JP2573797C - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION       [0001]     [Industrial applications]   The present invention relates to a genetic method for plant cells.       [0002]     [Prior art]Shuttle vector   Ruvkun and Ausubel (1981) Nature298: Shuttle bed developed by 85-88
The vector removes the foreign genetic material to a selected location on a large plasmid, virus or genome.
It allows a method to be inserted into the device. When dealing with large plasmids or genomes,
There are important issues. First, large plasmids have many sites for each restriction enzyme
Is Rukoto. Certain site-specific cleavage reactions are not reproducible,
Subsequent concatenation disrupts the order and direction of many fragments that you do not want to change
This creates major difficulties. Second, the transformation efficiency using the large DNA plasmid is
Very low. Shuttle vectors often carry small amounts of foreign genetic material in vitro.
It can be easily inserted into a plasmid and then transferred to a large plasmid, usually by in vivo techniques.
Thus, it is possible to overcome these difficulties.       [0003]   Shuttle vectors are usually plasmids that can be introduced into the ultimate recipient bacterium,
Consists of DNA molecules. It can also be a fragment of the recipient genome into which foreign genetic material can be inserted.
A copy of the fragment and a selection that is also inserted into the recipient genomic fragment
It contains a DNA segment that encodes quality. Selection traits ("markers")
Easily inserted by poson mutagenesis or restriction enzymes and ligases.       [0004]   The shuttle vector is the ultimate recipient cell, typically a bacterium of the genus Agrobacterium
, A three-parent cross (Ruvkun and Ausubel, supra), a self-movable vector in a two-parent cross
Direct transfer, direct uptake of external DNA by Agrobacterium cells (M. Holsters
et al. (1978) Molec. Gen. Genet.163: "Transformation" using the conditions of 181-187
), Spheroplast fusion of Agrobacterium with other bacterial cells, liposome encapsulation
Round D Shuttles on NA uptake or in vitro packaged virus
Introduced by vector infection. Triparental crosses relate to plasmid mobility and conjugative transfer.
Strain and a shuttle vector with a mobile plasmid containing
Includes crosses with strains. If shuttle vector can be moved by plasmid gene
If the shuttle vector is a large genome, such as the Agrobacterium strain Ti
Or transferred to recipient cells with the Ri plasmid.       [0005]   After the shuttle vector has been introduced into the recipient cell, either side of the marker
Double crossover with one recombination is expected. This phenomenon is due to the DN
The A segment can be transferred to the recipient genome and replaced with a homologous segment lacking the insert.
To select cells that lack the original shuttle vector, the shuttle vector
If replication is not possible in the ultimate recipient cell or the recipient cell has an existing, independently selectable
Must be incompatible with Rasmid. One common means for this is
Other plasmids that are incompatible with the vector and have other drug resistance markers.
Be prepared for the third parent.       [0006]   Therefore, when selecting for both drug resistances, surviving cells will have the shuttle vector marker in them.
Only those in which the car has recombined with the recipient genome. If the shuttle vector
Once between the shuttle vector and the recipient plasmid if you have the extra marker
Complete shuttle vector resulting from the transfer of the plasmid was integrated into the recipient plasmid
Cells with one can be selected and eliminated. If exogenous genetic material tries to select
If inserted within the marker or at a close location, the same double recombination will result in
It can be integrated into a recipient plasmid. Within or near a marker of a homologous fragment
If the foreign genetic material is inserted far away from the marker, but not
Recombination occurs between the foreign genetic material and the marker, making it impossible to transfer the foreign genetic material.
I will.       [0007]   Shuttle vectors are useful for manipulation of Agrobacterium plasmids
Has been proven: D.J.Garfinkelet al. (1981) Cell27: 143-153 、 A.J.M.Ma tzke and M.D.Chilton (1981) J. Molec. Appl. Genet.1: 39-49, and J. Leeman
set al. (1981) J. Malec. Appl. Genet.1: See 149-164, where
The shuttle vectors are called "intermedeate vectors".       [0008]Agrobacterium-Overview   Gram-negative bacteria, Agrobacterium belonging to the family Rhizobium
Aum tumefaciens species and Agrobacterium lyso
There are A. rhizogenes species. Each of these species is crown gall disease in plants,
It causes hairy root disease. Crown goal is an undifferentiated tissue
Is characterized by gall formation. Hair roots induce abnormal roots in infected tissues
A teratoma characterized by conduction. In both diseases, abnormally growing plant tissues are:
One or more commonly known as opines that are not normally produced by the plant
It produces more amino acid derivatives, which are catabolized by the infecting bacteria. Known
Pins are divided into three families, typical members of which are octopine, nopaline, and agro
It is a pin. Cells of abnormally proliferating tissue can be grown in culture, and
It is regenerated into whole plants while maintaining the phenotype transformed below.       [0009]   Agrobacterium virulent strain is Agrobacterium tumefaci
In Ens, Ti (Tumor-inducing) plasmid, Agrobacterium
In Rhizogenes, a large plasmid called Ri (root-inducing) plasmid is used.
Has a sumid. Elimination of these plasmids from the bacteria results in loss of pathogenicity. Ti plastic
Sumids are called T-DNA (transferred DNA), and in tumors they are found in the host plant genome.
Includes integrated regions. T-DNA encodes several transcripts. Sudden
Mutation studies indicate that some of these are involved in inducing tumor growth.
Was shown.tml,tmrandtmsThe mutations in the genes are large tumors (in tobacco),
The route appearance tendency and the shoot induction tendency are shown. T-DNA also contains at least one
Encoding the opin synthetase gene, the Ti plasmid often
Are classified by the opin that can be used. Each T-DNA gene is a T-DNA promoter
Is under the control of This T-DNA promoter has the structure of a eukaryotic promoter. Appear to function only in transformed plant cells. Ti plasmid also
Genes are also carried outside the T-DNA region. These genes are opin catabolism, carcinogenesis
It is involved in the functions of sex, agrosine sensitivity, replication, and self-transport to bacterial cells. Ri
The plasmid has a similar structure to the Ti plasmid. For transformation of plant cells
The series of genes and DNA sequences to be given are hereinafter referred to as transformation inducing factors (TIP).
