JP2001086994A - Transcription activator - Google Patents

Abstract

(57) [Summary] (Modified) [Problem] Transcription activator, especially gene effect by position effect
Provide factors that suppress the silencing (gene expression inactivation) phenomenon. Kind Code: A1 A transcriptional activator comprising one or more mobile factors, especially a MIT-like factor. The following (a) or (b) DN
A: (a) DNA consisting of a specific base sequence, (b)
Hybridizes with the DNA consisting of the nucleotide sequence of (a) under stringent conditions, and (A)
DNA encoding an MITE-like factor causing duplication of nG (A) n [n is an integer of 1 or more] and / or DNA of the following (c) or (d): (c) M that hybridizes under stringent conditions to DNA consisting of the base sequence and DNA consisting of the base sequence of (d) and (c) and causes TA duplication at the insertion position
A transcriptional activator having DNA encoding an ITE-like factor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a transcription activator, more specifically, when such an element is inserted into a gene, it promotes or enhances the transcription of a gene located near or near the inserted gene. The present invention relates to an agent having a property of suppressing the inactivation of gene transcription. Further, the present invention relates to a transgenic plant containing the transcriptional activator.

[0002]

2. Description of the Related Art In gene manipulation, most gene transfer into higher organisms has heretofore been carried out by nuclear genome insertion type gene transfer because there is no equivalent to a plasmid in a prokaryote.

[0003] In order to introduce such a nuclear genome insertion type gene,
A physical method of introducing a vector to which a desired gene to be inserted is ligated into a nuclear genome of a higher organism by a particle gun method, a lipofection method, an electroporation method, or the like, and once introducing the vector into a virus or a microorganism, There is a biological method of introducing the DNA into the nuclear genome of a higher organism using the DNA transfer / insertion system of the virus or microorganism.

[0004] However, both of these physical methods and biological methods have a problem that the expression activity of a gene inserted into a higher organism varies from individual to individual and is not constant. Many of this is due to silencing due to position effects (Peach et al., Plant. Mol. Biol. 17:
46-60 (1991)). Such a position effect is a phenomenon that is observed in many eukaryotes such as yeast.In spite of the fact that the nucleotide sequence of the inserted gene itself has not changed at all, the gene is inserted at any position on the chromosome. Is a phenomenon in which the expression activity is greatly changed depending on whether or not the gene expression is inactivated (gene silencing) (Molecular Biology of the Cell, No. 3). Edition, 434-435
(1995)).

[0005] Based on the gene silencing phenomenon, not all genes in the nuclear genome in higher organisms are transcribed uniformly, and the gene regions that are actively transcribed (active sites) ) And the non-transcribed gene region (inactive region (cryptic
site)) are mixed. The gene inserted into the inactivation region may be because the protein required for transcription cannot be approached at all, or genomic DNA may be methylated in this region, resulting in a change in nucleosome structure or heterochromatinization. It has been known that no transcription occurs at all, resulting in inactivation of gene expression (gene silencing) (Ng et al., Curr. Opin. Genet. Dev., 9: 158-1).
63 (1999); Matzke et al., Curr. Opin., Plant Biol. 1: 142-14.
8 (1999)).

[0006] This gene silencing is based on X
As seen in the chromosome, it is known that even in the same organism, it differs depending on the differentiation state of the cell. Furthermore, it is known that when gene silencing occurs by an unclear mechanism at present, it is transmitted not only to the individual but also to the next generation of offspring generated by mating (molecular biological organism of cells).学 、 Molecular Biology of the
Cell, 3rd edition, 434-453 (1995)).

[0007] By the way, in genetic engineering, a desired gene (foreign gene) is introduced into a higher organism cell by any one of a physical method and a biological method except for the homologous recombination method used in the gene knockout method. In that case, the region into the nuclear genome of the cell into which the gene is inserted is completely different from cell to cell, and it is almost impossible to artificially control it. For this reason, when a foreign gene is introduced into a higher organism cell, cells containing the foreign gene in the activated region and cells containing the foreign gene in the inactivated region of the nuclear genome are formed indefinitely.

[0008] It is known that when a foreign gene is inserted into an inactivated region, the expression of the foreign gene is significantly reduced or eliminated due to the influence of an inactive field in that region. It is known that even when a foreign gene is inserted into the activation region, gene silencing causes the expression of the foreign gene to become unstable and decrease as growth progresses due to repeated cell division (Peach Et al.
Plant Mol. Biol. 17: 46-60 (1991)).

[0009] It is the structure of nuclear DNA in the chromosome that is mainly considered as a cause of inactivation of the expression of a foreign gene. Factors involved in the structure of nuclear DNA include MAR (matrix attachment region, SAR).
(also called a scaffold attachment region). It has been found as a sequence that anchors nuclear genomic DNA to the nuclear matrix. In animals,
It has been shown that when the chicken A element containing MAR is transfected by binding to a foreign gene to be inserted, the expression of the inserted foreign gene is increased (Stief et al., Nature).
341: 343-345 (1989)), but later studies (Bonifer
Et al., Nucleic Acids Res. 22: 4202-4210 (1994), Poljak.
Et al., Nucleic Acids Res. 22: 4386-4394 (1994)) revealed that MAR contributes to an increase in gene expression efficiency but does not negate position effects by itself. In plants, studies have been conducted to examine the effect of MAR using transformants, but no reproducible results have been obtained, and it is thought that MAR alone can cancel the position effect. No (rice, plant genome science 153-160 (1996), Shujunsha).

[0010] Currently, transgenic plants are being developed as transgenic foods. However, the most important problem to be overcome is, as described above, the gene silencing phenomenon that leads to inactivation of expression of a foreign gene. In the development of genetically modified plants, in order to avoid the gene silencing phenomenon, select a plant individual into which a foreign gene has been inserted in a position that does not cause gene silencing as much as possible from a large number of plant individuals In addition, there is a need to select plant individuals that do not cause gene silencing even after repeated mating for many years. For this reason, such selection requires growing a large number of plant individuals and breeding many generations, which not only requires a great deal of labor and time, but also requires a large amount of land. There's a problem.

[0011] Therefore, as another strategy for avoiding gene silencing, a method for suppressing gene silencing by position effects or activating transcription of the inserted foreign gene itself, Development of a so-called “transcription activator” having any of the above has become the most important and urgent issue in genetic recombination of plants.

[0012]

DISCLOSURE OF THE INVENTION The present invention has the property of suppressing the gene inactivation phenomenon called gene silencing by the position effect, or activates the transcription of a gene located in the vicinity or in the vicinity thereof. It is an object of the present invention to provide a so-called “transcription activator” having properties.

[0013]

Means for Solving the Problems The inventors of the present invention have been diligently studying to solve the above-mentioned problems day and night. As a result, they insert a mobility factor into a region near or around a foreign gene introduced into plant genomic DNA. By this, the gene silencing phenomenon due to the position effect is found to be significantly suppressed, furthermore, by the gene silencing suppression effect or by the transcription factor of the foreign gene is activated by the mobile factor, It has been found that the expression of the foreign gene is enhanced. The present inventors have conducted further research based on such findings, and found that a vector constructed by further incorporating the above-mentioned mobility factor into a vector for gene recombination of a plant significantly reduces the gene silencing phenomenon due to the position effect. Can be suppressed to
We were convinced that it was extremely useful as a cassette for expressing foreign genes.

The present invention has been completed based on these findings.

That is, the present invention relates to a transcription activator listed in the following items 1 to 5: a transcription activator characterized by containing a mobilization factor of item 1.1 or more. Item 2. Item 2. The transcriptional activator according to Item 1, wherein the mobility factor is a MIT-like factor. Item 3. The mobilization factor is a MITE-like factor consisting of the following DNA (a) or (b): (a) DNA consisting of the nucleotide sequence of SEQ ID NO: 1 (b) Stringent condition with DNA consisting of the nucleotide sequence of (a) And (c) or (d) a DNA encoding a ITE-like factor that causes duplication of (A) nG (A) n [n is an integer of 1 or more] at the insertion position. (C) DNA consisting of the base sequence of SEQ ID NO: 2 (d) Hybridizing under stringent conditions with DNA consisting of the base sequence of (c), and causing ITE duplication at the insertion position D encoding like factor
Item 3. The transcription activator according to Item 2, which is NA 2. Item 4. The mobilization factor is a MITE-like factor consisting of the following DNA (a) or (b): (a) a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 (b) a stringent condition with a DNA consisting of the nucleotide sequence of (a) And a DNA encoding a MIT-like factor causing duplication of (A) nG (A) n [n is an integer of 1 or more] at the insertion position, and a DNA of the following (c) or (d): MITE-like factor: (c) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 (d) MITE-like factor which hybridizes under stringent conditions to DNA consisting of the nucleotide sequence and causes duplication of TA at the insertion position D to code
Item 3. The transcription activator according to Item 2, which is a tandem conjugate of NA. Item 5. A transcriptional activator comprising a DNA having the nucleotide sequence of SEQ ID NO: 3.

The present invention is also a transgene expression cassette containing the above-mentioned transcriptional activator. Specific examples include the cassettes listed in the following items 6 to 8: item 6. Item 7. A cassette for expressing a transgene in a plant, comprising the transcriptional activator according to any one of Items 1 to 5, and a DNA sequence operably linked to the factor. Item 7. Item 7. The transgene expression cassette according to Item 6, wherein the DNA sequence operably linked to the transcription activator is a promoter and / or a terminator. Item 8. Item 8. The transgene expression cassette according to Item 7, comprising a desired transgene sequence to be further expressed as a DNA sequence operably linked to a transcriptional activator.

