic plant of the present invention directly replicates and replicates DNA. Compared with the case of using other methods using mutants of factors involved in recombination and the like, it is considered that there is a low possibility that mutations are accumulated in genetic information itself. This is because the factors involved in the nucleosome formation reaction are not factors that affect the base sequence of the gene itself, but are factors involved in so-called epigenetic chromatin information such as DNA modification, histone modification, and chromatin structure. is there. Therefore, according to the present invention, safety against humans and the natural environment is further improved. For practical purposes such as food, feed, ornamental use, environmental use, experimental use, and resource use. The transgenic plant which can be used and its efficient manufacturing method can be provided.

  In addition to the mutant preparation step and the gene introduction step of obtaining a foreign gene introduction mutant, the gene introduction method of the present invention further removes suppression of functional expression of the gene in the mutant from the foreign gene introduction mutant, It is preferable to include a suppression and removal step of obtaining a foreign gene-transferred plant. The suppression removal step is preferably a step of returning the gene mutation causing the suppression of function expression to the wild type by crossing the foreign gene introduction mutant with the wild type plant.

  The one or more genes whose functional expression is suppressed in the mutant is one or more genes selected from the orthologous gene of the gene encoding three subunits of CAF-1 and the orthologous gene of the ASF1 gene It is preferable that

  In the mutant, suppression of the functional expression of the gene is preferably caused by mutation of the gene, expression of a dominant negative mutant of the gene product, or RNA interference with the gene.

  The introduction of the polynucleotide is preferably performed by means selected from electroporation, particle implantation, virus infection, and Agrobacterium infection.

  The plant is preferably a plant selected from food plants, feed plants, ornamental plants, environmental plants, experimental plants and resource plants.

  The first method for producing a transgenic plant of the present invention is a method for producing a transgenic plant comprising the step of incorporating a foreign gene into the genome of a plant by non-homologous recombination using the gene introduction method of the present invention.

  The second method for producing a transgenic plant of the present invention is a method for producing a transgenic plant comprising the step of incorporating a foreign gene into the genome of a plant by homologous recombination using the gene targeting method of the present invention.

  The first transgenic plant of the present invention is a foreign gene-introduced transgenic plant by non-homologous recombination produced by the method for producing the first transgenic plant of the present invention.

  The second transgenic plant of the present invention is a foreign gene-introduced transgenic plant by homologous recombination produced by the method for producing the second transgenic plant of the present invention.

  The mutant of the present invention is a plant mutant used in the gene introduction method of the present invention, the gene targeting method of the present invention, or the method of producing a transgenic plant of the present invention, and is used for a nucleosome formation reaction accompanying DNA replication. It is a mutant of a plant in which the expression of one or more genes encoding factors involved is suppressed.

  The one or more genes whose functional expression is suppressed in the mutant of the present invention is one or more selected from the orthologous gene of the gene encoding three subunits of CAF-1 and the orthologous gene of the ASF1 gene Preferably, the gene is

  The suppression of the functional expression of the gene of the mutant of the present invention is preferably brought about by mutation of the gene, expression of a dominant negative mutant of the gene product, or RNA interference with the gene.

  The mutant plant of the present invention is preferably a plant selected from food plants, feed plants, ornamental plants, environmental plants, laboratory plants and resource plants.

  As a specific example of the mutant of the present invention, there is a mutant of Arabidopsis thaliana in which the function expression of the ASF1a gene (accession number: AB078339) and the ASF1b gene (accession number: AB078340) is suppressed. As another example, a mutant of Arabidopsis thaliana, at least one gene of FAS1 gene (accession number: AB0272229), FAS2 gene (accession number: AB027230), and MSI1 (accession number: NM — 125208) Are mutants whose functional expression is suppressed.

