JP2006280282A - Method for producing gene-transferred cell, tissue or plant without being affected by selected marker gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the efficiency of production of a marker-free tissue in the method for producing a plant by utilizing a malformation-inducing gene. <P>SOLUTION: A target gene, a selected marker gene except a cytokinin-synthesizing gene, and a vector for introducing the target gene having a leaving site and the selected marker gene arranged at the leaving site are introduced to a plant cell, and the cell is selected by using the expression of the selected marker as an index. Thereafter, a vector for removing the selected marker, having the malformation-inducing gene, an eliminase gene and a leaving site, wherein the malformation-inducing gene and the eliminase gene are arranged at the leaving site is introduced to the plant cell, and the marker-free gene-introduced cell or tissue is selected by using the malformation of the cultured tissue caused by the expression of the malformation-inducing gene as an index. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a transgenic cell, tissue or plant in which the influence of a selection marker gene is excluded.

  In recent years, the progress of gene transfer technology in the field of plants has been remarkable, and recombinant crops with new traits have been created by gene transfer and have already been cultivated on a large scale in the field.

  In gene introduction into a plant, a gene intended to be introduced into a plant cell in order to impart a predetermined character as described above, that is, a target gene is introduced into the plant cell together with the selection marker gene, and the selection marker for the plant cell is introduced. The presence / absence of gene introduction, that is, the presence / absence of expression of a selection marker gene in the plant cell, is usually used as an index for introduction of the target gene.

  However, the selection marker gene used for the selection of the transgenic cell continues to exist in the cell, and is further transmitted to the cells of the transgenic plant created from such a cell. Give an obstacle.

  For example, in the case of plant breeding using gene transfer technology, once gene transfer is performed using a selected marker gene and a gene transfer plant is created, for this gene transfer plant, It is never possible to introduce a gene using the same selection marker gene as the previously used selection marker gene. Since this selection marker gene already exists in the plant cell, even if the same selection marker gene is introduced together with the new target gene, the new target gene will not be introduced. This is because it is always expressed in the cells and tissues of the plant and can no longer be used as an index for introducing a new target gene. Therefore, such a multiple introduction of genes must use a different selection marker gene for each introduction of each gene. However, there are only a limited number of selectable marker genes that can be used at present, and this limits the number of times that a gene can be introduced into a plant in multiple ways, giving it more useful and practical traits. Attempts to grant are difficult. In the first place, as long as the transgenic plant retains the selection marker gene, no matter how the gene transfer technology is used, no matter how a breakthrough trait is imparted, this should be used for food, etc. It is very difficult at present to obtain social consent.

  Therefore, after the selection of the transgenic cell, the selection marker gene used for the selection is removed from the DNA such as the chromosome in which it exists and functions, and the influence thereof is introduced into the transgenic cell, tissue, and plant body. It can be said that the technology excluded from the above is indispensable for new development of plant breeding using genetic engineering technology.

  As such a technique, the present applicant previously used a morphological abnormality-inducing gene as a selection marker gene, and combined this selection marker gene with a transposon, a site-specific recombination system, or the like when introducing a gene into a plant cell. In other words, a method for creating a so-called marker-free transgenic plant in which the influence of a selected marker gene is eliminated has been proposed (for example, Patent Document 1 and Patent Document 2).

  According to this method, the selection marker gene introduced into the chromosomal DNA of the plant cell together with the target gene is removed from the introduced DNA by the action of a transposon or site-specific recombination system after a certain period of time, Moreover, the introduction and removal of the DNA can be easily detected with the naked eye as a change in the shape of the tissue derived from the transgenic cell. In other words, in this method, plant cells after gene transfer treatment are first cultured under appropriate conditions, and a tissue exhibiting a morphological abnormality is selected from the tissues produced by the growth of the cultured cells. Plant cells and tissues in which a marker gene and a target gene are introduced into chromosomal DNA can be obtained, and then a tissue exhibiting this morphological abnormality is also cultured under appropriate conditions, and then arises from this tissue In this case, if a tissue showing normal morphology is selected, the selected marker gene is removed from the DNA and only the target gene remains in this chromosomal DNA, etc. In other words, the selected marker gene Marker-free transgenic cells and tissues in which the effects of the above are eliminated, and marker-free transgenic plants can be obtained from such cells and tissues. Rukoto can.

JP-A-9-154580 JP-A-10-327860

  In the above method, introduction and removal of the selection marker gene into the chromosomal DNA and the like result in a change in the shape of the tissue derived from the transgenic cell, since this selection marker gene is a morphological abnormality inducing gene. This is because, when introduced into and expressed in the chromosomal DNA or the like, the direction of proliferation / differentiation of the plant cell is distorted to cause unusual morphological differentiation.

  However, when a cytokinin synthesis gene, which is one of the morphological abnormality-inducing genes, is introduced into the chromosomal DNA of a plant, its expression product, cytokinin, also affects adjacent non-transgenic cells. Not only non-transgenic cells but also abnormal morphological differentiation may occur. For this reason, in the above method, when a cytokinin synthetic gene is used as a morphological abnormality induction gene that is a selection marker gene, a selection marker may be included in a tissue exhibiting morphological abnormality caused by a plant cell after gene introduction treatment. There is a high possibility that a tissue composed of non-gene-transfected cells into which not only the gene of interest is introduced but also a gene-transfected cell and such non-gene-transfected cells are mixed, and as a result, There has been a problem that the efficiency of creating marker-free cells and tissues is reduced.

  In view of such problems, the present invention has been made for the purpose of improving marker-free tissue creation efficiency in a marker-free gene-transfected cell, tissue or plant production method using a morphological abnormality-inducing gene. is there.