Furthermore, the present invention relates to a plasmid containing the above-mentioned transcriptional activator (for example, as a transgene expression cassette), and a transgenic plant into which a transcriptional activator has been introduced using such a plasmid. It is about.

[0018]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a transcriptional activator.

The transcriptional activator referred to in the present invention is:
A factor having the property of promoting the transcription of a group of genes located in the vicinity of or around the factor, and the fact that a desired foreign gene introduced into genomic DNA or the like is inactivated by gene silencing by a position effect. Includes factors that have a suppressive property. The factors that are known to be involved in transcriptional activation exist as enhancers in the vicinity of the promoter of a specific gene.
It activates transcription by acting directly on the promoter in cis. In contrast, as described above, the transcriptional activator of the present invention promotes the transcription of a single or a plurality of genes located in a nearby or peripheral region regardless of the position with respect to a promoter. Further, the term encompasses a concept which is substantially wider than the conventional concept of a transcription activator in that it includes a substance which substantially promotes transcription by suppressing an intrinsic transcription inactivation phenomenon.

[0020] The transcriptional activator of the present invention is characterized in that it contains one or more mobile factors.

Here, the mobility factor is largely classified into a DNA type (transposon and inserted sequence), an RNA type (retrotransposon) (edited by Berg et al., Moblie DNA (1989)), and those not belonging to any of them, that is, miniat.
ure-inverted repeat transposable element (MIT
E) (Wessler et al., Curr. Opin. Genet. Dev. 5: 914-8
21 (1995)).

The transcriptional activator of the present invention includes those containing at least one of these as a mobile factor, irrespective of whether they are the same or different. Preferably, it contains MIT as a mobility factor.

MITE is a mobility factor first discovered by Bureau et al. As a genus of Tourist in 1992 (B
ureau et al., Plant Cell 4: 1283-1294 (1992)). Since then, various MITs have been obtained from the genomes of plants, insects and animals.
E has been found. In general, MITE has (1) complete or incomplete inverted repeat sequences in both its 5'-terminal region and 3'-terminal region (similar in this respect to DNA-type mobile factors), (2) its At both ends of the inverted repeat sequence, a target-overlapping sequence-like sequence having two or more base pairs of the same orientation repeat sequence is found, as occurs when a DNA-type mobile element is inserted into genomic DNA. And (3)
It is defined as having a characteristic that its size is generally 2 kb or less (Wessler et al., Curr. Opin.
Genet. Dev. 5: 914-821 (1995)).

In the present invention, any ITE belonging to the category of the definition can be used as the mobility factor.

Among them, a preferable ITE is a mobility factor characterized in that (A) nG (A) n is duplicated at the insertion position of the genomic gene (hereinafter referred to as "IS2 for convenience"). Also referred to as "factor").

Here, n may be an integer of 1 or more and is not particularly limited, but is specifically 2 to 6, preferably 3 to
5, and more preferably 4. Such I
More specifically, the S2 factor is a DNA having a size of about 2 kb or less, preferably about 0.2 to 2 kb, and has a 5 ′ terminal region and a 3 ′ terminal region in which repetitive sequences opposite to each other (terminal reverse). (Repeated sequence).

The IS2 element has, as another structural feature, a formula (1): XttgcaYY (wherein X represents g or t and Y represents a or c) in its base sequence (SEQ ID NO: 4). To 7) or formula (2): Zatgcaa (wherein, Z
Represents t or a) (at least one of the base sequences represented by SEQ ID NOs: 8 to 9), which is repeated continuously or discontinuously. The position and the number of such repetitive sequences are not particularly limited, and may be sandwiched between the terminal inverted repeats located at both terminal regions of the IS2 element or between both terminal terminals (5 ′ terminal region and 3 ′ terminal region). May be included in the set intermediate region.

As the IS2 factor, specifically, FIG.
As shown in the above, a plurality of repetitive sequences represented by the above formulas (1) and (2) are contained in an intermediate region between terminal inverted repeats,
Further, there may be mentioned those having a structure containing a plurality of the repetitive sequences represented by the formula (1) in the terminal inverted repeat sequence.

The terminal inverted repeat sequence of the IS2 element does not need to be strictly complementary to each other, and the 5 ′ terminal region and the 3 ′ terminal region hybridize to each other under stringent conditions, As a result, it is sufficient that the IS2 factor has a stem structure as shown in FIG. In this sense, the IS2 element includes those having not only complete terminal repeats but also incomplete reverse repeats.

Specific IS2 factors used in the present invention include a nucleotide sequence of SEQ ID NO: 10 in the 5 'terminal region and a nucleotide sequence of SEQ ID NO: 11 in the 3' terminal region as a terminal inverted repeat sequence. Can be exemplified.
More specifically, the IS2 factor has the base sequence shown in SEQ ID NO: 1. As long as the IS2 element is a functional equivalent substantially having its own function or activity, the IS2 element may be in the 5 'or 3' terminal inverted repeat sequence or in a sequence located in an intermediate region interposed between these repeat sequences. One or more nucleotides may be substituted, added or deleted, and the MITE-like factors used in the present invention include functional equivalents of such MITE.

Preferred functional equivalents have substantially the function or activity of MITE (IS2 factor) having at least the nucleotide sequence of SEQ ID NO: 1 (at least the transcriptional activation effect in the present invention), and At the insertion position, (A) nG (A) n [n is an integer of 1 or more] may be caused to overlap and hybridize with the IS2 factor (SEQ ID NO: 1) under stringent conditions. As stringent conditions, 1 ×
For example, in SSC and 0.1% w / w SDS, conditions of 50 ° C. or more for 1 hour can be mentioned. More specifically, as a functional equivalent, it has the function of the above-mentioned IS2 factor, particularly the transcription activating effect of the present invention,
As compared with the S2 factor, those having a homology of 70% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more in the base sequence can be mentioned.

As another suitable ITE, TA is duplicated at the insertion position into the genomic gene, and the nucleotide sequence of SEQ ID NO: 12 is contained in the 5 'terminal region as a terminal inverted repeat sequence, and the 3' terminal region is A mobile factor characterized by having the base sequence of SEQ ID NO: 13 (hereinafter, such MITE is also referred to as “IS1 factor”). Specifically, the IS1 factor has a size of about 1 kb or less, preferably 100 bp to 500 bp.
About DNA.

As the IS1 factor, a factor having the structure shown in FIG. 2 can be specifically exemplified. More specifically, those having the base sequence of SEQ ID NO: 2 can be exemplified. As long as the IS1 element is a functional equivalent having substantially its own function or activity, the IS1 element may be in the 5 'or 3' terminal inverted repeat sequence or in a sequence located in an intermediate region interposed between these repeat sequences. One or more nucleotides may be substituted, added or deleted, and the MITE-like factors used in the present invention are intended to include such functional equivalents.

Preferred functional equivalents are those which substantially have the function or activity of the IS1 factor having the nucleotide sequence of SEQ ID NO: 2 (at least the transcription activating effect in the present invention) and which have TA overlap at the insertion position. Causes and hybridizes with the above-mentioned IS1 element (SEQ ID NO: 2) under stringent conditions. still,
As stringent conditions, as described above, 1
× SSC, 0.1% w / w SDS, at 50 ° C. or higher for 1 hour. More specifically, as a functional equivalent, it has the function of the above-mentioned IS1 factor, in particular, the transcription activating effect of the present invention, and has a base sequence of 70% or more when compared with the IS1 factor shown in SEQ ID NO: 2. , Preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more.

These IS2 and IS1 factors were newly discovered by the present inventors from the carrot genome, specifically the carrot phenylalanine ammonia-lyase gene, as described later, and are described in the present specification. It can be isolated and obtained as described in the examples.

The transcriptional activator of the present invention preferably contains at least one nucleotide sequence of the aforementioned IS2 factor, IS1 factor and their functional equivalents.

More specifically, the transcriptional activator of the present invention includes those having a base sequence of IS1 factor or a functional equivalent thereof, those having a base sequence of IS2 factor or a functional equivalent thereof, Those having a base sequence in which the base sequence of the factor or its functional equivalent and the base sequence of the IS2 factor or its functional equivalent are tandemly linked (the order of the IS1 and IS2 factors is not specified);
It has a base sequence obtained by binding the base sequence of S1 factor (or IS2 factor) or a functional equivalent thereof to the base sequence of IS2 factor (or IS1 factor) or a functional equivalent thereof via an arbitrary base sequence And the like. As a transcription activator, preferably IS2
A factor or a functional equivalent thereof, and a tandem conjugate of an IS1 factor (or an IS2 factor) or a functional equivalent thereof with an IS2 factor (or an IS1 factor) or a functional equivalent thereof. Specific examples include those having the nucleotide sequence described in SEQ ID NO: 3. In the above-described transcriptional activators, the nucleotide sequence located between the IS1 factor and the IS2 factor (in no particular order) is not particularly limited as long as the effects of the present invention are not hindered, and any nucleotide sequence may be used. it can. Specifically, there is no limitation, but one example is one derived from a carrot PAL promoter sequence. In addition, such a base sequence is usually 5 to 1000
bp, preferably 300-500 bp. Examples of the transcription activator of this embodiment include:
Specifically, those having the base sequence shown in SEQ ID NO: 14 can be exemplified.

[0038] The transcriptional activator of the present invention can be operably linked to a desired gene sequence (including a transgene sequence (including a foreign gene sequence)) to be introduced into a plant body. The resulting transcription activator can be operably linked to a functional DNA sequence such as a plant functional promoter or a plant functional terminator.

The term "operably linked" in the present invention means that the transcriptional activator is linked to the above-mentioned transgene sequence or various functional DNA sequences irrespective of the insertion position and direction of these sequences. Located sufficiently close to the sequence to affect the sequence.

