JP6374176B2 - Recombinant vector for use in plants and use thereof - Google Patents

Description

  The present specification relates to a recombinant vector used for a plant and its use.

  In plants, homologous recombination of genes on chromosomes, that is, introduction of foreign genes at the intended chromosomal site can occur, but the probability is low. In order to obtain the intended state of gene integration and gene expression, it is necessary to obtain homologous recombinants. In addition, such a homologous recombinant can be efficiently selected, and a selection marker gene that is not essentially necessary for a plant should be excluded from the chromosome.

  Therefore, various methods have been attempted that are intended to remove the selection marker for selecting a recombinant and to improve the efficiency of homologous recombination in the plant.

  By incorporating a positive selection marker that can select recombinants into the targeting vector for homologous recombination and a negative selection marker that can be deleted by killing non-homologous recombinants at high frequency, A technique capable of selecting a substitute is disclosed (Patent Document 1). In addition, by crossing a recombinant plant introduced with a positive selection marker with a recombinant plant introduced with a site-specific recombination system, a site-specific recombinant enzyme can be expressed in the cells of the hybrid plant. It is disclosed that a positive selection marker is excluded from a chromosome (Patent Document 1). Furthermore, in order to make removal of such a positive selection marker efficient, it is also disclosed that a site-specific recombination system is inducibly expressed with a chemical induction system promoter or the like (Patent Document 2).

  Although not intended for homologous recombination, recombination can be achieved by using a gene that forms adventitious buds under the control of a light-inducible promoter as a positive selection marker that can select recombinants for recombination vectors. It is also described to ensure safety and improve efficiency (Patent Document 3). This vector also discloses that a site-specific recombination system gene linked under the control of a chemical induction system promoter is retained to delete a positive selection marker by external expression induction (patent) Reference 3).

International Publication No. 2003/020940 JP 2008-253254 A JP 2000-83680 A

  However, the efficiency of obtaining homologous recombinants in plants has not always been sufficient. As a result of various studies by the present inventors, the site-specific recombination expression expression system such as Cre recombinase described in Patent Document 2 cannot sufficiently suppress the promoter activity before induction, and site-specific assembly It was found that the enzyme was expressed. Since a site-specific recombination system was unintentionally expressed, it was speculated that a positive selection marker essential for selecting a homologous recombinant could be lost during the positive-negative selection process. That is, it was speculated that despite the success of homologous recombination, the positive selection marker was lost before selection and growth of recombinant cells. In addition, it was also predicted that the positive selection marker was lost from homologous recombination vectors introduced into cells before low probability homologous recombination occurred, further reducing the probability of successful homologous recombination.

  Then, it was predicted that the unintended removal of such a positive selection marker resulted in the inability to efficiently obtain homologous recombinants.

  The present specification provides a technique for more efficiently producing a recombinant plant such as a homologous recombinant. More specifically, in a positive-negative selection system for obtaining a homologous recombinant, the control of expression of a site-specific recombination system for removing a positive selection marker is improved, and it is simple and frequently performed. Provides a technique capable of more effectively performing processes from homologous recombination to positive-negative selection.

  In the process of producing recombinants, the present inventors examined by focusing on the switching between suppression and promotion of expression of site-specific recombination systems. Finding that a vector can be constructed to retain a site-specific recombinase gene under the control of an activating promoter, so that positive selection markers can be reliably retained when necessary and then removed with high efficiency. Got. In addition, as a result, it was found that the efficiency of deletion of the positive selection marker by the site-specific recombination system can be dramatically increased while avoiding the reduction of the homologous recombinant production efficiency. This specification provides the following teachings based on these findings.

(1) A homologous recombination vector used for a plant body,
A positive selectable marker region that affirms that it is a recombinant, and
A negative selectable marker region that affirms and deletes a non-homologous recombinant,
A site-specific recombinase region;
A site-specific recombination system for removing the positive selectable marker region and the site-specific recombinase region;
With
A homologous recombination vector comprising the site-specific recombination system under the control of a tissue differentiation inducible promoter.
(2) The homologous recombination vector according to (1), wherein the tissue differentiation-inducing promoter is a light-inducible promoter.
(3) The homologous recombination vector according to (1) or (2), wherein the tissue differentiation inducible promoter is a greening tissue differentiation inducible promoter.
(4) The recombinant vector according to any one of (1) to (3), wherein the tissue differentiation-inducing promoter is an LP2 gene promoter.
(5) A random recombinant vector used for a plant body,
A region for expressing a positive selectable marker affirming that it is recombinant;
A site-specific recombination system for removing the positive selectable marker region and the site-specific recombinase region;
With
A recombinant vector comprising the site-specific recombination system under the control of a tissue differentiation-inducible promoter.

(6) A method for producing a homologous recombinant plant,
The homologous recombination vector according to (1), wherein the plant body can maintain a desired introduction region in an undifferentiated environment in which the plant body can maintain an undifferentiated state and does not activate the tissue differentiation-inducing promoter. Introducing into a plant and obtaining a first homologous recombination plant using the negative selection marker and the positive selection marker;
A second homologous product in which site-specific recombination has occurred by culturing the first homologous recombination plant in a differentiation-inducing environment capable of inducing differentiation of the plant and activating the tissue differentiation-inducible promoter. Obtaining a recombinant plant;
A manufacturing method comprising:
(7) A method for producing a random recombinant plant,
The random recombination vector according to (5), wherein the plant body is maintained in an undifferentiated state and retains a desired introduction region in an undifferentiated environment in which the differentiation-inducible promoter is not activated. Introducing into the body and using the positive selection marker to obtain a first random recombinant plant;
A second random plant in which site-specific recombination has occurred by culturing the first random recombinant plant in a differentiation-inducing environment capable of inducing differentiation of the plant and activating the tissue differentiation-inducible promoter. Obtaining a recombinant plant;
A manufacturing method comprising:

It is a figure which shows the outline | summary of a homologous recombination vector. It is a figure which shows the outline | summary of the recombination using a homologous recombination vector. It is a figure which shows the outline | summary of a random recombination vector. It is a figure which shows the outline | summary of the recombination using a random recombination vector. It is a figure which shows the structure of an estradiol induction type | mold Cre expression unit. It is a figure which shows construction | assembly of a light and greening tissue induction type | mold Cre expression unit. It is a figure which shows the structure of the vector for Cre-loxP site-specific recombination evaluation. It is a figure which shows the structure of the vector for a Cre-loxP site-specific recombination evaluation (positive control vector), and the evaluation test result in a rice cultured cell. It is a figure which shows the structure of the vector for Cre-loxP site-specific recombination evaluation (negative control vector), and the evaluation test result in a rice cultured cell. It is a figure which shows the evaluation test of an estradiol induction type | formula Cre expression unit. It is a figure which shows the examination result of the induction | guidance | derivation conditions of an estradiol induction type Cre expression unit. It is a figure which shows the detection of Cre-loxP recombination in the rice callus which introduce | transduced pZEN21 by PCR analysis. It is a figure which shows the structure and Cre expression of a Cre expression vector by a light and greening tissue induction type LP2 promoter. It is a figure which shows the basic vector for targeting gene modification. It is a figure which shows the vector for OsRacGEF1 gene modification | change, and a control vector. It is a figure which shows the targeting vector and control vector for self marker free type | mold OsRacGEF1 gene modification. It is a figure which shows the modification | change of OsRacGEF1 gene by a self marker free targeting system. It is a figure which shows the random insertion of the T-DNA area | region which has a self marker free type gene modification targeting vector for OsRacGEF1 gene modification. It is a figure which shows selection of the OsRacGEF1 targeting variant by PCR analysis. It is a figure which shows the success / failure determination result of marker gene deletion by PCR analysis.

  The disclosure of the present specification relates to a recombinant vector used in a plant and use thereof. Specifically, the present invention relates to a technique for more efficiently producing a recombinant plant such as a homologous recombinant. More specifically, in the positive-negative selection system for obtaining homologous recombinants, the control of the expression of the site-specific recombination system for removing the positive selection marker is improved to ensure positive-negative. The present invention relates to a technique capable of more effectively performing a process from selection to homologous recombination gene region to deletion of a positive selection marker gene.

  The disclosure of the present specification is characterized by comprising a site-specific recombination system under the control of a tissue differentiation-inducible promoter that is activated by inducing differentiation of a tissue in order to eliminate a selection marker used for recombination from a plant body. And

  Conventionally, a chemical induction promoter has been mainly used as a switch for inducing a site-specific recombination system, but the suppression of the expression of this promoter is insufficient in the homologous recombination process and the selection marker is lost unintentionally. It has not been known that, and as a result, homologous recombinants cannot be efficiently obtained. That is, the present inventors can achieve reliable suppression of site-specific recombination system and reliable switching of activation in order to efficiently obtain recombinant plants, particularly homologous recombinant plants. I found it more important than expected.

  Further, the inventors of the present invention also pay attention to that the process of producing a recombinant plant body induces tissue differentiation after introducing a gene into an undifferentiated plant body to cause recombination, It has been found that it is advantageous to use such a process to suppress the expression and switch the activation of the site-specific recombination system.

  This specification provides a recombinant vector for a plant, a method for producing a recombinant plant, and the like as means for realizing such knowledge.

  As used herein, “plant” is a general term for organisms belonging to the plant kingdom, and is characterized by chloroplasts, hard cell walls, the presence of abundant permanent embryonic tissue, and organisms that are not capable of motility. Typically, a plant body refers to a flowering plant having cell wall formation and anabolism by chloroplasts. “Plant” includes both monocotyledonous and dicotyledonous plants. Monocotyledonous plants include gramineous plants. Preferred monocotyledonous plants include corn, wheat, rice, oat, barley, sorghum, rye and millet, and more preferably, corn, wheat and rice are not limited thereto. Wheat includes wheat cultivar Norin 61, for which it was difficult to obtain transformants by conventional methods.

  Examples of dicotyledonous plants include cruciferous plants, legumes, eggplants, cucurbits and convolvulaceae, but are not limited thereto. Brassicaceae plants include but are not limited to Chinese cabbage, rapeseed, cabbage and cauliflower. Preferred cruciferous plants are Chinese cabbage and rapeseed. A particularly preferred cruciferous plant is rapeseed. Leguminous plants include, but are not limited to, soybean, azuki bean, kidney bean, and cowpea. A preferred legume is soybean. Examples of solanaceous plants include, but are not limited to, tomatoes, eggplants, and potatoes. A preferred solanaceous plant is tomato. Cucurbitaceae plants include, but are not limited to, cucumber, cucumber, melon and watermelon. A preferred cucurbitaceae plant is cucumber. Convolvulaceae plants include, but are not limited to, morning glory, sweet potato and convolvulus. A preferred convolvulaceae plant is morning glory.