  In the present invention, “factors associated with nucleosome formation reaction accompanying DNA replication” means a group of factors involved in a universal reaction in eukaryotes that rapidly convert replicated DNA into nucleosomes in the chromosomal DNA replication process of cells. Say. This group of factors is functionally linked to DNA replication devices (Shibahara and Stillman Cell 96: 575-85, 1999) and checkpoints (Takami et al. Under revision in MCB) to modify DNA, modify histones, chromatin structures, etc. Recent studies have revealed that it is also involved in the stable maintenance of information other than the base sequence of the DNA (so-called epigenetic chromatin modification information) (Kaya et al. Cell, 104, 131-142, 2001, Ono In preparation). Specific examples of the factor include histone-binding proteins such as CAF-1 and ASF1, but are not limited to these. In the future, it will be related to the nucleosome formation reaction accompanying DNA replication in cooperation with CAF-1. It includes factors that are shown to be As candidates for these factors, for example, tousled, PCNA, TopoI, TopoII, ACF and the like are expected.

  CAF-1 (chromatin assembly factor 1) protein is a protein purified from nuclear extracts of human cells using the activity of promoting nucleosome assembly of newly synthesized DNA as an index (Kaufman et al. (1995). Cell, 81, 1105-1114., Smith et al. (1989) Cell, 58, 15-25.). The CAF-1 protein is a complex composed of three subunits such as p150, p60, and p48, and is conserved in a wide variety of organisms from yeast to humans, and is also conserved in Arabidopsis (Kaya et al. Cell 104: 131-42, 2001). The orthologs of the CAF-1 protein of Arabidopsis are called FAS1 (accession number: AB027229), FAS2 (accession number: AB027230), and MSI1 (accession number: NM — 125208), respectively.

  The ASF1 (anti-silencing function 1) protein was a yeast gene product expressed in the late S phase, and was identified as an overexpression that caused derepression of position-dependent gene silencing, Subsequently, it was re-identified as a histone H3 and H4 binding protein having the activity of promoting the nucleosome formation reaction accompanying replication by CAF-1 (Tyler et al. Nature 402; 555-560, 1999). The ASF1 protein interacts with the p60 subunit of CAF-1. The ASF1 protein, like CAF-1, is conserved from yeast to human. However, the ASF1 ortholog of Arabidopsis thaliana has two genes, AtASF1a (accession number: AB078339) and AtASF1b (accession number: AB078340), due to gene duplication. (In the present invention, they are referred to as ASF1a and ASF1b, respectively). The ASF1a and ASF1b genes, which are Arabidopsis ASF1 orthologous genes, are genes that the present inventors have isolated for the first time.

  Various studies have been conducted on the factors involved in the nucleosome formation reaction associated with these DNA replications, and their functions are gradually becoming apparent. However, if the function of these factors is inhibited, the gene transfer frequency increases, gene silencing is released, and in addition, the homologous recombination frequency increases. It has not been done.

  In the present invention, “functional expression suppression” means that the functional expression of the gene and gene product is inhibited by mutation of the gene sequence, transcription inhibition, translation inhibition, protein activation inhibition, etc. Including. “Functional expression suppression” in the present invention also refers to a gene originally present in a plant genome, and is different from gene silencing of a foreign gene introduced into a plant. A method for suppressing the functional expression of a specific gene is not particularly limited, and a conventionally known method can be applied. For example, by introducing mutations such as substitution / deletion / addition (insertion) into the gene sequence to inhibit complete protein synthesis, by inhibiting transcription promoters or activating transcriptional repressors, Examples thereof include a method of inhibiting gene transcription, a method of inhibiting protein synthesis using RNA interference, and a method of overexpressing a dominant negative mutant of the gene product. Examples of methods for introducing mutations into the gene sequence include preparing a transgenic plant library in which T-DNA or transposon is inserted into a host plant, or screening an existing mutant library for the purpose. Examples thereof include a method of screening for a mutant in which the T-DNA or transposon is inserted at the gene locus. Examples of the RNA interference method include methods using antisense RNA, short inhibitory RNA (siRNA), micro RNA (miRNA) and the like. A “variant whose function expression is suppressed” refers to a plant individual, plant tissue or plant cell in which the function expression of the gene is stably or temporarily suppressed by the above-described method.

  In the present invention, the “foreign gene” is not particularly limited and refers to an arbitrary gene to be introduced into a host plant mutant. The foreign gene can include a gene for transforming the host plant, a promoter and a terminator operably linked in the host plant cell, and the like. The foreign gene may be a selectable marker gene or a selectable marker gene expression cassette. In the present invention, “gene” refers to any fragment of DNA associated with biological functions. For example, a gene includes a coding sequence necessary for gene expression, an expression regulatory sequence, and the like.

