JP2002291473A - Retrotransposon, dna fragment having promoter activity, and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an expression system-constructing material which can relatively safely and easily express a foreign gene in a plant (host) and can further artificially control the expression. SOLUTION: This DNA fragment contains a specific base sequence originated from Avena sativa L. or its homologous sequence. This retrotransposon contains two unidirectional repetitive sequences containing the base sequence or its homologous sequence and a transfer gene nipped between the two unidirectional repetitive sequences. The DNA fragment and the retrotransposon are used to express the foreign gene (an arbitrary gene fragment) in a host.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a retrotransposon, a DNA fragment having promoter activity, and a method for using the same. More specifically, a retrotransposon isolated from oats, a DN having a promoter activity contained in a repeat sequence of the retrotransposon
A fragment and a method for expressing a gene using the retrotransposon or DNA fragment.

[0002]

2. Description of the Related Art At present, as one of the basic techniques for efficiently expressing various genes in plants, a promoter of cauliflower mosaic virus 35S (hereinafter referred to as 3) is known.
5S promoter) is widely used. Also, a useful gene such as an antibody gene is selected as a foreign gene to be expressed under the control of the 35S promoter, and the gene is introduced into a plant to produce a large amount of a useful substance (translation product) (ie, the plant is used as a bioreactor). Attempts have been noted. As other techniques, attempts have been made to incorporate a useful gene (foreign gene) into a plant virus and infect the plant with the gene to produce a useful substance in large quantities.

[0003]

By the way, the above 3)
The 5S promoter is constantly activated and constantly expresses a foreign gene linked downstream thereof. That is,
Since the foreign gene is expressed from the time of seedling and the translation product is accumulated in the plant, the load is imposed on the plant and its growth is inhibited. In addition, plants respond to stress caused by the constant expression of a foreign gene by expressing a mechanism that suppresses this expression. As a result, translation products as expected cannot be obtained. It is considered that the allowable value of the translation product accumulation amount and the allowable value of the foreign gene expression amount are proportional to the size of the plant. Therefore, in order to solve the above-mentioned problems, development of a DNA fragment (including a retrotransposon) containing a promoter that is activated only when an exogenous stimulus is applied has been desired.

On the other hand, in a method of incorporating a foreign gene into a plant virus as a vector and infecting the plant with the foreign gene, 1) a large amount of the foreign gene can be expressed in the plant because the plant virus utilizes a high growth ability. ,Also,
2) By selecting the time of inoculation of the plant virus, there is an advantage that a translation product can be produced at any growth stage of the plant. However, many plant viruses exist without being integrated into the genomic DNA of the plant, and their expression is basically transient. Therefore, it is necessary to inoculate a plant with a plant virus every time a translation product is produced, which is very troublesome. In addition, there is a risk that the plant virus escapes out of the plant, which may hinder industrial use. In other words, there is a long-felt need for a construction material for an expression system that enables relatively safe and easy expression of a foreign gene in a plant (host), and that allows expression to be artificially controlled.

The present invention has been made in view of the above-mentioned conventional problems, and an object of the present invention is to provide a DNA fragment which exhibits a promoter activity by the application of an exogenous stimulus or has a further enhanced promoter activity. An object of the present invention is to provide a retrotransposon contained in a repetitive sequence and a method of using the same.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned conventional problems. As a result, 1) that a DNA fragment having a specific configuration exhibits promoter activity or is further enhanced by application of an exogenous stimulus; 2) a retrotransposon having a specific configuration (generally, the above-described DNA Fragment is included as a part of the composition), the transcription is activated or the transcriptional activity is further enhanced by the application of an exogenous stimulus. 3) A plasmid constructed using the DNA fragment or a retrotransposon The inventors have found that the expression of an arbitrary foreign gene in a host can be performed relatively safely and easily by using, and the expression can be artificially controlled, thereby completing the present invention.

[0007] That is, the retrotransposon according to the present invention is isolated from oats to solve the above-mentioned problems, and transcription in a host is activated by application of an exogenous stimulus, or transcriptional activity is increased. Is further enhanced.

[0008] In order to solve the above-mentioned problems, the retrotransposon according to the present invention further comprises two orthotopic repeats comprising the nucleotide sequence of SEQ ID NO: 1 or a homologous sequence thereof, and And a transgene sandwiched between repetitive sequences.

The retrotransposon according to the present invention comprises:
In the above configuration, it is more preferable that the isotropic repeat sequence has a configuration comprising the base sequence of SEQ ID NO: 2 or a homologous sequence thereof.

[0010] In order to solve the above-mentioned problems, the retrotransposon according to the present invention is characterized by comprising the nucleotide sequence of SEQ ID NO: 3.

Further, any of the above retrotransposons can be isolated from, for example, oats.

[0012] In order to solve the above-mentioned problems, the DNA fragment according to the present invention comprises the nucleotide sequence of SEQ ID NO: 1 or a homologous sequence thereof, and exhibits a promoter activity upon application of an exogenous stimulus, or It is characterized in that the promoter activity is further enhanced. Also, this DN
The A fragment can be isolated from, for example, oats.

[0013] In order to solve the above-mentioned problems, the plasmid according to the present invention has the above-mentioned DNA fragment inserted as a promoter, and further comprises a region for inserting an arbitrary gene fragment, and conferring host infectivity. Characterized by having a region to

According to the method for expressing a gene of the present invention, in order to solve the above-mentioned problems, an arbitrary gene fragment is inserted into any of the above retrotransposons, and the retrotransposon into which the gene fragment has been inserted is inserted into a host. Genome DN
A is characterized in that the above gene is expressed by incorporating it into A.

The method for expressing a gene according to the present invention also comprises
In order to solve the above-mentioned problem, the present invention is characterized in that the above-mentioned gene is expressed by infecting a host with the above-mentioned plasmid.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to the drawings. Needless to say, the present invention is not particularly limited only to the description of the present embodiment.

The retrotransposon according to the present invention comprises:
In the absence of an exogenous stimulus, the transcription of the host is relatively weak or almost non-existent, while the application of the exogenous stimulus activates transcription in the host, or the transcriptional activity is exogenous. It has been obtained as a novel DNA fragment that is more potent than in the absence of a strong stimulus. The extrinsic stimuli include chemical stress, physical stress, etc.
Various stimuli originating from outside the retrotransposon are included, and these stimuli are usually provided from the outside world via a host into which the retrotransposon is integrated.

The type of the extrinsic stimulus is not particularly limited. Specifically, for example, the application of a salicylic acid, a jasmonic acid, etc. (an elicitor) to a host into which a retrotransposon has been incorporated, and the conditions of overhumidity Application, ultraviolet irradiation, and damage to the host (including cuts, peeling of the epidermis, etc.).

One example of a specific configuration of the retrotransposon according to the present invention is shown in FIGS.
DNA portion having the base sequence of the 8th to 8665th positions),
And, as described in SEQ ID NO: 3, containing an isotropic repeat sequence arranged on both the 5 ′ side and the 3 ′ side, and a transgene (described below) interposed between the two isotropic repeat sequences. Is included. In FIGS. 1 and 4, 5 '
The orientation repeats arranged on both the 3 ′ side and the 3 ′ side are shown in order as 5 ′ LTR (long terminal repeat) and 3 ′ LTR, but in the following description, 5′-LT
The R sequence and the 3′-LTR sequence are sometimes referred to, and both may be collectively referred to as the LTR sequence. FIG. 6 shows a schematic configuration of the retrotransposon and its restriction enzyme recognition site.

The LTR sequence of the retrotransposon showing the full length in SEQ ID NO: 3 has a length of 1714 bp on both the 5 ′ side and the 3 ′ side, and has 98.7% homology with each other (FIG. 1). (See FIG. 4). The 5'-LTR sequence is indispensable as a promoter motif or an enhancer motif involved in the transcriptional control of a retrotransposon dependent on an exogenous stimulus (function (1)). In addition, by providing both the 5′-side and 3′-LTR sequences, the retrotransposon itself or a copy thereof can be used as a host genome DN.
A can be incorporated in A (function (2)).

In the LTR sequence, the minimum region necessary for performing the above functions (1) and (2) is the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in FIG. ~ Corresponding to the 1024th base sequence portion). As shown in FIG.
Various cis present in the promoter region of known disease resistance genes (including retrotransposons)
There are many sites with high homology to factors. Less than,
For sites with high homology to known cis factors,
Explanation will be made based on FIG.

In FIG. 5, "parsley PAL-1 L box", "pa
rsley 4CL-1 L box ", in order, pars
ley's cis factor found in PAL-1 and 4CL-1 (TCT
CACCTACCA: TCTCACCAACCC (Lois R et al. EMBO J 8: 1641-
1648 (1989) / Hauffe DK et al. Plant Cell 3: 435-443 (19
High homology with 91))). The site indicated by "AGC motif" is the AG found in various stress-inducing genes.
C motif (AGCCGCCT (Hart CM et al. Plant Mol. Biol. 21: 1
21-131 (1993))). "Ttol 13b
The site indicated by “p motif” is the cis factor (ATCTCACCTACCA (Taked
a et al. Plant J. 18: 383-393 (1999))). "Bean chs15 H box", "bean chs G box"
The sites shown in the order are, in order, the control region (CCTACC (N) ₇ CT: CATGTG (Loa
ke GJ et al. Proc. Natl. Acad. Sci. USA 89: 9230-9234 (19
It has high homology with 92))). The site indicated by “TCA motif” is the conserved region (TCATCTTCTT (Gold
sbrough AP et al. Plant J. 3: 563-571 (1993))). The site indicated by “asparagus AoPR-1 H box” is the cis factor (CACCTACCA (Warner SAJ et al. Plant J. 3: 191-201 (19
93))). See also "parsley PR
-2 HD motif '' indicates the elicitor-responsive cis factor (CTAATTG (K
orfhage U et al. Plant Cell 6: 695-708 (1994))). As described above, the DNA portion having the nucleotide sequence of SEQ ID NO: 1 has a number of sites that are not the same as known cis factors and have relatively high homology, and these sites are combined to form the retrotransposon (translocation). Factor) is considered to be involved in the transcription control. Further, the DNA portion represented by SEQ ID NO: 1 also includes a region considered to correspond to a TATA box.

