JP2003284561A - Recombinant plasmid for deleting plasmid, method for removing plasmid in agrobacterium, and plasmid-deleted microorganism obtained thereby - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for deleting and removing a plasmid such as a plasmid inducing disease in Agrobacterium without mutating a chromosomal DNA in high efficiency, and to provide a new Agrobacterium cell strain transformed by introducing a known plasmid to Agrobacterium from which the plasmid is deleted and removed, a method for producing the cell strain, a method for creating a gene-recombinant plant by using the method, and a method for investigating whereabouts of the gene governing useful characters. <P>SOLUTION: The replication gene having incompatibility with a replication gene possessed by a wild type Agrobacterium, the recombinant plasmid for deleting the plasmid, having a sensitive gene, and the method for producing the recombinant plasmid are provided. The method for deleting and removing the plasmid by using the recombinant plasmid for deleting the plasmid, and the cell strain from which the plasmid is removed by the method are also provided. A new plasmid can be introduced into the cell strain from which the plasmid is removed. The transformed cell strain obtained thus, and the method for using the cell strain are also provided. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a technique for genetically modifying Agrobacterium. Specifically, it relates to a method for introducing a foreign gene, and Agrobacterium and a plasmid used for the introduction. More particularly, the present invention relates to a method for producing agrobacterium deficient in a tumor-inducing plasmid and a hairy-root disease-inducing plasmid, and a method for producing Agrobacterium to which infection-injecting ability is imparted by introducing the plasmid.

[0002]

2. Description of the Related Art A technique for introducing a recombinant gene into a plant to produce a genetically modified plant is frequently used for the purpose of increasing the tolerance of crops, storability and imparting medicinal components.
Since Agrobacterium has a function of transferring a gene to the plant cell nucleus, it is an essential bacterium for a major technique for producing a transgenic plant. Agrobacterium is isolated from the natural world and stored as a root-carcinoma fungus and a hairy-root fungus. It is also known that these bacteria have various host ranges and have a high ability to induce infection and disease symptoms in specific plant species. However, not all of the collected and stored wild-type Agrobacterium is used for gene recombination.

As a method for incorporating a foreign gene into a plant using Agrobacterium, an intermediate vector method and a binary vector method (Hookemer et al., Patent No. 2,003,610) are known. As shown in FIG. 9 (1), the intermediate vector method is an Agrobacterium having a modified plasmid A ′ obtained by removing the onc gene in the T-DNA region from the Ti plasmid A of wild-type Agrobacterium (a). Create (b). Then, a plasmid vector (I: intermediate vector) having the required gene prepared in E. coli was introduced, and I was incorporated into the T-DNA region of plasmid A ′ by homologous recombination (d). Infect. Since this intermediate vector method has complicated procedures, it has many problems and is rarely used.

Next, the binary vector method is widely used because it is easy to operate. First, from the wild-type Agrobacterium (a) shown in FIG. 9 (2), the vir region essential for plant infection is retained and the total T-DNA region (LB
And RB, its internal region, and the onc gene are removed) to prepare a modified plasmid A '. It is replicable in both Escherichia coli and wild-type Agrobacterium, and infects plasmid B, which is a T-DNA region by incorporating the required DNA in the border region (c). When a plant is infected with Agrobacterium (c), the T-DNA region of plasmid B is introduced into the plant (p) by the action of the vir region in plasmid A '.

In the binary vector method, Agrobacterium for producing a genetically modified plant is Ti plasmid which is the main body of tumor formation in Agrobacterium tumefaciens, or Ri which is the main body of hairy root formation in Agrobacterium rhizogenes. Plasmid (Fig. 9
It is necessary to modify A) in (2) to remove the T-DNA region containing the symptom-expressing gene. Infect plants and enter the cell nucleus
Since it is not easy to make a strain having a functional region for injecting DNA but having a T-DNA region removed ((b) in FIG. 9 (2)), the number of Agrobacterium strains that make transgenic plants is Few.

Generally, in Agrobacterium, it has a functional region (vir) that infects plants and injects DNA into the cell nucleus.
It is not easy to make a strain in which the T-DNA region has been removed. Further, it is not easy to remove these plasmids because they have a replication mechanism that is stably maintained in Agrobacterium.

[0007]

The previously reported methods for removing plasmids include high temperature heating method, detergent addition method,
The method of adding ethidium bromide (EtBr) is known. Since these methods have low efficiency of removing the plasmid, not only a great deal of labor is required to obtain a cell line from which the plasmid has been removed, but also many cell lines cannot remove the plasmid. Furthermore, the conventional method for removing plasmids is characterized by culturing the cells in a culture medium at high temperature, with addition of detergent, with addition of EtBr for a long time, so that not only there are cell lines that cannot grow under these conditions, but The drawback is that accumulation of mutations in chromosomal DNA is unavoidable.

It is known that when a plurality of plasmids are present in Agrobacterium, the replication mechanism of the plasmids interferes with each other, making it difficult for the same kind of plasmid to replicate in the same cell. Existing plasmids can be removed by preparing a small plasmid having the same kind of replication mechanism and imparting drug resistance and introducing it into a cell line having the plasmid to be removed. However, since the newly introduced plasmid has the same replication mechanism and is stably maintained, it is inevitable that it is not easily removed.

On the other hand, since there are many cases in which it is difficult to produce genetically modified plants with these useful Agrobacterium, there is a demand for Agrobacterium having high infection and DNA injection ability. Agrobacterium genes that determine the ability to infect and inject DNA confers resistance to virulence genes (virulence region: vir) on Ti or Ri plasmids, chromosomal toxic genes (chv) on chromosomes, and various plant-carrying substances It is determined by the gene etc., but the detailed information is not yet known. Moreover, it is not easy to investigate whether these characteristics are determined by a plasmid or a chromosomal gene.

Therefore, the present invention relates to tumor induction (Ti) plasmid as well as hairy root disease induction (R) in Agrobacterium.
i) A method for easily and steadily deleting a plasmid such as a plasmid with high efficiency and without inducing mutation in the chromosomal DNA of a cell line, and also for a plasmid that is remarkably stably maintained in replication The purpose is to
Further, the present invention provides a method for easily producing a novel agrobacterium imparted with useful ability suitable for producing a transgenic plant by introducing a known plasmid into the thus deleted and deleted agrobacterium. With the goal.

[0011]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventor studied genetically the ability to induce infection / symptoms of Agrobacterium, and as a result, a plasmid for shedding that significantly promotes shedding of the plasmid. The present invention was completed and the present invention was completed by confirming that the cells eliminated at high frequency could be obtained by a steady and easy method, and that the plasmid had been removed. Therefore, the present invention relates to any of the following.