  In the present invention, the term “polynucleotide containing a foreign gene” is not particularly limited. For example, a vector in which the foreign gene is incorporated, for example, a conventionally known viral vector, Ti plasmid, binary vector, or the like is used. Can be prepared. The polynucleotide may contain a selection marker as appropriate, separately from the foreign gene. The nucleotide may be a foreign gene itself.

  In the present invention, “gene targeting” refers to introducing a foreign gene into a target gene locus on the genome of a plant via homologous recombination.

  In the present invention, a “transgenic plant” refers to a plant cell, plant tissue, or plant individual that retains a foreign gene integrated into the plant genome by gene transfer. The integration of the foreign gene may be random or by homologous recombination, but preferably a transgenic plant into which the foreign gene has been integrated by homologous recombination. This is because safety to the human body and the environment is improved.

  In the present invention, “orthologous gene” refers to a corresponding gene in a different species. An orthologous gene or protein is also simply referred to as an ortholog. Orthologs in different species corresponding to a certain protein can be determined using, for example, homology with the amino acid sequence or base sequence of the protein as an index. That is, the gene showing the highest homology among the genes of the different species can be determined as an orthologous gene. Here, homology refers to identity or similarity between two or more sequences.

In the present invention, that two polynucleotide sequences are “homologous” means that they have homology to such an extent that homologous recombination can occur in plant cells. Further, the phrase “homologous” in the polynucleotide sequence of a gene or the amino acid sequence of a protein means that the gene sequence is sufficiently similar to maintain its function. For example, even if there are differences due to point mutations, deletions or additions in the gene base sequence, they can be said to be homologous if they do not affect the function of the gene. The number of different bases is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1. Also, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% identity in two or more sequences These are said to be homologous. When two polynucleotides hybridize under stringent conditions, they can be said to be homologous. The stringent conditions are not particularly limited. For example, in a solution containing 6 × SSC, 0.5% SDS, 5 × Denhardt, 0.01% denatured salmon sperm nucleic acid, a temperature of [Tm−25 ° C.] And conditions for keeping the temperature overnight. The Tm is obtained by the following formula, for example.
Tm = 81.5-16.6 (log 10 [Na ⁺ ]) + 0.41 (% G + C) − (600 / N)
In the above formula, N is the chain length of the oligonucleotide probe, and% G + C is the content of guanine and cytosine residues in the oligonucleotide probe primer.

  In the present invention, identity and similarity refer to a relationship determined by comparing these sequences between two or more base sequences or amino acid sequences. Whereas the identity indicates the ratio of the same character string when aligned, the similarity includes not only the same character but also a similarly substituted character. Identity and similarity can be easily determined by various computer programs known in the art.

  In the present invention, the “plant” is not particularly limited, and for example, a plant used for food, feed, ornamental use, environment use, experiment use and resource use can be used. The host plant is, for example, a seed plant, and may be a gymnosperm or an angiosperm. The host plant may be a monocotyledonous plant or a dicotyledonous plant. Examples of plants for food and feed include, for example, monocotyledonous plants such as wheat, barley, sorghum, millet, rye, triticale, corn, rice, oats, sugar cane, rapeseed, rapeseed, Arabidopsis, cabbage or canola Legumes such as soybean, alfalfa, peas, kidney beans or peanuts, potatoes, tobacco, tomatoes, eggplants such as eggplant or pepper, asteraceae such as sunflower, lettuce or calendula, cucurbitaceae such as melon, pumpkin or zucchini, And dicotyledonous plants such as celery family such as carrot. Ornamental plants include roses such as roses, rhododendrons such as rhododendrons and azaleas, euphorbiaceae such as drosophila and croton, radishs such as redwood, solanaceae such as petunias, genus genus such as African violets, , Such as orchidaceae such as orchid, orchidaceae such as gladiolus, iris, freesia, crocus, asteraceae such as marigold, auraceae such as geranium, lilyaceae such as dracaena, mulberry such as figs. Examples of environmental and resource plants include various trees, and examples of experimental plants include Arabidopsis thaliana. In the present invention, the “plant” includes plant cells such as seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts and the like in addition to individual plants.

