JPH0549481A - Multicloning binary vector - Google Patents

Abstract

PURPOSE:To obtain a new multicloning vector used for introducing an extraneous gene into a plant cell. CONSTITUTION:A multicloning binary vector such as pKK 524 having a boundary sequence of T-DNA on a Ti plasmid derived from Agrobalterium tumebaciens, having a hygromycin B phosphotransferase gene and beta- glucuronidase gene between the boundary sequence and having a multicloning site between the hygromycin beta phosphotransferase gene and the beta-glucuronidase gene. An exemplified vector is obtained by a scheme expressed by the formula.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a multicloning binary vector used for plant gene manipulation. More specifically, the Agrobacterium tumef
A Tiens derived from Aciens has a border sequence in the T-DNA region and a hygromycin B phosphotransferase (hph) gene and a β-glucuronidase (GUS) gene between the border sequences, and further between the two genes. The present invention relates to a binary vector having a multi-cloning site, which can be used to introduce a foreign gene into plant cells.

[0002]

2. Description of the Related Art In recent years, research on plant breeding having a pesticidal resistance, virus resistance and other practical properties by introducing a foreign gene into plant cells has been actively conducted. As a method for introducing a foreign gene into plant cells, soil bacteria A. tune
T using a Ti plasmid carried by facies
There is an i plasmid vector system. There is a specific region called T-DNA on the Ti plasmid, and when soil bacteria infect plants, T-DNA is transferred from soil bacteria to plant cells and finally integrated into the chromosomal DNA of the nucleus of plant cells and stabilized. Maintained at. The Ti plasmid vector system utilizes such properties of T-DNA.

That is, the Ti plasmid vector system is
A foreign gene of interest is inserted into the DNA region, and the foreign gene is introduced into plant cells as a part of T-DNA. The binary vector method has been developed as one of such Ti plasmid vector systems. In the binary vector method, T-DNA is the chromosome DN of plant cells.
A binary vector was constructed in which the foreign gene of interest was inserted between the bordering sequences of T-DNA required for integration into A, while vir of Ti plasmid required for transfer of T-DNA into plant cells. This is a method of constructing separate plasmids containing regions, introducing these two plasmids into Agrobacterium, infecting the plants with the Agrobacterium, and incorporating the foreign gene of interest into the chromosomal DNA of the plant cell.

As an example of such a binary vector, for example, pBI121 [EMBO. J. , 6,390
1-3907 (1987)]. pBI121 is
It has a neomycin phosphotransferase gene and a GUS gene between the border sequences of T-DNA, has a cloning site for inserting a foreign gene between these genes, and can grow in Escherichia coli and Agrobacterium. It is a binary vector. pBI121 is the drawback that in the case of plants with kanamycin resistance is difficult to obtain a good efficiency transformants also have only only Hin dIII site as a cloning site.

Other binary vectors include, for example, pUCD2340 [E. Zyprian et a
l. , Plant Mol. Biol. , 15, 24
5-256 (1990)]. pUCD2340 has an hph gene between border sequences of T-DNA,
It has a multi-cloning site on the downstream side of the h gene. However, in pUCD2340, T-DN
Since the gene inserted between the border sequences of A is long, it is expected that the inserted gene has a low probability of being integrated into the chromosomal DNA of the plant. In addition, although it has a multi-cloning site, it does not have a marker gene on both sides of the multi-cloning site. Therefore, in order to confirm whether or not the foreign gene of interest was integrated into the chromosomal DNA of the plant, Southern hybridization was performed. I have to read.

[0006]

Therefore, the object of the present invention is to easily introduce a foreign gene into a plant having kanamycin resistance, and to easily integrate the foreign gene into the plant genome. Can be confirmed in
Another object of the present invention is to provide a binary vector having a multi-cloning site so that a foreign gene can be easily inserted.

[0007]

Means for Solving the Problems As a result of intensive studies aimed at obtaining a binary vector capable of achieving the above objects, the present inventors have found that T-DN of Ti plasmid is used.
The present inventors have completed the present invention by discovering that a binary vector having an hph gene and a GUS gene between the border sequences of A and having a multi-cloning site between the two can achieve the above object.

The present invention is directed to Agrobacterium
T- on Ti plasmid from tumefaciens
Multicloning having a DNA border sequence and a hygromycin B phosphotransferase gene and a β-glucuronidase gene between the border sequences, and a multicloning site between the hygromycin B phosphotransferase gene and the β-glucuronidase gene Regarding binary vector.