In the present invention, examples of the transgene include a DNA that is desired to be expressed in a plant irrespective of whether it is the same or different from the plant. Such transgenes include, for example, genes encoding β-glucuronidase; antibiotic resistance genes; genes encoding insecticidal and bactericidal protein toxins; pathogen resistant compounds; hypersensitivity responsive compounds such as peroxidase, glucanase, and chitinase, and phytoalexin Genes synthesizing agrochemicals; herbicides and fungicide resistance genes; genes synthesizing plant enzymes (eg, enzymes related to the content or quality of protein, starch, sugar and fat) and their regulator genes; Genes involved in plant hormone synthesis; genes involved in the synthesis of insect hormones and pheromones; drugs and nutritional compounds such as genes involved in β-carotene and vitamin synthesis; and present in plants. Nucleoti Including, but not limited to, antisense transcripts that interfere with the nucleotide sequence (TRANSGENIC PLANT, Volume 1, Academic Press 199).
3).

Further, the present invention provides the above-mentioned transcription activator,
And a gene expression cassette suitable for application to plants, comprising a DNA sequence operably linked thereto. In the present invention, the gene expression cassette means a plasmid used for introduction into a plant and a subfragment thereof.

Here, the DNA sequence operably linked to the transcriptional activator includes a functional DNA sequence such as a promoter or a terminator, and further includes the above-described transgene sequence.

Here, the promoter includes a DNA sequence capable of controlling the expression of a protein of interest in a plant cell when the structural gene of the protein is linked downstream of the promoter. Any plant functional promoter used in the art for transformation of E. coli is included. Conventionally, a large number of promoters have been used for plant transformation, such as Agrobacterium tumefaciens (Ag
octopine synthase (ocs) promoter (L. Comai et al., 1985; C. Waldron et al., 1985), mannopine synthase (mas) promoter, and nopaline, which are promoters isolated from Robacterium tumefaciens). A synthase (nos) promoter is included. Also,
Cauliflower Mosaic V
irus) 35S promoter is a promoter widely used for transformation of dicotyledons and can be suitably used in the present invention. Alterations of the 35S promoter, such as two parallel 35S promoters (R. Kay et al., 198
7) and the mas-35S promoter (L.
l., 1990). Furthermore, the cauliflower mosaic virus 19S promoter (J. Paszko
wski et al., 1984;) and the 34S promoter from Scrophularis mosaic virus (M. Sanger et al., 1990) can also be included. In addition, an actin promoter which is a plant-derived promoter, a ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) promoter and the like can also be exemplified.

The terminator includes a DNA sequence capable of efficiently terminating a target structural gene in a plant cell, and includes any plant used in the art for plant transformation. A functional terminator is included. Specifically, for example, a nopaline synthase (nos) terminator can be mentioned as a typical example.

The transcriptional activator of the present invention, or the cassette for expressing the transgene of the present invention containing the factor, can be used for inducing or regulating the expression of an introduced gene in a wide variety of plants. Can be.

The plant is not particularly limited, but particularly useful in agriculture is monocotyledonous plants and dicotyledonous plants. For example, monocotyledons include corn, rice, wheat, barley, sorghum, oats, rye, millet and other cereal crops, lilies, orchids, irises, palms,
Various houseplants such as tulips and sedges are included. Also, dicotyledons include chrysanthemum, snapdragon, carnation, magnolia, poppy, cabbage, rose, pea, poinsettia, cotton, cactus, carrot, lingonberry, peppermint, sunflower, tomato, elm, oak, maple, poplar, soybean, melon , Sugar beet, rape, potato,
Such as lettuce.

The present invention further provides a gene silencing phenomenon due to the position effect of a desired foreign gene introduced by including the transcriptional activator of the present invention or the transgene expression cassette of the present invention containing the factor. It is intended to provide a transgenic plant in which (inactivation phenomenon) is suppressed or transcription of a foreign gene is activated. The transgenic plants also include progeny of these plants.

The term "plant" used herein is intended to include not only complete plants but also parts of plants such as leaves, seeds, bulbs, cuttings, and the like. It also includes plant cells such as plant calli and mericlone proliferators.

The method for producing such a transgenic plant is not particularly limited, and any DNA introduction method commonly used in the art can be used. Specifically, it is a method for introducing DNA into plant cells using a transcriptional activator of the present invention or an expression plasmid containing a transgene expression cassette containing the factor. For example, an Agrobacterium method, an electric Known methods such as an introduction method (electroporation) and a particle gun method can be used.

The thus obtained plant cells containing the transcriptional activator of the present invention, a transgene expression cassette containing the factor, or an expression plasmid are, for example, SBGelv
in, RASchilperoot adn By DPSVerma: Plant Molecu
lar Biology Manual (1991), Kluwer Academic Publish
ers and Valvekens et al. Proc Natl. Acad. Sci., 85:
A plant derived from the plant cell or a part thereof can be obtained by regeneration according to the usual method used in the plant tissue culture technique described in 5536-5540 (1988).

The expression plasmid of the present invention is not limited as long as it contains a transcriptional activator and a functional DNA sequence such as a promoter and a terminator together with a desired DNA sequence (transgene) to be introduced into a plant cell. Are preferably operably linked to each other. Note that the operatively coupled here means that
It means that the plasmid acts for its intended purpose. Specifically, when the plasmid is introduced into a plant cell, the desired transgene (structural gene) is controlled under the control of a transcriptional activator without inactivating the promoter contained in the plasmid. ) Is expressed, and the expression is efficiently terminated by the action of a terminator.

The present invention also includes a method for expressing a transgene in a plant. Such a method can be carried out by at least a step of introducing a transgene expression cassette into which a transgene as described above has been incorporated into a plant, and a step of expressing the transgene in the plant. Note that a cassette for expressing a transgene into a plant (DN
Both the introduction of A) and the expression of the transgene can be carried out using methods known in the art (Plant Mo).
lecular Biology Manual 1991, Kluwer AcademicPublis
hers).

[0053]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. However, the present invention is not limited in any way by the examples. The genetic engineering techniques and molecular biological experimental procedures (restriction enzyme treatment conditions, ligation reaction conditions, transformation to Escherichia coli, etc.) used in the present invention are generally widely used, for example, J. Sambrook, E., F., Frisch, T., Maniatis
Author, Molecular Cloning 2nd Edition (Molecular Clon
ing 2nd edition), Cold Spring Harbor Laboratory pres
s) It can be carried out according to the method described in, for example, DNA cloning, published by IRL, 1985, published by D., M., Glover, 1989, and D., M., Glover.

Example 1 1) Target Plant and Target Gene In searching for a MITE-like factor, carrot (Daucus carota L.cv. Kurodagosun) was used as a target plant, and phenylalanine ammonia-lyase of the carrot was used as a target gene. : PAL) gene.

2) Cloning of carrot PAL genes gDCPAL3 and gDCPAL4 A carrot nuclear gene was cloned from a carrot nuclear DNA library. In addition, the carrot nuclear DNA library is based on the prior literature (Ozeki, Y., Davies,
E. and Takeda, J .; Structure and expression of cha
lcone synthase gene in carrot suspension cultured
cells regulated by 2,4-D. Plant Cell Physiol., 34
: 1029-1037 (1993)), carrot cultured cells (Ozeki, Y. and Komamine, A .; Induction of an
thocyanin synthesis in relationto embryogenesis in
a carrot suspension culture; Correlation of me
tabolic differentiation with morphological diffe
rentiation.Physiol.Plantarum, 53: 570-577 (198
1)) was prepared using a λEMBL3 vector (manufactured by Toyobo Co., Ltd.).

Specifically, carrot nuclear DNA was obtained by
ay and Thompson (1980) method (Murray, MG and Thomp
son, WF; Rapid isolation of high molecular weight
plant DNA.Nucl. Acids Res. 8: 4321-4325 (198
0)), cetyltrimethylammonium bromide (C
Prepared from lyophilized carrot using TAB) solution. The obtained nuclear DNA was partially digested with Sau3AI and size-fractionated by a sucrose concentration gradient method. DNA fractions ranging from 15-20 kbp were collected, ligated into BamHI digested λEMBL3 vector, and packaged into phage particles to construct a carrot nuclear library.

Next, a carrot PAL genomic clone was obtained from the carrot nuclear library by the literature of Ozeki et al. (1990) (Ozeki, Y., Matsui, K., Sakuta, M., Matsuoka, M.,
Ohashi, Y., Kano-Murakami, Y., Yamamoto, N. and T
anaka, Y .; Differential regulation of phenylalani
ne ammonia-lyase genes during anthocyanin synthesi
s and by transfer effect in carrot cell suspension
cultures.Physiol.Plantarum, 80: 379-387 (199
0)) and cloned PAL cDNA (ANT-
(PAL cDNA) was used as a probe for screening (Sambrook et al. 1989). In addition, the hybridization of the screening of the carrot nucleus library,
6 x SSC, 60 mM sodium phosphate (pH 6.8), 10 mM EDT
A, 1% SDS, 0.02% polyvinylpyrrolidone, 0.02% Fic
oll 400 and 100 μg / ml denatured salmon sperm DNA were treated overnight at 68 ° C. The membrane was washed at room temperature in a solution of 2 × SSC, 0.5% SDS.
The test was performed twice in 5 minutes, twice in a solution of 0.1 × SSC or 1 × SSC, 0.1% SDS at room temperature for 10 minutes, and finally twice in a 0.1 × SSC solution at 68 ° C. for 30 minutes.

As a result, eight positive clones were obtained. When a restriction enzyme map was prepared for such clones, they were classified into two types, and they were named gDCPAL3 and gDCPAL4, respectively.

