JP2992291B2 - Transcription in plants and bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial applications)   The present invention relates to the fields of genetic engineering and plant culture.
In addition, promotion of transcription and selection markers in both plants and bacteria
Provide the means for the (Conventional technology)   Disclose information on technical background closely related to the present invention
The publications are listed below. These publications are based on this prior art
Section discusses in detail. M.W.Bevan et al. (1983)
Nature304: 184-187, R.T.Fraley et al. (1983) Proc.
Natl.Acad.Sci.USA80: 4803-4807 and L. Herrera-Es
trella et al. (1983) Nature303: 209-213, is bacterial
May result in plant expression of a biomaterial resistance structural gene
disclosed the use of the nos promoter (manipulation of TIP plasmids).
See work). R.F.Barker et al. (1983) Plant Molec.B
iol.2: 335-350 and R.F.Barker and J.D.Kemp, U.S P
atent application ser.no.553,786 is an octopine type plastic
Discloses the complete sequence of the T-DNA from Smid pTi15955
Published; homologous to other Ti plasmid genes
The sequence is quoted there (genes on the TIP plasmid
Child). N.Murai and J.D.Kemp (1982) Nucleic Acids Re
s.Ten: 1679-1689 has a size of 1600 bases and its sequence
Open Reading Frame (ORF) 24
The 1450 base transcript identified there (1450
The existence and approximate location of bTx) were disclosed. S.B.Gelvin et
 al. (1981) Plasmid6: 17-29 is T_RIs Agrobacterium
Cells and plant cells (TIP
Gene on Rasmid). S.J.Karcher et al. (1984) Mo
l.Gen.Genet., determined the position of 1450bTx. Dual purpose
Since the features of the functions are disclosed and taught here,
Was not reported in any of the references. L. Herrera-Estrell
a et al. (1983) EMBO J.2: 987-995 with kanamycin
Structural gene encoding resistance to methotrexate
When placed after the nos promoter, bacteria and plants
It was reported that it was expressed in both cells. Shuttle vector   G.B.Ruvkun and F.M.Ausubel (1981) Nature298: 85
The shuttle vector invented by −88
Substitute the substance in a large plasmid, virus or genome.
It provides a method for insertion into a sharp position. Giant plasmid
Or there are two main problems encountered when working with genomes.
First, a large plasmid is used for each restriction enzyme.
Will have many sites. Unique site-specific cleavage reaction
Is not reproducible, multiple site cleavage reactions followed by binding
Compete for many pieces of order and direction that you do not want to change
Leads to great difficulties. The second is giant DNA plasmid
The efficiency of transformation in E. coli is very low. Shuttle Be
Are often small in vitro sources of foreign genetic material
Facilitates insertion into plasmids and is usually followed by in vivo techniques.
These difficulties due to surgical transfer to large plasmids
It is possible to overcome.   The shuttle vector must be introduced into the final host bacterium.
That can be maintained independently there
DAN molecules with con, usually consisting of plasmids. Ma
In addition, the shuttle vector contains the foreign genetic material.
And a copy of the host genome fragment
DNA encoding a selectable feature inserted into the nome fragment
Including small pieces. Selectable features (markers) in vivo
By transposon mutation or by restriction enzyme
Advantages of using ligase in vivo
Is inserted into.   The shuttle vector can be transferred to the final host cell, especially (Agro
Bacteria of the family Rhizobiasis (including bacteria)
It can be introduced in such a way. Three-parent cross
Law (Ruvkin and Ausubel, supra);
Agrobacterium, direct transfer of transposable vectors
Direct uptake of foreign DNA by cells (“transformation”, M. Holst
ers et al. (1978) Molec. Gen. Genet.163Articles 181-187
Agrobacterium with another bacterial cell)
Spheroplast fusion liposome-encapsulated DN
Uptake of A or can be packaged in vitro
Infection with a virus-based shuttle vector. Rizobi
By manipulating the large plasmid found in the family
Yes, three-parent crossing, a technique well known to those skilled in the art,
Strain holding the shuttle vector and plasmid start
Transferable Plasmids Containing Genes for Conjugation and Transfer
And crosses with strains carrying If the shuttle vector
Can be triggered by plasmid genes
The shuttle vector has a large genome, for example,
Retains the Ti or Ri plasmid of the Lactobacillus strain
Transferred to host cells.   After the shuttle vector has been introduced into the host cell,
A possible event is one recombination on either side of the marker.
Includes double intersections with the event. This homopartial diploidization
The elephant replaces the marker by replacing the homologous piece lacking the insertion.
Transfer of the containing DNA fragment to the host genome.
To select cells that have lost the original shuttle vector,
The shuttle vector cannot replicate in the final host cell
Ability or independently present in the host cell
Must be incompatible with the selectable plasmid.
One common means of arranging this is with a third host.
Drug resistant markers that are incompatible with the shuttle vector
Supplying another plasmid that holds the car
It is. Therefore, select resistance to both drugs.
Then, only the surviving cells are in it,
If the marker on the vector is recombined with the host genome
You. If the shuttle vector carries a special marker
If so, the shuttle vector and host
Plus from the result of a single crossover event with Sumid
Intact shuttle vector that eliminates cells containing mid
As a result of the integration of the host plasmid with the host plasmid. Also
Exogenous genetic material is inserted into the marker to be selected
Or adjacent, and the same double recombination
And will be integrated into the host plasmid. Also,
Inserted into a homologous fragment other than within or adjacent to the marker
Are retained together, but exogenous genetic material is
As the distance from the car increases,
Recombination events between genetic material and markers
Disturb the transfer of foreign genetic material.   If the shuttle vector is a phenotypically dominant
Signs (eg, a newly expressible insecticide structural gene,
Induces not an inactivated neoplastic T-DNA gene)
Need to rely on double homologous recombination when used to
Disappears. Single-stranded resulting in co-integrated plasmid
The cells obtained by the recombination event have the characteristics desired for plant cells.
Can metastasize (A. Caplan et al. (1983) Scien
ce222: 815-821, R.B.Horsch et al. (1984) ScienceTwo
twenty three: 496-498). Color with a single continuous T-DNA sequence
It may even be possible to use various shuttle vectors
No. However, the resulting T-DNA is now
And may contain repeated gene duplications,
Between two homologous sequences in either Cterium or plant cells
The shuttle vector due to the single homologous recombination event occurring
Be aware of rare deletions
Absent.   The shuttle vector is an Agrobacterium plasmid
Efficacy in operation. D.J.Garfinkel et al. (19
81) Cell27: 143-153, A.J.M.Matzke and M.-D.Chilto
n (1981) J. Molec. Appl. Genet.1: 39−49, and J. Leeman
s et al. (1981) J. Molec. Appl. Genet.1: 149-164
Shine They are shuttle vectors and intermediate vectors
Or "iV".   Shuttle vector for insertion of changes into large DNA molecules
A recently discovered variant of the system is the “suicide vector”.
You. In this system, Simon et al. Molecules of bacterial-plant interaction
Genetics, pp. 98-106 (ed. A. Puhler, (1983))
Vectors for in vivo and in vitro manipulation of negative bacteria
Tata plasmid "and R. Simon et al. (1983) Biotechn.
ol.1: 784-791
Vector cannot be maintained independently in host cells.
No. This trait is commonly used in three-parent crosses.
Incompatible with host cells to eliminate vector
It eliminates the need to introduce a plasmid. Already exists
All vector sequences that do not integrate into existing DNA are replicated
Suicide efficiently by not being done. Shuttle Beck
Two homologous regions, as can be done in the traditional pattern of
Screening for new antibiotic resistance genes
May distinguish between double and single homology
Not. Suicide to transfer DNA sequence to Ti plasmid
The use of vectors is described in E. Van Haute et al. (1983) EMBO J.
2: 411-417, L. Comai et al. (1982) Plant. Molec. Bio
l.1: 291-300, L. Comai et al. (1983) PlasmidTen:twenty one
-30, P. Zambryski et al. (1983) EMBO J.2: 2143−215
0, and also reported by A. Caplan et al., Supra.
I have. C.H.Shaw et al. (1983) Gene28: 315−330,
Can be used to select foreign DNA by means of single homologous recombination.
To Ti plasmid without introducing selectable marker
And then select for double homologous recombination
Reported the use of a suicide vector.   Homology sets for the introduction of new DNA sequences into T-DNA
An alternative to replacement use is the bacterial transposon.
Described in the section on Agrobacterium gene on TIP plasmid
As can be seen, the transposon is TIP plasmid T
-Dive into DNA. (For example, D.J.Garfinke
l et al. (1981) Cell27: 14-153). The tiger
Inspozones are modified in vitro by inserting new sequences.
So that the new DNA is
Can be transferred to the T-DNA of the TIP plasmid. TIP
When it is stably integrated, a new DN
A / transposon / T-DNA combinations can be transferred
Wear. Agrobacterium appearance   Gram-negative bacteria Rhizobiassaceae (including Rhizobium)
Agrobacterium spp.
Terium thiumefaciens and Agrobacterium
・ There is rhizogenes. Each of these species is a plant crop.
Causes Raungol's disease and hairy root disease. Crown go
Is characterized by the growth of the goals of the dedifferentiated organization
You. Hairy roots can cause inappropriate induction of roots in infected tissues.
It is a malformation characterized by guidance. Which disease
Inappropriately growing plant tissue
Not produced by the organism, but metabolized by the infecting bacteria.
One or more, known as opin
Usually produces amino acid derivatives of Known opines are
The three main components, kuppin, nopaline and agropine
Divided into groups. Tissues that grow inappropriately
The cells can be grown by culturing,
Below, a complete plant that maintains a transformed phenotype
Can be played back.   The pathogenic strain of Agrobacterium is Agrobacterium
Muti-Fume Science is Ti (Tumor Induction) Plasmid
De, Ri (root induction) in Agrobacterium rhizogenes
It holds a large plasmid known as a plasmid.
Deletion of these plasmids from the strain results in loss of disease.
Will be Ti plasmid is called T-DNA (transfer DNA)
Region, whose T-DNA is the genome of the host plant in the tumor
It is known that it is taken into. The T-DNA
Encodes several transcripts. Mutation research
Ultimately, some of these are involved in the induction of tumor growth.
It showed that. Genes for tml, tmr and tms
Mutants are large tumors (in tobacco),
Root-producing traits, leading to foliage induction
You. The T-DNA is at least one opin synthase
The Ti plasmid also encodes
Often caused by opine caused by synthesis
Divided. Each T-DNA gene is a T-DNA promoter.
Under control of the motor. T-DNA promoter is structurally
Similar to eukaryotic promoters, they are transformed
It appears to function only in isolated plant cells. The Ti plastic
Smids also carry genes other than T-DNA. These remains
The genes are opine metabolism, neoplastic, agrosine sensitive, replication,
And functions including autonomous transfer to bacterial cells. Ri
Plasmids are organized in a similar structure to Ti plasmids.
You. Genes and DNA sequences corresponding to plant cell transformation
The group has been focused on the transformation induction principle (TIP) since then.
Call. Therefore, the name TIP is not restricted, but T
Contains both i and Ri plasmids. Small pieces with embedded TIP
Is derived from Ti plasmid or Ri plasmid
Or T-DNA (transferred DNA),
You.   M.-D.Chilton (June 1983) Sci. Amer.248  (6): 5
0-59 has recently been developed for the use of Ti plasmids as vectors.
Introductory article was shown. Agrobacterium is the cause
D.J.Merlo (1982), Adv.Pl
ant Pathol.1: 139-178; L.W.Ream and M.P.Gordon (19
82), Science218: 854-859, and M.W.Bevan and M.-D.
Chilton (1982), Ann. Rev. Genet.16: 357-384; G. Kahl a
nd J. Schell (1982) Molecular Biology of Plant Tumo
r, K.A.Barton and M.-D.Chilton (1983) Meth.Enzymo
l.101: 527-539, and A. Caplan et al. (1983) Scienc.
e222: 815-821. Infection of plant tissue   Plant cells can be agglomerated in many ways known to those skilled in the art.
Can be transformed by bacteria,
Culturing plant cells with Agrobacterium
Plant with Agrobacterium spheroplast
Fusion of protoplasts, play with plant cell protoplasts
Direct transformation by incorporation of isolated T-DNA, complete bacteria
Of protoplasts with partially regenerated cell walls
Transformation, protoplasts by ribosomes containing T-DNA
Transformation, use of virus with T-DNA, microinjection
Injection, etc., but not limited to. Which
Method also ensures that genes are transmitted stably through mitosis and meiosis
That's enough to be reached.   Infection of plant cells by Agrobacterium should be
It is a well-known simple technique (for example, D.N.Butcher e
t al. (1980) in Tissue Culture Methods for Plant P
athologists, eds .: D.S.Ingram and J.P.Helgeson, pp.20
See 3-208). Typically, plants are many
Can also be hurt by the
Cut with a needle, stab with a needle, or rub with an abrasive
And Next, a solution containing tumor-inducing bacteria was applied to the wound.
Inoculate. An alternative to complete plant infection is potato stalks
Disc (D.K.Anand and G.T.Heberlein (1977) Amer.J.
Bot.64: 153-158) Alternatively, a small piece of inverted tobacco stem
(K.A. Barton, et al. (1983) Cell321033-1043)
There is inoculation of such a piece of tissue. After infection, the tumor is
Placed in tissue culture in medium lacking media. To hormones
Independent growth is typical of transformed tissues,
In sharp contrast to the normal conditions of tissue culture growth
You. (A.C. Braun (1956) Cancer Res.16: 53-56).   Agrobacterium grows in isolated cells and in culture.
Cultured cells (L. Maton et al. (1979) Nature277: 129−
131) and in isolated tobacco mesophyll protoplasts
Also have the ability to infect. The latter technique requires a new cell wall
After a period of partial regeneration, Agrobacterium cells
Was added to the culture for some time, and antibiotics were added
Killed by Agrobac carrying Ti plasmid
Exposure to Terium thyme fascient cells. This
Only these cells were on media lacking hormone
In particular, he had the ability to form calli. Most calli
Contains enzymatic activities involved in opine assimilation
Was. Other researchers (R.B.Horsch and R.T.Fraley (18 Ja)
nuary 1983) 15th Miami Winter Symposium)
A high proportion of calli that show mon-independent growth (10%
Above) 95% of those calli make opines,
Transformation by co-culture was reported. M.R.Davey et al.
(1980) in Ingram and Helgeson, supra, pp. 209-219,
Describes infection of old cells regenerated from protoplasts.
I'm eating.   Plant protoplasts for direct uptake of TIP plasmids
Thus, it can be transformed. M.R.Davey et al. (1980) Pl
ant Sci. Lett.18: 307-313 and M.R.Davey et al. (19
80) In Ingram and Helgeson, supra, Petunia
-Protoplasts in the presence of poly-L-α-ortinin
In the Ti plasmid, the phenotype of opine synthesis and
Can transform into a hormone-independent growth state
Was. Polyethylene glycol uptakes Ti plasmid
Stimulating, and T-DNA sequence is incorporated into the genome
It was shown later. (J. Draper et al. (198
2) Plant and Cell Physiol.twenty three: 451-458, M.R.Davey et
 al. (1982) in Plant Tissue Culture 1982, ed: A. Fuji
wara, pp. 515-516). F.A.Krens et al. (1982) Nature
296: 72-74 show that their data are based on the integrated T-DNA
Contains the adjacent Ti plasmid sequence,
Use polyethylene glycol with calcium shock
Have reported similar results.   Other methods of obtaining DNA uptake include the use of liposomes.