1. A recombinant plasmid for plasmid deletion, which has a replication gene incompatible with the replication gene of wild-type Agrobacterium and a susceptibility gene. 2. The susceptibility gene is the levansucrase gene,
A recombinant plasmid for plasmid deletion according to 1 above. 3. Furthermore, the recombinant plasmid for plasmid deletion according to 1 or 2 above, which has a conjugation origin gene. 4. Furthermore, the recombinant plasmid for plasmid deletion according to any one of 1 to 3 above, which has a drug resistance gene. 5. The recombinant plasmid for plasmid deletion according to the above 4, wherein the drug resistance gene is a gentamicin resistance gene or a kanamycin resistance gene. 6. A bacterium transformed with the recombinant plasmid for plasmid deletion according to any one of 1 to 5 above. 7. The transformed wild-type Agrobacterium described in 6 above. 8. The transformed wild type Agrobacterium tumefaciens described in 7 above. 9. The transformed wild-type Agrobacterium tumefaciens MAFF301001 strain described in 8 above. 10. A plasmid deficient Agrobacterium cell line characterized by introducing the recombinant plasmid for plasmid deletion according to any one of 1 to 5 above into a wild-type Agrobacterium strain and culturing in a sensitive medium How to create. 11. (i) Introduction of the recombinant plasmid for plasmid deletion according to any one of 1 to 5 above into a wild-type Agrobacterium strain, and (ii) transformation in which the recombinant plasmid for plasmid deletion is introduced Strains and strains that were not introduced are selected, and (iii) based on the incompatibility between the replication genes, the transformant strain that has lost the plasmid originally present in the wild-type Agrobacterium strain and has only the recombinant plasmid for plasmid deletion is selected. Obtaining, (iv) to determine a transformant having only the recombinant plasmid for plasmid deletion, and then
(v) The method according to the above 10, which comprises culturing a transformant having a recombinant plasmid for plasmid deletion in a sensitive medium. 12. (11) The preparation according to the above (11), which comprises selecting a transformant into which a recombinant plasmid for plasmid deletion has been introduced and a strain into which a recombinant plasmid for plasmid deletion has not been introduced by culturing in a medium containing a drug corresponding to the drug resistance gene. Method. 13. 13. The production method according to 11 or 12 above, wherein a transformant having only a recombinant plasmid for plasmid deletion is discriminated based on the presence or absence of assimilation of opine. 14. 14. The preparation method according to any one of 10 to 13 above, wherein the sensitive medium is a sucrose-containing medium. 15. A gene-recombinant Agrobacterium cell line lacking a plasmid. 16. 15. The Agrobacterium cell line for gene recombination according to 15 above, which is produced by the method according to any one of 10 to 14 above. 17. 17. The Agrobacterium cell line for gene recombination according to the above 15 or 16, which is the Agrobacterium tumefaciens MNS1 strain. 18. A method for producing an Agrobacterium cell line, which comprises introducing an exogenous plasmid into the Agrobacterium cell line for gene recombination in which the plasmid described in 15, 16 or 17 is deleted. 19. An exogenous plasmid is a plasmid in which the onc gene in the T-DNA region is removed from Ti plasmid A of wild-type Agrobacterium in the intermediate vector method, and instead the required gene is introduced (that is, "plasmid having vir gene"). (18) The method according to the above (18), characterized in that 20. An exogenous plasmid is a plasmid having a T-DNA region deleted in the binary vector method but having a vir region (that is, "plasmid having a vir region") and T
-The preparation method according to the above 18, which is an exogenous plasmid having a DNA region. 21. An Agrobacterium cell line in which an exogenous plasmid is introduced into the Agrobacterium for gene recombination according to the above 15, 16 or 17. 22. 22. The Agrobacterium cell line according to 21 above, which is produced by the method according to 19 or 20 above.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a general method for carrying out the present invention, examples of modifications, and preferred embodiments will be described.

FIG. 10 shows the foreign DNA for plants according to the present invention.
FIG. In FIG. 10, (a) is a wild-type Agrobacterium, and (b) is the plasmid B of the present invention.
(A) is the plasmid A
And B deleted Agrobacterium, (d) Agrobacterium having one foreign type plasmid, (e) T-DNA
Region-deleted plasmids and T-s containing foreign vir regions
Agrobacterium introduced with an exogenous plasmid having a DNA region, (f) Escherichia coli having the plasmid of the present invention,
(G) and (h) show E. coli having the above-mentioned foreign type plasmid, and (p) shows a plant into which the gene is introduced.

Agrobacterium (a) is shown in FIG.
This is shown in the example of Agrobacterium tumefaciens. As the wild-type Agrobacterium, any of those known to those skilled in the art, for example, Agrobacterium tumefaciens, Agrobacterium rhizogenes, and various types of Agrobacterium collected from soil and capable of infecting plants can be used.

Wild type Agrobacterium a has a replication gene (rep) in plasmid A. Therefore, the plasmid B of the present invention having a replication gene incompatible with its replication gene (rep) and a susceptibility gene is prepared in any bacterium, for example, Escherichia coli. In this specification,
The term “susceptibility gene” generally means a gene having a function of reducing cell growth ability or viability under a specific condition. For example, when the levansucrase gene is used as the sensitivity gene, it is used as a host cell. By imparting high sensitivity to sugar and culturing in a medium containing sucrose, a plasmid-deficient Agrobacterium (c) in which the plasmid B is removed can be obtained. Other susceptibility genes known to those skilled in the art include, for example, toxin genes and Fluoro-orotic acid processed so that their expression can be controlled.
For example, the pyrB gene that becomes supersensitive can be used.

Then, the plasmid B of the present invention is introduced into wild-type Agrobacterium a. As a method of introduction, any method known to those skilled in the art can be used. For example,
In the case of a plasmid having a mating origin gene such as incP-type oriT and incQ-type oriT, an introduction method by cell conjugation can be used. When prepared in E. coli, the conjugation efficiency with Agrobacterium is as high as about 10 ⁻² to 10 ⁻³ , so that the cell conjugation method can be widely applied. In addition to the cell conjugation method, the plasmid of the present invention may be introduced by a method such as an electric introduction method (electroporation method) or a DNA introduction method by chemical treatment, depending on the type and properties of the bacterium to be used. it can. In addition to Escherichia coli, for example, Salmonella typhimurium can be used as the bacterium.

By containing a drug resistance gene in the plasmid B of the present invention, by culturing cells with the drug, the Agrobacterium b into which the plasmid of the present invention has been introduced can be easily transferred from the non-introduced Agrobacterium. Can be sorted into As the drug resistance gene, any gene known to those skilled in the art, such as gentamicin resistance (Gm ^R ), kanamycin resistance (Km ^R ), and tetracycline resistance (Tc ^R ), may be contained alone or in combination. Since wild-type Agrobacteria show different drug resistances depending on the strains, the number of applicable Agrobacteria can be markedly increased in a plasmid having a plurality of plasmids than in a plasmid having a single drug resistance gene.

Agrobacterium b, which contains both the plasmid B of the present invention and the plasmid A originally present in wild-type Agrobacterium, has both plasmids because it has incompatibility between replication genes in cell culture. Agrobacterium cannot survive, and most of them have only one plasmid. By culturing in a medium to which a drug imparting resistance to plasmid B is added, cells deficient in plasmid A are highly concentrated and sorted. Then, the Agrobacterium deficient in the plasmid A originally present in the wild-type Agrobacterium (not shown in FIG. 10) is discriminated.

The method of discrimination is as follows, for example. (1) Discrimination method using opain Ti and Ri plasmids have genes for degrading and assimilating organic compounds collectively called opain. Since the nopaline type plasmid has the nopaline utilization gene,
The wild-type Agrobacterium A that retains this is a nopaline-utilizable type, and the Agrobacterium (c) that has lost this can be identified as a nopaline-incapable type. On the other hand, a wild-type Agrobacterium A that retains this
Is a nopaline-assimilating type, if a plasmid to which a nopaline-assimilating gene is added is used as the plasmid B of the present invention, Agrobacterium having the plasmid B of the present invention can be selected depending on the availability of nopaline. . When cultured in a medium supplemented with nopaline as the only carbon source, nopaline-unutilizable Agrobacterium (having plasmid A) cannot grow. (2) Discrimination Method by Southern Hybridization Method Wild type plasmid A contains a virulence gene (vir). Since the gene sequence is highly conserved among Agrobacterium, it is possible to detect Agrobacterium in which plasmid A is present by preparing a probe having this sequence. The plasmid B of the present invention does not have the vir gene. Therefore, the Agrobacterium for which no signal can be detected is the Agrobacterium lacking the plasmid A which will be used in the present invention. (3) Discrimination Method by Electrophoresis When the base sequence of the plasmid A of the present invention is known, or when the length of the restriction enzyme cleavage fragment is known, the length of the base sequence between the plasmids A and B is determined. It can be determined by the difference. Plasmids A and B are cleaved with appropriate restriction enzymes and the fragments are compared by electrophoresis. Regarding the sites that can be compared for the presence or absence, when the existing fragment is that of the plasmid B of the present invention, the clone is an agrobacterium deficient in the plasmid A used in the present invention.