Call it comprehensively. Thus, the name of TIP includes both Ti and Ri plasmids.
The segment in which TIP has been incorporated is referred to herein as T-DNA,
Alternatively, it is derived from the Ri plasmid. Recent Agrobacterium-induced diseases in general
For a review, see D.J.Marlo (1982), Adv. Plant Pathol.1: 139-178, L.W.Ream and M.P
.Gordon (1982), Science218: 854-859, and M.W.Bevan and M.D.Chilton (
1982), Ann. Reb. Genet.16: 357-384; G. Kahl and J. Schell (1982)Molecular
Biology of Plant TumorsIt is described in.       [0010]Agrobacterium-infection of plant tissue   Plant cells can be transformed by Agrobacterium by many known methods.
Eg co-culture of plant cells with Agrobacterium; direct infection of plants; plants
Fusion of protoplast and Agrobacterium spheroplast; plant cell proto
Direct transformation by uptake of free DNA by plasts; partially regenerates cell walls
Transformation of growing protoplasts with intact bacteria; TD of protoplasts
Transformation with NA-containing liposomes; use of virus carrying T-DNA;
Clo injection etc. Each method is sufficient as long as the gene is expressed
, Is transmitted stably through mitosis and meiosis.       [0011]   Infection of plant tissue with Agrobacterium is a simple technique known to those skilled in the art.
(For example, D.N.Butcheret al. (1980) inTissue Culture Methods for Plant
Pathologists, Eds .: See D.S. Ingrams and J.P. Helgeson, pp. 203-208). Planting
Objects can be hurt by any of a variety of methods, such as cutting with a blade or piercing with a needle
Or with an abrasive. The wound is then infected with a solution containing tumor-inducing bacteria.
You. Another way to infect complete plants is to use potato tuber pieces (D.K.
G.T. Herberlein (1977) Amer. J. Bot.64: 153-158) or tobacco stem fragments (Binnse
t al.) Is to plant a small piece of tissue. After induction, the tumor was
Tissue culture can be performed in a medium that does not contain glucose. Hormone-independent growth is a set of transformed plants
Typical of a tissue, in sharp contrast to the normal conditions of growth of cultured tissue (A.C.Br.
aun (1956) Cancer Res.16: 53-56).       [0012]   Agrobacterium is also isolated and cultured (Martonet al. (1979)
  Nature277: 129-131), and infected isolated tobacco mesophyll protoplasts
obtain. In the latter technique, after allowing time for some regeneration of the new cell wall,
In the meantime, Agrobacterium cells were added to the culture and then killed by adding antibiotics.
Contact Agrobacterium tumefaciens cells carrying Ti plasmid
Only Called Cells Formed Callus When Plated on Hormone-Free Medium
. Most calli had enzymatic activity for opine assimilation. Other researchers (R.
B. Horsch and R.T.Fraley (18 January 1983 15th Miami Winter Symposium)
Shows a high frequency of calli that transform and grow hormone-independently (10%
95% of the callus produced opine. M.R.Daveyet al. (1980) in Ing
ram and Helgeson, supra, pp. 209-219, describe the renal cell regeneration from protoplasts.
Talk about infection.       [0013]   Plant protoplasts can be transformed by direct uptake of TIP plasmid
. M.R.Daveyet al. (1980) Plant Sci. Lett.18: 307-313, and M.R.Daveyet
al. (1980) in Ingram and Helgeson, supra, is a petunia protoplast
Was transformed with the Ti plasmid in the presence of poly-L-α-ornithine, and
A phenotype of hormone synthesis and hormone-independent growth was demonstrated. Then polyethylene glycol
Promotes Ti incorporation and specific T-DNA sequences are incorporated into the genome.
Indicated. (J.Draperet al. (1982) Plant and Cell Physiol.twenty three: 451-458,
M.R.Daveyet al. (1982) inPlant Tissue Culture 1982, ed: A. Fujiwara, pp
.515-516). F.A.Krens et al. (1982) Nature296: 72-74, similar results
Reported by ethylene glycol and then by calcium shock, but they Results showed that the incorporated T-DNA contained a contiguous portion of the Ti plasmid sequence.
.       [0014]   Another method of incorporating DNA is to use liposomes. DNA-containing liposomes
The preparation of the arm is described in Papahadjopoulos U.S. Patent Nos. 4,078,052 and 4,235,871
You. A method for introducing Ti-DNA by liposome has been reported (T. Nagatae
t al. (1982) in Fujiwara, supra, pp. 509-510, and T. Nagata (1981) Mol. G.
en.Genet.184: 161-165). Melting of plant and bacterial cells with cell walls removed in a similar system
There is a case. An example of this technology is S. Hasezawaet al. (1981) Mol. Gen. Genet.182： 2
Agrobacterium spheroplast vine reported by 06-210
Transformation of periwinkle (Vinca). Plant protoplasts have incomplete cell walls
Can take up all Agrobacterium cells (S.Hasezawaet al. (198
2) in Fujiwara, supra, pp. 517-518).       [0015]   T-DNA is transferred to the regenerated tissue by fusion of the two protoplasts,
Is transformed (G.J. Wullems et al. (1980) Theor. Appl. Genet.56: 203-
208). As detailed in the section on plant regeneration, T-DNA is also transmitted in meiosis.
And transmitted to descendants according to the simple Mendelian law.       [0016]Agrobacterium-Plant regeneration   Differentiated plant tissue with normal morphology was obtained from crown gall tumors. A.C.Br
aun and H.N. Wood (1976) Proc. Natl. Acad. Sci. USA73: 496-500, a strange cigarette
Shoot teratomas on normal plants, shoots that look normal and flowering
I got When placed in culture, the shoots increase opin production and plant hormone-independent properties.