That is, the retrotransposon according to the present invention comprises a transposable sequence comprising two nucleotides comprising the nucleotide sequence of SEQ ID NO: 1 or a homologous sequence thereof, and a transposition sandwiched between the two nucleotides. And a retrotransposon comprising the nucleotide sequence of SEQ ID NO: 3 in this category.

In the present invention, the term "homologous sequence to the nucleotide sequence represented by SEQ ID NO: 1"
Refers to DNA consisting of a sequence having 8% or more homology.
This is because the corresponding portion of SEQ ID NO: 1 in the two LTR sequences has about 98% homology (exactly 98.2%) (see FIGS. 1 and 4). In addition, the same direction repeat sequence may be different on the 5′-side and 3′-side. When the homologous sequence is located at the 5 'end of the retrotransposon, the above functions (1) and (2)
It is necessary to perform the above function (2) when it is located at the 3 ′ end.

The above-mentioned "transgene" is a gene essential for integrating the retrotransposon itself or a copy thereof into the genomic DNA of the host (including transposition in the genomic DNA). And an integrase coding region (indicated by ED in FIG. 6). This transgene is, depending on the promoter activity of a sequence located on the upstream side and exhibiting promoter activity (one of two orthotopic repeat sequences comprising the base sequence of SEQ ID NO: 1 or a homologous sequence thereof) It needs to be transcribed and translated. In addition, the “transgene” includes, in addition to the integrase coding region, gag protein coding region (GAG), protease coding region (Prot), reverse transcriptase coding region (RT), RN
More preferably, it comprises an Hase coding region (RH) (see FIG. 6). The “transgene” does not need to have the same nucleotide sequence as the gene shown in FIGS. 2 to 4 and has the same or similar amino acid sequence as the gene (FIG. 2).
To FIG. 4). That is,
The “transgene” may include, for example, a gag protein coding region, a protease coding region, an integrase coding region, a reverse transcriptase coding region, and an RNase H coding region of a known retrotransposon.

In order to more reliably perform the function (2) of incorporating the retrotransposon itself or a copy thereof into the genomic DNA of the host, the full length of the LTR sequence must be determined.
That is, it is more preferable to include the nucleotide sequence shown in SEQ ID NO: 2 (corresponding to the nucleotide sequence of the 1st to 1714th nucleotides shown in SEQ ID NO: 3) or a homologous sequence thereof as an isotropic repeat sequence flanking the transgene. The category of such a retrotransposon also includes a retrotransposon comprising the nucleotide sequence of SEQ ID NO: 3.

In the present invention, the term "homologous sequence to the nucleotide sequence represented by SEQ ID NO: 2" refers to the nucleotide sequence represented by SEQ ID NO: 2 and 9
Refers to DNA consisting of a sequence having 8% or more homology.
This is because other retrotransposons having LTR sequences at both ends may have a homology of about 98% or less. In addition, the above-mentioned same direction repeat sequence may be different on the 5′-side and 3′-side (98.7% homology in the case of SEQ ID NO: 3). Further, the homologous sequence is 5 ′ of the retrotransposon.
When located at the side end, it is necessary to perform the function (1) of performing transcriptional control of a transgene depending on an exogenous stimulus and the above-mentioned function (2). Needs to fulfill the above function (2).

The DNA fragment according to the present invention (hereinafter, referred to as a promoter DNA fragment for discrimination) is represented by SEQ ID NO: 1.
Comprising a nucleotide sequence according to the above or a homologous sequence thereof
A fragment, as described above, exhibits a promoter activity upon application of an exogenous stimulus, or has a property of further enhancing the promoter activity (as described as function (1)). The category of the promoter DNA fragment includes:
A DNA fragment comprising the nucleotide sequence of SEQ ID NO: 2 or a homologous sequence thereof, or a DNA fragment comprising the nucleotide sequence of SEQ ID NO: 3 (DN including retrotransposon)
A fragment). In addition, these DNA fragments are required to exhibit a promoter activity by the application of an exogenous stimulus or to have a property that the promoter activity is further enhanced.

The method for isolating and cloning the DNA fragment containing the retrotransposon according to the present invention is not particularly limited. For example, taking advantage of the fact that the reverse transcriptase (RT) domain of a retrotransposon has a conserved region,
The oat genomic DNA library may be screened using a probe having a nucleotide sequence corresponding to the conserved region.

The probe may have any sequence and length as long as it has a base sequence capable of specifically hybridizing to at least a part of the sequence of the retrotransposon (or a neighboring region into which the retrotransposon is incorporated). May be used. In addition, each step in the above screening may be performed under generally used conditions.

The clones obtained by the above screening can be analyzed in more detail by creating a restriction enzyme map and determining its nucleotide sequence (sequencing). From these analyses, DNA fragments comprising the retrotransposon according to the present invention (FIGS.
DNA fragment shown in FIG. 4) can be obtained.

The DNA fragment comprising the retrotransposon obtained by the above-mentioned method is digested with an appropriate combination of restriction enzymes so as to contain the nucleotide sequence of SEQ ID NO: 1 (or a homologous sequence thereof). If the desired size,
The above-mentioned promoter DNA fragment having a terminus can be prepared. The combination of restriction enzymes to be used may be selected with reference to the restriction enzyme map shown in FIG. 6 according to the use of the promoter DNA fragment.

The retrotransposon according to the present invention comprises
The method for demonstrating that the transcription is activated or the transcriptional activity is further enhanced by the application of an exogenous stimulus is as follows. First, the retrotransposon is the genome D
A host incorporated in NA is prepared, and one of the hosts is given an exogenous stimulus (test body), and the other is not given an exogenous stimulus (control). Next, RNA was extracted from the control and the test sample, and the mR corresponding to the base sequence (particularly, coding region) of the retrotransposon was extracted.
By comparing the amount of NA (transcription amount) between the test body and the control (using Northern blot analysis or the like), it is confirmed that the test body has a higher transcription amount to mRNA. The method for proving that the DNA fragment for a promoter according to the present invention exhibits a promoter activity by applying an exogenous stimulus or that the promoter activity is further enhanced is basically the same. That is, a DNA portion to be transcribed (such as a retrotransposon transfer gene, which may be a coding region between the 5′-LTR sequence and the 3′-LTR sequence) is arranged downstream of the promoter DNA fragment. The amount of transcription of the DNA portion may be compared between the control and the test body.

The retrotransposon and the DNA fragment for promoter according to the present invention can be used for various purposes. For example, retrotransposons have the ability to integrate into genomic DNA of a host, and thus can be used for transposon tagging probes to isolate host genes and elucidate functions. In addition, if an arbitrary gene fragment (a DNA fragment containing an arbitrary gene) is inserted into the retrotransposon of the present invention, it can be used as a vector. In other words, by inoculating the host,
Since the retrotransposon (vector) into which the above gene fragment is inserted is integrated into the host genomic DNA,
Any of the above genes can be expressed in a host.

The type of the host is not particularly limited, but is preferably a monocotyledon or dicotyledon (angiosperm), more preferably a monocotyledon, and among the monocotyledons, wheats, especially oats. Is preferred. Specific examples of the above monocotyledonous plants include, for example, barley, wheat, rye, oats (wheat); grasses other than wheats such as rice, corn and sugarcane; lily plants such as onions and onions; But are not particularly limited to these. Also, as dicotyledonous plants, specifically, for example, cucumber, pumpkin and the like Cucurbitaceae plants;
Solanaceous plants such as tomato, eggplant and potato; legumes such as broad bean and soybean; and the like, but are not particularly limited thereto. Further, both monocotyledonous plants and dicotyledonous plants are not particularly limited to agricultural and horticultural crops.

The corn retrotransposon Ac factor was incorporated into the genomic DNA of dicotyledonous plants such as tobacco, Arabidopsis and carrot (B. Ba.
Keret al. Proc. Natl. Acad. Sci. USA, 83, 4844-4848 (1986)
Etc.), and retrotransposons isolated from genomic DNA of monocotyledonous plants can use dicotyledonous plants as hosts.

The type of any gene expressed in the host (in the cell of the host) is not particularly limited, but a gene encoding a useful protein such as various antibody genes and antigen genes (gene (1) ) And genes that induce changes in the appearance of the host (gene (2)), such as necrosis factor and luminescence-related genes (such as luciferase-encoding genes).

An arbitrary gene integrated into the genomic DNA of a host using the retrotransposon according to the present invention as a vector may be selected from the transcriptional activity of the retrotransposon (ie, the promoter of a DNA fragment having the nucleotide sequence of SEQ ID NO: 1). Is transcribed and translated in the host depending on the activity. In addition, the transcriptional activity of the retrotransposon is controlled by applying an exogenous stimulus. For this reason,
The production of the useful protein can be started after the maturation of the host, and a more efficient bioreactor system is constructed as compared with the conventional case (in the case of the gene (1)). Also,
A good environmental sensing system is constructed to monitor the presence or absence and the degree of external stimuli such as ultraviolet irradiation by changes in the appearance of the host (in the case of the gene (2)).