That is, the present invention is a method for producing a gene-introduced cell or tissue in which the influence of a selection marker gene is eliminated, and is characterized by undergoing the following steps (A) to (D).
(A) A process of introducing a vector comprising a target gene, a selection marker gene other than a cytokinin synthesis gene, and a detachment site, wherein the selection marker gene is present in the detachment site ( B) Process of culturing plant cells introduced with the vector in (A) and selecting plant cells introduced with the target gene using the expression of the selection marker gene as an indicator (C) Morphological abnormality induction gene, desorption enzyme A process of introducing a vector having a gene and a detachment site, wherein the morphological abnormality-inducing gene and the detachment enzyme gene are present in the detachment site into the plant cell selected in (B) (D) In the process (E) and (D) of culturing the plant cell introduced with the vector in (C), and as a result, selecting an abnormally shaped tissue generated from the tissue obtained by the proliferation of the cell. Selected Were cultured with tissue abnormal form, the process of selecting a resulting was normal morphology tissues

  In the present invention, first, a vector having a selection gene other than a target gene and a cytokinin synthesis gene and a desorption site, and having the selection marker gene arranged at the desorption site (hereinafter referred to as a target gene introduction vector). ) Is introduced into the plant cell, and the plant cell having the target gene introduced into its chromosomal DNA or the like is selected using the expression of the selection marker gene as an index, so that the gene-transferred cell can be reliably selected.

  Furthermore, the present invention has a morphological abnormality-inducing gene, a desorption enzyme gene, and a detachment site, and the morphological abnormality-inducing gene and the desorption enzyme gene are selected from the transgenic cells selected as described above. Since a vector (hereinafter referred to as a selection marker removal vector) arranged at the detachment site is introduced, the selection marker gene introduced into the chromosomal DNA of the gene-transferred cell together with the target gene is removed from the chromosomal DNA, etc. .

  Therefore, according to the present invention, it is possible to reduce the possibility of selecting a tissue composed of non-gene-transduced cells or a chimeric tissue in which gene-transfected cells and non-gene-transfected cells are mixed from plant cells after gene introduction treatment, In this process, the efficiency of creating marker-free cells and tissues can be improved.

  In addition, the removal of the selection marker gene can be easily detected with the naked eye as a change in the shape of the tissue derived from the transgenic cell by the function of the morphological abnormality inducing gene arranged in the vector for removing the selection marker. In addition, the organization can be easily created.

  In the present invention, the selection marker gene is introduced and removed in two stages. In other words, the selection marker gene is first introduced together with the target gene using the target gene introduction vector, and then the selection marker gene is removed using the selection marker removal vector. It is practically very convenient.

  Hereinafter, the present invention will be described in detail.

  First, the structure of the vector used in the present invention will be described.

  In the present invention, the target gene, a selectable marker gene other than the cytokinin synthesis gene, and a detachment site, the target gene introduction vector having the selection marker gene arranged at the detachment site, a morphological abnormality induction gene, A selective marker removal vector having a desorption enzyme gene and a desorption site, in which a morphological abnormality induction gene and a desorption enzyme gene are arranged at the desorption site, is used.

  There is no particular limitation on the type of target gene to be distributed in the target gene introduction vector, that is, the type of target gene that can be introduced into plant cells by the method of the present invention. For example, C2C1A gene (peroxidase gene) that imparts high growth potential, gals gene that imparts drought tolerance, codA gene that imparts salt and drought tolerance, desaturase gene that imparts cold tolerance, and other agriculturally superior traits A gene capable of imparting a gene and an agriculturally superior trait are not necessarily imparted, but various genes such as a gene required for the study of gene expression mechanisms can be selected. Furthermore, although not strictly a gene, a DNA factor having a predetermined structure and performing a predetermined function in a cell can also be introduced into a plant cell as a target gene in the present invention. For example, in such a case, for the purpose of suppressing the expression of a gene, a case where a structure in which the transcriptional regulatory factor of the gene is arranged in the antisense direction is introduced.

As the selection marker gene used for the target gene introduction vector, any marker gene can be used as long as it can be used as an index when the target gene is introduced into the chromosomal DNA of a plant cell. Can do. Examples of such selectable marker genes include antibiotic resistance genes such as a neomycin phosphotransferase gene (NPTII gene) that confer resistance to kanamycin, a hygromycin phosphotransferase gene that confer resistance to hygromycin, and bialaphos. ), A so-called drug resistance gene such as a herbicide resistance gene such as a phosphinothricin acetyltransferase gene (bar gene) that imparts resistance to), a green fluorescent protein gene (GFP gene), a β-glucuronidase gene (GUS gene) ) gene to allow visual selection of the luciferase gene and the like, Agrobacterium rhizogenes (Agrobacterium rhizogenes:.. hereinafter, the a rhizogenes abbreviated) exist on T-DNA of To rolA, etc. rol gene consisting rolB and / or rolC like, can be used as general-purpose. Further, two or more kinds of these selection marker genes may be used in combination. However, a cytokinin synthesis gene cannot be used as a selection marker gene used for the target gene introduction vector of the present invention. When a cytokinin synthesis gene is used as a selection marker gene, a tissue composed of non-gene-transferred cells or a chimera tissue in which gene-transferred cells and non-gene-transferred cells are mixed is selected from plant cells after gene transfer treatment. This is because the possibility of the creation of marker-free cells and tissues decreases in the subsequent process.

  On the other hand, as the morphological abnormality inducing gene used for the selection marker removing vector, all genes that cause morphological differentiation different from normal by deviating the direction of proliferation / differentiation of the host cell by its expression can be used. Such genes typically include plant hormone synthesis genes and plant hormone signals that cause the host plant to hatch, disrupt the bud dominance, change pigments, root cancer, hairy roots, leaf ripples, etc. Plant hormone-related genes such as transduction genes can be exemplified.

Here, the plant hormone synthesis gene means a gene that encodes a protein involved in the synthesis of plant hormones, such as a cytokinin synthesis gene, and is an ipt (isopentenyltoransferase) present in phytopathogenic fungi such as Agrobacterium. Gene (AC Smigocki, LD Owens, Proc. Natl. Acad. Sci. USA, 85: 5131, 1988 ), iaM (tryptophan monooxygenase) gene (HJ Klee et al., GENES & DEVELOPMENT, 1:86, 1987), gene 5 genes (H. kerber et al., EMBO Journal, 10: 3983, 1991), gene 6b gene (PJJ Hooyaas et al., Plant Mol. Biol., 11: 791, 1988), and rol gene group (FF White et al., J. Bacteriol., 164: 33, 1985), iaL (indoleacetic acid-lysine synthetase) gene (A. Spena et al., Mol.) present in Pseudomonas syringae subsp. savastanoi . Gen. Genet., 227: 205, 1991), Furthermore, homeobox genes and phytochrome genes of various plants are known.