For gDCPAL3, see Sambrook et al.
The λ phage of the positive clone was cultured by the method described in 9), λ DNA was extracted, cut with Bam HI,
Southern transfer was performed on the nylon membrane. Using a DNA fragment (984 bp) obtained by cleaving the ANT-PAL cDNA with EcoRI and labeling the 5 'end of the DNA fragment with [ ³² P] as a probe, Southern analysis is performed, and the probe hybridizes to a 2.77 kbp DNA. A DNA fragment was obtained. This DNA fragment was digested with BamHI and calf intestine alakaline pho
Subcloning into pBluescript SK plasmid treated with sphatase (CIP) yielded gDCPAL3-pro / SK (Fig. 3).
reference). Then, the obtained plasmid gDCPAL3-pro / SK was
After digestion with the restriction enzymes Sal I and Apa I, Sambrook et al.
Exonuclease III and Mun by the method described in
A series of deletion DNA fragments were prepared using g bean nuclease, and the nucleotide sequence of gDCPAL3 DNA was determined using these. Ozeki and Takeda (1994) (J. Regulatio
n of phenylalanine ammonia-lyase genes in carrot s
uspension cultured cells.Plant Cell, Tissue and O
rgan Culture, 38: 221-225 (1994)).
The transcription start point (+1) was determined from the position of the band found by the primer extension method using mRNA extracted from carrot.

Similarly, for gDCPAL4, a plasmid gDCPAL4-pro / SK corresponding to the above was prepared, and gDCPAL4
And the transcription start site thereof were determined.
Specifically, λ DNA obtained from λ phage of the positive clone gDCPAL4 was digested with Hind III and Bam HI, Southern analysis was performed using the same probe, and a 1.63 kbp DNA fragment to which the probe hybridized was hybridized with Hind III. III
And BamHI, and subcloned into CIP-treated pBluescript SK plasmid to obtain gDCPAL4-pro / SK. Next, the obtained plasmid gDCPAL4-pro / SK was digested with restriction enzymes XbaI and BstXI, and digested with Sambrook et al.
Exonuclease III and Mung b by the method described in 9)
A series of deletion DNA fragments were prepared using ean nuclease, and the nucleotide sequence of gDCPAL4 DNA was determined using these.

3) Results Comparison of the determined base sequences of gDCPAL3 and gDCPAL4 revealed that the gDCPAL3 promoter region had an incomplete inverted repeat sequence not found in gDCPAL4.
ure inverted-repeat transposable element (MIT
E) shows -1897 to -1599 (299 bp in length) and -1157
~ 389 (length 769 bp) at two sites (Fig. 4). These sequences are called IS1 and I, respectively.
Named S2.

Each of these sequences and the nucleotide sequence around the insertion position was sequenced using an auto sequencer (ABI). The nucleotide sequence of IS1 is shown in SEQ ID NO: 2, and the nucleotide sequence of IS2 is shown in SEQ ID NO: 1.

The properties of IS1 and IS2 were as follows. IS1 consisting of the nucleotide sequence of SEQ ID NO: 2 (total length: 299 bp)
The 5'-terminal region and the 3'-terminal region each had an incomplete inverted repeat sequence (32 bp), and a target overlapping sequence of TA was found at the insertion position into the genome as the target gene. Stowaway genus already reported from this
(Bureau, TE and Wessler, SR Stowaway: a new
family of inverted repeat elements associated wit
h the genes of both monocotyledonous and dicotyled
onous plants .; Plant Cell, 6: 907-16 (1994)). FIG. 2 shows the stem structure of the IS1 element and the structures of the terminal inverted repeat sequence region and the insertion site region.

Based on the obtained base sequence information, a homology analysis of the base sequence and the amino acid sequence was performed using a commercially available database (eg, GENETYX-MAC / CD1995). Genus (Bureau
and Wessler (1994)) have a homology of 70-90% in the gene sequence of the MITE element belonging to the MITE element and the terminal inverted repeat sequence, confirming that this element is a mobile element belonging to the genus Stowaway. (FIG. 5).

IS2: consisting of the nucleotide sequence of SEQ ID NO: 1 (769 bp in total length);
Incomplete inverted repeats (15
8 bp), and a target overlapping sequence of AAAAAGAAAA was found at the insertion position into the genome as the target gene. Homology comparison of nucleotide sequences was performed, but no homology with known mobility factors was observed. From this, mobility factors constituting a new family not belonging to the conventional mobility factor family, particularly MIT-like factors, It turned out to be. IS
FIG. 1 shows the overall structure (stem structure) of the two factors, and FIG. 6 shows the structure (base sequence) of the terminal inverted repeat sequence region and the insertion position region.

Example 2 (1) Cloning of IS1, IS2, IS12 and MU3 (i) Cloning of IS1 (FIG. 7) gDC prepared for determination of base sequence in Example 1
From plasmids with a DNA fragment deleted from the 3 'end of the PAL3 promoter region, select a plasmid (gDCPAL3-IS1 / SK) deleted to -1581, cut it with KpnI, and use T4 DNA polymerase. After blunting the ends, S
Cleavage with ca I, agarose gel electrophoresis
A 1 bp DNA fragment was cut out. This was subcloned into pBluescript SK plasmid which had been digested with Hinc II and treated with CIP. Plasmids were extracted from Escherichia coli colonies of multiple independent clones obtained by this subcloning, and the nucleotide sequence was determined to determine the direction of insertion of the DNA fragment, and the KpnI side of the multicloning site of the pBluescript SK plasmid was determined. At IS1
Select the one with the 5 'end inserted and insert it into IS1 / SK
And PBI221 (Clontech Inc.)
Cauliflower mosaic virus 35S promoter (35S) fragment obtained by digestion with SmaI and SmaI was excised by agarose gel electrophoresis, followed by HindIII and SmI.
It was subcloned into pBluescriptSK plasmid which had been digested with aI and treated with CIP, and this was designated as 35S / SK. IS1 /
SK was cut with KpnI and HindIII, the insert DNA fragment was cut out by agarose gel electrophoresis, and this was cut with KpnI and HindIII and treated with CIP-treated 35S / SK.
By subcloning into a plasmid, IS1-35S / SK having an IS1 region upstream of the 35S promoter region was obtained.

(Ii) Cloning of IS2 (FIG. 8) gDC prepared for determination of base sequence in Example 1
Plasmid deleted from the plasmid containing the DNA fragment deleted from the 3 'end of the PAL3 promoter region to -389
(gDCPAL3-IS12 / SK), cut it with KpnI,
After blunting the ends using T4 DNA polymerase, Dde I
And 797 bp by agarose gel electrophoresis.
Cut out the DNA fragment, cut it with Hinc II,
It was subcloned into the pBluescript SK plasmid treated with IP. Plasmids were extracted from Escherichia coli colonies of multiple independent clones obtained by this sub-cloning, and their nucleotide sequences were determined.
Check the direction of insertion of the fragment, select one with the 5 'end of IS2 inserted on the Kpn I side of the multicloning site of pBluescript SK plasmid, and call this IS2 / SK-1.
The one with the 3 'end of IS2 inserted in the opposite direction, that is, on the KpnI side, was designated IS2 / SK-2 (reverse).

IS2 / SK-1 was cut with Kpn I and Hind III, and the insert DNA was subjected to agarose gel electrophoresis.
The fragment was cut out, subcloned into the 35S / SK plasmid which had been digested with Kpn I and Hind III and treated with CIP, and the IS2 region (positive chain) was located upstream of the 35S promoter region.
IS2-35S / SK having the following formula:

(Iii) Cloning of IS12 (a tandem combination of IS1 and IS2) (FIG. 9) IS2 / SK-2 (reverse) obtained in (ii) was digested with KpnI and terminated with T4 DNA polymerase. After smoothing, Hi
After cutting with ndIII, the insert DNA fragment was cut out by agarose gel electrophoresis. This is digested with Pst I and blunt-ended with T4 DNA polymerase.
The plasmid was subcloned into the IS1-35S / SK plasmid digested with ndIII and treated with CIP.
I got SK.

(Iv) Cloning of MU3 (FIG. 10) MU3 cuts gDCPAL3-IS12 / SK with KpnI, and
After blunting the ends with lymerase, cut with ScaI, and agarose gel electrophoresis to detect a 1,514 bp DN
A fragment was cut out. Cut this with Hinc II and CIP
It was subcloned into the treated pBluescript SK plasmid. Plasmids were extracted from Escherichia coli colonies of multiple independent clones obtained by this sub-cloning, and the nucleotide sequence was determined to determine the direction of insertion of the DNA fragment. The plasmid was inserted into the KpnI side of the multicloning site of pBluescriptSK plasmid The one with the 5 'end of IS1 inserted was selected and designated as MU3 / SK. MU3 /
SK was cut with Kpn I and Hind III, and the insert DNA fragment was cut out by agarose gel electrophoresis.
This was cut with Kpn I and Hind III and treated with CIP.
Subcloned into the S / SK plasmid, the IS1 and IS2 regions were placed upstream of the 35S promoter region, and gDLPAL
MU having via the sequence of the region derived from 3 (441 bp)
3-35S / SK was obtained. (2) Incorporation of IS1, IS2, IS12 and MU3 into a plant gene expression vector (FIG. 11) pABN-Hm1 (Mita, S., Suzuki-Fujii, K. and Nakamura,
K. Sugar-inducible expression of a gene for β-am
ylase in Arabidopsis thaliana.Plant Physiol., 10
7: 895-904 (1995), distributed by Dr. Kenzo Nakamura, Nagoya University), cut with HindIII, and converted to β-amylase promoter.
(1.7 kb) was cut out, blunt-ended with T4 DNA polymerase, cut with XbaI, and treated with CIP.
i-plasmid region and kanamycin resistance gene (no
s-promoter, neomycin phosphotransferase II gene coding region (nptII), nos terminator), β-glucuronidase gene coding region (GU
A 10 kbp DNA fragment containing the (S) · nos terminator and the hygromycin resistance gene [35S promoter / hygromycin phosphotransferase gene coding region (HPT) · nos terminator] was separated by agarose gel electrophoresis. Add 35S / SK to Hind I
After digestion with II and blunting the ends with T4 DNA polymerase, the 35S fragment obtained by digestion with XbaI was
Cloning was performed to create pAB35S.