No. The preparation of liposomes containing DNA is well known to those skilled in the art.
ing. Control of liposome-mediated introduction of Ti-DNA
Has been reported. (T.Nagata et al. (1982) in Fu
jiwara, supra, pp. 509-510, and T. Nagata (1981) Mol.
Gen. Genet.184: 161-165). After removing the cell wall to a similar system
There is a fusion of plant and bacterial cells. An example of this technique is S.
Hasezawa et al. (1981) Mol. Gen. Genet.182: 206-210
Agrobacterium spheroplus reported by
Transformation of Vinca protoplasts. Planting
Protoplasts are aggro whose cell walls are demarcated
Can take up bacterial cells (S.Hasezawa
 et al. (1982) in Fujiwara, supra, pp. 517-518).   T-DNA is regenerated from the fusion of the two protoplasts
Can be communicated to other organizations. Of these two protoplasts
Only one of them was already transformed (G.J.Wull
ems et al. (1980) Theor. Appl. Genet. 56: 203-208).
As detailed in the section on plant regeneration, T-DNA
Can be transmitted to offspring as a simple, Mendelian trait
You. Plant regeneration   Dedifferentiated plant tissue in normal morphology is crown gall tumor
Obtained from the ulcer. A.C.Braun and H.N.Wood (1976) Pro
c.Natl.Acad.Sci.USA73: 496-500,
Transplant a malformation to obtain a normally appearing leaf stem that can flower
I was able to. When this leaf stem is cultured, it produces opine.
And the ability to grow independently of plant hormones.
In the plants screened, these tumor phenotypes
Transmission to offspring, apparently lost during meiosis, was observed
Did not. (R. Turgeon et al. (1976) Proc. Natl. Aca
d.Sci.USA73: 3562-3564). Spontaneously lost tumor characteristics
Or plants derived from malformed species
It was shown that all T-DNA had been lost. (F.-M. Yang et a
l. (1980) In Vitro16: 87-92, F. Yang et al. (1980)
Molec.Gen.Genet.177: 707-714, M. Lemmers et al. (198
0) J. Mol. Biol.144: 353-376). But hormone treatment
(1mg / kinetin), then revertant plant
Subsequent studies have shown that T-DNA remains in the transformed phenotype.
Plants that have lost the gene but have experienced meiosis are T-DN
Able to retain homologous sequences at both ends of A
(F. Yang and R. B. Simpson (1981) Proc. Nat)
l.Acad.Sci.USA78: 4151-4155). G.J.Wullems et al.
(1981) Celltwenty four: 719-724, are further included in the opine assimilation system.
The gene contained in the plant is
The ability to experience, seemingly invariant T-DNA
(G. Wullems et al. (19
82) in fujiwara, supra). L. Otten et al. (1981) Mole
c.Gen.Genet.183: 209-213, form tumors that proliferate foliage
Created with Tn7 transposon at the growing tms (foliage induction) site
A mutant of the Ti plasmid was used. These leaf stems are planted
Regenerated into self-pollinating flowers
Was. As a result, the resulting seeds germinate and contain T-DNA.
It became a plant that made pins. In further experiments, H.D.
eGreve et al. (1982) Nature300: 752-755,
Topin synthesis is inherited as a single dominant Mendelian gene.
I found that it could be. However, the T-DNA is
Extensive lack of non-ocs functions while playing from source
Lost. A similar experiment with the tmr (root-inducing) mutant
Full-length T-DNA is transmitted to progeny through meiosis
In the offspring, the nopaline gene was altered.
Yeast cells expressed at various levels and transformed together
That the cold dehydrogenase 1 gene is not expressed
(K.A. Barton et al. (1983) Cell32: 1033-104
3). Other experiments show that nopaline T-DNA is maintained during regeneration.
And the male flower is transmitted on T-DNA according to Mendel's rule.
(J. Memelink et al. (1983) Mol. Ge
n.Genet.190: 516-522). Functional foreign genes are also dominant
Inherited by Mendel's rule (R.B. Horsch et al. (1984) S
cience233: 496-498). Now, regeneration lacking the T-DNA sequence
Tissue is derived from non-transformed cells that are “mixed” with the tumor
Yes (G. Ooms et al. (1982) Cell30: 589-597). First
Epigenetic state of plant cells transformed for
(G.M.S.vanSlogteren et al. (198
3) Plant Mol. Biol.2: 321-333)
You. A.N.Binns (1983) Planta158: Recent by 272-279
Studies show that oncogenes, in this case tmr, are "stopped" during regeneration.
By placing the regenerated tissue in culture.
Indicates that it can be "started again".   Transformation from Agrobacterium rhizogenes
The roots obtained as fruits are relatively easy to regenerate directly into cultivated plants.
It proved simple (M.-D. Chilton et al. (1982)
Nature295: 432-434), and easily cloned
Was. The regenerative ability seems to depend on the copy number of T-DNA.
(C. David ed al. (1984) Biotechnol.2: 73-76). Genes in TIP plasmid   P of the octopine-type plasmid found in ATCC15955
The complete sequence of the T-DNA of Ti15955 has been reported,
14 open reads flanking the transcriptional control sequences of the organism
Frame (ORHs) (R.F.Barker a
nd J.D.Kemp.U.S.Patent application ser.no.553,786,
This is a reference, R.F.Barker et al. (1983) Plant Mole
c.Biol.2: 335-350).   Many genes have been identified in the T-DNA of the TIP plasmid.
ing. Many of the octopine plasmid T-DNA transcripts
A map has been created (S.B.Gelvin et al. (1982) Proc.
Natl.Acad.sci.USA79: 76-80, L. Willmitzer et al. (1
982) EMBO J.1: 139-146, N.Murai and J.D.Kemp (198
2) Nuclei Acids Res.Ten: 1679-1689, S.J.Karcher et a
l. (1984) Mol. Gen. Genet.)
(J. Leemans et al. (1982) EMBO J.1: 147-1
52). Some of these areas, especially tmr and tms
Encoding is also transcribed in prokaryotes (G. Schrod
er et al. (1983) EMBO J.2: 403-409). Transpo
Oct that is sufficiently clarified by mutation using a zon
Pin-type plasmid genes are tms, tmr, tml, and ocs.
Contains (D.J.Garfinkel et al. (1981) Cell27:14
3-153). Ti plastics with mutations in these genes
Sumids produce shoots, develop roots, and are normal
Stimulates the tumor callus of Nicotinatabacum larger than
You. In other hosts, mutants of these genes are divergent.
Phenotype (M.W.Bevan and M.-
D.Chilton (1982) Ann.Rev.Genet.16: 357-384). tmr
And tms phenotype depends on the level of plant hormones present in the tumor
Correlates with the difference. Difference in cytokinin: auxin ratio
Or anthers or roots of untransformed callus tissue
Is similar to that which can be induced in the medium (D.E.Akiyoshi
 et al. (1983) Proc. Natl. Acad. Sci. USA80: 407-411,
A.N.Binns (1983) Planta158: 272-279, A. Caplan et a
l. (1983) Science222: 815-821, R.M.Amasino and C.
O. Miller (1982) Plant Physiol.69: 389-392). function
Not only tml with tms but also tms or tmr
T-DNA containing genes promotes important tumor growth
can do. Shoot and root promotion function
Stimulated or inhibited by tml with
(L.W. Ream et al. (1983) Proc. Natl. Acad. Sci. USA8
0: 1660-1664). Mutations in the T-DNA gene are
Does not seem to affect insertion into the genome
(Before Leemans et al. (1982), before Ream et al. (1983)
Out). T-DNA gene promotes hormone-independent growth
Sequence (see TIP plasmid DNA)
(H. Joos et al. (198
3) EMBO J.2: 2151-2160).   Octopin Ti plasmid is octopine synthase
Ocs gene encoding (lysopine dehydrogenase)
Having. The ocs gene is an intron (a eukaryotic gene)
An intervening sequence that is normally found in mRNAs during mRNA maturation.
From the precursor after the transfer)
No. It is the eukaryotic transcription signal (“TATA” box)
Or a sequence resembling the poly-A addition site. ocs remains
All signals required for gene expression are located in the ocs transcription initiation site.
Found within 295 bp (C. Koncz et al. (1983) EMBO J.
2: 1597-1603). P. Dhaese et al. (1983) EMBO J.2:
419-426 is "transcript 7" (ORF3 of Barker et al., Supra).
Report on the use of various poly-A addition sites by ocs and ocs
did. Presence of the enzyme octopine citase in the tissue
From the toxic effects of various amino acid analogues
(G.A.Dahl and J.Tempe (198
3) Theor.Appl.Genet.66: 233-239, G.A.Dahl et al., US
 Pat.applicfation ser.no.532, 280, where references
Transfer).   Nopaline Ti plasmid is nopaline synthase (nos)
And its sequence is A. Depicker et al. (198
2) J.Mol.Appl.Genet.1: 561-573
You. nos is medium in introns as seen in the ocs gene
Not refused. It has two poly-A addition sites and an important
"TATA" box has a transcription start signal. nos compared to ocs
Is the transcription start signal, known as the “CAT” box.
There may be an array before it. Required for nos gene expression
All essential signals are found within 261 bp of the nos transcription start site
(C. Koncz et al., Supra). Agrosinopine sinter
Zea gene and genes equivalent to tms and tmr
On Rasmid (H. Joos et al. (1983) C
ell32: 1057-1067), many transcripts are determined on the map
(L. Willmitzer et al. (1983) Cell32: 1045
-1056). J.C. McPhersson et al. (1980) Proc. Natl. A
cad.Sci.USA77: Is 2666-2670 a crown gall tumor?
In vitro conversion of mRNA encoded by T-DNA
Reported a copy.   Root hair T-DNA transcription has been detected (L. Willmitze).
r et al. (1982) Mol. Gen. Genet.186: 16-22). function
Above, root hair syndrome is a Ti plasmid with a mutation in tms
And Ri plasmids can complement each other (G.M.S.van Slogte
ren (1983) Ph.D. thesis, Rijksuniversiteit te Leide
n, Netherlands)
Sumid (F.F.White and E.W.Nester (1980) J.Bacterio
l.144: 710-720) stimulated crown gall tumor
Seems to be equivalent to   In eukaryotes, DNA methylation (especially cytosine residues)
Group) is correlated with transcriptional inactivation, but relatively
Genes with low levels of transcription are transcribed into mRNA. S.B.Gelvin e
t al. (1983) Nucleic Acids Res.11: 159-174
T-DNA in Ungall tumors is always at least
Being present in one unchilled copy
Was found. T-DNA in which the same genome is methylated
That may contain many other copies of
Et al. That more than one copy of T-DNA is biologically inactive.
Suggests that there may be. (G.Ooms
 et al. (1982) Cell30: See also 589-597)
Treatment of tumor lines with thydin parallels the increase in transcription
Resulting in the demethylation of the T-DNA gene (A.G. Hepburn
 et al. (1983) J. Mol. appl. Genet.2: 315-329). 5
-Azacytidine treatment or cell transplantation is
Have been shown to activate genes (G.M.S.van Slog
teren (1983) Ph.D. thesis, Rijksuniversiteit te Leid
en, Netherlands).   Ti plasmid contains other genes outside the T-DNA region.
And is required for the infection process. (Nopaline
For plasmids, see M. Holsters et al. (1980) Plas
mid3: 212-230, for octopine plasmid, H.D.
eGreve et al. (1981) Plasmid6: 235-248, D.J.Garfi
nkel and E.W.Nester (1980) J. Bacteriol.144: 732-743 and
G. Ooms (1980) J. Bacteriol.144: 82-91)
What is important is the onc gene, which causes tumor formation when mutated
This results in an impossible Ti plasmid. (To Wilence
Thus, these places are known as vir. ) Some o
The nc gene is accurately located and has various Ti plus
Found to be in a conserved region between mids
(H.J.Klee et al. (1983) J. Bacteriol.153: 873-883,
V.N.Iyer et al. (1982) Mol.Gen.Genet.188: 418-42
Four). The onc gene has different plasmid types of T-DNA.
Plant cells and transform them physically into another plasmid.
Functioning as a trance (J. Hille et al.
(1982) Plasmid.7: 107-118, H.J.Klee et al. (198
2) J. Bacteriol.150: 327-331, A.J.de Framond et al.
(1983) Biotechnol.1: 262-269). Nopaline Ti DNA
Can be excised from the Ti plasmid or transferred to the host genome.
T-DNA that may be involved in either of the integrations
Approximately 25 base pairs immediately adjacent to the left and right boundaries of
It has a direction repeat sequence (N.S.Yadav et al. (1982) Proc. N
atl.Acad.Sci.USA79: 6322-6326), similar sequence is Oct
It is recognized adjacent to the boundary of the pin T-DNA (R.B.Sim
pson et al. (1982) Cell29: 1005-1014). Opin minutes
The solution is an octopine-type plasmid or a nopaline-type plasmid
Is specific to each of the occ and noc genes
You. Ti plasmids can also replicate themselves, including the origin of replication
Codes required functions.   Transcripts of the Ti plasmid were obtained from S.B. Gelvin et al. (1981) P
lasmid6: 17-29, Agrobacterium chew
Detected in the mefaciens, where he found that the T-DNA region was non-T
-Was found to be transcribed to a lesser extent than the DNA sequence. T
The characteristics specified in the i plasmid are Merlo,
able II), and Ream and Gordon, outlined above.
I have. TIP plasmid DNA   DNA hybridization (T.C.Currier and E.W.N.
ester (1976) J. Bacteriol.126: 157-165) or system
Restriction enzyme analysis (D. Sciaky et al. (1978) Plasmid1: 238
According to −253), different octopine type Ti plus
The mids are almost 100% similar to each other. Nopaline-type Ti-pu
Rasmids are only 67% similar to each other
(Currier and Nester, supra). Ri that looks different
Plasmids prove to be very similar to each other.
(P. Costantino et al. (1981) Plasmid
5: 170-182). N.H.Drummond and M.-D.Chilton (1978)
J. Bacteriol.136: 1178-1183 is octopine type and nopaline
Relatively small parts of the type Ti plasmid are similar to each other.
It showed that. G. Engler
et al. (1981) J. Mol. Biol.152: 183-208 in more detail
It is positioned on the map. They have four similar areas
Three of them are three (partially overlapping T-DNA
), Four (including some onc genes), and nine
Subdivided into one (having onc gene) similar sequences
Was found. Successive similarities have at least one tra
Gene (for transfer of conjugative transfer of Ti plasmid to cells of other bacteria)
And genes related to replication and incompatibility. This ream
The next region is lysozyme, a different gene in the family Rhizobium.
Sym plasmids from certain species of the genus Bium
(R.K. Prakash et al. (19
82) Plasmid7: 271-280). The order of the four areas is preserved
Although not done, they are all in the same direction.
Some parts of the T-DNA sequence are
Very well conserved between pin plasmids (M.-D.