Plant hormone signal transduction genes encode sensors that recognize the presence of plant hormones such as gibberellin, ethylene, auxin, and cytokinin, and proteins related to a series of signal transduction pathways that transmit information from the sensor. gene means that an ethylene receptor gene ETR1 gene (C. Chang et al, Science, 262:. 539,1993), believed to be a cytokinin receptor gene CKI1 gene (T. Kakimoto, Science 274: 982, 1996) and its mutants (ex. CKI2 gene) and GCR1 gene (S. Plakidou-Dymock et al., Current Biology, 8: 315, 1998), IBC6 gene and IBC7 gene (I. Brandstatter, JJ Kieber, The Plant Cell, 10: 1009, 1998).

Any of these morphological abnormality-inducing genes can be used in the selection marker removal vector of the present invention. However, in particular, the ipt gene that causes disruption of apical dominance causes a characteristic morphological abnormality. It is preferable as a morphological abnormality-inducing gene used for plants because it prevents re-differentiation of plant individuals.

  Further, in the target gene introduction vector and the selective marker removal vector of the present invention, the desorption site is desorbed from the DNA by the action of a specific desorption enzyme when it is incorporated into the DNA of the plant cell. It refers to a DNA region having a certain range. As such a desorption site and desorption enzyme gene, for example, those derived from site-specific recombination systems, transposons, and the like can be used.

  The site-specific recombination system recognizes a recombination enzyme recognition sequence having a characteristic DNA sequence and a recombination enzyme recognition sequence. When two or more recognition sequences exist, recombination between the sequences is performed. It consists of two elements: a catalytic recombination enzyme. When two recombination enzyme recognition sequences are present on the same DNA molecule at a certain interval in the same direction, the region between them is detached from the DNA molecule. Therefore, in the present invention, a region sandwiched between two recombination enzyme recognition sequences oriented in the same direction in the site-specific recombination system is recognized as a desorption site. At this time, the recombination enzyme recognition sequence is recognized. The recombinase acting as a desorption enzyme is used as a desorption enzyme, and the gene encoding the recombination enzyme is used as a desorption enzyme gene. In addition, in order to place a selection marker gene, or a morphological abnormality inducing gene and a desorption enzyme gene in a desorption site, these genes are sandwiched between the two recombination enzyme recognition sequences oriented in the same direction as described above. It may be arranged in a region that does not affect the detachment.

In addition, as a combination of a recombination enzyme recognition sequence and a recombination enzyme that recognizes and acts on this recognition sequence, Cre / lox system, R / RS system, FLP / FRT system, cer system, fim system, etc. Only those isolated from microorganisms such as phages, bacteria (eg E. coli), yeast, etc. (as a review, NL. Craig, Annu. Rev. Genet., 22:17, 1988) The existence is not yet known. However, even when these recombinase recognition sequences and recombinases isolated from microorganisms are introduced into a species different from the species from which they are derived, that is, when introduced into plants or animals, their behavior in the organism is the same. In the present invention, a combination of such a recombination enzyme recognition sequence and a recombination enzyme can be freely selected to form a desorption site and to be used as a desorption enzyme. be able to. Among these, the Cre / lox system and the R / RS system are preferred as such combinations because they are widely used in animals, plants, and the like and have high recombination efficiency. However, in the present invention, as the recombination enzyme recognition sequence, not only the wild type recognition sequence but also a mutant recognition sequence in which the DNA sequence is partially modified can be used. However, at this time, the mutant recognition sequence must be one that can be recognized by a recombinant enzyme used as a desorption enzyme.

  On the other hand, when a transposon is used as the desorption site and desorption enzyme gene of the present invention, a non-autonomous transposon is used. At this time, the transposon body can be used as a desorption site, its transferase as a desorption enzyme, and the transferase gene as a desorption enzyme gene. In order to arrange the selection marker gene, or the morphological abnormality inducing gene and the desorption enzyme gene in the desorption site, these genes may be arranged in the transposon body so as not to affect the desorption. In this sense, the position of these genes is preferably arranged in the transposon body and downstream of the terminal region to which the transferase is bound. A transposon is usually detached from its existing DNA by the action of a transferase, and then transferred to a new site on the DNA, but loses its function without being transferred with a certain probability, and disappears. In some cases, the present invention uses such a transposon transfer error.

Non-autonomous transposons include Ds and dSpm (H. -P. D〓ring and P. Starlinger, Ann. Rev. Genet., 20: 175, 1986), and many plants such as corn, snapdragon and morning glory. It has been isolated (eg, Y. Inagaki et al., Plant Cell, 6: 375, 1994). Even when these transposons are introduced into the chromosomal DNA of a plant of a different type from that of the plant from which they are derived, it is known in many cases that they exhibit their abilities and are detached and transferred (for example, B. Baker et al., Proc. Natl. Acad. Sci. USA, 83: 4844, 1986), so that in the present invention, such a non-autonomous transposon can be freely selected and used.

  Next, a method for producing a gene-introduced cell and tissue in which the influence of the selection marker gene according to the present invention, which is performed using the above vector, is described.

In this method, first, a target gene introduction vector having the above structure is introduced into a plant cell to introduce the target gene and a selectable marker gene into its chromosomal DNA or the like. As an introduction method into plant cells, for example, in addition to direct introduction methods by physical and chemical methods such as microinjection method, electroporation method, polyethylene glycol method, fusion method, high-speed ballistic penetration method, Indirect transfection methods can also be used via viruses and bacteria that infect plants (I. Potrykus, Annu. Rev. Plant Physiol. Plant Mol. Biol., 42: 205, 1991). In this case, as the virus, cauliflower mosaic virus, available geminivirus, tobacco mosaic virus, brome mosaic virus, and examples of the bacteria, Agrobacterium tumefaciens (Agrobacterium tumefaciense:.. Hereinafter abbreviated as A tumefaciens), A . Rhizogenes can be used.