This pAB35S was digested with XhoI and XbaI, treated with CIP, and then subjected to agarose electrophoresis.
The vector was prepared by removing the 35S DNA fragment. On the other hand, IS1-35S / SK, IS2-35S / SK,
Xho I and IS12-35S / SK and MU3-35S / SK
After cutting with XbaI, a DNA fragment for insertion (transduced gene expression cassette) was cut out by agarose electrophoresis. This DNA fragment was ligated to the above vector and transformed into Escherichia coli DH5α. The obtained Escherichia coli is transformed into L containing 25 μg / L
B. Spread on an agar medium (1% bactotryptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar powder for bacterial culture) and extract plasmids from the resulting colonies by rapid plasmid DNA extraction. By examining the restriction enzyme map of the plasmid thus obtained, as shown in FIG. 11, pIS1-35S / AB35S, pIS2-35S / AB35S, pIS12-35S /
AB35S and pMU3-35S / AB35S constructs, i.e., constructs in which the above-mentioned transgene expression cassettes are respectively incorporated between the nptII gene showing kanamycin resistance and the GUS gene which is a structural gene, have been created. Was confirmed.

(3) Preparation of Competent Cells of Agrobacterium tumefaciens Cells were taken from a glycerol stock of A. tumefaciens EHA101 with a platinum loop, and the cells were YEP solid medium (1% yeast extract, 1% bactopeptone, 0.5% sodium chloride).
Agar powder for bacterial culture was added to YEP medium at 1.5% and solidified by autoclaving. same as below)
And cultured at 28 ° C. for 2 days in the dark. A single colony of the grown A. tumefaciens was scraped with a toothpick, inoculated in 1.5 ml of YEP medium, and cultured at 28 ° C overnight with shaking. Place 80 ml YEP medium in a 500 ml flask, add 0.8 ml of the grown bacterial solution of A. tumefaciens,
With shaking until OD ₆₀₀ = 0.4. After cooling on ice, transfer to a pre-cooled centrifuge tube and
After centrifugation at 4 ° C for 5 minutes at rpm, the supernatant was removed, and 20 ml of 10% glycerol was added and suspended. This operation was repeated three times, and the medium was completely removed to remove A. tumefacie.
ns competent cells were obtained. 400 for stock
After suspending in μl of 10% glycerol, add
The solution was dispensed in aliquots and rapidly frozen in liquid nitrogen.

(4) Plasmid DNA of A. tumefaciens
Various constructs obtained in (2) (plasmid pIS1-3
5S / AB35S, pIS2-35S / AB35S, pIS12-35S / AB35S and pM
U3-35S / AB35S) was introduced into A. tumefaciens competent cells by electroporation (using GTE-10 manufactured by Shimadzu Corporation).

Specifically, 40 μl of the competent cells prepared in (3) were added to the plasmid pIS1-35S /
AB35S (IS1), pIS2-35S / AB35S (IS2), pIS12-35S / AB3
5S (IS12) and pMU3-35S / AB35S (MU3) and, for comparison, a transcriptional activator (IS1 and / or
2) About 100 ng of each of the plasmids pAB35S (35S), into which the plasmid was not incorporated, was transferred to a cell for electroporation. Electric pulse (1.2 kV, 35 μF
, 550 Ω), and quickly added 1 ml of YEP medium and cultured at 28 ° C for 1 hour. Take about 50 μl and add 50
The cells were spread on a YEP solid medium containing μg / L of hygromycin and cultured at 28 ° C. for 2 days in the dark. The single colony that has grown is gently spread again on another YEP solid medium containing 50 μg / L of hygromycin using a platinum loop, and then cooled to 28 ° C.
For 24 hours. Take a part of this cell, 50μg /
Inoculate 5 ml of YEP medium containing L of hygromycin
The cells were cultured with shaking at 28 ° C overnight.

Example 3 (1) Introduction of a construct containing a transcriptional activator into cultured tobacco cells pIS1-35S / AB35S (IS1), pIS prepared in Example 2 above
Using A. tumefaciens (hereinafter, referred to as transformed A. tumefaciens) into which each of the constructs of 2-35S / AB35S (IS2) and pIS12-35S / AB35S (IS12) was introduced, constructs IS1, IS2 and IS12 was introduced respectively. The same experiment was performed using A. tumefaciens into which pAB35S (35S) was introduced as a control.

First, the above-mentioned transformed A. tumefaciens cultured in 5 ml of YEP liquid medium was transferred to a 50 ml centrifuge tube, and then transferred to a 3,00 ml medium.
Centrifuged at 0 rpm for 10 minutes. Discard the supernatant and use Linsmaier
& Skoog medium (Linsmair, EM and Skoog, F .; Physio
l. Plantarum 18, 100-127 (1965); hereafter referred to as Lins medium) was added and suspended, and the mixture was centrifuged again at 3,000 rpm for 10 minutes at room temperature, and the supernatant was discarded. Do this 4
Repeated times. The collected A. tumefaciens was added to OD ₆₀₀ =
Suspend the Lins medium to 0.2 and add 10 to this.
Acetosyringone was added to give a μg / ml and resuspended.

On the other hand, the cultured tobacco cell BY-2 into which each construct is introduced (distributed from Dr. Toshiyuki Nagata, Graduate School of Science, The University of Tokyo) was previously prepared with 2,4-dichlorophenoxyacetic acid (2,4-D). Culture in 45 ml of Lins medium containing
Each week, 1 ml of the suspension culture was transferred to a new Lins medium, and approximately 100 hours had passed since the transfer, and cells that had entered the logarithmic growth phase were used.

The cultured tobacco cells BY-2 (4 ml) were mixed with 90 mm
100 μl of the transformed A. tumefaciens, which had been washed as described above, was added to the sterilized petri dish so as to be uniform. After gently mixing, the cells were co-cultured in the dark at 22 ° C for 3 days.

Thereafter, 12 ml of Lins was added to the culture cell solution.
Add medium, suspend, transfer to a 50 ml centrifuge tube, and add 1,000 rp.
After centrifugation at 1 m for 1 min, the supernatant was discarded. This operation was repeated four times. Then add 250 μg / m of claforan
12 ml of Lins medium containing l was added, and the same operation was performed once more. After discarding the supernatant, add about 25 ml to the precipitated cells.
Suspend by adding Lins medium, and use a hemocytometer to suspend the medium.
The number of cells in 0.25 μl was counted. The number of cells per plate is 4 x 10 ⁵ , 6 x 10 ⁵ , 8 x 10 ⁵ , 10 x 10 ⁵ ,
100 μg / ml or 300 μg to be 15 x 10 ⁵
Lins solid selective medium (containing 250 μg / ml claforan) containing 1 ml / ml kanamycin was uniformly seeded and cultured at 28 ° C in the dark. One month later, the number and formation rate of transgenic tobacco cultured cells (transformed callus) generated on the plate were measured. The results are shown in Table 1 and FIG.

[0080]

[Table 1]

From these results, it was found that the insertion rate of the transformed callus in the medium containing kanamycin was increased by inserting the construct IS2 or IS12 into the cultured tobacco cells, and that the formation rate was not attributable to the insertion of these constructs. It was found that the transformed callus formation rate was higher than that of the cultured tobacco cells (35S). Specifically, the rate of formation of transformed calli (transgenic tobacco cultured cells) into which the constructs IS2 or IS12 were introduced was determined by the control (35) to which these constructs were not introduced.
1.6 to 2.6 times higher than S).

In particular, when the concentration of kanamycin in the medium was 300 μg /
In the case of ml, construct IS2 or IS12
It was observed that the transformation callus formation efficiency was 10 times or more higher than that of the control (35S) by introducing E. coli. This suggests that the insertion of the construct IS2 or IS12 increases the expression of the kanamycin-resistant ntpII gene in the vicinity or in the vicinity thereof.

(2) G in transformed tobacco callus
Measurement of US activity Based on the above results, to confirm whether the inserted construct enhances the expression of the structural gene, construct pIS12-35S / AB35S (IS12)
Was used to examine the expression of the reporter GUS gene.

The expression of the GUS gene was determined by first randomly selecting independent calli from a plurality of transformed tobacco calli,
Nuclear DNA was extracted from the calli to confirm that gene transfer had been performed by Southern analysis, and then the callus protein was extracted and GUS activity was measured. Specifically, 0.75 g of the transformed tobacco callus was taken, and the protein was extracted using GUS-Light (Tropix). After measuring protein concentration using Bio-Rad Protein Assay Kit, GUS-Light
(Tropix) and Luminometer Lumat LB9501 (Bertold Japan) were used to measure GUS activity for 5 seconds. FIG. 13 shows the results. GUS shown on the vertical axis in the figure
The activity is expressed as the amount of luminescence per protein obtained by dividing the amount of luminescence obtained by the luminometer by the amount of protein.

As can be seen from FIG. 13, the transformed tobacco callus with the construct IS12 inserted was about 2.6 times GUS on average compared to the control with the construct-free pAB35S (35S) inserted.
The activity of construct IS12
Was found to significantly increase the expression of the GUS gene.