Fertility was maintained. In selected plants, these tumor phenotypes are not transmitted to progeny
Seemed to have disappeared during meiosis (R. Turgeonet al. (1976) Proc.Natl.Ac
ad.Sci. USA73: 3562-3564). Spontaneous loss of tumor characteristics or teratoma
Plants arising from the seeds initially appeared to have lost all T-DNA (F.
-M.Yanget al. (1980) In Vitro16: 87-92, F. Yang et al. (1980) Molec. Gen
.Genet.177: 707-714, M. Lemmerset al. (1980) J. Mol. Biol.144: 353-376). However, studies using plants recovered after hormone (1 mg / l kinetin) treatment
The plants that have undergone meiosis have lost the T-DNA gene related to the transformed phenotype.
However, a sequence having homology at both ends of T-DNA was maintained (F. Yang and R.B.S.
impson (1981) Proc. Natl. Acad. Sci. USA78: 4151-4155).       [0017]   G.J.Wullemset al. (1981) Celltwenty four: 719-724 further discusses opine assimilation
Genes that are transmitted through meiosis, even though the plant is male-sterile
Have shown that T-DNA can be inherited as it is according to Mendelian law
(G. Wullemset al(1982) in A. Fujiwara, supra). L.Ottenet al. (1981) M
olec.Gen.Genet.183: 209-213, is a tumor producing tumor that produces shootstms(Shoe
Induction) A Ti plasmid mutation at the Tn7 transposon at the locus was used. This
When these shoots regenerated in plants, they produced self-fertile flowers. The settled seeds germinate
Plants had T-DNA and produced opine.tmr(Route induction) using mutation
In a similar experiment, all T-DNA was transmitted to progeny through meiosis, where
To some degree, the nopaline gene is expressed and
Yeast alcohol dehydrogenase I gene was not expressed (K.A. Bart
onet al. (1983) Cell32: 1033-1043). Regenerating tissues lacking the T-DNA sequence
Probably, it is the progeny of non-transformed cells that were mixed in the tumor (G. Oomset al.
(1982) Cell30: 589-597). Transformation with Agrobacterium rhizogenes
It was shown that the resulting route regenerated into seedlings relatively easily. (M.-D.Chilt
onet al. (1982) Nature295: 432-434).       [0018]Genes on Agrobacterium-TIP plasmid   Numerous genes were identified in the T-DNA of the TIP plasmid. About half a dozen
The octopine plasmid T-DNA transcript is mapped (S.B. Gelvinet al.
(1982) Proc. Natl. Acad. Sci. USA79: 76-80, L. Willmitzeret al. (1982) EM
BO J.1: 139-146) and some functions have been clarified (J. Leemans et al.
1982) EMBO J.1: 147-152). The four genes of the octopine-type plasmidtms,
tmr andtmlTransposon mutagenesis including (D.J. Garfinkelet al. (1981) Cell27: 143-153). Ti with mutations in these genes
The plasmid stimulates the tumor callus of Nicotinia tabacum, causing shoots,
Causes a route and becomes larger than normal. In other hosts, mutations in these genes
Can induce different phenotypes (see Chilton, MD Ann. Rev. Genet. (1982)
).tmsWhentmrPhenotype correlates with differences in plant hormone levels present in tumors
I have. Differences in the cytokinin: auxin ratio were observed in nontransformed callus tissue
Differences and types of cytokine: auxin ratios in culture that induce gut or root formation
Similar (D.E. Akiyoshiet al. (1983) Proc. Natl. Acad. Sci. USA80: 407-4
11).tmsOrtmrT-DNA having only one of the functional genes (functionst
mlCan (but not only) stimulate obvious tumor growth. Shoots and routes
Stimulation is functionaltmlAre promoted and inhibited respectively (L.W. Reamet al. (1983) Proc
.Natl.Acad.Sci. USA80: 1660-1664). Mutation of T-DNA gene is plant genome
Does not appear to affect the insertion of T-DNA into J. Leemanset al. (19
82) Same as above; L.W. Reamet al(1983) Id.). Codes for octopine synthetase
DoocsThe gene is H. De Greveet al. (1982) J. Mol. Appl. Genet.1: 499-511, in
The nucleotide sequence was determined. This is an intron (common in eukaryotic genes)
Intervening sequence removes from messenger precursor during post-transcriptional mRNA processing
Not have). Eukaryotic transcription signals (TATA box) and poly
There is an adenylation site. Plant cells with octopine synthetase are homoargi
Because it detoxifies nin,ocsThe gene is a plant cell transformed with foreign DNA.
Can be an effective selection marker for cells (G.M.S.Van Slogterenet al. (1982) Plan
t Mol. Biol.1: 133-142).       [0019]   The nopaline Ti plasmid contains the nopaline synthetase gene (nos)
AndnosIs A. Depickeret al. (1982) J. Mol. Appl. Genet.1: 561-573, by
Sequenced.ocsSame as genenosNor introns. Two polyadenines
There is a sequence that can be a TATA box and a candidate for a site for conversion.ocsIn contrast tonosupon
In the stream there is a transcription-like sequence known as a CAT box. J.C.McPh
erssonet al. (1980) Proc. Natl. Acad. Sci.USA77: 2666-2670, the crown In vitro translation of mRNA encoded by T-DNA of goal tissue was reported.       [0020]   Transcription of hair root T-DNA was also detected (L. Willmitzer).et al. (1982) Mol.
Gen. Genet.186: 16-22). Functionally, hair root syndrometmrMutated Ti plasmid
Appear to be equivalent to crown gall tumors caused by (F.F.White and E.
W. Nester (1980) J. Bacteriol.144: 710-720).       [0021]   In eukaryotes, methylation of DNA (especially at cytosine residues) causes transcriptional inactivation.