After the Agrobacterium lacking the wild-type plasmid A is discriminated, the plasmid B remaining in the Agrobacterium is removed. At this time, by using the above-described susceptibility gene, the gene recombinant Agrobacterium cell line (c) of the present invention in which the plasmid B is deleted can be obtained.

The foreign plasmid C having foreign DNA is introduced into the thus obtained plasmid-deficient Agrobacterium cell line (c). Known methods can be applied to the introduction method. Known means can also be applied to select the introduced Agrobacterium (d). For example, when the recombinant plasmid (d) A ′ + I obtained by the intermediate vector method (FIG. 9 (1)) is used as the foreign plasmid C, the plant can be directly infected to transform the plant.

In addition to the above Agrobacterium (d),
It is also possible to introduce a foreign plasmid D having foreign DNA. Known methods can be applied to the introduction method. Outpatient D
Plant p is infected with agrobacterium (e) with NA C and D. As the method, known means can be applied. For example, as a combination of two types of exogenous plasmids C and D, the T-DNA region is deleted and
If a plasmid having a region and an exogenous plasmid having a T-DNA region are used, the binary vector method (Fig.
Since the conventional plant infection by (2)) can be used as it is,
The infection operation becomes easy.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

The strains and plasmids used in the following examples are shown in Table 1. Bacterial strains and culture conditions

[0026]

[Table 1]

Escherichia coli is Luri at 37 ° C
a-Bertani (LB) medium (1% Difco Bacto-trypton, 0.5%
Difco Bacto-yeast extract, and 0.5% NaCl). Agrobacterium tumefaciens (hereinafter AT
The strain was abbreviated) was grown at 28 ° C. in LB or mineral salt mixed medium (Petit and Tempe, (1993)). Antibiotics were added to the media at the following final concentrations (μg / mL): gentamicin (45), kanamycin (100),
Rifapicin (30). Either noparin (concentration 2 mg / ml in medium) or glucose (concentration 5 mg / ml in medium) was used as carbon source in the mineral salt mixture medium.

DNA Preparation and Analysis Plasmid DNA extraction from E. coli was performed by the alkaline SDS method (reference Bironboim et.al., 1979). The method for preparing intact genomic DNA in agarose gel is described in (Reference S
uzuki et.al., (2001)). Genomic DNA extraction by the liquid lysis method was carried out by making the following changes to the method of (Suzuki et.al. (2001)). 4m overnight culture
Bacterial cells recovered from L were used for extraction. The scale of the purification operation was reduced according to the volume of the culture. Instead of wrapping the DNA fiber around a glass rod, a brief spin down was performed. Agrobacterium conjugative transformants were selected by culturing in LB medium supplemented with antibiotics. Digestion and degradation of DNA and ordinary agarose electrophoresis were performed by standard methods. Transfer to positively charged nylon film by Southern method and hybridization are described separately (see Suzuki et al.
al, (2001)).

Example 1 Construction of Plasmid Deletion Recombinant Plasmid pMGTrep1 As shown in FIG. 2, the plasmid deletion recombinant plasmid (removal plasmid) was pK1 having oriT from RP4.
8mobsacB (reference Schafer et al (1994)) was used as the starting material. It is transferable from E. coli to both Gram-positive and Gram-negative bacteria including AT. The sacB gene on this plasmid has the effect of killing cells when the cells are cultured in a medium containing a high concentration of sucrose (Reference Steinmet).
z et al (1983), Gay et al (1985)), which is useful for isolating (counterselecting) cells in which the gene has been deleted. In addition, pK18mobsacB is a plasmid that automatically disappears because it cannot replicate in AT cells, and thus is maintained in Agrobacterium cells only when it is inserted into another replicon. E. coli JM109 was used as a host strain for plasmid construction. First, pMG plasmid was prepared. pM
G is pHRP309 (references Parales & Harwood et al, (1993))
PK1 added with gentamicin resistance gene Gm ^r from Escherichia coli
It is a derivative of 8mobsacB. This is at the PstI site of pK18mobsacB at the gentamicin resistance gene pHRP309 (Parale
s & Harwood (1993)) 2.9 kbp PstI fragment. Next, a HindIII fragment containing the repABC gene of pTi-SAKURA was inserted into pMG. pTi-SAKURA re
pABC segment (site between 17,363bp and 22,482bp in the entire nucleotide sequence) (Reference Suzuki et al (1998), Suzuki et al (20
00)) was amplified by PCR. The PCR product was digested with HindIII, and the 4.9 kbp HindIII fragment DNA containing the rep gene was inserted into pMG. The thus obtained plasmid for removal of drop according to the present invention was designated as pMGTrep1 (FIG. 2). This pMGTrep1 plasmid was introduced into E. coli S17-1λpir having conjugation ability by a transformation method. The nucleotide sequence of the used plasmid was determined by the primer extension method using a universal primer and a custom primer. First, pK18mobsac
Since the base sequence of B plasmid is not known, it was determined as follows. For the nucleotide sequences of the pK18mob and sacB genes, the data of DNA data accession number AF012346 (3793 bp) and the data of DNA data accession number X02730.1 (2007 bp) were used. Selbitschka is a part of the artificial sequence modification performed to reduce the restriction enzyme cleavage site in the sacB gene.
(Cited document Selbitschka et al (1993)). pK18mob and s at the AsuII cleavage site of pK18mob
The nucleotide sequence of 1 kb around the boundary (ligation) part of the fragment containing the acB gene was determined. The base sequence data obtained this time was adopted in the part where there is a discrepancy between the data on the literature and the database and the base sequence data determined this time. This is pK18
The entire base sequence of mobsacB was revealed. Furthermore, pMG is pK18mo
It is composed of a PstI fragment containing the bsacB and Gm ^R gene. For sequence of PstI fragment containing the Gm ^R gene not known, to determine the entire nucleotide sequence. The nucleotide sequence of 1 kb around the boundary (ligation) part of the PstI cleavage site of pK18mobsacB and the PstI fragment containing the GmR gene was also determined. As a result, the entire nucleotide sequence of pMG was 8623bp. pMGTrep1 is composed of pMG and a PCR-amplified HindIII fragment containing repABC of pTi-SAKURA. For the nucleotide sequence of the HindIII fragment containing repABC of pTi-SAKURA, the nucleotide sequence data of repABC of pTi-SAKURA (DNA data accession number AB006857 or AB016260) was used. Hind containing PCR amplified pTi-SAKURA repABC
The nucleotide sequence of 1.5 kb around the boundary (ligation) between the III fragment and the HindIII cleavage site of pMG was also determined. The total length of the base sequence is 13525b
It was p. The entire nucleotide sequence of pMGTrep1 is shown in Sequence Listing 1. The bases are indicated at the sites where base substitutions that are considered to be caused by PCR amplification were observed.