  Furthermore, examples of plant species include plants such as solanaceous, gramineous, cruciferous, rose, leguminous, cucurbitaceae, lily, lily, redwood, convolvulaceae, and chrysanthemum. . Furthermore, examples of plant species that can be used in the present invention include any tree species, any fruit tree species, mulberry plants (eg, rubber), and mallow (eg, cotton).

  Examples of cruciferous plants include plants belonging to Raphanus, Brassica, Arabidopsis, Wasabia, or Capsella, and include, for example, radish, rape, Arabidopsis thaliana, horseradish, and tuna.

  Examples of gramineous plants include plants belonging to Oryza, Triticum, Hordeum, Secale, Saccharum, Sorghum, or Zea, and include, for example, rice, barley, rye, sugarcane, sorghum, corn, and the like.

  The plant body means any plant individual, plant organ, plant tissue, plant cell, and seed. Examples of plant organs include roots, leaves, stems and flowers. Examples of plant cells include callus and suspension culture cells. In the expression of the first homologous recombination plant, the first random recombination plant, etc., the plant is understood as a plant cell, plant tissue, plant organ, etc. based on the technical common sense of those skilled in the art. .

  In the present specification, simply recombination includes both homologous recombination and random recombination unless otherwise specified.

  In this specification, the term “region” refers to a nucleic acid region such as DNA encoding a predetermined protein, a region encoding a predetermined protein, and other regions that can be expressed as a protein by translation of the region. A nucleic acid region that can carry out homologous recombination by itself, a nucleic acid region that encodes an intentional genetic modification or a foreign protein, DNA containing a transcription sequence of RNA having a predetermined function, etc. In the recombination in this specification, it uses for the nucleic acid region which may be functional by itself.

(Recombinant vectors for plants)
The recombinant vector used in plants disclosed in this specification includes a random recombinant vector intended for introduction of a random exogenous nucleic acid (transduction region) into a plant genome, and a foreign vector to a specific target site on the genome. And a homologous recombination vector (targeting vector) intended to introduce a sex nucleic acid (transduction region). The outline | summary of a homologous recombination vector and a random recombination vector is shown to FIG. 1A and FIG. 2A, respectively. As described later, the difference between the two is that the homologous recombination vector has a homologous recombination region for inserting an exogenous introduction region on the vector into the target site of the genome via homologous recombination. In contrast, random recombination vectors do not have a homologous recombination region for targeting. Targeting vectors have a positive selectable marker region that affirms recombination and a negative selectable marker that affirms nonhomologous recombination. Random recombination vectors differ at least in having only a positive selectable marker region that affirms recombination. Furthermore, the homologous recombination vector has a negative selection marker region, whereas the random recombination vector does not have a negative selection marker region.

  Hereinafter, the homologous recombination vector shown in FIG. 1 will be described first, and then the random recombination vector shown in FIG. 2 will be described.

  As shown in FIG. 1A, the homologous recombination vector includes a homologous recombination cassette. The homologous recombination cassette is generally provided in a part on a known vector. For example, when the homologous recombination vector is an Agrobacterium-mediated vector, it is provided between the RB that is the right boundary and the LB that is the left boundary of T-DNA.

(Positive selection marker area)
As shown in FIG. 1A, the homologous recombination vector has a positive selectable marker region that affirms that it is a recombinant.

  A selectable marker generally refers to a gene that functions as an indicator for selecting a host cell containing a nucleic acid construct such as a vector. Examples of selection markers include, but are not limited to, fluorescent markers, luminescent markers, and drug resistance selection markers. “Fluorescent markers” include genes encoding fluorescent proteins such as green fluorescent protein (GFP), blue fluorescent protein (CFP), yellow fluorescent protein (YFP) and red fluorescent protein (dsRed). It is not limited. “Luminescent markers” include, but are not limited to, genes encoding photoproteins such as luciferase. “Drug resistance selection markers” include hypoxanthine guanine phosphoribosyltransferase (hprt), dihydrofolate reductase gene, glutamine synthetase gene, aspartate transaminase, metallothionein (MT), adenosine deaminase (ADA), adenosine deaminase (AMPD1, 2) ), Xanthine-guanine-phosphoribosyltransferase, UMP synthase, P-glycoprotein, asparagine synthetase, and genes encoding ornithine decarboxylase.

  In general, as a positive selection marker, it is preferable that the positive selection marker is a marker that promotes the proliferation of cells and / or promotes the proliferation thereof when introduced into a chromosome of a plant cell and expressed. Examples of positive selection markers include hygromycin resistance gene, kanamycin resistance gene, imidazoline resistance gene, drug resistance gene such as neomycin resistance gene, glyphosate resistance gene, phosphomannose isomerase gene, blastoidin S resistance gene, Bar gene and Grifo Examples thereof include, but are not limited to, cinnate resistance genes.

  The positive selection marker region is provided in the homologous recombination cassette with an appropriate promoter region, terminator region, and the like. In addition, since the positive selection marker region is finally removed from the plant genome by a site-specific recombination system, it is provided at a position where removal does not interfere with the functional expression of the introduction region to be incorporated into a chromosome or the like. It is done. For example, as shown in FIGS. 1A and 1B, the homologous recombination cassette is preferably configured to be arranged in an intron or the like of the target genome.

(Negative selection marker region)
As shown in FIGS. 1A and 1B, the homologous recombination vector further comprises a negative selectable marker region that affirms that it is a non-homologous recombinant. The negative selectable marker region generally prevents cell growth when a region (introduction region) scheduled to be introduced into the genome by homologous recombination of the homologous recombination vector is introduced into a region other than the intended target region on the chromosome. By inhibiting and / or suppressing, it means a marker that enables selection of cells in which the introduction region of the homologous recombination vector is introduced into the target region. Typically, the negative selectable marker region inhibits and / or suppresses the growth of the cell when the homologous recombination vector is inserted into the genome without relying on homologous recombination at the target region. On the other hand, when the introduction region in the homologous recombination vector is introduced into the target region by the intended homologous recombination, for example, a negative selection marker as an additional sequence outside the introduction region is present in the genome. It will not be inserted. For this reason, cell growth is not inhibited or suppressed.

  Examples of the negative selectable marker include diphtheria toxin protein A chain gene (DT-A), Exotoxin A gene, Ricin toxin A gene, codA gene, cytochrome P-450 gene, RNase T1 gene and barnase gene. It is not limited.

  A negative selectable marker region is provided at one end of the homologous recombination cassette or at both ends as shown in FIGS. 1A and B, with an appropriate promoter and terminator. Negative selection marker regions are preferably provided at both ends of the cassette. This ensures that non-homologous recombinants are excluded by the negative selectable marker region. Two negative selection marker regions may be provided in opposite directions.

  Promoters, terminators, enhancers, etc. associated with the positive selection marker region and the negative selection marker region are well known in the art, and those skilled in the art can appropriately select from known elements to hold the selection marker functionally. Can do.

(Homology region)
As shown in FIGS. 1A and 1B, the homologous recombination vector can have a homologous region that enables homologous recombination with a target region of a plant chromosome. When the homologous recombination vector has a homologous region, the homologous region is provided at a site that allows functional expression of other elements of the vector and allows the functional expression of the other elements. Homologous regions are generally provided near both ends of the cassette. When a negative selection marker region is provided at both ends of the cassette, it is provided immediately inside each negative selection marker region. In this way, if homologous recombination is appropriately performed on the target region, the negative selectable marker region will not be integrated into the chromosome.

(Introduction area)
As shown in FIGS. 1A and 1B, the homologous recombination vector may have an introduction region (for example, a modified gene containing a base substitution or a completely different gene). Similar to the homologous region, the introduction region replaces a part of the plant chromosome. When the homologous recombination vector includes an introduction region, the introduction region is provided at a site where functional expression of other elements of the vector is allowed and functional expression of the other elements is allowed. For example, the introduction region is provided inside each of two homologous regions on both sides of the cassette as shown in FIGS. 1A and 1B, or inside one homologous region.

  As shown in FIGS. 1A and 1B, the introduction region or homologous region is preferably configured so that a site-specific recombination system and a positive selection marker region described later are arranged in an intron on the target genome. This facilitates the construction of a homologous recombination vector.

  As shown in FIG. 12, the homologous recombination vector may be in a form that does not have an introduction region or a homologous region. That is, it may have a form including a negative selection marker region, a positive selection marker region, and a site-specific recombination system so that a specific introduction region or homologous region can be inserted into the target genome. In this case, as shown in FIGS. 1A, 1B, and 12, a restriction enzyme site for incorporating the introduction region and the homologous region into the homologous recombination vector can be provided outside the identification site of the site-specific recombination system. . In the case shown in FIGS. 1A and 1B, restriction enzyme sites are identified between the 3 ′ negative selection marker region and the upstream identification site, and immediately downstream of the 5 ′ negative selection marker region. Can be provided at two locations.

(Site-specific recombination system)
As shown in FIGS. 1A and 1B, the homologous recombination vector can be provided with a site-specific recombination system for removing a positive selection marker region and a site-specific recombination enzyme region.

  A site-specific recombination system is a system comprising a site-specific recombination enzyme region and an identification site for the enzyme, and is well known to those skilled in the art. For example, the Cre / loxP recombination system derived from bacteriophage PI is a method of deleting unnecessary portions by a combination of Cre recombinase and loxP. The R / RS recombination system is a method of inducing deletion of a DNA region surrounded by two RS sequences by a combination of a recombinase encoded by an R gene and an RS sequence. The FLP-FRT recombination system derived from a 2μ circular plasmid is a method of inducing deletion of a DNA region surrounded by two FRT sequences by a combination of FLP recombinase and FRT sequences. Typically, such a recombination system is a Cre / loxP system that uses Cre recombinase as a site-specific recombinase and loxP as a recognition sequence for the site-specific recombinase, but is not limited thereto. Alternatively, it may be a combination of a transposase recognition sequence at both ends of the transposon and a gene encoding the transposase. As described above, a recombinant system derived from various organisms and applicable to plants is used. obtain. In addition, information on site-specific recombination enzymes in various recombination systems and the base sequences of their identification sites are well known to those skilled in the art, and those skilled in the art can use well-known information applicable to plants based on well-known information. The site-specific recombination enzyme and the identification site can be appropriately selected and applied to the disclosure herein.