Is correlated with Genes with relatively low methylation are transcribed into mRNA. Gelv
inet al. (1983) Nucleic Acids Res.1: 159-174, T of crown gall tumor
-DNA always finds at least one unmethylated copy present
did. That the same genome contains many other T-DNA copies that are methylated
This means that one or more copies of the excess T-DNA are biologically inactive.
Suggest (G. Oomset al. (1982) Cell30: 589-597).       [0022]   The Ti plasmid is outside the T-DNA region and contains other genes necessary for the infection process.
Encoding (for the nopaline plasmid, see M. Holsterset al. (1980) Pl
asmidThree: 212-230, H.De Greve for octopine plasmidet al. (1981
) Plasmid6: 235-248, D.J.Garfinkeland E.W.Nester (1980) J.Bacteriol144
: 732-743, and G. Ooms (1980) J. Bacteriol144: 82-91). the most important
IsoncMutation in this gene makes the Ti plasmid less carcinogenic (these
Locus is a word about virulencevirKnown as).oncGenes are
Acts on the lance and is physically located on another plasmid in a different plasmid type.
Can cause transformation of plant cells with different T-DNA (J. Hilleet al. (1982
) Plasmid7: 107-118, H.J. Kleeet al. (1982) J. Bacteriol150: 327-331,
M.-D.Chilton (18 January 1983) 15th Miami Winter Symp.). Nopaline TiDN
A appears to be involved in excision from the Ti plasmid or integration into the host genome.
, An approximately 25 base pair forward repeat that is very adjacent to the left or right border of T-DNA.
It has a repeat sequence (direct repeat) (N.S.Yadavet al. (1982) Proc.Natl.Aca
d. Sci.USA79: 6322-6326) and a similar sequence is adjacent to the octopine T-DNA border
(R.B.Simpsonet al. (1982) Cell29: 1005-1014)
. Opin assimilation occurs in octopine and nopaline-type plasmids, respectively.ocsandnosHeredity
Characterized by offspring. The Ti plasmid also propagates itself, including an origin of replication
It also codes the necessary functions. Ti plasmid transcript is S.B.Gelvinet al. (
1981) Plasmid6: 17-29, by Agrobacterium tumefaciens
Found in cells, where the T-DNA region is weak along non-T-DNA sequences.
Was transcribed well. The properties governed by the Ti plasmid are Merlo,
See above (especially in Table 2) and summarized by Ream and Gordon, supra.
I have.       [0023]Agrobacterium-TIP plasmid DNA   Various octopine-type Ti plasmids have nearly 100% homology to each other.
NA hybridization (T.C.Currier and E.W.Nester (1976) J. Bacterio
l.126: 157-165) or restriction enzyme analysis (D. Sciaky)et al. (1978) Plasmid1:twenty three
8-253). Nopaline-type Ti plasmids are at least 67%
Same sex (Currier and Nester, supra). The various Ri plasmids are very
It was found that there was homology (P. Costantinoet al. (1981) PlasmidFive:
170-182). N.H.Drummond and M.-D.Chilton (1978) J. Bacteriol.136： 1178-11
83, Octopin and nopaline-type Ti plasmids interact with each other in a relatively narrow area.
Showed that they have the same sex. These homologies are described in G. Engleret al. (1981) J. Mol B
iol.152: 183-208, mapped in more detail. They are 3 of 4 similar areas
One is 3 (over T-DNA), 4 (specificoncGene), and
And 9 (onc(Having the gene). Linked
At least the homologous regiontraGene (connection of Ti plasmid to other bacterial cells)
And genes involved in replication and incompatibility. This area
Was isolated from a species of rhizobium, another genus of the RhizobiassiaceaeSymplus
Mid (related to symbiotic nitrogen fixation) (R.K.Prakashet al. (1982)
Plasmid7: 271-280). The order of the four areas is not preserved, but all are the same It is arranged in the direction. Part of the T-DNA sequence is nopaline and octopine plus
It is very well preserved between mids (M.-D. Chiltonet al. (1978) Nature275
: 147-149, A. Depickeret al. (1978) Nature275: 150-153). Ri plasmid
Between them, and octopine (F.F.White and E.W.Nester (1980) J. Bacter
iol.144: 710-720) and nopaline (G. Risuleo)et al. (1982) Plasmid7: 45-51)
There is considerable homology between both Ti plasmids. The area is mainlyoncCode the gene
Area. RiT-DNA is weak against both types of T-DNA of Ti plasmid.
There is also considerable homology (L. Willmitzeret al. (1982) Mol. Gen. Genet.186
: 3193-3197). Uninfected Nicotinia glauca plant DNA is cT-DNA
Contains a sequence called (cell T-DNA) and is homologous to a portion of RiT-DNA.
(F.F.Whiteet al. (1983) Nature301: 348-350).       [0024]   Ti (M.-D.Chiltonet al. (1977) Cell11: 263-271) or Ri (M.-D.Chilto)
n (1982) Nature295: 432-434, F.F.Whiteet al. (1982) Proc. Natl.Acad.Sc
i. USA79: 3193-3197, L. Willmitzer (1982) Mol. Gen. Genet.186: 16-22)
A portion of the rasmid is found in the DNA of tumor plant cells. The transferred DNA is T
-Known as DNA. T-DNA is nuclear (M.P.Nutiet al. (1980) Pla
nt Sci. Lett.18: 1-6, L. Willmitzer et al. (1980) Nature287: 359-361, M.