  Plant cells into which a target gene introduction vector has been introduced are cultured using an appropriate medium, and selected using the expression of a selectable marker gene introduced into chromosomal DNA together with the target gene as an indicator. At this time, as the medium, Murashige and Skoog (MS (T. Murashige and F. Skoog, Physiol. Plant., 15: 473, 1962)), B5 of Gamborg, etc., a basic medium known as a plant tissue culture medium or the like If necessary, a solid or liquid medium to which a carbon source such as sucrose and / or a plant hormone such as cytokinin or auxin is added can be used. However, depending on the type of selection marker gene used, it is necessary to add necessary components to the medium in order to detect its expression. For example, when a drug resistance gene is used as a selection marker gene, it is necessary to detect the expression of the selection marker gene by adding a corresponding drug to the medium. When a GUS gene is used, 5-bromo When -4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt is used as a luciferase gene, it is necessary to add luciferin to the medium and detect the expression of the selection marker gene. In addition, what is necessary is just to employ | adopt suitably the conditions suitable for culture | cultivation of the plant cell, such as culture | cultivation temperature, humidity, and light intensity.

  Next, a selection marker removal vector is introduced into the plant cell selected by the above culture, and the cell is cultured. Then, the morphological abnormality-inducing gene arranged in the vector for removing the selection marker is introduced into the chromosomal DNA and expressed, and the direction of proliferation / differentiation is distorted, and there is indefiniteness without polyblasts or rooting ability. Differentiate forms such as bud differentiation and adventitious root differentiation. Therefore, when a tissue exhibiting such a morphological abnormality is selected and further cultured, the detachment enzyme gene introduced into the plant cell by the selective marker removal vector works together with the morphological abnormality induction gene. The morphological abnormality-inducing gene present in the isolated site and the selectable marker gene introduced by the target gene introduction vector are detached from the chromosomal DNA into which they have been introduced and lose their function, resulting in morphological abnormality. This time, a tissue showing a normal form is generated from the tissue showing, and this is selected. The probability that the selected tissue is a marker-free tissue is greatly improved as compared with the conventional method.

  The introduction of the selection marker removal vector and the culture of the cells after the introduction can be performed according to the introduction of the target gene introduction vector and the cultivation of the cells after the introduction. However, at this time, it is not necessary to add a component for detecting the expression of the selection marker gene to the medium. A marker-free plant can be obtained by growing and redifferentiating the marker-free tissue obtained as described above by a conventional method.

  In the present invention, applicable plant species are not particularly limited. For example, the present invention is also applied to herbaceous plants such as tobacco, chrysanthemum, carnation, rice, and woody plants such as eucalyptus, poplar, pine, acacia, cherry blossom, bayberry, kunugi, grape, apple, rose, camellia and ume. be able to.

  The details of the invention will be described below according to examples. However, the present invention is not limited to the following examples.

  In the following examples, molecular biological techniques among the more detailed experimental procedures are performed according to Molecular Cloning (Sambrook et al., 1989) or the manufacturer's instructions unless otherwise specified. It was.

[Example 1]
I. Construction of target gene introduction vector (pIGFP21) Plasmid pIG221 (Plant Cell Physio. 3 (6): 805-813. 1990) was digested with restriction enzyme Sse83871 (hereinafter abbreviated as SseI ), and T4 DNA polymerase was used. After the treatment, plasmid pIG221ΔSse lacking the restriction enzyme Sse I site was constructed by religation. A double-stranded oligonucleotide (5′-cctgcagg-3 ′) is ligated to the restriction enzyme Sma I site of pIG221ΔSse, and the restriction enzyme Sse I site is introduced between the CAMV35S promoter (35S-P) and the GUS structural gene. After constructing the plasmid pIG221sseΔsse, the GUS gene to which 35S-P and the polyadenylation signal of the nopaline synthase gene (hereinafter, all polyadenylation signals were derived from nopaline synthase) was restricted from this pIG221sseΔsse. cut at enzymes Hind III and EcoR I, which was arranged in restriction enzyme Hind III and EcoR I sites of plasmid pBI121 (clontech, Inc.), 35S-P, is replaced with GUS structural gene and a polyadenylation signal, a plasmid pBIG2 1sse (Figure 1) was constructed.

On the other hand, from the plasmid 128-35GFP, the site-specific recombination system recombination enzyme recognition sequence (hereinafter also simply referred to as the recognition sequence) of the nopaline synthase gene, sandwiched between two RSs, facing in the same direction. promoter cut at (Nos-P) and polyadenylation limit GFP structural gene linked to signal enzyme Sse I, and inserted into a restriction enzyme Sse I site of PBIG221sse, the desired gene transfer vector plasmids pIGFP21 of the present invention (Deposited on March 29 to the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center).

  The main structure of pIGFP21 is shown in FIG. This pIGFP21 has an NPTII gene as a model of a target gene and a GFP gene as a selection marker gene, and the selection marker gene is sandwiched between two recombination enzyme recognition sequences RS of a site-specific recombination system oriented in the same direction. It is arranged so as to be located at the desorption site. In the figure, a black triangle surrounded by a square frame indicates the direction of RS and its arrangement, and T indicates a polyadenylation signal (the same applies hereinafter). When this vector is introduced into a plant cell, the T-DNA region, that is, the region between the right border (RB) and the left border (LB) of T-DNA indicated by a small black triangle is integrated into the chromosomal DNA. .

