JP2003070477A - Vector for new plant gene trap and plant gene trap utilizing the same vector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a new gene-searching method useful for search of genes which participate in change with time of genes in a live plant individual and environmental response. SOLUTION: The vector for new plant gene trap comprising luciferase as a reporter gene and the plant gene-trapping method using the vector are provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel plant gene trap vector and a plant gene trap method using the same. More specifically, the present invention relates to a method of using a luciferase gene as a reporter gene to search for the expression of a gene in a living plant individual that corresponds to the temporal change of the gene or the environmental response.

[0002]

2. Description of the Related Art A gene trap is one of the methods for knowing information about genes in the genome, and a trap vector containing a reporter gene is randomly inserted into the genome to detect its expression pattern. Is a method of searching for expression information of an unknown gene. This method is characterized in that there is a high possibility that gene disruption mutants can be obtained simultaneously with gene expression information. Gene traps can be broadly classified into the following two types.

1) Gene trap in a narrow sense: A trap vector does not have a promoter structure that regulates transcription, and a reporter gene is transcribed as a fusion protein by its transcription initiation point only when inserted inside a structural gene on the genome. Express. Alternatively, it is expressed in some form without being a fusion protein. It also includes the case called a promoter trap.

2) Enhancer trap: The trap vector contains a minimal promoter such as TATA sequence. Therefore, transcription of the reporter gene is initiated from within the trap vector regardless of the orientation and position of insertion, but expression of the reporter gene is controlled by the enhancer in the vicinity of the insertion site, which reflects the expression pattern of genes in the vicinity. To be done.

Conventionally, as reporter genes used for gene traps, beta-galactosidase (lacZ) gene derived from bacteria and beta-glucuronidase (ui
The dA) gene and the green fluorescent protein (GFP) gene from luminescent jellyfish have been used. Each of these reporter genes has its own characteristics and is used properly according to the purpose. The reporter gene must be capable of easily measuring the activity of the reporter protein, and the activity of the protein can easily measure the expression status of the reporter gene.

Above all, it is possible to measure while keeping the living body alive.
GFP is extremely useful as a reporter. But GFP
Has the following problems as a reporter. 1) Since fluorescence is used for detection, the fluorescence from the original biological substance appears as noise, and the signal value against noise is weak. 2) It is not possible to measure expression in a plant cell neutrally because it is toxic (J. Haseloff and B. A)
mos, 1995, Trends Genet., 11, 328-329). 3) The GFP protein is extremely stable, making it unsuitable for observing changes in expression level over time (DC Baulcombe, S. Chapman, a
nd SS Cruz, 1995, Plant J., 7, 1045-1053).

On the other hand, another reporter, the firefly luciferase gene (LUC), can be measured while keeping the living body alive, like GFP, and has the following advantages (AJ Millar
Et al., 1992, Plant Mol. Biol. Rep., 10, 324-337). 1)
The value of the signal against noise is extremely high because detection is performed by light emission. 2) No toxicity in plant cells. 3) Since LUC proteins are unstable in plant cells, changes in gene expression over time are directly reflected in LUC activity. 4) Unlike the case of GFP, it is not necessary to irradiate excitation light to measure LUC, so the effect of excitation light on the living body can be ignored.

On the other hand, the choice of reporter in a gene trap varies considerably depending on the species. For example, the lacZ reporter is a commonly used reporter in Escherichia coli, mouse, and Xenopus, but it has not been used in plants. This is probably because the lacZ protein is very unstable in plant cells, does not fold correctly in plant systems, or the environment such as intracellular pH is inappropriate for the lacZ reporter. It is considered that the activity is unlikely to occur inside. Instead, the GU
The S (beta-glucuronidase) reporter is commonly used.

In addition, the translation efficiency differs between plants and animals due to the difference in the optimal codon usage and the like. Therefore, it is extremely difficult to predict in advance whether or not the reporter activity will reach a practically sufficient level simply by changing the reporter gene or promoter having the same nucleotide sequence for animals and plants.

[0010] Japanese Patent Application Laid-Open No. 11-332564 discloses a Drosophila gene trap vector using an LUC reporter, but there has been no report on an example of using the LUC reporter for a gene trap in plants. This is because LUC tends to lose its activity in plants when it becomes a fusion protein (CK Worley, N Zenser, J Ramos,
D Rouse, O Leyser, A Theologis, J Callis, Plant J,
2000, 21: 553-562) Therefore, it is believed that it is not suitable for gene trap. In fact, the question as to whether the LUC fusion protein produced by randomly inserting the LUC gene into the genome using a translation fusion type trap vector has an activity has not yet been solved.