  By using a site-specific recombination system, a region such as DNA once inserted into the genome by recombination can be deleted. That is, for example, in a system using Cre-loxP, a region sandwiched between two recognition sites such as loxP is caused to undergo homologous recombination with a site-specific recombinase such as Cre recombinase to remove the region between loxPs.

  According to the disclosure herein, homologous recombination vectors can be site-specific so as to remove the site-specific recombination enzyme region itself that is part of the site-specific recombination system in addition to the positive selectable marker region. It has a recombination system. In this way, by expressing a site-specific recombination enzyme in a plant cell transformed by introducing a homologous recombination vector, the positive selection marker region and the site-specific recombination enzyme region are autonomously removed. Will be. For example, in the case of using the Cre-loxP system, as shown in FIG. 1B, by sandwiching the positive selection marker region and the site-specific recombination enzyme region at the identification site of the site-specific recombination system, Removal is possible.

(Tissue differentiation inducible promoter)
A site-specific recombination system is provided under the control of a tissue differentiation-inducible promoter. That is, the site-specific recombination enzyme region is provided such that the expression is controlled by a tissue differentiation-inducible promoter, and site-specific recombination can be performed between identification sites. A tissue differentiation-inducible promoter is a promoter that is induced and activated by differentiation of a tissue according to the type of the promoter and promotes the expression of a gene under control. By providing a site-specific recombination system under the control of a tissue differentiation-inducible promoter, the tissue differentiation-inducible promoter can be induced by placing it in a differentiation-inducing environment that can induce differentiation and activate the tissue differentiation-inducible promoter. Activate to promote expression of site-specific recombinant systems. That is, the tissue differentiation-inducible promoter can be activated by inducing tissue differentiation, express a site-specific recombination enzyme, and perform site-specific recombination between identification sites. In particular, by using such a tissue differentiation-inducible promoter for callus, its suppression and activation can be accurately controlled.

  Various chemically-inducible promoters are well known, but according to the present inventors, in the callus state where gene transfer operation is performed, this promoter is activated and under control even in the absence of specific induction conditions. It was found that a gene can be expressed. That is, with this promoter, it was found that even if gene expression could be inductively activated, suppression of promoter activity before induction was insufficient.

  Examples of the tissue differentiation inducible promoter include a tissue-specific promoter. This is because the activity of a tissue-specific promoter is suppressed in an undifferentiated callus where targeting occurs due to its tissue specificity, and it is specifically activated in a differentiated specific tissue. Examples of the tissue-specific promoter include a greening tissue inducible promoter specific for greening tissues such as leaves. Such greening tissue inducible promoters can generally also be induced by light. Examples of the greening tissue light-inducible promoter include ribulose diphosphate carboxylase small subunit gene (rbcs) promoter (R. Fluhr et al., Proc. Natl. Acad. Sci. USA 83: 2358, 1986). . Other greening tissue light-inducible promoters include a fructose-1,6-bisphosphatase gene promoter (Japanese Patent Publication No. 7-501921), and a light-harvesting chlorophyll a / b binding protein gene promoter (Japanese Patent Laid-Open No. Hei 5). -89).

  Furthermore, examples of the greening tissue light-inducible promoter include rice LP2 leucine-rich repeat receptor kinase gene promoter (Plant Biotechnology Journal (2009) 7, pp. 1-16). Since promoters of similar genes exist in other plant species and are expressed in the same tissue-specific light-inducible manner, promoters of similar genes can be used for the same species in other plant species ( Plant Biotechnology Journal (2009) 7, pp.1-16). An example of a DNA base sequence that can be used as a rice greening tissue light-inducible promoter is represented by SEQ ID NO: 1.

  In the site-specific recombination system, the identification site is provided in the forward direction so as to sandwich the tissue differentiation-inducible promoter, the site-specific recombination enzyme region under its control, and the positive selection marker region. By doing so, the site-specific recombination enzyme operated by the tissue differentiation-inducible promoter removes the site-specific recombination system and the positive selection marker region all at once.

  As already described, the region to be removed between the identification sites is provided in the homologous recombination cassette in a state of being sandwiched between the homologous recombination region and the introduction region. Such a removal region itself, that is, a removal region comprising a sequence of a site-specific recombination enzyme region under the control of a tissue differentiation-inducing promoter and a positive selection marker region under the control of an appropriate promoter, is removed. It can be used independently as a cassette. The cassette to be removed can further include identification parts at both ends thereof.

  Such removed regions are preferably placed in intron or UTR regions within the gene where such removed regions are ultimately replaced. This is because within these regions, the function expression of the introduction region is not inhibited after removal.

(Random recombination vector)
Next, the random recombinant vector shown in FIG. 2 will be described. The random recombination vector includes a recombination cassette. The recombination cassette is generally provided on a known vector. For example, in the case of an Agrobacterium-mediated vector, a random recombination vector is provided between RB, which is the right boundary of T-DNA, and LB, which is the left boundary.
As shown in FIG. 2, the random introduction vector comprises a positive selection marker region and a site-specific recombination system. The random recombination vector removes the positive selection marker region and the site-specific recombination enzyme region, which is a part of the site-specific recombination system, by the action of the enzyme in the same manner as the homologous recombination vector. Are arranged to be. That is, a positive selection marker region and a site-specific recombination enzyme region are provided so as to be sandwiched between two identification sites that are part of the site-specific recombination system.

  The random recombination vector can comprise an introduction region, as shown in FIG. 2A. The random recombination vector may have an introduction region in any region as long as it is outside the site-specific recombination system. As with the homologous recombination vector, the random recombination vector does not necessarily have an introduction region. That is, it suffices to have a positive selection marker region and a site-specific recombination system so that the introduction region can be inserted. In this case, as shown in FIG. 2A, a restriction enzyme site for incorporating the introduction region into the random recombination vector can be provided outside the identification site of the site-specific recombination system. In the case shown in FIG. 2A, the restriction enzyme site can be provided either downstream of the 3 ′ identification site or upstream of the 5 ′ identification site.

  The homologous recombination vectors and random recombination vectors described above can be constructed as plant vectors and plasmids well known to those skilled in the art. In addition, various elements used in these recombinant vectors can be obtained by those skilled in the art using well-known gene recombination techniques. It is also possible for a person skilled in the art to select a gene to be homologously recombined with a target gene in the plant genome for homologous recombination and introduce the 5 ′ region and 3 ′ region into appropriate sites, respectively, by those skilled in the art. Easy to do with engineering.

  For example, each recombination cassette shown in FIG. 1 and FIG. 2 is replaced with a T-DNA region of a vector for plant transformation by the Agrobacterium method or a region between RB and LB by an appropriate restriction enzyme site. Can do.

  According to the recombinant vector described above, it is possible to reliably control the suppression and expression of the site-specific recombination system that removes the positive selection marker region and the like. For this reason, a recombinant plant can be efficiently obtained by using this vector.

(Recombinant plant production method)
(Method for producing homologous recombinant plant)
A method for producing a homologous recombination plant disclosed in the present specification includes a region for expressing a positive selection marker that affirms that the plant is a recombinant, and the positive selection marker region and the site-specific recombination enzyme region. A site-specific recombination system for removing the first homologous recombination plant that has been homologously recombined so as to provide the site-specific recombination system under the control of a tissue differentiation-inducing promoter. Obtaining the plant body in an undifferentiated environment in which the plant body can maintain an undifferentiated state and does not activate the differentiation-inducing promoter, and the first homologous recombinant plant body can induce tissue differentiation of the plant body. And a second homologous recombination plant that is cultured in a differentiation-inducing environment capable of activating the differentiation-inducing promoter and does not retain the positive selection marker and the site-specific recombination system. That may comprise the steps of obtaining a positive selection marker and the site-specific recombination system is a first homologous recombinant dropped.), The. In the second homologous recombination plant, generally, the positive selection marker and the site-specific recombination enzyme region are deleted from the first homologous recombination plant by site-specific recombination, and one loxP that becomes a trace is obtained. This results in a second homologous recombinant in which only the sequence is left.

  According to this method, since the site-specific recombination system is provided under the control of the tissue differentiation-inducible promoter, the expression of the site-specific recombination system is sufficiently suppressed in the undifferentiated environment. On the other hand, since it is in the undifferentiated environment, homologous recombination with respect to the plant genome is likely to occur. For this reason, the 1st homologous recombination plant body which is the homologous recombination plant body in which the positive selection marker was expressed reliably was produced. Thereafter, by placing the first homologous recombination plant in a tissue differentiation-inducing environment, the expression of the site-specific recombination system was promoted, and as a result, further modification in which the positive selection marker was surely removed was generated. 2 recombinant plants can be obtained efficiently.

  This method comprises the steps of introducing the already described homologous recombination vector into a plant body in the undifferentiated environment to obtain a first homologous recombination plant using a negative selection marker and a positive selection marker, A step of obtaining one homologous recombination plant, and the first homologous recombination plant was cultured in the tissue differentiation-inducing environment to cause modification by site-specific recombination within the homologous recombination sequence. And obtaining the second homologous recombination plant. According to this method, a homologous recombinant plant in which the target gene sequence is efficiently modified can be obtained.

(Method for producing random recombinant plant)
Further, the method for producing a random recombinant plant disclosed in the present specification includes a region for expressing a positive selection marker that affirms that the plant is a recombinant, the positive selection marker region, and site-specific recombination. A site-specific recombination system for removing the enzyme region, and the first random recombinant plant modified so as to comprise the site-specific recombination system under the control of a differentiation-inducible promoter. Obtaining a plant body in an undifferentiated environment in which the plant body can maintain an undifferentiated state and does not activate the tissue differentiation-inducing promoter; and the first random recombinant plant body can induce tissue differentiation of the plant body. And modified so as not to retain the positive selection marker and the site-specific recombination system by culturing in a differentiation-inducing environment capable of activating the differentiation-inducing promoter. Obtaining a second random recombinant plants which may comprise. According to this method, a random recombinant can be obtained by causing random recombination to the plant genome.