II. Construction of a selection marker removal vector (pTSspsMRSrubipt35R) From the plasmid 128-35Rrubit (Japanese Patent Application No. 2004-382394), a Rubisco promoter (Rub-P) sandwiched between two recognition sequences RS facing in the same direction was linked. ipt gene (promoter only; use Rub-P in place of the original promoter), and site-specific recombination recombination enzyme linking 35S-P and a polyadenylation signal (hereinafter simply referred to as recombination enzyme). The R structural gene was excised with the restriction enzyme Sse I, and this was inserted into the restriction enzyme Sse I site of the plasmid pTSsps (Japanese Patent Application No. 2004-382394) to construct a selection marker removal vector plasmid pTSspSMRrubipt35R ((Germany). )industry Operative Research Institute, has been deposited with the Patent Organism Depositary Center on March 29 with the addressed).

The main structure of pTSspsMRSrubipt35R is shown in FIG. This pTSspsMRSrubipt35R is morphological abnormality induction gene as ipt gene includes a gene for the recombinant enzyme R site-specific recombination system as lyase gene, these genes, site-specific recombination facing the same direction It is arranged so as to be located at the elimination site sandwiched between two recombination enzyme recognition sequences RS of the system.

III. Introduction of a target gene introduction vector (pIGFP21) and a selection marker removal vector (pTSspsMRSrubipt35R) into Agrobacterium Tumefaciens 4404 strain was added to 10 ml of YEB liquid medium (beef extract 5 g / l, yeast extract 1 g / l, peptone 1 g / l, sucrose 5 g / l, 2 mM MgSO ₄ , pH 7.2 at 22 ° C. (hereinafter not particularly indicated) In this case, the pH is set at 22 ° C.))) and inoculated at 28 ° C. until the OD 630 is in the range of 0.4 to 0.6. The culture was collected by centrifugation at 6900 × g, 4 ° C. for 10 minutes, and then the cells were suspended in 20 ml of 10 mM HEPES (pH 8.0) and centrifuged again at 6900 × g, 4 ° C. for 10 minutes. The cells were collected and then suspended in 200 μl of YEB liquid medium, which was used as a bacterial solution for plasmid introduction.

  An electroporation method was used to introduce the vector into Agrobacterium. That is, in a 0.5 ml tube, 50 μl of the plasmid-introducing bacterial solution prepared as described above and 3 μl of pIGFP21 were mixed, and electroporation was performed on this mixed solution (Gene Pulser II system [BIORAD, Inc.]). ) Thereafter, 200 μl of YEB liquid medium was added, and this was cultured with shaking at 25 ° C. for 1 hour. The obtained bacterial cells were added to 50 mg / l kanamycin-added YEB agar medium (agar 1.5 w / v%, The other composition was the same as above.), And cultured at 28 ° C. for 2 days to form bacterial colonies. Confirmation of the introduction of pIGFP21 into Agrobacterium was performed by transplanting this bacterial colony into a YEB liquid medium and culturing, and then extracting the plasmid from the bacterial cell by the alkaline method and performing the standard method. In this way, p. Tumefaciens 4404 strain was designated as LBA4404 (pIGFP21).

  In addition, pTSspsMRSrubipt35R was similarly introduced into Agrobacterium 4404 strain, and this was designated as LBA4404 (pTSspsMRSrubipt35R).

IV. Introduction of vector for introduction of target gene into tobacco (pIGFP21) About 8 mm by removing the middle vein from the leaves of Nicotiana tabacum SR1, which is aseptically grown in a culture container. 36 pieces of leaf pieces obtained by cutting so as to form corners were cultured overnight in the above-mentioned LBA4404 (pIGFP21) bacterial solution (OD630 = 0.25, YEB liquid medium), then diluted with sterilized water to obtain the bacterial cell concentration. Incubate for about 1 minute to infect this, place it on a sterilized filter paper to remove the excess bacterial solution, and then remove the phytohormone (hormone free) MS supplemented with 50 mg / l acetosyringone Place on an agar medium (agar 0.8 w / v%) so that the back of the leaf is on top, 25 ° C, all bright (unless otherwise stated, culture of explants and plant tissues / plants On condition The culture was performed for 3 days.

  The cultured leaf pieces were transplanted to an MS agar medium supplemented with 6-benzylaminopurine 1 mg / l, naphthalene acetic acid 0.1 mg / l, carbenicillin 500 mg / l and kanamycin 200 mg / l. Since 13 shoots had been redifferentiated, the adventitious buds were isolated and rooted by transplanting and culturing onto a hormone-free MS agar medium containing 500 mg / l carbenicillin and 200 mg / l kanamycin. Eventually, it was possible to obtain 13 kanamycin-resistant recombinant lines derived from 13 adventitious shoots.

V. Chromosomal DNA was extracted from the 13 kanamycin-resistant recombinant lines (pIGFP21-1 to 13) obtained by Southern analysis IV of tobacco into which the target gene introduction vector (pIGFP21) was introduced , using the CTAB method. After digestion with restriction enzyme Bam HI, it was subjected to 0.8% agarose gel electrophoresis. The agarose gel after electrophoresis was transferred to a nylon membrane after alkali / acid treatment, and this nylon membrane was previously labeled with a DIG PCR labeling kit (purchased from Boehringer Mannheim). Hybridization was performed using a probe P1 having homology with the GUS gene. After hybridization, chemiluminescence detection was performed using DIG Wash and Block Buffer (purchased from Boehringer Mannheim). As a result, one copy of pIGFP21 was introduced into two lines (pIGFP21-10 and 13) subjected to Southern analysis. It was confirmed.

VI. The leaf piece of pIGFP21-13 prepared in the same manner as the introduction IV of the selection marker removal vector (pTSspsMRSrubipt35R) into tobacco into which the target gene introduction vector (pIGFP21) has been introduced, was added to the bacterial solution of the LBA4404 (pTSspsMRSrubipt35R) (OD630 = Incubate overnight in 0.25, YEB liquid medium and dilute with sterile water to adjust the bacterial cell concentration.) Infect with this for about 1 minute, place it on sterile filter paper and remove excess bacterial solution After removing, place it on hormone-free MS agar medium (agar 0.8 w / v%) supplemented with 50 mg / l acetosyringone so that the back of the leaf is on top, 25 ° C., 2 days in total darkness Culture was performed.