-D. Chilton et al. (1980) Proc. Natl. Acad. Sci. USA77: 4060-4064) Host D
NA (M.F.Thomashowet al. (1980) Proc. Natl. Acad. Sci. USA77: 6448-6452
, N.S.Yadavet al. (1980) Nature287: 458-461).       [0025]   M.F.Thomashow et al. (1980) Proc. Natl. Acad. Sci. USA77: 6448-6452, oh
And M.F.Thomashowet al. (1980) Cell19: 729-739 is an octopine type Ti
Rasmid T-DNA was found to be TL-DNA and TR-
It was found to be incorporated in two different places in the DNA. Copy of TR and TL
Numbers can vary (D.J.Merloet al. (1980) Molec.Gen.Genet.177: 637-643)
. The core of T-DNA is highly homologous to nopaline T-DNA (Chilton
et al(1978) supra, and Depickeret al(1978, supra), required for tumor maintenance
so, Found in TL, generally present at one copy per cell, andtms,tmr and tml
Encodes a gene. On the other hand, TR has a large copy number (D.J.Merloet al. (1980) ago
Out) but completely unnecessary (M.De Beuckeleeret al. (1981) Molec.Gen.Gene
t.183: 283-288, G.Oomset al. (1982) Cell30: 589-597). G.Oomset al. (
1982) Plasmid7: 15-29 shows that even if TR was deleted from Ti plasmid,
Bacterium tumefaciens keeps virulence, but TR
-It is assumed to be involved in DNA uptake. G.Oomset al. (1982) Cell30:
Due to 589-597, T-DNA is sometimes deleted after being integrated into the plant genome
However, tumors that are generally stable and contain mixed cells with different T-DNA compositions are
Was shown to be the result of multiple transformation events.ocsExists in the TL. I
However, they can be deleted from the plant genome without losing the phenotype associated with tumor growth.
The left end of the integrated TL consists of a forward or reverse repeat T-DNA sequence (
R.B.Simpsonet al. (1982) Cell29: 1005-1014).       [0026]   In contrast to the octopine-type tumor situation, nopaline T-DNA is a series of fragments.
Integrated into the host genome (M. Lemmerset al. (1980) J. Mol. Biol.144： 35
3-376, P. Zambryskiet al. (1980) Science209: 1385-1391). Forward repeat
The sequence was observed. The T-DNA of the plant resulting from the teratoma is located at the end of the inserted DNA.
Fragments have slight modifications (Lemmerset al., Supra). Between the right and left edges
Analysis of the sequence at the junction reveals many forward repeats and one reverse repeat
Became. The latter straddles the joint (Zambryskiet al(1980) supra).
The left junction has at least 70 base pairs (bp) variation, while the right junction has 1
bp only (P. Zambryskiet al. (1982) J. Molec. Appl. Genet.1: 361-
370). The left and right ends of the junction of the repetitive sequence are longer than 130bp.
It is divided by Pacer. The origin of this spacer is unknown and specific T
-Contains a DNA sequence. T-DNA consists of repeat sequences and low copy number host sequences.
It is built into both.       [0027]   N.S.Yadavet al. (1982) Proc. Natl. Acad. Sci. USA79: 6322-6326, T -In the nopaline Ti plasmid just outside the left edge of the DNAchiFind the site, this
Increases general recombination with DNA around 10 Kbp apart in bacteriophage λ
Make it bigger. R.B.Simpsonet al. (1982) Cell29: 1005-1014, is Octopin Ti
In the plasmidchiNo sequence found, but outside of sequenced region
I cannot exclude the possibility of doing it. With the Ti plasmidchiThe significance of is unknown. ifchi
Has a function,chiIs not in the T-DNA, so probably in plants
And will be used in Agrobacterium cells.       [0028]Manipulation of Agrobacterium-TIP plasmid   As detailed in the shuttle vector section, the altered DNA sequence was
Techniques have been developed to introduce the plasmid into the desired location. Transposons use this technique
Easily inserted by surgery (D.J.Garfinkelet al. (1981) Cell27: 143-153). J.
-P.Hernalsteenet al. (1980) Nature287: 654-656 is the T-plasmid of Ti plasmid.
The DNA sequence inserted into the DNA (here, a bacterial transposon) is
Has been shown to be transferred and incorporated into the genome. Many foreign DNAs of various origins are inserted
So far, the gene has not been expressed under the control of its own promoter.
Was. These genes include yeast alcohol dehydrogenase (Adh) (K.A. Bartonet
al(1983)), maize AdhI (J. Bennetzen, unpublished) and zein, suckling
Interferons and globins, the mammalian virus SV40 (J. Schell, unpublished)
and so on. M. Holsterset al. (1982) Mol. Gen. Genet.185: 283-289, according
For example, the bacterial transposon (Tn7) inserted into the T-DNA is integrated into the plant genome.
After incorporation, it was recovered in a fully functional and possibly unchanged form.       [0029]   Deletions can be made in the TIP plasmid in a variety of ways. Mark the shuttle vector
It can be used to introduce deletions made by semi-recombinant DNA techniques (Cohen
and Boyer US Pat. 4,237,224). A predetermined deletion at one end
Can be created by incorrect sponson clipping (B.P.Koekmanet al. (1979) Plasmi
dTwo: 347-357, G.Oomset al. (1982) Plasmid7: 15-29). J. Hille and R. Sch
ilperoot (1981) Plasmid6: 151-154 has both ends at predetermined positions It was shown that a deletion could be made using two transposons. This technology
Is also used in vivo to construct "recombinant DNA molecules".       [0030]   Nopaline synthetase gene used to select transformed plant cells
Used to insert a DNA segment encoding the desired drug resistance. M.Bevan (M
.D.Chiltonet al(18 January 1983) Reported by 15th Miami Winter Symp.