In addition, the retrotransposon according to the present invention forms a large number of copies, which are transferred to the host genome DN.
A is stably incorporated in A (see Examples). In other words, the ability to proliferate in the host is extremely high, so that the above-mentioned arbitrary gene can be expressed in large amounts. In addition, it is not necessary to inoculate the host with the above-mentioned retrotransposon every time an arbitrary gene is expressed. Also, unlike retroviruses, they do not have an envelope-encoding region, and there is no danger of escape outside the host. In other words, the retrotransposon of the present invention is a material for constructing an expression system that enables relatively safe and easy expression of a foreign gene (any of the above-mentioned genes) in a host, and that allows artificial control of the expression. Is very suitable.

The position where the above-mentioned arbitrary gene is inserted in the retrotransposon of the present invention is not particularly limited as long as the basic function of the retrotransposon is not impaired. The “basic function” here means
1) that at least a part of the 5′-oriented orthotopic repeat exhibits promoter activity in response to an exogenous stimulus; 2) transposition sandwiched between two orientation repeats in response to this promoter activity. 3) at least the integration of the retrotransposon into the genomic DNA of the host by coordination of the 5′- and 3′-originated repeats. For example,
In the retrotransposon shown in FIG. 6, it is particularly preferable to insert the above-mentioned arbitrary gene into a non-coding region between the RNase H coding region (RH in the figure) and the 3′-LTR sequence. It is not limited to this position.

As a method for inserting the above-mentioned arbitrary gene (fragment) into the retrotransposon according to the present invention, a conventionally known method such as a method using an appropriate combination of a restriction enzyme and a ligase can be adopted. In addition, a retrotransposon into which an arbitrary gene has been inserted can be implanted into a leaf disk of a host (plant) using a particle gun.
2) Introduced into host cells by a conventionally known method such as incorporation into protoplasts of a host (plant) by electroporation, callus induction according to a method established for each host, and a plant (transformed plant: host) What is necessary is just to redifferentiate into. Thereby, any gene can be expressed in the plant obtained by the regeneration.

Since retrotransposons have the property of incorporating themselves into genomic DNA of a host, they exhibit relatively high gene transfer efficiency even by a gene transfer method using particle gun or electroporation.

The DN for a promoter according to the present invention
The use of the fragment A (except for the fragment containing the retrotransposon according to the present invention for convenience of explanation) is also advantageous in that any gene linked downstream can be expressed in a transformed plant in response to an exogenous stimulus. Common in. In addition, a method for preparing a transformed plant by using an insertion DNA fragment obtained by linking an arbitrary gene (fragment) to the downstream side of the promoter DNA fragment, the type of the aforementioned arbitrary gene, the type of the transformed plant (the type of host) Is described in common with the case where the retrotransposon according to the present invention is used as a vector, and a detailed description thereof will be omitted.

A more specific use of the promoter DNA fragment is that the promoter DNA fragment is inserted as a promoter, and further, a region (region (1)) for inserting any of the above-mentioned genes. And constructing a plasmid having a region (region (2)) that confers host infectivity. For example, the T-DNA region of the Ti plasmid is defined as the above region (1), the vir region is defined as the above region (2), and the DNA fragment for the promoter is further modified such that the expression of any of the above genes can be controlled.
If inserted into a plasmid, the plasmid according to the present invention can be constructed. When the plasmid is infected to a host, any of the above genes can be expressed in response to an exogenous stimulus.

That is, the plasmid of the present invention can infect a host, relatively easily and easily express a foreign gene (any of the above-mentioned genes) in the host, and furthermore, the expression can be artificially controlled. It is very suitable as a construction material for an expression system.

[0046]

The present invention will be described in more detail with reference to the following examples. Needless to say, the present invention is not particularly limited only to the description of the present embodiment.

(Retro in the oat genomic DNA
Search for transposons) Retrotransposons are widely distributed in living organisms as transposable elements having a structure similar to that of retroviruses, and are mediated by transcription into RNA and cDNA biosynthesis by reverse transcriptase using this as a template. Self-proliferate. It has been reported that the reverse transcriptase domain of the retrotransposon is highly conserved and can be detected based on the conserved region (Hirochika et al. Jpn Agr
Res Quart 25: 230-237 (1992)), and in this example, amplification of a nucleic acid having a corresponding nucleotide sequence was performed by RT-PCR (assuming that the oat-derived retrotransposon had this conserved region). reverse-transcribed-PCR). The RT-PCR method uses the mRNA, which is the transcription product of the DNA encoding the above conserved region, as a template for cDN
A method for obtaining A and amplifying the cDNA, which enables searching for a retrotransposon having activity contained in oat genomic DNA.

First, a plant and a callus (plant material) from which mRNA was to be extracted were prepared by the following method. In addition, oats (Avena Sat
ivaL.), a seed of IowaX469. First, in order to obtain the above-mentioned plants, the filter paper was placed in a petri dish with moistening, and seeds were germinated on the filter paper for one day. The germinated seeds were planted in vermiculite arranged in a growth chamber, and grown at 20 ° C. for 16 hours with a photoperiod of 8 days. From the multiple plants thus obtained, the intact (intact) first leaf, the first leaf whose epidermis was stripped, and treated with HST victorin C (0.25 ng ml-1) (Mayama
and Tani.Physiol Mol Plant Pathol 29: 1-18 (1986))
, Respectively, were obtained and used as samples for mRNA extraction. On the other hand, the callus contains 2,4-D at 4 ppm
Seed embryos were cultured for 3 weeks in Murashige-Skoog medium (MS medium) containing
D was obtained by transplantation into an MS medium containing 2 ppm of D.

The extraction of total mRNA from the intact first leaf, the first leaf from which the epidermis was stripped, the first leaf subjected to HST victorin C treatment, and callus cells was performed using the QuickPrep Micro mR
NA Purification Kit (Amersham Pharmacia Biotech
Was performed according to the description in the manual. cDNA
Is Ready-To-Go You-Prime First-Strand Beads (Amer
sham Pharmacia Biotech) and ReverTra Ace α-TM
(TOYOBO) and reverse transcription using total mRNA as a template.

As described above, the RT-PCR of this example
The reaction is performed for the purpose of amplifying a nucleic acid having a nucleotide sequence corresponding to the conserved region of the reverse transcriptase (RT) domain of the retrotransposon described by Hirochika et al. (1992). Therefore, as an oligonucleotide primer to be used, Tos-F (QMDVKT peptide chain, 5′-CARATGGAYGTNAARAC) was used as described in Hirochika et al. (1992).
-3) and Tos-B (YVDDM peptide chain, 5'-CATRTCRTCNACRTA
-3 '). In the base sequence of each primer, N = A, C, G, T; R = A, G; Y = T, C. In the RT-PCR method of this example, a PCR reaction is performed by adding 2 μl of an aliquot of each cDNA obtained by the above method to 10 μl of a reaction solution for PCR. The PCR reaction solution was prepared by combining the above two primers at a concentration of 2 μM each.
Each dNTP was added at a concentration of 0.1 mM in rTaqpolymerase (TOYOBO
PC containing 0.2 U and MgCl ₂ at a concentration of 2.5 mM.
Includes buffer for R (TOYOBO). Also PCR
The reaction was carried out by repeating a cycle of a denaturation step at 94 ° C. for 1 minute, an annealing step at 45 ° C. for 1 minute, and a chain extension step at 72 ° C. for 1 minute, which were repeated 30 times. By this reaction, if a Ty1-copier retrotransposon is present in the genomic DNA of the plant material, a nucleic acid having a base sequence corresponding to the conserved region of its reverse transcriptase domain (D
NA) is amplified.

The RT obtained by the above RT-PCR method
Transfer the amplified product (amplicon) to 1x TAE buffer (Tris-ace
tate, EDTA buffer) and electrophoresis was performed. As a result, in all plant materials including the untreated and intact first leaf, a DNA of about 280 bp indicating the presence of the coding region of the conserved region was obtained.
The fragment was obtained as an amplification product (see FIG. 7). In addition,
In FIG. 7, 1 to 4 are intact first leaves, first leaves from which epidermis has been stripped, first leaves subjected to HST victorin C treatment,
And callus data, C indicates negative control (absence of template).

Next, the above DNA fragment of about 280 bp was ligated with the plasmid vector pBluescript SKII + (STRATAGENE).
And cloned into the EcoRV recognition site to obtain 140 clones. When the sequence of a DNA fragment of about 280 bp of these 140 clones was determined and cross-hybridization analysis was performed, the oat retrotransposon was classified into seven groups based on the homology of the DNA fragment of about 280 bp. It has been suggested. Therefore, these seven groups are referred to as RT fragments Oa for convenience.
trt1 to Oatrt7. RT fragments Oatrt1 to Oatrt7
When compared at the nucleotide sequence level of NA, nucleotide identity between groups is 43% to 8%.
Nucleotide identity within the group was 87% to 99%.

(Source of oat genomic DNA library)
Cleaning) RT fragment Oatrt2 is 48 out of 140 clones
Make up clones and be the largest group. But 4
36% of 0 clones contain a stop codon in the translation region, and it is estimated that relatively many are inactive.
So we have the second largest group, RT fragments
Focusing on the clone containing Oatrt1, a genomic library of IowaX469 constructed using a λ FIX II phage vector (STRATAGENE) was screened using an RT fragment Oatrt1 (RT region probe: pOatrt1).

