JPH0258917B2 - - Google Patents

Abstract

(1) Acceptor tumour-inducing (Ti) plasmid comprises (a) the 2 border sequences of the T-region of the wild-type Ti plasmid; (b) non-oncogenic DNA segment derived from a cloning vector located between the 2 border sequences contg. a DNA sequence homologous with at least part of a DNA sequence in an intermediate cloning vector permitting a single cross-over event; and (c) segment of the wild-type Ti plasmid contg. DNA sequences essential for the transfer of Agrobacterium of the T-region of the T-region of wild-type Ti plasmids into plant cell genomes.

Description

[Detailed description of the invention]

The present invention relates to recombinant molecules, their preparation methods, and their use in plant cells.
Its method of introduction, and foreign DNA sequences into the genome
Relating to a plant cell or plant containing.
More specifically, the present invention provides for
Concerning the expressed DNA sequence. Set according to the present invention
Engineered DNA molecules can be used for plant growth and as nutrients.
improvement of its quality or useful metabolites (e.g.
production of lucaloids or steroid precursors)
production of useful amino acids and polypeptides, etc.
Its characteristic is that it has a sequence that encodes something
It is said that The terms used in this specification are explained below.
Ru. bom site Mob functional bodies interact specifically
DNA region that initiates autonomous DNA translocation Boundary sequence DNA sequence that includes the end of T-DNA Wide range of host replicons Transferable to a wide variety of host cells
can be transferred and retained
dna molecule Callus tissue A mass of unorganized, undifferentiated cells Cloning Asexual reproduction of one organism or
is a group of organisms or DNA sequences from a DNA sequence.
The operation process to obtain the
For example, by isolating a specific organism or part thereof,
Propagating subfractions as a homogeneous population
operation process Cloning medium A medium that can replicate in host cells.
Sumid, Phage DNA or other DNA sequences.
sequence, the DNA sequence of which is e.g.
essential functions of its DNA, such as the production of outer shell proteins.
physical function or promoter or binding
correct in place without incidental loss of the part.
one or more that can cut the sequence exactly
has a small number of endonuclease recognition sites.
and the cells into which it has been introduced (transformed).
markers useful for confirming the identity of
– (e.g. tetracycline resistance or
Characterized by having picillin resistance)
It will be done. Cloning medium is often a vector
Also called. Coding sequence Determine the polypeptide amino acid sequence
dna sequence Mutual integration body (cointegrate) 2 rings
Structure obtained by single crossover between DNA molecules Trans-complementarity Physically binds to another replicon
DNA molecules (replicons) that have not been
Is it necessary for other unbound replicons?
It is possible to supply the missing diffusible substances.
process Conjugation: Contact between cells
This allows the separation of bacteria from one type of bacteria to another type.
Transfer of DNA to bacteria Crossover When genetic material is exchanged between homologous DNA sequences
thing Deletion substitution One DNA sequence is removed and its replacement
be replaced with a different DNA sequence as differentiation The descendants of a cell acquire specialized structures and functions
and further maintain it DNA sequence or DNA segment Adjacent pen
Phosphoric acid diester between the 3′ and 5′ carbons of tosose
A group of nucleotides connected to each other by bonds
straight line array Double transfer Mutual integration (cointegrate) structure
The process by which DNA is broken down into two circular DNA molecules. this
Processes are used to exchange genetic information. child
one of the DNA circles thereby recombining
It has two regions homologous to the target DNA that can occur.
These two regions intersect with the target DNA.
sandwiching the non-homologous DNA sequence to be exchanged. too
The first crossover and the second crossover are in the same DNA region.
When this occurs in the region, the original DNA ring is generated.
When this second crossover occurs in the second homologous region,
Gene exchange occurs between the two toroids.
Become. Expression A polypeptide is produced by a structural gene.
The process of This is a combination of transcription and translation. Expression regulation (control) sequence present in structural genes
expression of their structural genes.
Nucleotide sequences that regulate and control F-type plasmid F factor (F stands for fertility)
It is a plasmid with the F factor
transfer a copy of the plasmid into a host that does not have
Plasmids that can be Gene Two parts, namely (1) for the gene product;
the coding sequence of the gene and (2) whether the gene is expressed.
The sequence within the promoter region that regulates
a DNA sequence consisting of a sequence Genome The entire DNA of a cell or virus. this is,
First, the structural genes that encode polypeptides
child, further operator, promoter, rebozo
binding and interacting sequences of the system (e.g.
Shine-Dalgarno sequence). Genotype All of the genetic information contained in an organism
hand Homologous (sexual) recombination Contains homologous sequences
Recombination between two regions on DNA Type I plasmid Incompatibility group different from F
A group of autonomously transposable plasmids Incompatibility No selective pressure
and two pieces of DNA cannot coexist in the same cell.
thing Insertion Insert one DNA sequence into the DNA sequence of another molecule.
to add to Leader sequence From less than 5′ to the first structural gene
The region on the mRNA up to the end. This includes
Important for initiating translation of coding sequences of synthetic genes
Contains parts. Meiosis Initially, 4n chromosomes are divided into two consecutive cycles.
Each of the four cells generated by division
A process in which 1n pieces are distributed in each. This passing
The process is important in sexual reproduction. mob (passive function body) In combination with tra function body
A series of products that only promote DNA transfer. mob
transfers the plasmid containing the bom site
can be encouraged. Mobilization Transfer to another cell
A DNA molecule that cannot interact with another DNA molecule
The process of metastasis with help Passive helper plasmid carried by other plasmids
Not spread to metastasize to another host cell
Plasmids capable of supplying sexual products Nonconjugative recombinant plasmid
By itself, it cannot be transferred from the original host cell to other hosts.
A DNA molecule that cannot be transferred to the main cell.
To metastasize, other DNA, e.g.
The functional body provided by the lasmid is not required.
Ru. Nucleotide sugar moiety (pentose), phosphate
It is composed of a nitrogen-containing heterocyclic base.
A monomeric unit of DNA or RNA. This base is
It is linked to the sugar moiety through a glycosidic bond (pen
carbon at position 1′ of tos), this base and sugar bond
The result is a nucleoside. nucleotide
The properties of are determined by this base. DNA 4
The bases are adenine (“A”), guanine
(“G”), cytosine (“C”) and thymine
(“T”). The four bases of RNA are A, G,
C and uracil (“U”). Phenomenal traits Interrelationship between developmental environment and genetic traits
Observable characteristics of the resulting individuals The plasmid itself is replicated in the host cell.
unstained, consisting of complete (intact) replicons.
Chromosomal double-stranded DNA sequence. This plasmid
When placed in a unicellular organism, the plasmid
DNA changes the properties of an organism, i.e.
transformed. For example, tetracycline resistance
Sex (Tc^R) plasmid carrying the gene for
Therefore, those who are originally sensitive to tetracycline
resistant cells are transformed into resistant cells. P
Transforming cells transformed by lasmids
It's called transmutation. Polypeptide Adjacent amino acids are α-a
Due to the peptide bond between the mino group and the carboxyl group,
linear amino acid chains connected to each other promoter region controls gene transcription
DNA sequence upstream from the start of the coding sequence Promoter sequence RNA polymerase binds to
This is the downstream sequence of the polymerase.
Facilitates faithful transcription of sequences. recombinant DNA molecule or hybrid
DNA from a sequence of at least two nucleotides
In nature, one of the sequences is usually the second sequence.
A miscellaneous array consisting of such an array that does not coexist with an array of
species dna sequence recombinant new DNA molecule or part of a DNA molecule
Creating new conjugates Homologous region Same sequence as in another region of DNA
DNA region with DNA sequence Replicon DNA replication initiation site and replication
Genes specifying the functions necessary to control
Self-replicating gene unit that has offspring Restriction fragments recognize specific target DNA sequences
produced by double-strand cleavage by an enzyme that
dna molecule RNA polymerase responsible for transcription of DNA into RNA
Enzymes that grow Selectable Marker Gene A certain DNA sequence
Therefore, when it is expressed in a cell, its
Easier to proliferate than cells that do not contain DNA sequences
A DNA sequence that confers an advantage to that cell. cell
When placed in a suitable selective growth medium, these two
Different types of cells can be distinguished. regular messenger
The selectable marker gene used is an antibiotic
This is a gene that encodes resistance. Single crossover Two circular DNA molecules are recombined to
Operation to form large inter-incorporated toroids
process Structural gene A gene that encodes a polypeptide
Child T-DNA Stably integrated into plant cell genome
Part of the Ti plasmid that has been found to be T-region DNA sequence transferred to plant cell genome
The part of the Ti plasmid that contains Ti plasmid Tumors (crown moths) on sensitive plants
Contains genetic information to induce
Large amounts present in Agrobacterium tumefaciens strains
Kii plasmid TL-DNA and TR-DNA Octopine Crow
Ngaru tumor cells contain two T-DNA sequences, viz.
Left T-DNA (TL-DNA) and Right T-DNA
(TR-DNA). TL-DNA is no
A sequence common to the T-DNA of palin tumor cells.
sequence, but not TR-DNA. tra (transfer function) encoded in a plasmid
diffusible products, and intercellular DNA
Refers to both sites of action used during metastasis.
For example, the energy needed to create a bridge between two cells.
product and the site where DNA transfer begins. Transcription The process by which mRNA is produced from structural genes;
Or, by forming base pairs,
Based on the genetic information contained in DNA
A process in which RNA strands with complementary base sequences are formed.
degree Transformation To the DNA complement of cells
Caused by the introduction of foreign DNA
genetic modification The process by which polypeptides are produced from translated mRNA.
or the genetic information present in the mRNA molecule.
information on specific amino acids in polypeptide synthesis.
Process of specifying acid order undifferentiated phenotypic trait, without any unique parts, a group of
The appearance of the cells in the tissue is uniform. Vector To transfer between different host cells
engineered DNA molecule Advances in recombinant DNA technology have made it possible to
A new outlook has opened up in electronic engineering. If one person is thin
If we could regenerate a complete living thing from a vacuole,
These techniques will extend to multicellular eukaryotes.
Dew. Cells from certain higher plants have excellent regenerative abilities.
and therefore for genetic engineering of higher organisms.
It becomes the material for katsuko. The main problem with plant genetic engineering is foreignness.
Utilization of the system for introducing DNA into plant genomes
be. Such systems include Gram-negative soil bacteria.
Tumors caused by Agrobacterium tumefaciens
There is an inducing (Ti) plasmid. This microorganism is
Crown gal for a wide range of damaged tissues in dicotyledonous plants.
a neoplastic trait called crowngall (crowngall)
It is known to be the cause of conversion.
These proliferating tumors are called opines.
Synthesize new metabolites specific to Ti. this
At the molecular level, transformation involves Ti plasmid
The true nature of the T-DNA (translocation) is known.
DNA) fragments are transferred to the plant cell genome.
This is caused by stable integration. Let me rephrase it
For example, crown gall tumors have chromosomal DNA that
in the Ti plasmid resulted in a swollen cell line.
A DNA segment called T-DNA that is homologous to the DNA sequence.
Contains components. In all cases,
This T-DNA is a continuous series of Ti plasmids.
It corresponds to DNA and is colinear with this.
be. This is therefore called the T-region. Ti plasmid is synthesized in crown gall cells
It is classified according to the type of opine. Crow
Nopaline [N-α-(1,3-dica)
synthesis of L-arginine]
The Agrobacterium strain that is induced is called the nopaline strain.
octopine [N-α-(N-1-carboxylic acid)
What synthesizes ethyl)-L-arginine]
It is called Octopine strain. These are the most commonly used
It is an Agrobacterium strain that can be eaten. Using T-DNA as a vector for plant genetic surgery
Attempts to use it were made in model experiments. This fruit
In our experiments, in vivo, Agrobacterium
Right border of T-DNA from Ti plasmid of T37 strain
14kb bacterial transposon near the boundary
(transposon) Tn7 was inserted. Then this
Agrobacteria carrying Ti plasmid
The resulting nopaline synthesis in the tumor is abolished.
Ta. In addition, Southern Blotting Hybrid
As a result of dazation, such insertions should not be performed.
As part of the T-DNA sequence, which is normal if
The presence of all Tn7 in the chromosomal DNA of this tumor
(Hernalsteens et al., Nature287
(1980), 654-656; Holsters et al., Mol.Gen.
Genet. 185 (1982), 283-289). In this way, 23kbT
- Even if a 14kb DNA fragment is introduced into the DNA,
The ability of 23kbT-DNA to transfer to the plant cell genome
No changes were observed. Nopaline strain, Agrobacterium T37 Ti Plus
The boundaries of the mido T-DNA have been investigated very precisely.
It is. This is the pole of all nopaline Ti plasmids.
The smallest part is only about 23kb. Furthermore, this T-
The boundaries of DNA are known: i.e. this T-
The nucleotide sequences that determine the boundaries of DNA
The same region of the nopaline Ti plasmid and
compared (Zambryski, Science 209 (1980),
1385−1391; Zambryski et al., J.Mol.Appl.Genet.1
(1982), 361−370). The boundary of this T-region is
Most relevant to the integration of T-DNA into the plant cell genome.