  The plant mutant of the present invention can be used as the mutant in the plant mutant preparation step of the gene introduction method of the present invention. The method for producing the plant mutant of the present invention is not particularly limited, and for example, it can be produced using a method capable of suppressing the functional expression of the aforementioned gene.

  The gene introduction process of the gene introduction method of the present invention can be roughly divided into two stages. First, a first step of introducing a foreign gene into the mutant and a second step of selecting a host mutant cell into which the foreign gene has been introduced and redifferentiating it into an individual. The gene introduction means in the first step is not particularly limited, and a conventionally known method, for example, direct / physical means such as a particle gun method or indirect / biological means such as an Agrobacterium method is used. Can do. Once a mutant cell into which a foreign gene has been introduced is obtained, a plant mutant can be regenerated from the cell in the next second stage, and offspring can be obtained by sexual reproduction or asexual reproduction. . In addition, when the suppression of gene function expression in the mutant is temporary, the mutant naturally returns to the wild type, and thus a plant into which the foreign gene has been introduced is obtained in the second stage. You can also.

  However, in the case where the suppression of the functional expression of the gene in the mutant is due to, for example, mutation of the gene sequence, the gene introduction method of the present invention is a third step following the preparation step and the gene introduction step. Furthermore, it is preferable that the method further includes a removal step of removing suppression of functional expression of the gene from the foreign gene introduction mutant obtained in the gene introduction step. By this removal step, the exogenous transgenic transgenic plant having a wild-type background can be obtained. Examples of the method for removing the suppression of functional expression and returning to the wild type background include backcrossing between a mutant into which a foreign gene has been introduced and a wild type strain. Can be adopted.

  The gene targeting method of the present invention comprises a target gene prepared in the polynucleotide preparation step and / or a polynucleotide comprising a foreign gene containing a foreign gene and a region homologous to a region adjacent to the target gene and the gene of the present invention. It can implement by introduce | transducing into the variant of this invention using the introduction method. The homologous part is preferably located at both ends of the foreign gene to be introduced. In order to facilitate selection of cells in which homologous recombination has occurred, it is preferable that the polynucleotide further includes two selectable marker genes capable of two-step selection known in the art. The first selectable marker is a positive selectable marker that can confirm that the polynucleotide has been integrated into the genome, and the second selectable marker is a negative selectable marker that can exclude those that have been integrated by non-homologous recombination. is there. In this case, the introduced polynucleotide may be linear or circular. The positive selection marker can be used as a foreign gene.

  The first method for producing a transgenic plant of the present invention is a production method comprising the step of incorporating a foreign gene into a plant genome by non-homologous recombination using the gene introduction method of the present invention, A foreign transgenic transgenic plant by non-homologous recombination, which is the first transgenic plant, can be produced.

  The second method for producing a transgenic plant of the present invention is a production method comprising the step of incorporating a foreign gene into the genome of a plant by homologous recombination using the gene introduction method of the present invention. It is possible to produce an exogenous transgenic transgenic plant by homologous recombination, which is two transgenic plants.

  The background of the transgenic plant of the present invention may be that of the mutant of the present invention or may have been returned to the wild type. Moreover, the transgenic plant of the present invention is a progeny, clone, and propagation material (eg, seed, fruit, cut ear, tuber, tuberous root) obtained from the transgenic plant produced by any of the above two methods. , Strains, callus, protoplasts, etc.).

  When Arabidopsis thaliana is used as a plant, the first preferred embodiment of the mutant of the present invention is the functional expression of both of two ASF1 orthologous genes, ASF1a (accession number: AB078339) and ASF1b (accession number: AB078340). Is a double mutant in which is suppressed. The ASF1 orthologous gene of Arabidopsis thaliana has two genes, ASF1a and ASF1b, which are duplicated, and either one of the mutations is not sufficient for use as a mutant of the present invention. Must be a heavy mutant.

  Moreover, the 2nd preferable aspect of the variant of this invention is FAS1 gene (accession number: AB027229) which is a CAF-1 orthologous gene, FAS2 gene (accession number: AB027230), and MSI1 (accession number: NM_125208). ) Is a mutant whose function is suppressed.