This means that each factor (IS1 factor, IS2 factor and a combination of these factors (such as IS12 factor)) contained in the above-mentioned construct is a transcriptional activator.

Example 4 Introduction of a Construct Containing a Transcription Activating Factor into Tobacco Plants (Leaf) (Leaf Disk Method) The construct pIS1-35S / AB35S prepared in Example 2
(IS1), pIS2-35S / AB35S (IS2) and pIS12-35S / AB35S
A. tumefaciens (hereinafter, referred to as a transformed A.
tumefaciens) using the leaf disk method.
The constructs IS1, IS2 and IS12 were introduced into tobacco leaves, respectively. A. tumefaciens transfected with pAB35S (35S) not containing the above construct as a control
An experiment was similarly performed using.

Specifically, first, leaves of tobacco (SR 1)
It was immersed in a 0% hypochlorous acid solution, air bubbles were removed with a spoon for 2 minutes while gently stirring with a stirrer, the liquid was refreshed, and the same operation was performed for another 5 minutes. Leaves were removed and immersed in sterile water and gently stirred. The same operation was performed a total of three times, replacing the hypochlorous acid solution with a new solution. After gently removing water with a sterilized paper towel, the leaves were hollowed out with a cork borer (excluding leaf veins), and immersed in 10 ml of sterilized water. The leaf disk thus prepared was transformed with the above transformed A. tumefaciens cultured in 5 ml of YEP medium (OD ₆₀₀ = 0.25 with sterile water).
For 1 minute. Next, the bacterial solution together with the leaf disk was poured onto a sterilized paper towel, and the water was removed with another sterilized paper towel.

The MS medium (Murashige,
T. and Skoog, F.; Physiol. Plantarum 15, 473-497
(1962)) on an MS infection medium containing 40 mg / L acetosyringone and 0.2% gellan gum, and cultured at 25 ° C for 2 days in the dark. Then, wipe each leaf disk with MS regeneration medium (MS medium containing 0.1 mg / L naphthaleneacetic acid, 1 mg / L benzyladenine, 150 mg / L kanamycin, 500 mg / L claforan, and 0.2% gellan gum). After the eradication, the leaves were similarly cultivated at 25 ° C in another MS regeneration medium with the back side up. The plants were replaced with a new MS regeneration medium every two weeks, and one month later, the number of regenerated shoots was counted. Table 2 shows the results.

[0090]

[Table 2]

As can be seen from the results, in the case of tobacco plants, by including the construct IS1 or IS2,
The shoot regeneration efficiency was about 1.4 times that of the control without the construct (35S), especially when both IS1 and IS2 were included in tandem (IS12).
Double shots were obtained. From this, the factors of the present invention (IS1 factor, IS2 factor and their conjugates (IS12
)) Also enhances the activity of the kanamycin resistance gene (ntpII gene) in the vicinity of or around the factor in tobacco plants. This result supports the judgment of Example 3 that the factor of the present invention is a transcriptional activator.

Example 5 Introduction of a Construct Containing a Transcriptional Activator into Carrot Somatic Embryos Each of the constructs pIS1-35S / AB35S prepared in Example 2
(IS1), pIS2-35S / AB35S (IS2), pIS12-35S / AB35S (I
A. tumefa with S12) and pMU3-35S / AB35S (MU3)
Each construct IS1, IS2, IS12 was isolated from carrot somatic embryos using ciens (hereinafter referred to as transformed A. tumefaciens).
And MU3 were introduced respectively. The same experiment was performed using A. tumefaciens into which pAB35S (35S) not containing the above-mentioned construct was introduced as a control.

Specifically, carrot hypocotyls were cut into pieces of about 1 cm, and MS containing 4.5 × 10 ⁻⁶ M of 2,4-D (2,4-dichlorophenoxyacetic acid) was used. After culturing in the medium for 24 hours in the dark,
The cells were cultivated for 3 days in the dark in an MS medium containing no.
The culture medium was changed and cultured in the same manner for 7 days to prepare a carrot somatic embryo.

On the other hand, the above-mentioned transformed A. tumefaciens cultured in 5 ml of YEP medium was centrifuged at 3,000 rpm for 10 minutes, and the supernatant was removed to suspend about 30 ml of MS medium. This operation was repeated twice to completely remove the YEP medium. Then centrifuged 10 minutes at 3,000 rpm The supernatant was removed and was adjusted to around OD ₆₀₀ = 0.3 by adding MS medium containing acetosyringone 10 mg / l.

The carrot somatic embryo collected using a net was added to this and shaken lightly for 5 minutes. The adventitious embryos were dehydrated with a sterile paper towel, immersed in MS medium containing 10 mg / L acetosyringone, and cultured at 22 ° C for 3 days in the dark. Remove the water from the adventitious embryo with a sterile paper towel.
Immerse in an MS medium containing μg / L carbenicillin and wash by gently shaking.
Was cultured in the dark on an MS agar medium (containing 0.8% agar) containing carbenicillin and 100 μg / L kanamycin. After 1.5 to 2 months, the number of hypocotyls that regenerated callus was counted. Table 3 shows the results.

[0096]

[Table 3]

As can be seen from Table 3, carrot somatic embryos were also subjected to dedifferentiated growth conditions cultured in a medium containing 2,4-D (+ 2,4-D) as in the case of tobacco callus. In addition, under any of the differentiative growth conditions cultured in a medium containing no 2,4-D (−2,4-D), the regeneration efficiency was improved by inserting the constructs IS1, IS2, IS12 and MU3. Improvements were seen. Carrot somatic embryos with the construct IS2 inserted showed the greatest improvement in regeneration efficiency.

Example 6 Introduction of a Construct Containing a Transcription Activator into Rice Construct pIS1-35S / AB35S prepared in Example 2
A. tume with (IS1) and pIS2-35S / AB35S (IS2)
Using faciens (hereinafter, referred to as a transformed A. tumefaciens), constructs IS1 and IS2 were introduced into rice seeds, respectively. The same experiment was performed using A. tumefaciens into which pAB35S (35S) not containing the above-mentioned construct was introduced as a control.

Specifically, first, rice ripe seeds (Nipponbare)
Among them, those with normal shape and color were selected and rubbed lightly with a mortar to remove the paddy. The seeds without paddy were placed in a 50 ml Falcon tube, 2.5% sodium hypochlorite was added, and the mixture was shaken at 100 to 120 rpm for 20 minutes. Thereafter, the supernatant was discarded, sterile water was added, and the mixture was gently shaken. After repeating this procedure three times, the seeds were placed in a callus induction medium. This was cultured at 28 ° C. in the dark.

After 3 to 4 weeks, the scutellum turned into callus, and the yellowish shoots grew in the growing callus with a diameter of 2-3 mm.
Only those that had spilled out were placed in a new callus induction medium and cultured at 28 ° C for 7 days in the dark.

On the other hand, the above transformed A. tumefaciens was
The cells were inoculated on P solid medium and cultured at 28 ° C for 3 days in the dark. A. tumefaciens was scraped off using a spoonful of medicine, and AAI medium supplemented with acetosyringone at 40 mg / l (Toriyam
a, K. and Hirata, K .; Plant Science 41 , 179-183
(1985)) in and adjusted to OD ₆₀₀ = .18 to 0.2 in addition. This was cultured with shaking at 25 ° C for 1 hour in the dark.

The rice callus cultured as described above was placed in a sterilized tea strainer, and the cultured A. tumefaciens suspension was added. After shaking the tea strainer occasionally for 3 minutes so that the entire callus was immersed, the tea strainer and the strainer were placed on a sterilized paper towel to remove excess bacterial solution. The calli were placed in a co-culture medium and cultured at 25 ° C for 3 days in the dark.

Then, the co-cultured calli were collected in a tea strainer, immersed in sterilized water containing 500 mg / l of claforan, and shaken to shake off the A. tumefaciens. Was. The same operation was performed four times in total. The calli were placed on a selection medium and cultured at 28 ° C in the dark. Three to four weeks later, the callus was randomly selected from a large number of calli, a portion of the callus was removed, and a GUS staining solution (0.75 mM X-Gulc (5-bromo-4-choloro-3-i
ndolyl-β-D-glucronic acid), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.3% Trit
on X-100, 20% methanol, 50 mM phosphate buffer (pH
7.0)) and allowed to react overnight at 37 ° C to detect GUS activity. Transgenic rice calli in which the callus stained blue and GUS activity was detected were transfected. Table 4 shows the results.

[0104]

[Table 4]

As can be seen from the results in Table 4, it was confirmed that the introduction of the constructs IS1 and IS2 increased the transformation efficiency about 1.5 times and about 2 times of the control (35S), respectively. This indicates that IS1 and IS2 alone enhance the transformation efficiency. From the above Examples 1 to 6, it was shown that the use of the transcriptional activator of the present invention (IS1, IS2, IS12, MU3) increases the regeneration efficiency (transformation efficiency). The following three possibilities are considered as the reason. Possibility that transfection efficiency of Ti-plasimid into plant cells is increased, and the activity of nos-promoter is IS1 or / and IS2
Increased transcription by the factor promoted transcription of the nptII gene and produced a large amount of gene product, increasing the number of cells that could grow on the kanamycin-containing medium used for selection, resulting in higher regeneration efficiency from cells Possibility, the possibility that the IS1 or / and IS2 factor activates the nearby or surrounding gene region containing them or prevents the gene region from being inactivated.