At the time of screening, 5000 plaques of λ FIX II phage per NZY medium were formed on the colony of Escherichia coli strain XL1-Blue MRA (lysogenized with P2 phage: STRATAGENE). Was. These plaques are then separated according to the instructions in the Hybond
TM-N + nylon membrane (Amersham Pharmacia Biotec
h) and denatured the copied genomic DNA fragment, followed by immobilization on a membrane by irradiation with ultraviolet light. Next, Gene ImagesTM kit (Amersham Pharmacia
Biotech) and a fluorescein-labeled probe (ie, RT fragment Oatrt1) as described in the manual.
Was prepared, and the membrane on which the genomic DNA fragment was immobilized was hybridized with the probe all day and night. After hybridization, the above membrane was washed twice with a 1 × SSC solution containing 0.1% SDS at 60 ° C. for 15 minutes, and then with a 0.5 × SSC solution containing 0.1% SDS at 60 ° C.
The washing was performed twice under the condition of minutes. Then, a detection operation was performed using the above kit, and among 5000 plaques, 210
It was confirmed that plaques (positive plaques) hybridized with the probe. Subsequently, 40 plaques were arbitrarily selected from the positive plaques, and the second screening was performed. In the second screening, the nucleotide sequence corresponding to the conserved region of the reverse transcriptase domain was determined using the D
This is for confirming whether or not NA has the above-mentioned RT-
RT amplification product desired according to PCR method (amplicon)
Were selected (positive clones).

Next, the QIAGEN Lambda Mini Kit (QIAGEN
Λ DNA of the above positive clone was extracted from the lysate according to the instructions of the kit. The extracted λ DNA was used for restriction enzymes ApaI, BamHI, EcoRI, Kpn
Digestion was performed with I, PstI, SacI, SalI, XbaI, and XhoI, respectively, and a restriction map was created based on the results. In addition,
The conditions for digesting λ DNA with each restriction enzyme, the method for creating a restriction enzyme map, and the like follow conventional methods.

From the created restriction map, it was found that all 21 positive clones contained a factor having an orthotopic repeat sequence called LTR at both ends. Next, one positive clone was selected from these 21 positive clones and subjected to sequencing. One selected positive clone has a restriction enzyme recognition site with the highest commonality, and was determined to be the most typical. For sequencing, BigDye Terminator Cycle Sequencing
Perform a cycle sequencing reaction using the Ready Reaction Kit (Perkin-Elmer), purify the reaction product as ethanol precipitate, and then use the ABI PRISM 310 Geneti
The nucleotide sequence was determined using c Analyzer (Applied Biosystems). As a result of sequencing, it was found that this positive clone contained an almost complete LTR-type retrotransposon (8665 bp in length). This LTR
Retrotransposon OARE-1
It was named. FIG. 6 shows the restriction map, and FIGS. 1 to 4 show the full-length nucleotide sequence and the amino acid sequence encoded by the open reading frame (ORF).

(Details of the isolated retrotransposon
Thin) retrotransposons are widely distributed in the living world. In addition, retrotransposons self-proliferate via RNA intermediates like retroviruses, and furthermore, LT
It is divided into two subgroups, R-type retrotransposons and non-LTR-type retrotransposons. LTR
Retrotransposons are also Tyl-copier and T
It is classified into two types, y3-gypsy type. LTR-type retrotransposons are very similar in structure to retroviruses. Both comprise LTR sequences located at both ends, the core protein (gag protein) of virus-like particles (VLPs), and the coding region (gene) encoding the pol polyprotein. Also, the coding region encoding the pol polyprotein comprises the coding regions encoding each of the four proteins, namely, protease, integrase, reverse transcriptase, and ribonuclease H (RNase H). The retrotransposon isolated in this example is a Tyl-copier retrotransposon because it contains the coding regions of the above four proteins in this order. Hereinafter, the detailed configuration will be described for each region.

LTR sequence of retrotransposon OARE-1 The LTR sequence of retrotransposon OARE-1 is 5 '
The 3 'side is 1714 bp long, 98.7% of each other
Constitute a repetitive sequence in the same direction with homology of In addition,
The base sequence constituting the 5′-LTR sequence is as described in SEQ ID NO: 2, and the base sequence constituting the 3′-LTR sequence is as described in FIG. The 5 'end of each LTR sequence is "5'-TGTTGG-3'", and the 3 'end is "5'-CCTTCA-3'", which is not complete but an inverted repeat. Shows the characteristics of The inverted repeat beginning with TG and ending with CA is
It is commonly found in retroviruses and retrotransposons. In addition, two LTs of retrotransposon OARE-1
The R sequence has one repetitive sequence, pAs17.

The LTR sequence of the retrotransposon OARE-1 is composed of a U3 region, an R region and a U5 region. U3 area is generally RN such as TATA box or CCAAT box.
A promoter II comprises a promoter motif for binding. Retrotransposon OARE-1 LT
In the R sequence, a region presumed to be a TATA box is formed at a position starting from position 958 of the nucleotide sequence. But CCAA
No area that could be a T-box is found. The R region is
Defined by the region to be transcribed, and more specifically, 5 '
Side-The site where transcription starts in the LTR sequence and the 3 'side
-Corresponds to the region between the poly A cap site in the LTR sequence. The terminator required for polyadenylation (poly A) is located at the 8212nd position of the base sequence in the 3′-LTR sequence, and is located at the 8212 position of the base sequence in the 5′-LTR sequence.
It corresponds to the 1261st part. Retro Transposon OARE
-1 potential R region is from position 992 to 1300 in the LTR sequence.
It is likely to be a region with a nucleotide length of 309 bp, corresponding to the third base. The U5 region is a region between the 3 'end of the R region and the end of the LTR sequence. The U5 region of the retrotransposon OARE-1 is a region having nucleotides of 414 bp in length, corresponding to bases 1301 to 1714. In addition, adjacent to both sides of the retrotransposon OARE-1 sequence, a 5 bp long direct repeat sequence (direct
repeats) exists. This tandem repeat is 5'-GGGAC
-3 ', which is part of the oat's original genome. This direct repeat is a retrotransposon OARE-1
It is thought that the target site was formed when the oat copy was inserted into the oat's original genome (see Figs. 1 and 4).

Retrotransposon OARE-1 Leader Region Retrotransposon OARE-1 has two noncoding regions. One is the 5 'side-a region that is continuous downstream of the LTR sequence
(Corresponding to 1225 bp between bases 1715 to 2939), and the other one is a continuous region downstream of the gag-pol coding region (corresponding to 28 bp between bases 6921 to 6948). is there. The former comprises a primer binding site (PBS) adjacent to the 5′-LTR sequence (FIG. 1). PBS is the binding site for the tRNA primer and is a retrotransposon RNA.
Is essential for reverse transcription. The latter comprises a polyprint lacto (PPT) flanking the 3'-LTR sequence. PP
T is a factor essential for initiating the synthesis of (+)-stranded DNA.

The coding region of the retrotransposon OARE-1 is a region having a length of 3981 bp corresponding to the region between bases 2940 and 6920, and coding region of gag protein.
(GAG), one open reading frame (ORF) comprising a protease coding region (Prot), an integrase coding region (ED), a reverse transcriptase coding region (RT), and an RNase H coding region (RH) in this order. (See FIGS. 1 to 4 and FIG. 6). From this sequence, retrotransposon OARE
-1 is a copier-type retrotransposon. As a result of sequence analysis of retrotransposon OARE-1, this clone
It was confirmed that the ORF contained two stop codons. One is a TAG located at the 3825th base, and the other is a TAG located at the 5670th base.
Therefore, we performed RT-PCR according to the procedure shown below.
We tried to amplify the coding region of retrotransposon OARE-1 by the method.

First, in order to amplify a part (about 3.5 kb) of the coding region (CDS) of the retrotransposon OARE-1, a set of primers, OARE / M03 (5'-ATCGGATTAGGTGTGG
TGAA-3 ') and OARE / R03 (5'-TCGTCAAACTAAAGAGCATT-3') were synthesized. Next, Ready-To-Go You-Prime First-St
rand Beads (Amersham Pharmacia Biotech) and Rev
Using erTra Ace α-TM (TOYOBO), cDNA was obtained by reverse transcription using the whole mRNA of oats as a template, and this solution was added to 50 μl of a reaction solution for PCR. PC
For the reaction solution for R, the above two primers were
M d, each dNTP at a concentration of 0.4 mM, LA Taq polym
2.5 U of erase (TAKARA) and 2.5 mM of MgCl ₂
Buffer for LA PCR (TAKARA). The PCR reaction is performed using a thermal cycler PE 480 (Perki
n-Elmer), a denaturation step at 94 ° C for 1 minute, an annealing step at 54 ° C for 1 minute, a chain elongation step at 72 ° C for 6 minutes were repeated for 30 cycles, and finally at 72 ° C. 1
A 0 minute chain extension step was performed. And the obtained RT-PCR
The amplification product was applied to a 0.9% agarose gel placed in 1 × TAE buffer, subjected to electrophoresis, and the obtained fragment was cloned by inserting it into the EcoRV site of plasmid pBluescript SKII +, and the sequence was determined.

When the sequence of the genomic clone of the retrotransposon OARE-1 was compared with the sequence of the RT-PCR amplification product ((pRTM03-R03)), 13 nucleotides were different. When these 13 bases were replaced with those of the RT-PCR amplification product, it was found that the two stop codons in the genomic clone also changed to TGG (W) and CAG (Q) in order. From this result, a complete ORF without stop codon was constructed. FIGS. 2 and 3 also show the base substitution in the 13 nucleotides and the state in which the encoded amino acid residue changes due to the base substitution.

Gag protein coding region The gag gene (corresponding to the gag region) is a gene encoding a gag polypeptide. The gag polypeptide is essential for encapsidation of the retrotransposon (Emori et al. Nature 315 (27): 773-776 (1985)),
Contributes in packaging transcripts and forming virus-like particles (VLPs). Retrotransposon OARE-1
The gag protein coding region is the first to
Encodes the 277th amino acid residue (29 in the base sequence)
40th to 3770th). As shown in FIG. 8A, the translation product of the gag protein coding region of the retrotransposon OARE-1 is a known retrotransposon BA.
RE-1, RIRE1, Sto-4, Tnt1 and translation products of the gag protein coding region of Ta1-3, 51%, 37%, 22%, 21
%, 17%, and, in turn, 88
%, 81%, 68%, 67%, 69% similarity
ity). Furthermore, a conserved motif corresponding to the nucleic acid binding region, CX2CX4HX4 (Berg.Science
232: 485-487 (1986)) is a retrotransposon OARE
-1 was also found in the gag sequence (C250-C263).