It seems that they are involved. A vector for transferring DNA to plant cells.
To use the Ti plasmid, transfer
Learn about the T-DNA sequence that determines the boundaries of DNA
This is basically necessary. Then, the extraneous
Insert DNA within this boundary to ensure plant cell germination.
Can be transferred to Nomu. Furthermore, this system
If you want to use transformed plant cells
However, its growth characteristics do not have the characteristics of a tumor,
Being normal is important. T-DNA
After metastasis, T-DNA is required to produce normal cells.
need to know what features are encrypted by itself
be. Therefore, which regions are associated with tumor phenotype?
In order to investigate whether the T-region of the Ti plasmid
A comprehensive genetic analysis of the area was conducted. T-DNA is responsible for crown gall phenotypic traits
The functional body is encoded. The gene is T
-localized to specific regions of DNA
(Leemans et al., EMBO J.1 (1982), 147-152;
Willmitzer et al., EMBO J.1 (1982), 139-146).
Generally governs the non-differentiated phenotype of tumor callus tissue
There are at least four genes that
Ru. Mutants of these genes (Myutan
g) is a transformation with a shoot-like or root-like appearance.
It is possible to form a replacement tissue. This latter formation
The fruit is expressed in normal plant tissue but not in tumor tissue.
If you want to transfer DNA to plants to
is particularly important. Recently, it can be regenerated into a completely normal plant
Ti plasmid mutants that induce transformed shoots
Found it. These plants are highly prolific.
and transmit the T-DNA specific sequence through meiosis.
even the T-
contained DNA-specific sequences (Otten et al., Mol.
Gen. Genet. 183 (1981), 209-213). However, this
The transformed plant tissue has in its chromosomal DNA,
The T-DNA region that controls tumor phenotype is removed.
Due to the occurrence of a large-scale loss,
It contained significantly smaller T-DNA. child
The defect may have occurred during the initial transformation, but the
It is unclear whether this occurred during the subsequent process that led to its formation.
It is. The Ti plasmid is large (200kb) and its Ti plasmid
Many genes present in various locations in Sumid
are involved in plant transformation. Therefore, T-
Specialized endonucleases in appropriate cases within the region
Contains recognition sites that allow T-DNA to be used in plant cell genomes
All the machines necessary for stable insertion and transfer to the
A small clone derived from a Ti plasmid with the ability to
It is not possible to assemble a vector.
Add the desired DNA fragment to the T-
for introduction into specific restriction enzyme cleavage sites in the region.
One known method is to use Escherichia coli
As in Agrobacterium, there are multiple
The desired restriction flag of T-DNA can be
cloning plasmid containing the
It is about assembling. This kind of cloning vector
vector was named "intermediate vector." Like this
The intermediate vector is encoded by the T-region.
was used to analyze the features
(Leemans et al., J. Mol. Appln. Genet. 1 (1981), 149
−164). The present invention provides expressionable genes in plant cell genomes.
It concerns how to introduce 1 of the present invention
One purpose is to introduce any desired gene(s).
Improved acceptor Ti plastic that can
The aim is to provide Sumido. desired to be introduced
The gene(s) has its acceptor Ti plasmid
A new medium with a region homologous to the corresponding region of the
contained within an intermediate cloning vector. this
This project also provides intermediate cloning vectors.
This is one of the purposes of Ming. Acceptor Ti plus of desired gene(s)
Introduction to Mido is retained in Agrobacterium
Acceptor Ti plasmid and intermediate clone
between two homologous DNA segments of a vector
This is achieved by a single transfer occurring at this middle
Cloning vector contains helper plasmid
From Escherichia coli it grows using
Induced by Agrobacterium. Helper like this
– plasmids and their functions for transfer are known.
(Fibbegan et al., Mol. Gen. Genet. 185 (1982),
344−351; Figurski et al., Proc. Natl. Acad. Sci.
USA76 (1979); Ditta et al., Proc. Natl. Acad. Sci.
USA77 (1980), 7347). High as a result of a single transfer in Agrobacterium
A hybrid Ti plasmid vector is obtained. child
Hybrid Ti plasmids such as
This is one of the targets. This hybrid held in Agrobacterium
plasmid vector (hereinafter referred to as vector composition)
) was used to directly infect plant cells, and then
Screen for expression of desired gene product
do. Plant cells are infected with the vector composition to form
This method of preparing transformed plant cells, their transformation
converted plant cells and plants developed from them
It is also an object of the present invention to provide. This technique
is the entire plant-transferable plasmid of Agrobacterium.
It can be applied to The attached drawings will be described in detail below. Figure 1 shows the T-region except for boundary arrays 1 and 2.
The access of the present invention obtained by removing the internal part of the area
One embodiment of the PterTi plasmid is shown. child
The border sequence of the T-region integrates into the plant cell genome.
It is essential to Area 3 between boundary arrays 1 and 2
is the DNA segment that will be transferred to the plant.
It is. This acceptor Ti plasmid is
Inter-cloning vectors are assembled by a single crossover.
An intermediate cloning vector that allows
homologous to at least a portion of the DNA sequence within the vector
Contains DNA segment 3 with DNA sequence.
Ru. Ti plasmid region 4 is in Agrobacterium
Therefore, the T-region is transferred to the plant cell genome.
Necessary functions are encrypted. This area is vir
- called a region. Figure 2 shows the access of Figure 1 by a single transfer.
Intermediate of the invention inserted into a PterTi plasmid
A cloning vector is shown. this vector
is an acceptor that allows the desired single transfer.
-At least DNA segment 3 of the Ti plasmid
Cloning that also has some homologous DNA sequences
Contains media DNA segment 3'. Furthermore, this
The intermediate cloning vector of
Gene or gene group 5 with motor sequence
Contains. In this assembly, generally
Plant genes can be used. it is,
Because it seems to be more easily expressed than other things.
It is. However, in principle, any desired genetic
Children can be inserted. This intermediate cronin
The vector contains selectable marker gene 6.
Good too. In plant cells, this gene
Contains a promoter sequence that allows expression of
There must be. Contains this marker gene
Plant cells that contain it are more likely to contain it than cells that do not contain it.
Must have a growth selection advantage.
This is because, in this way, this marker gene
Plant cells transformed with DNA containing
Can cells be distinguished from non-transformed cells?
It is et al. Figure 3 shows the intermediate cloning vector of Figure 2.
The acceptor Ti plasmid in Figure 1, similar to
The medium according to the present invention inserted by a single transfer into
Another embodiment of the inter-cloning vector is shown.
ing. This is a cloning medium DNA segment
3′, which allows for controlled expression of the desired gene.
exogenous promoter sequence 8 that enables
Optionally, marker gene 6 is included. Figure 4 shows the acceptor Ti plasmid in Figure 1.
and intermediate cloning of Figures 2 and 3.
According to the present invention by a single transfer from a vector
Demonstrates the preparation of hybrid Ti plasmid vectors.
FIG. Figure 5 shows the acceptor Ti plasmid from E.coli.
intermediate clones to Agrobacterium containing the code.
Processes related to gene transfer of vectors
This is a summary. The first stage is the intermediate cron
E. coli strain containing the vector (1) and then
Two helpers for conjugation with Agrobacterium
- Another E. coli strain containing the plasmid and
It is a junction of One helper plasmid is a plasmid.
Contains a DNA sequence (tra) that is important for smid transition.
The other helper plasmid contains important components for motility.
Contains columns (mob). By joining these
Helper plasmid is introduced into E. coli strain (1)
and the intermediate cloning vectors it contains
can be transferred to other bacterial strains.
Ru. tra and mob helper plasmids are intermediate
Antibiotic resistance of cloning vectors
Marker (Ab^r1), a marker different from Ab^r2oh
Call Ab^r3Each of them has Therefore, everything
on the selective medium to see if the plasmid is present.
can be monitored. In this way, the endowed stock (3)
can get. This vested stock (3) is transferred to the acceptor in Figure 1.
A. tumefaciens strain containing the Ti plasmid (4) and
Conjugate and Intermediate Cloning Vector Antibiotics
Select with resistance marker. intermediate cloning vector
Agrobacterium cannot replicate in Agrobacterium, so
Acceptor Ti plasmids and mutual integrants
can only be held if formed
Ru. This mutually integrated structure in Agrobacterium
(5) to transfer DNA into the plant cell genome.
Final hybrid Ti plasmid used
It is. Figure 6 shows a similar model to that shown in Figure 1.
Assembly of acceptor Ti plasmid (type A)
It shows the position. Here, Ti plasmid and
This replaces part of the Ti plasmid.
with another plasmid containing the DNA sequence.
Double transfer occurs. To be more specific, small
The other plasmid is T in cloning medium 3.
- Contains region boundary arrays 1, 2. double transfer
As a result, the T part inside the T area is removed and the
and then replaced with a cloning medium. obtained
The acceptor Ti plasmid A has border sequences 1,
Convert the DNA contained between 2 into the plant cell genome
It can be transferred. obtained, transformed
Ti plasmid A inhibits tumor growth.
Tumor-like because the controlling gene has been removed
Do not create a crown gar organization. Ti plasmid
Code A has homology to cloning vehicle 3.
Extremely universal for all intermediate cloning vectors
This is a typical acceptor Ti plasmid. This horn
Loning medium 3 can be a regular plasmid, e.g.
e.g. by pBR322 or its derivatives.
Can be replaced. Figure 7 shows the intermediate cloning vector
Schematic illustration of the assembly process in host cells
It is. Restriction endonuclease site R₁to
Surrounded desired gene 5 and restriction endonucleus
Azesite R₂selectable marker surrounded by
gene 6, enzyme R₁and R₂1 each for
Cloning medium containing one restriction site
Insert into 3'. Restriction enzyme R for all three molecules₁oh
and/or R₂and using DNA ligase
Intermediate cloning with ligation
Allow vectors to form. This cloning medium
3' is used as a selection marker in bacterial genetics
Antibiotic resistance (Ab^r1) is also encrypted.
It must contain another DNA sequence.
The desired gene 5 may be linked to its natural promoter or
Exogenous promoters outlined in Figures 2 and 3
- is under the control of Figure 8 shows the acceptor Ti Plus according to the present invention.
Another specific aspect of mido (type B) is assembled
This is a schematic diagram for setting it up. In this aspect, the boundary
Homologous to the Ti sequence immediately outside Examples 1 and 2,
clusters containing DNA sequences 9 and 10, respectively.
Double multiplication between loning medium and Ti plasmid
A change occurs. By this double transfer, the boundary array
The entire T-area T containing 1 and 2 is deleted.
and it is replaced by cloning medium 3.
Ru. Ti plasmid B contains border sequences 1 and 2.
contains the desired gene cloned between
Acceptor for intermediate cloning vectors
(See Figure 9). Figure 9 shows the acceptor Ti plasmid in Figure 8.
In the present invention inserted into the deB by a single transfer
Figure 3 illustrates an intermediate cloning vector. this
are border sequences 1 located at both ends of the desired gene 5
and 2. This also includes two plastic
To enable homologous recombination between smids,
Cloning medium in Scepter Ti plasmid B
Cloning that is at least partially homologous to a sequence
It also includes a media array 3'. Figure 10 shows the acceptor Ti plus in Figure 8.
Mid and its corresponding intermediate clone in Figure 9
The hybrid Ti plate of the present invention is
Schematic diagram showing assembly of lasmid vectors.
Ru. Intermediate cloning in Figure 9 by single crossover
The acceptor Ti plasmid shown in Figure 8 is
Introduced to DeB. Figures 11 to 20 show more specific examples of the present invention.
It is meant to show. Figure 11 shows the 5.2kb Hind fragment AcgB
insertion into pBR322 (Zambryski
et al., Science 209 (1980), 1385-1391). This hula
The component AcgB is located on the left and right sides of the nopaline Ti plasmid.
Contains border areas. This clone pAcgB is
“A-type” acceptor tape shown in Figure 6
lasmid, used for assembly of pGV3850
Ru. Contains the left and right border regions of the wild-type Ti plasmid.
This cloned restriction fragment containing
to obtain clones similar to clone pAcgB.
It is readily understood by those skilled in the art that
It should be possible. Figure 12 shows nopaline Ti plasmid pGV383.
9 T-region is shown. Hind limit end
The nuclease site is marked H. mutated
Hind fragment 19 is shown as (19').
Ru. Confers kanamycin or neomycin resistance
The acetylphosphotransferase gene is
apt, which exists in the blacked out area.
There is. The boundaries of the T region are indicated by arrows.
The nopaline synthase gene is nos.
expressed. Values are from Depicker et al. (plasmid, 3
(1980), 193-211).
It represents the size of the Ti plasmid pGV
3839 is based on Example 1 and the two listed therein.
It can be assembled according to the literature. Figure 13 shows acceptor Ti plasmid pGV
3850 assembly is shown. plasmid
pBR322-pAcgB (Figure 11) was linearized.
It is depicted in the form. The sequence of pBR322 is indicated by diagonal lines.
ampicillin resistance of pBR322.