  Also in this method, the intended random recombinant plant (second random recombinant plant) can be efficiently obtained as in the method for producing a homologous recombinant plant.

  The above method comprises the steps of introducing the already described random recombination vector into a plant body in an undifferentiated environment and selecting it using a positive selection marker to obtain a first random recombinant plant body, The step of culturing the random recombinant plant in a tissue differentiation-inducing environment to obtain a second random recombinant plant can be carried out.

  Hereinafter, a method for producing a homologous recombination plant will be described. Regarding the random recombinant plant, the first random recombinant plant was obtained by the same operation except that the random recombinant vector was used instead of the homologous recombination vector in the above step, and the second recombinant plant was obtained. Random recombinant plants can be obtained.

(Step for obtaining first homologous recombination plant)
In this step, a first homologous recombination plant is obtained by introducing a homologous recombination vector into the plant. Such a first homologous recombination plant can be carried out based on a well-known method for obtaining a gene recombination plant.

  The plant body is preferably a plant cell or a plant tissue. Plant cells include callus and suspension culture cells. In addition to the dormant tissues including ripe seeds, immature seeds, winter buds, and rhizomes, plant tissues include germplasm, growth points, flower buds, and the like.

  Vectors can be introduced into plant cells using viruses such as cauliflower mosaic virus and geminivirus, Tsumefaciens, A. Any of the methods of indirect introduction through bacteria such as Rhizogenes, the method of direct introduction by physical and chemical methods such as microinjection method, electroporation method, polyethylene glycol method, and particle gun method are used. be able to.

  The introduction of the homologous recombination vector can typically be performed via T-DNA using Agrobacterium. T-DNA is a part of the Ti plasmid retained in Agrobacterium. When a plant is infected with Agrobacterium, the cell itself does not enter the plant cell, but it is a part of the Ti plasmid present in the cell. Certain T-DNA regions are transferred into plant cells. T-DNA is delimited by BR (right border sequence) and BL (left border sequence). Each border sequence consists of an incomplete repeat of about 25 bp.

  Gene transfer into a plant can be carried out according to conventional methods well known in the art. For example, when Agrobacterium is used, the homologous recombination vector is present in Agrobacterium containing Ti plasmid, the Agrobacterium is infected with plant cells, tissues or callus, and the like by positive-negative selection. A first homologous recombinant is selected. The method of transformation and selection can also be appropriately selected and carried out by those skilled in the art according to well-known techniques and positive selection markers used.

  This step is performed in an environment where the plant body can maintain an undifferentiated state. This is because the efficiency of gene recombination is ensured and the tissue differentiation inducible promoter is not activated. Environmental conditions (culture conditions) for maintaining the plant body in an undifferentiated state are well known to those skilled in the art. In general, plant tissues after gene introduction are cultured using a suitable basic medium or a gene-introduced tissue selection medium selected according to the tissue, and at a temperature suitable for the growth of the tissue (usually 20 ° C. to 30 ° C. (C, dark place). The culture conditions for preventing activation of the differentiation-inducible promoter using the tissue differentiation-inducible promoter can be easily obtained as long as the promoter is used. For example, when a greening-specific light-inducible promoter is used, the light condition is appropriately set according to the type of differentiation-inducible promoter and suitable for suppressing its activation. By combining these culture conditions, the undifferentiated state of plant cells and the like is maintained.

(Second homologous recombination plant acquisition step)
The site of the first homologous recombination plant selected in the previous step is induced by a tissue differentiation-inducible promoter that induces differentiation of the first homologous recombination plant in the tissue differentiation-inducing environment and is activated by differentiation induction. A specific recombination system is expressed. Thereby, the 2nd homologous recombination plant which does not hold | maintain a positive selection marker and a site-specific recombination enzyme area | region simultaneously with the differentiation induction of a 1st plant body is acquirable.

  The tissue differentiation induction environment is well known to those skilled in the art as a regeneration medium or regeneration medium. Typically, a regeneration medium in which a plant hormone (generally 0 to 10 mg / l auxin and 0 to 10 mg / l cytokinin) is added to a basic medium according to the type of plant is applied. Furthermore, the conditions for activating the differentiation-inducible promoter are determined according to the type of the differentiation-inducible promoter. For example, in the case of a greening tissue-specific light-inducible promoter, predetermined light irradiation is a condition in addition to general culture conditions that can induce differentiation of a greening tissue. A person skilled in the art can select an appropriate differentiation-inducing environment according to the type of differentiation-inducing promoter used.

  The removal of the positive selection marker and the site-specific recombination system by culturing in a differentiation-inducing environment can be performed by, for example, PCR, Southern blotting, base sequencing for regions specific to homologous recombinants after removal of these regions. It can be confirmed by sequence analysis or the like. Thus, a second homologous recombinant plant can be obtained. The second homologous recombination plant can be a homologous recombination from which both the positive selection marker and the site-specific recombination system have been removed.

  The obtained second homologous recombination plant can be regenerated to a plant by a known method. The second homologous recombination plant in which removal of the positive selection marker and the site-specific recombination system has been confirmed is grown to a plant individual, and then a homo-type recombinant plant individual, etc. by segregation inheritance in the next generation seeds, etc. Can be obtained.

  Hereinafter, examples describing the disclosure of the present specification in more detail will be shown, but these examples are for explaining the disclosure of the present specification, and limit the disclosure of the present specification. is not.

(Evaluation of inducible Cre-loxP site-specific recombination unit incorporated into rice gene targeting self-marker free system)
In this example, in the positive-negative selection targeting of rice, in order to construct a system that causes targeted homologous recombination in the callus genome in one step and further deletes the extra sequence on the genome, it is controlled by an inducible promoter. Cre-loxP site-specific recombination (Cre-loxP system) was used. In order to realize genetic modification by self-marker-free targeting, appropriate control of Cre-loxP system activation is essential so that site-specific recombination by Cre-loxP system occurs after target gene homologous recombination targeting is established. Become. Therefore, the enzyme gene Cre that recombines between loxP sequences was combined with an inducible promoter and incorporated into an evaluation vector, and appropriate activation control conditions for Cre-loxP recombination were studied to select the optimal promoter.

(1) Construction of inducible Cre expression unit
As an inducible promoter for initiating the mechanism of excess sequence deletion by Cre-loxP site-specific recombination, we used estradiol-inducible promoter (Zuo et al. 2001, Usuda et al. 2009) and rice photosynthesis-related tissues. The promoter region (pLP2) of the Leaf Panicle 2 gene (Thilmony et al. 2009), which is expressed in a specific manner, was evaluated. As a Cre-expressed gene, an artificial gene (Cre-int) in which the fifth intron of Arabidopsis KORRIGAN1 gene was incorporated into the gene region based on the sequence of pCKF1 (Terada et al. 2010) was prepared. The preparation of Cre-int and ligation with an inducible promoter proceeded by the overlap extension method via PCR.

(Construction of estradiol-inducible Cre expression unit)
First, the following four DNA fragments were prepared as 1st PCR under the following conditions.
Fragment 1 (O _LexA region, 463 bp): pBIMFN (Usuda et al. 2009) was used as a template, and OLexA-F and OLexA-Cre 1st-R were used as primer sets.
Fragment 2 (Cre gene 5 ′ region, 473 bp): pCKF1 (Terada et al. 2010) was used as a template, and OLexA-Cre 1st-F and Cre-iKOR1-R were used as primer sets.
Fragment 3 (iKOR1 region, 210 bp): pBIMFN was used as a template, and Cre-iKOR1-F and iKOR1-Cre-R were used as primer sets.
Fragment 4 (Cre gene 3′-t35S, 892 bp): iCKOR1-Cre-F and Cre-t35S-R were used as a primer set with pCKF1 as a template.

Subsequently, the following two DNA fragments were synthesized as 2nd PCR under the following conditions.
Fragment 5 (O _LexA -Cre gene 5 'region, 896 bp): Fragments 1 and 2 were diluted 1/1000 and mixed in equal amounts as a template, and OLexA-F and Cre-iKOR1-R were used as a primer set. Using.
Fragment 6 (iKOR1-Cre gene 3'-t35S region, 1062bp): Fragments 3 and 4 were diluted 1/1000 and mixed in equal amounts to form a template, and primer sets, Cre-iKOR1-F, Cre-t35S -R was used.

  Finally, as 3rd PCR, fragments 5 and 6 were diluted 1/1000 and mixed in equal amounts as a template, and a reaction was performed using OLexA-F and Cre-t35S-R as a primer set. By these operations, an inducible Cre expression unit, OLexA-Cre (iKOR1) -t35S was synthesized. Since the attB1 site was added to the 5 'end and the attB2 site was added to the 3' end, this DNA fragment was cloned into the pDONR / Zeo vector by the BP recombination reaction of the Gateway cloning system (Life Technologies). Furthermore, after removing the restriction enzyme recognition site (EcoRI, SalI, SbfI, SphI) between the Cre (iKOR1) gene and t35S, the full-length nucleotide sequence was analyzed with a DNA sequencer (Applied Biosystems 3130xl DNA Analyzer) to ensure accurate detection. A clone having a simple nucleotide sequence was selected to prepare OLexA-Cre (iKOR1) -t35S / pDONR Zeo (FIG. 3B).

  On the other hand, using the homologous recombination mechanism in Escherichia coli, we subcloned the expression unit of XVE, a synthetic transcriptional activator that encodes the binding domain from pBIMFN to the human estrogen receptor and LexA. First, a linear vector was synthesized by PCR using pBlueScript SK (-) as a template and XVE cloning-F and XVE cloning-R as a primer set. Each vector was designed to have 50 bp of homologous regions on the 5 'and 3' sides of the XVE expression unit at both ends of the vector, and a restriction enzyme recognition site was added during cloning. Using the Red / ET Recombination System BAC Subcloning Kit (Gene Bridges), the XVE expression unit was cloned into pBlueScript to prepare pAct-XVE-tE9 / pBS (FIG. 3A). Next, pAct-XVE-tE9 was cut out with XhoI-SbfI and incorporated into OLexA-Cre (iKOR1) -t35S / pDONR Zeo to prepare XVE-OLexA-Cre (iKOR1) -t35S / pDONR Zeo (FIG. 3C).