[0106] Introduction of a gene by Ti-plasimid is not inserted at a fixed position in a plant chromosome, and where it is inserted is not determined. Therefore, it is inserted into the active site determined by the chromosome structure,
Or a cryptic site in which a gene in the vicinity has been inactivated due to methylation of genomic DNA, etc.
It depends on whether or not it is inserted. If the transgene is cry
When inserted into a ptic site, the “place” of that chromosome
It is thought that the transgene is also inactivated because of the effect of

The construct used in the above Examples is a transcription activator of the present invention (IS1 or / and / or IS2).
Factor) is the terminator side of the nptII gene (kanamycin resistance gene),
It is inserted on the opposite side of the nos-promoter. Therefore, these factors do not affect the promoter of the kanamycin resistance gene in cis.

However, from the above examples, it can be seen that the number of kanamycin-resistant cells (transformed tobacco callus) in cultured tobacco cells can be increased even when the transcriptional activator of the present invention is inserted at a position that cannot directly act on the nos-promoter. It was shown that the number of kanamycin-resistant cells increased even when the kanamycin concentration in the medium was as high as 300 mM (Example 3 (1)). The result obtained was that the efficiency of regeneration of a plant exhibiting kanamycin resistance was increased by the introduction into Examples (Examples 4 to 6). This result indicates that the activity of the kanamycin resistance gene is maintained by acting on the kanamycin resistance gene located in the vicinity of the IS1 or / and IS2 element, instead of directly causing cis activation on the nos-promoter, but on the factor. This shows that the number of cells (including the meaning of both activation and suppression of inactivation) increased. That is, this is different from the conventional transcription activator in which the transcription activator of the present invention exists near the promoter of a specific gene as an enhancer and activates transcription by acting on the promoter in cis. By inserting the factor into a genomic gene, acting on one or more genes in the vicinity and surrounding regions where the factor has been inserted (regardless of the position and direction of the acting gene with respect to the promoter); This indicates that it is a transcriptional activator having the above-mentioned mechanism of promoting transcription.

Further, the construct used in the above-mentioned examples is one in which IS1 and / or IS2 elements are inserted into the GUS gene at the side of the 35S-promoter. Example 3
In (2), since the GUS activity of the cultured tobacco cells into which the construct was introduced was increased, the transcriptional activator of the present invention also acted on the 35S promoter located near the downstream thereof to increase the transcriptional activity of the GUS gene. It became clear to let. This result supports the above-mentioned judgment, and the transcriptional activator of the present invention has the above-mentioned effects,
In other words, it indicates that "IS1 or / and IS2 factor activates or inhibits inactivation of a nearby gene region containing these".

In the above examples, not only the cultured cells (Example 3) but also the plants were actually regenerated by using the transcriptional activator of the present invention in the tobacco leaf disk method. Increased efficiency (Example 4), enhanced formation efficiency of embryogenic callus, which is the source of plant regeneration from carrot hypocotyl (Example 5), callus, which is the source of plant regeneration in rice (Example 6) was shown to increase the efficiency of the formation of. These results indicate that, for a plant cell in which a foreign gene introduced into a plant genomic gene is gene silenced (inactivated) by a position effect and plant regeneration and callus formation are no longer possible, This shows that the transcriptional activator of the present invention can increase the regeneration efficiency and formation efficiency of the plant, and is useful in practical use.

It has also recently been shown that MITE binds to the nuclear matrix in the same manner as MAR (Tikhonov et al., Plant Ce).
ll 12: 249-264 (2000)).
It seems that it plays the same role as R. Thus, according to the transcriptional activator MITE of the present invention, not only can it be used alone for the production of transgenic plants, but also it can be used in combination with factors such as MAR.
It can be expected that the regeneration efficiency and formation efficiency of the plant can be further improved.

[0112]

The most important problem to be overcome in the production of transgenic plants is the gene silencing phenomenon which leads to the inactivation of the expression of foreign genes. In the development of transgenic plants, it is necessary to select a plant in which a foreign gene has been inserted at a position that does not cause gene silencing from a very large number of plant individuals in order to avoid the gene silencing phenomenon There is.

In one aspect, the transcriptional activator (IS1 and / or IS2) of the present invention is a position effect (position effec) in gene transfer in plants.
It is considered to be a factor that has the effect of suppressing and eliminating the inactivation phenomenon (gene silencing phenomenon) of gene expression due to t). Therefore, by using the transcriptional activator of the present invention alone or in combination with other factors such as MAR (matrixattachment region) involved in nuclear DNA structure in the production of transgenic plants, stable gene expression can be achieved. As a result, it is expected that the number of screenings usually performed after gene transfer and the number of transgenic plants to be bred can be significantly reduced.

Plants with a large genome size, such as lilies, chrysanthemums, and wheat, have a large inactive region (cryptic site) in the genome, so that most of the introduced foreign genes are inserted into the inactivated region. I will. For this reason, it has been extremely difficult and practically impossible to produce genetically modified plants for such plants. However, according to the transcriptional activator of the present invention, silencing of a foreign gene introduced into a plant genomic gene can be significantly suppressed, so that conventional gene recombination has been difficult as described above. It is expected that a foreign gene can be efficiently introduced into a plant species that has been used, and that a transgenic plant can be produced.

The transcriptional activating factor of the present invention is also considered to be a factor having an effect of activating (including activating transcription) a gene located near or around the gene region into which it has been inserted. Therefore, according to the transcriptional activator of the present invention, gene recombination in plants in which it was difficult to produce transgenic plants due to frequent silencing due to the large genome size as described above was put to practical use. In addition to normalization, in gene recombination in plant species such as soybean, corn, potato, and tomato that have already been put into practical use, for example, the promoter upstream of useful trait genes such as herbicide resistance genes and insecticidal protein genes Alternatively, by inserting this transcriptional activator in the vicinity thereof, the gene transfer efficiency can be increased and the transcriptional activity of these useful trait genes can be increased. To produce a higher-production, higher-quality transgenic plant than the method of producing a transgenic plant It is expected that it is.

[0116]