Protease coding region Protease gene (corresponding to protease coding region)
Is a gene encoding an aspartic protease. This aspartic protease cleaves pol polyprotein into multiple functional units. The protease coding region of the retrotransposon OARE-1 encodes a region corresponding to amino acid residues 1 to 151 (bases 3771 to 4223) of the pol polyprotein. As shown in FIG. 8B, the amino acid residues (translation products) encoded by the protease coding region are 19% to 74% identical to the amino acid residues encoded by the protease coding region of the known retrotransposon. ,
And 69% to 91% similarity. When comparing translation products of protease coding regions between retrotransposons, substitutions between similar amino acids are frequently observed.
Compared to known retrotransposons Sto-4 and Tnt1, the identity between amino acid residues is low (in order)
33%, 19%), similarity is relatively high
(Both 69%). The translation product of the active aspartic protease coding region has a DSG or DTG motif (Pearl et al. Nature 329: 351-354 (1987)) as a conserved region. And retro transposon OA
The translation product of the protease coding region of RE-1 also has a DTG motif as residues D22-G24.

Integrase coding region Integrase is essential when the retrotransposon is integrated into the host genome, and is well conserved in many retrotransposons. The integrase coding region of the retrotransposon OARE-1 encodes a region corresponding to amino acid residues 152 to 451 of the pol polyprotein (corresponding to bases 4224 to 5123). Amino acid residue encoded by this region (translation product)
Is a known retrotransposon BARE-1, RIRE1, Sto-
4, 80%, 74%, 47%, 41%, and 80%, 74%, 47%, 41%
It shows 40% identity, and shows 74% or more similarity with translation products of other retrotransposons (FIG. 8 (c)). In the integrase, the HHCC motif located at the N-terminus of the zinc finger region and the D (35) E motif involved in recognition of the LTR sequence are well conserved in many retrotransposons (Khanet al.
Nucleic Acids Res 19: 851-860 (1990)). The translation product of the retrotransposon OARE-1 integrase coding region has both of the above motifs, and the HHCC motif is 160
From the amino acid residue and the D (35) E motif is 28
It starts at the fifth amino acid residue.

Reverse transcriptase coding region The reverse transcriptase coding region of the retrotransposon OARE-1 is a region corresponding to amino acid residues 452 to 884 of the pol polyprotein (corresponding to bases 5124 to 6422). Code. Also known retrotransposons
The translation products of the reverse transcriptase coding regions of BARE-1, RIRE1 and Sto-4 have the identity of 70%, 67% and 50%, respectively, and the translation products of the reverse transcriptase coding regions of other retrotransposons. It has a similarity of 76% or more (Fig. 9). The translation product of the reverse transcriptase coding region has a YXDD motif corresponding to amino acid residues 721 to 724, and specific mutations occurring at three amino acids in this motif rapidly increase reverse transcriptase activity. There is a report that falls.

RNase H coding region The RNase H coding region of the retrotransposon OARE-1 encodes amino acid residues located at positions 885 to the end of the coding region (corresponding to bases 6423 to 6920).
The translation product of the RNase H coding region is the same as the translation product of the RNase H coding region of retrotransposon RIRE1.
% Identity and 93% similarity, but relatively low homology with the translation product of the RNase H coding region of retrotransposon BARE-1 (identity 32%, similarity 75%) (FIG. 8 (d)). ). Also,
The translation product of the RNase H coding region contains a DEDD motif involved in metal binding (Davies et al. Science
252: 88-95 (1991)).

(Stress of Retrotransposon OARE-1)
Responsiveness) To elucidate the change in the transcriptional activity of the retrotransposon OARE-1, stress was applied to oats and the change in the transcriptional activity of the retrotransposon OARE-1 was observed by Northern blot analysis as shown below. .

As a plant for stress treatment, IowaX469, a cultivar of oats, was used. The wet filter paper was placed on a Petri dish, and the seeds were allowed to germinate on the filter paper for one day. Subsequently, the germinated seeds were planted in vermiculite packed in a seedling case (seedling case: size 5.5 × 15 × 10 cm). Then, the cells were grown for 8 days in a growth chamber at 20 ° C. for 16 hours. Next, 1) a jasmonic acid solution (chemical stress) at a concentration of 100 μM, 2) a salicylic acid solution at a concentration of 1 mM (chemical stress), and 3) 2 minutes on the first leaf of oats grown for 8 days in the seedling raising case. (Irradiation of ultraviolet light (wavelength: 254 nm: physical stress)). After the treatment, the seedling raising case was returned to the growth chamber set to the condition of 16 hours photoperiod, and the seedling case was stored at 20 ° C.
Incubation was further continued for 2 or 24 hours.

As another treatment, 4) the first leaf of oats grown in the above seedling raising case for 8 days was cut to 5
After being cut into pieces of mm square or 5) After peeling off the epidermis of the first leaf, it was floated in distilled water placed in a Petri dish (physical stress). The petri dish was then 16
It was returned to the growth chamber set to the condition of the time day length, and further incubated at 20 ° C. for 12 hours or 24 hours.

Then, the QuickPrep Micro mRNA Purificatio was obtained from the first oat leaves treated with the above 1) to 5) and the untreated first oat leaves (control).
RN using Kit (Amersham Pharmacia Biotech)
A was extracted and subjected to Northern blot analysis described below.

In the Northern blot analysis, first, all RNs were
A (10 μg) containing 3% formaldehyde 2.0
It was applied on a% agarose gel and fractionated by electrophoresis. After electrophoresis, the RNA was copied onto a nylon membrane and fixed according to the description in the manual. The nylon membrane was then hybridized with a plurality of fluorescein-labeled RNA probes. In addition, the plurality of RNA probes are
POARES / S region containing SacI-SacI fragment (see FIG. 6) and victorin C-dependent subtracted cDNA
CDNA fragments screened from the library (PA
L fragment), respectively, using T3 RNA polymerase
It consists of a transcript transcribed by itro. The PAL fragment is 95
5 bp long, plasmid vector pBluescript SK
It was cloned into the EcoRI-XhoI site of II + and sequenced.

Next, 1) The membrane after hybridization was heated to 70 ° C. using a 1 × SSC solution containing 0.1% SDS.
For 15 minutes (low stringency conditions) twice, and then using 0.1 × SSC solution containing 0.1% SDS
Washing was performed twice at 70 ° C. for 15 minutes. Alternatively, 2) 0.1% SDS is included in the hybridized membrane
Wash twice with 1xSSC solution at 72 ° C for 15 minutes (high stringency conditions), then contain 0.1% SDS
Washing was performed twice at 72 ° C. for 15 minutes using a 0.5 × SSC solution.

As shown in FIG. 11, in the control (untreated, intact first leaf), the retrotransposon
OARE-1 was hardly transcribed. On the other hand, chemical inducers (inducers) of defense reactions against pathogens
When exogenously given jasmonic acid (JA) or salicylic acid (SA), the expression of the retrotransposon OARE-1 was induced. Also, salicylic acid activated the retrotransposon OARE-1 more efficiently than jasmonic acid. From this, retrotransposon OARE
The signaling system that induces -1 expression appears to be related to the salicylic acid-related signaling system rather than the jasmonic acid-related signaling system.

Further, when irradiated with ultraviolet light (UV), the expression of the retrotransposon OARE-1 was activated 12 hours after the treatment. When the first leaf was cut or the epidermis was peeled, expression of the retrotransposon OARE-1 was activated up to 24 hours after the treatment.

Further, as shown in FIG. 11, the expression pattern of the retrotransposon OARE-1 was compared with the expression pattern of phenylalanine ammonia lyase (PAL).
As a result, PAL expression was detected in all the first leaves treated with 1) to 5) except for the control, and the pattern of expression was almost identical to that of the retrotransposon OARE-1. . In particular, both genes (PAL, OARE-1) were similarly activated by jasmonic acid or salicylic acid treatment. PAL is a key enzyme involved in phenylpropanoid metabolism when oats synthesize phytoalexin (Mayama et al. Physiol Mol Plant Pathol 2
9: 1-18 (1986)). Therefore, the retrotransposon OARE
-1 is thought to be activated by pathogen infection.

(Retrotrans for plants other than oats
Distribution of Poson OARE-1) Retrotransposon OARE-1
The possibility of distribution in plants other than oats (barley, wheat, rice) was examined by Southern hybridization analysis described below. First, a method for preparing a plant material will be described.

First, for oats, barley, and wheat, a filter paper moistened in a Petri dish was placed, and one filter was placed on the filter paper.
The seeds were allowed to germinate over the days. Then, the germinated seeds are
The plants were planted in vermiculite arranged in a growth chamber and grown for 8 days at 20 ° C. and 16 hours photoperiod. On the other hand, as for rice, moistened filter paper was placed in a petri dish, and seeds were germinated on the filter paper over 3 days. Then
The germinated seeds were planted in vermiculite arranged in a growth chamber, and grown at 25 ° C. for 16 hours under a photoperiod for 3 weeks.

From the first leaf of each plant material prepared as described above, Liu et al. (Jpn. J. Genet. 65: 367-38)
0 (1990)). Next, the whole genomic DNA (10 μg) was
This was digested with I, and this was fractionated by electrophoresis on a 0.5% agarose gel placed in 0.5 × TBE buffer. After the electrophoresis, the DNA was copied onto a nylon membrane and fixed according to the description in the manual. The nylon membrane was then hybridized with a fluorescein-labeled RNA probe. The RNA probe was obtained by in vitro transcription of pOARES / P digested with an appropriate restriction enzyme using T3 RNA polymerase. pOARES / P comprises the SacI-PstI fragment of the screened OARE-1 clone (FIG. 6).