  In this vector, attL1 is located on the 5 'side of the inducible Cre expression unit, and attL2 is located on the 3' side, so it functions as an entry clone in the LR recombination reaction, and the targeting vector has been improved as the destination vector. It can be used when incorporating into pGEF1-S549D-D and pGEF1-Con-D.

(Construction of light-induced Cre expression unit)
The rice light-inducible promoter, pLP2, and the Cre-expressed gene were linked according to the following procedure advanced by the overlap extension method. First, two DNA fragments were prepared as 1st PCR under the following conditions.

Fragment 1 (pLP2 region, 2329 bp): pGPro1-LP2 (Thilmony et al. 2009) was used as a template, and pLP2-F and pLP2-Cre 1st-R were used as primer sets.
Fragment 2 (Cre (iKOR1) region, 1468 bp): OLexA-Cre (iKOR1) -t35S / pDONR Zeo (described above) was used as a template, and pHSP-Cre 1st-F and Cre-t35S-R were used as primer sets.

  In the subsequent 2nd PCR, fragments 1 and 2 were diluted 1/1000, mixed in equal amounts to form a template, and a light-induced Cre expression unit was synthesized by a reaction using pLP2-F and Cre-t35S-R as a primer set. An entry clone, pLP2-Cre (iKOR1) -t35S / pDONR Zeo, was prepared by the BP recombination reaction of the Gateway cloning system (FIG. 4).

(Addition of restriction enzyme site and transcription termination region)
In order to incorporate the above inducible Cre expression unit into a basic vector for evaluation of Cre-loxP site-specific recombination, pZEN20 (described later), the following operation was performed. A linear vector with a recognition site added was synthesized by PCR using pBlueScript SK (-) as a template and pBS for Cre-loxP-F and pBS for Cre-loxP-R as a primer set, and between AvrII and KpnI. An estradiol-inducible Cre expression unit was incorporated to prepare XVE-OLexA-Cre (iKOR1) -t35S / pBS (FIG. 4). Similarly, a light-inducible Cre expression unit was incorporated to produce pLP2-Cre (iKOR1) -t35S / pBS. Furthermore, cloning was performed by adding a restriction enzyme recognition site from pINA134 (Terada et al. 2002) to the transcription termination region En / spm via PCR, and between the AbsI-SbfI sites of pLP2-Cre (iKOR1) -t35S / pBS. Insertion was performed to prepare En-pLP2-Cre (iKOR1) -t35S / pBS (FIG. 4).

(2) Construction of Cre-loxP site-specific recombination vector series for self-marker-free targeting To achieve self-marker-free targeting, cells that have undergone targeted homologous recombination in the rice callus genome have grown to some extent. At the stage, it is essential to control the inducible promoter so that Cre-loxP site-specific recombination occurs at an appropriate frequency. In order to select an inducible Cre expression unit suitable for this experimental system, an evaluation vector using a reporter gene (GUSPlus) was constructed. The basic vector pZEN20 is a vector derived from pZEN11 (Shimatani et al. 2009) .There are two copies of loxP sites arranged in the forward direction between the 35S promoter and GUSPlus gene in T-DNA, and between the loxP sites. An evaluation vector can be constructed by incorporating each inducible Cre expression unit into a restriction enzyme site. An inducible Cre expression unit was excised from XVE-OLexA-Cre (iKOR1) -t35S / pBS with AbsI-PmeI and incorporated into pZEN20 to obtain pZEN21 (FIG. 5A). Similarly, pZEN24E was obtained by incorporating En-pLP2-Cre (iKOR1) -t35S (FIG. 5B). In these vectors, an inducible Cre expression unit is inserted between the 35S promoter and GUSPlus gene to suppress the expression of GUSPlus, but estradiol-inducible type (XVE-OLexA) linked to Cre gene, and light and greening tissues The induction of each inducible (pLP2) promoter causes a Cre-loxP site-specific recombination reaction, and the GUSPlus expression is restored when the insertion region is removed (FIG. 5C). That is, based on the expression of the GUSPlus gene in plant cells, the timing and frequency of Cre-loxP site-specific recombination by each inducible promoter can be visualized and evaluated. In addition, using a specific sequence on T-DNA, Cre-loxP site-specific recombination can be verified at the DNA level such as PCR and sequencing. When loxP is detached, only one copy of the loxP site remains between the 35S promoter and the GUSPlus gene, and GUSPlus can always be expressed, but this state is a positive control vector that artificially reproduces this state. Was designated as pZEN20PC (FIG. 6A). In addition, En / spm, which is a transcription termination region, was inserted between loxPs, and a negative control vector pZEN20NCEG in which GUSPlus expression by the 35S promoter was suppressed was prepared and used as an index in this evaluation test (FIG. 7A).

(3) Introduction of a vector for evaluating Cre-loxP site-specific recombination into Agrobacterium
pZEN21, pZEN24E, pZEN20PC and pZEN20NCEG were introduced into Agrobacterium (Agrobacterium tumefaciens EHA105 strain) by electroporation (BioPrader MicroPulser electroporation system). First, competent cells for Agrobacterium were prepared according to the following procedure. Agrobacterium strain is applied to YEB agar medium (Beef Extract 5g / L, Yeast Extract 1g / L, Bacto Pepton 1g / L, Sucrose 5g / L, MgSO ₄ 2mM, Bacto Agar 12g (1.2%)), 28 ° C -Cultured for 2 days in the dark. After inoculating the obtained single colony in 5 mL of YEB liquid medium, shake culture at 28 ° C in the dark for 12 hours. Add 200 μL of the suspension to 200 mL of YEB liquid medium and shake culture in the dark at 28 ° C. OD600 = 0.2-0.4. The cells were then collected by centrifugation (3000 rpm, 4 ° C., 10 minutes), suspended in 20 mL of 10 mM HEPES (pH 8.0), and centrifuged 2-3 times. The cells recovered by centrifugation were suspended in 2 mL of a sterile 10% glycerol aqueous solution to obtain a competent cell. Next, each vector was introduced into Agrobacterium by the procedure described below. Each vector was dissolved in sterile water at a concentration of 1 μg / μL, mixed with 50 μL of the above Agrobacterium suspension, transferred to a micropulcer cuvette (0.1 cm gap, BioRad), and electroporation (2.2 kV, 5.8 ms). Next, 800 μL of YEB liquid medium was added to this solution and cultured with shaking at 28 ° C. in the dark for 2 hours, applied to a YEB agar medium containing 50 mg / L kanamycin, and cultured at 28 ° C. in the dark for 36 to 48 hours. The obtained bacterial colonies were grown in 5 mL of YEB liquid medium containing 50 mg / L kanamycin, dispensed into microtubules as glycerol (final concentration 35%) stock, and stored at −80 ° C.

(4) Introduction of Cre-loxP site-specific recombination vector series into rice cultured cells Rice transformation was performed according to the technique of Terada et al. (2002).

(Preparation of rice callus for transformation)
About 100 seeds of rice (Oryza sativa.L Japonica variety; Nipponbare) were removed and shaken in 70% ethanol for 1 minute, then immersed in 2.5% sodium hypochlorite for 20-30 minutes for sterilization did. 2N6 medium (mixed salt for N6 medium (Sigma-Aldrich)) 4.0g / L, Casamino acid 300mg / L, Myo-inocitol 100mg / L, Nicotinic acid 0.5mg / L, Pyridoxine HCl 0.5mg / L, Thiamine HCl 0.5 mg / L, L-Proline 2878 mg / L, Sucrose 30.0 g / L, Gelrite 4.0 g / L, pH 5.8), and cultured in the dark at 31.5 ° C for 4 weeks. A sufficient amount of callus derived from scutellum cells was induced.

(Preparation of Agrobacterium for transformation)
Dissolve the Agrobacterium solution containing each vector for evaluation of Cre-loxP site-specific recombination on ice, and add 300 μL of AB medium (NH ₄ Cl 1 g / L, MgSO ₄ 7H ₂ 0.3g / L, KCl 0.15g / L, CaCl ₂・ 2H ₂ O 0.012 g / L, FeSO ₄・ 7H ₂ O 0.0025g / L, K ₂ HPO ₄ 3g / L, NaH ₂ PO ₄・ H ₂ O 1.15 g / L, Sucrose 5.5 g / L, agarose 6.0 g / L, pH 7.2) and cultured at 28 ° C. in the dark for 3 days. After that, the grown Agrobacterium was added to 40 mg / L of Acetosyringone (3 ', 5'-Dimethoxy-4'-hydroxy-acetophenone, Aldrich, Cat No. D13, 440-6) in AAI liquid medium (MgSO ₄・ 7H ₂ O 5g / L, CaCl ₂・ 2H ₂ O 1.5g / L, NaH ₂ PO ₄・ H ₂ O 1.5 / L g, KCl 29.5g / L, MnSO ₄・ 4H ₂ O 10g / L, ZnSO ₄・ 7H ₂ O 2g / L, H ₃ BO ₃ 3g / L, KI 0.75g / L, Na ₂ MoO ₄・ 2H ₂ O 0.25g / L, CoCl ₂・ 6H ₂ O 25mg / L, CuSO ₄・ 5H ₂ (O 25mg / L, FeSO ₄・ 7H ₂ O 13.9g / L, Na ₂ EDTA 18.7g / L, Myo-inocitol 100mg / L, Thiamine HCl 0.01g / L, Nicotinic acid 1mg / L, Pyridoxine HCl 1mg / L) The suspension was then cultured with shaking at 25 ° C. for 2 hours, and this suspension was diluted with an AAI liquid medium containing 40 mg / ml of Acetosyringone to prepare 120 ml of a suspension having an OD600 = 0.1.