The sex gene is Ap^RIt was shown in pGV shown in Figure 12
Part of the T-region of 3839 is depicted here.
: Hind involved in homologous recombination with pAcgB
Contains fragments 10 and 23 and the apt gene
It is. By double transfer, pGV3850 and
and another replica containing the lost apt gene
Con is assembled. FIG. 14 shows the intermediate cross section described in detail in Example 2.
lonning vector pGV700 assembly.
This is shown formally. restriction endonuclea
The following abbreviations were used to indicate zesite: B=
BamH, Bg=Bgl, E=EcoR, H=Hind
, S=Sal, Sm=Sma. antibiotic resistance
The following abbreviations were used to indicate: Ap = Ampicillary
Cm = chloramphenicol, Sm = strain
Ptomycin, Tc = tetracycline. TL−
The numbers at the bottom of the figure shown in DNA are for this region.
showing RNA transcripts (Willmitzer et al.
EMBOJ.1 (1982), 139-146). Figure 15 shows intermediate cloning vector pGV7
50 structures are shown. The assembly is an example
2. restriction endonuclease site
is its relative in number of kilobase pairs (kb)
It is shown in the target position. Although the Pst site is not shown
Km^R/Nm^R3 in the area, Cb^RThere is one in the gene
Ru. The left and right border areas are also shown. pGV750
Bgl/BamH site used for assembly of
and Hpa/Sma sites are shown,
This is not present in pGV750. shaded
The area is TL-DNA, the black area is Km^R/Nm^Rterritory
The white part is the adjacent Ti plasmid sequence.
and the line is in the cloning medium pBR325
They correspond to each other. Other abbreviations have the following meanings:
Has: Ocs = octopine synthase, Cm^R= Ku
Loramphenicol resistance, Cb^R= carbenicillin
(ampicillin analog) resistance, Km^R/Nm^R= kana
Mycin resistance/neomycin resistance. FIG. 16 shows the intermediate vector detailed in Example 3.
The assembly of target pGV745 is shown. pGV
745 is the "B type" acceptor shown in Figure 8.
target plasmid, used for construction of pGV2260.
used. Restriction endonuclease sites are:
Indicated by abbreviations: B=BamH, H=Hind,
R=EcoR. The ampicillin resistance gene is Ap^R
It was shown in The shaded area is the Octopin Ti plate.
DNA homologous to the left side of the T-DNA region of the lasmid
The white region is the T of the octopine Ti plasmid.
- DNA homologous to the right side of the DNA region is shown.
Starting material plasmid pGV0219 and
Physical location and description of pGV0120
found in De Vos et al., Plasmid6 (1981), 249-253.
It will be done. Figure 17 shows acceptor plasmid pGV22
60 assembly is shown. pGV2217 medium
deletion substitution for neomycin and kanamycin
Acetyl phosphotransferase confers resistance to
Contains the enzyme gene (denoted as apt).
This is shown in black. intermediate vector pGV7
45 (see FIG. 16) is drawn linearly.
This is the Hind support of pGV745 shown in Figure 16.
It was cleaved at the site. Sequence of pBR322
The ampicillin resistance gene is shown as the shaded area.
The transmission is Ap^RIt is shown. By double transfer,
pGV2260 was assembled and the apt gene was lost.
It will be done. Restriction endonuclease site stands for
Indicated by numbers: B=BamH, H=Hind, R=
EcoR. Figure 18 shows the nopaline synthase gene
(nos)
Construction of plasmid pLGV2381 to
It shows. 5′ and 3′ are transcription initiation and
means the end of transcription, ATG and TAA are the beginning of translation
and represents the codon used to terminate translation.
Ru. The thick line is the nos promoter region, the white part is
It shows the nos cryptographic area. Ap^Ris ampicillin
Resistance, Km^Rshows resistance to kanamycin. Figure 19 shows the complete octopine synthase
(ocs) Plasmid pAGV4 containing the coding sequence
0 assembly, and plasmid pLGV2381
(See Figure 18) The rear part of the middle nos promoter
It shows its insertion. The thick line is the promoter region
area, the white part indicates the OCS encryption area. So
Other symbols are the same as in FIG. Figure 20 shows the nopaline synthase (nos) gene
Nucleotide sequence surrounding the child promoter region
and octopine synthase gene coding region and fusion.
Example of nucleotide arrangement around the same region after combining
It shows. The fusion point is indicated by an asterisk (*). go
Some restriction endonuclease sites, i.e.
BamH, Hind, and Sac are also shown.
5' and 3' refer to the start and end of transcription.
ATG is the most truncated codon used for translation, and TAA is the most truncated codon used for translation.
It represents the termination codon used for translation. Shiranuki
The large arrow indicates the coding region of the nopaline gene, and the stripe
The arrow with a mark represents the octopine gene.
Ru. The present invention will be explained in detail below. Figure 1 shows the acceptor Ti plasmid easily
It is shown diagrammatically. This acceptor Ti Plus
The midpoint of the wild-type tumor-inducing Ti plasmid is
Contains boundary arrays 1, 2 or regions. this boundary
The field sequence connects the T-region of the Ti plasmid to plant cells.
Essential for integration into the genome. In other words, a
Any DNA sequence 3 or T-region can be combined with these
Incorporating into the plant cell genome that exists between the sequences
This bounds array is absolutely necessary. This acceptor Ti plasmid DNA sequence 3
is the intermediate clonin shown in Figures 2 and 3.
Compatible with at least part of the 3' DNA sequence of the vector.
Contains the same DNA segment. This homology
Intermediate cloning vector and acceptor
Ti plasmid undergoes single crossover (homologous recombination)
Therefore, it is necessary for mutual integration. Mutual integration
The frequency of homologous bodies is basically determined by the length of the homologous region.
That's it. To cause homologous recombination to occur at a high frequency,
Regions of 1 to 14 kb are usually used (Leemans et al.
J. Mol. Appl. Genet. 1 (1981), 149−164). The acceptor Ti plasmid further
T-region of Ti plasmid by Agrobacterium
Sequence 4 necessary for the movement of the region into the plant cell genome
Contains. Construction of such an acceptor Ti plasmid
and intermediate claws shown in Figures 2 and 3.
Regarding its mutual incorporation with nuking vectors,
This will be explained in detail below with reference to FIG. Figures 2 and 3 show the immediate
It is transcribed in plant cells under the control of promoters.
the desired prokaryotic or eukaryotic gene to be translated
intermediate cloning vector for cloning
is shown in a simplified diagram. These intermediate croni
The vector contains the acceptor Ti plasmid.
homologous to at least a portion of DNA segment 3
and thus the DNA sequence that allows for single crossover.
DNA segmentation from cloning media containing
3'. Additionally, this intermediate clone
A vector is a natural or foreign vector.
at least one desired promoter sequence containing
Contains genes 5 and 7. This promoter arrangement
column allows expression of inserted gene sequences
It is. Adjust the expression of the desired inserted gene(s).
In order to
It is also possible to use a modified promoter). Examples of various types of adjustments include:
(i) tissue-specific expression, i.e., leaves;
roots, stems, flowers, etc. (ii) expression level, i.e., expression intensity;
weak, (iii) inducible expression, i.e., temperature, light or added
expression by chemical factors. of the desired gene for intermediate cloning vectors
Examples include synthesis of products like amino acids and sugars
control to improve plant nutritional value and growth.
A DNA fragment or sequence containing genetic information
columns, protection against pathogenic substances from the outside, e.g.
Resistance to biological or stressful environmental factors
control the synthesis of products that impart
DNA fragments or sequences containing genetic information,
Basics of plants to be improved through genetic engineering
control the synthesis of products that inform processes
DNA fragments containing genetic information that
or an array. Figures 2 and 3 show selectable marker residues.
Intermediate cloning that may include gene 6
It represents a vector. selectable marker remains
Genes include, for example, antibiotics or toxic analogues.
Encodes substances (e.g. amino acid analogs)
genes, genes that compensate for defects in the recipient host cell, etc.
Can be mentioned. Figure 4 shows the hybrid Ti plasmid vector.
It shows the configuration involved in the assembly of the fifth part.
The figure shows the hybrid Ti plasmid vector.
involved in the isolation of Agrobacterium harboring
This shows the actual joining process. This process is
Intermediate cloning vector assembled in E.coli
This intermediate cloning vector can be
to the acceptor plasmid in Agrobacterium
Necessary for metastasis. Part of the T-region is replaced with an altered sequence
used to prepare improved Ti plasmids with
Known transfer techniques consist of a number of steps.
Usually, most DNA recombination operations are specially designed
cloning medium, e.g. pBR322
(Boliver, Gene2 (1977), 75-93)
Ru. However, this cloning vehicle itself
Unable to move to Agrobacterium. child
The problem is solved in known ways as follows:
ing: (a) Another widespread species that can also replicate in Agrobacterium
cloning medium for host hosts, e.g. mini-Sa
Plasmids (Leemans et al., Gene19 (1982), 361
−364) to replace the pBR cloning vehicle sequence.
Ru. This operation is performed in E.coli and an intermediate clone
A vector is obtained. (b) intermediate clonin containing the desired DNA;
E.coli strain carrying vectors,
Although it cannot be replicated in Agrobacterium, it
Transfer of body and other DNA to Agrobacterium
A helper plasmid that can mediate
Conjugation with another retained E. coli strain. (c) Containing E. coli and Ti plasmids obtained in step (b).
Agrobacterium conjugation. helperp
Lasmid is lost. (d) Intermediate cloning vectors are independent replicates.
Replicates and lives in Agrobacterium as a recon.
The contact obtained in step (c)
The merging involves the intermediate cloning vector and the Ti plastic.
Cells containing mutual integrants with Sumid, or
or intermediate cloning vectors and reciprocal integration.
Contains a Ti plasmid that did not cause
It is a mixture of different cells. only mutual integrants
Ti plasmid free for specific isolation
Repeat mating with another Agrobacterium strain.
have to jump. This transition is caused by Ti plastic
The functionality is encrypted by Sumid itself.
It is mediated by This second Agrobacterium
Transfer of the intermediate cloning vector to the strain
Only in the form of mutual integrants with Ti plasmid
be called. (e) Final modified Ti plastic with desired substitutions.
A second transfer was made to get Sumido.
(Leemans et al., J.Mol.Appl.Genet.1
(1981), 149−164). Just one more known method is to add step (d) above to
is not compatible with the intermediate cloning vector
Introducing another plasmid into Agrobacterium
This method is basically the same as the above method except for this. child
If the intermediate cluster remains an independent replicon,
All rowing vectors are lost, so
You can choose to include (single transfer)
(Matzke et al., J.Mol.Appl.Genet.1 (1981), 39−
49). Here, the present inventors have discovered that Agrobacterium activator
Intermediate cloning vector into Scepter Ti plasmid
A new, highly simplified
The present invention provides a method. Simply put, this
There are many commonly used cloning methods
Plasmids (e.g. pBR322) directly
It takes E. coli help to transfer to Agrobacterium.
found that par plasmids are useful.
Based on facts. These plasmids are
Since none of them can be replicated in Agrobacterium,
Can interintegrate with acceptor Ti plasmid
Only things will be retained. In addition, the main
The authors show that this mutual integration in Agrobacterium
direct vector assembly for infection of plant cells.
It is used as a product. In this way, the book
The inventors omitted steps (d) and (e) above. child
This allows us to create an improved hybrid Ti plasmid.
Reduces assembly time and increases possible assembly
This increases the flexibility of the plant cells, thus increasing the plant cell genome.
This is used as a vector to transfer DNA to
Possibility of using acceptor Ti plasmids
has increased significantly. That is, as shown schematically in Figure 5, the acceptor
-Intermediate cloning vector into Ti plasmid
The introduction is carried out in two steps. First, intermediate claw
This intermediate E. coli strain (1) carrying the
Transfer of cloning vector to Agrobacterium
Another E. coli strain with two plasmids that promote motility
Join with (2). of these helper plasmids
A typical and preferred example includes mob functionality.
pG28 containing daR64 drd11 and tra functions
(Finnegan et al., Mol.Gen.Genet.185
(1982), 344−351). intermediate cloning vector
bom site on a cloning medium (Warren et al.,
Nature 274 (1978), 259-261), but the other two
By the functional body encrypted by Sumido
Recognized and able to transfer. all plasmids
antibiotic-resistant antibiotics to detect their presence.
Preferably, it includes a marker. Then get
E. coli strain containing all three plasmids
The endowed strain (3) is used as an intermediate cloning vector.
The acceptor Ti plasmid has a region homologous to
Merge with Agrobacterium that holds the code.
Intermediate cloning vectors and acceptor tips
Whether a single transfer with Lasmid was made
is an intermediate cloning vector for antibiotic resistance.
This can be detected by selecting a marker. Figure 6 shows the acceptor Ti plasmid in Figure 1.
Schematic diagram of the DNA molecules used to assemble the
This is what is shown. Herein, this plasmid
with acceptor Ti plasmid (type A)
Other acceptor Ti plasmids (type
B) (see Figure 8). this
Assembly, Ti plasmid and cloning
Another one with border arrays 1 and 2 in medium 3
Double crossover with one plasmid
is necessary. As shown in the figure, the cloning medium
The field array 3 is the left boundary array 1 and the right boundary array 2.