Example 2 Conjugate transfer of plasmid pMGTrep1 for removal of pMGTrep1 was carried out by introducing pMGTrep1 into C58rif and MAFF301001rif.
It was carried out by conjugation with E. coli S17-1 λpir retaining 1. Other methods of joining have been reported (reference Simon et al.
(1983)). The donor and recipient cells were cultured to a cell density of 1.0D ₆₀₀ . The cells are 10 mM Mg
Washed with SO ₄ . Agrobacterium cells were mixed with an equal volume of donor E. coli cells and then added dropwise to a nitrocellulose membrane filter (Millipore) on LB agar. 28
After incubating for 15 hours at ℃, the cell mixture on the filter was washed, dispersed in 0.9% (w / v) NaCl, and then spread on LB agar medium supplemented with rifampicin, kanamycin, and gentamicin. The number (colonization efficiency) per number of donor cells using the number of colonies (zygotes) generated in the culture for 3 days is shown in Table 2.

Example 3 Removal efficiency of Ti plasmid by conjugation The presence of Ti plasmid in zygotic cells was tested. pTi-SAKURA and pTiC58 are MAFF301001 and C respectively
It is a nopaline-type Ti plasmid in 58. Therefore,
Wild-type Agrobacterium cells carrying these plasmids can use nopaline as the sole carbon source.
However, it was expected that the Ti deletion zygotes would not be able to grow on minimum salt medium containing noparin. The zygote colonies were cultured on mineral salt medium (noparin medium) agar supplemented with noparin (2 mg / ml) as the sole carbon source.
The Agrobacterium conjugate was obtained by mating AT MAFF301001rif with Escherichia coli S17-1λpir (pMGTrep1).
(a), and AT C58rif and d E. coli S17-1λ pir (pMG
The result of crossing with Trep1) is shown in FIG. 3 (b).
In FIG. 3, among the colonies inoculated into the Noparin medium at equal intervals, the colonies that did not grow were observed in a relatively small shape, and the grown colonies were observed in a clearer white color and in a relatively large shape.

[0032]

[Table 2]

In the C58rif zygote, as shown in FIG. 3 and Table 2, 99% of the colonies did not grow on the nopaline medium. On the other hand, 32% of MAFF301001rif zygote colonies did not grow. The tumor-inducing ability of a zygote colony that did not grow on wild-type MAFF301001rif and nopaline medium (nopaline assimilation) was examined. AT was infected by injuring a stalk of Ceylon lanceolata with an injection needle. When it was cultivated in a greenhouse after being kept at 25 ° C for 30 hours, no tumor developed at the site of inoculation of the nopaline-unutilizable strain (Fig. 4 Ti-les.
s). On the other hand, a tumor developed in the wild-type AT before deletion (Fig. 4 WT).

Example 4 Demonstration of removal of the Ti plasmid by biochemical analysis In order to confirm whether the Ti plasmid was deleted in the above-mentioned nopaline-unutilizable conjugate, Southern hybridization was applied. Since the virulence gene (vir) is encoded by the Ti plasmid, the 4.4 kb fragment of the virB region of pTi-SAKURA is transformed into T
i Used as a probe for detecting plasmid. As shown in FIG. 5, the virB fragment was clearly detected in the wild-type strain and the nopaline-utilizable conjugate. The virB gene of pTiC58 is pTi-
Since it has a high homology of more than 99% to the virB gene of SAKURA, wild-type C58rif carrying pTiC58 could be detected with the same probe as efficiently as wild-type MAFF301001rif carrying pTi-SAKURA. On the other hand, as shown in FIG. 5, all of the nopaline-unutilizable zygotes showed no signal, so it can be said that the Ti plasmid is lost. The deletion of the Ti plasmid in the nopaline-unutilized zygote was further confirmed by pulsed field gel electrophoresis analysis of total zygotic cell DNA. All digested with the restriction enzyme SwaI
DNA is 24% at 200V using 1% (wt / vol) agarose gel.
Analysis was carried out by pulse field gel electrophoresis under the conditions of time, pulse time of 40 seconds and 0.5 × TBE buffer. The DNA fragment in the gel was stained with ethidium bromide (0.5 μg / ml) for 1 hour. After staining, the gel was rinsed with distilled water, and a UV light excitation fluorescence image was taken using a digital camera system FasII (Toyobo). The result is shown in FIG. Ten fragments were detected in the SwaI digestion product of total genomic DNA prepared from MAFF301001. The ninth band in lane 1 of Figure 6 was previously pT
It has been identified as a 203-kbp fragment of i-SAKURA (Reference Su
zuki et al (2001)). As shown in lane 2 of FIG. 6, M
In the nopaline-assimilating conjugate of AFF301001rif, this 9th band was clearly deleted. These results are
It is shown that pMGTrep1 introduced into Agrobacterium cells successfully removed the Ti plasmid by shedding. The above results indicate that the Ti plasmid was frequently deleted in the wild-type Agrobacterium colonies into which pMGTrep1 had been introduced in terms of the loss of nopaline assimilation ability using pMGTrep1 and the loss of the virB gene. Dropout frequency is 2
Although there were differences between the three Agrobacterium strains, at least 30% of the drug resistant colonies lacked the Ti plasmid. That is, Ti plasmid-deficient cells can be obtained at a ratio of one in several agrobacterium colonies into which pMGTrep1 was introduced.

Example 5 Removal of pMGTrep1 The above Ti deletion conjugate was replaced with pMGTrep1 instead of Ti plasmid.
have. PM to utilize Agrobacterium cells
Although GTrep1 needs to be removed, pMGTrep1 has a repABC gene and thus replicates stably, so it is not easy to remove by existing methods. As shown in FIG. 2, pMGTrep1 has a sacB gene that makes host cells highly sensitive to sucrose. Therefore, it was expected that pMGTrep1 removal from zygote cells could be performed by high-concentration sucrose treatment. The Ti-deficient strain was cultured overnight in LB medium and then in new LB containing 10% sucrose. Whether or not pMGTrep1 was present in the cells recovered after this treatment was determined by the gentamicin resistance gene (Gm ^r ).
(See FIG. 2) was tested in a Southern hybridization experiment using the probe. As shown in lanes 1 to 3 of FIG. 7, no signal was detected in cells obtained by culturing at a high sucrose concentration. This result indicates that a MAFF301001rif strain lacking the pMGTrep1 plasmid was obtained. This cell line is ready to introduce a new foreign Ti plasmid. MAFF301001rif-derived MNS-1 is one of the strains confirmed to lack the plasmid. The thus obtained Agrobacterium tumefaciens MNS1 strain, which is an Agrobacterium cell line for gene recombination according to the present invention, was dated March 22, 2002, National Institute of Advanced Industrial Science and Technology Deposited at the deposit center, deposit number FE
RM P-18794 is assigned.

Comparative Example 1 Plasmid Deletion Efficiency According to Conventional Method On the other hand, an attempt was made to isolate a Ti deficient strain after inducing Ti deficiency by high temperature culture treatment. MAFF301001 in LB medium
It was shake-cultured at 37 degrees for 2 days. The culture solution was diluted and then spread on LB agar medium to form colonies. When the colonies were transplanted onto Nopaline medium and cultured, all colonies grew. This result indicates that the Ti plasmid is highly stable in MAFF301001 cells and it is not easy to produce a plasmid-deficient strain by the high temperature culture method.