Chilton et al. (1978) Nature275: 147-149, A.Depick
er et al. (1978) Nature275: 150-153). Ri Plasmi
The octopine Ti plasmid (F.F.White)
and E.W.Nester (1980) J. Bacteriol.144: 710-720)
Nopaline Ti plasmid (G. Risuleo et al. (1982) Plas
mid7: 45-51) mainly for the onc gene
Has broad similarity in coding regions
It has been shown. Is RiT-DNA a Both Type of Ti Plasmid?
Have broad but weak similarity to T-DNA
(L. Willmitzer et al. (1982) Mol. Gen. Genet.186: 16
-22). Plant DNA from uninfected Nicotiana grauka
Is called cT-DNA (cellular T-DNA).
Showing similarity in the parts (F.F.White et al. (1983)
Nature301: 348-350, L.Spano et al. (1982) Plant Mo
lec.Biol.1: 291-300). G.A. Huffman et al. (1983)
J. Bacteriol., Is the region of cross-hybridization
Is located on the map, Ri plasmid pRiA4b is from pTiT37
Is very similar to pTiA6 (octopine type)
Ri plasmids have a region similar to tms but not tmr.
It shows that it seems to be. Their results are Ri T
-DNA is discontinuous and similar to that of octopine T-DNA
Suggest that.   Ti plasmid (M.-D. Chilton et al. (1977) Cell1
1: 263-172) or Ri plasmid (M.-D.Chilton (1.
982) Nature295: 432-434, F.F.White et al. (1982) P
roc.Natl.Acad.Sci.USA79: 3193-3197, L. Willmitzer
(1982) Mol.Gen.Genet.186: 16-22) is part of tumor plants
It was shown to be found in the DNA of cells. transfer
The resulting DNA is known as T-DNA. T-DNA is
Nuclear (M.P.Nuti et al. (1980) Plant Sci. Lett.18: 1
−6, L. Willmitzer et al. (1980) Nature287: 359-36
1, M.-D.Chilton et al. (1980) Proc.Natl.Acad.Sci.U
SA77: 4060-4064) at many sites (D. Ursic et al.
(1983) Mol. Gen. Genet.190: 494-503, J. Memelink et a
l. (1983) Mol. Gen. Genet.190: 516-522), for host DNA
(M.F.Thomashow et al. (1980) Pro
c.Natl.Acad.Sci.USA77: 6448-6452, N.S.Yadav et al.
(1980) Nature287: 458-461). Many non-T-DNA Ti
Plasmid DNA is transferred to plant cells before T-DNA integration
(H. Joos et al. (1983) EMBO J.2: 2
151-2160).   M.F.Thomashow et al. (1980) Proc.Natl.acad.Sci.U
SA77: 6448-6452 and M.F.Thomashow et al. (1980) C
ell19: 729-739 is derived from the octopine-type Ti plasmid.
T-DNA has two different parts of T_L−DNA and T_R-DNA, it
The left T-DNA and the right T-DNA are incorporated into
I found that. T_RAnd T_LTumors with different copy numbers
May vary in the series (D.J. Merlo et al. (1980) Molec. Ge
n.Genet.177: 637-643). T-DNA core is nopaline TD
Very similar to NA (Chilton et al. (1978)
, Depicker et al. (1978) supra), necessary for tumor maintenance
Yes, T_LAnd generally there is one copy per cell
And encodes tms, tmr, and tml genes
ing. On the other hand, T_RCan be omitted as a whole
(M.De.Beuckeleer et al. (1981) Molec.Gen.genet.18
Three: 283-288, G. Ooms et al. (1982) cell30: 589-59
7), usually at higher copy numbers (Merlo et al. (1
980) supra). G. Ooms et al. (1982) Plasmid7: 15-2
9 they are T_RIs deleted from the Ti plasmid,
Bacterium tumefaciens a certain Willen
Admitted to have_RIs for T-DNA integration
I hypothesized that they were involved. G. Ooms et al. (198
2) Cell300: 589-597 is T after integration into the plant genome.
-DNA is often deleted by the Ti plasmid,
In terms of stability and T-DNA formation
Tumors that contain a mixture of different cells
Is the result of the occurrence of The osc gene is T_LAt the inner
Has been found but does not lose a phenotype similar to tumor growth
Can be deleted from the plant genome. T_LLeft and right border of
And T_RThe left and right boundaries of are here T_LLB (A), T
_LRB (B), T_RLB (C), T_RRB (D), respectively.
The sequence has been determined (R.F. Barker et al. (1983) Pla
nt Mol.Biol.2: 335-350, and R.F.Barker and J.D.K
emp, U.S.Patent application ser.no.532,280), each
Is a 24-base pair imperfectly oriented repeat sequence.
And the same orientation found in either less than nopaline T-DNA
Similar to repetitive sequences. Embedded T_LAround LB (A)
Whether the plant DNA on the side is in the same or opposite direction
Is observed to consist of a repeat of the T-DNA sequence
(R.B.Simpson et al. (1982) Cell29: 1005−
1014). M. Holsters et al. (1983) Mol. Gen. Genet.19
0: 35−41 is T_LArises from both plant and T-DNA sequences
Aligned co-separated by about 400 bp "linker"
Found that it is incorporated into the pea.   Compared to the situation in octopine tumors, nopaline T-DN
A is integrated into the host genome in a contiguous fragment
(M. Lemmers et al. (1980) J. Mol. Biol.144: 353-37
6, P.Zambryski et al. (1980) Science209: 1385-139
1). Directed tandem repeats were observed. Regenerate from malformed species
T-DNA of the isolated plant is a minor fraction in the fragment at the border of the inserted DNA.
(Lemmers et al., Supra). Right and left
Analysis of contact points between multiple boundaries allows more co-repetitions
One reverse iteration was revealed (Zambrysk
i et al. (1980) supra). Left contact is at least 70bp
It is shown to be diverse up to
Contacts do not change except for a single nucleotide (P. Zambry
ski et al. 1982) J. Mol. Appl. Genet.1: 361-370). Vertical
The left and right borders of the contacts in the queue are over 130bp
Separated by possible gaps. The gap is of unknown origin
And contained several T-DNA sequences. T
-DNA is repetitive and incorporated into the sequence of low molecular weight hosts.
Was found to be embedded. H. Joos et al. (198
3) Cell32According to 1057-1067, Wilrence is usually
After deletion of either one of the nopaline T-DNA border sequences
It is not shown to be caused.   Simpson et al. (1982) supra and Zambryski et al. (19
80) In the above, the co-directional repetition of the border region is due to
-Suggested to be involved in DNA integration. two
The boundaries of T-DNA from two different Ti plasmids are similar
The less specific integration of similar boundaries is that
We support this suggestion (G. Ooms et al. (1982) Plant
 Molec.Biol.1: 265-276).   N.S.Yadav et al. (1982) Proc.Natl.Acad.Sci.USA7
9: 6322-6326 is 10 kilobases in bacteriophage λ.
Also increase universal recombination in distant surrounding DNA
The chi site to be made is located just outside the left end of the T-DNA.
It was found to be on the Palin Ti plasmid. R.B.Simpso
n et al. (1982) Cell29According to: 1005-1014, Oct
If a chi sequence is found at a similar position in the Pin Ti plasmid
Was not. The importance of chi in the Ti plasmid is known
Not in. Manipulation of TIP plasmid   As detailed in the section on shuttle vectors,
Introduce modified DNA sequence to desired location on TIP plasmid
The technology for doing so was developed. With this technology,
Can be easily inserted (D.J.Garfin
kel et al. (1981) Cell27: 143-153). J.-P. Hernals
teen et al. (1980) Nature287: 654: 656 is Ti plus
DNA sequence inserted into the T-DNA (here, bacterial
Ranspozone) translocates and integrates into the genome of the recipient plant
It was shown that. M. Holsters et al. (1982) Mo
l.Gen.Genet.185: 283-289 are bacteria inserted into T-DNA
Transposon (Tn7) is integrated into the plant genome
Is still fully functional and re-separated in an appearance-invariant manner
Showed that you get. Several genes from different origins
Was used to insert foreign DNA, but until now, foreign DNA has been inserted.
The foreign gene usually promotes its own promoter in plant cells.
Is not expressed under the control of a protein. The origin of these genes
The source is rabbit β-globin (C.H. Shaw et al. (1983) G
enetwenty three: 315-330), yeast alcohol dehydrogenase
(Adh) (K.A. Barton et al. (1983) Cell32: 1033-104
3), corn Adh I (J. Bennetzen, unpublished) and
Zein, mammalian interferons and globins,
And the mammalian virus SV40 (J. Schell, unpublished)
It is. However, the nopaline synthase gene
When plant tissue was transformed by insertion into T-DNA,
Was found to be fully functional (C.L.Fink (198
2) M.S.thesis, University of Wisconsin-Madison). beans
Phaseolus vulgaris L.
Phaseolin, a storage protein found in the seeds of
The introduced gene is introduced into the sunflower tumor, where it is released.
Revealed. Transcription is started and terminated at the correct location, and
Intron processed correctly after transcription
(N. Murai et al. (1983) Science222: 476-482, and
And T.C. Hall et al. US application ser.no.485,613,
Which is incorporated herein by reference). A. Caplan et al.
(1983) Science222: 815-821 is pea blow blow
Small 1,5-bisphosphate carboxylase
DNA from the 5 'upstream region of the subunit gene
Of light-induced expression of tobacco by the bacterial chloramph
Enicol acetyltransferase structural gene
Claims that it is enough to be awarded.   Deletion of TIP plasmids by several methods
Can cause. Standard recombinant DNA technology
Shuttle vectors to introduce more created deletions
Can be used. One end that has already been determined
Truncated deletions can lead to inappropriate transposon excision
(B.P.Koekman et al. (197
9) Plasmid2: 347-357, and G. Ooms et al. (1982)
Plamid7: 15-29). J. Hille and R. Schilperoot (19
81) Plasmid6: 151-154 is the predetermined position
Deletion at both ends is due to the use of two transposons
Proven that can be caused. This technology is
Used to construct "recombinant DNA" molecules in vivo
Can be P. Zambryski et al. (1983) EMBO J.
2: 2143-2150 shows very small callus in tobacco
Promotes the formation and leads to the regeneration of plants of normal morphology
Of nopaline T-DNA genes other than nos and transcript a
The use of all deleted vectors has been reported.   The nopaline synthase gene is
DN encoding drug resistance that can be used for cell sorting
Used for A-piece insertion. In plant cells, Tn
The bacterial kanamycin resistance gene from 5 is itself
Is not transcribed under the control of the promoter (J.D. Kemp et
al. (1983) in Genetic Engineering: Applications to
Agriculture, (Beltsville Symp.Agric.Res.7), Ed .:
Before L.D.Owens, pp.215-228; and C.L.Fink (1982)
Out). M.W.Bevan et al. (1983) Nature304: 184-187,
R.T.Fraley et al. (1983) Proc. Natl. Acad. Sci. USA80:
4803-4807, and L. Herrera-Estrella et al. (198
3) EMBO.J.2: 987-995, is Kanamycin resistance from Tn5
Gene (neomycin phosphotransferase
II) after the nopaline synthase promoter
Was. The construct is used to transform plant cells,
Plant cells are better than kanamycin and G418 in culture.
Una showed resistance to the analog. Herrera-Est
rella et al., supra, is Tn7-derived methotrexate resistance
Gene (dihydrofolate reductase) is nopaline sinter
A similar structure, such as one behind a promoter
Reported. Transformed cells are antagonistic analogs of folic acid
It was resistant to certain methotrexate. Similarly, L. Herre
ra-Estrella et al. (1983) Nature303: 209-213 is
Octopine synthase and chloram phenylcholoa
Cetyltransferase (chloramfe in bacteria)
Expression of the enzyme activity of
Under the control of the nos promoter, the structural genes of the two enzymes
At the plant cells.   N. Murai et al. (1983) Science222: 476-482, and
And T.C. Hall et al. US application ser.no.485,614,
Here, the ocs promoter and the ocs promoter are incorporated into the literature.
Bean seed protein at the 5 'end of the structural pin gene
Reported the fusion of phaseolin to the structural gene.
Retains the amino terminus of octopine synthase and
A fusion protein that deletes the amino terminus of phosphorus is
Produced under motor control. Phaseolin sequence
Given intron is correctly processed after transcription.
Singed.   A.J.de Framond et al. (1983) Biotechnol.1: 262−
269 on the construction of "mini-Ti plasmids"
Has been reported. In nopaline T-DNA, the restriction enzyme K
Usually only one site is cleaved by pn I
You. Create a mutation that deletes the site
A Kpn I fragment containing T-DNA was isolated. This fragment is Kana
Inserted into pRK290 together with the mycin resistance gene,
Fruit, Agrobacterium tumefaciens (A.tu
mefaciens) and can retain most of the non-T-DNA Ti sequences.
This resulted in a plasmid that deleted almost everything. By itself,
This plasmid cannot transform plant cells.
won. However, the octopine Ti plasmid carrying
Placed in G. tumefaciens
And the tumor is induced to induce both octopine and nopaline
Was synthesized. The mini-Ti plasmid also has its own T-
When complemented with a DNA-deleted Ti plasmid,
Cells have been introduced. These results indicate that non-T-DNA
Ability to act on T-DNA and trans, deleted nopaline Ti
Rasmid function complemented by octopine Ti plasmid
And nopaline "mini-Ti plasmid"
It has been shown to function in the transformation of target cells. Each
Having octopine T-DNA or onc gene
A similar pair is A. Hoekema et al. (1983) NatureThree
03: 179-180.   Chilton et al. (18 January 1983) 15th Miami Wint
er Symp. is the nopaline synthase gene and the right and left borders.
To delete substantially all T-DNA except for the kingdom,
“Mi-Ti” created by subdivision of “Mini-Ti” using Sma I
We reported on the construction of "clo-Ti".
Inserted into the modified pRK290 plasmid, which deletes the I site,
Used in a manner similar to the Ni-Ti plasmid,
The fruit was obtained. G.A.Dahl et al.US application ser.n
o.532,280 is the T of the octopine-type plasmid pTi15955._LTerritory
Micro-Ti plasmid carrying ocs gene constructed from
Smid has been announced. Overview of the present invention   The method for genetically modifying cells of the present invention comprises the steps of (a)
Has the target promoter region / foreign structural gene conjugate
Transforming a prokaryotic cell with a DNA molecule, resulting in
Detecting the expression of this conjugate in a prokaryotic strain that has been
And (b) dual purpose promoter region / foreign structural gene
Transform plant cells with the DNA molecule containing the conjugate.
The expression of this conjugate in plant tissues that have emerged
Process. The plant of the present invention comprises: (a) a dual purpose plant;
DNA containing the promoter region / foreign structural gene conjugate
Transformation of prokaryotic cells with offspring
Detecting expression of the conjugate in an organism strain; and
(B) Binding of dual purpose promoter region / foreign structural gene
Transforming a plant cell with a DNA molecule having a body
To detect the expression of this conjugate in plant tissue
Inherited by a method of genetically modifying cells, including
Qualified. The DNA vector of the present invention can be identified
One or more dual purpose promoters that can confer a phenotype
Vector having a promoter region / foreign structural gene conjugate
The conjugate is transformed with the vector
The vector that confers an identifiable phenotype on the bacterial strain
It is the only means. The plant tissue of the present invention has an identifiable table.