  Furthermore, another preferred embodiment of the mutant of the present invention is a double mutant of suppressing the functional expression of the ASF1a gene or ASF1b gene and suppressing the functional expression of any one of the FAS1, FAS2 and MSI1 genes.

  The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.

Inhibition of transgene silencing in Arabidopsis fas2 mutants Expression of introduced genes in higher plants is often suppressed by either post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS). Yes. The transgenic Arabidopsis thaliana L5 strain has a GUS (β-glucuronidase) gene introduced therein, but the GUS gene is silenced at the transcriptional level and is hardly detected in the wild type background. Therefore, the expression of the GUS gene was confirmed by crossing the wild type L5 strain with the fas2 mutant or the mom1 mutant. The mom1 mutant is a control mutant that lacks transcriptional gene silencing (TGS). Expression of GUS was detected in the L5 strain fas2 homozygote and L5 strain moml homozygote of the F2 and F3 generation progeny obtained by crossing. Detection of GUS expression was performed by conventionally known histochemical staining.

  As a result, in the wild type L5 strain, the expression of GUS could not be confirmed with few exceptions. This is shown in FIG. 1A. The photograph in the box in the figure is a photograph of an exceptional cell expressing GUS. In addition, the mom1 homozygote was expressed uniformly throughout the whole plant as shown in the previous report (not shown). On the other hand, in the fas2 homozygote, although the expression of GUS was confirmed, in contrast to the mom1 homozygote, the size of the expressed region was very small, and the size and size of the region were the fas2 homozygote. It varied among zygotes. This is shown in FIGS. 1B and 1C. These results indicate that gene silencing of the transgene is accidentally derepressed in the fas2 mutant.

Derepression of gene silencing in Arabidopsis asf1a; asf1b mutants Identification and isolation of ASF1 orthologs of Arabidopsis ASF1 family of proteins are conserved in a variety of organisms, from yeast to humans. Thus, two types of ASF1 arabidopsis orthologs were predicted from the database based on the known ASF1 sequence. The shorter ortholog on the first chromosome is named AtASF1a and the longer ortholog on the fifth chromosome is named AtASF1b, and are hereinafter referred to as ASF1a and ASF1b, respectively.

  ASF1a and ASF1b cDNAs were isolated by reverse transcription PCR (RT-PCR) using primers designed based on the database. About each isolated cDNA, the cDNA end rapid amplification method (RACE-PCR) was performed, and 5 'and 3' untranslated region was determined. As a result, each of the cDNAs is a full-length cDNA of ASF1a containing an ORF of 196 amino acids (predicted molecular weight 22.1 kDa), and a full-length of ASF1b containing an ORF of 218 amino acids (predicted molecular weight 24.7 kDa). It was confirmed to be cDNA. The sequences of the ASF1a gene and the ASF1b gene are shown in SEQ ID NOs: 1 and 2, respectively.

2. Screening and isolation of ASF1 function-deficient mutants In order to obtain ASF1 function-deficient mutants in Arabidopsis thaliana, screening and database search using a transgenic plant library into which T-DNA had been inserted were performed. As a result, an asf1a-1 mutant in which T-DNA was inserted into the genomic region of the ASF1a gene and an asf1b-2 mutant in which T-DNA was inserted into the genomic region of the ASF1b gene were isolated. The insertion position in the genome of T-DNA in each mutant is shown in the schematic diagram of FIG. As shown in the figure, in the asf1a-1 mutant, T-DNA was inserted between exon 2 and exon 3 of the ASF1a gene, and 60 bp of genomic DNA was deleted. In the asf1b-2 mutant, T-DNA was inserted between exon 1 and exon 2 of the ASF1b gene, and 12 bp genomic DNA was deleted. The asf1a-1 mutant is a mutant in which T-DNA derived from pD991 is inserted into Wassilewskija (Ws) ecotype Arabidopsis thaliana isolated from the library of AKF (arabidopsis Knockout Facility) at the University of Wisconsin. The asf1b-2 mutant is a mutant in which T-DNA derived from pPCVICEn4HPT is inserted into Columbia (Col) ecotype Arabidopsis thaliana isolated from a library of Kazusa DNA Laboratory. The asf1a and asf1b mutants used in this example are homozygous mutants that are progenies of these F2 generations.