And J.L. Marx (1983) Science219: 830) and R. Horschet al. (
(18 January 1983) 15th Miami Winter Symp., And Marx, supra, see)
Tn5 kanamycin resistance gene (neomycin phosphotransferase)
Was inserted (under its control) after the nopaline promoter. To make it, culture
Transforms plant cells to become kanamycin and anagro-resistant like G418
An alternative approach was taken. J. Schellet al. (18January 1983) 15th Miami Winter S
ymp. (see also Marx, supra) reports a similar construction, in which Tn7 methotrex
A xate resistance gene (dihydrofolate reductase) is
Tethered after the promoter. Transformed cells are methotrexate resistant
there were. Plant cells with octopine synthetase are toxic chemicals
G.M.S.Van Slogterenet al. (1982) Plant Mol. Biol.1:13
3-142, proposed using this enzyme as a washing marker.       [0031]   M.-D.Chilton et al. (1983), supra, described A. Defremeu as "mini-Ti
Nopaline T-DNA is usually restricted to one place.
enzymeKpnThere is an I cleavage site. A mutation lacking this site was created, and complete nopaline T
-Contains DNAKpnThe I fragment was isolated. This fragment is
Inserted into pRK290 along with the Agrobacterium tumefacier
A plasmid was obtained which was maintained in the sense and lacking all non-T-DNA sequences. So
By itself, this plasmid failed to transform plant cells. But Ok
Put into the Agrobacterium tumefaciens strain carrying the Topin Ti plasmid
This induced a tumor that synthesized both octopine and nopaline. This is nopaline
Loss of Ti plasmid function was complemented by octopine Ti plasmid, and And nopaline "small Ti" indicate that it was capable of transforming plant cells. Chilton
et al. (1983), supra, also used small TiSmaCut with I, nopaline synthetase inheritance
"Micro-Ti" from which all T-DNA was deleted except for the child and its left and right ends
It was created. This fine TiSmaInsertion into the pRK290 plasmid derived pair lacking the I site
Used similarly to Ti, with comparable results.       [0032]   H. Lorzet al. (1982) inPlant Tissue Culture 1982, Ed: A. Fujiwara, pp.
511-512 are plasmid vectors whose TIP system is apparently unrelated to DNA uptake and maintenance.
And a nopaline synthetase gene was used as a marker.       [0033]Phaseolin and gene regulation   Generally, higher eukaryotic genes are highly regulated. Multicellular like plant
Objects have many differentiated tissues, each of which has unique functions that require unique gene products
have. One such tissue is cotyledon. In legumes
Cotyledon leaves fats, carbohydrates, minerals and proteins until needed during germination.
It is a storage organ of the species that stores throat. Fosseolas vulgaris L. (French bi
Bean, kidney beans (kidny bean), dried white beans (navy bean), green beans (green bean)
)), The major storage protein is phaseolin. This protein
White is very similar and consists of a small number of molecular species equal to each other. Phaseolin is dry
It is responsible for the main nutritional value of beans, often accounting for more than 10% of their dry weight.       [0034]   Phaseolin is highly regulated during the life cycle of Phaseolus vulgaris.
You. This protein is made only while the seed is growing in the pod, and its level is
According to a fixed synthesis schedule, occupy half of the species protein from the lowest detection limit
Rises until it stops. At its peak, phaseolin synthesis is 80% of cotyledon cell protein synthesis.
% Or more. Phaseolin synthesis detected at other times and in other tissues
Can not. With the importance of global nutrients, the mechanism of phaseolin regulation is
I am very intrigued by the study of aseolin, its properties and its regulation.       [0035]     [Summary of the Invention]   The invention disclosed herein introduces a plant gene and expresses the genetically modified
And a plant having the plant cell obtained. In addition, the present invention provides a method for
A plant tissue provided with a plant cell having T-DNA containing a gene is provided. This plant
Genes are expressed in plant cells. In addition, T-DNA (which can be expressed in plant cells here)
(Defined as T-DNA modified to include a functional inserted plant gene).
A novel bacterial strain of Agrobacterium that can replicate is provided. In addition,
Ming is capable of replicating in Escherichia coli (E. coli), contains T-DNA,
To contain a plant gene inserted into the T-DNA contained in the plasmid.
A standard plasmid is provided.       [0036]   The experimental studies disclosed herein demonstrate that plant cells can be planted in plant cells after introduction via T-DNA.
The gene is expressed, that is, a plant gene is inserted into T-DNA by a known method, and
The first example was performed by introducing T-DNA containing the insert of
I believe The experiments disclosed herein also show that plant genes containing introns have T
-To provide the first example of expression in plant cells introduced via DNA
Cheating. These results indicate that all known genes expressed in T-DNA (T-DN
A endogenous gene or inserted foreign gene)
This is surprising given the fact that it does not. This result is also
Had previously inserted a promoter outside the T-DNA gene into the T-DNA.
Could not provide an example of fulfilling the function of controlling expression in plants.
In fact, it is not easily conceived.       [0037]   The present invention provides for introducing useful plant genes from other plant species or strains,
Useful for genetically modifying plant tissues or whole plants. Such a useful plant
Product genes are storage proteins, lectins, disease, insect and herbicide resistance factors,
Genes such as factors conferring resistance to environmental stress, and inheritance of specific fragrances
There are children, but not limited to these. The present invention relates to the storage protein of the main seeds of legumes.
Example of introduction and expression of certain phaseolin gene into sunflower and tobacco plant tissues Is shown. Once plant cells express the plant gene introduced via T-DNA,
Once obtained, plant tissue and whole plants can be removed therefrom by methods known in the art.
Can be regenerated. The regenerated plants are then propagated in the usual way and the transferred genes
Is transferred to other plants by normal plant improvement techniques. For example, the phaseolin gene
Introduction and expression are based on the protein content and nutrition of forage crops such as alfalfa.
It can be used to improve nutritional value. Other uses of the invention, ie introduction into other plant species
Exploring the properties of other genes that have been made is easy for those skilled in the art. Departure
Ming says that essentially any plant gene can be introduced and stably transfected with T-DNA.