The basic vector used to construct the binary vector of the present invention may be any vector so long as it can grow in Escherichia coli and Agrobacterium. For example, pBI121 or pGAH [protein nucleic acid enzyme, 35, no. 4,2476-2489 (1
990)] and the like. The binary vector of the present invention has, in such a vector, a right border sequence (RB) and a left border sequence (LB) of T-DNA which is a border region of T-DNA on the Ti plasmid. Between RB and LB, there are two marker genes, hph gene and GUS gene. The GUS gene is one of the reporter genes and is a marker gene that allows the expression site to be easily identified by a color reaction. A promoter for expressing these genes is provided on the upstream side of each of the hph gene and the GUS gene. Examples of the promoter include the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthase gene (NOS) promoter, and the like. Also, a terminator is provided on the downstream side of the hph gene and the GUS gene. CaM as a terminator
Examples thereof include V terminator and NOS terminator.

There is a marker between the hph gene and the GUS gene.
It has a multi cloning site. Multi cloning
Regardless of which restriction enzyme site is present as a site
Well, for exampleKpnI,ApaI,XhoI,Sal
I,ClaI,HindIII site etc.
It The binary vector of the present invention is multicloning
Easy insertion of various foreign genes due to the site
It is possible to

The positional relationship between the hph gene and the GUS gene is not particularly limited, and the GUS gene may be present on either the upstream side or the downstream side of the hph gene. When constructing the binary vector of the present invention, vectors for E. coli, such as pUC19 and pUC18 [Yan
isch-Perron, C.I. et al. , Gen
e, 33, 103-119 (1985); Messin.
g, J. , Methods in Enzymolog
y, 101, 20-78], etc., and the hph gene, 35S promoter, NOS
A desired vector can be constructed by introducing a terminator, CaMV terminator, etc. and amplifying in E. coli. hph gene, 35S promoter, Ca
When preparing genes such as MV terminator, p
GL2 [Gritz, Let al. , Gene, 2
5,179-188 (1983)], and these genes already existing in a known plasmid can be used. For the RB and LB genes of T-DNA, GUS gene and the like, the binary vector of the present invention can be constructed by appropriately utilizing these genes existing in the known vector pBI121. To introduce a multi-cloning site, for example, pBSI
The multi-cloning site already existing in known plasmids such as IK + and pBSIIK- can be used as it is. Alternatively, in order to introduce a cloning site, the same restriction enzyme site existing in other parts is cleaved, the ends are blunted and re-ligated to remove the restriction enzyme sites existing in other parts to create a cloning site. It can also be introduced.

[0012] Escherichia coli harboring a vector obtained by introducing an appropriate foreign gene into the multicloning site of the binary vector obtained in the present invention and Agrobacterium harboring a plasmid having a vir region on the Ti plasmid are ligated. Then, by introducing a binary vector into Agrobacterium and infecting the plant with this Agrobacterium, the foreign gene of interest can be incorporated into the chromosomal DNA of the plant cell. As a method for introducing a binary vector into Agrobacterium, a triparental mating method [Ditta,
G. et al. , Proc. Natl. Acad. S
ci. , USA, 77, 7347-7351 (198).
0)] and the like are adopted, and as a method for infecting a plant with Agrobacterium, the leaf disk method [Horsc
h, R.H. B. et al. , Science, 227,
1229-1231 (1985)], a protoplast coculture method [Marton, L. et al. et al. , Natu
re 277,129-131 (1979)] and the like are adopted.

[0013]

As described above in detail, according to the present invention,
A foreign gene can be easily introduced into a plant having resistance to Kanamai, integration of the foreign gene into the plant genome can be easily confirmed, and the foreign gene can be easily inserted. A binary vector having a multi-cloning site is obtained. As shown in the examples below, in the present invention, specifically, a binary vector pKK having a multi-cloning site is used.
524 and pKK525 are obtained, and these binary vectors can be used when introducing a foreign gene into the genomic DNA of plant cells, and are extremely significant in plant gene manipulation.