[Sequence List] <110> Ozeki, Yoshihiro <110> San-Ei Gen FFI, Inc. <120> Enhancer of Transcription <130> 14200JP <150> JP 1999/206320 <151> 1999-07-21 <160> 14 <210> 1 <211> 769 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 1 gggatctttt taaaaatacc catctgtaaa attatttttt taaaaatact accatctttt 60 tcattgtttt taaaaatacc ttttcattgttcagcatcagca tgttcagca tca tca tca tca tca tca tca tca tca tca t gttca tca aaagttgcaa atgagtttgt aaaagttgca aatgaggttg caaaagttgc aaataaaaat 240 ggaaagttgc aacagttgca actgcaattg caactagttc aactgaaaac tgtaagttgc 300 aaaagttgca aatgaggttg caactaaatg caactgaaaa ctgtaagtaa caacagatgt 360 atggtgtgcc cctggcgggg ccgttagatt acaatagaat caactgaatg caatcatatg 420 caactgaata caactatatg caatcatata tgcaattaca aatcctgatt tcaagttcca 480 gttttcgaat gtcattttcg aaatcgatat atatatatat atatatatat cgatttcgaa 540 aatgacattc gaaaactgga acttgaaatc aggaattcag ctgcatatga agttgcaaaa 600 gaggttgcaa cacggctggc gccgcctgta gttgcaaatg aggttgcaaa agtt gcaaac 660 agtatttttg aaaaaaagat tttatgaaaa ggtattttta aaaataattc tggaaggtag 720 tatttttgaa aacaataaaa gaaaaggtag gtagttttgt agatttccc 769 <210> 2 <211> 299 <212> DNA <213> ct gt tt tt cc ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct ct gt cc 60 cctagaatac ttactatttt ttaacactat ttttcactat tacacccacc aactctatat 120 tttatactat tttattatta aataaacact attacaccca ctacttttct ccactatctc 180 aaatctatta ttaaatattg ataggtccac cactttaccc acttttcaac tacatttact 240 acatttttct taatctccgt gaaagtcaaa ctcattcaca taaaatggga cagagggag 299 <210> 3 <211> 1192 <212> DNA <213> Carrot (Daucus carota L. cv.Kurodagosun) <400> 3 ggtaccgggc cccccctcga ggtcactccc tacgtcccat tttatgtgac ctcattttct 60 ttttgggacg tctcaaaaaa aataacctag aatacttact attttttaac actatttttc 120 actattacac ccaccaactc tatattttat actattttat tattaaataa acactattac 180 acccactact tttctccact atctcaaatc tattattaaa tattgatagg tccaccactt 240 tacccacttt tcaactacat ttactacatt tttcttaatc tccgtgaaag tcaaactcat 300 t cacataaaa tgggacagag ggagtaatta ttaattttaa tagacggtat cgataagctt 360 atcgataccg tctcagattc gcaaacataa aaagaaaagg gatcttttta aaaataccca 420 tctgtaaaat tattttttta aaaatactac catctttttc attgttttta aaaatacctt 480 ttcataaatt ttttttttca aaaatacgat ttgcaacttt tgcaacctca tttgcaacct 540 tgggcggcgc agccgtaaaa gttgccagtg aggttgcaaa agttgcaaat gagtttgtaa 600 aagttgcaaa tgaggttgca aaagttgcaa ataaaaatgg aaagttgcaa cagttgcaac 660 tgcaattgca actagttcaa ctgaaaactg taagttgcaa aagttgcaaa tgaggttgca 720 actaaatgca actgaaaact gtaagtaaca acagatgtat ggtgtgcccc tggcggggcc 780 gttagattac aatagaatca actgaatgca atcatatgca actgaataca actatatgca 840 atcatatatg caattacaaa tcctgatttc aagttccagt tttcgaatgt cattttcgaa 900 atcgatatat atatatatat atatatatcg atttcgaaaa tgacattcga aaactggaac 960 ttgaaatcag gaattcagct gcatatgaag ttgcaaaaga ggttgcaaca cggctggcgc 1020 cgcctgtagt tgcaaatgag gttgcaaaag ttgcaaacag tatttttgaa aaaaagattt 1080 tatgaaaagg tatttttaaa aataattctg gaaggtagta tttttgaaaa caataaaaga 1140 aaaggtaggt agttttg tag atttcccaga cctcgagggg gggcccggta cc 1192 <210> 4 <211> 8 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 4 gttgcaaa 8 <210> 5 <211> 8 <212> DNA < 213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 5 gttgcaac 8 <210> 6 <211> 8 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 6 tttgcaaa 8 < 210> 7 <211> 8 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 7 tttgcaac 8 <210> 8 <211> 7 <212> DNA <213> Carrot (Daucus carota L .cv.Kurodagosun) <400> 8 tatgcaa 7 <210> 9 <211> 7 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 9 aatgcaa 7 <210> 10 <211> 158 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 10 gggatctttt taaaaatacc catctgtaaa attatttttt taaaaatact accatctttt 60 tcattgtttttt taaaaatacc ttttcataaa tttttttt caaaaatacg <ttgcaacgtt ttgcaacg cat ttggcaacct 120 tttgcaacct cat tgg > DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 11 acggctggcg ccgcctgtag ttgcaaatga ggttgcaaaa gttgcaaaca gtatttttga 60 aaaaaagatt ttatgaaaag gtatttttaa aaataattct ggaaggtagt atttttgaaa 120 acaataaaag aaaaggtagg tagttttgta gatttccc 158 <210> 12 <211> 32 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> gttccttcat 13 <211> 32 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 13 aaactcattc acataaaatg ggacagaggg ag 32 <210> 14 <211> 1553 <212> DNA <213> Carrot (Daucus carota L.cv.Kurodagosun) <400> 14 ggtaccgggc cccccctcga ggtcactccc tacgtcccat tttatgtgac ctcattttct 60 ttttgggacg tctcaaaaaa aataacctag aatacttact attttttaac actatttttc 120 actattacac ccaccaactc tatattttat actattttat tattaaataa acactattac 180 acccactact tttctccact atctcaaatc tattattaaa tattgatagg tccaccactt 240 tacccacttt tcaactacat ttactacatt tttcttaatc tccgtgaaag tcaaactcat 300 tcacataaaa tgggacagag ggagtaatta ttaattttaa taaattatat gtgattttga 360 ttatgtgtgg attcttgata aaatatcaca gagttgatat aaattctaaa gatttaacca 420 caatgtttca aaatctcata gattttagta ggattcaaaa aatttaaaat acgttcaaaa 480 atctcataaa attcatcatt ttattaaatc caaaaaaatc cagtaatatt tgacaatcag 540 attttaatga attttaaatt gaacaatctc agttgaatac catgagattt taaaatataa 600 tttaaaattc taattgaata ccaccagatt ttgtaaaata atttaaaatt ttaattgaat 660 attcaagatt ttaatatatt ttaaataatc tcgatctgaa ttacaaaaaa tgcttaaaat 720 ctgaatccca tacgtcctct cagattcgca aacataaaaa gaaaagggat ctttttaaaa 780 atacccatct gtaaaattat ttttttaaaa atactaccat ctttttcatt gtttttaaaa 840 ataccttttc ataaattttt tttttcaaaa atacgatttg caacttttgc aacctcattt 900 gcaaccttgg gcggcgcagc cgtaaaagtt gccagtgagg ttgcaaaagt tgcaaatgag 960 tttgtaaaag ttgcaaatga ggttgcaaaa gttgcaaata aaaatggaaa gttgcaacag 1020 ttgcaactgc aattgcaact agttcaactg aaaactgtaa gttgcaaaag ttgcaaatga 1080 ggttgcaact aaatgcaact gaaaactgta agtaacaaca gatgtatggt gtgcccctgg 1140 cggggccgtt agattacaat agaatcaact gaatgcaatc atatgcaact gaatacaact 1200 atatgcaatc atatatgcaa ttacaaatcc tgatttcaag ttccagtttt cgaatgtcat 1260 tttcgaaatc gatatatata tatatatata tatatcgatt tcgaaaatga cattcgaaaa 1320 ctggaacttg aaatcaggaa ttcagctgca tatgaagttg caaaagaggt tgcaacacgg 1380 ctggcgccgc ctgtagttgc aaatgaggtt gcaaaagttg caaacagtat ttttgaaaaa 1440 aagattttat gaaaaggtat ttttaaaaatagattag tttagaa ggtagtt ttttaaaaattaggttagttagtttag tttagaaa ggtagtt 1500g

[Brief description of the drawings]

FIG. 1 shows the structures of MIT-like factor and IS2 factor of the present invention.

FIG. 2 shows the structure of the MIT-like factor and IS1 factor of the present invention,
And its terminal inverted repeat sequence, inserted duplication sequence (underlined T
A) is shown.

FIG. 3 is a schematic diagram showing a method for constructing gDCPAL3-pro / SK.

FIG. 4. Carrot PAL genes gDCPAL3 and gDCPAL4
It is a figure which compared the structure with.

FIG. 5 shows the results of comparison of the nucleotide sequence of the MIT-like factor (IS1 factor) of the present invention with a conventionally known nucleotide sequence of the genus Stowaway (Bureau and Wessler, Plant Cell, 6: 907-917 (1994)). FIG. The sequence in black and white is the terminal inverted repeat sequence with homology.

FIG. 6 shows an incomplete inverted repeat sequence found at the end of the MIT-like factor and IS2 factor of the present invention, and an inserted duplication sequence (AAAAAGAAAAA underlined with an arrow).

FIG. 7 is a schematic diagram showing a method for constructing IS1-35S / SK.

FIG. 8 is a schematic diagram showing a method for constructing IS2-35S / SK.

FIG. 9 is a schematic diagram showing a method for constructing IS12-35S / SK.

FIG. 10 is a schematic diagram showing a method for constructing MU3-35S / SK.

FIG. 11: pIS1-35S / AB35S, pIS2-35S / AB35S, pIS12-35
It is the schematic which shows the construction method of S / AB35S and pMU3-35S / AB35S.

FIG. 12 shows constructs pIS1-35S / AB35S (IS1) and pIS2-35S / AB35 in a selective medium (a medium supplemented with kanamycin).
S (IS2), pIS12-35S / AB35S (IS12) and pAB35S (35
S) (Transformed) transgenic tobacco BY-2 (control)
It is a figure which shows the result of Example 3 (1) which compared the reproduction | regeneration callus number of the cultured cell. The upper and lower parts of the figure show the results of using a selective medium containing kanamycin at 100 μg / ml and 300 μg / ml, respectively.

FIG. 13 shows GUS activity (left panel) of pAB35S (35S) -introduced tobacco callus (control) and pIS12-35S /
FIG. 9 shows the results of Example 3 (2) in which the GUS activity (right figure) of tobacco callus into which AB35S (IS12) was introduced was compared.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yoshihiro Koseki 2-3-6-302, Omon-cho, Higashi-Kurume-shi, Tokyo (72) Inventor Takatoshi Koda 1-1-11, Miwa-cho, Toyonaka-shi, Osaka Gen Sanei Inside FFI Co., Ltd.

Claims (11)

[Claims] 1. A transcriptional activator comprising one or more mobile factors. 2. The transcription activator according to claim 1, wherein the mobility factor is a MIT-like factor. 3. The method according to claim 1, wherein the mobility factor is D of the following (a) or (b):
ITE-like factor consisting of NA: (a) DNA consisting of the base sequence of SEQ ID NO: 1 (b) Hybridizing under stringent conditions with DNA consisting of the base sequence of (a), and (A) D) encoding a MIT-like factor causing duplication of nG (A) n [n is an integer of 1 or more]
NA or MI comprising DNA of the following (c) or (d)
TE-like factor: (c) hybridizes with DNA consisting of the nucleotide sequence of SEQ ID NO: 2 under stringent conditions with DNA consisting of the nucleotide sequence of (c), and causes TA duplication at the insertion position of the genomic gene The transcriptional activator according to claim 2, which is at least one kind of DNA encoding a MIT-like factor. 4. The method according to claim 1, wherein the mobility factor is D of the following (a) or (b):
ITE-like factor consisting of NA: (a) DNA consisting of the base sequence of SEQ ID NO: 1 (b) Hybridizing under stringent conditions with DNA consisting of the base sequence of (a), and (A) D) encoding a MIT-like factor causing duplication of nG (A) n [n is an integer of 1 or more]
NA comprising the DNA of the following (c) or (d):
TE-like factor: (c) hybridizes with DNA consisting of the nucleotide sequence of SEQ ID NO: 2 under stringent conditions with DNA consisting of the nucleotide sequence of (c), and causes TA duplication at the insertion position of the genomic gene The transcriptional activator according to claim 2, which is a tandem conjugate of a DNA encoding a MIT-like factor. 5. A DNA having the nucleotide sequence of SEQ ID NO: 3.
A transcription activator consisting of 6. A cassette for expressing a transgene in a plant, comprising the transcriptional activator according to any one of claims 1 to 5, and a DNA sequence operably linked to said factor. 7. A DN operably linked to a transcriptional activator.
The transgene expression cassette according to claim 6, wherein the A sequence is a promoter and / or a terminator. 8. A DN operably linked to a transcriptional activator.
8. The transgene expression cassette according to claim 7, further comprising a desired transgene sequence to be expressed as the A sequence. 9. A plasmid containing the transcriptional activator according to any one of claims 1 to 5. 10. A plasmid comprising the transgene expression cassette according to any one of claims 6 to 8. 11. A transgenic plant comprising the transgene expression cassette according to any one of claims 6 to 8.