It can be applied to introduction into any plant species that can maintain replication. Generally these species
Include, but are not limited to: That is, sunflower (composite com
positeae), tobacco (Solanaceae), alfalfa, soybean and
And other legumes (leguminose leguminoseae) and most vegetables (
Dicotyledonous) is a dicotyledonous plant.       [0038]     Configuration of the Invention   The present invention relates to (a) a plant having a plant-derived promoter and a plant structural gene.
Inserting the gene into T-DNA, whereby the T-DNA / plant
A gene conjugate is formed, and the promoter derived from the plant is 5 'of the structural gene of the plant.
Adjacent to the end, the structural gene of the plant is in the transcription direction from a promoter derived from the plant
Downstream and (b) transferring the T-DNA / plant gene conjugate to plant cells
And a method for genetically modifying a plant cell.       [0039]   In another aspect of the present invention, after the step (b) is performed, (c) the T-DNA /
Detects the expression of structural genes of plants in plant cells containing the gene conjugate of the plant
And a method for genetically modifying a plant cell.       [0040]   Unclear ambiguities regarding the intent and scope of use herein and in the appended claims
The following definition is made to exclude.       [0041]   T-DNA: D derived from transformation inducer (TIP) integrated into plant genome
NA segment. As used herein, the term Agrobacterium tumef
And Agrobacterium, including Agrobacterium rhizogenes
Includes DNA derived essentially from any tumor-derived strain.       [0042]   In the case of the latter strain, previous researchers sometimes called it R-DNA. Further
The term T-DNA, as used in the textbook, refers to naturally occurring or laboratory operations.
And also includes changes, modifications, mutations, insertions and omissions. The only structural requirement is natural
The right and left ends of the T-DNA generated in the above are the characteristics of all T-DNAs.
It is sufficient to ensure the expected functionality of stable integration.
And       [0043]   Plant gene: As used herein, the structural and regulatory elements of plant genes, ie,
Includes elements that are foreign to the gene for T-DNA itself. Plant remains used here
The gene provides a promoter of plant origin (providing initiation of transcription and initiation of translation) to T-DNA.
And the region of the gene that can be regulated) and the structural gene (one or
Region containing or not including a plurality of introns). This plant genetics
Offspring may have the ability to control transcription termination and post-transcriptional RNA processing
3'-untranslated region. The promoter and the structural genetic element are the same or
Will be from different existing genes and from the same or different plant sources
I can do it. For example, a plant gene is a plant gene, which has its own promoter.
Or the same as the coding region of one gene (presence or absence of introns)
It may be an in vitro construct consisting of other promoters from plant species. In this specification
The coding region of the plant gene to be defined is a cDNA copy of the structural gene of the plant gene.
including. Promoters and coding regions can also be spontaneously or artificially induced
It may also include modifications, and may include chemically synthesized segments. Segume to code
Are originating from multiple sources that encode hybrid proteins, naturally occurring or synthetic.
It may be a hybrid product derived therefrom.       [0044]   Plant tissue: Crown gall-like roots, buds, pollen, seeds, tumor tissue, and embryos,
Or higher plants containing various forms of aggregates of cultured plant cells such as callus
Includes differentiated and undifferentiated tissues derived therefrom.       [0045]   Plant cells: Includes cultivated plant cells, cultured plant cells and protoplasts.       [0046]   Production of genetically modified plants expressing plant genes introduced via T-DNA
Combines currently known, specific techniques with various techniques and means known to technicians
It was done. In many instances, alternative means will be present at each stage of the overall process. hand
The choice of stages depends on the choice of the basic TIP, the plant species to be modified and the desired regeneration method.
All of which are selected by the skilled person to achieve the desired result.
Provides another process step that can be used. The basic feature of the present invention is the sex of plant genes.
Quality and structure, and how to insert it into T-DNA. Getting genetically modified plants
The remaining step is to transfer the modified T-DNA to the plant cells (where
Indicates that the modified T-DNA is stably integrated into the plant cell as part of the plant cell genome.
), Which involve in vitro culture and the actual regeneration to whole plants.
There is technology to do. This technology involves the steps of selecting and detecting transformed plant cells.
And transfer the transgene from the first transformant to a commercially viable culture.
Steps may be included.       [0047]   A major feature of the present invention is the construction of a T-DNA having an inserted plant gene as defined above.
It is. The location of the plant gene insertion site is determined by the sequence transfer near the T-DNA border.
As long as the signaling function is not disrupted, these regions indicate that modified T-DN
Since it is essential for insertion of A into the plant genome, it can be anywhere. Preferred insertion site
Located in the most actively transcribed region, especiallytmlGenes, and
, Spans map segment 13 as shown in FIG.HindCalled "1.6" in III-f fragment
Area. No phenotype correlates with the latter transcript. The term "1.6" is used here
Means this region of T-DNA that is actively transcribed. Any T-DNA
It can also be obtained from the TIP plasmid. Plant genes are standards well known to those skilled in the art.
Skill It is inserted by operation. Inserted plant residue regarding the direction of transcription and translation of endogenous T-DNA gene
The direction of the gene may be either, and one of the two possible directions functions. A certain inheritance
When the offspring are inserted at different positions in the T-DNA, factors such as chromatin structure may occur.
Therefore, differences in the expression levels will occur. Phaseolin gene is Agrobacterium
PTi15955, an octopine-type plasmid from TumefacienstmlWithin the gene
ofSmaWhen inserted at the I site, it reaches an easily detectable expression level.       [0048]   A simple method for inserting a plant gene into T-DNA is the shuttle vector described above.
This is because T-D incorporated into a replicable plasmid in Escherichia coli
It has an NA segment (a segment expected to be inserted here). This T-DNA
The segment preferably has one restriction site specific for the shuttle vector.
Plant genes can be inserted at specific sites within the T-DNA segment. And this
The shuttle vector is a suitable Agrobacterium strain, preferably a T-
DNA is transferred to cells of a strain having homology to the T-DNA segment of the shuttle vector.
Is done. Transform the transformed Agrobacterium strain into an existing segment of the Ti plasmid.