[0014]

EXAMPLES The present invention will be described in more detail below with reference to examples. Example 1 Construction of binary vector pKK524 [A] SacII site on the upstream side and KpnI site on the downstream side
Of the CaMV35S promoter having the ITE and pBI121 [E having the CaMV35S promoter
MBO J. , 6 , 3901-3907 (1987)].
And pUC19 [Yanish-Perron, C .; e
t al. , Gene, 33, 103-119 (198).
5); Messing, J .; , Method in E
ngymology, 101, 20-78],
As shown in FIG. 1 and below, CaMV35S having a Sac II site on the upstream side and a KpnI site on the downstream side.
A plasmid with a promoter was constructed.

(1) Preparation of DNA fragment Alkaline SDS method ( minipreparation method) [Man
iatis, T .; et al. , J. (1982) in
Molecular Cloning, ALabora
tory Manual, Cold Spring
Harbor Laboratory] was used to prepare pBI121 (2 μg) and pUC19 (2 μg),
Restriction enzymes Bam HI (Takara Shuzo) and Hin dI, respectively
Completely decomposed with II (Takara Shuzo). The decomposed product of pBI121 was separated by agarose gel (0.7% agarose) electrophoresis, and ethidium bromide (0.5 mg / m 2
After staining with 1), the portion containing a fragment of about 0.8 kb was cut out with a razor blade under 365 nm UV light. The agarose gel was dissolved in 500 μl of NaI solution at 50 ° C .; the DNA therein was adsorbed with 5 μl of glass milk (Geneclean II kit; BIO101In).
c. Manufactured by Vogelstein, B. et al. ，
Proc. Natl. Acad. Si. , USA, 7
6,615 (1979)], a DN of about 0.8 kb containing a CaMV35S promoter was excised from the excised gel.
The A fragment was recovered. Solution 10 containing degradation product of pUC19
10 μl of 3M-NaAc and 275 μl of ethanol in 0 μl
1, and frozen at -80 ° C for 20 minutes to give 12,000 rp.
It was centrifuged at m for 10 minutes. The resulting DNA pellet is 7
It was rinsed twice with 500 μl of 0% ethanol and once with ethanol, and dried under vacuum.

(2) Rebinding of DNA 5 μl of an aqueous solution containing 0.3 μg of a pUC19 degradation product and C
About 0.8 kb DN with aMV35S promoter
Prepare 5 μl of an aqueous solution containing 0.2 μg of the A fragment, and
NA Ligation Kit (Takara Shuzo) [Hayash
i, K. et al. , Nucleic Acids
Res. , 14, 7617-7631 (1986)]. The reaction was carried out at 16 ° C. for 30 minutes to transform Escherichia coli JM109 [Maniatis, T. et al. e
t al. , J. (1982) in Molecular
Cloning, A Laboratory Man
numeric, Cold Spring Harbor L
It was kept on ice until used for laboratory. (3) Transformation of E. coli JM109 Competent cell of E. coli JM109 (Takara Shuzo) [Me
ssing, J. et al. , Gene, 33, 119 (198
5)] and the DNA solution 20 re-bonded to and kept on ice
μl was added and left at 0 ° C. for 45 minutes. After treatment at 42 ° C for 3 minutes, the mixture was left at 0 ° C for 5 minutes. 2 ml of LB medium was added, and the mixture was shake-cultured at 37 ° C for 1 hour. 1.5 ml of it
Was transferred to an Eppendorf tube and centrifuged at 10,000 rpm for 10 seconds. The supernatant was removed and resuspended in 100 μl of LB medium, and ampicillin IPTG and X-Gal (5-
Bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was added to the agar medium, and the mixture was incubated at 37 ° C for 1 hour.
Cultured at night. The produced colonies were all white colonies that lost the X-Gal degrading activity. 5 colonies
ml LB-medium (ampicillin 50 μg / ml) was inoculated and cultured overnight. The transformed E. coli JM1
09 was collected by centrifugation, and the plasmid was recovered from it by the alkaline SDS method. A portion of the recovered plasmid was completely degraded with Bam HI and Hin dIII. The recovered plasmid with Bam HI and Hin dIII in degraded plasmid was confirmed that the recombined DNA was obtained by observing the size of the agarose gel electrophoresis perilla respectively. The plasmid thus obtained was designated as plasmid a. Next, as shown below, the plasmid a is cleaved with Pst I and Kpn I to obtain CaMV.
The DNA fragment containing the 35S promoter was cloned into pBSIIS
K + was ligated to the Pst I / Kpn I fragment.