[0011]

[Problems to be Solved by the Invention] The plant gene trap method is widely used as a part of gene mining not only in the field of basic research but also in the field of applied research with a view to genetic engineering of plants / agricultural products. , Has an important value as a method for analyzing genomic functions. Therefore, it is desired to develop a novel gene trap useful for plant genome analysis.

[0012]

[Means for Solving the Problems] The present inventors have prepared several types of trap vectors incorporating the LUC reporter gene and analyzed them. As a result, some of them were found to be extremely useful as trap vectors. Heading out, the present invention has been completed. That is, the present invention provides the following (1) to (8).

(1) A plant gene trap vector containing a luciferase gene as a reporter gene. (2) The vector according to the above (1), which further contains a marker gene in an order in which it can function. (3) The vector according to (1) or (2) above, which further comprises a splicing donor sequence, an intron, and a splicing acceptor sequence in this order upstream of the luciferase gene.

(4) The vector according to (1) or (2) above, which further comprises a stop codon and an IRES sequence in this order upstream of the luciferase gene. (5) The vector according to (1) or (2) above, which further comprises a minimal promoter sequence upstream of the luciferase gene. (6) The vector according to any one of (2) to (5) above, wherein the marker gene is at least one selected from kanamycin resistance, hygromycin resistance, gentamicin resistance, and herbicide resistance.

(7) A plant gene trap method comprising the following steps. 1) Step of introducing the vector according to any one of (1) to (6) above into a plant 2) Step of selecting a transformed plant from the above plant 3) Adding luciferin to the above transformed plant, Step 4 of measuring generated light emission) From the measured light emission pattern,
Step (8) of obtaining information on plant genomic genes A method for gene trapping a living plant individual, which comprises the following steps.

1) Step of introducing the vector according to any one of (1) to (6) above into a plant 2) Step of selecting a transformed plant from the above plant 3) Utilizing the above transformed plant Luciferin is added as it is, and the resulting luminescence is measured 4) A step of obtaining temporal information on the genomic gene of a living plant individual from the measured luminescence pattern

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. In the present invention, “gene trap” means a method in which a reporter gene is randomly inserted into the genome and the expression control information of the gene near the insertion site is obtained from the expression pattern of the reporter gene, which includes a promoter trap and an enhancer. It includes all known gene traps such as traps.

The gene trap of the present invention is a gene trap for plants. The target plant species is not particularly limited, and may be a transformable plant such as Arabidopsis thaliana, tobacco, rice, moss, Lotus japonicus, and the like. The plant gene trap vector of the present invention,
Contains a luciferase gene as a reporter gene,
Furthermore, it is preferable to include the marker genes in a functional order.

The “reporter gene” is a marker gene incorporated for investigating transcriptional activity of promoters and enhancers, and the luciferase (LUC) gene is used in the present invention. The origin of the luciferase is not particularly limited, and in addition to the luciferase gene derived from bacteria and firefly, LUC ⁺ (US Pat. No. 5,583,0
24, 5,674,713, etc.) artificially synthesized luciferase gene may be used.

The "marker gene" is a gene for gene recombination, that is, a marker gene incorporated for the detection of transformants, and includes, for example, a kanamycin resistance gene, a hygromycin resistance gene, a gentamicin resistance gene, and a herbicide. A drug resistance gene such as a resistance gene can be used. In the present invention, the "functional order" means that the integrated genes are present in an order that can achieve the purpose.

[0021] In one embodiment, the plant gene trap vector of the present invention comprises a splicing donor sequence, an intron, and a splicing acceptor sequence in this order upstream of the LUC reporter gene. By including such a sequence, the reading frames of the gene on the genome and the reporter gene are easily matched, and the reporter gene is expressed as a fusion protein with the gene on the genome regardless of the insertion position. The origin of the splicing donor sequence, intron and splicing acceptor sequence used is not particularly limited, but preferably derived from a species close to the plant to be transformed.
For example, when transforming monocotyledonous plants, those derived from monocotyledonous plants are preferable, and when transforming dicotyledonous plants, those derived from dicotyledonous plant are preferable.

In another embodiment (usually referred to as the "enhancer trap"), the vector of the invention comprises:
It contains a minimal promoter upstream of the LUC reporter gene.
Here, the "minimal promoter" cannot express a downstream gene by itself, but it can express a downstream gene if an enhancer is nearby.
A core promoter containing TATA sequence, etc.
NOS101, 35S90, etc. (Puente et al., 1996, EMBO J., 15, 3732
-3743) can be mentioned. When the vector is used, the LUC reporter gene is expressed under the control of an enhancer existing in the vicinity of the insertion site, but the mRNA containing the reporter gene alone encodes the LUC protein, so fusion with the gene on the genome No protein is expressed.