One or more dual purpose promoters that can give a genotype
Vector having a chromosome region / foreign structural gene conjugate
Wherein the conjugate was transformed with the vector
Only those vectors which confer an identifiable phenotype to the bacterial strain
One means is to include DNA derived from a DNA vector. Book
The plant of the invention may also provide an identifiable phenotype.
Or more dual purpose promoter regions / foreign structures
A DNA vector having a gene conjugate, wherein the conjugate
Can be identified as a bacterial strain transformed with the vector.
DNA is the only means for the vector to confer a phenotype
It is derived from a plant tissue containing DNA derived from a vector. Sa
Furthermore, the bacterial strain of the present invention may confer an identifiable phenotype
One or more dual purpose promoter regions / foreign
Bacterial strain containing DNA vector with structural gene conjugate
Wherein the conjugate confers on the strain the identifiable phenotype
The only means, including DNA vector.   One of the objects of the present invention is that the gene
And is exogenous and not expressed in other ways,
Expression of structural genes in both plant and bacterial cells
To provide a way to promote it. For this purpose
In the execution, the dual purpose promoter region / foreign structural remains
A gene conjugate is provided. It is shaped by its union
Confers an identifiable phenotype on transformed cells
In plant and bacterial cells linked to foreign structural genes
Is a DNA sequence that can restrict the transcription of the structural gene. Already
One of the purposes is to make the protein encoded by the foreign structural gene
And, if it is an enzyme, its gene
Is seen in cells that have
Metabolites or compounds not found or found
Special plant tissues that are retained or deleted or
To serve plants. For other purposes, eukaryotic organisms
Predictions in prokaryotes of structures designed to
Providing a method for pilot experiments and, alternatively, for selection
And thereby both eukaryotic and prokaryotic transformations
The purpose is to provide a method by which cells can be identified. It
Other purposes and advantages will become apparent from the following description.
Will.   The present invention conserves introduced and expressed foreign structural genes.
A plant consisting of genetically modified plant cells
Offer. And the foreign structural gene is easy to understand.
For example, a transcriptional regulatory element capable of expressing T-DNA in a plant derived from 1450bTx.
Expressed under row control. Furthermore, the present invention relates to T-DNA
Transcriptional sequences that can be expressed in some plants
Direction that can be expressed in plant cells under the control of rows
And genomes containing foreign structural genes inserted at intervals
A plant tissue comprising a plant cell having Also, T−
A new strain of bacteria that retains and replicates DNA is provided
You. And the T-DNA is plants and bacteria derived from T-DNA.
For promoter regions that can be expressed in plants,
Can be expressed in bacterial cells under the control of the above promoter region
Foreign structural inheritance inserted in the orientation and spacing such that
It is qualified to hold children. In addition, books
The invention retains the ability to replicate in bacteria and contains T-DNA.
In addition, a new and convenient tool for transforming plants
A new vector. In addition, the vector is
Plant cells under the control of the native promoter region or
The T contained in the vector so that it can be expressed in bacteria
-Contains a foreign structural gene inserted into the DNA. Even better
Also disclosed are bacterial strains carrying the above vectors.   The experimental work presented here is for both eukaryotic and prokaryotic organisms.
That cause transcription of a single copy of the structural gene
Describes a DNA molecule with motor activity. Dual purpose
The effectiveness of the promoter / regional foreign gene combination
A person skilled in the art of transforming a product
And to facilitate other manipulations of the DNA sequence.
Prior to transformation into eukaryotes, foreign structural inheritance in prokaryotes
The ability to express offspring means that the structure is correctly assembled
To verify that the recombinant DNA structure
It allows you to examine functionally. Genetic marker generation
This promoter region, when used to control
Yet other benefits of the area become apparent. Markers are plants
For selective drugs that are toxic to both bacteria, eg antibiotics
Promoter or resistance
Single DNA sequence containing region and foreign structural gene is transformed
Identify or select whether the replacement cell is bacterial or plant-derived
Can be used for Dual purpose promoter region /
Structural gene conjugation means that the conjugation
For the expression of identifiable phenotypes conferred by cells in cells
This is particularly useful if this is the only method. Two
Purpose, ie selection (or selection) in plants and
Single DNA sequence for selection (or selection) in bacteria
The use of sequences can be used for DNA carrying such markers.
It is possible to reduce the size of the molecule,
To facilitate recombinant DNA manipulation and cell transformation procedures
You.   The present invention provides a method for controlling a dual purpose promoter region.
Foreign structural gene and a polyadenylation site,
Binding of the above promoter / gene / polyadenylation site
Can be applied to cells by any method known to those skilled in the art.
Inserted. More clearly, to be clear, here
The present invention as represented, after introduction via T-DNA,
The foreign structural gene is controlled under the control of T-DNA 1450bTxPR and
Insert before the polyadenylation site and use known methods
By introducing T-DNA containing the insert into plant cells
Transcriptional regulatory elements that can be expressed in certain T-DNA-derived plants
Sequence, ie, foreign structural gene under the control of 1450bTxPR
Further includes expression in plants and bacteria. Once, two
Generating foreign structural genes under the control of the original target promoter region
Once the plant cell to be obtained is obtained, it is well known to those skilled in the art.
Using methods and techniques, from which plant tissues and plants
The whole can be played. The regenerated plants are then
Genes that are propagated and transduced in the usual way
Transfer to other strains and cultivars using normal plant breeding techniques
Can be. In principle, the present invention relates to foreign DNA (for easy understanding)
To put it another way, T-DNA) can be introduced using the DNA
To any plant that can exist stably replicated,
Applies to any introduction of a foreign structural gene. one
In general, these classifications are currently not limited to this
Igari, sunflower (compositae), tobacco (solanasa)
C), purple coconut palm, soybean, and other beans
Family (Regminosaceae), cotton (Marbassaceae),
And most plants.   The present invention relates to useful structural genes from other species, organisms and strains.
Inserts bacteria, plant cells, plant tissues,
It is also useful for genetically modifying whole plants. So
However, useful structural genes such as
But not to communicate an identifiable phenotype such as:
Genetics included: improved against extreme heat or cold
Resistance; anaerobic conditions (eg, flooding), drought, or immersion
Improved resistance to osmotic pressure; insects (eg Bacillus
Insecticidal poisons like the crystalline protein of Thuringiensis
Element), spiders, nematodes, or parasitic pests,
Improved resistance to fungal or viral diseases
Or tolerance; yeast that is not normally present in the tissue or plant
Production of elemental or secondary metabolites; improved nutrition (eg
For example, lectin, or zein or phaseolin
Storage protein), flavor (eg, thaumatin)
Sweet protein), or fiber or human or animal food
Properties in the processing when used; altered forms
Scientific characteristics or modes of growth (eg, from insects)
Leaves to protect plants, discoloration and change pleasing to aesthetics
Growth habits of corrected plants, corrective plants, it takes to mature
Reduced time for gene expression or communication in tissues
Expression at a time when it is not normally expressed, etc .; male sterility;
Improved photosynthetic capacity (including reduced light respiration);
Nitrogen fixation; improved nutrition intake; against plants
Improved resistance to harmful substances (eg glyphosate
Or triazine); increased cereal yield; to other plants
Improved competitiveness; genetically modified cells
A new genetic marker; One genetic marker
Or more characteristic nucleic acid sequence; protein, heredity
The presence of offspring products or phenotypes that were somehow identified
Can be used to improve the identification of germ cells by
Wear. Genetic markers can be genetically linked or
When transformed, artificially introduced DNA sequences, or other
Phenotypic transfer to other genotypes by (eg, sexual) methods
To facilitate movement or by patent or plant line
Promote the identification of plants protected by protection certificates
Is an unmodified plant, plant cell, or bacterium
Et al., The genetically modified plants and plant cells of the present invention.
Vesicles or bacterial cells can be distinguished. If cell
Resistance to selected drugs in tissue culture (or
Is resistant) (ie selectable marker), and during selection
Easily recognizable markers (eg clear cell surface
Easily recognizable, like the original or enzyme, or visually
(A selectable marker such as β-galactosidase).
It is also a useful genetic marker. Neomish from Tn5
Encodes phosphotransferase II (NPT II)
The effect of kanamycin and its analog compounds
Genes that confer a resistant phenotype (kan
), A promoter that naturally controls 1450bTx expression
The invention is illustrated by being under the control of the
You. The promoter / kan conjugate is characterized by this conjugate.
Can be used for detection and selection of transformed cells
You. As will be appreciated by those skilled in the art, eukaryotic and prokaryotic organisms
Choose any DNA sequence linked to this conjugate
Cells transformed with the linked DNA sequence
Therefore it will be identified. Lectin structural gene 14
By placing it under the control of the 50bTx promoter region,
The invention is further illustrated. Lectins are nutritionally important,
Phaseolus vulgaris (legume) seed cotyledon tampa
It is. Introduction and expression of lectin structural gene
Increases protein content and changes the nutritional value of various cereals
Can be used to Other uses of the invention
Develop characteristics of other structural genes introduced into various plant species
Emissions will be readily apparent to those skilled in the art.   The present invention further provides for transcription under the control of the 1450 dTx promoter.
Sub-Ti plasmid containing the foreign structural gene
It is.   Direct repeats involved in incorporation of T-DNA into plant genome
Sub-Ti substrate having a sequence and one or more opine synthesis genes
The use of rasmid has the following advantages: 1, onc genetic
Transformed tissue culture or pro
It is easy to obtain regenerated plants from toplasts. 2, opin
Synthetic genes are used to identify plant cells or tissues with integrated T-DNA.
Can be used to determine
Other articulated or co-transformed
The gene that is). 3, plant cells are T_L− DNA alone, T_R
-DNA alone or T_L, T_R-Transformed with both DNA
You. In transformed plant cells, T_R-See multiple copies of DNA
Is actively transcribed, so T_L-All or one piece of DNA
The deletion of the part is due to the absence of various onc genes,
Is efficiently expressed. 4, The sub-Ti plasmid is small.
Therefore, many operations required for transformation are simplified.
it can.   FIG. 1 shows S.J.Karcher et al. (1984) Mol.Gen.Gene.
T-DNA region of octopine-type Ti plasmid obtained from T.
FIG.   BamHI fragment 30 'is located between BamHI fragments 8 and 30.
You. The line under the restriction map is E9 tobacco tumor
The DNA region contained in the tumor cell lineage is shown.   Fig. 2 shows the T paper taken from Karcher et al._RDNA
Is a restriction map of 5 T_RTranscript location on map
And the polarity. The restriction enzymes shown are BamH I,
EcoR I, Hinc II, Xba I, Sst I, Cla I, Hind III, Pst I,
And Sal I. T by the above enzyme_RAll cuts in
The site is not included in the map.   FIG. 3 shows the DNA experimental procedures used in Examples 2.2 to 3.4.
It is a schematic diagram of the work, and the size is not accurate. To restriction enzymes
Therefore, the site to be cleaved indicates the name of the enzyme. To restriction enzymes
Therefore, the site that is no longer cleaved is the name of the restriction enzyme.
This is indicated by parentheses before and after. Promoter
Or the size and polarity of the structural gene are indicated by arrows.
Was. Plasmid names should be written circularly
I wrote in that time. “Ex” means an example, a special
The operation is described. These uses are illustrated in FIG.
I used it.   Figure 4 shows the 1450bTx promoter region and NPT II structural inheritance.
A graph showing the growth characteristics of bacterial cells having an offspring conjugate
It is. This promoter region is
It is active in mu
Combination with the gene confers kanamycin resistance.   Fig. 5 shows the outline of the DNA experiment procedure described in Example 6.1.
It is a schematic diagram. Detailed description of the invention   The following terms are defined in the patent specification and claims.
Ambiguous range of meaning and meaning of these terms in the range
It is for removing the height. Promoter; located at the 5 'end of the structural gene
Sequence involved in the initiation of transcription. The present invention has two
Promoter activity (eukaryotic promoter activity and prokaryotic promoter
Activity is present in the promoter region of the T-DNA sequence
It consists of doing. As a result, the foreign
Transcription of the structural gene DNA sequence in eukaryotes and prokaryotes
That is, the smell of certain plants and Gram-negative bacteria
It became possible. Naturally, the T-DN illustrated here
A promoter region sequence in the crown gall of 1450bTx
Causes transcription. Expression under the control of the promoter is directly
It may take the form of indirect expression or fusion protein expression. former
In the type, the structural residues that are normally governed by the promoter
For removing part or all of the gene and inserting foreign structural genes
Therefore, it is replaced. And the initiation codon is T-DNA structural inheritance
The remaining part of the child or the inserted structural remains
The one of the gene is used. In the latter type, the existing T-DN
A Make sure that the reading frame fits within the structural gene.
The whole or a part of the gene is inserted. At this time, the translation
The product is a fusion protein. Eukaryotic promoter sequence
Is about 10-30 bases of the 5 'end (cap site) of mRNA
5 '... TATAA ... 3' on the pair (bp) 5 'side
The presence of a DNA sequence homologous to the basic sequence
It is commonly recognized. About 30 bp 5 'of the TATAA sequence
Another promoter of this sequence 5 '... CCAAT ... 3'
You can see the sequence. Initiation of translation is generally performed at the cap site.
The most efficient is from the first 3 'AUG. Polyadenylation site; this term is transmitted in eukaryotes
Nucleic acid sequence capable of inducing polyadenylation of RNA (mRNA)
Means That is, after transfer is completed, polyadenylic acid
A "tail" is added to the 3 'end of the pre-mRNA. Poly
The DNA sequence at the adenylation site may be natural or artificial, prokaryotic,
Or eukaryotic genomic DNA or cDNA derived from mRNA.
It is a mixture of sequences derived from the report. Polyadenylation unit
The position has homology to the basic type 5 '... AATAAA ... 3'
Is recognized by the presence of the DNA sequence. However, transcription
Partial variation in distance from 5 'to 3' end of product
It is not uncommon for "read" and tandem duplication of basic sequences. “Po
The basic type of “readenylation site” is polyadenylation itself.
Determine the position of the 3 'end of the mRNA, not the position of the body
(N. Proudfoot (1984) Nat
ure307: 412-413). Transcription regulatory sequence; this term is adjacent to a structural gene
Refers to the promoter / polyadenylation site combination
You. Promoter adjacent to a specific foreign structural gene
And polyadenylation DNA sequences are genes of the same origin (eg,
For example, a set of two T-DNA transcripts) or
Genes of origin (for example, sequences derived from T-DNA
Products, molds, yeasts, eukaryotic viruses, bacteria and synthetic sequences
A set of non-T-DNA derived DNA sequences)
There is no. Foreign structural gene; the term is used here for foreign RNA, protein
Quality of the gene including the DNA sequence encoding the polypeptide
Means a part or a part of gene including translation initiation codon
I do. A foreign structural gene is a cell into which the gene has been introduced.
May encode a gene product not normally found
is there. The term also refers to the genetics that naturally exist in the cell.
A copy of a structural gene introduced artificially, even in the offspring
Also means Foreign genes include prokaryotic DNA, eukaryotic DNA, and episomal DNA.
DNA, plasmid DNA, plastic DNA, gene DAN,
cDNA, viral DNA, viral cDNA, chemically synthesized DNA, etc.