  In the ASF1a mutant, the full-length cDNA of the ASF1a gene was not confirmed by RT-PCR. Moreover, the incomplete exon 1 and the exon 2 confirmed by RT-PCR were trace amounts. Therefore, it was shown that the asf1a mutant is a function-deficient mutant of ASF1a in which the ASF1a gene lacks function or is very weak. Similarly, even in the ASF1b mutant, the full-length cDNA of the ASF1b gene could not be confirmed by RT-PCR, and the incomplete exon 1 confirmed by RT-PCR was a trace amount. Therefore, it was shown that the asf1b mutant is a function-deficient mutant of ASF1b in which the ASF1b gene lacks function or is very weak.

3. Production of asf1a; asf1b mutant These double mutants were prepared using the Ws ecotype asf1a-1 mutant and the Col ecotype asf1b-2 mutant. An asf1a-1 (wB3) homo mutant backcrossed three times to the Ws type, or an asf1a-1 (cB3wB3) homo mutant back crossed three times to the Ws type and then back crossed three times to the Col type; Multiplying the Asf1b-2 (wB3) homozygous mutant backcrossed to the Col type three times, and using the PCR, the homozygous type of T-DNA was identified using PCR in the F2 / F3 generation. Was made.

4). Derepression of partial transcriptional gene silencing (TGS) in asf1a; asf1b mutants. TSI repeats are sequences located in the region near the centromere. TSI repeats are silenced at the transcriptional level in wild-type Arabidopsis, but are activated and transcribed in many TGS variants. As the TGS mutant, for example, the mom1 mutant, bru1 mutant, and ddm1 mutant are known. Thus, when wild-type, asf1a mutant, asf1b mutant, and asf1a; asf1b mutant were confirmed by using RT-PCR of total RNA, whether or not TSI repetitive sequences were transcribed. Only asf1a; asf1b mutants showed very weak expression. Next, total RNA was prepared from seeding on day 15 of each mutant of wild type and ddm1, fas2, asf1a, asf1b, asf1a; asf1b, and after DNase treatment, RT-PCR (+ RT) or PCR ( -RT) to detect TSI transcripts. The result is shown in FIG. 3A. In the figure, AP2 is an amplification reaction control.

  As shown in FIG. 3A, TSI transcripts were reproducibly detected from the two seedlings of ddm1 and fas2 mutants (FIGS. 3 lanes 3 to 6). On the other hand, no signal was detected from the wild type, asf1a and asf1b mutants (lanes 1, 2 and 7 to 10 in FIG. 3). In the asf1a; asf1b mutant, a weak signal was detected only from one seeding. Since no signal is detected in PCR (-RT), this signal does not originate from genomic DNA contamination. Therefore, it was confirmed that in some of the asf1a and asf1b mutants, suppression of transcription gene silencing (TGS) was canceled.

  Next, it was confirmed that the proportion of seeding individuals to which the TSI sequence was transcribed increased with time. For each of the wild type, ddm1, fas2, and asf1a; asf1b mutants, total RNA was prepared from seeding on day 5 or 15 and similarly, RT-PCR was used to transcribe the TSI sequence. confirmed. The result is shown in FIG. 3B. As shown in FIG. 3, in the asf1a; asf1b mutant, the transcription of the TSI sequence was confirmed only in 1 out of 22 individuals by seeding on the 5th day, but on the 15th day, 22 individuals were confirmed. It became 5 of them. Thus, the tendency that the rate of derepression of TGS increases with time was consistent in the fas2 and asf1a; asf1b mutants.

  As described above, according to the method of the present invention, a method for introducing a gene into a plant with improved introduction efficiency and de-inhibition of gene silencing is possible. Furthermore, according to the present invention, a plant gene targeting method is possible, and a safer transgenic plant can be produced. Therefore, the present invention is useful in a wide range of industrial fields including agriculture, commerce, and industry, and is particularly useful in the field of development of genetically modified (GM) crops.