  The cultured leaf pieces were transplanted to a hormone-free MS agar medium supplemented with 500 mg / l carbenicillin, and the cultivation was continued. As a result, 65 adventitious buds were re-differentiated. The culture was continued in the medium. During this culture, multi-buds were formed from 29 adventitious buds, and 28 adventitious buds of normal morphology were elongated from the multi-buds. The adventitious shoots thus produced from the multi-buds were rooted by culturing on a hormone-free MS agar medium supplemented with 100 mg / l of cefotaxime, and 28 rooted individuals could be obtained. These rooted individuals were subjected to the following analysis while being subcultured and maintained.

VII. Analysis of tobacco into which a target gene introduction vector (pIGFP21) and then a selection marker removal vector (pTSspsMRSrubipt35R) were introduced Confirmation of GFP gene removal by PCR analysis The chromosomal DNA of 28 rooting individuals prepared in VI was extracted with Fast DNA kit (BIO 101 Inc), and about this chromosomal DNA, primer 35s60 binding to 35S-P and GUS structure PCR analysis was performed using the primer EXGUS that binds to the gene. In this case, the GFP gene (selection marker gene) introduced together with the NPTII gene (target gene) into tobacco chromosomal DNA by the target gene introduction vector (pIGFP21) is introduced by the selection marker removal vector (pTSspsMRSrubipt35R). When the DNA is removed by the action of the R gene (eliminase gene) and removed from the DNA, a 900 bp DNA fragment is amplified (FIG. 4).

In the PCR reaction, 1 μg of the chromosomal DNA extracted as described above, 10 mM Tris-HCl (pH 8.8 at 25 ° C.) containing 50 μM of this primer, 50 mM KCl, 1.5 mM MgCl ₂ , 1 w / v% After dissolving in 50 μl of a mixture having a composition of Triton X-100, 0.1 mM dNTP, and 1.25 units of Taq polymerase (purchased from CETUS), this was heated at 94 ° C. for 1 minute 30 seconds, and then 94 A heating cycle of 30 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 2 minutes was repeated 30 times. The obtained reaction mixture was analyzed by agarose gel electrophoresis to detect amplification of DNA fragments.

  As a result of PCR analysis, amplification of a 900 bp DNA fragment was observed in 7 out of 28 individuals subjected to the analysis. Therefore, these 7 individuals are affected by the desorption enzyme gene once introduced into the chromosomal DNA by the target gene introduction vector (pIGFP21) and introduced by the selection marker removal vector (pTSspsMRSrubipt35R). , Considered to have been removed.

  At this time, amplification of a 1.9 kbp DNA fragment was also observed in one individual together with the 900 bp DNA fragment. This 1.9 kbp DNA fragment corresponds to the size of the DNA fragment that is amplified when the GFP gene continues to be present on the chromosomal DNA while being introduced by the target gene introduction vector (pIGFP21). To do. Therefore, this individual is considered to be a chimeric individual in which cells from which the selection marker gene has been removed and cells from which the selection marker gene has not been removed exist in the same individual.

B. GUS activity test In the target gene introduction vector (pIGFP21), the GFP gene is located at a position where 35S-P and the GUS structural gene are separated. Therefore, the GFP gene (selection marker gene) introduced into the chromosomal DNA of tobacco together with the NPTII gene (target gene) by the target gene introduction vector (pIGFP21) is introduced by the selection marker removal vector (pTSspsMRSrubipp35R). When it is desorbed by the action of a gene (eliminase gene) and removed from the DNA, 35S-P and the GUS structural gene are linked and the GUS gene is expressed (FIG. 4).

  Therefore, a part of the plant body was collected from 7 lines derived from 7 individuals in which removal of the GFP gene was confirmed by the PCR analysis, and a GUS activity test was performed according to the method of Jefferson et al.

  As a result, staining with GUS was observed in all of the individuals subjected to the GUS activity test of the above seven lines, and it was confirmed that the GFP gene was removed. On the other hand, the pGGFP21-13 prepared after the introduction of the target gene introduction vector in the above IV was similarly tested for GUS activity, but no staining with GUS was observed in individuals derived from this strain. .

C. Southern analysis Chromosomal DNA was extracted from 8 individuals derived from 8 individuals (including individuals considered to be chimeric individuals) whose removal of the selection marker gene was confirmed by PCR analysis, and cleaved with the restriction enzyme EcoR I. Southern analysis was conducted in the same manner as V. In this case, the GFP gene (selection marker gene) introduced together with the NPTII gene (target gene) into tobacco chromosomal DNA by the target gene introduction vector (pIGFP21) is introduced by the selection marker removal vector (pTSspsMRSrubipt35R). When the DNA is removed from the DNA by the action of the R gene (eliminase gene), about 7.0 kbp, while the GFP gene was introduced by the target gene introduction vector (pIGFP21) If the chromosomal DNA continues to exist in the state, a band of about 2.7 kbp will be detected.

  As a result, a band of about 7.0 kbp was observed in all the 8 strains subjected to the analysis, and it was confirmed that the GFP gene was removed. In addition, as a result of PCR analysis, about one individual whose chimerism was suggested, a band of about 2.7 kbp was detected in addition to the band of about 7.0 kbp, and this line is a line of a chimeric individual. Was confirmed. On the other hand, in the above IV, only about 2.7 kbp band was detected in individuals of the strain derived from pIGFP21-13 prepared after introduction of the target gene introduction vector.

VIII. Analysis of next generation marker-free tobacco As a result of the above PCR analysis, GUS activity test and Southern analysis, 7 lines in which the removal of the GFP gene from the chromosomal DNA was confirmed. It is considered that this is a marker-free individual line consisting of marker-free cells, in which the target gene (NPTII gene) has been introduced and the selected marker gene (GFP gene) has been removed from its chromosomal DNA.

  Therefore, in order to analyze the next generation of these lines, seedlings obtained by self-pollination were grown from seedlings, and PCR analysis and GUS activity tests were conducted in the same manner as in the above VII-A and -B. went.