It is between. Show the correct polarity of this DNA strand
Therefore, we can draw this on a ring. deer
and to indicate homologous regions used for double crossing over
The ring is shown broken open. I understand this
It is important as a way to help people, and is shown in Figure 8.
Assembly of acceptor Ti plasmid B in
It is also used in i.e. if the bounds array
1 and 2 are simply in the cloning vehicle sequence 3.
If inserted, T-region can be transferred by double transfer.
This boundary array 1 and 2, although the area has been deleted,
Ti plasmid without cloning medium sequence between
This means that you will get . As shown in Figure 6,
By double crossing, between boundary arrays 1 and 2
A circular DNA molecule with the original T-region is generated.
Ru. This is not a replicon, so it is destined to disappear.
It is in. Whether this double transfer occurred or not can be determined by e.g.
For example, antibiotics contained within the T-region of the Ti plasmid.
Select for missing material markers or
Regarding the antibiotic resistance marker in the cleaning medium array 3
Genetic selection
Can be done. Intermediate cloning in Figures 7, 2 and 3
FIG. 2 is a schematic diagram showing the assembly of a vector. limit
endonuclease site R₁or R₂And each one
Desired gene 5 and selectable marker surrounded by
Car gene 6 is enzyme R₁and R₂unique for
Cloning media sequences containing restriction sites
3', the digestion and ligation of all these molecules.
Inserted by Shion. The resulting recombination
DNA molecules transform E.coli host cells
The transformant is used as a cloning medium.
Antibiotic resistance marker (Ab^R1)
selected. FIG. 8 shows another aspect of the invention, namely the access
used to assemble PtaTi plasmid B.
FIG. In this case, the boundary
DNA sequences located just outside of field sequences 1 and 2
Cloning medium array 3 between columns 9 and 10
between the containing plasmid and the Ti plasmid.
Double transfer occurs. Homologous regions used for crossover
The smaller plasmid was cleaved to demonstrate
(Same as Figure 6). The product of double crossing is
Acceptor Ti plasmid B and original Ti plasmid
T-region and DNA sequence from Sumid2,10,
Another ring that disappears, containing 9 and 1
It is a shaped DNA molecule. Genetic selection is shown in Figure 6.
This can be done in the same manner as described for
Wear. Figure 9 shows the acceptor Ti plasmid of Figure 8.
Intermediate cloning vector used in combination with
FIG. Here, the desired gene
5 is contained in the cloning vehicle sequence 3'
is inserted between boundary arrays 1 and 2. Figure 10 shows the intermediate cross section of Figure 9 with a single transfer.
How does the structuring vector become an acceptor?
Schematically shows how it is inserted into Ti plasmid B.
ing. In this case, the intermediate cloning vector
Antibiotic resistance marker in cloning vehicle sequence 3'
- Select. Mutual integration between two plasmids
The resulting hybrid Ti plasmid
you can definitely find it. Thus the boundary array
with the desired genes contained within 1 and 2
A hybrid Ti plasmid is obtained. this
Hybrid plasmids assembled in the same way
For example, in the T-region, the hybrid shown in FIG.
Direct sequences such as sequences 3 and 3' in the Ti plasmid
Contains no repetitive sequences and therefore has no intramolecular combinations.
As a result of the change, the hybrid vector or
DNA introduced into the plant cell genome becomes unstable
The possibility of this happening can be avoided. From the experimental results conducted in the laboratory of the present inventors,
To assemble the intermediate vector in Figure 9, the boundary array
It turns out that it is not necessary to own both 1 and 2.
Katsutata (unpublished). However, it is difficult to extract the desired DNA from plants.
To incorporate it into the nom, at least the right border array 2
(See Figures 1 and 9)
This is a minute condition. Agrobacterium Ti plasmid, e.g. Nopa
Restriction enzymes for phosphorus or octopine Ti plasmids
Knowledge about the Donuclease Map (Depicker
et al., Plasmid3 (1980), 193-211; De Vos et al.
Plasmid6 (1981), 249-253) and T-DNA boundaries
for restriction fragments containing boundary sequences.
Findings (Zambryski et al., J.Mol.Appl.Genet.1
(1982), 361−370; De Beuckeleer et al., Mol.
Gen. Genet. 183 (1981), 283-288), those skilled in the art
Anyone can access by following the method of the present invention.
TarTi plasmid can be assembled.
In addition, common recombinant DNA techniques and basic
Requires the ability to perform genetic manipulation of bacteria
It's nothing more than that. The present invention is a hybrid Ti plus
be effective for assembling midvectors.
Acceptor Ti type described in this specification
Unique in that it specifically proposes lasmid
It is something. Furthermore, these acceptor Ti
Lasmids introduce genes into plant cell genomes
designed to form part of a method for
It is. The previously described acceptor Ti plasmid, intermediate clone
Rowning vector, hybrid Ti plasmid
further exemplify vectors and vector compositions;
Expression of foreign genes integrated into the plant cell genome
of providing transformed plant cells and plants exhibiting
exemplify the effectiveness of this vector composition in
Examples are given below. Example 1 Acceptor Ti plasmid pGV3850 (A
Type) assembly Starting strain and plasmid: Agrobacterium tumefaciens (wild type
Rifuapicin-resistant strain C58C1 from Agrobacterium
and chloramphenicol-erythromycin
Resistant strain C58C1) Ti plasmid = pGV3839 Plasmid in Figure 11 = pAcgB Ti plasmid pGV3839 is nopaline plus
Mido pTi C58tra^c(pGV3100; Holsters et al.
Assemble from Plasmid3 (1980), 212-230).
This is a deletion substitution mutant near the center of the T-region.
(mutant): i.e. Hind
Sma fragment 24 inside fragment 19
(Depicker et al., Plasmid3 (1980), 193-211)
Tn5 apt (acetyl phosphotransferase)
Hind fragment of pKC7 containing the gene
(substituted by Rao et al., Gene7 (1979, 79-82))
Ru. This gene is the aminoglycoside neomycin
and encodes resistance to kanamycin.
Ru. The restriction map of the T-region of pGV3839 is
Shown in Figure 2. Plasmid pAcgB contains only the T-DNA border
The AcgB insert in pBR322 containing
(See Figure 11). This boundary is the end of T-DNA.
These regions are defined as the ends of T-DNA
plays a role in stable integration into the plant cell genome.
vinegar. For more information on the origin and analysis of this clone
has been described (Zambryski et al., Science 209
(1980), 1385−1391). This clone is transformed
Re-dividing the T-DNA part from the converted tobacco DNA
obtained by separating. pAcgB is T-
Two lines arranged in a vertical line to include the left and right borders of DNA
It contains the junction of T-DNA copies of . Furthermore,
pAcgB has its genetic information at the right T-DNA border.
Nopalinecine because it is located close to
Contains the tase gene. This plasmid
pAcgB is a “type A” acceptor Ti plasmid
used for the construction of pGV3850. No.
Figure 6 shows an outline of the structures involved, and Figure 13 shows pGV.
DNA region involved in double crossing giving 3850
This shows the area more accurately. The above plasmid pAcgB is its pBR322
It has a ColE1-specific bom site in the
Ruper plasmid R64drd11 and pGJ28
Transferring E.coli to Agrobacterium using
I can do that. Plasmid R contained in E.coli
64drd11 and pG28 are conjugated to
introduced into an E. coli strain carrying pAcgB. Trance
The zygote is ampicillin resistant (pBR3 of pAcgB
22 sequences), streptomycin resistance (R6
4drd11), and kanamycin resistance (pGJ
28) as a colony. E.coli carrying all three plasmids
strain, rifampicin resistant and Ti plasmid
Agrobacterium strain C containing pGV3839
Join it to 58C1. Nopaline Ti plasmid
pBR32 to choose the first single transfer with
Utilizing ampicillin resistance of 2. ampicillin
can be stabilized in Agrobacterium
The only way is to use one of the homologous regions near the T-region boundary
crossed by homologous recombination with pGV3839.
It is to be. Second transfer in another homologous region
contains the apt gene (kanamycin resistance).
The central part of the T-region of pGV3839 containing
The pBR322 sequence of the clone pAcgB is substituted.
Therefore, the second recombinant is ampicillin resistant,
Sensitive to namycin. Isolate the second recombinant
In order to increase the probability of
(pAcgB::pGV3839)
Ampicin-resistant Agrobacterium, Ti plasmid
A second chloramphenicol that does not have a
Mating with erythromycin-resistant Agrobacterium strain
let In this way, one out of about 600 colonies
Ronnie's rate of ampicillin resistance, Kanamaysi
sensitive to chloramphenicol/eriomyelin
Obtaining syn-resistant Agrobacterium GV3850
I can do that. Of course, pGV3850 type acceptor Ti
Can be used to assemble plasmids
There are other Ti plasmids as well. T-inside the area
Ti with a selectable marker gene near the center
All plasmids can be used as receptors. Change
In, left border fragment-pBR322-right border
Left and right bounding fragments in the direction of the fragment
pBR322 so that the pBR sequence is located in the middle of
by inserting a T-region boundary fragment into
It is now possible to assemble a pAcgB-like plasmid.
can. For example, the left side of the nopaline Ti plasmid
and right boundary fragment are Hind flags, respectively.
10 and 23 (Depicker et al.
Plasmid3 (1980), 193−211). By a single transfer, pBR322 or its
The medium containing the desired gene is inserted into the derivative.
The inter-cloning vector is an improvement of pGV3850.
is introduced into the T-DNA region. only one needed
The thing is, is the intermediate cloning vector E.coli?
Means for selecting transfer from Agrobacterium to Agrobacterium
In order to be used as
Another resistance in addition to that present in pBR322
This means that it contains sex marker genes.
Ru. This resistance marker is contained within the pBR sequence.
(For example, Cm of pBR325^R,Also
is Km of pKC7^R), tested in plant cells
May be contained within DNA. Furthermore, acceptance
Ap in tar Ti plasmid pGV3850^RLegacy
The gene pBR322 is Km^RAnother resistance marker such as
- May be replaced with a gene. In this way, Ap^R
An intermediate cloning vector containing pBR322, which is
- Even this pGV3850 type acceptor
It can be directly injected into the TarTi plasmid. pGV3850 type acceptor Ti plus
Another advantage of Mido is that transformed plant cells
This means that the cells do not form tumors. pGV
The shortened T-DNA region of 3850 is still
Gene encoding nopaline synthase as
Contains offspring, so transform with pGV3850
The treated cells can tell whether nopaline is present or not.
Easy selection from non-transformed cells by analysis
It can be separated. Of course, acceptor Ti
Intermediate clone integrated into plasmid pGV3850
The training vector contained a marker gene.
However, it is also subjected to direct screening, that is, selection.
can be done. The above intermediate clone containing the pBR322 sequence
acceptor by a single transfer of the switching vector
In addition to insertion into the Ti plasmid, this acceptor
-Ti plasmid is a “shotgun” type fruit.
In our experiments, pBR322 or its derivatives
Also used as a receptor for cloned DNA banks
can be used. Everything in Agrobacterium
Hybrid plasmid vectors can be used in plant cells.
can be used to infect and then select the desired
Screening for possible gene(s) expression
be logged. For example, a deficiency of selected amino acids
By applying the whole bank to the plant cells in
A brief explanation of the genes encoding amino acid synthesis.
It can simply be selected. The acceptor Ti plasmid pGV3850 is
It has two distinctive phenotypic traits: (i)
(ii) if T-
Once the DNA has been transferred into the plant cell genome, Nopari
It has the ability to synthesize compounds. pGV3850
Various plant sets infected with Agrobacterium containing
Various experiments were conducted to investigate these characteristics of textiles.
I did it. (a) Using potato and carrot discs
test Accept potato and carrot sections
When inoculated with TarTi plasmid pGV3850,
A small amount of induration tissue is produced. Nopari to this organization
The test was positive for the presence of
I found out that This mutant has a small amount of hardness.
It is interesting that it can produce connective tissue.
However, it is important to note that these discs are
Contains both auxin and cytokinin
Obtained only when grown in a suitable medium. (b) Whole plant acceptor Ti plasmid pGV3
Inoculated at 850 Grown on sterile agar medium that does not contain hormones.
pGV3 in tobacco and petiunia seedlings
Inoculate 850. Small amount of tissue growth after several months
has only been observed (usually after 2 weeks)
“wild-type” tumors are detected). This organization
Although it does not grow in a medium that does not contain mons, oak
Sterile tissue in syn- and cytokinin-containing media
Further growth can be achieved in culture. this
The tissue was also found to be positive for nopaline. (c) Additionally, pGV3850 “transformed” cells
Because they are not neoplastic, these cells cannot metastasize.
DNA segments that have been added to its genome are still
Can be regenerated into a normal plant while retaining
Ru. The transformed cells were grown in normal regeneration medium (
(see Example 5), normal plants were obtained.