Next, 0.1% SDS was applied to the nylon membrane after hybridization with the above RNA probe.
Was performed twice at 68 ° C. for 15 minutes (low stringency conditions) using 1 × SSC solution containing 0.1% SD
Washing was performed twice at 68 ° C. for 15 minutes using a 0.5 × SSC solution containing S. Alternatively, the hybridized nylon membrane is washed twice with a 1 × SSC solution containing 0.1% SDS at 72 ° C. for 15 minutes (high stringency conditions), and then washed with a 0.5 × SDS solution containing 0.1% SDS. Washing was performed twice at 72 ° C. for 15 minutes using the SSC solution.

As shown in FIG. 10, when the above nylon membrane was hybridized with pOARES / P (OARE-1 probe), the four oat cultivars (oat1 to 4) were prepared under low stringent conditions (68 ° C.). ), A signal indicating the presence of OARE-1 was confirmed under both high stringency conditions (72 ° C.). In addition, barley (barley5,6), wheat (w
heat7,8) also detected a signal. More specifically,
A 0.5 kb band was detected in the barley genome, and a 0.8 kb band and a weak smear band were detected in the wheat genome. On the other hand, no signal was detected in rice (rice 9, 10). Note that oat1 to 4 shown in FIG. 10 indicate IowaX469, IowaX424, shokan1, CW-491-4, and barl
ey5,6 indicates Nakaizumi-zairai, Russian 66 in order, wh
eat7,8 indicate Chinese Spring, Norin-4 in order, rice9,10
Indicates Toride1 and CO39 in order.

(Copy of Retrotransposon OARE-1)
Number) In Southern hybridization of oat genomic DNA using a probe prepared from pOARES / P (FIG. 6), formation of a considerable hybrid was observed even under high stringency conditions (FIG. 10). This suggests that the oat genome has a considerable number of copies of the retrotransposon OARE-1. Oats (Avena sativa L.)
The copy number of the retrotransposon OARE-1 contained in the genome was estimated as follows.

As a primary screening, the retrotransposon OARE-1 was screened from the genomic library of the oat cultivar IowaX469. As described above, about 210 plaques out of the 5000 plaques on average showed the Oatrt1 probe. (The above RT fragment Oatrt1) showed a positive signal.

Average insert of the above genomic library
Since the length is 20kb, 5000 plaques are 10⁸equivalent to bp
You. The genome size of oats is about 10^Tenbp
So the retrotransposon OARE-1 copy per genome
The number is 210 × 10^Ten/ 10 ⁸= 20000
It is.

[0086]

As described above, the retrotransposon according to the present invention is isolated from oats, and the transcription in the host is activated or the transcriptional activity is further enhanced by the application of an exogenous stimulus. Configuration.

The retrotransposon according to the present invention comprises:
As described above, a configuration comprising two identical repeats comprising the nucleotide sequence of SEQ ID NO: 1 or a homologous sequence thereof, and a transgene sandwiched between the two identical repeats. is there.

The retrotransposon according to the present invention comprises:
In the above-mentioned configuration, the above-described orthotopic repeat sequence comprises the base sequence of SEQ ID NO: 2 or a homologous sequence thereof.

The retrotransposon according to the present invention has a structure comprising the nucleotide sequence of SEQ ID NO: 3, as described above.

According to any of the above constitutions, the effect is obtained that the transcription can be activated by the application of an exogenous stimulus, or a novel retrotransposon whose transcription activity is further enhanced can be provided. In addition, any of the above retrotransposons can be isolated from, for example, oats.

As described above, the DNA fragment according to the present invention comprises the nucleotide sequence of SEQ ID NO: 1 or a homologous sequence thereof, and exhibits a promoter activity upon application of an exogenous stimulus, or exhibits a lower promoter activity. The configuration is enhanced. This DNA fragment can be isolated from, for example, oats.

[0092] According to the above configuration, there is an effect that it is possible to provide a novel DNA fragment which exhibits a promoter activity by application of an exogenous stimulus or has a further enhanced promoter activity.

As described above, the plasmid according to the present invention comprises the above-described DNA fragment inserted as a promoter, and further comprises a region for inserting an arbitrary gene fragment and a region imparting host infectivity. It is a configuration to have.

According to the above configuration, it is possible to provide a novel plasmid which can infect a host and express the gene fragment by giving an exogenous stimulus.

As described above, the method for expressing a gene according to the present invention comprises inserting an arbitrary gene fragment into any of the above retrotransposons, and incorporating the retrotransposon into which the gene fragment has been inserted into genomic DNA of a host. This is a method for expressing the above gene.

The method for expressing a gene according to the present invention
As described above, this method is to express the gene by infecting a host with the above plasmid.

According to any one of the above-mentioned methods, it is possible to provide a method that allows relatively safe and easy expression of an arbitrary foreign gene in a host, and that allows the expression to be artificially controlled. This has the effect.

[Brief description of the drawings]

FIG. 1. D containing retrotransposons according to the invention.
It is a figure which shows a part of base sequence of NA fragment.

FIG. 2 is a diagram showing a part of a base sequence that is continuous with the base sequence shown in FIG. 1;

FIG. 3 is a diagram showing a part of a base sequence that is continuous with the base sequence shown in FIG. 2;

FIG. 4 is a diagram showing a nucleotide sequence that is continuous with the nucleotide sequence shown in FIG. 3;

FIG. 5 is a diagram showing a main part of a 5′-side homo-directional repeat sequence in the DNA fragments shown in FIGS. 1 to 4;

FIG. 6 is a diagram showing a restriction enzyme map of the DNA fragment shown in FIGS.

FIG. 7 shows the results of amplification by the RT-PCR method.

8 (a) to 8 (d) show an amino acid sequence encoded by a retrotransposon coding region contained in the DNA fragment shown in FIGS. 1 to 4 and an amino acid sequence encoded by a known retrotransposon coding region. FIG. 3 is a diagram showing the homology of

FIG. 9 is another diagram showing homology between the amino acid sequence encoded by the coding region of the retrotransposon contained in the DNA fragment shown in FIGS. 1 to 4 and the amino acid sequence encoded by the coding region of a known retrotransposon. is there.

FIG. 10 shows the results of confirming the distribution of retrotransposons contained in the DNA fragments shown in FIGS.

FIG. 11 is a diagram showing the responsiveness of a retrotransposon contained in the DNA fragment shown in FIGS. 1 to 4 to an exogenous stimulus.