Part or whole origin. The foreign structural gene is
Has one or more modifications at the coding or untranslated site
Are also considered. This modification affects the biological activity of the expression product.
Effects on the sex, chemical structure, rate of expression and mode of expression regulation.
It is thought that it will be lost. This qualification may be one or more
Mutations, insertions, deletions, substitutions, and expression products of the above nucleic acids
Localization and translocation of expression products without changing the chemical structure of
"Silent" affecting transport, secretion or stability
Including, but not limited to, modifications. Structural genes are coordinate
May consist of only the
One or more points, synthetic or natural
May contain Ron. This intron is appropriate
Functional splices at both ends. Structural gene
Is a constituent protein, whether chemically synthesized or natural.
It consists of a site that encodes white matter. This constituent protein
Is external to cells in which the gene has been introduced and expressed.
Or is partially derived from a foreign protein.
You. Foreign structural genes are fusion proteins, especially transcription regulatory sequences.
Fused with part or all of the structural gene from which
May be available. Dual purpose promoter region / foreign structural gene conjugate; book
The term is used for multiple promoter activities, such as eukaryotic promoters.
And prokaryotic promoter activity
Means a foreign structural gene. This promoter activity
The sex sites need not overlap, ie, eukaryotic and
Prokaryotic promoter activity is not physically identical
May or may be. For example, eukaryotic promoters
DNA sequences with different activities and prokaryotic promoter activities
May be on top. The distance between both promoter activities is
In the present invention, the relationship between prokaryotic promoter activity and structural genes
Unless there is a prokaryotic transcriptional endpoint in the
Eukaryotic mRNA transcription termination between the site and the coding sequence
Insignificant as long as no signal is present. Plant tissue; the term includes the differentiated and undifferentiated tissue of a plant. This
Like roots, foliage, pollen, seeds, crown gall
Plant cultures such as embryos and calli as well as tumor tissues
Various aggregates of cells are also meant. Plant tissue is a plant
In or in organs, tissues or cultured cells
U. Plant cells; in the context of plant cells in plants, in plant culture
Plant cells and protoplasts. Bacterial cells; for use herein as biologically pure cultures and
Including but not limited to those dispersed in the environment
Includes cells in culture.   The definition of the DNA fragment is defined in FIG. 1 and FIG.   Generates a dual purpose promoter region / foreign gene conjugate
The generation of the resulting genetically modified cells is disclosed herein.
Special knowledge and various technologies and means in the field.
It is a combination. In most cases, the entire program
Other means exist at each stage of the process. The choice of means is two
Basic measures for the introduction and stable maintenance of the original target conjugate
Selection of the plant system, the plant species to be modified and the desired regeneration
The means and special foreign structural genes or
Depend on variables such as the motor
Can be selected and used by the vendor to achieve the desired result.
There is another process step. For example, a dual purpose promotion
The starting point for obtaining the data region is pT
Illustrated in T-DNA isolated from iA6, other phases
Appropriate modifications are promoted in the DNA sequence of the homosexual Ti plasmid.
As far as isolation and manipulation procedures of the
You can. (Often pTi15955 is used without modification
It is. ) Binary not derived from 1450bTx gene shown here
The promoter region of interest may be found or constructed
U. Homology genes may be determined by one of ordinary skill in the art under conditions of appropriate stringency.
Under the cross-hybridizing ability of homologous nucleic acids, or
Identified by nucleic acid or protein sequences familiar in the art
Will be. Genetics used or disclosed in this application
It is understood that there are slight sequence variations within the child
Would. These variations are standard techniques that can be manipulated by those skilled in the art.
And the promoter of such homology gene
-Area will be available. Exogenous genes to plant cells
If new means for stable insertion of
In order to achieve the desired result, the
You will be able to choose between stages. The basic aspect of the invention is binary
Properties of the target promoter region and a single copy of the foreign structural gene
To produce expression in prokaryotic and eukaryotic regenerants
Is its use. Another aspect is the sex of foreign structural genes
Quality and structure, and insertion and expression in bacterial and plant genomes
There are means for Getting Genetically Modified Plants
The remaining step in the implementation that is desired for
Insertion of 450bTxPR / structural gene conjugate, monitoring bacterial expression
Visual and modified T-DNA stably as part of plant cell genome
Transfer and transformation of modified T-DNA into plant cells to be incorporated
Stages of selection and detection of transgenic plant cells, and first transformation
Of transgenes and other coexistence from cultivated strains to practical cultivation
Including the step of transferring the transferred or simultaneously transferred DNA sequence.
Techniques for in vitro culture and regeneration to whole plants, and
There is monitoring of expression in transgenic plants.   The basic aspect in its desired embodiment of the present invention is the insertion
Gene is under the control of 1450bTxPR, ie
T between the active promoter region and the polyadenylation site
-The construction of DNA derivatives, these terms being defined above
Was. Structural gene is in correct position with respect to promoter region
And must be inserted in the direction. There are two perspectives on location
is there. First, the structural gene is inserted on either side of the promoter.
It is about whether it is entered. Most promoters
Controls initiation of transcription and translation in only one direction along DNA
It is known that DNA under promoter control
The region is "downstream" of the promoter, or other expression
It is said to be "after" or "on the 3 'side." Accordingly
In order to be controlled by the promoter,
Correct Insertion Position Should Be "Downstream" of Promoter
Must. A second aspect of location is the promoter
Known functional elements, such as the transcription initiation site,
It relates to the distance in base pairs between translation initiation sites.
The promoter has substantial variation in this distance.
It seems to be. Therefore, the structural requirements in this regard
Is best described by the word functional. the first
Approximately, sensible action is based on promoter and insertion
Promoters in which the distance between foreign structural genes normally controls
Obtained when the distance between the protein and the gene is close. Orientation
Is related to the direction of the structural gene. Eventually
Part of the structural gene encoding the amino terminus of the foreign protein
Is called the 5 'end of the structural gene, while the protein carb
The end encoding the amino acid near the xyl end is a structural gene
Called the 3 'end. The correct orientation of the foreign structural gene is
5 'end near the promoter. Fusion protein
Additional requirements for expression-driven construction are:
The fusion of two structural genes means that the coding sequences of the two genes are the same.
Must be in the same reading frame phase
And the structural requirements are well known in the art.
I have. Exceptions to the requirement of this phase are two introns
When the coding sequence derived from the structural gene of
Exist. In that case, both structural genes are compatible splices
Site must be retained, and intron
The chair part has the correct reading frame, but the intron
Is removed by post-transfer processing,
It must be in a position that will be preserved in the stage. Departure
Differences in control of current speed or development are due to another foreign structural gene
50bTxPR derivative or other dual purpose promoter region
Will be observed when inserted under control. Expression speed
Degree also depends on the secondary structure of the resulting mRNA, especially the stem-loop structure.
It is greatly influenced by the details of the structure. Familiarity in the field
In addition, the translation rate in prokaryotic cells depends on the AUG translation initiation site.
5 'ribosomal RNA binding sequence (J. Shine and L. Dalga
rno (1974) Proc.Natl.Acad.Sci.USA71: 1342-1346)
And will be affected by the presence of
Translation speed of a special nucleotide near the AUG (M. Kozak
(1981) Nucl.Acids Res.9: 5233-5252)
Will be. Stability of the expressed protein itself, between cells or
Subcellular localization or secretion, solubility, target specificity, and
Properties that include and are not limited to other functional properties
Differences in the insertion site and fusion protein in the case of the fusion protein.
Length and properties of the foreign protein part
It will be observed that it depends on the effect of the folding structure
And all of them contain plant cells, plant tissue and
Depending on the physiological properties required of the whole plant
Manipulate and control the functional properties of protein products
There are numerous opportunities. Similar to the promoter region, poly
The adenylation site is also correct for the 3 'end of the coding sequence.
Must be in position and direction. Foreign structural gene
Provides the origin of the 3 'end of the protein and the polyadenylation site
Fusion proteins between polypeptides encoded by different DNA
It is possible.   As will be appreciated by those skilled in the art,
Some sequences to be compatible with translation and transcription functions
More specifically used Cla I promoter / structural inheritance
Replaced by child structure. Of the fragment that carries the structural gene
The restriction sites at the 5 'and 3' ends may be the same
It may be different. Specific at both ends of gene fragment
The use of sites with cohesive ends of different sexes is automatically configured.
The gene in the correct orientation after 1450bTxPR.
And If the restriction site has an incompatible end,
Convert them to blunt ends using methods well known in the art
And the blunt ends are ligated together. Suitable linker, ada
The use of a putter or coupler is also compatible with 1450bTxPR.
In some cases, it is effective in forming connections between
And this has been proven to those skilled in the art and is shown again here.
You.   People who actually perform plant transformation have structural genes
It is expressed in bacterial hosts as well as in plant cells
You will notice a number of situations. However, transformed cells
As genetic markers for identification and / or selection of
Use of 1450bTxPR to bring about expression of a functional gene
Is particularly useful and is exemplified here (examples
4). Transformation of cells, both prokaryotic and eukaryotic, is
Special recombinant D that retains Kerr and contiguous DNA sequences
A method for easily identifying cells transformed by NA molecules
This is most easily achieved when Single marker
The use of 1450bTxPR for effective expression is
Eliminate the need to introduce a second marker into the conversion vector
Let Furthermore, the absence of a second marker is important for transformation.
Vectors can be miniaturized to make them, and DNA
This results in ease of operation and an increase in transformation efficiency. I
However, if a special marker is placed after 1450bTxPR,
As is known in the art, a positive effect on gene rearrangement
You have to be careful about homologous sequences with other parts of the mid
No. For example, T_Lonc gene and T_R1450bTxPR / kan combination
The kan gene used to select for deleted homozygous diploids
The offspring have undergone recombination and have no intervening T-DNA sequence
Produces a reversal. More than 1 copy of 1450bTxPR
Used to effect expression of a large number of different foreign structural genes
The same is the case when you have to pay attention.   As will be apparent to those skilled in the art,
Region / foreign structural gene conjugate is the plus
Any convenient way to remove the combination from the mid
Restriction sites and plant transformants or selected shuttle vectors
Between the restriction sites convenient for insertion into the
U. The position of the insertion site of the dual purpose combination in T-DNA is
The transfer function of the sequence at the adjacent T-DNA boundary is not impaired
As long as it is not strict. Because, in previous research,
These regions are used to insert the modified T-DNA into the plant genome.
It seems to be essential to the entry. The preferred insertion site is
Also actively transcribed regions, especially T_R, And 1450bTx
It is in the area that includes. The dual purpose combination is inserted
T-DNA can be obtained from any TIP plasmid, and
This conjugate is inserted by standard techniques familiar to those skilled in the art.
You. Transcription and translation of endogenous T-DNA or vector genes
The orientation of the inserted plant gene with respect to orientation is not exact,
Both of the possible directions are functional. Expression speed in plants
The difference in degrees is due to the fact that a gene is inserted at a different position in T-DNA.
And DNA methylation and
And chromatin structure.   Binary target conjugate and any desired linked DNA
-A convenient method for insertion into DNA, as described in the background section
First, the plasmid was cloned into Escherichia coli.
Small pieces of T-DNA inserted (there is a desire to insert between these pieces)
Are available. this
A piece of T-DNA is a restriction site, preferably a shuttle vector.
Including special parts in the Binary target conjugate is T-DN
It can be inserted at a special site in the A sequence, and
Shuttle vector is a suitable Agrobacterium strain,
If possible, the T-DNA will be combined with the T-DNA fragment of the shuttle vector.
Transfer to cells with homology. Transformed agroba
Cuterium strain shuttles existing small pieces of Ti plasmid
Double homologous set such that it is replaced by a small piece of T-DNA in the vector
Under conditions that allow the selection of the transmutation (homopartial diploidization) phenomenon
Propagate if possible. However, the present invention does not
Is limited to a double homologous recombination mechanism
It should be noted that there is no shuttle vector (probably
Has only one continuous region of homology to T-DNA)
Single site homologous recombination (co-integration), with
Bacterial transposer with promoter region / structural gene
Dzon insertion is also effective in inserting this conjugate into T-DNA
Means.   In accordance with the policy just described, apply modified T-DNA
Can be transferred to plant cells by any technique in the field.
You. For example, this translocation may be a foreign structure incorporated into T-DNA.
Of Agrobacterium strains
Direct infection or plant cells and this Agrobacterium
Most easily achieved by either co-culture with the strain
You. In the former technique, direct infection, the tumor site or
Brings the appearance of a crown goal. Crown gall cells
Can then be grown in culture, and
Complete plants containing inserted T-DNA fragments under known suitable circumstances
Can be regenerated to the body. The technology of co-culture enables plant cells
Is transformed, ie, the transferred T-DNA is
And inserted into the plant cell genome. In both cases,
Transformed cells are selected or distinguished from non-transformed cells.
I have to. Selection is in addition to TxCS / foreign structural gene
To prepare a selection marker incorporated into T-DNA
More easily achieved. Examples of published markers and
And methotrexate-resistant dihydrofolate resin
Dactase or nopaline synthase promoter
Neomycin phosphotransfer expressed under control
There is Rase II (NPT II). Each of these markers
Methotrexate or kanamycin or its
Selected by growth on a Nalog-containing medium. Heavy metal io
Toxic effects of metallothinine are reduced by the presence of metallothionein.
obtain. In fact, the invention is suitable for the selection of transformed plant cells.
Selection marker, 1450bTxPR / NPT II structural gene conjugate
An example of creation was given. In addition, T-DNA is
Ri, a gene that controls hormone-independent growth of induced tumors
-Genes controlling abnormal morphology of induced tumor roots, and
Controls resistance to toxic substances such as amino acid analogs
Gene, such resistance can be determined by opin synthase (eg, oc
s), but possesses intrinsic markers such as
I do. Selection methods familiar to those skilled in the art include analysis of opine production,
Specific hybridization to characteristic nucleic acid sequences
Or ELISA (“enzyme linked immunosourcing”
"Bunt assay"), radioimmunoassay,
Specific protein immunity, including and Western blots
Includes, but is not limited to, epidemiological analysis. Further out of expression
The phenotype of the foreign gene can also be used to identify transformed tissues
Yes (for example, antibiotic resistance or Bacillus
Insecticidal activity of Ngensis crystal protein).   Dual-purpose conjugates inserted as an alternative to shuttle vector tactics
Containing modified T-DNA or modified T-DNA,
There is the use of plasmids that can replicate independently in Lium strains.
Recent evidence in the background suggests that Agrobacterium strains
-Has the function of promoting the transfer of DNA to plant cells.
Including trans-acting genes, such plasmids
Transfer of plant cells from Agrobacterium strains to plant cells
showed that. Contains T-DNA and is among Agrobacterium strains
The plasmid that can be replicated independently at
Or called a sub-Ti plasmid. Sub-TIP Plasmi
Changes in the amount of T-DNA it contains
Exists. Is one extreme of this variation a TIP plasmid?
It retains all of their T-DNA and is sometimes
-TIP "or mini-Ti plasmid.
Extreme is that all but the amount of DNA near the T-DNA border is deleted
In the remaining part, the sub-TIP plasmid is transferred to the host cell.