Good morning. pGV3850 acceptor plasmid and
In order to prove the usefulness of
Ivy. Octopine T-DNA tumor in pBR325
Intermediate cloning vector containing tumor functions
, holds pGV3850
Incorporated into Agrobacterium. With a single transfer
Hybrid in Agrobacterium obtained by
The Ti plasmid was inoculated into injured tobacco plants.
Ta. Tumor tissue appeared two weeks later. this thing
The tumor-inducing DNA was reintroduced into pGV3850.
and was properly expressed in transformed plant cells.
It shows. Example 2 Intermediate cloning vector pGV7000 and
Assembly of pGV750 The outline of this assembly is schematically shown in Figure 14.
Ta. TL- of Octopine Ti plasmid B6S3
This is the right part of the DNA, pGV0201 (De
Vos et al., Plasmid6 (1981), 249-253)
First, Hind fragment 1, which is
Principal vector pGV1122 (Leemans et al.
Gene19 (1982), 361-364) Hind site
Enter. Recombinant plasmid pGV0201 contains multiple
Copy vector pBR322 (Bolivar et al., Gene2
(1977), 95−113) into the unique Hind site of
Contains Hind fragment 1.
pGV0201 and pGV1122 DNA are
Prepared as described by Betlach et al.
(Fed.Proc.35(1976), 2037−2043). Final amount 20μ
Medium, 2 μg of pGV0201 DNA, 2 units of Hind
(All restriction enzymes were from Boehringer Mannheim.
(purchased) for 1 hour at 37°C.
Ta. The incubation buffer was described by O′Farrell et al.
(Mol.Gen.Genet179 (1980),
421−435). 2 μg of pGV1122 DNA under the same conditions.
was completely digested with Hind. T4 ligase (Boehringer
Mannheim) using 0.02 units and 0.1 μg of Hind quenching.
Label pGV0201 with Hind-digested pGV1122.
It was ligated. incubation
Buffers and conditions were according to the manufacturer's instructions.
(Brochure “T4 Ligase”, Boehringer
Mannheim, August 1980, #10.M.880.486). La
Igesion mixture competent E.coli
K514hsr^-hsm⁺cells (Colson et al., Genetics52
(1965), 1043-1050)
The method of Dagert and Ehrlich (Gene6 (1980), 23
-28). cells, streptoma
Icin (20μg/ml) and spectinomycin
LB medium (Miller,
Experiments in Molecular Genetics (1972),
Cold Spring Harbor Laboratory, New
York) was smeared. Contains recombinant plasmid
transformants with tetracycline resistance.
Inactivation of the encoding gene by insertion into it
(Leemans et al., Gene 19 (1982), 361-364).
and tetracycline sensitivity (10 μg/ml).
Cleaned (sorted). Streptomyces
resistant to spectinomycin and spectinomycin;
Physically identify clones sensitive to tracycline
was identified. Microscale DNA preparation
According to the method of Klein et al. (Plasmid3 (1980), 88-91)
It was carried out. In the Hind site of pGV1122
The orientation of Hind fragment 1 in Sal digestion
I have finally decided. Digest the recombinant plasmid
(Conditions of O'Farrell et al., Mol. Gen. Genet. 179 (1980),
421−435), when subjected to agarose gel electrophoresis
Two fragments were obtained. For α-orientation
0.77kb and 22.76kb fragments, β-orientation
has fragments of 10.33kb and 13.20kb.
Ivy. The α-oriented recombinant plasmid was
used for loning and named it pGV1168.
I attached it. The left part of TL-DNA (including the left border sequence)
Bgl-Sal fragment containing
was introduced into pGV1168 cleaved with Bgl-Sal.
Enter. This fragment is vector pBR32
From the T region of pTiB6S3 inserted into 2,
Recombinant protein containing BamH fragment 8
Plasmid pGV0153 (De Vos et al., Plasmid6
(1981), 249-253). pGV015
3 and pGV1168 DNA according to the method of Betlach et al.
(Eed.Proc.35 (1976), 2037-2043).
10 μg of pGV0153 DNA was added to 10 units of Bgl and
1 at 37°C in a final volume of 100 μ using 10 units of Sal.
The time was completely digested. Prepare the digestion mixture
Eve on 0.8% agarose gel, method of Allington et al.
(Anal. Biochim. 85 (1978), 188-196)
2.14kb Blg−Sa fragment from the gel
was collected. 2 units of 2 μg of pGV1168 DNA
of Bgl and 2 units of Sal.
Use 0.02 units of T4 DNA ligase in a final volume of 20μ.
0.02μg of BglSal fragment DNA 0.1μg
Bgl-Sal digested pGV1168 and ligate
I turned on. Compete this ligation mixture.
E.coliK514hsr^-hsm⁺cells (Dagert and
Erhlich, Gene6 (1980), 23-28).
Cells were treated with streptomycin (20 μg/ml) and
LB supplemented with spectinomycin (50μg/ml)
Media (Miller, Experiments in Molecular
Genetics (1972), Cold Spring Harbor
Loboratory, New York). Streptomycin and spectinomycin
from micro-scale resistant transformants.
DNA preparation (Klein et al., Plasmid3
(1980), 88-91). 2.1kb Bgl−
Sal fragment is Bgl-Sal digested pGV11
Bgl the recombinant plasmid inserted into 68
-Identified by Sal digestion. 2.14kb with this digestion
Two fragments of 1 and 21.82 kb were obtained.
Equivalent to these molecular weights (2.14kb and 21.82kb)
A plasmid with a digestion pattern of pGV1
Named 171 and used for further cloning.
Ta. 12.65kb fragment from pGV1171
are the left and right TL-DNA boundary sequences (De Beuckeleer
et al., in Proceedings Vth International
Conference on Plant Pathogenic Bacteria,
M. Ride′ (ed.) (1978), I.N.R.A., Angres, 115
−126) and genes that enable neoplastic growth.
(Leemans et al., EMBO J. (1982), 147-152)
Contains. plus this Hind fragment
inserted into mid pBR325 (Bolivar, Gene4
(1978), 121−136). pGV1171 and pBR3
25 was prepared by the method of Betlach et al. (Fed.
Proc. 35 (1976), 2037−2043). each
2 μg of DNA was added using 2 units of Hind at 37°C for 1 hour.
Completely digested (incubation buffer is
As described by O′Farrell et al. (Mol.Gen.
Genet. 179 (1980), 421-435)). 0.1μg Hind
Digested pGV1171 was linearized with Hind.
0.05 μg of pBR325 and 0.02 monotonic T4 DNA ligase
Ligation was performed using ligation
Competent E.coli K514hsr by mixture^-hsm⁺
Transformation was performed by the method of Dagert and Ehrlich.
Ivy (Gene6 (1980), 23-28). cells, carve
LB medium supplemented with Nicilin (100μg/ml)
(Miller, Experiments in Molecular Genetics
(1972), Cold Spring Harbor Laboratory,
New York). Carbenicillin resistance
Colonies encoded with tetracycline resistance
to inactivate the gene by inserting it into the gene.
Based on tetracycline (10 μg/ml) sensitivity
(Bolivar, Gene4 (1978),
121-136). Carbenicillin resistance, tetracyc
Prepare a phosphorus-sensitive colony from that colony.
Microscale DNA is generated by restriction enzyme digestion of the DNA.
method (Klein et al., Plasmid3 (1980), 88-91)
Physically characterized by That is, BamH
Digestion yields four DNA fragments.
: 0.98kb, 4.71kb, 5.98kb and
and 7.02 kb fragments were obtained, and in the case of β orientation
is 0.98kb, 4.71kb, 1.71kb and 11.20kb
can be obtained. The α orientation obtained in this way
The recombinant plasmid was named pGV700,
It was further used in subsequent experiments. pGV750 is a TL-
The tumor inside the region encodes essential functions.
Instead of the 3.49kb Bgl−Sma fragment,
2.81kb encoding kanamycin resistance
Inserting the BamH−Hpa fragment
is derived from pGV700. Kanamai
BamH-Hpa file with thin-resistance encryption
The fragment is λ::Tn5 (Berg et al., Proc. Natl.
Acad.Sci.USA72 (1975), 3628−3632)
It will be done. Preparation of λ::Tn5 was described by Miller
Experiments in Molecular Genetics
(1972), Cold Spring Harbor Laboratory,
New York). pGV700 DNA is from Betlach et al.
(Fed.Proc.35 (1976), 2037-
2043). Add 2 μg of pGV700 DNA to 2 units of Bgl.
and 2 units of Sma. λ::
2 μg of Tn5DNA with 2 units of BamH and 2 units
Completely digested with Hpa. 1μg BamH−
Hpa digested λ::Tn5 in a final volume of 10μ;
0.2 μg Bgl using 0.5 units of T4 DNA ligase
−Ligation with Sma-digested pGV700
(according to manufacturer's instructions). This la
Competent E.coli mixture
K514hsr^-hsm⁺cells (Dagert and Erhlich,
Gene6 (1980), 23-28). cells, cells
Rubenicillin (100μg/ml) and kanamycin
LB medium (Miller,
Experiments in Molecular Genetics (1972),
Cold Spring Harbor Laboratory, New
York). microscale techniques
(Klein et al., Plasmid 3 (1980) 88-91).
By restriction enzyme analysis of the prepared DNA, Cb^Rand
Km^RColonies were physically characterized. this DNA
When double digested with Bgl/BamH, 3.94kb,
Three fragments of 5.89kb and 8.09kb are
Digested with Hind: 2.68kb, 5.99kb and
Three fragments of 9.25kb are obtained. This erasure
The plasmid showing this pattern was named pGV750.
and is schematically shown in FIG. pGV700 and pGV750 are Octopin Ti plates.
Left and right border configuration of TL-DNA in lasmid pTiB6S3.
Two different intermediate cloning vectors containing
It is a tar. Furthermore, these two plasmids
The extent of deletion within the T-region is different. pGV
700 is octopine synthase (transcriptase)
3) and three other products, namely 4,6
a and 6b (for T-region products
(See Willmitzer et al., ENBOJ.1 (1982), 139-146)
Contains a reduced T-region with genetic information for
There is. of these three products (4, 6a and 6b)
The combination promotes shoot formation in transformed plants.
vinegar. pGV750 has a small T-region, i.e.
Contains only the octopine synthase gene.
Information for products 4, 6a, and 6b can be found in Kanama
Antibiotics encoding icin (neomycin) resistance
replaced by biological resistance marker genes.
It is worshiped. pGV700 and pGV750 are B type
Acceptor Ti plasmid (Figure 8 and below)
Intermediate clonins that can be used with (see Example 3)
This is an example of a vector. These vectors
except that they do not contain the desired gene.
For example, it is partially similar to that shown in Figure 9.
Ru. These vectors have within their improved T-regions
1 restriction endonuclease for cloning
site, so you can easily find the desired gene.
can be inserted into those vectors (first
(See Figures 4 and 15). Example 3 Acceptor Ti plasmid pGV2260 (Ta
Assembling type B) Starting strain and plasmid: Agrobacterium tumefaciens (wild type
Riampis derived from Agrobacterium
C58C1 and erythromycin-resistant strain C58C1 and erythromycin-chlora
Mufenicol resistant strain C58C1) Ti plasmid = pGV2217 Intermediate vector (Figure 16) = pGV745 Regarding the assembly of Ti plasmid pGV2217
are described in detail (Leemans et al.
FMBO J.1 (1982), 147−152). This is Otato
Deletion and substitution of the entire TL-region of the PinTi plasmid
Contains mutants: i.e. BamH fragment
8, 30b, 28, 17a and BamH
The 3.76kb BamH−EcoRI fragment on the left of fragment 2
fragment (De Vos et al., Plasmid6 (1981), 249
-253), apt (acetylphosphotrans) of Tn5
EcoR of pKC7, which contains the (Ferase) gene
−BamH fragment (Rao & Rogers,
Gene7 (1979), 79-82).
This gene contains aminoglycosides, neomycin and
and encodes resistance to kanamycin.
Ru. Assembly of intermediate vector pGV745
It is schematically shown in the figure. This is detailed below.
Ru. Recombinant plasmid pGV713 in α orientation
Contains Hind fragments 14, 18c, 22e and 33c.
Octopine Ti plasmid subclone pGV
0219 (De Vos et al., Plasmid6 (1981), 249-
253). pGV0219DNA
Completely digested with BamH and then self-ligated
Ligames under conditions favorable to Ligames
ligation mixture (of DNA in the ligation mixture)
final concentration <1 μg DNA/ml). Resistant to ampicillin
Transformants are selected and physically digested with restriction enzymes.
characterized. Thus present in pGV0219
no longer contains the 6.5kb BamH fragment
Isolate a clone that does not contain pGV713.
was named and used for subsequent cloning (hereinafter referred to as
(see description below). Contains BamH fragment 2
I'm here. pGV0120 (De Vos et al., Plasmid6
(1981), 249-253) recombinant plasmid pGV
738 was induced. EcoR pGV0120DNA
and digested in the same way as for pGV713.
Self-ligated. Make transformants resistant to ampicillin
The samples were then selected and analyzed by restriction enzyme digestion.
EcoR fragments 20, 21 and EcoR flag
Contains part of ment 19a and part of pBV322.