(Inoculation of Agrobacterium to rice callus, co-culture and sterilization)
About 5-40g of callus derived from the rice seed scutellum was collected in a sterilized glass beaker, added with the Agrobacterium suspension introduced above (see above), and inoculated with shaking for 3-5 minutes It was. The suspension was filtered with a stainless mesh (joint opening 1.5 mm) to remove excess Agrobacterium. Next, N6 co-culture medium (mixed salt for N6 medium (Sigma) 4.0g / L, Casamino acid 300mg / L, Myo-inocitol 100mg / L, Nicotinic acid 0.5mg / L, Pyridoxine HCl 0.5mg / L, Thiamine HCl 0.5mg / L, Sucrose 30.0g / L, Glucose 10g / L, 2,4-D 2mg / L, Gelrite 4.0g / L, Acetosyringone 40mg / L, pH5.2) They were arranged at regular intervals with tweezers and co-cultured for 3 days in the dark at 25 ° C. Thereafter, in order to sterilize Agrobacterium from the callus after co-cultivation, the callus was collected in a 500 ml beaker and washed with 300 ml of sterilized water added with 100 mg / L of vancomycin with stirring for 20 minutes. Thereafter, the callus was collected on a stainless mesh, water around the callus was removed with a paper towel, and the same sterilization operation was repeated again using sterilized water containing vancomycin as described above. Next, the callus after sterilization was subjected to selective medium N6SE (mixed salt for N6 medium [Sigma) 4.0 g / L, Casamino acid 300 mg / L, Myo-inocitol 100 mg / L, Nicotinic, containing 50 mg / L of hygromycin. acid 0.5mg / L, Pyridoxine HCl 0.5mg / L, Thiamine HCl 0.5mg / L, L-Proline 2878mg / L, Sucrose 30.0g / L, Gelrite 4.0g / L, Vancomycin 100mg / L, Claforan 400mg / L, pH5 .8) were arranged at regular intervals and cultured in the dark at 31.5 ° C. for about 4 weeks. In the transformation into which pZEN21, pZEN24E, pZEN20PC and pZEN20NCEG were introduced, growth of multiple calli showing hygromycin resistance was confirmed on the 30th day after the start of selective culture. All the work of callus selection from the above-mentioned transformation regarding pZEN24E was performed in a weak light environment (150-200 lux) in order to maintain the suppression of LP2 promoter activity, and all the callus were cultured in the dark.

In random transformation, since the genomic region into which T-DNA is inserted and the copy number of a foreign gene cannot be controlled, the expression level of the foreign gene is often different for each transformant. On the other hand, each callus colony is likely to be an independent cell lineage derived from a different transformed cell. Therefore, it is possible to more accurately investigate the frequency of Cre-loxP site-specific recombination with an inducible promoter by using as many lines as possible for analysis and increasing the number of independent event repetition trials. For the above reasons, each callus from each of the grown colonies of 60-100 callus was subcultured into 24-well plates containing N6SE selective medium containing 50 mg / L of hygromycin, and Isolated culture. Of the obtained transformed calli, 82 lines were introduced with pZEN21, 22 lines were introduced with pZEN24E, 96 lines were introduced with pZEN20NCEG, and 24 lines were introduced with pZEN20PC.

(4) Evaluation of inducible Cre expression unit In order to select an inducible Cre expression unit suitable for self-marker-free targeting and to optimize the induction / suppression conditions, callus introduced with the aforementioned evaluation vector series X -The frequency of Cre-loxP site-specific recombination was assessed by Gluc staining and PCR analysis.

  First, X-Gluc staining was performed on 24 individual transformed calli into which the positive control vector pZEN20PC had been introduced, and as a result, a blue signal due to GUSPlus expression was confirmed in 23 callus (FIG. 6B). It is speculated that gene silencing occurred in one line without blue coloration. On the other hand, as a result of X-Gluc staining and PCR analysis, 89 of the 96 callus lines into which the negative control vector pZEN20NCEG was introduced had GUSPlus expression suppressed by En / spm inserted between loxPs (FIG. 7B). . As for the remaining 7 lines, although no detachment between loxP was observed as a result of PCR analysis, very slight expression of GUSPlus was detected for some reason (FIGS. 7C to 7E). Based on the above, this experimental system combining PCR analysis that detects the presence or absence of Cre-loxP site-specific recombination with a visual marker (GUSPlus) that can detect the occurrence and frequency of recombination is It was judged to be effective for the evaluation of Cre-loxP system and the setting of optimum conditions.

  As a result of X-Gluc staining of 82 independent transformed calli introduced with the estradiol-inducible vector pZEN21, 46 or 56 (56%) already stained part or all of the callus in blue. (FIG. 8A, B). In addition to Cre-loxP recombination, such GUSPlus expression cannot be denied that the GUSPlus gene on pZEN21 accidentally acquires a rice genome endogenous promoter during random transformation. Therefore, of 32 strains in which GUSPlus reporter expression was confirmed, DNA was extracted from a part of the callus by an automatic nucleic acid extraction device (Kurabo PX-80), primer set pCAM-nDart No.2-F and As a result of PCR analysis using pCAM-nDartNo.2-R, a 666 bp DNA fragment was obtained that was amplified when Cre-loxP recombination occurred in all of them (FIG. 10). Therefore, in this inducible Cre expression unit, it is considered that Cre-loxP recombination occurred even though estradiol was not treated.

  Next, as an examination of conditions for appropriately inducing the promoter activity intensity, an evaluation test of estradiol treatment concentration was performed using the expression of GUSPlus by Cre-loxP recombination as an index. 82 lines of transformed callus introduced with pZEN21 were placed on N6SE medium for each line, grown at 31.5 ° C for 2 weeks, and then transferred to N6SE selective medium containing estradiol at 0, 25, or 50 μM concentration respectively. And cultured at 31.5 ° C. for one week. Subsequently, some of these calli were stained with X-Gluc to examine the expression status of GUSPlus. As a result, an increase in the proportion of cells expressing GUSPlus in proportion to the estradiol concentration was confirmed in 19 lines (23%) of callus (FIG. 9). On the other hand, as a result of PCR analysis using DNA extracted from a part of the callus of each strain as a template, 666 bp by Cre-loxP recombination was detected regardless of estradiol treatment and treatment with 25 μM and 50 μM ( FIG. 10). From the above, it was shown that the estradiol-inducible Cre expression unit can regulate the frequency of Cre-loxP recombination depending on the concentration at the time of estradiol treatment, but loxP in more than half of the callus regardless of estradiol treatment. Since the detachment was confirmed, Cre-loxP recombination could not be suppressed when it was unnecessary, which revealed a difficulty in application to self-marker-free targeting. In 17 strains (20%) of callus, no GUSPlus expression was observed regardless of the estradiol treatment concentration, and no detachment between loxPs was detected by PCR analysis. These results are due to loss of function of the inducible Cre expression unit due to T-DNA breakage during random transformation, inactivation due to insertion of T-DNA into the heterochromatin region, or silencing of the transgene. It is presumed that the unit did not function sufficiently.

  As a result of X-Gluc staining of cultured calli of 22 independent transformed calli into which pZEN24E (FIG. 11A) containing a light and greening tissue inducible promoter (pLP2) promoter was introduced, 5 lines (23%) Spot-like blue and small sector-like blue staining were observed, but when comparing the number of blue cell sectors with the estradiol-inducible vector pZEN21, the expression activity of the light and greening tissue-inducible (pLP2) promoter was estradiol-induced It was estimated that it was suppressed to about half of the type vector pZEN21. The pZEN24E green tissue-inducible (pLP2) promoter contains a cis-factor region whose promoter activity is induced by light, so that the fluorescent lamp can be used while minimizing it during the transformation and callus selection processes. Since the work under irradiation was necessary, it is presumed that the Cre expression unit worked in response to light at a low level. It was confirmed that the expression of the light and greening tissue-inducible (pLP2) promoter was rapidly induced during the culture period in which roots and leaves redifferentiated in the state of callus redifferentiation reaction in which expression was suppressed (Fig. 11B).

  From the above evaluation tests, in rice positive-negative selection targeting, after target homologous recombination occurred in the rice callus genome, surplus such as positive selection markers placed between loxP by Cre-loxP site-specific recombination To delete the sequence, we predicted that the light and greening tissue promoter pLP2 would function effectively.

(Development of self-marker-free technology after gene targeting modification in rice)
In this example, for rice blast resistance gene OsRacGef1 in the rice genome, base substitution via homologous recombination was introduced by rice positive-negative selection targeting, and then induced Cre using light and greening tissue promoter (pLP2). A “self-marker-free targeting gene modification technique” was established to delete positive selection markers inserted into the modified OsRacGef1 gene region using the -loxP site-specific recombination system.

  As an example, Akamatsu A et al. (2013) An OsCEBiP / OsCERK1-OsRacGEF1-OsRac1 Module is an essential early component of chitin-induced rice immunity. According to the report of Cell Host Microbe 13: 465-476 ) ”OsRacGef1 was able to induce a variety of disease-resistant functions such as“ rice blast ”by inducing mutations to active OsRacGEF1 (OsRacGEF1 S549D) by inducing single-base substitution using“ self-marker-free targeting gene modification technology ” There is a high possibility of producing practical rice seeds in as little as 10 months to 1 year.

(1) Construction of basic vector for modifying targeting gene In rice targeting by positive-negative selection in this case, pINA135D (FIG. 12) was used as a basic vector. pINA135D is an advanced version of pINA134 (JPW02003-020940), and has a function as a destination vector corresponding to the LR recombination reaction of the Gateway cloning system.

  pINA135D is a pVS1 vector and T-DNA that has a replication origin (Ori) that can replicate in both Escherichia coli and Agrobacterium tumefaciens, a selectable marker that is resistant to spectinomycin and ampicillin. Constructed from the area. Inside the RB (Figure 12; RB) and LB (Figure 12; LB) of the T-DNA region, two DT-A (Diphtheria toxin A fragment; Figure 12; DT-A) genes, which are negative selection markers, are opposite to each other. It is arranged in the direction. These DT-A genes have a 35S terminator (Fig. 12; t35S) and are respectively located in the ubiquitin promoter and ubiquitin intron (Fig. 12; pUbi-iUbi) and in the 35S promoter and Himacatalase intron (Fig. 12; p35S-iCat). Connected. In the center of T-DNA, hpt (hygromycin phosphotransferase (Fig. 12; hpt) gene as an actin promoter, actin intron (Fig. 12; pAct) and 35S terminator (Fig. 12 t35S) as a positive selection marker in plant cells. A transcription termination sequence derived from a corn transposon (Fig. 12; En / Spm) is linked downstream of the positive selection marker, and further downstream of the Gateway vector conversion system (Life Technologies) Reading. The sequence derived from frame A is incorporated, and the internal attR1 and attR2 sequences function as target sequences during the LR recombination reaction.2 of the Cre-loxP site-specific recombination system at both ends of the positive selection marker and En / Spm sequences Two loxP (34bp) sequences (Figure 12; loxP) are ligated in the forward direction, and between the loxP and the negative selectable marker, there is a targeting homologous recombination sequence. Two multiple cloning sites (FIG. 12; I-CeuI, PacI, HindIII, SrfI) and (FIG. 12; PmeI, SpeI, AscI, AvrII, I-SceI) for inserting the sequence are arranged.