Is the minimum necessary to be captured. This
Plasmids such as "Micro-TIP" or Micro-Ti
Call. Sub-TIP plasmids are small and directly manipulated
Is relatively easy, and shuttle vector
The advantage that there is no need to transfer genes from
There is a point. After inserting the desired structural gene, the T-DNA
Plants containing a trans-acting vir gene that facilitates translocation
It can be easily introduced directly into cells. Agrobacterium strain
Introduction to the technology is well known to those skilled in the art,
Transformation of terium strains or conjugation transfer from donor bacterial cells
Easily accomplished by any of New DNA sequence
For the purpose of introduction into the plant genome, TIP plasmids and
And sub-TIP plasmids are considered functionally equivalent.
Should be. Example 6 generally uses T_RBased on
Disclosure of the TIP plasmid and further discussion
U.   A preferred embodiment of the present invention is a dual-purpose promoter region.
/ The foreign structural gene conjugate to the plant to be transformed
Agrobacterium based on T-DNA for introduction into the genome
In the lium-mediated system, but the transition and incorporation of this conjugate
Other means for this are also included in the scope of the present invention. Second element
For stable integration of a genetic conjugate into the plant genome
Means include virus genome, mini chromosome
Homologues to chromosomes, transposons and plant chromosomes
There is the use of vectors based on heterologous recombination.
Not limited to Another introduction of these vectors into plant cells
The method may be liposome containing vector or bacterial sphero.
Fusion with plast, microinjection, virus coat protein
Encapsulation followed by a process similar to infection,
Of plasma membrane by laser, laser or chemical agent
Direct uptake of DNA after induction, but not limited to
No. Means of transfer uptake and / or expression are also provided by the present invention.
Included in the outlook. Agrobacterium cells and TIP
Based systems rely on the transfer of DNA from bacteria to plant cells.
Can be used to transform cotyledons; other vectors
Based systems or vector transfer means are monocotyledonous and
All gymnosperms, including cotyledons, and all angiosperms
Can be used for transformation of the product.   Regeneration of transformed cells and tissues achieved by known techniques
Is done. The purpose of the regeneration phase is normal, but incorporated
Obtain a complete plant that grows and proliferates while retaining T-DNA
That is. Regeneration technology follows T-DN
Depending on the origin of A, the nature of the modification and the transformed plant species
Changes to some extent. Transformed with Ri-type T-DNA
Plant cells can be obtained without any additional experimentation using techniques familiar to those skilled in the art.
Can be easily reproduced. Transformed by Ti-type T-DNA
Plant cells may, in some cases,
It can be reproduced by an appropriate operation. But hope
Alternatively, the Ti-transformed tissue may have a T-DNA of tmr and t-DNA.
Most mutations in one or both ms genes
Played easily. Inactivation of these genes is
Orient the hormonal balance of the tissue normally, and
In order to be able to manipulate the Lemon level very easily,
Regenerates easily due to its more normal hormonal physiology
Become a plant. If mutations in tmr and tms are shuttle vectors
Introduced into T-DNA by double homologous recombination using
If the mutation is introduced, the introduction of the mutation
Selection in a different manner than the introduction of the region / structural gene conjugate
It is important to note that this must be done. For example, before
Tmr and tms inactivation in chloramphene
Selection based on call tolerance
Foreign gene selection is performed with kanamycin resistance. tms
Inactivation of the tmr and tmr sites
Or insertion of one or more bases in the promoter
Insertion, deletion, or substitution, ie, inactivation of the promoter
Achieved by mutations that measure degradation or disruption of protein structure
Will be done. (Generation of suitable mutations is described in T.C. Hall et al.,
US application ser.nos. 485,613 and 485,614 and back
Examples are given in the literature cited in the scenery. In one example,
Tumor cells have integrated T-DNA and nopaline syn
Expresses a T-DNA gene such as a
Expresses the inserted plant structural gene.
Can be played. This shoot has roots
It can be maintained by vegetative cells by grafting to plants, and
To produce fertile flowers. This shot is this
Thus, the foreign structural remains having T-DNA inserted therein
Provide parent plant material for normal progeny plants that express the gene
Offer.   The genotype of the plant tissue to be transformed
It can grow and regenerate in vitro and is sensitive to the selective agent used.
As it is, it is often chosen for ease. Agriculture
Culturally interested crops are not suitable for this operation and
Variants are transformed first. After playback,
Introduced dual-purpose promoter region / foreign structural remains
Gene conjugates and any articulated and / or joint
Transferred DNA to easily cultivated agricultural cultivation, plant breeding
And techniques well known to those skilled in the field of plant genetics.
You. First by sexual crossing of agricultural and transformed plants
Hybrids are formed. These hybrids are then desirable genetic background
Backcrossed with plants with Descendants are always inserted foreign DN
Consecutive presence of A or caused by inserted foreign DNA
New phenotypes as a result of the expression of
Selection and / or sorting. Like this
Agriculturally desirable after multiple backcrossing and sorting
Have essentially the same genotype as the parent, and
Combined plants can be created. Example   In the following example, TIP and Agrobacterium
For those skilled in biology and manipulation,
Utilizes many of the techniques that have been developed and are easy to do; this
These methods are cited unless detailed here.
Detailed in one or more of the references
You. Enzymes are purchased from commercial sources and recommended by the seller or
Used by other variations of the art. Reagents, buffers and
Also, culture conditions are known to those skilled in the art. Such a mark
Reference materials including quasi-technology include the following:
R. Wu, ed. (1979) Meth. Enzymol.68, R. Wu et al. (1
983) Meth. Enzymol.100and101, L. Grossman and K.M
oldave, ed. (1980) Meth. Enzymol.65, J.H. Miller (197
2) Experiments in Molecular Genetics, r. Davis et a
l. (1980) Advanced Bacterial Genetics, R.F.Schleif
And P.C. Wensink (1982) Practical Methods in Mole
cular Biology, and T. Maniatis et al. (1982) Molec
ular Cloning. In addition, R.F.Lathe et al. (198
3) Genet.Engin.4: 1-56, useful for DNA manipulation
I have an explanation.   Under the name of isolated restriction endonuclease
Use in the text, for example “Bcl I”,
Sequence sites that are susceptible to the action of the enzyme
The enzyme in an enzymatic reaction, unless it represents a restriction site
Represents the use of In the text, the restriction site is called "site"
Additional use of terms such as the term "Bcl I site"
More displayed. Additional use of the word “fragment”,
For example, the “Bcl I fragment” is produced by the action of its naming enzyme.
Represents a linear double-stranded DNA molecule that retains its terminus (eg,
Restriction fragment). Phrases such as “Bcl I / Sma I fragment” are 2
On the action of two different enzymes, here Bcl I and Sma I
Indicates that a restriction fragment has been generated,
It has two ends generated as a result of the action of the resulting enzyme.
The ends must be “sticky” (ie, complementary
Can be paired with any single-stranded oligonucleotide,
Have single-stranded protrusions) or be “smooth”
Characteristics and the specificity of sticky ends
Is determined by the specificity of the enzyme that produces it.
Keep in mind.   Plasmids, and only plasmids, such as pT
The initials of “p” are added like i15955 or pUC13,
The name of the strain is, for example, Agrobacterium thymefa
Science (pTi15955) or Ethelicia Collie JM83
As in (pUC13), the parentheses indicate that the plasmid
Indicates that it is retained.   The following strains have been deposited: Etherica Collie K12 RRI (pRK290Kan-1)  NRRL B-15736 (this strain is a national
This is a deposit. ) Agrobacterium thiumefaciens (pTi1595
Five)                                  ATCC15955 Ethelicia Collie C600 (pKS4)                              NRRL B-15394 Etherica Collie HB101 (pPVL134)                                  ATCC39181   Other plasmids and strains are widely available to those of skill in the art and
Easy to obtain. Example 1   This example determines the location of the 1450 base transcript (1450bTx).
The results of transcription mapping experiments
Also teaches the method used to obtain the above results
You. These experimental results are essentially S.J.Karcher et a
l. (1984) Molec. Gen. Genet.,
It is included here as a background necessary to understand the invention.
You. 1.1 Results   Fig. 1 shows stable integration into plant DNA (T-DNA).
Restriction region of pTiA6, which indicates the region of the Ti plasmid
3 shows a nuclease map. Sub-fragment of BamHI fragment 2 (primary
(See map in Figure 2) is the plasmid pBR325, pMK2004, if
Or T-DNA cloned into pUC13_RCode to region
Localization of RNA transcribed in plant tumors
Used as a hybridization sample. E9
Suspension tumor cell line, ie, well known to those skilled in the art
Lactobacillus tumefaciens (pTiB₆806)
(M.F.Thomashow et al. (1980) Cell19: 729-739)
More stimulated Nicotiana Tabacam series, more isolated
Total cell RNA or poly A⁺Northern blot of RNA
The sample b_Five, C and d₁Hybridizes to about
An RNA of 1450 bases in size was discovered. This RNA is sample b_Four
And b_ThreeSeems to hybridize to a low degree
(FIG. 2).   1450bTx boundaries and orientation can be determined more accurately
S₁A nuclease mapping experiment was performed. S₁Nuku
One fragment was used as a sample for the lase protection experiment.
In both directions in the vector derived from the bacteriophage M13 chain
Cloned. (See Example 1.3b) Such M1
Using single-stranded DNA from 3₁Nuclease
As a sample for analysis, it is more effective than using double-stranded DNA.
there were. Hybridization of DNA and RNA at 65 ° C
It can be performed in an aqueous solution in a relatively short time.
Was. In addition, both strands of DNA are
Are separated, so S₁By RNA from nuclease degradation
Each strand can be tested separately for protection.
there were. Which cloned DNA strand is protected
And the diversity cloning site of M13
By determining the orientation of the cloned insert
Therefore, it was possible to infer the direction of transcription of RNA.
Since both strands in a given region are protected, bidirectional
A photo was shown.   Figure 2 shows the results of such an analysis using E9 total cellular RNA.
Is shown in d₁+ D_TwoS₁For nuclease protection sample
That 1450bTx is C and d₁Between Hind III
It was decided to start at about 240bp on the right. C and b
_FiveBoth fragments of the₁Nuclease degradation
They were completely protected. b_FiveIs S₁Nuclease protected sample
When used as a fragment, a fragment of about 280 bp is recovered.
Was. b_Four+ B_FiveIs used, fragments of about 20 bp or more
S₁Protected from nuclease degradation. From this data, 1
450bTx is_FourAnd b_FiveB just to the left of the Cla I site_Fourso
It was shown to end. 1.2 Discussion   Northern blotting and S₁Nuclease analysis
Octopine-type crown gall tumor
T of next T-DNA_RFive transcripts encoded by region
Has been localized. Polyadenylated RNA is a host plant
Promoter inside T-DNA, not from promoter
More transcribed. E9 tumor by Northern blot analysis
In the series, T including 1450bTx_RMost coded
The amount of transcript is T_LGreater than the transcript encoded by
There were many. Hence T_RFor use in plant genetic engineering experiments
Is interested in Because it is a powerful professional
Including motor, but directly involved in tumorigenesis
It is not.   Determination of the direction of transcription of these RNAs, and 5 'and
Localize 3 'end more than blotting experiments
S₁Nuclease mapping operation
Used. S₁1450 bases R from nuclease protection data
The gene encoding NA contains no detectable intervening sequences.
It was shown not to be. For Northern blotting analysis
The determined transcription size is S₁For nuclease analysis
It was larger than the indicated size. In size
This difference is easy due to the addition of poly (A) sequence after transcription.
It is explained in.   Northern blot and S₁Nuclease protection data
Is the DNA sequence of this region inferred by others.
Data (R.F.Barker et al. (1983) Plant Molec. Biol.
2: 335-350, R.F.Barker and J.D.Kemp.US Patent App
lication ser.no.553,786). Above
The direction and length predicted by the mapping experiment
Open reading frame with ORF24
Existed). In addition, during 1450bTxPR,
TATAA or goats involved in promoting eukaryotic transcription
Goldberg-Hognes Box
s box) (J.E.Darnell (1982) Nature297: 365-371)
There was a sequence similar to The TATTA box has an accurate
Has been shown to be required for in vitro transcription (B. Wasy
lyk et al. (1980) Proc. Natl. Acad. Sci. USA77: 7024-7
028) However, the sequence upstream of TATAA is an efficient transcription in vivo
It is known to be necessary. The sequence CCAAT is T
It is commonly found upstream of the ATAA box (C. Benoist et al.
(1980) Nucleic Acids Res.8: 127-142, A.Efstratiad
is et al. (1980) Celltwenty one: 653-668, T. Shenk (1981) Cu
rr.Topics Microbiol.Immunol.93: 25-46), 1450bTxPR
There were similar sequences in it. Near the 3 'end of the transcript,
Sequence signals required for proper determination of the 3 'end of many mRNAs
(N. Proudfoot (1984) Nature307: 412-413), AAT
The AAA hexanucleotide has three similar sequences
Exists. 1.3 Experimental materials and methods 1.3a Culture conditions   Crown Gall tumor lineage is 1.0% Fight Agar (M.
F.Thomashow et al. (1980) Cell19: 729-739)
MS3 without (for suspension culture) or added (for callus culture)
The cells were cultured at 25 ° C. under continuous light irradiation in the medium. Control and
XSR, a non-neoplastic tobacco line used as
Naphthalene acetic acid and 0.1 mg / benzyl aminop
The cells were cultured in an MS3 medium supplemented with phosphorus.   Escherichia coli strain carrying the recombinant plasmid
Is cultured in L liquid medium supplemented with 0.2% casamino acid.
Was. The antibiotic concentrations used were determined by Escherichia coli.
For: ampicillin, 50-100 μg / ml; tetracycline
Phosphorus, 10 μg / ml; kanamycin, 20 μg / ml; and agroba
For Cterium Tumefaciens: Kalbe
Nicilin, 100 μg / ml; Tetracycline 5 μg / ml; Kanama
Isin, 100 μg / ml; Rifampicin, 10 μg / ml; Gentama
Isine, 100 μg / ml. 1.3b Construction of recombinant DNA plasmid and M13   BamHI fragment 2 (FIG. 1) was known to those of ordinary skill in the art.
Agrobacterium tumefaciens by standard operation
Ens plasmid pTiB₆PB from 806 (cultured in strain A277)
Cloned into R322. The sub-fragment of BamHI fragment 2 is p
BR325, pMK2004, or pUC13 (F. Bolivar et al.
(1977) Gene2: 93-113, M. Kahn et al. (1979) Meth.
Enzymol.68: 268-280, and J. Messing (1983) Meth.En
zymol.101: 20-78). Restriction endonu
All of the creatase reactions are performed by the supplier (Bethesda Researc
h Laboratories (BRL), P.L.Biochemicals, or New
 England Biolabs). T4 DNA
Ligase was purchased from BRL. The recombinant plasmid is
Cleared lysate operation (D.G.Blair et al. (197
2) Proc.Natl.Acad.Sci.USA69: 2518-2522) or
Alkaline lysis procedure (N.C. Birnboim and J. Doly (1979)
Nucleic Acids Res.7: 1513-1523)
Separated from A Collie.   S₁Of the BamHI fragment 2 used in the nuclease protection reaction.
The region can be duplicated using techniques well known to those skilled in the art.