  As a result, amplification of a 900 bp DNA fragment by PCR analysis and GUS staining by GUS activity test were observed in all the young plants of the next generation subjected to analysis here, as in the parent generation. Therefore, with respect to these lines, the traits of the parent generation are surely transmitted to the next generation, and these parent generation 7 lines are individual lines composed only of marker-free cells and are chimeric. It is thought that it has no sex at all.

[Example 2]
I. Construction of target gene transfer vector (pIRol21)
A plasmid 128-rol having a structure in which the rol gene is sandwiched between two recognition sequences RS of the site-specific recombination system oriented in the same direction and then sandwiched between the recognition sequences RS The rol gene is excised from the 128-rol with the restriction enzyme Sse I, and inserted into the restriction enzyme Sse I site of the plasmid pBIG221sse constructed in Example 1-1, and the vector plasmid pIRol21 for introducing the target gene of the present invention. (Deposited on March 29 to the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary).

The main structure of pIRol21 is shown in FIG. This pIRol21 has the same structure as the target gene introduction vector pIGFP21 used in Example 1, except that it has the rol gene as a selection marker gene.

II. Introduction of target gene introduction vector (pIRol21) into Agrobacterium In the same manner as in Example 1-III, pIRol21 was transformed into A. coli. This strain was introduced into Tumefaciens 4404 strain, and this strain was designated LBA4404 (pIRol21).

III. Introduction of target gene introduction vector (pIRol21) into tobacco The above LBA4404 (pIRol21) was infected with tobacco in the same manner as in Example 1-IV.

  The 105 tobacco leaf pieces after the infection treatment were transplanted to MS agar medium supplemented with 1 mg / l 6-benzylaminopurine, 0.1 mg / l naphthalene acetic acid, 500 mg / l carbenicillin and 200 mg / l kanamycin, and the culture was continued. However, since 11 adventitious buds were redifferentiated, the adventitious buds were isolated and further rooted by transplanting and culturing them on a hormone-free MS agar medium containing 500 mg / l carbenicillin and 200 mg / l kanamycin. . Eventually, 11 kanamycin-resistant recombinant lines each derived from 11 adventitious buds could be obtained.

IV. Southern analysis of the 11 kanamycin-resistant recombinant lines (pIRol21-1 to 11) obtained in Southern analysis III of tobacco into which the target gene introduction vector (pIRol21) was introduced was carried out in the same manner as in Example 1-V. As a result, it was confirmed that one copy of pIRol21 was introduced into two of them (pIRol21-7, 11).

V. Introduction of selection marker removal vector (pTSspsMRSrubipt35R) into tobacco into which the target gene introduction vector (pIRol21) has been introduced In the same manner as in Example 1-VI, pIRol21-11 confirmed the introduction of one copy of pIRol21 in IV. Infected with the LBA4404 (pTSspsMRSrubipt35R), cultured leaf pieces were cultured to redifferentiate 84 adventitious buds, and 109 rooted individuals were obtained from the adventitious buds via multi-buds. . These rooted individuals were subjected to the following analysis while being subcultured and maintained.

VI. Analysis of tobacco into which a target gene introduction vector (pIRol21) and then a selection marker removal vector (pTSspsMRSrubipt35R) were introduced For rooting individuals 109 chromosomes DNA created at check V removal of rol gene by PCR analysis, primer 35s60 and EXGUS, and bind to rol genes, the primers rolc1 designed to amplify a part rolc2 PCR analysis was performed in the same manner as in Example 1-VIIA. In this case, the rol gene (selection marker gene) introduced together with the NPTII gene (target gene) into the chromosomal DNA of tobacco by the target gene introduction vector (pIRol21) is introduced by the selection marker removal vector (pTSspsMRSrubipt35R). When the DNA is removed by the action of the R gene (eliminase gene) and removed from the DNA, a 900 bp DNA fragment is amplified, and the rol gene is introduced by the target gene introduction vector (pIRol21). If the DNA fragment continues to exist on the chromosomal DNA, a 1.0 kbp DNA fragment will be amplified (FIG. 6).

  As a result of PCR analysis, amplification of a 900 bp DNA fragment was observed in 17 out of 109 individuals subjected to the analysis. Therefore, in these 17 individuals, the selection marker gene once introduced into the chromosomal DNA by the target gene introduction vector (pIRol21) is caused by the action of the desorption enzyme gene introduced by the selection marker removal vector (pTSspsMRSrubipt35R). , Considered to have been removed.

  At this time, amplification of a 1.0 kbp DNA fragment was also observed in 9 individuals along with the 900 bp DNA fragment. Therefore, this individual is considered to be a chimeric individual in which cells from which the selection marker gene has been removed and cells from which the selection marker gene has not been removed exist in the same individual.

B. Southern analysis Chromosomal DNA was extracted from 26 individuals derived from 26 individuals (including individuals considered to be chimeric individuals) whose removal of the selection marker gene was confirmed by PCR analysis, and cleaved with restriction enzyme EcoR I. Southern analysis was performed in the same manner as in Example 1-V. In this case, the rol gene (selection marker gene) introduced together with the NPTII gene (target gene) into the chromosomal DNA of tobacco by the target gene introduction vector (pIRol21) is introduced by the selection marker removal vector (pTSspsMRSrubipt35R). About 7.0 kbp or about 20.0 kbp when the DNA is removed from the DNA by the action of the R gene (eliminase gene), while the rol gene is the target gene introduction vector (pIRol21 ), A band of about 2.7 kbp will be detected if it continues to exist on the chromosomal DNA in the state introduced by (1).

As a result, a band of about 7.0 kbp or about 20.0 kbp was observed in all of the above-mentioned 26 individuals subjected to the analysis, except for the individual of the strain whose chimerism was suggested by PCR analysis. It was confirmed that the rol gene was removed. In addition, about 9 individuals whose chimerism was suggested by PCR analysis, a band of about 20.0 kbp and a band of about 2.7 kbp were detected in all of them, and this line was a line of chimeric individuals. Was confirmed. On the other hand, in III, only about 2.7 kbp band was detected in pIRol21-11 prepared after introduction of the target gene introduction vector.