In yet another embodiment, the plant gene trap vector of the present invention comprises a stop codon, IRES (internal ribosome entr), upstream of the LUC reporter gene.
y site) array in this order. This type of trap vector has been reported in the mouse system (BPZambro
wicz, GAFriedrich, ECBuxton, SLLilleberg, C.
Person, ATSands, 1998 Nature 392, 608-611), and its application in plants has not been reported yet.

The "IRES sequence" refers to a specific sequence on the mRNA to which the ribosome binds in the internal initiation mechanism where the ribosome directly binds to a specific region inside the mRNA to initiate translation. IRES sequences that can function in plants (tobacco) include
For example tobamovirus IRES-MP228, IRES-MP75 and IR
ES-CP148 (MVSskulachev et al., 1999, Virology, 263,13
9-154), and IRES-EM of emcephalomyocarditis virus
CV (P. Urwin et al., Plant J., 2000, 24, 583-589) has been reported. Normally, in eukaryotes, the second coding region is not translated in the case of polycistron, which has two coding regions on one mRNA, but if the IRES sequence is placed between the two, the second coding region is Is also translated (M. Ko
zak, 2001, Mol. Cell Biol., 21, 1899-1907). Therefore, when the vector is used, the introduced LUC reporter gene is fused with the gene on the genome at the mRNA level, but is translated as the LUC reporter alone as the protein product.

In the plant gene trap vector of the present invention, the vector used is not particularly limited, and known vectors such as plasmid, phage, cosmid, P1 vector can be appropriately selected and used. The vector can be introduced into a plant by a method commonly used, for example, a PEG method, an electroporation method, a microinjection method, a retrovirus introduction method, etc., but especially the method of Clough et al. Using Agrobacterium. (Yamamoto Y.
Y. et al 2001, Plant Cell, 13 399-411; SJClough and
AFBent, 1998Plant J. 16 735-743) is preferred.

In the plant gene trap method of the present invention,
Expression of the LUC reporter gene in transformed plants is
It can be detected by incorporating luciferin, which is a substrate of luciferase, into a plant, and measuring the generated luminescence.
The light emission can be measured by using a known method such as a CCD camera, a photo-counting type VIM camera (Hamamatsu Photonics), or a scintillation counter.

The detected luminescence data directly reflects the expression information of genes near the reporter gene insertion site,
This makes it possible to obtain information on unknown gene functions and expression control. In particular, if the luciferase activity is measured using a method in which a substrate is administered to a living plant individual and measurement is performed using a video camera or a photomultiplier tube, the gene expression of the living plant individual can be measured over time in a manner close to real time. The excellent effect of being able to monitor can be obtained.

[0028]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Example 1: Development of a fusion genotype plant trap vector
And its application example 1.1) Construction of yy322 and yy323 (LUC) Vector yy3 that produces a fusion protein with LUC when a trap vector is inserted into a gene on the genome.
Created 22 and yy323. yy322 and yy323 are constructs designed to generate multiple types of fusion mRNAs having different reading frames so that the reading frames of the gene on the genome and the reporter gene can be easily matched at the time of fusion. As a principle, the reference by Nussaume et al. (L. Nussaume et al., 1995, Mol. Gen. Genet., 24
9, 91-101), but in this Example, Arabidopsis CIP4 (Yamamoto YY et al 2001, Pla
nt Cell, 13 399-411) splicing donor site (SEQ ID NO: 1), Arabidopsis CIP7 (Yamamoto YY et al.
1998 1001083-1094) intron (SEQ ID NO: 2) and
A splicing acceptor site (SEQ ID NO: 3) designed by Nussaume et al. Was used in combination. In other words, by combining these, two synthetic DNAs that partially overlap are created, annealing, complete double stranded by Klenow enzyme treatment, end treatment by HindIII / BamHI enzyme treatment, and H
Synthetic DNA with a shape that can be inserted into the indIII / BamHI site (Hint
B) was prepared (see FIG. 1). Thus, HintB is a modified CIP7 intron with overlapping splicing donor and acceptor sites. The HintB sequence (SEQ ID NO: 4) is shown below.

HintB: 5'- AAG CTT CAA GGT CTC TCT TC
A AGG TGA GTT TTT TTC TGT TCA CTCTCT TAG ATG CCA A
AA CTT GAG TTA TTG CTT AAT GTT TCA ATT GTT GTG GAC
TCTGTG TAT GTG TAG GTT ATA TGC AGG TTA TAT GCA GG
T TAT ATG GGA GGT GGA G GGATC C -3 '(underlined restriction enzyme site) The above HintB was added to the plasmid pPZP200A (P. Hajdukiewicz et al.
1994, Plant Mol. Biol., 25, 989-994) HindIII / Bam
It was inserted into the HI site to obtain yy306. In addition, pPZP200A is Dr. Pal
Granted by Maliga (The State University of New Jersey).

Next, the plasmid pBIN19 (M. Bevan, 19
84, Nucleic Acids Res., 12 8711-8721)
OS / NPTII / TNOS (SEQ ID NO: 5) and PNOS / NPTII (SEQ ID NO: 6) were amplified by PCR. PNOS / NPTII is a sequence encoding a PNOS plant promoter and a kanamycin resistance gene, and PNOS / NPTII / TNOS is a sequence having a poly A addition signal added thereto. In addition, pBIN19 was granted by Professor Aoyama of Kyoto University. The reaction conditions of the primers and PCR are shown below.

PNOS / NPTII / TNOS Primer: Forward: 5'-CGG GGT ACC TGA TCA TGA GCG GAG AAT TA
A GGG A-3 '(SEQ ID NO: 7) Reverse: 5'-GGA ATT CCC GAT CTA GTA ACA TAG ATG AC
A-3 '(SEQ ID NO : 8) PNOS / NPTII primer: Forward: 5'- GGA ATT CTC AGA AGA ACT CGT CAA GAA G
GC GAT A-3 '(SEQ ID NO: 9) Reverse: 5'- GGA ATT CTC AGA AGA ACT CGT CAA GAA G
GC GAT A-3 '(SEQ ID NO : 10) Reaction conditions: Reaction solution: dNTP mix 0.4 mM each, primers 0.25 uM eac
h, template DNA (pBIN19) appropriate amount (1-100ng), 1x reaction buffer (supplied with Stratagene PfuTurbo enzyme), Pfu Turbo enzyme 1-1.5 units.

Cycle conditions: 94 ^° C. 20 seconds, 50 ^° C. 30 seconds,
72 ^o C 4 minutes 30 cycles.

The PCR reaction product was extracted with phenol / chloroform and then digested with KpnI / EcoRI to give yy30.
It was inserted into KpnI / EcoRI site of 6. Yy309 when PNOS / NPTII / TNOS is inserted, yy when PNOS / NPTII is inserted
It is 310. On the other hand, the plasmid pG6LUC (T.
Aoyama and N.-H Chua, 1997, Plant J., 11, 605-61
The SalI site in 2) was removed by digestion with SalI and then Klenow treatment, and KpnI,
Then, the BamHI site was removed to obtain yy319. This yy319 L
The UC / T3A portion was amplified by PCR. PG6LUC was granted by Professor Aoyama of Kyoto University. The primers and reaction conditions are shown below.

LUC / T3A Primer: Forward: 5'-CGG GAT CCA AAC AAT GGC TAT GGC TGA AG
A CGC CAA AAA CAT AAAGAA AG-3 '(SEQ ID NO: 11) Reverse: 5'-GGG GTA CCA TCG ACA CAA AAA GCC TAT AC
T GTA C-3 '(SEQ ID NO : 12) Reaction conditions: Reaction solution: dNTP mix 0.4 mM each, primers 0.75 uM eac
h, template DNA (yy319) appropriate amount (about 1-100 ng), 1x reaction buffer (provided with Stratagene PfuTurbo enzyme), Pfu Turbo enzyme 1-1.5 units. Cycle conditions: 94 ^o C 30 seconds, 55 ^o C 30 seconds, 72 ^o C 4 minutes 3
0 cycles.

The obtained PCR product was extracted with phenol / chloroform and then digested with BamHI / KpnI to obtain yy309 and yy310.
Were inserted into the BamHI / KpnI site of γ320 to obtain yy320 and yy321, respectively. Inserted into the plant genome at yy320 and yy321
Right end RB of T-DNA and multiple splicing unit HintB
ScaI / Hin included in the vector to reduce the distance between
The dIII portion was replaced with the PCR product of pPZP100. The primers and reaction conditions are shown below.

ScaI / HindIII primer: Forward: 5'-CCC AAG CTT TGA CAG GAT ATA TTG GCG GG
T A-3 '(SEQ ID NO: 13) Reverse: 5'-ATA GCA GCG GAG GGG TTG GAT CA A-3' (SEQ ID NO : 14) Reaction conditions: Reaction solution: dNTP mix 0.4 mM each, primers 0.25 uM eac
h, template DNA (pPZP100) appropriate amount (about 1-100ng), 1x
Reaction buffer (supplied with Stratagene PfuTurbo enzyme), Pfu Turbo enzyme 1-1.5 units. Cycle conditions: 94 ^o C 30 sec, 55 ^o C 30 sec, 72 ^o C 2 min 3
0 cycles.

The resulting PCR product was extracted with phenol / chloroform, digested with ScaI / HindIII, and incorporated into yy320 and yy321. The clone derived from yy320 is yy322, and the clone derived from yy321 is yy323. The outline of the T-DNA portion of yy322 and yy323 is shown in FIG. 1.2) Introduction of yy322, yy323 into Arabidopsis thaliana yy322, and yy323 were introduced into Agrobacterium GV3101, pMP90 by electroporation method, and Arabidopsis thaliana was transformed by a reduced pressure wet method or a floral dip method using the obtained Agrobacterium. (Yamamoto
YY et al 2001, Plant Cell, 13 399-411; SJ Clough an
d AFBent, 1998 Plant J. 16 735-743).

Transformed T1 generation seeds with kanamycin
Sprouting was performed on GM medium containing 30-50 ug / ml, and several hundred transformants were selected and grown to obtain T2 generation selfed seeds. The transformed individuals obtained by this method are independently transformed. T-DNA at different positions on the genome
(G.-N. Ye et al. 1999, Plant J., 19 24
9-257).

1.3) Analysis of LUC expression pattern of T2 generation i) Analysis using ARGUS video system LUC activity can be measured while utilizing plant material, and one method is visualization of LUC activity with a high-sensitivity video camera. There is. Millar et al.'S method (AJ Millar et al. 1992 Plan
T. Mol. Boiol. Rep., 10 324-337), the LUC activity of the produced transformation line was detected. An Argus 50 VIM-CCD camera system manufactured by Hamamatsu Photonics was used for detection.

T2 seeds were treated with GM medium (D. Valvekens et al., 1988,
Proc. Natl. Acad. Sci. USA, 85, 5536-5540), the LUC activity was measured using the ARGUS system after germinating for 1-2 weeks. As a result, LUC activity was detected from about 20% to 30% of the lines, and as shown in FIG. 2, the line was observed in the whole individual, the line was observed only in the green tissue part, etc. Various tissue-specific expression patterns were obtained. From this, it was confirmed that the LUC reporter gene was inserted at a position on a different genome in each line, and that LUC expression was regulated according to the position, that is, that it effectively functions as a gene trap system. Was done.

Ii) Time-dependent measurement of LUC activity using TopCount Using a scintillation counter capable of repeated and automatic measurement, LUC activity can be time-measured for about a week while the plants are kept alive (J. Sai and CH Johnson,
1999, Proc. Natl. Acad. Sci. USA, 96, 11659-1166
3). The seeds of the trap line obtained in the previous section were placed on GM agar medium containing 5 mM of luciferin, which is the substrate of LUC, and germinated and grown on the trap line for about a week.
C activity was automatically measured by a scintillation counter. TopCount of Packard was used for the measurement. At this time,
When grown under bright conditions for 7 consecutive days (Fig. 3) and 3 in the dark
The LUC activity was measured in the case of irradiating with light after growing and growing for 3 days and growing for 3 days under bright conditions (Fig. 4). As a result, it was found that each line shows various LUC expression patterns, such as a line that transiently expresses during one period of germination and a line that shows a light response (Fig. 5). As described above, the results of examining the time-dependent changes for each line also confirmed that LUC effectively functions as a gene trap system. Also,
It was also confirmed that the LUC reporter can be used to analyze the temporal changes of trapped genes and environmental responses.

Example 2: Trap vector using LUC ⁺
Development of yy327 (fusionLUC ⁺ ) Firefly luciferase protein has a peroxisome translocation signal at the C-terminus. Remove this array,
A modified luciferase, LUC ^+, whose codon has been optimized for mammalian cells has been created. LUC ⁺ is expected to have stronger specific activity than conventional LUC, so LUC ⁺
An attempt was made to create a trap vector using C ⁺ . yy327 is y
The LUC / T3A part of y323 is replaced with LUC ⁺ / TNOS.
As the plasmid 221-LUC ⁺ containing LUC ⁺ , the one given by Professor Hiratsuka of Nara Institute of Technology was used. This 221-LUC ⁺ LUC ⁺ / T
The NOS part was amplified by PCR. The primers and reaction conditions are shown below.

LUC ⁺ / TNOS Primer: Forward: 5'-CGG GAT CCA AAC AAT GGC TAT GGC TGA AG
A CGC CAA AAA CAT AAAGAA AG-3 '(SEQ ID NO: 15) Reverse: 5'-GGG GTA CCC TAT CTA GTA ACA TAG ATG A
CA-3 '(SEQ ID NO : 16) Reaction conditions: Reaction solution: dNTP mix 0.4 mM each, primers 0.75 uM eac
h, template DNA (yy319) appropriate amount (about 1-100 ng), 1x reaction buffer (provided with Stratagene PfuTurbo enzyme), Pfu Turbo enzyme 1-1.5 units. Cycle conditions: 94 ^o C 30 sec, 50 ^o C 30 sec, 72 ^o C 4 min 3
0 cycles.

The obtained PCR product was extracted with phenol / chloroform, digested with BamHI / KpnI, and incorporated into yy323 digested with BamHI / KpnI to obtain yy327. When yy327 was used to transform Arabidopsis thaliana in the same manner as in Example 1, various luciferase expression patterns which were different depending on the transformation line were obtained as in yy323 (FIG. 6).

Example 3: Trap vector using IRES sequence
Although schematically mRNA levels in the development of Tar 3.1) yy375 (IRES ^EMCV) and yy376 (IRES ^CP) is a fusion type of gene in the genome as a trap vector to produce only luciferase alone as a translation product, Yy375 and Yy376 It was created. yy375 is IRES ^EMCV (SEQ ID NO: 17) as IRES, y
y376 has a construct incorporating IRES ^CP (SEQ ID NO: 18). These vectors contain a stop codon from the 5'end of the T-DNA region, an IRES sequence (CUTHellen and P Sarnow, 2
001 Genes Dev 15: 1593-1612) and the luciferase gene are arranged in this order, and the gene is trapped.
It produces mRNA with a dicistronic structure such as 7. The coding region for luciferase is the second cistron, but IRES activity allows luciferase to be translated alone.

3.2) Preparation of yy376 yy371 which is a derivative of yy327 (NPTII on the outside of LB of yy327)
Antisense sequence was introduced so that resistance to kanamycin disappears when a sequence 3'downstream from LB is inserted into the plant genome). Tabamovirus at HindIII / BamHI site
Derived from IRES ^CP (MVSkulachev, PAIvanov, OVKarpo
va, T.Korpela, NPRodionova, YLDorokhov, JGAt
abekov, 1999 Virology 263, 139-154) was chemically synthesized and inserted. The nucleotide sequence of IRES ^CP is shown in FIG. This plasmid was digested with HindIII and filled in with Klenow enzyme, and then ligated again.
And When Arabidopsis thaliana was similarly transformed with yy376, various luciferase expression patterns were obtained by the transformation line as with yy323 and yy327 (Fig. 9).

3.3) Preparation of yy375 IRES ^EMCV is pIRES2-EGFP (CLONTECH Co .; http: //www.clo
ntech.com; US Pat. No. 4,937,190) was used as a template and amplified by PCR reaction. The primers and reaction conditions are shown below.

IRES ^EMCV Primer: Forward: 5'-GCT CTA GAT AGT TAG TTA GAT CCG CCC CTC
TCC CTC CCC-3 '(SEQ ID NO: 19) Reverse: 5'-CGG GAT CCT GTG GCC ATA TTA TCA TCG TG
T TT-3 '(SEQ ID NO : 20) Reaction conditions: Reaction solution: dNTP mix 0.25 mM each, primers 0.75 uM eac
h, template DNA (pIRES2-EGFP) appropriate amount (about 1-100 ng), 1x reaction buffer (Boehringer ExpandHiFideri
ty kit), ExpadHiFiderity enzyme 1 ul /
100ul reaction solution). Cycle conditions: 94 ^o C 1 min, 60 ^o C 30 sec, 72 ^o C 1.5 min
30 cycles.

After the reaction, the PCR product was purified using a QIAquick column (Qiagen) according to the instructions of the kit. Purification D
After NA was digested with XbaI / BamHI, only cytosine (C) and thymine (T) were filled-in, extracted with phenol, and used for ligation reaction. On the other hand, after digesting yy371 with HIndIII, adenine (A)
And guanine (G) were filled-in, extracted with phenol, digested with BamHI, dephosphorylated and ligated with the prepared IRES ^EMCV.
It was 375. The structure of yy375 is shown in FIG.

When yy375 was used to transform Arabidopsis thaliana in the same manner as in Example 1, yy323, yy327 and yy37 were obtained.
Similar to 6, the transformation line produced various luciferase expression patterns (FIG. 11). As mentioned above,
It was confirmed that the vector of the present invention also functions effectively in a vector using LUC ⁺ and a vector incorporating an IRES sequence. It was also confirmed that the LUC reporter can be used to analyze the time course of the trapped gene and the environmental response.

[0051]

INDUSTRIAL APPLICABILITY By using the plant gene trap vector of the present invention, the conventional trap reporter gene (lac
Z, uidA, GFP, etc.) makes it possible to analyze genes over time. Further, it becomes possible to search for genes that respond to stress such as injury and pathogenic bacteria and gene-disrupted strains thereof.

[0052]

[Sequence list]                                SEQUENCE LISTING <110> RIKEN Genomic Sciences Center <120> A new vector for plant gene trapping comprising the       luciferase reporter gene and the use thereof <130> RJH13-064T <160> 20 <170> PatentIn Ver. 2.1 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificial Splicing Donor <400> 1 aaggtctctc ttcaaggtga gttttt 26 <210> 2 <211> 88 <212> DNA <213> Arabidopsis thaliana <400> 2 gtgagttttt ttctgttcac tctcttagat gccaaaactt gagttattgc ttaatgtttc 60 aattgttgtg gactctgtgt atgtgtag 88 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificial Splicing Acceptor <400> 3 taggttatat gcaggttata tgcaggtta 29 <210> 4 <211> 157 <212> DNA <213> Artificial Sequence <220> <221> intron <222> (24) ... (111) <223> Description of Artificial Sequence: HindB (modified intron of CIP7) <400> 4 aagcttcaag gtctctcttc aaggtgagtt tttttctgtt cactctctta gatgccaaaa 60 cttgagttat tgcttaatgt ttcaattgtt gtggactctg tgtatgtgta ggttatatgc 120 aggttatatg caggttatat gggaggtgga gggatcc 157 <210> 5 <211> 1771 <212> DNA <213> Artificial Sequence <220> <221> promoter <222> (13) ... (326) <221> polyA_signal <222> (1123) ... (1764) <223> Description of Artificial Sequence: PNOS / NPTII / TNOS <400> 5 ggtacctgat catgagcgga gaattaaggg agtcacgtta tgacccccgc cgatgacgcg 60 ggacaagccg ttttacgttt ggaactgaca gaaccgcaac gttgaaggag ccactcagcc 120 gcgggtttct ggagtttaat gagctaagca catacgtcag aaaccattat tgcgcgttca 180 aaagtcgcct aaggtcacta tcagctagca aatatttctt gtcaaaaatg ctccactgac 240 gttccataaa ttcccctcgg tatccaatta gagtctcata ttcactctca atccaaataa 300 tctgcaccgg atctggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc 360 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 420 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 480 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 540 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 600 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 660 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 720 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 780 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 840 ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt cgtgacccat ggcgatgcct 900 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 960 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 1020 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 1080 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga 1140 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 1200 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 1260 cggggatctc atgctggagt tcttcgccca cgggatctct gcggaacagg cggtcgaagg 1320 tgccgatatc attacgacag caacggccga caagcacaac gccacgatcc tgagcgacaa 1380 tatgatcggg cccggcgtcc acatcaacgg cgtcggcggc gactgcccag gcaagaccga 1440 gatgcaccgc gatatcttgc tgcgttcgga tattttcgtg gagttcccgc cacagacccg 1500 gatgatcccc gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc 1560 cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa 1620 catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata 1680 catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc 1740 ggtgtcatct atgttactag atcgggaatt c 1771 <210> 6 <211> 1127 <212> DNA <213> Artificial Sequence <220> <221> promoter <222> (13) ... (326) <223> Description of Artificial Sequence: PNOS / NPTII <400> 6 ggtacctgat catgagcgga gaattaaggg agtcacgtta tgacccccgc cgatgacgcg 60 ggacaagccg ttttacgttt ggaactgaca gaaccgcaac gttgaaggag ccactcagcc 120 gcgggtttct ggagtttaat gagctaagca catacgtcag aaaccattat tgcgcgttca 180 aaagtcgcct aaggtcacta tcagctagca aatatttctt gtcaaaaatg ctccactgac 240 gttccataaa ttcccctcgg tatccaatta gagtctcata ttcactctca atccaaataa 300 tctgcaccgg atctggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc 360 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 420 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 480 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 540 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 600 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 660 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 720 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 780 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 840 ccaggctcaa ggcgcgcatg cccgacggcg atgatctcgt cgtgacccat ggcgatgcct 900 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 960 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 1020 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 1080 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agaattc 1127 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 cggggtacct gatcatgagc ggagaattaa ggga 34 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 ggaattcccg atctagtaac atagatgaca 30 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 ggaattctca gaagaactcg tcaagaaggc gata 34 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 10 ggaattctca gaagaactcg tcaagaaggc gata 34 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 11 cgggatccaa acaatggcta tggctgaaga cgccaaaaac ataaagaaag 50 <210> 12 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 12 ggggtaccat cgacacaaaa agcctatact gtac 34 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 13 cccaagcttt gacaggatat attggcgggt a 31 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 14 atagcagcgg aggggttgga tcaa 24 <210> 15 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 15 cgggatccaa acaatggcta tggctgaaga cgccaaaaac ataaagaaag 50 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 16 ggggtaccct atctagtaac atagatgaca 30 <210> 17 <211> 156 <212> DNA <213> Tobamovirus Ob <220> <223> Internal ribosome entry site <400> 17 agcttagata gatagcgatt cggttgcagc atttaaagcg gttgacaact ttaaaagaag 60 gaaaaagaag gttgaagaaa agggtgtagt aagtaagtat aagtacagac cggagaagta 120 cgccggtcct gattcgttta atttgaaaga agaaac 156 <210> 18 <211> 590 <212> DNA <213> emcephalomyocarditis virus <220> <223> Internal ribosome entry site <400> 18 gatccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg 60 ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag 120 ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc 180 caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg 240 aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag 300 gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca 360 gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt 420 caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc 480 tcggtacaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa 540 ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca 590 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 19 gctctagata gttagttaga tccgcccctc tccctcccc 39 <210> 20 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 20 cgggatcctg tggccatatt atcatcgtgt tt 32