M13mp8 and M13mp9, which are functional forms of DNA (from Dr. N. Jones
M13mp10 and M13mp11 (P.L. Bioche
cloned in both directions into
Was. 1.3c RNA separation, electrophoresis, blotting, and
Hybridization   Tumor RNA was refrigerated 1.5 times after phenol extraction.
Add 0% ethanol and keep at constant temperature on ice for 15 minutes.
Except for precipitating the polysaccharide, it was described previously (S.
B. Gelvin et al. (1981) Plasmid6: 17-29)
Released. RNA is precipitated after centrifugation at 10,000 xg for 10 minutes.
Add 2 volumes of 100% ethanol to the supernatant
Was.   Agarose gel electricity by denatured formaldehyde gel
Electrophoresis, blotting on nitrocellulose, and
Hybridization is as described (Gelv
in et al. (1981) supra).
There was: gel containing 2% agarose, washing solution 1 × SSC
(0.15M sodium chloride, 0.015M sodium citrate),
0.1% SDS, and 10 mM Na_TwoIt contained EDTA. Nick To
The translation is Nick Tranley from Amersham
This was performed using an application kit. Nuclease protection analysis of 1.3d E9 tumor RNA   Hive between recombinant M13 single-stranded DNA and E9 suspended RNA
Redidation is 5 × SSC20-30μ, 20mM Tris-HCl
(PH 7.4) at 65 ° C. Typically, 500n
g of recombinant phage DNA was isolated from the E9 suspension culture.
And 20 μg of total RNA. Refrigerated S after 5 hours
₁Nuclease degradation buffer (280mM sodium chloride, 50m
M sodium acetate, 4.5 mM zinc sulfate, 20 μg / ml denatured calf thymus
DNA, pH 4.6) to a total volume of 150μ and 100 units of S₁Nuclea
(Sigma) was added. Incubate this sample at 37 ° C for 45 minutes
Was done. 50μ refrigerated S₁Termination mixture (2.5 M sodium acetate
50mMNa_TwoEDTA) and add 20 μg of the protected fragment.
Precipitation by addition of mother tRNA and 2.5 volumes of 100% ethanol
Let it settle.   After incubating at -20 ° C, collect the precipitate and dilute with a 20μ alkaline buffer.
Immersion liquid (30 mM sodium hydroxide, 2 mM Na_Two Dissolved in EDTA)
Then cut the fragments into 1.2% or 2.0% alkaline agarose
Gel electrophoresis (M.W.McDonnell et al. (1977) J. Mol.
Biol.110: 119-146). DNA nitrocellulose
Transfer, blot hybridization and washing
For purification, the sample concentration is usually 50 ng / ml or less,
Previously, except that it was only washed with 0.3 × SSC for 5 hours
(M.F.Thomashow et al. (1980) before)
Out). Example 2   This embodiment is suitable for octopi such as pTiA6 and pTi15955.
Plasmid T_R14 suitable for homopartial diploidization to
Teaching the construction of a mediator for the 50bTxPR promoter. 2.1 T_RCloning   BamHI fragment of p-TiA6 T-DNA in the BamHI site of pBR322
Two recombinant DNA clones were completely digested with EcoR I
(High homology with pTi15955 isolated from ATCC15955
PTi6A is Agrobacterium tumefaciens A
6NC). 5.4 kilobase pair (Kbp) DNA
The digestion mixture containing the fragment, EcoR I13, is linearized with EcoR I.
PRK290 DNA (G. Ditta et al. (1980) Proc. Natl. A
cad.Sci.USA77: 7347-7357)
And transform the mixture into Escherichia coli K12PR1
Changed. Plasmid DNA is transformed into tetracycline resistance
EcoR I13T- isolated from the transformant and named pRK290Eco13
Colonies containing plasmids containing DNA fragments are restricted
Identified by elementary analysis. 2.2 Deletion of 1450bTx structural gene   pRK290Eco13 DNA is completely separated by Cla I and religated
And transformed into PRI. Tetracycline resistance
Restriction analysis of plasmid DNA isolated from sexual invertants
And named pRK290Eco13 ΔCla
A colony carrying the isolated plasmid was identified,
Fig. 2b_Five, C and d₁Deletion of the Cla I fragment encompassing the fragment
Was. Foreign structural gene is 1450bTx promoter region
At the only Cla I site of pRK290Eco13 ΔCla
It can be easily inserted (Fig. 3). Of the Cla I fragment
Deletion causes the first polyadenylation site 3 'of 1450bTx
Position removed; but two other polyadenylations
The sequence of the site is maintained downstream of the only remaining Cla I site.
Have. 2.3 Substitution of other restriction sites for the Cla I site   The following shows the only Cla I part of pRK230Eco13 ΔCla.
The substitution by the Hind III site at position is described. pRK290Ec
o13 Isolate ΔCla DNA and completely degrade with Cla I
You. The resulting sticky end of Cla I is
By adding the Klenow fragment of Limerase I and keeping it warm
Buried, and Double-stranded linker with the structure
A blunt-end bond is made in the Cla I site. as a result
The resulting mixture was completely digested with Hind III, recombined, and
To transform into PR1. Transformation of tetracycline resistance
Plasmid DNA isolated from the recombinant was deleted and 1450bTx
Lack of Cla I site and Hind at structural gene location
Selection by restriction analysis due to the presence of the III site
The plasmid is named pRK290Eco13ΔClaHind.   As will be apparent to those skilled in the art, the Cla I site
By changing the restriction enzyme other than II to the desired specific sequence.
It is possible to substitute the above linker with another linker.
Wear. For example, With BamHI linker (obtained from BRL)
Experiments with d III linkers substituted, others essentially as described above
Incorporated during the procedure. Here, pRK290Eco13 ΔClaBam
B at the location of the deleted 1450bTx structural gene, named
An RR1 strain carrying a plasmid with an amHI site was identified.
Was. Example 3   In this example, the antibiotic was used as shown in FIG.
Kanamycin and its analogs, such as
In both plants and bacteria for neomycin and G418
The construction of a selectable marker that confers resistance is taught. 3.1 Preparation of kan gene   Neomycin phosphotransferase II
Bacterial transposon T encoding an enzyme_FiveKana of origin
The DNA sequence of the mycin resistance (kan) gene is E. Beck e.
t al. (1982) Gene19: 327-336,
Present on plasmid pKS4, which is Escherichia coli
(PKS4) isolated from NRRL B-15394. Bgl pKS4DNA
Complete degradation by II and Sma I
The NPTII-containing fragment of the base pair was already digested with Sma I and Bam
Mixed and bound with pUC13 degraded by HI. BamH
 The sticky ends of I and Bgal II have the same specific sequence (5'G
ATC ... 3 ') and both can be easily combined
But the resulting BamH I / Bgl II suture site,
IeIs not affected by either enzyme. Ery the binding mixture
Transformed into Shea Collie K12 JM83 (J. Messing (197
9) Recomb.DNA Tech.Bull.2(2): 43-48, NIH Publ. N
o. 79-99), and white-colored colonies were selected.
Plasmid DNA is separated from the selected transformants,
Characterization by restriction site mapping was performed. pU
Colonies containing a plasmid named C13KanBgl / Sma
Was identified.   The kan gene-containing fragment of pUC13KanBgl / Sma has kan structural inheritance.
PUC13 immediately upstream of the BamH I / Bgl II suture site
Polylinker (Polylinker is a restriction enzyme
(This is a short sequence that includes the site that is affected by the element)
Has an Acc I site inside. The kan gene is Sma I and
Degradation of the 1 Kbp DNA fragment by Acc I resulted in pUC13KanB
Removed from gl / Sma. In particular, this Acc I cleavage site
It has a sticky end of 5'CG ... 3 ', which is the enzyme Cla I
Can be easily bonded to the terminal generated by 3.2 Insertion of kan behind the 1450bTx PR promoter   pUC13KanBgl / Sma and pRK290Eco13 ΔCla were
Linearize by complete decomposition using c I and Cla I,
Ligated and transformed into E. coli RR1.
Ampicillin and tetracycline resistance (pUC13
And the sequence of pRK290).
Plasmid DNA characterized by restriction analysis
It was done. a plasmid named pRK290Kan-1
Was identified and the plasmid contained 1450bTx
Behind the promoter, the 1450bTx
Kan gene inserted in the same orientation and position as the code sequence
Was holding a child. The kan gene of pRK290Kan-1 is transcribed.
RNA polymerase II is located 3 'of the kan gene
Reaches the first T-DNA polyadenylation site at
Beforehand, all Tn5 and all pUC13 sequences must be transcribed.
I have to. However, on the 3 'side of the kan / pUC13 suture site,
There are other sequences that serve as polyadenylation sites. 3.3 Deletion of pRK290 from pRK290Kan-1   The plasmid on which pRK290 is based is quite large
(20Kbp or more), and therefore when performing recombinant DNA manipulation,
Often difficult to handle. By the replicon of pUC13
The construction of the two plasmids to be replicated is described below.
And is schematically illustrated in FIG.   pRK290Kan-1 DNA is completely digested with EcoR V, and
They were ligated and transformed into JM83. Ampicillin resistance and
Plasmids from transformants sensitive to tetracycline
Separated, characterized by restriction analysis, and pVic
Colonies containing the plasmid named 1 were identified.
The plasmids were pUC13, kan, and EcoR V T-DNA
The fragment retains the 1450bTx promoter (as shown in FIG. 2).
Fragment d_TwoOf polyadenylate) and 1450bTx
Site (b_FourAnd b_Three1450bTx structure with
The gene was deleted. T on both sides of 1450bTx structural gene
-PVicl, which is homologous to DNA, is
Transformation of carbohydrates and direct transformation of umefacine S cells
After selection by sylin resistance, double homologous recombination
Suitable for integration into octopine-type Ti plasmids
You. After homopartial diploidization, the T-DNA is functionally functional
The transgenic plant cells that do not retain the gene and
To produce the pin mannopine or agropine
And not. After co-localization, the structure is
It confers nicillin resistance and agropine and
And synthesis of mannopine.   pRK290Kan-1 DAN was separated by Sst I, EcoR I and Hind III.
I understand. pUC13 DNA was digested with SstI and EcoRI. Minute
Mixing the solved pRK290Kan-1 and pUC13 DNA, and
After binding to each other, the ligation mixture was transformed into JM83.
Was. Plasmid DNA is transformed into a white, ampicillin-resistant colony.
And characterized by restriction enzyme analysis.
And containing the plasmid named pUC13Kan-1
Knees were identified and the plasmids contained pUC13, kan, and 14
T-DNA fragment d carrying 50bTx_Two(As in Figure 2)
Was held. One side of the 1450bTx structural gene
PUC13Kan-1 which is homologous to T-DNA
Direct transformation of C. tumefaciens cells
And homology after selection by carbenicillin resistance
Into octopine-type Ti plasmid by sexual recombination
Suitable for embedding. After co-uptake, the T-DNA was
_TwoAnd maintained under the screening of carbenicillin
Must have a functional 1450bTx gene and
Opin, mannopine and
And / or produce agropine. 3.4 Construction of activator vector   Plasmids of the pUC series (eg pUC13-based plasmids)
Sumid) Ag from Etherica Collie by pRK2013
For conjugative transmission to Lactobacillus tumefaciens
On the other hand, it cannot be activated.
Must be transformed directly into the cells. Only
And the pBR322-based plasmid is activated by pRK2013
But not replicated in Agrobacterium.
Therefore, such a plasmid can be selected 1450bTxPR / kan
Transfer of functional markers to octopine-type Ti plasmids
And a useful suicide vector.   PVic1 and HindIII and EcoRI digested respectively
And pBR322 DNA are mixed, bound to each other, and transformed into PR1.
Transformed and isolated from ampicillin-resistant transformants.
Rasmid DNA is characterized by restriction mapping
Colonies carrying the plasmid named pVic2
And its plasmid is a substitute for the pUC31 sequence of pVic1.
As a copy of pBR322. pVic2 coexists
Capture or T_RThat can be homodiploidized
It is a suicide vector.   PUC13 Kan-1 digested with Hind III and EcoRI, respectively
 The DNA and pBR322 DNA are mixed, bound together, and
To transform into RR1. Ampicillin resistant transformants
Plasmid DNA isolated by restriction mapping
Characterized, plus named pBR322Kan-1
A colony carrying the mid was identified and the plasmid was
Copy of pBR322 as a substitute for the pUC13 sequence of pUC13kan-1
Holding. pBR322Kan-1 is T_RCoexistence import into
This is a viable suicide vector that can only be used. Example 4   This example shows that after the 1450bTx promoter region
Structural genes of kanamycin-resistant bacteria are eukaryotic cells, especially
Plant cells and prokaryotic cells, especially Agrobacterium and Escherichia
Unexpectedly expressed in both Lithia coli cells
Results, and therefore a dual purpose program
Lomomotor region and foreign structural gene
Previously clear that 1450bTxPR could be used
Explain the facts that you did not. 4.1 Kanamycin resistance in prokaryotes   Agrobacterium previously known in the art
Tumefaciens A348 is an octopine-type plasmid
Non-toxic induction of pTiA6 shed by the heat of nopaline-type strain C58
Guided to rifampicin resistant derivative A136 of conductor A114 (NTI)
Produced by entering. Transform pRK290Kan-1
Introduced to A38, but in that case, pTiA6
Did not homo-diploidize. In the resulting stock
One A348-pRK290-Kan-1 contains 100 μg / ml kanamycin.
It was observed that the cells grew when placed in a medium containing the cells. Liquid medium
The growth curve of this strain in YEP medium (30 ° C) was generally tested.
Growth was comparable at all kanamycin concentrations, but the highest
At a concentration of 200 μg / ml the curve is observed at low drug concentrations
It quickly reached its peak (Fig. 4). 4.2 Kanamycin resistance in eukaryotes   A348-pRK290-Kan-1 is homopartially diploidized to pTiA6
And used to transform plant cells. Inverted sunflower
The upper edge of the section of the hypocotyl section of K. (K.A. Barton et al. (198
3) Cell321033-1043) and 2-4 weeks later
The resulting callus was placed on a solid MS3 medium lacking hormones
(Example 1.3a). Agrobacterium cells contain 1 mg / ml
Killed with carbenicillin and 200 μg / ml vancomycin
The callus grew to about 2.5 cm in diameter. Callus
A small fragment of the protein into a solid MS3 medium lacking hormone
Syrin, vancomycin, and 25 μg / ml G418 Kanama
Added Isin analog. Many fragments remain green
Untransformed, possibly growing callus
Other fragments from the transformed cells were killed. kan structural remains
A sequence consisting of a gene and a zein sequence substituted in either direction
All controls died on G148. From this, 1450bTx
PR / kan is a plant cell transformed with a structural gene conjugate
Has been shown to be resistant to namycin action. Example 5   This example was placed after the 1450bTx promoter region.
Eukaryotic structural genes are expressed in both eukaryotic and prokaryotic cells.
Teach the unexpected consequences of manifestation.   Lectins are nutritionally important seed proteins.
Considered important in establishing plant and rhizobium symbiosis
Have been. Etherica Corey HB101 (pPLV134), ATC
PPVL134 obtained from C39181 is
Including the cDNA structural gene of squirrel L. seed lectin (L.M.
Hoffman et al. (1982) Nucl. Acids Res.Ten: 7819−782
8). Sequences encoding cDNA are separated by introns.