(4) Preparation of DNA fragment Recombinant plasmid a obtained by the above operation and pBSII
SK + (Toyobo), Pst I (Takara Shuzo) and Kpn I
(Takara Shuzo) completely decomposed. The digestion product of the recombinant plasmid a was treated in the same manner as the digestion product of pBI121 shown in (1) above to recover a DNA fragment of about 0.8 kb. This DNA fragment, as shown in FIG.
Eco R of and from pUC19 has 5S promoter
I site and Sca I site and derived from pBI121
Sph I site and Hin dIII sites in which has been removed. The degradation product of pBSIISK + was treated in the same manner as the degradation product of pUC19 shown in (1) above. (5) a DNA fragment of approximately 0.8kb was obtained by recombination above DNA (4), pBS
The degradation product of IISK ⁺ was recombined in the same manner as in (2) above. (6) Transformation of E. coli JM109 recombined E. coli JM109 obtained in (5) above
After transformation with NA, a recombinant plasmid b was obtained in the same manner as in (2) above. (7) Cleavage of plasmid b and end blunting Recombinant plasmid b (2 μg) was completely digested with restriction enzymes Not I (Takara Shuzo) and Pst I and purified by the ethanol precipitation described in (1) above, followed by DNA Blunting.
Kit (Takara Shuzo) [Maniatis. , T. , Fr
itschE. F. and Sambrook, J. et al.
(1982) in Molecular Clonin
g, A Laboratory Manual, Co
ld Spring Harbor Laborato
ry] was used for blunting the ends. (8) Rebinding of DNA The self-ligation of the blunt-ended DNA obtained in (7) above was performed according to the method of (2) above. (9) Transformation of E. coli JM109 E. coli JM109 was transformed with the DNA obtained in the above (8) to obtain a recombinant plasmid c. The thus obtained plasmid c is a plasmid containing the CaMV 35S promoter having a Sac II site on the upstream side and a Kpn I site on the downstream side. Further, as shown in FIG. 1, the plasmid c is the No derived from the plasmid b.
t I site, Xba I site, Bam HI site, Sm
It is a plasmid in which the a I and Pst I sites have been removed. Further, the plasmid c was completely digested with Bam HI and Kpn I and purified by ethanol precipitation. DNA obtained
The fragment was used in the next step.

[B] Construction of hph from plasmid pGL2
Preparation of Gene Gene The hph structural gene was prepared from pGL2 as shown in FIG. That is, pGL2 [Gritz, which is a plasmid in which the CaMV35S promoter, the hph structural gene and the CaMV terminator are integrated into pUC18.
Let al. , Gene, 25, 179-188 (1
983)] (2 μg) is completely digested with a restriction enzyme Bam HI, and about 1. containing hph by the method of (1) of [A] above.
Approximately 3.8 kb without 1 kb DNA fragment and hph
Fragments were collected.

[C] CaMV terminator from pGL2
As shown in FIG. 3, Bam was added to the 5 ′ side from pGL2.
Ca with HI site and Kpn I site on the 3'side
An MV terminator was prepared. (1) Preparation of DNA pGL2 was cleaved with Bam HI, and then self-ligation was performed according to the method of (2) of [A] above, and h
A DNA in which the ph structural gene portion was removed was obtained. (2) Transformation of E. coli JM109 With the obtained DNA, E. E. coli JM109 was transformed to obtain a recombinant plasmid d. (3) Cleavage of plasmid d and blunt- ended plasmid d (2 μg) were digested with restriction enzymes Xba I and Sph I
Was completely decomposed with, and the end was blunted according to the method shown in (7) of the above [A]. (4) Religation of DNA The obtained blunt-ended DNA was self-ligated according to the method of (2) of [A] above. (5) Transformation of E. coli JM109 E. coli JM109 was transformed with the DNA obtained in (4) above to obtain a recombinant plasmid e. (6) Preparation of CaMV terminator The plasmid e obtained in (5) above was digested with the restriction enzyme Bam H
Completely digested with I and Kpn I. The decomposed product was separated by agarose gel electrophoresis and the C of about 0.2 kb shown in FIG.
The DNA fragment containing the aMV terminator was recovered by the GeneClean method.