(2) Construction of OsRacGEF1 gene modification vector pGEF1-S549D-D using targeting basic vector (pINA135D) As shown in FIG. 13A, two multiple cloning sites of pZEN-LP2, PmeI and SrfI, homologous to OsRacGef1 and surrounding region PGEF1-S549D-D was constructed by incorporating the correct sequence. The 5 ′ and 3 ′ homologous recombination regions were prepared by nested PCR using genomic DNA common to rice varieties “Kinnamaze” and “Nippon Hare” as templates. In all PCRs, PrimeStar GXL (Takara Bio) was used as the DNA polymerase, and the reaction conditions followed the manufacturer's protocol.

  The 5 ′ homologous recombination region is composed of a region corresponding to 5′UTR to 3′UTR of OsRacGef1, and by modifying the nucleotide sequence in the 5th exon, the GT variant is mutated OsRacGEF1 (S549D ). Furthermore, a PacI site (5 ′ end) and a SrfI site (3 ′ end) used for incorporation into pINA135D are added. For the 5 ′ homologous recombination region, first, a sequence including the OsRacGEF1 gene and a neighboring region was amplified using the primer sets GEF1 5′1st-F and GEF1 5′1st-R as 1st PCR. The obtained amplification product was diluted 1/1000 and used as a template for DNA sequence modification by site-directed mutagenesis by PCR. About 1000 bp amplified product obtained by primer set GEF1 5'-F and GEF1 S549D-R and about 90 bp amplified product obtained by GEF1 S549D-F and GEF1 5'-R were mixed after dilution of 1/1000 respectively. And used as a 3rd PCR template. In the subsequent 3rd PCR, the 3024 bp DNA fragment obtained using the primer sets GEF1 5'-F and GEF1 5'-R was cloned into pCR BluntII TOPO (Zero Blunt TOPO PCR cloning kit, Life Technologies) and the DNA sequencer After confirming the nucleotide sequence, a clone having the sequence as designed was selected.

  The 3 'homologous recombination region consists of a region downstream from the 3'UTR of OsRacGef1, with the addition of the PmeI site (5' end) and AscI site (3 'end) used for integration into pINA135D. ing. For the 3 ′ homologous recombination region, first, a sequence including the 3 ′ end side of the OsRacGEF1 gene and a neighboring region was amplified using the primer sets GEF1 3′1st-F and GEF1 3′1st-R as 1st PCR. The obtained amplification product was diluted 1/1000 and used as a template for nested PCR. The 3270 bp DNA fragment amplified by nested PCR using the primer sets GEF1 3'-F and GEF1 3'-R was cloned into PCR BluntII TOPO and analyzed by nucleotide sequence analysis. A clone matching the genome sequence was selected. The obtained 5 'and 3' homologous recombination regions were incorporated between the PacI-SrfI sites and the PmeI-AscI sites of pINA135D to construct pGEF1-S549D-D (Fig. 13A).

(3) Construction of control basic vector pGEF1-Con-D.
In order to identify the targeted homologous recombination mutant sequence by PCR, targeting homologous recombination is confirmed using the homologous recombination fragment obtained by amplifying the genomic sequence region linked to the targeting insertion sequence region and the homologous recombination region as a marker. There is a need. In such a PCR method, a template vector artificially having a homologous recombination region is used as a positive control for the PCR reaction. Therefore, a control vector pGEF1-Con-D was constructed in which several hundred bp of genomic sequences outside the 5 ′ and 3 ′ homologous recombination sequences of the targeting vector pGEF1-S549D-D were extended. This vector was prepared according to the above-mentioned procedure for preparing pGEF1-S549D-D, and the steps up to the preparation of the 1st PCR product at the time of cloning of the 5 ′ and 3 ′ homologous regions were made common. The 5 'sequence to be incorporated into pGEF1-Con-D is the primer set GEF1 5' Con-F, which is a 1/1000 dilution of 1st PCR product using GEF1 5'1st-F and GEF1 5'1st-R. And GEF1 5′-R was amplified by nested PCR. The 3'-side sequence was amplified by nested PCR using primer sets GEF1 3'-F and GEF1 3'Con-R using 1/1000 dilution of GEF1 3'1st-F and GEF1 3'1st-R as a template. did. The obtained DNA fragments of 5 ′ side sequence 3820 bp and 3 ′ side sequence 4360 bp were cloned into pCR Blunt II TOPO to confirm the base sequence, and clones were selected. Subsequently, each homologous recombination region was integrated between the PacI-SrfI sites and the PmeI-AscI sites of pINA135D to construct pGEF1-Con-D. This vector has a 5 ′ sequence extended by about 400 bp and a 3 ′ sequence extended by about 660 bp relative to pGEF1-S549D-D. The PCR reaction conditions for detecting the mutated mutant sequence are optimized (FIG. 13B).

(4) Preparation of self-marker-free genetically modified targeting vector pGEF1-S549D-pLP2-Cre and control vector pGEF1-Con-pLP2-Cre
pGEF1-S549D-D and pGEF1-Con-D are destination vectors in which attR1 and attR2 sequences are located downstream of En / Spm. Due to the LR recombination reaction of Gateway cloning system, between entry clones attL1 and attL2 sequences Of DNA fragments can be incorporated. As a result, pGEF1-S549D-pLP2-Cre and pGEF1-Con-pLP2-Cre were prepared by incorporating an inducible Cre expression unit from pLP2-Cre (iKOR1) -t35S / pDONR Zeo (previous [Example 1]) (FIGS. 14A and 14B).

(5) Introduction of pGEF1-S549D-pLP2-Cre vector into Agrobacterium
pGEF1-S549D-pLP2-Cre was introduced into Agrobacterium by electroporation according to the procedure described in [Example 1]. After selecting clones with pGEF1-S549D-pLP2-Cre, they were grown in 5 mL of YEB liquid medium containing 125 mg / L spectinomycin and dispensed into microtubules as glycerol stocks (final concentration 35%), Stored at -80 ° C.

(6) Modification of OsRacGEF1 gene by targeting (preparation of rice callus for transformation)
Using rice (Oryza sativa.L Japonica varieties) Nipponbare seeds about 800 and Kinnan style “Kinmaze” seeds about 500 seeds, sterilized in the same way as in [Example 1] and then placed on 2N6 medium. The cells were cultured in the dark at 31.5 ° C. for 4 weeks to induce a sufficient amount of callus derived from scutellum cells.

(Preparation of Agrobacterium for transformation)
Agrobacterium introduced with pGEF1-S549D-pLP2-Cre was streaked in AB medium supplemented with 125 mg / L spectinomycin and grown for 3 days. As in [Example 1], AAI containing acetosyringone A bacterial solution used for inoculation was prepared by suspending in a liquid medium.

(Inoculation of Agrobacterium to rice callus, co-culture and sterilization)
All transformation and callus transfer operations were performed in a weak light environment (150-200 lux) to maintain the suppression of LP2 promoter activity, and all callus were cultured in the dark.
Callus derived from “Nipponbare” and Kinnanze “Kinmaze” scutellum, respectively, 194.72g and 145.09g (fresh weight, FWg) were collected in a sterile beaker and inoculated with Agrobacterium as in [Example 1]. Co-culture with N6 co-culture medium was performed. In order to sterilize Agrobacterium from the callus, it was washed for 20 minutes while mixing in a beaker with 2 L of sterilized water added with 100 mg / L of vancomycin. Thereafter, the callus was collected on a stainless mesh, water around the callus was removed with a paper towel, and the same sterilization operation was repeated again using sterilized water containing vancomycin as described above. Subsequently, the callus after sterilization was placed on a selective medium N6SE containing 50 mg / L of hygromycin as in [Example 1] and cultured at 31.5 ° C. in the dark for 7 weeks.

After 50 days from the start of positive / negative selection, it became possible to visually distinguish proliferating or dead callus. 812 proliferating callus of “Nipponbare” were individually selected and transferred to 24-well plates dispensed with N6SE selective medium containing 50 mg / L of hygromycin. Since 486 callus out of 812 continued to grow in a 24-well plate, a line number was assigned to each callus, and an automatic nucleic acid extraction device (PX-80, Kurashiki Boseki Co., Ltd.) ) To extract DNA. In Kinnanze “Kinmaze”, 192 proliferating calli were obtained and DNA was extracted in the same manner. These recombinant calli are expected to correspond to one of the following four types of genome variants.
1) Homologous recombination with targeting vector pGEF1-S549D-pLP2-Cre at the target gene OsRacGef1 (Figure 15C)
2) After homologous recombination with the targeting vector pGEF1-S549D-pLP2-Cre at the position of OsRacGef1, the LP2 promoter was activated by light and redifferentiation induction, and the Cre gene was expressed, and pGEF1-S549D-pLP2-Cre LoxP internal sequence deleted (Figure 15E)
3) A target vector pGEF1-S549D-pLP2-Cre in which the negative selection markers at both ends are mutagenized and inserted at random positions in the genome (FIG. 16A)
4) After random insertion as in 3) above, the Cre gene downstream of the LP2 promoter was expressed, thereby deleting the loxP internal sequence of pGEF1-S549D-pLP2-Cre (FIG. 16B)
Among these, 1) and 2) are the variants that are likely to succeed in targeting the OsRacGEF1 gene and successfully introduce the S549D mutation. In addition, in 2) and 4), the two loxPs derived from pGEF1-S549D-pLP2-Cre are deleted, and hygromycin resistance is changed to sensitivity, so that cells that cannot grow on hygromycin selective medium N6SE are expected to appear. The

In addition, the following genome variants are expected to be obtained, albeit at a low frequency.
5) Only one 5 'or 3' homologous region of the targeting vector pGEF1-S549D-pLP2-Cre causes homologous recombination with the target region in the genome, while non-homologous end joining occurs at the other end of the vector. The occurrence of recombination (OSI; One sided invasion) was also predicted.
6) In addition, ectopic recombination (Ectopic targeting), in which OsRacGef1 and the region of similar base sequence of rice genome are incorporated into pGEF1-S549D-pLP2-Cre by homologous recombination and then inserted at random positions in the genome. ) Was predicted to be included in rare cases.