Example 6 The low plasmid shedding frequency observed with MAFF301001rif is a useful property as a host vector system. However, it was unclear whether this characteristic was determined by the plasmid or the chromosomal gene. In general, if you can steadily and easily determine whether the traits of a cell line are the characteristics of a plasmid or the characteristics of a chromosomal gene, analyze the characteristics in more detail to find the causative gene and clone it. It is a starting point for the widespread application of attributes. If plasmids are once removed from multiple cell lines with different plasmid stability, and then the same plasmid is introduced into each cell line to measure the stability, the difference in plasmid stability will result in the characteristic of the chromosome. It is determined whether the cause or the nature of the plasmid is the cause. Plasmid deletion A prepared in the present invention
Using T, the plasmid of another cell is plasmid deleted AT
Then, the frequency of dropping of the plasmid between cell lines due to the introduction of pMGTrep1 was measured and tested. As shown in Example 3 (Table 2), the Ti plasmid shedding frequency at C58rif is clearly higher than at MAFF301001rif. MAFF301001rif-derived Ti plasmid-free MNS1 cells were treated with C58 by a known method.
Was introduced. This new cell line MNS1 (pTiC5
If the plasmid loss frequency of 8) is close to the frequency of wild-type MAFF301001rif, it is considered that the chromosome contributes to stability,
On the contrary, if it is close to the frequency of C58, it is considered that the plasmid is the cause. As shown in FIG. 8, it was revealed that the plasmid loss frequency of the novel cell line was close to the C58 plasmid loss frequency. Therefore, it was revealed that the difference in the plasmid dropout frequency was caused by the difference in the genes existing on the plasmid. The thus obtained Agrobacterium tumefaciens ( Agro
bacterium tumefaciens) MNS1 (pTiC58) strain was deposited on March 22, 2002 at the Japan Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, with the deposit number FERM.
P-18793 is assigned.

[0038]

INDUSTRIAL APPLICABILITY According to the above-mentioned method, the yield of obtaining plasmid-deficient Agrobacterium from wild-type Agrobacterium is a high ratio (b / c) exceeding 30% to 90% of zygotic colonies. Further, Agrobacterium C deficient in T-DNA can be obtained without adding heat or an organic medium. C does not suffer from the damage such as the destruction or mutation of genetic material such as chromosomal DNA, and that various Agrobacterium strains that could not remove T-DNA can be used for gene transfer into plants. Become. Furthermore, according to the present invention, any foreign plasmid can be introduced into Agrobacterium at high frequency. Therefore, it is possible to compare the abilities among the host Agrobacterium by preparing a cell line that has the same plasmid in common with a plurality of different host Agrobacterium and comparing the traits. Similarly, the ability between plasmids can be compared by preparing cell lines in which different plasmids were introduced into the same host Agrobacterium and comparing the traits. Therefore, it becomes easy to scrutinize whether the useful trait is on the plasmid or on the chromosome side. It becomes possible to select a host Agrobacterium cell having high capacity and an efficient plasmid, and combine them to construct a novel Agrobacterium strain having a remarkably excellent quality.

References Gay, P., Le coq, D., Steinmetz, M., Berkelmann,
T., and Kado, CI 1985. Positive selection pr
ocedure for entrapment of insertion sequenceelemen
ts in Gram-negative bacteria. J. Bacteriol. 164, 9
18-921 Petit, A., and Tempe, J. (1978) Isolation of Agrob
acterium Ti-plasmid regulatory mutants. Mol. Gen.
Genet. 167: 147-155. And Puhler, A. (1993) Constru
ction of gene replacement vectors for Gram- bacter
ia using a genetically modified sacRB gene as a po
sitive selection marker. Appl. Microbiol. Biotech.
38: 615-618.Schafer, A., Tauch, A., Jager, W., Kalinowski, J.,
Thierbach, G., an dPuhler, A. 1994. Small mobili
zable multi-purpose cloning vector deri ved from
the Escherichia coli plasmid pK18 and pK19: select
ion of defined deletions in the chromosome of Coryn
ebacterium glutamicum. Gene 145: 69-73 Simon, R., Priefer, U., and Puhler, A. 1983. A bro
ad host range mobilization system for in vivo ge
netic engineering: transposon mutagenesis in Gram
-negative bacteria.Bio/ Technology 1: 784-794 Steinmetz, M., Le Coq, D., Ben Diemia, H., and Ga
y, P. 1983. Analysis genetique de sacB, gene de st
ructure d'une enzyme secreetee, la leva nesacchar
ase de Bacillus subtilis Marburg. Mol. Gen. Genet.
191: 138-144 Suzuki, K., Hattori, Y., Uraji, M., Ohta, N., Iwat
a, K., Murata, K., Kato, A., and Yoshida, K. 200
0. Complete nucleotide sequence of a plant tumor-i
nducing Ti plasmid. Gene 242: 331-336 Suzuki, K., Iwata, K. and Yoshida, K. 2001.Genome
analysis of Agrobacterium tumefaciens: constructio
n of physical maps for linear and circular chromos
omal DNAs, determination of copy number ratio and
mapping of chromosomal virulence genes. DNA Re
s. 8: 141-52. Suzuki K., Ohta N., Hattori Y., Uraji M., Kat
oh A. & Yoshida K. (1998) Novel structural differ
ence between nopaline- and octopine- type trbJ gen
e: construction of genetic and physical map and se
quencing of trb / traI and rep gene clusters of a ne
w Ti plasmid pTi-SAKURA. Biochim. Biophys. Acta 1
396: 1-7.27. Lin, BC, and Kado, CI (1997) Studies on Agro
bacterium tumefaciens.VII. Avirulence induced by t
emperature and ethidium bromide. Can. J. Microbio
l.23, 1554-1561. Parales, RE, and Harwood, CS (1993) Construct
ion and use of a new broad-host-range lacZ transcr
iptional fusion vector, pHRP309, for Gram bacteri
Gene 133, 23-30. Yanisch-Perron, C., Vieira, J., and Messing, J. (1
995) Improved M13 phage cloning vectors and host s
tarins: nucleotide sequence of the M13mp18 and pUC
19 vectors. Gene 33, 103-119.

[0040]

[Sequence list]                          SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> Plasmid having ability to delete other plasmid, the method of del eting plasmid in Agrobacterium by this plasmid, and Microorganism lossin g plasmid by this method. <130> PA900327 <160> 1 <170> PatentIn version 3.1 <210> 1 <211> 13525 <212> DNA <213> recombinant <220> <221> misc_feature <223> pMGT rep1 <400> 1 cgataagcta gcttcacgct gccgcaagca ctcagggcgc aagggctgct aaaggaagcg 60 gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg 120 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct 180 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc 240 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc 300 gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt 360 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 420 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 480 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 540 actccaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 600 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 660 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 720 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 780 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 840 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcggatgcc 900 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 960 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 1020 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 1080 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 1140 tcttgacgag ttcttctgag cgggactctg gggttcgcta gaggatcgat cctttttaac 1200 ccatcacata tacctgccgt tcactattat ttagtgaaat gagatattat gatattttct 1260 gaattgtgat taaaaaggca actttatgcc catgcaacag aaactataaa aaatacagag 1320 aatgaaaaga aacagataga ttttttagtt ctttaggccc gtagtctgca aatcctttta 1380 tgattttcta tcaaacaaaa gaggaaaata gaccagttgc aatccaaacg agagtctaat 1440 agaatgaggt cgaaaagtaa atcgcgcggg tttgttactg ataaagcagg caagacctaa 1500 aatgtgtaaa gggcaaagtg tatactttgg cgtcacccct tacatatttt aggtcttttt 1560 ttattgtgcg taactaactt gccatcttca aacaggaggg ctggaagaag cagaccgcta 1620 acacagtaca taaaaaagga gacatgaacg atgaacatca aaaagtttgc aaaacaagca 1680 acagtattaa cctttactac cgcactgctg gcaggaggcg caactcaagc gtttgcgaaa 1740 gaaacgaacc aaaagccata taaggaaaca tacggcattt cccatattac acgccatgat 1800 atgctgcaaa tccctgaaca gcaaaaaaat gaaaaatatc aagtttctga atttgattcg 1860 tccacaatta aaaatatctc ttctgcaaaa ggcctggacg tttgggacag ctggccatta 1920 caaaacgctg acggcactgt cgcaaactat cacggctacc acatcgtctt tgcattagcc 1980 ggagatccta aaaatgcgga tgacacatcg atttacatgt tctatcaaaa agtcggcgaa 2040 acttctattg acagctggaa aaacgctggc cgcgtcttta aagacagcga caaattcgat 2100 gcaaatgatt ctatcctaaa agaccaaaca caagaatggt caggttcagc cacatttaca 2160 tctgacggaa aaatccgttt attctacact gatttctccg gtaaacatta cggcaaacaa 2220 acactgacaa ctgcacaagt taacgtatca gcatcagaca gctctttgaa catcaacggt 2280 gtagaggatt ataaatcaat ctttgacggt gacggaaaaa cgtatcaaaa tgtacagcag 2340 ttcatcgatg aaggcaacta cagctcaggc gacaaccata cgctgagaga tcctcactac 2400 gtagaagata aaggccacaa atacttagta tttgaagcaa acactggaac tgaagatggc 2460 taccaaggcg aagaatcttt atttaacaaa gcatactatg gcaaaagcac atcattcttc 2520 cgtcaagaaa gtcaaaaact tctgcaaagc gataaaaaac gcacggctga gttagcaaac 2580 ggcgctctcg gtatgattga gctaaacgat gattacacac tgaaaaaagt gatgaaaccg 2640 ctgattgcat ctaacacagt aacagatgaa attgaacgcg cgaacgtctt taaaatgaac 2700 ggcaaatggt acctgttcac tgactcccgc ggatcaaaaa tgacgattga cggcattacg 2760 tctaacgata tttacatgct tggttatgtt tctaattctt taactggccc atacaagccg 2820 ctgaacaaaa ctggccttgt gttaaaaatg gatcttgatc ctaacgatgt aacctttact 2880 tactcacact tcgctgtacc tcaagcgaaa ggaaacaatg tcgtgattac aagctatatg 2940 acaaacagag gattctacgc agacaaacaa tcaacgtttg cgccgagctt cctgctgaac 3000 atcaaaggca agaaaacatc tgttgtcaaa gacagcatcc ttgaacaagg acaattaaca 3060 gttaacaaat aaaaacgcaa aagaaaatgc cgatggtacc gagcgaaatg accgaccaag 3120 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg 3180 gcttcggaat cgttttccgg gacgccctcg cggacgtgct catagtccac gacgcccgtg 3240 attttgtagc cctggccgac ggccagcagg taggccgaca ggctcatgcc ggccgccgcc 3300 gccttttcct caatcgctct tcgttcgtct ggaaggcagt acaccttgat aggtgggctg 3360 cccttcctgg ttggcttggt ttcatcagcc atccgcttgc cctcatctgt tacgccggcg 3420 gtagccggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg cgaataaggg 3480 acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct gccccgctga 3540 cgccgttgga tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt 3600 gcgaaaaagg atggatatac cgaaaaaatc gctataatga ccccgaagca gggttatgca 3660 gcggaaaagc gctgcttccc tgctgttttg tggaatatct accgactgga aacaggcaaa 3720 tgcaggaaat tactgaactg aggggacagg cgagagacga tgccaaagag ctcctgaaaa 3780 tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga 3840 acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg gcttcccggt 3900 atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca caggtattta 3960 ttcggcgcaa agtgcgtcgg gtgatgctgc caacttactg atttagtgta tgatggtgtt 4020 tttgaggtgc tccagtggct tctgtttcta tcagctcctg aaaatctcga taactcaaaa 4080 aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga 4140 tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac agggacacca 4200 ggatttattt attctgcgaa gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg 4260 tcgggtgatg ctgccaactt actgatttag tgtatgatgg tgtttttgag gtgctccagt 4320 ggcttctgtt tctatcaggg ctggatgatc ctccagcgcg gggatctcat gctggagttc 4380 ttcgcccacc ccaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 4440 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 4500 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 4560 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 4620 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 4680 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 4740 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 4800 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 4860 acctacaccg aactgagata cctacagcgt gagcattgag aaagcgccac gcttcccgaa 4920 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 4980 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 5040 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 5100 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 5160 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 5220 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca 5280 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 5340 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat 5400 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgtgg aattgtgagc 5460 ggataacaat ttcacacagg aaacagctat gaccatgatt acgaattcga gctcggtacc 5520 cggggatcct ctagagtcga cctgcaggac aaactggcgc ataacgagcg cttcgtggtg 5580 cgctccaagc acccacgcaa ggacgtagag tctgggttgt atctgtacga ccatctgaag 5640 aaccaaccgg agttgggtgg ctatcagatc agcattccgc aaaagggcgt ggtggataaa 5700 cgcggtaaac gtaaaaatcg accagcccgc aaggcgagct tgagcctgcg cagtgggcgc 5760 atcacgctaa aacaggggaa tatcacgctc aacgcggtgc tggccgagga gattaacccg 5820 cccaagggtg agaccccgtt gaaatggttg ttgctgacca gcgaaccggt cgagtcgcta 5880 gcccaagcct tgcgcgtcat cgacatttat acccatcgct ggcggatcga ggagttccat 5940 aaggcatgga aaaccggagc aggagccgag aggcaacgca tggaggagcc ggataatctg 6000 gagcggatgg tctcgatcct ctcgtttgtt gcggtcaggc tgttacagct cagagaaatt 6060 tcgatggtca ccgctaccct catgatgtct aacggccaag gtaagcggcc cgcagaatgc 6120 gggtccgctt gaccgcagag ttagacccga aggccaaaag ggagcgcccg gccttgaaga 6180 agggcgcagc ccaagaaaaa ccgctggacc acgaaagcgg cgcagcgctg caagaaggat 6240 agctggtggc gcttgctgca caagcgctgg aggcccaaaa ggctagaaag cctttggaac 6300 agggcggagc gctgaaataa gtggctgcgc gcataaagcg ggtgatggga ctaacgcctg 6360 agttaagccg gagcgctttg cggccgcggc gttgtgacaa tttaccgaac aactccgcgg 6420 ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa 6480 agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac 6540 cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga 6600 gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat 6660 agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc 6720 caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt 6780 tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac 6840 gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg 6900 tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg 6960 taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg 7020 gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa 7080 caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac 7140 cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc 7200 aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc 7260 agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg 7320 catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg cacggatctg 7380 ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc 7440 ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt gttcgcccag 7500 cttctgtatg gaacgggcat gcggatcagt gagggtttgc aactgcgggt caaggatctg 7560 gatttcgatc acggcacgat catcgtgcgg gagggcaagg gctccaagga tcgggccttg 7620 atgttacccg agagcttggc acccagcctg cgcgagcagc tgtcgcgtgc acgggcatgg 7680 tggctgaagg accaggccga gggccgcagc ggcgttgcgc ttcccgacgc ccttgagcgg 7740 aagtatccgc gcgccgggca ttcctggccg tggttctggg tttttgcgca gcacacgcat 7800 tcgaccgatc cacggagcgg tgtcgtgcgt cgccatcaca tgtatgacca gacctttcag 7860 cgcgccttca aacgtgccgt agaacaagca ggcatcacga agcccgccac accgcacacc 7920 ctccgccact cgttcgcgac ggccttgctc cgcagcggtt acgacattcg aaccgtgcag 7980 gatctgctcg gccattccga cgtctctacg acgatgattt acacgcatgt gctgaaagtt 8040 ggcggtgccg gagtgcgctc accgcttgat gcgctgccgc ccctcactag tgagaggtag 8100 ggcagcgcaa gtcaatcctg gcggattcac tacccctgcg cgaaggccat cggtgccgca 8160 tcgaacggcc ggttgcggaa agtcctccct gcgtccgctg atggccggca gcagcccgtc 8220 gttgcctgat ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 8280 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 8340 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 8400 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg catgcaagct 8460 tcgataccgt tcgtattggt ccggcgaaac tgtgagtatc cacatcgtaa tctccgcatg 8520 aacaggtcat gcgaacagaa atcatctcac ggtgcgtttg cctacgtgca gatttgcacc 8580 tcaggtgatt ctaccgagtc ggtgttccaa ggcgcgaaat gcgagcgggt gaggccggcc 8640 agacgccgac aaggttgtgc agatctgcac ttggtgccac gtcgcacaga agaagggaat 8700 cggtctaact cacagatagc atttgaagaa tcgggattta gtgtgatttc gattgaaacg 8760 cgcgtaaccg ttcattaacc aaaaacgtct tgcaacctca cccgcattag gtaatcgtca 8820 cggataaatg gcaatacgcg ccaattaacc gtgacaagag ataacaccgt gagcaaagcc 8880 gctgccatat cccgaaatga tcgcccgtcg gtagatgtta ccattggtga gcatgctgag 8940 cagctcagct ctcagcttca agcgatgagc gaggctttgt ttcctccgac gtcgcacaag 9000 agcttgcgca aattcacctc gggtgaagcc gcacgcttga tgaaaatatc tgactcaact 9060 cttcgaaaga tgacactggc tggcgaaggg ccgcaacctg aactcgccag caacggacgg 9120 cgcttttaca ccctcggtca gataaacgaa atccggcaga tgcttgccgg ctcgactcga 9180 ggacgtgaaa gcattgattt tgtgcctcat cgccgaggtt ctgagcattt gcaagtcgtt 9240 gctgtaacca acttcaaagg tggctctggg aagacgacga cgtccgctca tcttgcacag 9300 tatctggcgt tgcaaggtta cagggttctc gcagtcgatc tcgatccgca ggctagtctt 9360 tcagcactcc tcggcgttct gccagaaact gatgtcggtg caaacgaaac gctctatgcg 9420 gctattcggt acgacgacac acgtcgtccg ttgcgagatg tgatccgacc gacgtatttt 9480 gatggtcttc accttgttcc tggaaatctc gagcttatgg agttcgagca taccaccccg 9540 aaagcattga ctgacaaagg tacgcgcgac ggattgttct tcactcgcgt ggcccaagcc 9600 tttgatgagg tcgccgacga ttacgatgtc gtggtcatcg actgccctcc tcagcttggg 9660 tttttgactc tcagcgggtt gtgtgctgca acatcaatgg taatcaccgt acatcctcag 9720 atgctggata tcgcttccat gagccagttt ctcctcatga cacgcgacct tctgggtgtc 9780 gtgaaagagg cggggggcaa tctccagtac gatttcatac gctatctctt gacgcgctat 9840 gagccccagg acgcgccgca gacgaaagtg acggcactgc tgcgcaacat gttcgaggat 9900 cacgtcctta caaatcctat ggtcaagtcg gcagcggtat ctgatgccgg tttaaccaag 9960 cagacgctct atgagatagg gcgagagaac cttacgcgat cgacatacga ccgggcgatg 10020 gaatctttag atgcggtgaa ttcggagatc gaggctttga tcaagatggc gtgggggcgg 10080 gtctaatgaa aggctttgcg ttcctcacag atctgttggg agctcccaac agacaggtgt 10140 tgattcgccc cctggacatg gggcactgga gaagccgggg taatttgaga cgacgacgca 10200 cgcccatcgc taattggcca gggtgcagtt gtcttgtctt gttgggagct cccaaccaag 10260 cgcatttgca atcaaaaatg cgacgccacg acgccaaacc caagaggctg atatcatgag 10320 ccgcaaagac gcaatcgata ctttgttcct caagaagcaa cctgcgaccg atagagcagc 10380 agtcgacaag tcgaccgctc gtgttcgtac cggagcgatt tcggccatgg gttcgtcttt 10440 gcaagagatg gctgagggcg caaaggctgc agctcggctg caggatcaac tggctacagg 10500 cgaagccgtc gtgtccctgg atccatccat gatcgacggg tcgccgatcg cggatcggct 10560 gccctcagac gtggatccga aattcgagca gcttgaggcg agcatttcgc aggaggggca 10620 gcaggtgccg gttcttgtca gaccgcaccc tgaggctgcc ggtcgatatc agatcgtata 10680 tggaaggcgg cggctgcgcg cggcagtaaa tctgcggaga gaggtttctg ccattgttcg 10740 aaatctcacg gactgtgaac tggtcgtggc ccagggccgc gaaaatctta accgtgctga 10800 cctctcgttc attgagaagg ctctcttcgc cctgcgcctc gaagatgcgg gttttgatag 10860 agccaccatc attgccgcgc tatccactga caaggccgac ctcagccgct acataactgt 10920 agcaaggggc ataccgctga acctcgccac acaaatcggc ccagcgtcga aagcgggtcg 10980 atcgcgttgg gtcgcacttg ccgaggggct tgggaagcct aaggcaacgg acgcaatcga 11040 agcgatgctt gggtcagagc agttcaagca atctgatagc gatacccgct ttaacctcat 11100 tttcaacgcc gtttcaaggc cacctgcgaa gactccaaaa aaggtaaggg cctggagcac 11160 gccaaagggg aaaaaggcag cgacgatccg acaagaaact ggacgaacgg cgctggtttt 11220 cgacgagaga ctggtgccaa cttttggcga atatgtcgct gaccagttgg acagtctgta 11280 cgcccagttc attgaaacca acggaggagg caagctcgac caatagtcag ggtttcatcc 11340 aatttaaagc tccgctcgac tgagatggac tggctctcac cgcaaaagaa aaaggccccc 11400 gaaacggcgt tccggaagac cttctctgta gtctcgcagc taagagaatc gcatttccag 11460 gaatcgtagt caagggtccc gtaagggaaa gcgtcatttc gacgggcgga tttcaattgc 11520 ctaacaaaag gtaaaaggaa atgcagacgc atatctcaac gacgtccttt gggcggcggc 11580 cgatgacact cggccatatt gcaagccaga tggcagcaaa agcggtcgca tcagacactg 11640 tcgcccacaa atggcaggtc ttccagcaca tccgtgaatc ccggggactg atcggagcca 11700 cggaccgctc actctcgatc ctgaacgcgc tgttgacgtt ttacccggag accgccttga 11760 ctggtggtgc cgaactggtc gtatggcctt ctaacgaaca gctgatggct cgcgccaacg 11820 gcatgcccgc cacgacactg cgccggcatc ttgccatact ggttgattgc gggctcatca 11880 ttcgccgcga cagccccaat ggcaagcggt tcgcccgcaa gggaagggga ggggagattg 11940 agcaggccta tgggttcgat ctgtcgccga tcgtcgcgcg ggccgaggag ttccgagatc 12000 tggcccagac agtgcaagct gaaaaaaagg ccttccgggt ggccaaggag cgcttgactc 12060 ttcttcgtcg tgacattgtc aaaatgatcg aaactggcgt cgaagagagc gttcctggaa 12120 actggggaag agttacccag acctatcagg ggatcatcgg ccgcctgcca cgctcggcac 12180 ctcggcagct tgtcgagagt attgggcaag agcttcagga actttgcatc gagatccgtg 12240 acgtattgga atctttcaca aaaacgatga atctggacgc caatgagtcc catatcggtc 12300 gccacaaaca gaattcaaat ccagactcta aatttgaatc tgaatacggc tctggaaaaa 12360 aagatgaagc gggcggcagc gttgcggaaa ccgacaatgt acggagcttg ccgaaacgcg 12420 agctgccttt gggaatcgtg ctggatgcct gccccgaaat gcgggaattg gcccagggag 12480 gtccaattcg acattggcgc gacttgctgg cggcggctga gcttgcccgg ccgatgctgg 12540 ggattagtcc gagcgcctgg cgggaggccc gcgaaaccat gggcgagcaa cacgcggcga 12600 tcacgctggc ttcgatctat cagcgggccg gtcagatcaa taacgctggg ggctatctgc 12660 gcagcctgac cgaccgggcc aaggatggga agttttcgac ctggccgatg gtcatggcgt 12720 tgctccgggc gaagctggac gagcagaaga atgcagtcgg cgctggaaag ccgcgaactg 12780 ctgaggaggt cgaggatgac agccgcctcc acgtatcgga atcgctgctc aaaaacctgc 12840 gaaagccgag atcttggtga tcctctcgct attcagccgc ggcgatgtcg acgtcggtga 12900 tcaacccgga acgtcgggcg cggtcgatca ggttcttgac ggacgagggc gcccatttgg 12960 atccaccgcg cggcgtgcgc tcgtgcagtc tttcaagctg gccggcgatc tcgcggagct 13020 tcaggtcggg gttcgaagaa tggatgccgg ccacaagcgt catcaggcga tcttcgggaa 13080 gacggggagg agattttttc aggagcgctg catccaccag gcgttccgtc accatccact 13140 tcacggcgcg gcgaagacgt tccggcgtcc agtcgaggcc ccgctgcttg agcattcggg 13200 cgatgtcgtc ccatgtgtga tccggtcgca tgcgacgaac ggtaggaagc cattggttcg 13260 cggacgcctg aatcctatcg ccatatgccg ctttctgggc cgcggtcatc cttgccagtg 13320 cctccgggcg cttttcccgg atgcccgggt tgccggaaag cttggcactg gccgtcgttt 13380 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 13440 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 13500 tgcgcagcct gaatggcgaa tggcg 13525

[Brief description of drawings]

FIG. 1 is a diagram showing a novel shedding and removal method using the incompatibility of plasmids of the present invention. This method consists of the following three steps. 1) Introducing an incompatible plasmid for removal of occlusion into an Agrobacterium strain. 2) Identify the Ti-deficient zygote using the cell traits such as nopaline (opin) assimilation ability. Alternatively, it is determined by a biochemical method such as a nucleic acid hybridization method. 3) Remove the removal plasmid.

FIG. 2 is a plasmid pMGTre for removing a drop of the present invention.
It is a figure explaining the structure of p1.

[Fig. 3] Fig. 3 is a photograph showing selection of opaque assimilation-type conjugates of the present invention.

FIG. 4 is a photograph showing selection of non-tumorigenic conjugates of the present invention.

FIG. 5 is a photograph showing the result of analysis of Ti plasmid in a pMGTrep1-derived zygote by the Southern hybridization method of the present invention.

FIG. 6 is a photograph showing the results of analysis of Ti plasmid deletion in a conjugate with pMGTrep1 of the present invention using pulsed field gel electrophoresis.

FIG. 7 is a photograph showing removal of pMGTrep1 loss by treatment of a Ti-deficient strain with a high-concentration sucrose by the Southern hybridization method of the present invention.

FIG. 8 is a comparison of traits between cells of the present invention having a common plasmid but different chromosomal DNA, and
FIG. 3 shows a comparison of traits between cells of different plasmids that have a common chromosomal DNA.

FIG. 9 is a diagram showing the concept of conventional methods (intermediate vector method and binary vector method) for incorporating a foreign gene into a plant using Agrobacterium.

FIG. 10 is an exotic D to a plant according to the method of the present invention.
It is a figure which shows the introduction of NA.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B024 AA08 AA20 CA01 DA02 DA05                       EA04 FA10 FA15 FA20 GA11                       GA27 HA20                 4B065 AA11X AA11Y AA88X AA99X                       AA99Y AB01 AC20 BA01                       BA02 BA25 BA30 CA53

Claims (22)

[Claims] 1. A recombinant plasmid for plasmid deletion, which has a replication gene incompatible with the replication gene of wild-type Agrobacterium and a susceptibility gene. 2. The recombinant plasmid for plasmid deletion according to claim 1, wherein the susceptibility gene is a levansucrase gene. 3. The method according to claim 1, further comprising a zygotic origin gene.
Or a recombinant plasmid for plasmid deletion according to 2. 4. The method according to claim 1, further comprising a drug resistance gene.
A recombinant plasmid for plasmid deletion according to any one of 1 to 3. 5. The recombinant plasmid for plasmid deletion according to claim 4, wherein the drug resistance gene is a gentamicin resistance gene or a kanamycin resistance gene. 6. A bacterium transformed by the recombinant plasmid for plasmid deletion according to any one of claims 1 to 5. 7. The transformed wild-type Agrobacterium according to claim 6. 8. The transformed wild type Agrobacterium tumefaciens according to claim 7. 9. The transformed wild-type Agrobacterium tumefaciens MAFF301001 according to claim 8.
stock. 10. A plasmid characterized by introducing the recombinant plasmid for plasmid deletion according to any one of claims 1 to 5 into a wild-type Agrobacterium strain and culturing in a sensitive medium. A method for producing a deleted Agrobacterium cell line. 11. (i) The recombinant plasmid for plasmid deletion according to any one of claims 1 to 5 is introduced into a wild-type Agrobacterium strain, and (ii) the recombinant plasmid for plasmid deletion. A transformant having the plasmid and a strain not having the plasmid were selected, and (iii) a transformant having only the plasmid originally present in the wild-type Agrobacterium strain, and the plasmid, based on the incompatibility between the replication genes. Obtain a transformant having only the recombinant plasmid for deletion, (iv) identify a transformant having only the recombinant plasmid for plasmid deletion, and then (v) have only the recombinant plasmid for plasmid deletion The method according to claim 10, which comprises culturing the transformant in a sensitive medium. 12. A method of culturing in a medium containing a drug corresponding to a drug resistance gene to select a transformant into which a recombinant plasmid for plasmid deletion is introduced and a strain not to be introduced, The creating method according to claim 11. 13. The method according to claim 11 or 12, wherein a transformant having only a recombinant plasmid for plasmid deletion is discriminated based on the presence or absence of assimilation of opine. 14. The method according to claim 10, wherein the sensitive medium is a sucrose-containing medium. 15. A plasmid-deficient Agrobacterium cell line for gene recombination. 16. The agrobacterium cell line for gene recombination according to claim 15, which is produced by the method according to any one of claims 10 to 14. 17. Agrobacterium tumefaciens M
The agrobacterium cell line for gene recombination according to claim 15 or 16, which is an NS1 strain. 18. A method for producing an Agrobacterium cell line, which comprises introducing an exogenous plasmid into an Agrobacterium cell line for gene recombination in which the plasmid according to claim 15, 16 or 17 is deleted. 19. The method according to claim 18, wherein the exogenous plasmid is a plasmid having a vir gene. 20. The method according to claim 18, wherein the foreign type plasmid is a plasmid having a vir region and a foreign type plasmid having a T-DNA region. 21. An Agrobacterium cell line in which an exogenous plasmid has been introduced into the Agrobacterium for gene recombination according to claim 15, 16 or 17. 22. The Agrobacterium cell line according to claim 21, which is produced by the method according to claim 19 or 20.