FIG. 1A is a photograph showing an example of GUS expression in the wild type, and FIGS. 1B and 1C are photographs showing an example of GUS expression in the fas mutant (Example 1). FIG. 2 is an explanatory diagram showing the insertion positions of T-DNA in the asf1a mutant and the asf1b mutant (Example 2). FIG. 3A is a diagram showing the results of detecting TSI transcripts by RT-PCR (Example 2). FIG. 3B is a graph showing the percentage of individuals in which TSI sequence transcription was detected over time (Example 2).

Claims (17)

A gene introduction method for introducing a foreign gene into a plant,
A preparation step of preparing a plant mutant in which the expression of one or more genes encoding a factor involved in a nucleosome formation reaction associated with DNA replication is suppressed,
A gene introduction method comprising introducing a polynucleotide containing a foreign gene into the mutant to obtain a foreign gene-introduced mutant.   The gene transfer method according to claim 1, further comprising a suppression and removal step of removing a function expression suppression of the gene from the foreign gene transfer mutant to obtain a foreign gene transfer plant.   The gene introduction method according to claim 2, wherein the suppression and removal step is a step of returning the gene mutation causing the suppression of function expression to the wild type by crossing the foreign gene introduction mutant with a wild type plant.   The one or more genes whose functional expression is suppressed in the mutant are selected from the group consisting of an orthologous gene of a gene encoding three subunits of CAF-1 and an orthologous gene of the ASF1 gene. 4. The gene transfer method according to any one of items 1 to 3.   The gene introduction method according to any one of claims 1 to 4, wherein the functional expression suppression of the gene is caused by mutation of the gene, expression of a dominant negative mutant of the gene product, or RNA interference with the gene.   The gene introduction method according to any one of claims 1 to 5, wherein the introduction of the polynucleotide is performed by means selected from the group consisting of electroporation, particle implantation, virus infection, and Agrobacterium infection.   The gene transfer according to any one of claims 1 to 6, wherein the plant is a plant selected from the group consisting of food plants, feed plants, ornamental plants, environmental plants, experimental plants, and resource plants. Method. A gene targeting method in which a foreign gene is incorporated by homologous recombination into a target gene locus of a plant genome,
A polynucleotide preparation step of preparing a polynucleotide comprising a target gene and / or a portion homologous to a region adjacent to the target gene and a foreign gene;
A gene introduction step for obtaining a foreign gene-introduced mutant or a foreign gene-introduced plant by introducing the polynucleotide into the plant mutant by homologous recombination by the gene introduction method according to any one of claims 1 to 7. Gene targeting method including.   A method for producing a transgenic plant, comprising the step of incorporating a foreign gene into the genome of a plant by non-homologous recombination using the gene introduction method according to any one of claims 1 to 7. Method.   A method for producing a transgenic plant, comprising the step of incorporating a foreign gene into a plant genome by homologous recombination using the gene targeting method according to claim 8.   A transgenic plant, which is produced by the method according to claim 9 or 10.   A plant mutant used in the gene introduction method according to any one of claims 1 to 7, the gene targeting method according to claim 8, or the transgenic plant production method according to claim 9 or 10, comprising a DNA A mutant of a plant in which the function expression of one or more genes encoding factors involved in a nucleosome formation reaction accompanying replication is suppressed.   The plant according to claim 12, wherein the one or more genes whose functional expression is suppressed is selected from the group consisting of an orthologous gene of a gene encoding three subunits of CAF-1 and an orthologous gene of an ASF1 gene. Mutants.   14. The plant mutant according to claim 12 or 13, wherein the suppression of gene expression is caused by mutation of the gene, expression of a dominant negative mutant of the gene product, or RNA interference with the gene.   The mutant according to any one of claims 12 to 14, wherein the plant is a plant selected from the group consisting of food plants, feed plants, ornamental plants, environmental plants, experimental plants, and resource plants.   13. A mutant of Arabidopsis thaliana, wherein the ASF1a gene (accession number: AB078339) and the ASF1b gene (accession number: AB078340) are functionally suppressed.   A mutant of Arabidopsis thaliana, wherein at least one of the FAS1 gene (accession number: AB0272229), FAS2 gene (accession number: AB027230), and MSI1 (accession number: NM — 125208) is suppressed in functional expression. The mutant according to claim 12.