[Sequence List] SEQUENCE LISTING <110> TLO Hyogo TLO Hyogo <120> Retrotransposon, DNA fragment with promoter activity, and its use <130> 130313 <160> 3 <210> 1 <211> 903 <212> DNA <213> Avena sativa L. <400> 1 gtgaacacca ccgtgtccct agtacgcctc tatggactat ctcattgatc aatgatggtt 60 aaggtttcct aaccatagac atgagttgtc aattgataat gagatcatat catggagaat 120 gatatgatgg acagacccaa actaagcgaa gcaattagat cgtgtcatcg tgttcgtctt 180 tgcgaagctt ttcttatgtc caatattgta ttccttagac catgagataa tacaactccc 240 tggtatcgga agaaagcttt gaggtcatca aacatcatcc tatgagaggg tgatcataaa 300 ggtgtctctc aggtgatcgg aaagtatctg ttgggttgta tgtatcgaga tgggatttgt 360 ccctcccaag ttggagagat atacttcggg cccactcggt aatcgatatc accaccgctt 420 gcaagcatgt ggttaaggag ttaatcacaa agtgatatcg gattacggaa cgagtaaagt 480 acttgctgtg aacgagattg aactaggtat gacggtaccg acgatcgaat ctcgggcaag 540 taaaatatcg cgagacgaag ggact atata tatgggattg tttgaatcct gacatcgtgg 600 ttcatccgat atgatcatcg atgaatgtgt gggagtcaat atgggtgccc agaccccgct 660 gttgattatt gatagaagat catgtctgca tattctcgaa cccgcagggt cacacactta 720 acgtgtcttg acgctaggat agattggata attggtttat gaatttttgt tcggagtctc 780 ggatgggatc cgggatatca cgagaggttc cggaatggtc cggagaataa gattcatata 840 aggcagtgca gtttttggag ttcggaattg ttccgggtta atcggtattg taccggaagc 900 ttc 903 <210> 2 <211> 1714 <212> DNA <213> Avena sativa L. <400> 2 tgttggaatt atgccctaga ggaaataata aagatgttat tattataatt cttgttcatg 60 ataatgttta tattccatgc tagaattgta ttaagcggaa actttaatac ttgtgtggtt 120 tgtgaacacc accgtgtccc tagtacgcct ctatggacta tctcattgat caatgatggt 180 taaggtttcc taaccataga catgagttgt caattgataa tgagatcata tcatggagaa 240 tgatatgatg gacagaccca aactaagcga agcaattaga tcgtgtcatc gtgttcgtct 300 ttgcgaagct tttcttatgt ccaatattgt attccttaga ccatgagata atacaactcc 360 ctggtatcgg aagaaagctt tgaggtcatc aaacatcatc ctatgagagg gtgatcataa 420 aggtgtctct caggtgatcg gaaagtatct gttgggttgt atgtatc gag atgggatttg 480 tccctcccaa gttggagaga tatacttcgg gcccactcgg taatcgatat caccaccgct 540 tgcaagcatg tggttaagga gttaatcaca aagtgatatc ggattacgga acgagtaaag 600 tacttgctgt gaacgagatt gaactaggta tgacggtacc gacgatcgaa tctcgggcaa 660 gtaaaatatc gcgagacgaa gggactatat atatgggatt gtttgaatcc tgacatcgtg 720 gttcatccga tatgatcatc gatgaatgtg tgggagtcaa tatgggtgcc cagaccccgc 780 tgttgattat tgatagaaga tcatgtctgc atattctcga acccgcaggg tcacacactt 840 aacgtgtctt gacgctagga tagattggat aattggttta tgaatttttg ttcggagtct 900 cggatgggat ccgggatatc acgagaggtt ccggaatggt ccggagaata agattcatat 960 aaggcagtgc agtttttgga gttcggaatt gttccgggtt aatcggtatt gtaccggaag 1020 cttctagaac cttccgaaag gggaccaacg tggccccacc ttccgggagg gggcatgggc 1080 ctatgagtga gtgtcctggc cgcattggcc aggggcacaa ggccccatag ggcccagccg 1140 atcctagaga gtttaaatcg tttaaactag agatagggtt tctagaagag gggagagtcc 1200 atgcccccca ctccttatcc tttgggcttg ggggaggggc tagggctgcg ccccccctct 1260 atataaagag gggtgggggc gcaccaaccc cccacacctc tcctccaaac cctagccgcc1320 ttgctagctt tcctctctcc ctcccgactg tttgtaggcg aagccctact gcagaaattc 1380 tccaccatat acaccacgcc gtcgtgttgt agatccaatc tatctctcca ccatacttgc 1440 tggtccgagg aaggaggaga tgtcgaggtg ctgcacgtgt gcatctcacg gaggcaccgc 1500 cacttgtggt gctagatcgg atcggatcgc gaggaggaga agcgagtacg actacatcac 1560 ccatgttccg acgaacgttc cgctgtgcga atcttcaagg gtatgaagat ctaatccaat 1620 ctctctcgtt gtttgcatct cctagattag atcttggctt tccctctgtt cgctccgtag 1680 aaaattttga tttactatgc cgcgaaaccc ttca 1714 <210 > 3 <211> 8665 <212> DNA <213> Avena sativa L. <400> 3 tgttggaatt atgccctaga ggaaataata aagatgttat tattataatt cttgttcatg 60 ataatgttta tattccatgc tagaattgta ttaagcggaa actttaatac ttgtgtggtt 120 tgtgaacacc accgtgtccc tagtacgcct ctatggacta tctcattgat caatgatggt 180 taaggtttcc taaccataga catgagttgt caattgataa tgagatcata tcatggagaa 240 tgatatgatg gacagaccca aactaagcga agcaattaga tcgtgtcatc gtgttcgtct 300 ttgcgaagct tttcttatgt ccaatattgt attccttaga ccatgagata atacaactcc 360 ctggtatcgg aagaaagctt tgaggtcatc aaacatcatc ctat gagagg gtgatcataa 420 aggtgtctct caggtgatcg gaaagtatct gttgggttgt atgtatcgag atgggatttg 480 tccctcccaa gttggagaga tatacttcgg gcccactcgg taatcgatat caccaccgct 540 tgcaagcatg tggttaagga gttaatcaca aagtgatatc ggattacgga acgagtaaag 600 tacttgctgt gaacgagatt gaactaggta tgacggtacc gacgatcgaa tctcgggcaa 660 gtaaaatatc gcgagacgaa gggactatat atatgggatt gtttgaatcc tgacatcgtg 720 gttcatccga tatgatcatc gatgaatgtg tgggagtcaa tatgggtgcc cagaccccgc 780 tgttgattat tgatagaaga tcatgtctgc atattctcga acccgcaggg tcacacactt 840 aacgtgtctt gacgctagga tagattggat aattggttta tgaatttttg ttcggagtct 900 cggatgggat ccgggatatc acgagaggtt ccggaatggt ccggagaata agattcatat 960 aaggcagtgc agtttttgga gttcggaatt gttccgggtt aatcggtatt gtaccggaag 1020 cttctagaac cttccgaaag gggaccaacg tggccccacc ttccgggagg gggcatgggc 1080 ctatgagtga gtgtcctggc cgcattggcc aggggcacaa ggccccatag ggcccagccg 1140 atcctagaga gtttaaatcg tttaaactag agatagggtt tctagaagag gggagagtcc 1200 atgcccccca ctccttatcc tttgggcttg ggggaggggc tagggctgcg ccccccctc t 1260 atataaagag gggtgggggc gcaccaaccc cccacacctc tcctccaaac cctagccgcc 1320 ttgctagctt tcctctctcc ctcccgactg tttgtaggcg aagccctact gcagaaattc 1380 tccaccatat acaccacgcc gtcgtgttgt agatccaatc tatctctcca ccatacttgc 1440 tggtccgagg aaggaggaga tgtcgaggtg ctgcacgtgt gcatctcacg gaggcaccgc 1500 cacttgtggt gctagatcgg atcggatcgc gaggaggaga agcgagtacg actacatcac 1560 ccatgttccg acgaacgttc cgctgtgcga atcttcaagg gtatgaagat ctaatccaat 1620 ctctctcgtt gtttgcatct cctagattag atcttggctt tccctctgtt cgctccgtag 1680 aaaattttga tttactatgc cgcgaaaccc ttcagtggta tcagagctag atctatgcgt 1740 agatgtgttt cacgagtaga acgcaacatg gttgttgcgg gcgttgatgt tttctgttgc 1800 aagcctcttg tttgtgttct tcttatgacg cgggatagtg agattgaagc ggtccaggcc 1860 aaccttacgc gacgtcgttc gcgagaccgg ctccgcactt gacgtgtggc ttgttccaca 1920 catgcggctg gtggatgtca gtttctctca ctatagttgt catgggatta acaccggagt 1980 ttggcaacca taatatcacc gttgtggctt tccgtaggta agaatagttc ttagcttcta 2040 ctcccataaa gtcacgtaaa acatgcaaca acaaagtaga ggcgtctaac ttgtttttgc 2100 agggagtgtt ttctgtgata tggtatggcc caagatgtga tgtgtataat gtatgcgatg 2160 atcatgtttt gtaatgtttc acgacttgca tgtcgatgag tacacaaccg gcagaagcca 2220 tatggttgtc tttataattg tgtatgacct gtgtgtcaac ttcttatacg ccatgtaata 2280 gctttacttt attgctaagt agttagcaat agttgtagtt gctattcgtg gtggaggcaa 2340 tcatggtgga gtgatggaga tcatgccgga gcgcacaaga tggagatcat tgcgcgaaga 2400 ttggctatac catgtcacat aaagttatat gcatgtgatg tttatccttt tatgtatctt 2460 attatgctta gatcgcgacg gtagcattat aagatgattc ctcacttaaa gatcaagata 2520 aaaactgtgt tctccccaag tatgcaccgt tgcgaaagtt cgtcgtgtcc aagcatcacg 2580 tgatgatcgg gtgctataga ctctacgctc acatacaacg ggtgcaagcc agtttttgca 2640 cacgcggaaa tacttgggtc tagcttgacg agcctagcat gtacagacat ggcctcggaa 2700 cacaagaacc gaaaggttaa gcatgagtca tatgaatgat atgatcgaca cttcgttgtc 2760 caccattgaa accacatctc ttctcgtgat gatcggatta ggtgtggtga atttggatcg 2820 tgttcactaa acaacttgag ggatgttgaa tttagtggga gttcatctag taatttgatt 2880 aagttgaact atttatcatg aacatagtct aaattgtact tgcaaatact gtagatcaca 2940 tggac aagtc caacttcaac ttcaacgttt ttgctgagaa ggaaaagctt agaaacaatg 3000 ggtctaactt caccaattgg ttccgcaatc taagaatcat tctgtctggc gaacaaaagg 3060 actatgtcct tgggaaagcg ttaggtaacc cccctgctgc aactgcgctg gctgatgaga 3120 aagctgtcta tcagtcgcgc aaagatgatt atggtgtgat taagagcacc atgttgtttg 3180 ctatggaaac ggagcttcaa aatcgttttg aagagtatcc cgggcccttc gagattctcg 3240 aggagctgaa aaccatgttt cagactcagg cccgttctga gagatatgag atctccgaga 3300 agttctttaa ctgcaagatg gaggaaggga gttccgttag tgagaacgcc attaagatga 3360 cgggctacac tcaacgcctt gagcaactgg aatgcacgat tccagaggag ctcaagattg 3420 acagggttct gcaatcactc cctcccagct acaaaagttt tgtagtgact cataatcaga 3480 ttggttccac tgatatgatc accgagctct tcgcaaagct gaaggctacg gaagtggaca 3540 tcaagaaggt taatcatgtg ttgatggtca acaagccctc ctttaagaaa ggtaagggag 3600 ctaagaagcc cttcaagaag ggcggcggca agcgcaatgc ccctgctgag aagaagctca 3660 agtctggtcc caagccggac actaagtgct tctattgcaa ggacaagggt cactggaagc 3720 gcaactgtcc gaagtactta gcggacaaga aggctggcac catcaaaggt atatttgata 3780 tacatgttat tgatgttttc cttactagcc ctcgttgtag ttcctgggta tttgataccg 3840 gttcagttgc taacatttgt aactcgaaac aggggctacg gaatagacgg cggcttgcga 3900 gggatgaggt gacgatgcgc gttggcaacg gatcaagagt tgatgtgatg gccgtcggca 3960 cgctctcctt agttttacct tcgggattag ttttaaacct taataagtgt tataatgtgt 4020 ctgcgttgag catgaacatt atttctggat cgtgtttatt gcaagacggt tattcattta 4080 aatcggagaa taatggttgt tctatttata tgagtaacat cttttatggt catgcacctc 4140 ttgtaaatgg tttatttctt ctaaatctcg aaagggatga aacacacatc cataacattg 4200 aggctaagag atgcaaagta gatagtgata atactactta tttgtggcac tgtcgtctag 4260 gtcatattgg agtaaagcgc atgaagaaac tccattctga tggacttttg gagtctcttg 4320 attttgaatc atttgacaca tgcgaaccat gcctcatggg caagatgatc aaaacaccgt 4380 tctccggtat gatggagcga gcaacggact tgttggaaat catacatacc gatgtatgcg 4440 gaccgatgag tatttcgacg cgcggtggat atcgctattt cctcactttt accgatgact 4500 taagtagata tgggtatatc tatttaatga aacataaatc tgaaacattt gaaaagttca 4560 aggaatttca gaatgaagtg gagaaccatc gtaacagaaa gattaagttt ctacgatccg 4620 atcgtggagg tgaata tttg agttatgatt ttctcacgca tcttaaagca agtggaatcg 4680 tttcacaact cacgccaccc ggaacaccac aacgcaatgg tgtgtctgaa cgccggaatc 4740 gaaccctatt agatatggtc cgatcaatga tgtctcttac tgatttgccg ttatcatttt 4800 ggggctatgc attagaaacc gctgcattca ctttaaatag ggctccatct aaatccgttg 4860 agacgacacc gtatgacctg tggtttggtg ctaaacctaa gttgtcgttt cttaaagttt 4920 ggggatgcga agcttacgtc aagaagttga tgccggacaa gctcgaaccc aaagcggaga 4980 aatgcgtctt cataggatac cctaaggaaa ctattgggta taccttctat cacagaaccg 5040 aaggcaaagt ttttgtcgca aagaacggtt cctttcttga gaaggagttt ctctcgaaag 5100 aagtgagtgg gacgaaagta gagctagacg aggttattga accttctctt gagttggaga 5160 gtagcgcagc acctgaaatt gttccagtgc cgcctgcacc aacaatagag gaagctaatg 5220 acaaggatca tgaagcttcg gacgatgctg ctactgaacg tcgcaggtcg acaaggtcgc 5280 gtgcccctcc tgagtggtac ggcaaccctg tcttaactgt cttgttagta gaagacagtg 5340 aacctacgaa ctatgaggaa gcgatgatga tcccagactc tgagaaatgg ctagaagcca 5400 tgaaatccga attgggatcc atgagcgaaa accaagtatg gactttggta gaccctccta 5460 gcgaccgaaa ggccgttgag t gcaaatgga tcatcaagaa gaaaacggac gcggatggta 5520 atgtcaccgt ctataaagca cggcttgtcg cgaaaggttt tcgacaagtt caaggtgttg 5580 actacgatga gactttctct cccgtagcga tgtttaagtc cattcggatt ttattagcaa 5640 ttgctgcgtt tcatgatgat gaaatatggc agatggatgt caagactgcg ttcctcaatg 5700 gcaacattga tgaagagttg tatatgatgc aacccgaagg ttttgtcgat cctaaggaag 5760 ctagtaaagt atgcaaactt cagtgttcca tttatggact gaagcaagca tctcggagtt 5820 ggaaccttcg ttttgatcaa gtgatcaaga gctttggatt tgttccaaat tgctacgaag 5880 cttgtatcta caagaatgtg agtgggagct cggtgacgtt tctggtacta tatgtggatg 5940 acatattgat tatcggaaat gacatcaaca tgcttaacga tgtgaagagt tatttgaaca 6000 agtgtttttc aatgaaagac ctaggtgaag ctgcttatat attgggcatc aaaatatata 6060 gagatagaca aagacgcctg atagccttaa gccagagtac atacctagac aaagttctga 6120 aaaggttcag aatggataag gcaaagaaag gttcagtgcc tatgttgtca ggtaagatat 6180 ctagcaaggc tcagtgtcta gctacggtaa atgataggga agctatgagc aaagtgcctt 6240 atgcctcggc cataggttcc atcatgtatg ccatgctgtg tactagaccg gatgtgtcca 6300 atgctcttag tttgacgagc agatatc aaa gtgatccagg agtggaacac tggacagccg 6360 tgaagaacat cctaaagtac ctaaatagga ctaaagaaat gttcttagtc tatgggggtg 6420 atgaacagct tgtcgtaaaa ggttacgtcg acgctagttt tgacaccgac aaggatgatt 6480 ccaaatctca gaaaggatac gtttatattt tgaatggagg agctgtgagc tggagaagct 6540 gcaagcaaag cgtgatcgcg ggatctatag tggaagcgga atacatggct gctgcagaag 6600 cgtcaagtga aggagtttgg atcagaaact tcatcactga gcttggcatg gttccaagtg 6660 cattggaccc actagagata ttatgtgaca acactggcgc catagcactc gctaaggaac 6720 caagatatca tccaaagacc aagcatatca actgcaagtt tcatataatc cgagattata 6780 tagcagatgg agatctgaag attagcaaag tacatacaga tctgaatgtc gcagatccat 6840 tgactaaacc tcttccacgt gtaaaacatg atcaacacct cagtgccatg ggtgtgagat 6900 tgttgccaaa tgtaaactag attattgact ctagtgcaag tgggagactg ttggaattat 6960 gccctagagg caataataaa gatgttatta ttataattct tgttcatgat aatgtttata 7020 ttccatgcta gaattgtatt aagcggaaac tttaatactt gtgtggtttg tgaacaccac 7080 cgtgtcccta gtacgcctct atggactagc tcattgatca atgatggtta aggtttccta 7140 accatagaca tgagttgtca attgataatg ag atcatatc attggagaat gatatgatat 7200 acagacccaa actaagagaa gcaattagat cgtgtcatcg tgttcgtctt tgcgaagctt 7260 ttcttatgtc aaatattgta ttccttagac catgagataa tacaactccc tggtatcgga 7320 agaaagcttt gaggtcatca aacgtcatcc tatgagaggg tgatcataaa ggtgtctctt 7380 aggtgatcgg aaagtatctg ttgggttgta tgtatcgaga tgggatttgt ccctcccaag 7440 ttggagagat atacttcggg cccactcggt aatcgatatc aacaccgatt gcaagcatgt 7500 ggttaaggag ttaatcacaa agtgatatcg gattacggaa cgagtaaagt acttgctgtg 7560 aacgagattg aactaggtat gacggtaccg acgatcgaat ctcgggcaag taaaatatcg 7620 cgagacgaag ggaatatata tatgggattg tttgaatcct gacatcgtgg ttcatccgat 7680 atgatcatcg atgaatgtgt gggagtcaat atgggtgcct agaccccgct gttgattatt 7740 gatagaagat catgtctgca tattctcgaa cccgcagggt cacacactta acgtgtcttg 7800 acgctaggat agattggata attggtttat gaatttttgt tcagagtctc ggatgggatc 7860 cgggatatca cgagaggttc cggaatggtc cagagaataa gattcatata aggcagtgca 7920 ggttttggag ttcggaattg ttccgggtta atcggtattg taccagaagc ttctagaacc 7980 ttccagaagg ggaccaacgt ggccccacct tccgggag gg ggcatgggcc tatgagggag 8040 tgtcctggcc gcattggcca ggggcacaag gccccatagg gcccagccga tcctagagag 8100 tttaaatcgt ttaaactaga gatagggttt ctagaagagg ggagagtcga tgccccccac 8160 tccttatcct ttgggcttgg gggaggggct agggctgcgc cccccccctc tatataaaga 8220 ggggtggggg cgcaccaacc ccccacacct ctcctccaaa ccctagccgc cttgctagct 8280 ttcctctctc cctcccgact gtttgtaggc gaagccctgc tgcagaaatt ctccaccata 8340 tacaccacgc cgtcgtgttg tagatccaat ctatctctcc accatacttg ctggtccgag 8400 gaaggaggag acgtcgaggt gctgcacgtg tgcatctcac ggaggcaccg ccacttgtgg 8460 tgctagatcg gatcggatcg cgaggaggag aagcgagtac gactacatca cccatgttcc 8520 gacgaacgtt ccgctgtgcg aatcttcaag ggtatgaaga tctaatcgg tattctgcc 8580 tgtctgcc 8580 tgtttgcc 8580 tgtttgcc