The entire 2.95kb EcoR fragment was removed.
The clone was named pGV738, and further
Used for cloning. This plasmid
Naturally, it is the right part of BamH fragment 2.
The 5.65kb EcoR−BamH fragment of
(De Vos et al., Plasmid6 (1981), 249
−253). pGV713 DNA was then transformed into Hind and BamH
Digest the digested product with prepared agarose.
Pour into gel. After electrophoresis, into pGV713
2.30kb Hind−BamH fragment included
The components were purified by electroelution (Allington et al., Anal.
Biochem. 85 (1975), 188-196). This fragmen
Completely digested with Hind and BamH
It was ligated with pGV738. After transformation,
Ampicillin-resistant colonies were isolated by restriction enzyme digestion.
physically characterized. For example, EcoR−
By BamH digestion, each 3.98kb (=vector
- part) and 7.95kb (= inserted part).
You should be able to get a ment. Having this property
The recombinant plasmid was named pGV745 and was
Assemble receptor Ti plasmid pGV2260
was used as an intermediate vector for Plasmid pGV745 is in pBR322 part
It has a ColE1-specific bom site, and Example 1
(acceptor Ti plasmid) as described in
Regarding assembly of pGV3850), Helperp
Using lasmid R64drd11 and pGJ28
It is possible to infect Agrobacterium from E.coli.
Wear. pGV745 is rifampicin resistant and Ti
Contains plasmid pGV2217
Agrobacterium strain C58C1 was inseminated. first power
Recombination is carried out in Example 1 (acceptor Ti plasmid
Assembling pGV3850)
Using the same method, ampicillin resistance of pBR322 was used.
I chose it. With the second transfer, pGV
The deletion substitution mutant present in 2217 is positive
Replaced by pBR322 sequence of midpGV745
be done. Mutual combination of pGV745 and pGV2217
The resulting ampicillin-resistant transformer
Direct selection of combinations by lack of kanamycin resistance
A second recombinant was obtained by doing this. like this
pGV2260 (ampicillin resistant, canada)
rifampicin containing (mycin-sensitive)
Agrobacterium strain C58C1 was obtained. This Ti plasmid pGV2260 is pGV70
0- or pGV750-type intermediate clones
acceptor plasmid (B type) for
It is used as a type. these are,
(i) ampicillin resistance gene, origin of replication and
A DNA fragment with the bom site of pBR322.
(ii) just outside the left and right border sequences of TL-DNA.
DNA sequences located in and intermediate cloning
Transfer of vector from E.coli to Agrobacterium
and its acceptor Ti plasmid pGV226
can be genetically screened for reciprocal integration into 0;
resistance markers already present in pBR322 and
contains another resistance marker
Composed of DNA fragments. For example, we found that pGV2260 and pGV
Has mutual integration between 700 and 700
Agrobacterium has a desired DNA sequence (T-DNA
contained between the boundaries) into the plant cell genome.
It has been proven that it can be transferred. This transformed
A plant cell can contain pG700 if
genetic information (4, 6a, 6b; Willmitzer et al.
EMBO J.1 (1982), 139-146)
is a tumor that produces the expected phenotype, i.e., sprouts.
shows. In this way, the present inventors have discovered that type B
The receptor Ti plasmid is shown in Figure 9 and further updated.
Intermediate cloning of the type described in Example 2
When used as a mutual integrant with vectors,
Being able to transfer DNA to plant cells
proved. Example 4 Intermediate containing the gene to be expressed in plants
Cloning vector assembly Until the present invention is completed, the Ti plasmid T
− All the data are placed at more or less random locations within the region.
Even if a gene is inserted, the foreign sequence will remain in the plant genome.
It is not expressed after metastasizing to Nome.
Ta. According to the method of the present invention, the desired exogenous gene
(group) of coding regions to function in plant cells.
are coupled to known transcription start and stop signals.
can be done. The utility of this method is that nopaline
The DNA sequence encoding the synthase gene is
Illustrated by experiments involving the present invention. child
The complete sequence of the gene and the exact start and end of transcription.
is known (Dep-icker et al. J. Mol. Appl.
Genet.1 (1982), 561-574). According to the present invention, outpatient
The protein coding region of the sex gene is the nos promoter
It can be inserted next to foreign gene sequence
As an example of a column, the dark side of the octopine synthase gene
No. area (De Greve, J.Mol.Appl.Genet.1 (1982),
499−512) inserted adjacent to the nos promoter
do. This construct is the acceptor Ti plasmid
used to infect plants.
Ru. Is octopine present in the generated tumor tissue?
After analyzing the results, it was found to be positive.
Ta. Contains a chimeranopaline promoter
Inter-cloning vector assembly: octopine
The synthase structural genes are shown in Figures 18 to 20.
vinegar. Briefly, a restriction block containing the nos gene
In vitro processing of fragment Hind-23
removes most of the nos cipher sequence, while limiting the
adjacent to the endonuclease site BamH
The nos promoter is retained (Figure 18).
10 μg of pGV0422 (containing the complete nos gene)
pBR322 with Hind-23 fragment
Derivatives; Depicker et al., Plasmid (1980), 193−
211) was digested with Sau 3A and contained the nos promoter.
Preparative 5% of the 350bp fragment
Separate on polyacrylamide gel. this promo
ter fragment with a 5′-terminal phosphorylated ester group.
Pre-bacterial alkaline phosphatase to remove
Bg1-cleaved pKC7 treated with enzyme (BAP)
(Rao et al., Gene7 (1979), 79-82).
20 μg of the obtained plasmid (pLGV13) was added to Bgl.
Digest with 400μ 12mM MgCl₂, 12mM
CaCl₂, 0.6M NaCl, 1mM EDTA and 20mM
Bal31 exon at 30°C in Tris-HCl (pH 8.0)
Crease (Biolabs, New England) 7 units
for 4 to 10 minutes. During this time, about 20~
50bp of DNA is removed. This Bal31−processing amount
After digesting the offspring with BamH, DNA polymerase
Klenow fragment and four deoxynucleotides
together with ocide triphosphate (10mM each)
incubate to fill its ends. filling
Ligate the BamH end with the Bal31 removed end.
- Have a playback BamH site that can be obtained from
Select ivy plasmids. some candidates
Size of BamH-Sac fragment in 6% urine
Estimated size in plain polyacrylamide gel
Candidate nuclei in the range of 200 to 280 nucleotides
Determine the leotide sequence. I had a promoter
Contains a 203bp Sac-BamH fragment
The clone pLGV81 was cloned into the nos of pGV0422.
Replacement with Sac-BamH fragment in gene
Use this final promoter vector to:
- is called pLGV2381. All recombinant
Lasmids were selected by transformation of E. coli strain HB101
do. Contains the nos promoter processed in this way
Digest the plasmid vector with BamH,
ocs cipher contained in BamH fragment
Insert an array into this site. This ocs cipher array
were also treated in vitro, as shown in Figure 19.
at the BamH restriction endonuclease site.
Make yourself surrounded. Octopine Ti plasmid
10 μg of BamH fragment 17a of B6S3 (De
Vos et al., Plasmid6 (1981), 249-253) with BamH
and Sma and contains the ocs − cipher sequence
The fragments were separated from a 1% agarose gel and
Large BamH-Pvu fragment of pBR322
The resulting plasmid, pAGV8
28 (20μg) was digested with BamH and shown in Figure 18.
treated with exonuclease Bal31 as indicated,
Then digested with Hind, filled in the ends, and self-ligated.
Match. The size of the Bal31-removed body is 6% polyac
Estimate in Rylamide gel. Some candidates
Determine the nucleotide sequence and determine the 5′-untranslated leader sequence.
Select the candidate with only 7bp remaining in the column and do the following
Subject to manipulation (pOCS△). ocs array to BamH size
To surround the Cla−Rsa fragment with
Filled with pLC236 (Remaut et al., Gene15
(1981), 81-93) subcloned to the Bal site of
do. Obtained plasmid Digest pAG40 with BamH and have ocs sequence
Preparate the fragments in 1% agarose.
Separated by electroelution from the gel and pre-filled with BamH
Digested with BAP (bacterial alkaline phosphatase)
ligated to pLGV2381 treated with ocs distribution
By inserting the column into pLGV2381, both orientations
are obtained (pNO-1 and pNO-2). nos: nucleo indicating the exact junction of the ocs fusion
The sequence is shown in Figure 20. Furthermore, it contains a processed nos promoter.
The plasmid vector contains the enzyme dihydrofluoride.
Plasmid R6 encoding reductase
Used to insert DNA from 7. Jihee
Contains the dolphiolate reductase gene
The coding sequence is contained in BamH (O′Hare
et al., Proc. Natl. Acad. Sci. USA78 (1981), 1527−
1531), therefore, as mentioned above, the promoter region
nos pro containing the BamH site adjacent to
Easily inserted into motor vectors. this inheritance
The child is expressed and treated with the antibiotic methotrexate.
selectable markers that confer resistance to
One example of a gene (2nd, 3rd, 4th, 5th and
(See Figures 7 and 7). This intermediate cloning vector
Containing the wild-type nopaline acceptor Ti plasmid.
When indoctrinated with Agrobacterium, a single
Crossover occurs and the hybrid Ti plasmid vector
can be obtained. This vector composition can be applied to plants.
Use for infection. The obtained tumor tissue was 0.5μg/
continued growth in the presence of ml of methotrexate.
I found out what I could get. Dihi after ocs and above nos promoter
Contains the dolphiolate reductase coding region
Assemble an intermediate cloning vector to
Mutual integration with Agrobacterium Ti plasmid
After that, it can be transferred and expressed in transformed plant cells.
According to the method of the present invention, foreign genes can be introduced into plants.
This means that it can be metastasized to cells and expressed.
is proven. Example 5 Plant cells containing the desired inserted gene in their chromosomes
and plant separation The present inventors decided to use one of the following three methods.
Using the non-tumor acceptor Ti plasmid inducer
transformed with a conductor (e.g. pGV3850)
Plant cells and whole plants were obtained. (1) Inoculation of whole plants in vivo, then re-inoculation of shoots.
Cultivation in vitro on viable media; (2) Other methods that promote the production of new shoots directly at the damaged site
in vivo in the presence of Agrobacteria strains.
cross-infection of whole plants, (3) Single plant cell protoplasts in vitro
Symbiotic culture of. These methods are detailed below. The first method involves the production of crown gall tissue.
wild-type Agrobacterium strain of whole plant tissue.
improved methods commonly used to obtain infected organisms.
It is something that pGV3850 does not form tumors.
Since it is an Agrobacterium derivative, the infection site
No tumor growth was observed in the patients. but infected
When the tissue is removed and grown in tissue culture, the shape
Transformed tissue can be easily obtained. first time
After the initial culture period (simply to increase the amount of tissue),
The damaged tissue is grown under conditions that allow sprout formation.
let Non-transformed cells and pGV3850-form
Both transformed cells generate sprouts. transformation new
Buds are determined by a simple analysis for the presence of nopaline.
can be easily distinguished. The inventors followed the following protocol:
Decapitated Nicotiana tabacum Wisconsin38
pGV3850-transformed tobacco seedlings
Obtained callus and shoots (all operations were performed on the lamina).
- performed under sterile conditions in a flow hood). (1) In a small bottle (diameter 10cm, height 10cm),
Solid Murashige containing 0.8% agar &
Skoog (MS) medium (Murashige and Skoog,
Physiol.Plant.15 (1962), 473-497).
Use tobacco seedlings that are 6 weeks old. (2) Cut out the youngest parietal lobe with a scalpel.
dispose of (3) Selective conditions (e.g. Ti plasmid pGV38
50 containing rifampicin resistance, ampicin
For syrin-resistant Agrobacterium strains,
100 μg/ml rifampicin and 100 μg/ml
Use YEB medium containing rubenicillin.
YEB medium = 5g/Bacto beef extract,
1g/Bacto yeast extract, 5g/peptone,
5g/sucrose, 2×10^-3M MgSO_Four,
Fresh flats grown in pH 7.2, 15g/agar)
Agrobacterium from plate culture, spatula
Or inoculate the damaged surface with a toothpick. each
At least 8 pGV3850 assemblies
inoculate seedlings. (4) Incubate for 2 cycles. At the vaccination site
There should be little or no reaction.
However, sometimes very small calli are observed.
be done. (5) Cut a thin section with a thickness of 1 mm or less from the damaged surface.
take. Treat the damaged surface with auxin and cytotoxic
Nin (1mg/NAA, 0.2mg/BAP) and
Linsmaier & with 1% sucrose added
Skoog (LS) agar (Linsmaier and
Skoog, Physiol.Plant.18 (1965), 100−127)
Culture on a plate containing (6) After about 6 weeks, part of the callus is removed and nopa
large enough to test for the presence of phosphorus
(at least about 5mm in diameter). all
Not all damaged callus produces nopaline.
stomach. Approximately 1 in 4 plants has nopaline positive damage
Create a callus. (7) Regenerate nopaline-positive callus using agar containing medium.