[D] Construction of plasmid f As shown in FIG. 4, the CaMV35S promoter,
A plasmid f containing the hph gene and the CaMV terminator was obtained. (1) Recombination of DNA [A] Degradation product of plasmid c obtained in (9) (about 3.8)
kb) 5 μl of an aqueous solution containing about 0.2 μg, and 5 μl of an aqueous solution containing about 0.1 μg of the about 1.1 kb DNA fragment containing the hph gene obtained in [B] were obtained in [C] (6). 5 μl of an aqueous solution containing about 0.03 μg of a fragment containing about 0.2 kb CaMV terminator was prepared and ligated using a DNA ligation kit. (2) Transformation of Escherichia coli JM109 With the DNA obtained in (1) above, E. E. coli JM109 was transformed to obtain the recombinant plasmid f. The direction of hph was confirmed as follows. Plasmid f to S
When completely digested with ac I and Eco RI, a section of about 1.1 kb was obtained. If hph was bound in the opposite direction to that shown in Figure 4, an approximately 1.5 kb fragment should be obtained. Similarly, when the plasmid f was completely digested with Sac I and Pst I, a fragment of about 1.2 kb was obtained. If h
If the ph was ligated in the opposite direction to that shown in Figure 4, a fragment of approximately 1.4 kb should be obtained.

[E] Construction of plasmid h As shown in FIG. 5, CaMV35S promoter,
A plasmid h containing the hph gene, CaMV terminator and multiple cloning site was obtained. (1) was completely degraded in removing plasmid f about 2μg restriction enzyme Xba I in XbaI site of plasmid f, was blunt-ended according to the method (7) of [A]. Self-ligation of the obtained blunt-ended DNA was performed according to the method (2) of [A]. (2) Transformation of E. coli JM109 E. coli JM109 was transformed with the DNA obtained in (1) above to obtain a recombinant plasmid g. (3) Attachment of multi-cloning site to plasmid g
Limit the given pBSIISK + about 2μg enzyme Kpn I and Bss
After complete digestion with HII and separation by agarose gel electrophoresis, a DNA fragment of about 0.14 kb containing a multicloning site was recovered by the Geneclean method. The plasmid g was completely digested with the restriction enzyme Kpn I, purified by the ethanol precipitation method, and then partially digested with Bss HII. The partial decomposition conditions were an enzyme concentration of 1 unit / ml, a temperature of 50 ° C. and a decomposition time of 5 minutes. The partially decomposed DNA was separated by agarose gel electrophoresis, and a fragment of about 5.1 kb was recovered by the Genecoon method. Thus obtained about 0.
A 14 kb DNA fragment and an about 5.1 kb fragment [A]
Rebinding was carried out in the same manner as in (2) above. E. coli J
M109 was transformed with the recombined DNA obtained, and about 5.
A 24 kb plasmid h was obtained.

[F] Construction of pKK524 As shown in FIG. 6, a target binary vector pKK524 was obtained. (1) Cleavage plasmid of pBI121 About 2 μg of pBI121 was completely digested with a restriction enzyme Sma I and then purified by an ethanol precipitation method.
It was partially digested with Sac II, separated by agarose gel electrophoresis, and a fragment of about 10 kb was obtained by the Geneclean method. (2) Cleavage of plasmid h About 2 μg of the plasmid h was completely digested with the restriction enzyme Not I, and the ends were blunted according to the method of (7) of [A]. The blunt-ended DNA was further partially digested with Sac II, and about 2.2 kb DNA fragment was obtained by agarose gel electrophoresis and the Geneclean method. The reaction conditions for partial decomposition with Sac II are: enzyme concentration 0.05 unit / ml,
The temperature was 37 ° C. and the reaction time was 10 minutes. (3) Religation of DNA The two DNA fragments obtained in (2) above were religated in the same manner as in (2) of [A]. (4) Transformation of E. coli HB101 E. E. coli HB101 competent cell (Takara Shuzo) [Hanahan, D. , J. Mol. Bio
l. , 166, 577 (1983)] was transformed with the recombined DNA obtained in (3) above to obtain a recombinant plasmid i of about 10.2 kb. The transformants were selected on an agar medium containing kanamycin. As shown in FIG.
Plasmid i is, Sac II site and S of pBI121
CaMV3 derived from plasmid h between the ma I site
It is a plasmid in which the 5S promoter, hph gene and CaMV terminator are integrated.

[0023] (5) was completely degraded cleavage of DNA pBI121 and (2 [mu] g) of plasmid i2μg with restriction enzymes Hin dIII and Xba I. (6) Religation of DNA The DNA obtained in (5) above was religated according to the method of (2) of [A]. (7) Transformation of E. coli HB101 With the recombined DNA obtained in (6) above, E. coli HB1
01 was transformed to obtain the desired plasmid pKK524. As shown in FIG. 6, pKK524 contained T-DNA.
RB, CaMV35S promoter, hph gene,
Contains CaMV terminator, CaMV35S promoter, GUS gene and NOS terminator, and CaMV terminator and CaMV35S
Multiple cloning sites ( K
pr I, Apa I, Xho I, Sal I, Cla I and
Hin dIII) are present.

Example 2 Construction of Binary Vector pKK525 The binary vector pKK525 shown in FIG. 7 in which the hph gene and the GUS gene were replaced in the pKK524 obtained in Example 1 was constructed as follows. (1) Cleavage of pKK524 2 μg of pKK524 was partially digested with Eco RI and subjected to agarose gel electrophoresis and Geneclean method to obtain 10.4.
The kb DNA was recovered. Next, the recovered DNA is Sa
The fragment was partially decomposed with cII and the end was blunted. A fragment of about 5.2 kb was recovered by electrophoresis and the Gene Clean method. (2) Re-ligation of DNA The DNA obtained in (1) was re-ligated according to the method of (2) of [A] of Example 1. (3) Transformation of E. coli HB101 With the recombined DNA obtained in (2), E. coli HB101
Was transformed. (4) E. coli transformed with pKK525. coli H
Selection of B101 Transformants colonized on an agar medium containing kanamycin were cultivated in 5 strains independently, and alkali S
The plasmid was recovered by the DS method. The recovered plasmid was cleaved with Sac II and analyzed by electrophoresis. As a result, two strains having pKK525 which gave a fragment of about 6.4 kb were obtained. This pKK525 had a structure as shown in FIG.

Example 3 Triparental mating method [Ditta, G. e
t al. , Proc. Natl. Acad. Sc
i. , USA, 77, 7347-7351 (198).
0)] according to A. tumefaci
It was introduced into ens as follows. Recipien
A as t. tumefaciens LBA4404
Alternatively, R1000, E. coli having the pKK524 obtained in Example 1 as Donor was retained. coli HB101
(PKK524), E. coli H
B101 (pRK2013) was used to perform overnight culture in the following liquid medium (3 ml). Bacterial medium A. tumefaciens LBA4404 or R1000 LB E. E. coli HB101 (pKK524) LB + Km (100 μg / ml) E. E. coli HB101 (pRK2013) LB + Km (100 μg / ml) Then, each culture was transferred to an Eppendorf tube and centrifuged at 10,000 rpm for 60 seconds, and the supernatant was discarded. Next, 1 ml of 10 mM MgSO ₄ was added and stirred with a vortex mixer. Repeat this operation twice,
A thick suspension was prepared. A suspension of each of the above three strains was added dropwise onto the LB plate in an amount of 30 μl, and left at 28 ° C. overnight. The mixture of bacteria was scraped off with a platinum loop, suspended in a 10 mM MgSO ₄ solution in an Eppendorf tube, and this was diluted to 10 ^-2 to 10 ^-8 to prepare a dilution series. Mi with 400 μg / ml kanamycin
Spread the above diluted solution on nA selective medium with a spreader, and
It was left still at 8 ° C.

After about 3 days, the A. tumefa
ciens LBA4404 or R1000 with pKK
524 introduced A. Only tumefaciens grew in MinA selective medium and formed colonies. The colonies were transferred to LB plates and isolated. In addition, single colonies on LB plates were isolated on MinA medium with kanamycin. From the obtained colony, plasmid DN
A is extracted and pK is obtained by Southern hybridization.
It was confirmed that K524 was introduced. A holding the pKK524. tumefaciens LBA4404
Alternatively, R1000 was designated as 4404 + 524 or 1000 + 524, respectively.

Example 4 A. Infection of Fumefaciens with plant chimney: 1000 + 524 single colonies obtained in Example 3 were added to 5 ml of LB containing 100 mg / ml of kanamycin.
The cells were inoculated and cultured at 28 ° C. for 24 hours, and the bacterial solution was transferred to a plastic petri dish. Brassica juncea cv Kikar developed after aseptic seeding
ashina) cotyledons were cut, the petiole part of the cut cotyledons was dipped in the bacterial solution, and the excess bacterial solution was sucked with Kimwipe. Cotyledon is placed on MS Gelrite (0.2%) medium, 2
The cells were cultured in a culture room (3,000 lux, 16 hours light period, 8 hours dark period, 25 ° C.) for 3 days. Then 0.5 g / cotyledon
The cells were transferred onto an MS gellite medium (selection medium) containing 1 Claforan and 0.5 mg / L hygromycin and cultured in a culture room. After 1 week, the cotyledons were transferred to a fresh selection medium and the culture was continued. Hairy roots were formed about 2 weeks after infection. This hairy root with 0.5 g / l Claforan, 20 m
The cells were transferred to MS gellite medium containing g / l hygromycin, and the culture was continued. Completely Agrobacterium
After confirming that m has been removed, X-
GUS assay was performed using Gluc. GUS Assay 1. Approximately 7 mm of root tip is cut and placed in a microtiter well. 2.50 μl of X-Gluc solution (1.02 mg / m
1:50 mM phosphate buffer: pH 7.0) is added. X-Gluc: 5-Bromo-4-chloro-3-indolyl-β-D-glucuronic acid 3. Wrap the microtiter well with wrap film,
Incubate at 37 ° C overnight. 4. Observe with a stereomicroscope. The cells expressing the GUS gene are stained blue. When GUS assay was carried out on 10 individuals (hairy roots) which were confirmed to have Agrobacterium completely removed, GUS activity could be detected in all the individuals. GUS activity is not found in the roots of normal individuals.

Example 5 A. Infection of tobacco with tumefaciens leaf disk method [Horsh, R. et al. B. et a
l. , Science, 223, 496-498 (19.
A by 84)]. tobacco was infected with tumefaciens . That is, 4404 + 52 obtained in Example 3
4 in 2 ml of LB liquid medium supplemented with Km (100 μg / ml)
It was cultured overnight at 8 ° C. This culture solution was placed in a sterile petri dish, and leaf pieces of 1 cm square of a sterile individual of tobacco were immersed in this petri dish. Then remove excess liquid with sterile filter paper and
It was placed on a solid medium and cultured for several days in the light at 25 ° C. Claforan (500 mg / l) for each leaf disc,
It was transferred to an MS solid medium supplemented with hygromycin (20 mg / l) and benzyladenine (1 mg / l) and cultured for several weeks in the light to eradicate the bacteria and induce adventitious buds. The adventitious buds that emerged were transferred to MS solid medium (without hormone addition) containing claforan and hygromycin, and allowed to stand in the light.
Roots are growing. After sterilization was completed, the cells were transferred to MS solid medium containing hygromycin and subjected to GUS assay. 6
When GUS assay was performed on redifferentiated individuals, GUS activity could be detected in all individuals. still,
GUS activity is not found in normal individuals.

[Brief description of drawings]

FIG. 1 shows the steps for constructing plasmid c.

FIG. 2 shows the preparation of the hph gene from pGL2.

FIG. 3 shows the preparation of CaMV terminator from pGL2.

FIG. 4 shows the steps for constructing plasmid f.

FIG. 5 shows the construction process of plasmid h.

FIG. 6 shows the construction steps of pKK524.

FIG. 7 shows the structure of pKK525.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display area C12N 15/56 // (C12N 1/21 C12R 1:01) (C12N 1/21 C12R 1:19)

Claims (4)

[Claims] 1. Agrobacterium tume
A border sequence of T-DNA on a Ti plasmid derived from Faciens , and a hygromycin B phosphotransferase gene and a β-glucuronidase gene between the border sequences, and the hygromycin B phosphotransferase gene and the β-glucuronidase gene A multi-cloning binary vector having a multi-cloning site between them. 2. A multi-cloning site, Kp
n I, Apa I, Xho I, Sal I, Cla I and H
multicloning binary vector of claim 1 having in dIII site. 3. The multicloning binary vector according to claim 1, which has a CaMV35S promoter upstream of the hygromycin B phosphotransferase gene and the β-glucuronidase gene, respectively. 4. The multicloning binary vector according to claim 1, which is pKK524 or pKK525.