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yosuke Kimura 3-11-7 Okamoto, Higashinada-ku, Kobe-shi, Hyogo F-term (reference) 4B024 AA08 CA01 CA09 GA11 HA08

Claims (10)

[Claims] 1. A retrotransposon isolated from oats and characterized in that transcription in a host is activated or transcriptional activity is further enhanced by application of an exogenous stimulus. 2. A method comprising two identical repeats comprising the nucleotide sequence of SEQ ID NO: 1 or a homologous sequence thereof, and a transgene sandwiched between the two identical repeats. Characterized retro transposon. 3. The retrotransposon according to claim 2, wherein the orthotopic repeat comprises the nucleotide sequence of SEQ ID NO: 2 or a homologous sequence thereof. 4. A retrotransposon comprising the nucleotide sequence of SEQ ID NO: 3. 5. The retrotransposon according to any one of claims 2 to 4, wherein the retrotransposon is isolated from oats. 6. A DNA fragment comprising the nucleotide sequence of SEQ ID NO: 1 or a homologous sequence thereof, wherein the DNA fragment exhibits promoter activity or is further enhanced in promoter activity by application of an exogenous stimulus. 7. The DNA fragment according to claim 6, which is isolated from oats. 8. The DNA fragment according to claim 6 or 7, wherein the DNA fragment is inserted as a promoter, and further has a region for inserting an arbitrary gene fragment and a region imparting host infectivity. Plasmid. 9. An arbitrary gene fragment is inserted into the retrotransposon according to any one of claims 1 to 5,
A method for expressing a gene, which comprises expressing the gene by incorporating the retrotransposon into which the gene fragment is inserted into genomic DNA of a host. [10] A method for expressing a gene, which comprises expressing the gene by infecting a host with the plasmid according to [8].