Transfer to plate: LS medium above + 1% sucrose
and 1 mg/BAP cytokinin (8) New shoots of good size (height
1cm) will appear. grow further and form roots
In order to make this sprout, hormone-free LS
Medium + new containing 1% sucrose
Transfer to an agar plate. (9) A portion of it to test for the presence of nopaline.
(1-2 small leaves)
Let the sprouts grow for 1 to 2 weeks. (10) Nopaline-positive sprouts were grown in the same MS medium as in (1).
A slightly larger container (same as above, 10 cm long)
) and grow further. (Note) All plant culture media for stained tissue
For Agrobacterium containing pGV3850,
The antibiotic cefota is used as a selective poison against
cefotaxime, Claforan^R, Hekis
g) Contains 500μg/ml. this drug
All Agrobacterium (carbenicillin)
(including resistant species). In our laboratory, transformed sprouts were
Alternative methods have been developed to obtain tissue for release. this method
is a type of mutant Ti of Agrobacterium
Plasmid strain stimulates budding crown gall tumors
Based on the observation that
uttered. Regarding this kind of sprout-inducing (shi)
The mutation is T in the Ti plasmid of A. tumefaciens.
- specific regions of DNA (transferred DNA segments)
located (Leemans et al., EMBO J.1 (1982),
147-152; Joos et al., Cell32 (1983), 1057-1067).
The induced sprouts are completely normal non-transformed cells.
Often configured. Therefore, the inventors
are two different Agro-bacteria, i.e. one
Octopine Ti plasmid release (shooter) Miyu
-The one with tanto, the other one is pGV385
Plants were inoculated with a mixture of 0 and 0. child
The octopine release mechanism is
John induced roots transformed with pGV3850.
Give them a good chance to wake up. Ti plastic
Sumid pGV3850 and octopin sprout induction
Contains Ti plasmid at a ratio of 5:1
Plants were inoculated with Agrobacterium. Do this
pGV3850-transformed shoots were obtained by.
This sprout can be analyzed for the presence of nopaline.
Therefore, it can be easily sorted. This person
The method does not require sophisticated tissue culture methods. Nopali
For further growth of the positive shoots, the length control hormone was added.
Transfer to a medium containing simple salts and sucrose along with rumon.
vinegar. Easily propagated after the shoots reach sufficient size
can be transferred to soil for This co-infection method
can easily transform plant types that are difficult to tissue culture.
It is particularly useful for converting Therefore, any range
Agronomically or economically important plants in the enclosure, e.g.
leguminous plants, medicinal plants and ornamental plants.
It could be treated with Agrobacterium. The third method is Nicotiana tabacum protop.
Isolation of last and hormone-independent T-
The selection of DNA-transformed cell clones
Rotoplast-derived cells and tumorigenicity
What can be done after co-cultivation with Agrobacterium strains
It is. Other dominant selection markers, e.g.
Antibiotics engineered to be expressed in biological cells
If resistance genes are used (see Example 3), the trait
Similar methods can be used to select transformed cells.
I can do that. However, in this case, each case
It is necessary to find the optimal conditions for selection regarding
(Concentration of selection agent, time between transformation and selection,
Protoplast-derived cells or cells in selective media
colony concentration, etc.). Selection of transformed cells is possible.
For example, pGV3850 or pGV2
217 (Leemans et al., EMBO J.1 (1982), 147−
Using a non-toxic T-DNA mutant such as (152)
If selection is not possible because of
After transformation, cells are treated with auxin- and cytokini-
-containing medium (e.g. 2 mg/NAA (alpha
phthaleneacetic acid) and 0.3mg/kinetin.
Murashige and Skoog medium (Murashige and Skoog medium)
and Skoog, Physiol.Plant15 (1962), 473−
497)), and the transformed colonies were cultured with their opiates.
It can be identified by its opine content. child
Agropine and macerate
Electrophoretic analysis of mannopine synthesis
Leemans et al., J. Mol. Appl. Genet. 1
(1981), 149-164), approximately 660 colonies were
obtained after infection with pGV2217, TR-encrypted
Pine Manopin (N²(1-manityl)-gluta
Nicotiana tabacum SR1 cells synthesize
It turned out to be a line. This cell line
Regenerate callus sections with medium (with only plant growth regulator)
BAP (6-benzylaminopurine) (1 mg/
) on Murashige and Skoog medium) containing
When cultivated, numerous new shoots formed. 20 analyzed
all of the shoots still synthesize manopine
I was able to do that. Murashige without hormones
and After transferring to Skoog medium, these sprouts
Still containing manopin and morphologically normal
It grew into a tobacco plant. Protoplastic described below for N.tabacum
The isolation and transformation method for N. plumbagini
-Can also be used for folia. 2 Experimental method 2.1 Sprout culture conditions Under sterile conditions in the culture room (16 hours a day, 1500
Lutx white fluorescence (“ACEC LF58W/
24300〓 Economy”), 24℃, relative humidity 70
%), hormone-free in a 250ml glass bottle.
Contains Murashige and Skoog medium
(Murashige and Skoog, Physiol.Plant15
(1962), 473−497) on Nicotiana
Maintain tabacum sprout culture. 5 week old new
Bud cultures are used for protoplast isolation. 2.2 Isolation of protoplasts In protoplast isolation and culture
All steps are performed using aseptic techniques. Mixed enzyme method
Separate protoplasts by length 2cm
Process all leaves except for the very young leaves below.
It can be used to separate toplasts.
Using a sharp scalpel, cut the leaves into pieces about 2-3 mm wide.
Cut into long strips. 2-3g of this leaf material
in 50 ml of enzyme mixture in the dark at 24°C for 18
Incubate for an hour. This enzyme mixture is
0.5% cellulase in K3 medium without Mon.
Onozuka R-10 and 0.2% Macerozyme
OnozukaR-10 (Nagy and
and Maliga, Z. Pflanzenphysiol.78 (1976),
453−455). This mixture produces a 0.22μm pore membrane
over-sterilization and a significant decrease in activity.
Store for at least 6 months at -20°C without
can do. 2.3 Protoplast culture Release protoplasts after 18 hours of culture
Gently stir the mixture to next
Pass this mixture through a 50 μm sieve.
and transfer the liquid to a 10 ml centrifuge tube. vibrating bucket
Place in a rotor and centrifuge at 60-80g for 6 minutes.
The protoplasts then turn into dark green floating bands.
Form a do (band). Lower layer of protoplast
peristaltic pump to remove liquid and pelleted debris.
Remove using a capillary tube connected to the proto
Collect the plasts in one centrifuge tube and incubate with culture medium.
Wash twice. This culture medium contains NAA (0.1
mg/) and kinetin (0.2 mg/)
K3 medium containing as a growth regulator
(Nagy and Maliga, Z.Pflanzenphysiol.78
(1976), 453−455). This medium was adjusted to pH 5.6.
Section and sterilize through a 0.22 μm membrane.
After the second wash, Thoma hemocytometer
(“Assistant”, obtained from West Germany)
Measure protoplasts and final density 10^FiveProfessional
Suspend toplasts/ml in culture medium.
Ru. High quality petri dish for tissue culture with a diameter of 9cm
Culture protoplasts in a 10 ml volume.
Parafilm this Petri dish^RSeal at 24℃
Then, in the dark and then in the faint light (500 to 1000 ruts)
culture for 24 hours. 2.4 Transformation by symbiotic culture Infect protoplast cultures 5 days after isolation
let Agrobacterium in liquid LB medium
(Miller, Experiments in Molecular
Genetics (1972), Cold Spring Harbor
Laboratory, New York) for 18 hours.
After, 2×10⁹K3 medium to a density of cells/ml.
Resuspend in nutrient medium. Inoculate 50μ of this suspension.
In addition to protoplast cultures, Parafilm^R
After sealing with
Cultivate under conditions. After 48 hours, transfer the culture to 10 ml.
Transfer to a centrifuge tube and place in a vibrating bucket rotor.
Centrifuge for 6 minutes at 60-80g. floating band
and pellets, and antibiotics (carbenzol)
Syrin 1000μg/ml or cefotaxime
K3 medium (Nagy and
and Maliga, Z. Pflanzen physiol.78 (1976),
453−455) Resuspend in 10 ml. After 2 weeks of culture, protoplast-derived microorganisms
Centrifuge the crocallus and collect the same concentration of product as above.
Contains long-term regulators and antibiotics
As for the base, K3 culture containing 0.3M instead of 0.4M
Earth (Nagy and Maliga, Z.
Pflanzenphysiol.78 (1976), 453-455).
suspend The cell density of this medium is approximately 25 x 10³
Adjust to micro callus/ml. under the same conditions
After culturing for another two weeks, the callus was cultured as described above.
Contains the same concentration of antibiotics, but with a lower concentration
Euclose (0.2M) and growth regulators
(NAA0.01mg/, kinetin 0.02mg/)
Transfer to K3 medium containing Culture for another 2-3 weeks
Afterwards, the presumed transformants are
green dense appearance, and better growth rate?
It can be recognized from these colonies
and lower concentrations of antibiotics (carbenicillin).
500μg/ml or cefotaxime 250μg/ml)
0.6% agar solid medium containing but without hormones
(Linsmaier and Skoog, Physiol.Plant.18
(1965), 100-127). Transformants and
When the defined object reaches a diameter of about 3-4 mm,
Those growing in hormone-free media
Opine tests can be administered. each colony
octopine and nopaline.
(Aerts et al., Plant Sci. Lett. 17 (1979), 43−
50) or agropine and manopin
(Leemans et al., J. Mol. Appl. Genet. 1 (1981),
149−164). This test
Colonies selected in hormone-free medium
to confirm the transformed nature of the
Wear. The selected colonies are then treated with antibiotics.
It can be cultured in a medium without nutrients. 2.5 Symbiotic culture without selection on hormone-free media No selection for transformed cells (e.g.
If a non-toxic T-DNA mutant is used,
or if it is not necessary (for
Dominant selectable markers such as biological resistance genes
(because the marker exists in T-DNA), the professional
To simplify the processing of toplast-derived cells
(hormone reduction step is no longer necessary)
(not). protoplasts until the infection stage.
Process as described above. 48 hours after adding bacteria
Afterward, the protoplast-derived cells were centrifuged.
(6 min, 60-80g), cell growth at very low density.
AG medium that can maintain long
(Reproduced in Caboche, Planta 149 (1980), 7-18).
suspend Fuchs−Rosenthal counting chamber
- (obtained from “Assistant” West Germany)
Measure the density until it reaches the density required for the following operations.
Resuspend in Colony for Opine Test
If you need to operate the
Low cell density (100 protoplasts per ml)
- inoculated with derived cells and cell colonies)
Then, a large cell colony was formed after one month of culture.
can get. Transformed cells can be selected with drugs
If the cells are dense (10³−10^Four/ml)
Optimal timing and concentration for each type of cultivation
Then, add the selective agent to be used to the medium. 2.6 Regeneration of whole plants from callus tissue Easily obtain normal plants from callus tissue
(e.g. protoplast transformation)
or from whole plant inoculation (see 2.7).
). Callus tissue was cultured with 1 mg/ml BAP.
Grow in Murashige and Skoog medium containing
Allow: This medium will form new shoots after 1-2 months.
make it happen Place these sprouts on a hormone-free medium.
Transfer, allow roots to form, and create a complete plant.
can be done. 2.7 Tumor induction in tobacco seedlings Tobacco seeds (e.g. cultivated varieties)
Wisconsin38) surface with 70% denatured ethanol
Le/H₂O for 2 minutes, then 10% commercial standard
Whitening agent and 0.1% sodium dodecyl sulfate (SDS)
sterilize by treating with water and washing 5 times with sterile water.
Ru. These sterilized seeds are used as Murashige and
Large sample containing salts on Skoog (0.7% agar) medium
Test tube (width 25 mm, polycarbonate kit)
(attached). This test tube is then placed in an incubation chamber.
(12000 Lux, 16 hours irradiation/8 hours non-irradiation
culture at 70% relative humidity, 24°C).
Ru. Plants are ready for use after 4-6 weeks
become. Optimal for at least the next month
maintain the condition. Seedlings should be at least 3cm tall
and should have four or more leaves.
It is. The youngest of the plants with a new scalpel
Cut the head transversely through the internodes. on the plant
Remove the part from the test tube and place it on the fire.
Damage bacteria from plate agar culture with partel surface
smear on. In the case of wild type, after 2 weeks,
In the case of degenerated mutant strains of
A tumor appears. This method uses tobacco
(Nicotiana tabacum), Nicotiana
plumbagini folia and Petunia hybrida
used to inoculate. As described above, the present invention provides wild-type Ti plus
Hives lacking tumor function in the mid T-region
Contains LidTi plasmid
Transform your first plant with Agrobacterium
This is what made it possible. Ti plasmid
The effect of T-region on the transfer of DNA from plant cells to plant cells
The influence of regional tumor function is unknown;
However, the implantation of an improved T-region containing the desired gene
It is surprising that metastasis to physical cells occurs.
Ru. This transferred DNA reciprocally integrates into the plant cell genome.
and is maintained stably. Furthermore, the selected desired
The gene has a suitable promoter sequence.
contains or is constructed to contain
It can be expressed when it occurs. containing the desired gene.