  As a result of observing 486 proliferating callus derived from “Nihonbare” and 192 proliferating callus derived from “Kinnan-style” that moved to the 24-well plate after 50 days from the start of positive / negative selection, whitening and cell division rate were observed. Cells that were reduced or completely stopped dividing were observed. As shown in 2) and 4) above, these changes were achieved when OsRacGEF1 gene targeting was successful or the negative selection marker on the T-DNA region of pGEF1-S549D-pLP2-Cre was inactivated in some way. It was speculated that the mutants randomly inserted into the rice genome, due to Cre-loxP site-specific recombination, deleted the hpt sequence between loxPs, reflecting the change to hygromycin sensitivity. Rice callus has a high redifferentiation ability, and it is known that when callus growth is continued, redifferentiation occurs even in the absence of plant hormones. LP2 promoter activation was caused by the fact that callus growth was continued in a 24-well plate with a small amount of medium as described above. As a result, medium components such as auxin were metabolized with callus growth, and the physiological state of callus was This is thought to be due to a change from proliferation to regeneration. For this reason, calli in such a state are distinguished and referred to as “proliferation redifferentiation callus”.

  Subsequently, PCR selection conditions were set in order to select OsRacGEF1 targeting variants. First, several sets of primers designed to select both callus that succeeded in targeting (FIG. 18B) and callus from which extra sequences such as the hpt gene were deleted by Cre-loxP recombination (FIG. 18C) were prepared (FIG. 18). 17B). Next, PCR was performed using the above primer set using the control vector pGEF1-Con-pLP2-Cre as a template, and the most suitable primer set and PCR reaction conditions were determined. As a result, primer sets GEF1 5-Junction No. 1-F and GEF1 5-Junction No. 1-R were used for detection of homologous recombination on the 5 ′ side. Expected to be amplified (FIG. 17B). On the other hand, GEF1 3-Junction No. 1-F and GEF1 3-Junction No. 1-R were used to detect homologous recombination on the 3 'side, and it was predicted that a 3884 bp DNA fragment would be amplified (Fig. 17B). In all selected PCRs, Tks Gflex (Takara Bio) is used as the DNA polymerase, and the PCR reaction conditions are 98 ° C for 30 minutes as initial denaturation, 98 ° C for 30 seconds, and 68 ° C for treatment. 15-second annealing and 68 ° C 4-minute extension was 40 cycles.

  Results of detection of 5'-side homologous recombination under the above PCR conditions using genomic DNA extracted from 486 lines of growth-redifferentiation callus derived from "Nipponbare" and 192 lines of growth-regeneration callus derived from "Kinnanfu" From the regenerated callus derived from “Nipponbare”, a band of 3324 bp was detected in 30 lines (6.2%). Next, PCR was performed to detect 3 ′ homologous recombination of these 30 strains. As a result, 18 strains (3.7%) were considered that homologous recombination occurred on both the 5 ′ and 3 ′ sides. %) Selected (Figure 17C, Table 3). For these 18 lines, the success or failure of deletion of the internal hpt sequence between loxPs was confirmed by PCR analysis using a primer set, GEF1 3 ′ 1st-F and GEF1 5 ′ 1st-R. As a result, 6 lines (6/486; 1.2%) were targeted as shown in [1] above, and the 6924 bp region retaining the sequence between loxP such as hpt resulted in PCR amplification products due to repetitive sequences. On the other hand, only the 369 bp band representing the wild type of OsRacGef1 was detected, indicating that these were heterozygotes that were successfully targeted to OsRacGEF1 (FIGS. 18A, B, C, D). On the other hand, in 12 lines (12/486; 2.5%), after targeting occurred as in [above 2]], the sequence between loxP such as hpt was deleted and one loxP (34bp) sequence was left in the 3'UTR A band of 419 bp indicating this and 369 bp indicating OsRacGef1 wild type were detected (FIGS. 18A, B, C and D). In addition, the 419 bp PCR product was cloned into pCR4 Blunt TOPO (Life Technologies), the base sequence of multiple clones was analyzed with a DNA sequencer, and the marker gene was successfully deleted by Cre-loxP site-specific recombination. confirmed. Therefore, these 14 lines were shown to be heterozygotes that succeeded in targeting OsRacGEF1 followed by Cre-loxP site-specific recombination. Similarly, 44 strains (23.0%) that were thought to have undergone homologous recombination on both the 5 'and 3' sides were selected from 192 strains of growth-redifferentiated callus derived from "Kinnankaze", of which 20 strains were selected. (20/192; 10%) after targeting occurred, the sequence between loxP such as hpt was deleted and one loxP (34bp) sequence was left in 3'UTR, and 369bp indicating the wild type of OsRacGef1 Bands were detected (Table 3).

  Based on the above results, 195.72g fresh heavy rice callus derived from 808 Nihonbare seeds was self-marker-free targeting vector pGEF1-S549D-pLP2-Cre, and Agrobacterium-targeted transformation was performed. As a result of obtaining 486 redifferentiated callus lines and performing PCR screening, targeting occurs in the OsRacGef1 gene region, and further callus culture results in the expression of Cre under the control of the LP2 promoter that is activated by light and induction of redifferentiation. As a result, 12 lines of regenerative callus with successful deletion of the loxP internal hpt sequence were obtained. Similarly, targeting transformation of 145.09g fresh heavy rice callus derived from 500 seeds of "Golden style" resulted in 192 positive and negatively selected growth redifferentiated callus, and targeting occurred in the OsRacGef1 gene region, 20 lines of proliferative redifferentiation callus were obtained in which Cre was expressed under the control of the LP2 promoter and the loxP internal hpt sequence was successfully deleted. These 12 lines derived from “Nipponbare” and 20 lines derived from “Kinnankaze” were further differentiated in MSRE medium (MS mix (Sigma) 4.4 g / L, casamino acid 2000 mg / L, shout). Claus 30.0g / L, Sorbitol 30.0g / L, NAA 0.02mg / L, Kinetin 2mg / L, 4.0g / L, pH5.8), 25 ℃, 11 hours light (8000lux) / 13 hours dark By culturing for 3-4 weeks, we succeeded in plant regeneration from all 12 growth-redifferentiated callus derived from "Nipponbare", which preceded the experiment, and obtained OsRacGEF1-modified rice plants with normal fertility .

  The sequences of primers and the like used in the above examples are shown in the following table.

The entire contents of the following documents are incorporated herein by reference.
(1) Zuo J, Niu QW, Moller SG, Chua NH (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nature Biotechnology 19: 157-161
(2) Usuda K, Wada Y, Ishimaru Y, Kobayashi T, Takahashi M, Nakanishi H, Nagato Y, Mori S, Nishizawa NK (2009) Genetically engineered rice containing larger amounts of nicotianamine to enhance the antihypertensive effect. Plant Biotechnology Journal 7 : 87-95
(3) Guan JC, Jinn TL, Yeh CH, Feng SP, Chen YM, Lin CY (2004) Characterization of the genomic structures and selective expression profiles of nine class I small heat shock protein genes clustered on two chromosomes in rice (Oryza sativaL .). Plant Molecular Biology 56: 795-809
(4) Thilmony R, Guttman M, Thomson JG, Blechl AE (2009) The LP2 leucine-rich repeat receptor kinase gene promoter directs organ-specific, light-responsive expression in transgenic rice. Plant Biotechnology Journal 7: 1-16
(5) Terada R, Nagahara M, Furukawa K, Shimamoto M, Yamaguchi K, Iida S (2010) Cre-loxPmediated marker elimination and gene reactivation at the waxy locus created in rice genome based on strong positive-negative selection. Plant Biotechnology 27 : 29-37
(6) Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S (2002) Efficient gene targeting by homologous recombination in rice. Nature Biotechnology 20: 1030-1034
(7) Itoh H, Nonoue Y, Yano M, Izawa T (2010) A pair of floral regulators sets critical day length for Hd3a florigen expression in rice. Nature Genetics 42: 635-638
(8) Shimatani Z, Takagi K, Eun CH, Maekawa M, Takahara H, Hoshino A, Qian Q, Terada R, Johzuka-Hisatomi Y, Iida S, Tsugane K (2009) Characterization of autonomous Dart1 transposons belonging to the hAT superfamily in rice. Molecular Genetics and Genomics 281: 329-344
(9) Akamatsu A et al. (2013) An OsCEBiP / OsCERK1-OsRacGEF1-OsRac1 Module is an essential early component of chitin-induced rice immunity. Cell Host Microbe 13: 465-476

Claims (6)

A homologous recombination vector for use in a plant,
A region for expressing a positive selection marker affirming that it is a homologous recombinant,
A region for expressing a negative selectable marker that affirms being a heterologous recombinant;
A site-specific recombination system comprising a site-specific enzyme region having a region encoding a site-specific enzyme and an identification site for the site-specific enzyme ;
With
The region for expressing the positive selection marker and the site-specific recombination system are configured to be capable of homologous recombination, and the coding region of the site-specific enzyme is arranged under the control of a tissue differentiation-inducible promoter. And a homologous recombination vector configured to delete the region for expressing the positive selection marker and the site-specific enzyme region when the tissue differentiation-inducible promoter is activated .   The homologous recombination vector according to claim 1, wherein the tissue differentiation-inducible promoter is a light-inducible promoter.   The homologous recombination vector according to claim 1 or 2, wherein the tissue differentiation inducible promoter is a greening tissue differentiation inducible promoter.   The recombinant vector according to any one of claims 1 to 3, wherein the tissue differentiation-inducible promoter is an LP2 gene promoter.   The recombinant vector according to any one of claims 1 to 4, wherein the identification site is configured to sandwich the site-specific enzyme region and the positive selection marker region. A method for producing a homologous recombinant plant, comprising:
The homology according to any one of claims 1 to 5, wherein the plant body is capable of maintaining a desired differentiation region in an undifferentiated environment in which an undifferentiated state can be maintained and the tissue differentiation-inducible promoter is not activated. Introducing a recombinant vector into the plant, and obtaining a first homologous recombinant plant using the negative selection marker and the positive selection marker;
Culturing the first homologous recombination plant in a differentiation-inducing environment capable of inducing differentiation of the plant and activating the tissue differentiation-inducing promoter to obtain a second homologous recombination plant. When,
A manufacturing method comprising: