JP3350753B2 - New large capacity binary shuttle vector - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a large-capacity shuttle vector used for transformation of seed plants and the like. In particular, it is used for transformation of monocotyledonous crops that are difficult to transform despite having high economic value among seed plants, and are also suitable for analysis of complementation of genomic function and construction of genomic library for genomic function analysis High-capacity binary shuttle vector that can be used for

[0002]

BACKGROUND OF THE INVENTION Despite their agricultural importance, monocot crops are more difficult to transform than dicots. Until now, these plants have been transformed by forced transfer using a gene gun, electroporation after protoplasting, or PEG (polyethylene glycol).
Law, and so on. However, electricity introduction method and PE
In the case of the G method, it is difficult to regenerate from protoplasts, and in the case of using a gene gun, an expensive apparatus is required, and it is difficult to simultaneously process a large number of samples. In addition, there has been no example of introducing a large gene fragment of 10 kb or more into monocotyledonous crops efficiently and without impairing its structure by using any of the methods.

Recently, Hinaga et al. Reported that it is possible to transform rice, which is a monocotyledonous crop, using a pBI vector and Agrobacterium (Hiei et al., Plant
Journal, 6; 271-282 (1994)), thereby greatly facilitating the transformation of rice. However, Hinaga et al. Used
With a pBI-based vector, only about 10 kb of DNA could be stably introduced into rice. In addition, when a DNA of a larger size is incorporated as a shuttle vector, it becomes difficult to stably maintain the vector even in Escherichia coli.

[0004]

On the other hand, with the recent progress of genomic (chromosome) research, as a method for isolating useful genes that determine agricultural traits, genes have been isolated based on positional information on chromosomes. Positional cloning methods have quickly gained attention as promising methods. When using this method, to identify a gene that truly carries the trait among a plurality of genes narrowed down from positional information, a chromosome fragment containing the gene is introduced into a plant to express the trait. You need to make sure. At this time, if a large chromosome fragment can be introduced, efficient gene narrowing can be performed. Therefore, it is desirable to introduce a large chromosome fragment as much as possible.

[0005] As a method for introducing a chromosome fragment of 10 kb or more into plants, the binary cosmid method and the PEG method described below have been known, both of which involve the following problems. (1) In the binary cosmid method, the size of the chromosome fragment that can be introduced is limited to about 20 kb, and RK2 of the pBI system is used as the replication origin, so the chromosome is actually first introduced. In many cases, maintenance of the plasmid in E. coli was difficult.

(2) In the PEG method, since advanced technology is required for reproduction from protoplasts, and the introduced fragments often fall apart and are not introduced as they are,
There were many problems in testing the function of the gene contained in the introduced chromosome fragment.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances. An object of the present invention is to introduce a large chromosome fragment of 10 kb or more into higher plants, especially monocotyledonous crops which contain many agriculturally important crops and are difficult to transform. Also, the present invention
By efficiently and stably introducing DNA of 10 kb or more into the above plants into the nuclei of these plants, which has been particularly difficult so far, it has become possible to analyze the complementarity of genes in large chromosomal regions. An object of the present invention is to facilitate the isolation of a gene that controls an agricultural trait by efficiently indicating where on the chromosome the gene governing the trait is located. It is another object of the present invention to introduce a large gene complex in a lump so that complex traits which have been difficult so far can be collectively introduced into these plants.

[0008] That is, the present invention is a large-capacity binary shuttle vector having a T-DNA region and a Ri origin of replication. The large-capacity binary shuttle vector of the present invention comprises:
It is a pBI binary shuttle vector. In addition, the large-capacity shuttle vector contains the T-
A gene having a multiple cloning site in the DNA region,
Specifically, the lacZ gene is introduced.

Further, the large-capacity binary shuttle vector is characterized in that an antibiotic resistance gene has been introduced between the lacZ gene introduced into the T-DNA region and the boundary region of the T-DNA. The border region is preferably a left border region, and the antibiotic resistance gene is preferably a hygromycin resistance gene. Another selectable marker antibiotic gene may be a kanamycin resistance gene, which can be used as a selectable marker in E. coli and monocots.

[0010] Furthermore, the large-capacity shuttle vector is characterized in that it can integrate from a small DNA fragment to a large chromosome fragment. It has been confirmed that those chromosome fragments having a size of at least up to 40 kb can be efficiently incorporated, and even larger ones may be incorporated.

The present invention is a genomic library capable of transforming plants, particularly monocotyledonous plants. Each clone constituting the genomic library capable of transforming plants is a large-capacity binary shuttle vector of the present invention, T-DN.
It is characterized by having an insert in the A region.

The present invention relates to a binary shuttle vector having a lox site in the T-DNA region, a parC gene, and Riori as an origin of replication, wherein the lox site is formed by the cre enzyme in Escherichia coli or Agrobacterium as a host. This is a binary vector that can be integrated with a clone plasmid of a circular genomic library having: The present invention also provides T-
A multiple cloning site flanked by a plant promoter and terminator in the DNA region, and Ri as the replication origin
It is a binary vector for transformation of a plant having ori.

[0013] The present invention is a complementation assay for evaluating the function of a gene in a chromosome fragment inserted into a clone of a genomic library. The method comprises the steps of introducing the plasmid having the genomic insert into Agrobacterium cells, and transferring the plasmid to a plant. Further, the present invention is another complementation assay for evaluating the function of a gene in a chromosome fragment inserted into a clone of a genomic library. This method includes the steps of integrating a plasmid constituting a library having a genomic insert with the binary vector, introducing the integrated vector into Agrobacterium cells, and transferring the integrated vector into a plant. And a step. Here, the library is constructed by using a circular vector having a lox site and using Escherichia coli as a host.

[0014] The present invention is a gene obtained by the above-described complementation assay. The present invention is also a method for screening a useful gene, comprising a step of using the above-mentioned large-capacity binary shuttle vector.

Further, the present invention is a transgenic plant transformed with the large-capacity binary shuttle vector. Plants to be transformed include all plants, mainly seed plants, especially monocots.

The present invention, recA in order to stably maintain a binary vector with large insert ^- a high conversion rate production method of Agrobacterium strains having a. This method comprises the steps of generating a site-specific mutation on the recA gene of a strain having a high transformation rate and transforming it into a recA ^- strain, the recA ^- vector and the recA ⁺ gene transformed in the cell. In between, a step of introducing homologous recombination into a strain having the recA ⁺ gene, a step of selecting a recombinant by creating a replica of a plate on which the strain is expanded, and irradiating the plate with radiation Screening for clones that cannot grow under UV irradiation.

[0017] The present invention provides recA which stably maintains the large-capacity binary shuttle vector having a large insert.
^- a high transformation efficiency Agrobacterium strain may be transformed plants having a variety of conventional agronomic traits for transformation.

[0018]

FIG. 1 shows a large-capacity shuttle vector.
FIG. 3 shows the construction of pBIGRZ and a similar vector pBIGZ without Ri origin of replication. Large capacity shuttle vector Ri ori
Construction of pBIGRZ without A. Ri ori arrows indicate the direction of the gene and the corresponding number of the plasmid. 1 shows a restriction enzyme having a unique cloning site. RB indicates the right border region of T-DNA, and LB indicates the left border region of T-DNA. NPT
II indicates the neomycin resistance gene, NP and NT indicate the promoter and terminator of nopaline synthase, la
cZ indicates the β-galactosidase gene, and MCS indicates a multiple cloning site. P35S has 35S promoter, iGUS
Is a β-glucuronidase gene having an intron,
HPT indicates the hygromycin resistance gene, and Ri ori indicates the origin of replication of the Ri plasmid.

FIG. 2 is a diagram showing a process from pBI121 to pBIHG to pBIGRZ production. FIG. 3 is a gel electrophoresis photograph showing the results of Southern blotting of two human 40 kb chromosome fragments introduced into rice by pBIGRZ. A: When compared with the control at the time of introduction (R ₀ ), the introduced genomic fragment shows almost no change, indicating that the introduced chromosome fragment remains as it is. B: No change was observed even in the transgenic progeny (R ₁ ), indicating that the introduced chromosome fragment was stably transmitted to the progeny.

FIG. 4 is a drawing substitute photograph showing rice flowers (left) and ears (center) into which a human chromosome fragment of about 40 kb has been introduced by pBIGRZ. The fertility is extremely high, and the ears that hang down their heads. FIG. 5 shows a rice genomic library made using pBIGRZ. These were cut with the restriction enzyme NotI. A indicates a digested fragment into which a chromosome fragment having a size of 45 kb or more has been introduced, and B indicates a digested fragment into which a fragment having 30 to 50 kb has been introduced. In A and B, the average size of the introduced fragments was 51 kb and 39 kb, respectively.

This indicates that a genomic library useful for complementation assay was actually constructed. The average insert size is approximately 20
Obtained by subtracting kb. The two lanes on the right are size markers.

FIGS. 6A and 6B show various derived vectors utilizing the properties of the large-capacity binary vector of the present invention. A shows a single vector utilizing specific recombination at the lox site by primary expression of the cre enzyme. pBRC is an example of a vector for performing complementation assays in which a BAC vector with a genomic insert and a lox site was integrated by primary expression of the cre enzyme in host cells. PBRCs with non-integrated BACs are rapidly removed if the parC sequence is present therein. B is the vector, pB
An example of a PRPT is shown. This vector is used in the step of introducing a single gene having expression characteristics from a promoter and a terminator, and is a large-capacity vector of the present invention having high transformability.

Hereinafter, the present invention will be described in more detail. The large-capacity binary shuttle vector of the present invention is
It was constructed as follows. The large capacity binary shuttle vector of the present invention was constructed as follows. (1) Development of a binary vector capable of holding a large chromosome fragment as an insert (see FIGS. 1 and 2) Based on a binary vector system satisfying the following requirements. (I) T-cells with various chromosomal fragments introduced as inserts
The vector must have a DNA region. This T-DNA region can be efficiently introduced into various plant cells. (Ii) Easy construction of a genomic library. (Iii) Plasmid DNA can be easily prepared and Escherichia coli can be used as a host. (Iv) The constructed plasmid can be easily transferred to Agrobacterium, and can be stably maintained to efficiently transform plants.

The large-capacity binary shuttle vector of the present invention is required to transfer to Agrobacterium as described above. Agrobacterium
um tumefaciens) is a soil bacterium, has a Ti plasmid of about 200 kb in size, and 10 to
There is a region called T-DNA of 20kb. When Agrobacterium infects a plant, the T-DNA region is excised from the Agrobacterium by the gene product of the vir (virulence: pathogenicity) region on the Ti plasmid. The excised T-DNA is transferred to the plant, and finally the plant nuclear DNA
It is known to be incorporated therein.

At this time, the T-DNA region and the vir region may be present in different vectors, and therefore, a binary vector having only the T-DNA region, at least one replication origin, and a marker gene is prepared. It becomes possible. 25 bp at each end of T-DNA for T-DNA transfer and integration
Only the border sequence consisting of
The genes above are unnecessary. The border sequences at both ends of the T-DNA are referred to as a left border sequence (left border, LB: left border) and a right border sequence (right border, RB: right border), respectively.

The binary shuttle vector that satisfies the above requirements is not particularly limited as long as it has a suitable origin of replication and can use two or more types of bacteria as hosts and can function as a binary vector. Specifically, pBI vectors such as pBI101 and pBI
121 and the like are commercially available, and these can be used.

As an origin of replication used for a binary vector, RK2 is generally used. However, when RK2 is used as an origin of replication, it is often difficult to stably maintain a large insert of 10 kb or more in E. coli or Agrobacterium. Therefore, it is necessary to increase the capacity of the vector in order to stably hold such a large insert in any of the above-mentioned hosts. By introducing the Ri origin of replication of the Rh plasmid of the Rhizobium bacterium as the origin of replication, the capacity of the vector can be increased. In this case, the Ri origin of replication may be present in the vector along with RK2.

Examples of the selection marker to be provided in the vector plasmid include various antibiotic resistance genes. Specifically, the kanamycin resistance gene (NP
T), hygromycin resistance gene (HRT) and the like. These markers can be introduced into the above-described plasmid by a known method, if necessary. The pBI-based basic vector plasmid has only the kanamycin gene as a selectable marker in the introduced plant, but it is preferable to introduce the hygromycin resistance gene. In rice, kanamycin does not work very well as a selectable marker.

When there is a portion (site) in the selection marker to be introduced that hinders the introduction of various chromosome fragments, such site may be removed or modified. For example, from the above-mentioned hygromycin resistance gene, the EcoRI site in this gene is modified so as not to be cleaved by a restriction enzyme. Here, the chromosome or DNA fragment to be introduced into the binary vector can be obtained from viruses, various bacteria, filamentous fungi, protozoa, animals and plants, and all other organisms by known methods.

The DNA or chromosome fragment to be introduced into the above-described binary vector can be integrated at least about 40 kb or more in size, and can be selected according to the purpose. Transformation of rice using a 40 kb chromosome fragment can be performed with an efficiency that is not different from that of a several kb gene, and there is a possibility that a larger fragment can be introduced without difficulty. It is preferable to confirm the introduction of a chromosome fragment by introducing a cloning site into the marker gene. As a marker gene, lacZ (β
-Galactosidase) in the gene, HindIII, SpeI,
Those having a cloning site such as Not I can be used.

The following genes can be further introduced into this vector. That is, when a plant is transformed using the binary shuttle vector, a gene for monitoring the introduction and expression pattern of T-DNA in the transformed plant may be introduced.

As such a gene, the NPT or HRT gene among the antibiotic resistance genes mentioned above can be used, but in particular, the expression site can be visualized.
The β-glucuronidase (GUS) gene is preferred. GUS containing introns (iGUS) is expressed only in eukaryotic cells, ie, the plant cells used herein, and not in Agrobacterium. For this reason, when GUS containing an intron is used, there is no blue color due to expression in bacteria, and noise can be avoided. However, since the expression of the target gene may be affected by the presence of a large number of such markers in the vector, it is not always necessary to include the marker.

(2) Introduction of a chromosome fragment into a vector and transformation of E. coli (i) Introduction of an arbitrary DNA fragment into a vector The vector obtained in (1) and an appropriate size suitable for the cloning site A DNA fragment (chromosome fragment) or a fragment adapted via a linker is ligated using a commercially available ligase or the like. The obtained reaction product is introduced into Escherichia coli DH10B by electroporation. The introduced bacteria were treated with hygromycin B (50 μg / m
L), X-gal, IPTG (isopropyl thiogalactoside (is
spread on LB agar plate containing opropyl thiogalactoside)). Under normal conditions, the cells were cultured overnight, and white colonies expressed on an Lb agar plate were selected as those having the DNA fragment incorporated as an insert.

(Ii) Construction of Genomic Library with Transformation Vectors Chromosomal DNA from plant cells or cells of other materials can be partially purified using a suitable restriction enzyme, often Hind III, as appropriate. Or digest completely. The thus obtained fragment was subjected to CHEF (contour-clamped hexagonal election).
ric field) Select size by electrophoresis. The DNA fragment thus obtained is ligated to the vector (1) by ligation. The obtained reaction product was introduced into E. coli DH10B strain by electroporation, and hygromycin B (50 μg / mL) and X-ga
l, spread on an LB agar plate containing IPTG, and select a strain containing the insert in the same manner as in (i) above.

(3) Transfer of vector from E. coli to Agrobacterium Transfer of the vector from E. coli to Agrobacterium
It can be suitably performed using the following electroporation method and tripalent method (triple mating method).

(I) Electroporation A binary vector is recovered from Escherichia coli by a usual alkaline miniprep method. Here, when there is an automatic plasmid extractor, a large number of vectors can be extracted very easily and efficiently. Agrobacterium for transferring the vector includes EHA101 and the like. In particular, when a large fragment is inserted into these cells, a recA ⁻ strain deficient in recombination ability derived from a strain having high transformation ability can be suitably used. From such Agrobacterium, an electro-competent cell is prepared by a known method.

The binary vector recovered by the miniprep method is introduced into the above competent Agrobacterium cells by electroporation. Competent cells into which this vector was introduced were replaced with Hygromycin B
On a medium containing (50 μg / mL) and kanamycin (50 μg / mL), the cells are cultured under the same conditions as described above, and a transformant is selected.

(Ii) Triparent method The desired binary vector is transformed from E. coli into Agrobacterium by mixing and culturing E. coli into which the binary vector described in the above (2) is introduced, E. coli having a helper plasmid, and Agrobacterium. Can be transferred to. Escherichia coli having these helper plasmids and Escherichia coli into which the above-described binary vector was introduced were subjected to Agrobacterium and an appropriate medium at 28 ° C.
For about 12 to 24 hours. Suitable media include:
YEP medium and the like can be mentioned.

Here, the helper plasmid refers to a plasmid that assists in transferring a vector to a target bacterium when the vector is transferred to the target bacterium, and specifically includes pRK2013 and the like. it can. When Escherichia coli having a helper plasmid is used during mixed culture,
Efficient transfer from the bacteria into which the binary vector has been introduced to Agrobacterium during mixed culture. From the Agrobacterium mixed and cultured as described above, a bacterium containing the large-capacity binary shuttle vector of the present invention was kanamycin,
Selected by tetracycline, hygromycin,
It is used for transformation of the following plants.

(4) Transformation of plant with Agrobacterium into which binary shuttle vector has been introduced Next, T-DNA is introduced into the plant from Agrobacterium into which the binary vector has been introduced to transform the plant. That is, the Agrobacterium strain into which the binary shuttle vector was introduced as described above (about 3 × 10 ⁸
(3 × 10 ⁹ cells / mL) is allowed to coexist with a plant cell callus or tissue fragment for about several minutes, and then co-cultured in a medium such as 2N6-AS or N6CO at 25 to 28 ° C. for about 3 days. Here, as a plant to be co-cultured, a seed plant is used although the degree of difficulty in co-culture varies. In particular, monocotyledonous crops that have been difficult to transform, that is, rice, wheat, barley, corn, and the like, can also be targeted. However, among them, rice is most preferably used because of its ease of individual regeneration.

After the above co-culture with Agrobacterium, the callus or tissue piece is selectively cultured in a medium containing an appropriate antibiotic. As described above, when a hygromycin resistance gene is introduced as a selection marker into the binary shuttle vector, hygromycin (10 to 100
μg / mL) and 2N6-CH or N6Se medium containing cefotaxime (250 μg / mL) or carbenicillin (500 μg / mL) for the removal of Agrobacterium. The converted callus body can be obtained selectively.

The callus obtained selectively was converted to N6S3-CH, MSre
Induction of regeneration is performed using an appropriate regeneration medium such as the above, to obtain a regenerated individual. As described above, when a binary vector having the β-glucuronidase gene on T-DNA is used for transformation, x-gluc (x-glucuronide) is given blue color by giving selected callus or regenerating individuals. Can be seen. Transformation can be confirmed by this blue coloration. As described above, a large chromosome fragment can be introduced into a plant and transformed using the large-capacity binary shuttle vector of the present invention.

Therefore, a large chromosome fragment can be introduced into a plant by using the large-capacity binary shuttle vector of the present invention and transformed. For this purpose, genomic DNA is obtained from a suitable plant or other organism according to a standard method, and fragmented with a suitable restriction enzyme as described above,
The genomic DNA library can be prepared by incorporating the gene into the large-capacity binary shuttle vector of the present invention as described above.

Since the vector of the present invention is a large-capacity binary shuttle vector, the genomic DNA library thus prepared has the ability to transform various cells. Therefore, using this genomic DNA library, a plant DNA fragment contained in each clone constituting this library can be introduced into cells of another plant, and these can be transformed.

In particular, since the large-capacity binary shuttle vector of the present invention can incorporate a large DNA or chromosome fragment of about 40 kb, useful genes can be efficiently searched for. That is, if there is a clone that restores its function by transforming into a mutant in which the function of a specific gene has been lost, the clone contains the target gene (complementation test).

In the vector, PBRGRZ, the gene is only introduced with a chromosomal fragment or with a promoter and terminator. A gene or cD from which a promoter specific to a vector having a multiple cloning site between an appropriate promoter and terminator has been removed
The full length NA can be introduced to express the properties of any gene (FIG. 6B). After insertion of the gene or cDNA, such a vector can be inserted into a plant for transformation. Generally, a combination of an actin promoter and an actin terminator is preferable because the expression level of the gene is increased. Repeated use of the same promoter or terminator in one vector should be avoided due to the possibility of homologous recombination in the host cell.

The recA ^- strains high conversion of Agrobacterium having to produce in order to maintain a large plasmid made from large binary vector of the present invention.
Since this large plasmid is a single or low copy number plasmid, it is generally stably maintained in both E. coli and Agrobacterium. In Escherichia coli, recA ⁻ strains that cannot undergo genetic recombination are, for example, DH1
0B is easily obtained. Also, some recA in Agrobacterium ^- are known strains. However,
As for the high conversion rate strain essential for the transformation of difficult-to-transform plants, no recA ^- strain which is desirable for stable maintenance and transformation of large plasmids has been reported.

[0048] Thus, recA of such a high conversion rate ^-
The strain is created by site-directed mutagenesis on the recA gene of EHA101 strain. This mutation causes a chromosome re
Agrobacterium is introduced by homologous recombination between the cA gene and a transforming plasmid having the recA ^- gene.
A high conversion Agrobacterium strain is transformed with a plasmid containing the recA ^- gene created by site-directed mutagenesis and then spread on plates. After preparing the replica plate, the plate is irradiated with ultraviolet light, and Agrobacterium cells are cultured on the plate. recA ^- for mutants with can not grow under UV irradiation, selecting such clones. With the use of mutants, clones containing large chromosomal fragments are more stably maintained in Agrobacterium cells. Such mutants can also be negatively selected under the above-mentioned UV irradiation after the usual mutagenesis.

[0049]

EXAMPLES Hereinafter, the construction of a large-capacity binary shuttle vector of the present invention and the use of the same in human rice DN
Although an example in which A is introduced is shown, the present invention is not limited to the following example.

Example 1 A large-capacity binary shuttle vector pBIGRZ (shown in FIG. 2) of the present invention was prepared according to the following steps (1) to (3). (1) Preparation of pBH A hygromycin resistance gene (HPT, obtained from Hatsuyama of Hokkaido University) having a 35S promoter and a nopaline terminator and having no EcoRI cleavage site therein was converted into a binary vector pBI121 according to a standard method. Introduced into the EcoRI site of the left border sequence (indicated by LB in FIG. 2) of T-DNA
Was prepared. In FIG. 2, the 35S promoter is P35S
And the nopaline terminator was labeled NPT.

(2) Preparation of pBHG β-glucuronidase (iGUS) containing an intron was introduced between the NPT and HPT of the T-DNA of pBH prepared in (1) above to prepare pBHG. did.

(3) Insertion of Replication Origin of Ri Plasmid The Ri plasmid of Rhizobium was inserted into the NotI site outside the T-DNA by ligation according to a standard method.

(4) The lacZ gene having multiple cloning sites of HindIII, SpeI and NotI in the middle thereof was inserted into pB
The plasmid was prepared from the lacZ portion of luescript and introduced between NPT and iGUS to prepare a plasmid. The plasmid completed here was named pBIGRZ. As described above, since pBIGRZ has a cloning site in lacZ, whether or not a fragment such as a chromosome is inserted into the plasmid is determined by X-gal and I-gal.
It can be confirmed in a medium containing PTG.

Example 2 In order to examine whether the binary vector pBIGRZ prepared in Example 1 can actually efficiently and stably introduce a chromosome fragment of 10 kb or more into monocotyledonous crops, a human chromosome fragment was tested. An example in which 40 kb was introduced into rice is shown. (1) The NotI site of pBIGRZ in lacZ was cut with a restriction enzyme, and the terminal was dephosphorylated. Then, Not I fragment DNA of human chromosome inserted in two cosmids
(About 40 kb each) were inserted into a binary vector by ligation. The vector having this insert was introduced into E. coli.

(2) Introduction of human chromosome fragment was confirmed.
The pBIGRZ vector was introduced from E. coli into Agrobacterium by electroporation. That is, a binary vector was recovered from Escherichia coli by a miniprep method using a normal alkali. The obtained binary vector is electroporated, Agrobacterium having a Ti plasmid without T-DNA (EHA101)
Into a competent cell, and add hygromycin (50μ).
g / mL) and kanamycin (50 μg / mL).

(3) The pBIGRZ vector into which the human chromosome fragment stably introduced into Agrobacterium was inserted was introduced into rice scutellum calli as follows. That is, the Agrobacterium strain into which the binary vector was introduced was co-cultured with the callus at 25 ° C. for 3 days, and the transformed callus was selectively cultured using a hygromycin medium (30 to 50 μg / mL). Thereafter, individuals were regenerated in a regenerating medium, shoots were formed in a hormone-free medium, and regenerated individuals were obtained. After acclimation of this individual, it was cultivated in a greenhouse pot.

Here, the direction of the Ri origin of replication depends on the retention of the plasmid having the human chromosome fragment in Agrobacterium (EHA101 strain) in the above process, and the transient GUS expression rate after co-culture with rice callus. PBIGRZ1 and pBIGRZ2, which differ in the direction of the Ri origin of replication, were used to examine whether or not they affect the regeneration rate of regenerated individuals. Table 1 shows the results.

[0058]

[Table 1] In the table, + indicates acceptable (possible), ± (possible but difficult) and-indicates poor (impossible).

(4) In parallel with this, the same operation was performed by introducing the same human chromosome fragment into pBIGZ (FIG. 1) to attempt to introduce this chromosome fragment into rice callus cells. pB
IGZ is a vector having the same structure as pBIGRZ except that it does not have the Ri origin of replication and has only the RK2 origin of replication of pBI121. Table 1 shows the plasmid retention in Agrobacterium and the regeneration rate of individual plants.

(5) Comparing pBIGRZ with the Ri replication origin with pBIGZ without it, it can be seen that the plasmid retention in Agrobacterium and the rate of plant regeneration are significantly improved with pBIGRZ. It was revealed. In particular, in the regeneration rate of individuals, a dramatic improvement was observed in pBIGRZ.

(6) Depending on the orientation of the Ri replication origin in the plasmid, the retention rate in Agrobacterium,
No significant difference in individual regeneration rate was observed. (7) Extract chromosomal DNA from green leaves of regenerated individuals and
Digested. Cosmid (H7
8C10, H605) was used for Southern analysis using the NotI insert of the human genome. The results are shown in FIGS. 3A and 3B.

3A and 3B, panel A is H605
R _0, which is transformed with (approximately 44 kb) or H78C10 (about 38 kb)
It is the result of Southern hybridization of generations. The control lane in each panel contains the original human chromosome fragment
The fragment cut in III was run at a density of one copy. Lanes 1 to 10 of each clone show the results of Southern hybridization performed by cutting DNA extracted from the leaves of one regenerated individual with EcoRI. As a result of Southern hybridization of the DNA fragment obtained from each transformant, almost the same pattern as that of the control was observed. Panel B
Shows Southern analysis of three plant individuals obtained from the same parent individual. All plants contained approximately one copy of the same fragment as the control. The bands indicated by dots are those hybridized to the random marker.

As shown in FIGS. 3A and 3B, one copy or more of the chromosome fragment in the human cosmid fragment H78C10 has a ratio of 6/10 individuals and the fragment H605 has a ratio of 5/10 individuals. It was clarified that it was introduced into the rice genome in a state where it had been transformed. It was also proved that such a chromosome fragment was stably transmitted to offspring.

As described above, when a large chromosomal DNA fragment is inserted into a vector and used for plant transformation, the integrated DNA fragment rarely causes fragmentation or rearrangement in the vector of the present invention. It was shown that it is stably retained not only in this generation but also in the progeny, and that it is efficiently introduced into plant cells. Therefore, it has also been clarified that the large-capacity binary shuttle vector of the present invention is useful for complementing a target gene function with a chromosomal DNA fragment as large as possible to narrow down the target gene on a chromosome.

In this example, results using rice were shown. However, according to conventional knowledge, transformation of Agrobacterium is generally easier in dicotyledonous plants than in rice, and is expected to be widely applied to dicotyledonous plants as well as in rice and other important monocotyledonous plants. It is expected that crops can be applied to a wider range of crops by examining some conditions. Furthermore, recently, it has been shown that transformation is performed on yeast rather than Agrobacterium, and it is considered that application is possible not only to higher plants but also to all plants.

[0066]

According to the present invention, the above object of 10 kb
The above chromosome fragments, easily, efficiently, good stability, under conditions that do not cause chromosomal rearrangement, higher plants,
In particular, a method for introducing rice into monocotyledonous crops such as rice is provided. In addition, using the large-capacity binary shuttle vector of the present invention, a genomic library having an insert size of about 40 kb and capable of transforming a target plant can be prepared. Therefore, this vector is useful for isolating a useful gene using a positional cloning method or the like.

Further, since the large-capacity binary shuttle vector of the present invention can incorporate such a large chromosome fragment, it is necessary to efficiently introduce a gene or a complex of genes into plants more efficiently than before. The high capacity binary shuttle vectors of the present invention are useful.

Further, the present invention provides a means for extremely efficiently testing the genomic function by a complementation test in rice, which is one of the crops representing higher plants, especially monocotyledonous crops. Thereby, various gene groups which are biologically important but have low expression level, such as regulatory genes, genes involved in signal recognition / transmission, and genes involved in expression of agriculturally important traits Can be considerably facilitated.

By using only Ri ori as an origin of replication of a vector, a large capacity can be achieved. Further, by using the large-capacity binary shuttle vector of the present invention, the efficiency of searching for a useful trait gene can be greatly improved. This vector may be modified to have the ability to transform a plasmid having an insert of a conventional genomic library having a lox site, such as a component of a BAC library, into a form that can transform plants.

To date, a large number of genomic libraries have been prepared, and it is very useful if these libraries can be used for complementation testing of plants. For example,
A vector having only Ri ori or both Ri ori and RK2 ori, that is, the large-capacity vector described above, is very stable in Escherichia coli and Agrobacterium and has excellent plant transformation ability. Based on these characteristics,
The vector of the present invention having Riori, a lox site, and a parC site gene as a replication origin in a T-DNA region can be prepared. This vector is then introduced into an E. coli strain that has been genetically engineered to express the cre enzyme primarily under special conditions, along with a circular plasmid that constitutes a clone of a genomic library with lox sites such as BAC. be able to. The resulting transformant is then subjected to antibiotic selection of a library vector and subsequent selection of a binary vector. In this way, it is possible to obtain a vector in which a component vector of a library having a lox site such as BAC is integrated into a lox site in the T-DNA region of another vector (FIG. 6A).

In the integrated vector obtained as described above, a large genomic insert is inserted basically in the same manner as in the large-capacity binary shuttle vector described above. Therefore, introduction of the integration vector into Agrobacterium using the procedure described above resulted in Table I
Enables the introduction of the insert into plants having a high conversion rate. To remove the binary vector with Ri ori that is not integrated with the library plasmid,
Insert the parC sequence near the lox site.

Therefore, without reconstructing the library using the large-capacity binary vector of the present invention, the lo
A constructed genomic library consisting of a vector such as BAC or P1 having x sites can be effectively used for complementation assays. As shown in Table I, Ri ori and RK2
Vectors having both ori can transform plants with much higher efficiency than vectors having only RK2 ori. Based on this result, the gene can be introduced by using the large-capacity binary shuttle vector for transformation of agronomic traits of plants.

[Brief description of the drawings]

FIG. 1 shows the construction of a large-capacity shuttle vector pBIGRZ and a similar vector pBIGZ without Ri origin of replication.

FIG. 2 is a diagram showing a process from pBI121 to pBIHG to pBIGRZ production.

FIG. 3 shows two types of humans introduced into rice by pBIGRZ.
It is a gel electrophoresis photograph showing the result of Southern blot of a chromosome fragment of 40 kb. A: When compared with the control at the time of introduction (R ₀ ), the introduced genomic fragment shows almost no change, indicating that the introduced chromosome fragment remains as it is. B: No change was observed even in the transgenic progeny (R ₁ ), indicating that the introduced chromosome fragment was stably transmitted to the progeny.

FIG. 4 is a drawing substitute photograph showing rice flowers (left) and panicles (center) into which a human chromosome fragment of about 40 kb was introduced by pBIGRZ. The fertility is extremely high, and the ears that hang down their heads.

FIG. 5 is a drawing substitute photograph showing the result of gel electrophoresis of a fragment obtained by cleaving a rice genomic library prepared with pBIGRZ with a restriction enzyme NotI.

FIG. 6 shows various derived vectors utilizing the properties of the large-capacity binary vector of the present invention. A: Integration into a single vector using specific recombination at lox sites by primary expression of the cre enzyme. B: An example of a vector, pBRPT.

Claims (18)

(57) [Claims] 1. A T-DNA region and Riori and RK2 at the origin of replication.
Large capacity binary shuttle vector with ori. 2. The binary shuttle vector is pBI
The large-capacity binary shuttle vector according to claim 1, which is a system vector. 3. The large-capacity binary shuttle vector according to claim 1, wherein a gene having a multiple cloning site is introduced into the T-DNA region. 4. The large-capacity binary shuttle vector according to claim 1, wherein the gene having the multiple cloning site is a lacZ gene. 5. The lacZ introduced into the T-DNA region and the lacZ
5. The large-capacity binary shuttle vector according to claim 4, wherein an antibiotic resistance gene is introduced between the DNA and the boundary sequence of the DNA. 6. The large-capacity binary shuttle vector according to claim 5, wherein the antibiotic is hygromycin. 7. The large-capacity binary shuttle vector according to claim 1, wherein a large chromosome fragment can be integrated. 8. A binary vector having a lox site in a T-DNA region, a parC gene, and Riori as an origin of replication, and can be integrated with a clone constituting a circular genomic library having a lox site by a cre enzyme. Claims 1-7
A large-capacity binary shuttle vector according to any one of the above. 9. A large-capacity binary shuttle vector for transforming a plant having a multi-cloning site flanked by a plant promoter and a terminator in a T-DNA region and having a replication origin of Ri ori and RK2 ori. 10. A genomic library capable of transforming a plant, comprising the large-capacity binary shuttle vector according to claim 1, wherein a chromosomal fragment is inserted into a T-DNA region. 11. A method comprising the steps of introducing the large-capacity binary shuttle vector according to any one of claims 1 to 9 into Agrobacterium cells, and transferring the Agrobacterium into a plant. A complementation test method for evaluating the function of a gene in a chromosome fragment inserted into the clone of the genomic library according to 10. 12. A step of integrating a plasmid plasmid clone of a library constructed using a circular vector having a lox site and having a host of Escherichia coli with the binary vector according to any one of claims 1 to 9. The step of introducing the integrated vector into Agrobacterium cells, and the step of transferring the Agrobacterium into a plant, wherein the chromosome fragment inserted into the clone of the genomic library according to claim 10 A complementation test method for evaluating the function of a gene. 13. A step of introducing the large-capacity binary shuttle vector according to any one of claims 1 to 9 wherein a chromosomal fragment is inserted into a T-DNA region, into Agrobacterium cells; And a method for searching for a gene in a chromosome fragment. 14. A DNA of 10 kb or more is inserted into a T-DNA region.
A large-capacity binary system according to any one of claims 1 to 9.
Transformation characterized by introducing a shuttle vector
How to make plants. 15. The plant is a seed plant or all plants.
15. The method of claim 14, wherein: 16. The method according to claim 14, wherein the plant is a monocotyledonous plant.
The described method. 17. recA by site-specific mutations on the recA gene ^- a step of preparing a plasmid having a gene of the plasmid recA ^- gene of Agrobacterium rec
A step of performing homologous recombination with the A ⁺ gene, a step of preparing a plate in which the Agrobacterium strain after homologous recombination has been spread and a replica plate thereof, and irradiating the plate with ultraviolet light to grow under UV irradiation. and a step of screening can not clones, recA for introducing large binary shuttle vector according to claim 1-9 ^- the method of producing Agrobacterium strains. 18. obtained by introducing a large binary shuttle vector according 10kb or more DNA to claims 1 to 9 inserted into the T-DNA region recA ^- Agrobacterium strain.