As with unrestricted genes, it is the same as that of the genes themselves.
is there. 5.1 Construction of expression vector   Esercia Cory HB101 methylates DNA, so D
NA is not cleaved by the enzyme Bcl I, but Escherichia coli
-GM33 and other strains known to those skilled in the art
The Bcl I site is not protected. Isolated from HB101 (pBVL134)
The transformed pPVL134 DNA transforms GM33,
Resistant transformants are identified. From GM33 (pPVL134)
Isolated pPVL134 DNA is linearized by complete digestion with Bcl I
After BAP treatment, BamHI digested pRK290Eco13 ΔClaBa
Mix with ligation and transform into RR1. Tetracycline
Plasmid DNA isolated from transfection-resistant transformants
Immediately upstream of the lectin-encoding sequence.
With the pPVL134 insert in the orientation where there is 50bTxPR, p2RK9
Select colonies having a plasmid designated as 0Lec-1
You. The orientation of the lectin gene is less than 5 'of the insert.
From less than 3 'to 0.09 kbp and 0.78 kbp of the Cla I site, respectively.
Determined by existence. Both ends are pRK290Eco13 ΔClaBa
After connection to m, form a suture that cannot be disassembled with BamH I and Bcl I.
come.   pRK290Lec-1 DNA is either transformed or conjugated
Is converted to A348 (pTiA6), followed by independent pRK29
0Excludes selection of replicons and tetracycline resistant cells
To introduce pPH1J1. Lectin gene expression
It is not necessary to isolate homopartial diploids for
If necessary, tetracycline resistance by restriction enzyme analysis
It can be identified by selecting progeny of the co-integrant. pR
K290Lec-1 and pTiA6 co-integrate or homo-part 2
The TIP plasmid generated by either of the diploids is
Expressed as TiA6Lec-1. 5.2 Expression in prokaryotes   RR1 (pRK290Lec-1) and A348 (pTiA6Lec-1)
Select by electrophoresis and hybridization, and
It is observed that the plant RNA sequence contains various plant RNA sequences. 5.3 Expression in eukaryotes   A348 (pTiA6Lec-1) inoculated in the sunflower axis inverted
The resulting crown gall tumors contained lectin mRNA and protein.
It is commonly known to those skilled in the art that
Hybridization, electrophoresis and immunological procedures
Observed by the method. Example 6   This example shows that all T_ROf a sub-Ti plasmid containing
Teach construction. T_RIs hormone-independent of transformed cells
Have no genes that exhibit a growth phenotype. Also, o
cs is selectable on some of the plasmids discussed here
Selectable markers if they can function as
Use 1450bTxPR to promote expression of (eg, kan)
Note that the need to stay is eliminated. 6.1 T_RConstruction of sub-Ti plasmid   Transfer pRK290Kan-1 to A348 (pTiA6) by transformation
I let it. 1450bTx structural gene deleted, deficient in opin synthesis
After phenotypic homodiploidization, kanamycin resistance
Restriction of Ti plasmid isolated from Globacterium cells
Characterize by enzyme analysis. Homopartial diploid than co-integration
Digestion of the DNA sample resulting from the digestion with BamHI and ligation by itself
I let it. The resulting mixture is transformed into JM83. Kanamai
Purification of syn and / or ampicillin resistant transformants
Rasmid DNA characterized by restriction analysis, non-transmissible
In Escherichia coli, pUC13Bam2Kan-1
A colony carrying a plasmid designated as
(Fig. 5)   pUC13Bam2Kan-1 and pRK290 DNA were respectively transferred to BamHIBgl
Decompose with II, mix and connect. Concatenated blend
Decompose the mixture with BamH I and Bgl II to linearize unmixed molecules
Then, RR1 was transformed. (If the plasmid is digested with BamHI
When divided and linearized, pBR322Bam2Kan-1 becomes pUC13Bam2K
can be replaced with an-1. ) Ampicillin and / or
Is resistant to kanamycin and tetracycline
Plasmid DNA isolated from recombinants is characterized by restriction analysis
Attached. At two hybrid Bgl II / BamHI sites
That of the two parental plasmids sewn in either direction
With each single copy, pRK290Bam2Kan-1 (Fig. 5)
Identify colonies that carry the plasmid. pRK290Bam2Kan
-1 for Agrobacterium (vir) strain, Escherichia coli
Lee RR1 (pRK290Bam2Kan-1) and Escherichia coli
-(PRK2013) aggro containing vir gene by triparental cross
Transfer to bacterial strain. Changes in the normal three-parent crossing process
Form, pPH1J1 is independent in Agrobacterium (vir) strain
PRK290 replicon, designed to replicate in pRK
Since it is incompatible with 290Bam2Kan-1, it was not introduced.
No. 6.2 T_RSub-Ti plasmid variants   The vector described in Example 6.1 was based on the BmaHI fragment 2
And therefore T_LBoth boundaries (T_RLB (C) and T_RRB
(D)) plus T_LRight border (T_LRB (B)). Ok
T for the topin-type plasmid pTi15955_RIs not cleaved and the sub-Ti
Other enzymes that have shown efficacy in plasmid construction are Apa I and Sm
a I (part of ocs and tml), Mlu I and Hpa I (part of ocs) and
And Kpn I (ocs, tml, ORF9). (T in parentheses
_LStructural gene or open reading frame (O
RFs) are contained on fragments generated by the aforementioned enzymes.
You. ) T successfully_ROther enzymes that cut DNA, such as Hind III
(Part of ocs, tml, ORF9, tmr, ORF5 / tms) and Bgal I (o
cs) is the fragment b in FIG._Five, C, and d₁Cla covering
 T from I fragment_RDo not cut the derivative. Those skilled in the art will recognize that
Including a Bgl I site and ensuring that pBR322 and pUC1
You will find that there is a Hind III site. Partial Bgl I
Or wise use of Hind III restriction enzyme digestion conditions
Is described here, as is well known to those skilled in the art.
Of sub-Ti plasmid based on construction of selective marker
Will be needed in the event of   Other enzymes such as Ata II and Cla I_LRB (B) included
Not based on pTi15955 T_RFor construction of sub-Ti plasmid
May be used. In particular, properly methylated
Host, such as Esercia coli 8K02 (W.B.Wo
od (1966) J. Mol. Biol.16: 118) when grown in pRK290
Homopartial diploidized T of Kan-1, pVic1 and pVic2
-DNA derivative is T_RNot methylated within, can be cleaved
Has no Cla I site, but T_RLB (C) and T_LRB (B)
It has a Cla I site that can be cleaved in between (see FIG. 5). K
PUC13Bam2Kan-1 or pBR322Bam2K grown in 802
an-1 DNA is digested with BamHI and ClaI.
This DNA may be used with appropriate linkers or
Less than the adhesion of Klenow fragment of Collie DNA polymerase I
Are connected by blunting, so that T_LRB
This results in the deletion of (B). BamH I by nuclease Ba131
Of pUC13Bam2Kan-1 linearized under control conditions
And T_LRB (B) can also be removed. 6.3mm Ti plasmid   The mini-Ti plasmid was cloned using the enzymes Eco K and Mst II.
It is built like. This Eco K cleaves pTi15955T-DNA.
Absent. Mst II is b_Five, c and d₁Of the Cla I fragment covering the fragment
A single pTi15955T-DNA cleavage site removed by the deletion
Have. Mst I sticky end is Klenow digestion of DNA polymerase I
It must be blunted before ligation on the piece and Eco K-terminal
Is due to the reaction of both the Klenow fragment and T4 DNA polymerase.
Must be smoothed.   The size of the sub-Ti plasmid discussed here was reduced.
Smaller vectors replace pRK290 instead.
Can be used. Maintained with Agrobacterium in the prior art section
What is extraordinary plus that mentions the shuttle vector
Mid was prepared by R.C.Tait et al. (1982) Gene20: 39-49, J.Lee
mans et al. (1982) Gene19: 361-364 and J. Hilla
nd R. Schilperoort (1981) Plasmid6: 360-362
Including those mentioned. However, it is not limited to this.
No. Example 7   Triparental crossing was performed according to the following procedure;   Other variations known to those skilled in the art are possible.   E.Coli RR1 (with shuttle vector based on pRK290)
E.Coli K802 (shuttle vector based on pRK290)
E.Coli RR1 (with pRK2013) and A3
48 (Rifampicin resistant agro with Ti plasmid
(Bacterium tumefaciens strain)
Let it. pRK2013 is a shuttle vector with strains
Transfer and transfer shuttle vector to Agrobacterium
Let it. The minimum medium in which E. coli cannot grow (that is,
Shuttle vector by the growth of bacteria
Agrobacterium cells with rows are selected. this
The medium is resistant to rifampicin and the shuttle vector.
Drug, often kanamycin or carbenic
Includes any of the cylins (ampicillin analogs). This
Hybridization of these cells with E. coli (pRH1J1) 2104 strain
Causes 1J1 to translocate to Agrobacterium cells. p
Shuttle vector based on PH1J1 and pRK290 is the same cell
Can not coexist for a long time. Gentamicin and Kanamaishi
Growth on a medium containing Ti plasmid
The vesicles are sorted. This Ti plasmid can be single or double
Homologous recombination (co-incorporation or homologous part, respectively)
Diploidization) between the shuttle vector and
It has a desired structure. Antibiotic concentration used for selection
Is as described in Example 1.3a. E. coli strain
Is an L-medium supplemented with 0.2% kaza amino acid at 37 ° C
Growing Agrobacterium tumefaci
Ensu is grown at 30 ° C in YEP medium. pRK290 and pR
K2013 is based on G. Ditta et al. (1980) Proc. Natl. Acad. Sci. U
SA77: 7347-7357, and pPH1J1 is P.R.Hirs (1978) Th
esis, Univ. E. Anglia. Summary of the Invention   1450-based T in octopine-type crown gall tumors_R
Promoter regions that direct transcript expression are also bacterial.
That can promote the expression of foreign structural genes within
And disclosed. One of the foreign structural genes in both plants and bacteria
This dual-purpose promoter for directing pea expression
The use of regions is taught. Works in eukaryotic and prokaryotic functions
Construction of a selectable marker is effective in trying to transform plants
Illustrated as a simple vector.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a restriction map of the T-DNA region of the octopine-type Ti plasmid, FIG. 2 is a restriction map of the T _R DNA, and FIG. Fig. 4 is a schematic diagram of the DNA experiment procedure used for the experiment. Fig. 4 is a graph showing the growth characteristics of bacterial cells having a conjugate of the 1450bTx promoter region and the NPT II structural gene. FIG. 2 is a schematic diagram of a DNA experiment operation being performed.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (C12N 1/21 C12R 1:01) (72) Inventor Stanton B. Gerbin United States Indiana 47906 West Lafayette, Moores Bay Road 5251 (56) References JP-A-60-256383 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 C12N 5/00-5/28 C12N 1/00-1/38 A01H 1/00-5/12 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] A method of making a plant transformation vector and genetically modifying a plant cell with it, comprising: (a) a prokaryotic cell with a DNA molecule comprising a dual purpose promoter region / foreign structural gene conjugate. Cloning the dual purpose promoter region / foreign structural gene conjugate by detecting the expression of the conjugate in the resulting prokaryotic strain; and (b) the cloning dual purpose Transforming a plant cell with a DNA molecule containing a promoter region / foreign structural gene conjugate, and then detecting such transformation. The dual purpose promoter region is shown in the following restriction enzyme map. Agrobacterium tumefaciens AT
14 containing the T-DNA fragment d _1, c and b ₅ derived CC15955
Derived from a 50 base DNA fragment, And a method wherein the transformation of the plant cell is detected by detecting the expression of the binary promoter region / foreign structural gene complex in the plant cell. 2. 2. The method of claim 1, wherein said structural gene confers an identifiable phenotype in a plant transformed to contain said structural gene. 3. 3. The method of claim 2, wherein said identifiable phenotype is resistance to kanamycin, neomycin, G418, or an analog thereof. 4. 4. The method according to claim 3, wherein the structural gene encodes Tn5-derived neomycin phosphotransferase II. 5. 5. The method according to any one of claims 1 to 4, wherein the binary target conjugate is linked to a replicon that cannot be maintained independently in a Rhizobiasiaceae bacterium. 6. 5. The method according to any one of claims 1 to 4, wherein the binary target conjugate is linked to a replicon that can be maintained independently in a Rhizobiasiaceae bacterium. 7. The promoter region / structural gene conjugate, T _L or T right or left border repeat sequence of _R, or one or more linked to repetitive sequences of such boundaries, the first term claims The method according to any one of claims 4 to 4. 8. The structural gene, the following restriction enzymes are coupled to the left end of the T-DNA fragment d ₂ shown in the map, the method described in paragraph 1 claims: 9. The structural gene, the following restriction enzymes are coupled to the right end of the T-DNA fragment b ₄ shown on the map, the method described in paragraph 8 claims: 10. A method for producing a genetically modified plant tissue, comprising the steps of: (i) creating a plant transformation vector and using it to genetically modify a plant cell. (A) transforming a prokaryotic cell with a DNA molecule comprising a dual target promoter region / foreign structural gene conjugate, and then detecting expression of the conjugate in the resulting prokaryotic strain; Cloning the binary target promoter region / foreign structural gene conjugate; and (b) transforming a plant cell with a DNA molecule comprising the cloned binary target promoter region / foreign structural gene conjugate. The dual target promoter region is Agrobacterium tumefaciens AT shown in the following restriction map.
14 containing the T-DNA fragment d _1, c and b ₅ derived CC15955
Derived from a 50 base DNA fragment, And genetically modifying the plant cell by a method characterized in that the transformation of the plant cell is detected by detecting the expression of the binary target promoter region / foreign structural gene complex in the plant cell. And (ii) growing the genetically modified plant cell to produce the genetically modified plant tissue. 11. Genetically modified plant cells comprising a plant transformation vector, wherein said plant transformation vector is a plant transformation vector comprising a dual purpose promoter region / foreign structural gene conjugate. Agrobacterium tumefaciens AT whose promoter region is shown in the following restriction map
14 containing the T-DNA fragment d _1, c and b ₅ derived CC15955
Genetically modified plant cells containing a plant transformation vector derived from a 50 base DNA fragment: 12. A method for producing a genetically modified dicotyledonous plant comprising the steps of: (i) creating a plant transformation vector and using it to transform a dicotyledonous plant cell (A) transforming a prokaryotic cell with a DNA molecule containing a dual target promoter region / foreign structural gene conjugate and detecting the expression of the conjugate in the resulting prokaryotic strain Cloning the binary objective promoter region / foreign structural gene conjugate; and (b) transforming a dicot plant cell with the DNA molecule containing the cloned binary objective promoter region / foreign structural gene conjugate. Detecting the transformation, and wherein the dual-purpose promoter region is Agrobacterium tumefaciens as shown in the following restriction map. Nsu AT
14 containing the T-DNA fragment d _1, c and b ₅ derived CC15955
Derived from a 50 base DNA fragment, Transforming the dicotyledonous plant cell by detecting the expression of the binary target promoter region / foreign structural gene conjugate in the dicotyledonous plant cell. Genetically modifying; and (ii) growing said genetically modified dicotyledon cells to produce said genetically modified dicotyledonous plant.