Intermediate cloning vectors containing
A single link between the determined acceptor Ti plasmid and
The concept of the present invention (idea) of making one transfer
ia) is a hybrid for the transformation of plant cells.
Significantly simplifies assembly of deTi plasmid vectors.
It makes things simple. This specially designed
The acceptor Ti plasmid contains the desired gene
(This is the same as part of the intermediate cloning vector
Cloning that is or relates to
inserted into the medium) by a single transfer.
Usually so that mutual integration can be formed
Cloning medium containing DNA segments of
There is. Two segments of this cloning medium
provides the necessary regions of homology for recombination. Microorganisms prepared by the method of the present invention, intermediate creams
Rowning Vector, Acceptor Ti Plasmi
and hybrid plasmid vectors.
December 21, 1983, German Collection of
Deposited at Microorganisms (DSM) (Goettingen)
exemplified by the following confirmed culture strains: (1) Intermediate vector in Escherichia coli K12HB101
tar plasmid pAcgB, (2) Carbenicillin-resistant acceptor Ti plus
Possesses Mido pGV3850
Agrobacterium tumefaciens C58C1 Lihuan
Picin resistant strain (3) Escherichia coli K12 strain K514 (thr len thi
intermediate vector plasmid pGV in lac hsdR)
700 (4) Escherichia coli K12 strain 514 (same as (3))
intermediate vector protoplast pGV750, (5) Carbenicillin-resistant acceptor Ti plus
Possesses Mido pGV2260
Agrobacterium tumefaciens C58C1 Lihuan
picin-resistant strains, (6) Nopalli in Escherichia coli K12 HB101
octopine synthase under the control of the promoter
Intermediate vector plasmid that holds the cryptographic domain
depNO−1, (7) Transferring intermediate vectors to Agrobacterium
Strains used = transfer plasmid pGJ28 and
R64drd11 (Van Haute et al., EMBO J.2
(1983), 411-418);
GJ23 is Escherichia coli K12, JC2926,
It is a recA derivative of AB1157 (Howard-
Flanders et al., Genetics 49 (1964), 237-246). The accession numbers for these cultures are 2792, respectively.
(1), 2798(2), 2796(3), 2797(4), 2799(5), 2833(6),
and 2793(7). Although various aspects of the present invention have been described, the basic
By changing the configuration, the method and assembly according to the present invention can be realized.
It goes without saying that other ways of utilizing the composition can be obtained.
It's no good.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram of the acceptor Ti plasmid.
Figures 2 and 3 are schematic diagrams of the intermediate cloning vector inserted into the acceptor Ti plasmid, Figure 4 is a schematic diagram showing the method for preparing the hybrid Ti plasmid vector, and Figure 5 is a schematic diagram of the gene transfer process of the intermediate cloning vector. Figure 6 is a schematic diagram showing the assembly of the A-type acceptor Ti plasmid; Figure 7 is a schematic diagram showing the assembly of the intermediate cloning vector; Figure 8 is a schematic diagram showing the assembly of the intermediate cloning vector.
The figure is a schematic diagram showing the assembly of the B type acceptor Ti plasmid, Figure 9 is a schematic diagram of the intermediate cloning vector inserted into the B type acceptor Ti plasmid, and Figure 10 is a schematic diagram showing the assembly of the B type acceptor Ti plasmid.
Schematic diagram showing the assembly of the Ti plasmid vector,
Figure 11 shows the 5.2kb Hind fragment AcgB.
A schematic diagram showing the insertion into pBR322, FIG. 12 is a schematic diagram showing the T-region of nopaline Ti plasmid pGV3839, and FIG. 13 is a schematic diagram showing the assembly of acceptor Ti plasmid pGV3850.
Figure 14 shows intermediate cloning vector pGV70.
Figure 15 is a schematic diagram showing the structure of intermediate cloning vector pGV750, Figure 16 is a schematic diagram showing the assembly of intermediate vector pGV745, and Figure 17 is a schematic diagram showing the assembly of acceptor plasmid pGV2260. figure,
Figure 18 is a schematic diagram showing the assembly of plasmid pLGV2381, Figure 19 is a schematic diagram showing the assembly of plasmid pAGV10.
and the construction of plasmid pLGV23
FIG. 20 is a schematic diagram showing the nucleotide sequence around the promoter region of the nopaline synthase gene before and after fusion with the octopine synthase gene encoding region.

Claims (1)

[Scope of Claims] 1 (a) two border sequences 1 and 2 of the T-region of the wild-type Ti plasmid; (b) homologous to at least a portion of the DNA sequence in the intermediate cloning vector capable of single crossover;
3, a DNA sequence derived from a cloning medium, comprising a DNA sequence and placed between two border sequences;
and (c) a tumor in a plant containing DNA segment 4 of the wild-type Ti plasmid, which contains DNA sequences essential for the transfer of the T-region of the wild-type Ti plasmid into the plant cell genome by Agrobacterium. An acceptor Ti plasmid that does not induce T-DNA and does not substantially contain the internal T-DNA sequence of the wild-type Ti plasmid [herein, the intermediate cloning vector refers to (a) a promoter controlled by a promoter capable of expressing the gene in plants; (b) a cloning vehicle segment 3' containing a DNA sequence homologous to that in the acceptor Ti plasmid]. 2 Along with intermediate cloning vector
The acceptor Ti plasmid according to claim 1 inserted into Agrobacterium. 3 (a) DNA segment 4 of the wild-type Ti plasmid without the T-region and the two border sequences of the T-region, and (b) an intermediate containing the two border sequences of the T-region of the wild-type Ti plasmid. a DNA sequence from the cloning vehicle that is homologous to at least a portion of the DNA sequence of the cloning vector, does not induce tumors in plants, and contains the border sequences and internal T of the wild-type Ti plasmid.
- Acceptor Ti plasmid that does not contain a DNA sequence [here, the intermediate cloning vector refers to (a) the two border sequences 1 and 2 of the T-region of the wild-type Ti plasmid, and (b) the DNA sequence in the acceptor Ti plasmid. (3) a cloning vehicle segment 3' containing a DNA sequence homologous to , and (c) control of a promoter capable of expressing the gene in the plant, placed between the two border sequences so as to be integrated into the plant genome. Refers to a cloning vector containing at least one desired gene 5 below]. 4. The acceptor Ti plasmid according to claim 3, which is pGV2260 (DSM2799). 5 With intermediate cloning vector
The acceptor Ti plasmid according to claim 3 or 4 inserted into Agrobacterium. 6. In the presence of a helper plasmid: (a) the two border sequences 1 and 2 of the T-region of the wild-type Ti plasmid; (b) at least part of the DNA sequence in the intermediate cloning vector capable of single crossover: and (c) the transfer of the T-region of the wild-type Ti plasmid into the plant cell genome by Agrobacterium. DNA segment 4 of the wild-type Ti plasmid, which contains DNA sequences essential for The Agrobacterium containing the Ti plasmid contains (a) at least one desired gene 5 under the control of a promoter capable of expressing the gene in plants, and (b) a DNA sequence homologous to the acceptor Ti plasmid. A method for the preparation of a hybrid Ti plasmid, which comprises injecting an intermediate cloning vector containing a cloning vehicle segment 3', containing a DNA sequence, and interintegrating the acceptor Ti plasmid and the intermediate cloning vector by a single crossover. 7 In the presence of a helper plasmid, (a) DNA segment 4 of the wild-type Ti plasmid without the T-region and the two border sequences of the T-region, and (b) two of the T-region of the wild-type Ti plasmid. A border sequence of a wild-type Ti plasmid that does not induce tumors in plants and contains a DNA sequence from a cloning vehicle that is homologous to at least a part of the DNA sequence of the intermediate cloning vector described below containing the border sequence. and internal T
- Agrobacterium containing an acceptor Ti plasmid that does not contain any DNA sequences, (a) two border sequences 1 and 2 of the T-region of the wild-type Ti plasmid, (b) a DNA sequence homologous to the above DNA sequence in the acceptor Ti plasmid. a cloning vehicle segment 3' containing a DNA sequence, and (c) under the control of a promoter capable of expressing the gene in the plant, placed between the two border sequences so as to be integrated into the plant genome. A method for the preparation of a hybrid Ti plasmid, which comprises injecting an intermediate cloning vector containing at least one desired gene, and interintegrating the acceptor Ti plasmid and the intermediate cloning vector by a single crossover. 8 (a) the two border sequences 1 and 2 of the T-region of the wild-type Ti plasmid; (b) a DNA sequence homologous to at least a portion of the DNA sequence in the intermediate cloning vector A described below that allows single crossover; DNA from the cloning medium containing and placed between the two border sequences
sequence 3, and (c) DNA segment 4 of the wild-type Ti plasmid containing DNA sequences essential for the transfer of the T-region of the wild-type Ti plasmid into the plant cell genome by Agrobacterium; an acceptor Ti plasmid A that does not induce tumors in plants and does not substantially contain the internal T-DNA sequence of the wild-type Ti plasmid; one desired gene 5, and (b) DNA in the acceptor Ti plasmid A above.
with an intermediate cloning vector A comprising a cloning vehicle segment 3' comprising a DNA sequence homologous to the sequence;
or (a) DNA segment 4 of the wild-type Ti plasmid without the T-region and the two border sequences of the T-region, and (b) the following containing the two border sequences of the T-region of the wild-type Ti plasmid. a DNA sequence 3 from a cloning vehicle that is homologous to at least a portion of the DNA sequence of intermediate cloning vector B of
- acceptor Ti plasmid B that does not contain a DNA sequence; (a) two border sequences 1 and 2 of the T-region of the wild-type Ti plasmid; (b) DNA in the above acceptor Ti plasmid B.
a cloning vehicle segment 3' containing a DNA sequence homologous to the sequence; and (c) a promoter capable of expressing the gene in the plant, placed between the two border sequences so as to be integrated into the plant genome. A hybrid Ti plasmid vector with an intermediate cloning vector B containing at least one desired gene 5 under the control of at least (i) two border sequences 1 of the T-region of the wild-type Ti plasmid and 2. (ii) non-neoplastic DNA sequences 3 and 3' from the cloning vehicle; (iii) DNA sequences that are essential for the transfer of the T-region of the wild-type Ti plasmid into the plant cell genome by Agrobacterium; wild type
DNA segment 4 of the Ti plasmid, and (iv) at least one promoter placed between the two border sequences 1 and 2 that is capable of expressing the gene in plants.
A hybrid Ti plasmid vector containing two desired genes. 9. The hybrid Ti according to claim 8, further comprising at least one selectable marker gene adjacent to the desired gene 5.
Plasmid vector. 10. The hybrid Ti plasmid vector according to claim 8, characterized in that the desired gene 5 is under the control of its natural promoter. 11. The hybrid Ti according to claim 8, wherein the desired gene 5 is under the control of a promoter foreign to the gene.
Plasmid vector. 12 (a) T-region 2 of wild-type Ti plasmid
(b) A cloning vector containing a DNA sequence homologous to at least a part of the DNA sequence in the intermediate cloning vector A described below capable of single crossover and located between the two border sequences. DNA from the medium
sequence 3, and (c) DNA segment 4 of the wild-type Ti plasmid containing DNA sequences essential for the transfer of the T-region of the wild-type Ti plasmid into the plant cell genome by Agrobacterium; an acceptor Ti plasmid A that does not induce tumors in plants and does not substantially contain the internal T-DNA sequence of the wild-type Ti plasmid; one desired gene 5, and (b) DNA in the acceptor Ti plasmid A above.
with an intermediate cloning vector A comprising a cloning vehicle segment 3' comprising a DNA sequence homologous to the sequence;
or (a) DNA segment 4 of the wild-type Ti plasmid without the T-region and the two border sequences of the T-region, and (b) the following containing the two border sequences of the T-region of the wild-type Ti plasmid. a DNA sequence 3 from a cloning vehicle that is homologous to at least a portion of the DNA sequence of intermediate cloning vector B of
- acceptor Ti plasmid B that does not contain a DNA sequence; (a) two border sequences 1 and 2 of the T-region of the wild-type Ti plasmid; (b) DNA in the above acceptor Ti plasmid B.
a cloning vehicle segment 3' containing a DNA sequence homologous to the sequence; and (c) a promoter capable of expressing the gene in the plant, placed between the two border sequences so as to be integrated into the plant genome. A hybrid Ti plasmid vector with an intermediate cloning vector B containing at least one desired gene 5 under the control of at least (i) two border sequences 1 of the T-region of the wild-type Ti plasmid and 2. (ii) non-neoplastic DNA sequences 3 and 3' from the cloning vehicle; (iii) DNA sequences that are essential for the transfer of the T-region of the wild-type Ti plasmid into the plant cell genome by Agrobacterium; wild type
DNA segment 4 of the Ti plasmid, and (iv) at least one promoter placed between the two border sequences 1 and 2 that is capable of expressing the gene in plants.
Agrobacterium harboring a hybrid Ti plasmid vector containing the three desired genes.