VII. Analysis of next-generation marker-free individuals and chimeric individuals Self-pollination was performed on 26 individuals (including chimeric individuals) in which removal of the rol gene from the chromosomal DNA was confirmed as a result of the PCR analysis and Southern analysis. Then, seedlings were grown from the obtained seeds, and the following experiment was conducted.

A. PCR analysis PCR was performed on the seedlings having the above 26 strains as the parent generation in the same manner as in Example 1-VIIA. As a result, the selection marker gene ( An amplification of a 900 bp DNA fragment indicating the removal of the rol gene) was observed. In other words, in this next-generation analysis, amplification of a 1.0-kbp DNA fragment was observed along with a 900-bp DNA fragment in the parent generation, and a 900-bp DNA fragment was also amplified in a seedling obtained from a chimeric individual line. However, it was observed that the chimerism of this individual and strain disappeared.

As described above, the chimera individuals obtained by the method of the present invention all showed the common result that the chimera disappeared in the next generation. These chimeras are not irregular. It is very interesting, suggesting that it has a certain regularity. The reason for this is not clear, but the tissues that form the plant are largely divided into three layers: the L1 (development in the epidermis) layer, the L2 (development in the reproductive organs) layer, and the L3 (development in the skeletal structure) layer. In the above-described chimeric individuals, the rol gene introduced into the chromosomal DNA by the target gene introduction vector (pIRol21) is introduced only by the selection marker removal vector (pTSspsMRSrubipt35R) only in the L2 layer cells. The R gene is removed from the DNA by the action of the R gene, and in the cells of the L1 and L3 layers, the rol gene continues to be present on the chromosomal DNA while being introduced by the target gene introduction vector (pIRol21). It is thought that there is.

B. GUS activity test Also in the target gene introduction vector (pIRol21) used in this example, the rol gene is located at a position where 35S-P and the GUS structural gene are separated, and the target gene introduction vector (pIGFP21) is used. The rol gene (selection marker gene) introduced together with the NPTII gene (target gene) into the chromosomal DNA of tobacco is removed by the action of the R gene (elimination enzyme gene) introduced by the selection marker removal vector (pTSspsMRSrubipt35R). When separated and removed from the DNA, 35S-P and the GUS structural gene are linked and the GUS gene is expressed (FIG. 6).

Therefore, here, among the 26 lines in which removal of the rol gene from the chromosomal DNA was confirmed as a result of the PCR analysis and the Southern analysis, 17 seedlings excluding the line suggested to have chimerism were used as parent plants. The GUS activity test was conducted in the same manner as in Example 1-VIIB. As a result, in all the seedlings subjected to the analysis here, staining with GUS was observed, and it was confirmed that the rol gene was removed. Therefore, the parental generation traits are reliably transmitted to the next generation of these strains, and these parental generation 17 strains are individual strains composed only of marker-free cells and have no chimerism. It is thought that it does not have.

It is a figure which shows from construction of plasmid pIG221 to construction of plasmid pBIG221sse among the preparation schemes of the vector plasmid pIGFP21 for target gene introduction | transduction. It is a figure which shows from construction of plasmid pBIG221sse to construction of pIGFP21 among the preparation schemes of the vector plasmid pIGFP21 for target gene introduction | transduction. It is a figure which shows the preparation scheme of the vector plasmid pTSspsMRSrubipt35R for selection marker removal. It is explanatory drawing which shows the structure of the chromosomal DNA in which the T-DNA area | region was integrated by the vector plasmid pIGFP21 for target gene introduction | transduction, and the structure after a GFP gene was removed from this chromosomal DNA. It is a figure which shows the preparation scheme of the vector plasmid pIRol21 for target gene introduction | transduction. It is explanatory drawing which shows the structure of the chromosomal DNA in which the T-DNA area | region was integrated with the vector plasmid pIRol21 for target gene introduction | transduction, and the structure after a rol gene was removed from this chromosomal DNA.

Claims (8)

A method for producing a gene-introduced cell or tissue in which the influence of a selection marker gene is eliminated, which comprises the following steps (A) to (D).
(A) A process of introducing a vector comprising a target gene, a selection marker gene other than a cytokinin synthesis gene, and a detachment site, wherein the selection marker gene is present in the detachment site ( B) Process of culturing plant cells introduced with the vector in (A) and selecting plant cells introduced with the target gene using the expression of the selection marker gene as an indicator (C) Morphological abnormality induction gene, desorption enzyme A process of introducing a vector having a gene and a detachment site, wherein the morphological abnormality-inducing gene and the detachment enzyme gene are present in the detachment site into the plant cell selected in (B) (D) In the process (E) and (D) of culturing the plant cell introduced with the vector in (C), and as a result, selecting an abnormally shaped tissue generated from the tissue obtained by the proliferation of the cell. Selected Were cultured with tissue abnormal form, the process of selecting a resulting was normal morphology tissues The desorption site is a region sandwiched between two site-specific recombination enzyme recognition sequences oriented in the same direction, and the desorption enzyme gene recognizes the two site-specific recombination enzyme recognition sequences. The method according to claim 1, wherein the gene encodes a site-specific recombinase that acts. The method according to claim 1 or 2, wherein a drug resistance gene is used as a selection marker gene. The method according to claim 1 or 2, wherein a GUS gene, a GFP gene or a luciferase gene is used as a selection marker gene. The method according to claim 1 or 2, wherein a rol gene is used as a selection marker gene. The method according to claim 1, 2, 3, 4 or 5, wherein a cytokinin synthetic gene is used as the morphological abnormality inducing gene. The method according to claim 6, wherein an ipt (isopentenyltransferase) gene is used as a cytokinin synthesis gene. After creating a transgenic cell or tissue in which the influence of the selection marker gene is eliminated by the method according to claim 1, 2, 3, 4, 5 or 6, this is proliferated and the plant body is redifferentiated. A method for producing a transgenic plant in which the influence of a selection marker gene is excluded.

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