[Brief description of drawings]

FIG. 1 shows a schematic of the T-DNA portion of yy322 and yy323.

FIG. 2 shows luciferase activity (video camera image) of yy322 and yy323 transformed plants.

FIG. 3 shows luciferase activity for 7 days in the light.

FIG. 4 shows luciferase activity for 3 days in the dark and 3 days in the light.

FIG. 5 shows the luciferase activity of lines transiently expressed during seedling development (upper graph) and classification of transformed plants by environmental response (lower box).

FIG. 6 shows the luciferase expression pattern of yy327-transformed plants.

FIG. 7 shows an outline of a gene trap using IRES.

FIG. 8 shows the structure of yy376.

FIG. 9 shows the luciferase expression pattern of yy376-transformed plants.

FIG. 10 shows the structure of yy375.

FIG. 11 shows a luciferase expression pattern of yy375-transformed plants.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 2B030 CA15 CA17 CA19                 4B024 AA08 BA11 CA01 DA01 FA10                       GA11 GA17                 4B063 QA01 QA18 QQ04 QQ09 QQ30                       QR58 QS26 QX02

Claims (8)

[Claims] 1. A vector for a plant gene trap, which contains a luciferase gene as a reporter gene. 2. The vector according to claim 1, further comprising a marker gene in a functional order. 3. The vector according to claim 1, further comprising a splicing donor sequence, an intron, and a splicing acceptor sequence in this order upstream of the luciferase gene. 4. The vector according to claim 1, further comprising a stop codon and an IRES sequence in this order upstream of the luciferase gene. 5. The vector according to claim 1, further comprising a minimal promoter sequence upstream of the luciferase gene. 6. The vector according to claim 2, wherein the marker gene is at least one selected from kanamycin resistance, hygromycin resistance, gentamicin resistance, and herbicide resistance. 7. A plant gene trap method comprising the following steps. 1) A step of introducing the vector according to any one of claims 1 to 6 into a plant, 2) a step of selecting a transformed plant from the plant, and 3) adding luciferin to the transformed plant to generate luminescence. Measuring step 4) A step of obtaining information on plant genomic genes from the measured luminescence pattern 8. A gene trap method for a living plant individual, which comprises the following steps. 1) A step of introducing the vector according to any one of claims 1 to 6 into a plant, 2) a step of selecting a transformed plant from the plant, and 3) a luciferin in a state where the transformed plant is kept alive. Is added to measure the generated luminescence 4) A step of obtaining temporal information on the genomic gene of a living plant individual from the measured luminescence pattern