Homologous recombination expression that replaces a DNA fragment with a shuttle vector T-DNA segment
Elephants are grown under conditions of choice.       [0049]   In accordance with the policies set forth herein, the modified T-DNA may be combined with any technique known in the art.
More can be transferred to plant cells. For example, this transfer is incorporated into T-DNA.
Direct infection of plasmids of a novel Agrobacterium strain containing isolated plant genes
Or most easily achieved by co-cultivation of Agrobacterium strains and plant cells.
It is. The former technique, direct infection, eventually causes the tumor site or crown
Results in the appearance of rules. Crown gall cells are then expanded in culture and
Under suitable circumstances known to the trader, a complete plant with an inserted T-DNA segment
Play it. Certain parts of the plant cells are transformed by the co-cultivation method. Immediately
The T-DNA is transferred into the cell and inserted into the genome of the plant cell. In any case,
Must select or search for transformed cells to distinguish them from untransformed cells
. Selection is performed using a selectable marker incorporated into the T-DNA together with the plant gene.
To This is easily achieved. For example, the nopaline synthetase promoter
Dihydrofolate reductase or neomycin fas
There is a photransferase. Each of these markers is methotrexate or
Or by growth on media containing kanamycin or their analogs
. In addition, T-DNA can be used as an endogenous marker, such as in hormone-
Genes or genes that regulate dependent growth and abnormal forms of the Ri-induced tumor route
Genes or groups of genes that you control, and toxic compounds such as amino acid analogs.
Genes that control the resistance (the resistance is provided by opin synthetase)
With groups. Search methods familiar to those skilled in the art include measuring opine production,
Specific hybridization to RNA or T-DNA sequences, or
ELISA (short for enzyme-linked immunosorbent assay), radioimmunoassay, and
There are immunological analyzes of specific proteins such as Estan's blot.       [0050]   Another method of shuttle vector operation involves inserting T-DNA or plant genes into it.
Plasmid containing the modified T-DNA, ie, independently in the Agrobacterium strain.
There is the use of a replicable plasmid. Recent sources show that Agrobacterium strains
Acts on a trans which has the function of promoting the transfer of T-DNA to plant cells.
If the T-DNA of such a plasmid is
Has been shown to be transferable from plant strains to plant cells. Including T-DNA
Plasmids that can replicate independently in Agrobacterium strains are referred to herein as "sub-
TIP "(sub-TIP) plasmid. There is a range of variation, where" sub-TIP "
IP "plasmids differ in the amount of T-DNA they contain.
Indicates that all of the T-DNA from the TIP plasmid remains, especially "small TI
It is called P "(mini-TIP) plasmid. The other end of the range is the T-DNA border
Everything is missing except for a minimal amount of DNA around. The remaining part is the host
This is the minimum amount that can be transferred and integrated in cells. Such a plasmid is
The sub-TIP plasmid is small and directly manipulated.
It has the advantage of being relatively easy to make. After inserting the desired gene,
Easy to plant cells containing trans-acting genes that promote T-DNA transfer Can be introduced. Introduction into Agrobacterium strains is transformation of Agrobacterium strains
Alternatively, simplicity can be achieved by conjugation transfer from donor bacterial cells, a technique well known to those skilled in the art.
Achieved in flight.       [0051]   Regeneration is achieved by known techniques. The purpose of the regeneration step is normal, but
Obtaining the whole plant to grow and reproduce while retaining the integrated T-DNA.
You. The technique of regeneration is, according to principles known in the art, the origin of T-DNA, where
Will vary somewhat depending on the nature of the modification of E. coli and the species of the transformed plant. Ri-type
Plant cells transformed with T-DNA can be used without any additional experiments by known techniques.
Is easily reproduced. A plant cell transformed with Ti-type T-DNA is an example.
Can be regenerated by appropriately manipulating the hormone levels in the culture. However
Preferably, the Ti-transformed tissue is one in which the T-DNA is one of the Tmr and Tms genes.
Or, if both are mutated, it's the easiest to regenerate. Failure of these genes
Activation restores the hormonal balance of the transformed tissue to normal and reduces the
Can be manipulated very easily so that playback is easy.
Resulting in plants with normal hormonal physiology. In some cases, the tumor cells are
Having an integrated T-DNA such as nopaline synthetase and
Regenerate sheets expressing the gene, and also shoots expressing the inserted plant gene
Can be made. This shoot is vegetatively thin by cutting it to a plant with roots.
Spores and can produce fertile flowers. The shoot has T-DNA in this way.
Then, it becomes a parent plant of a normal progeny plant expressing the plant gene inserted therein.       [0052]   The genotype of the transformed plant tissue is often found in cells grown in vitro.
Easy to choose by being nurtureable and reproducible. Cultural changes of agricultural interest
If the cultivar is not adapted to these operations, there is more room for variation.
Transformation is done first. After regeneration, the newly introduced foreign plant genes
The desired agronomic cultivar can be produced using techniques known to those skilled in the art of plant genetics.
Easily populated. Exchange of the transformed plants with these agronomically cultivated varieties
The distribution gives the first hybrid. These hybrids are then transformed into the desired genetic background Can be crossed with other plants. The progeny were then ligated with the integrated T-DNA
The presence or absence of a new phenotype as a result of the expression of the inserted plant gene
Searched and selected sequentially. In this way, after many backcrosses and selections
Together with the inserted plant gene, a genotype that is essentially identical to the agriculturally desirable parent strain.
Thus, plants can be produced.       [0053]     【Example】   The following examples are provided by those skilled in the molecular biology and manipulation of TIP and Agrobacterium.
It makes use of many well-known and acceptable techniques;
, Not always detailed. Enzymes are obtained from commercial sources.
It is used in accordance with Underwood's advice or other variations known in the art. Reagents, buffers
Liquid and medium conditions are also known in the art. About such standard technology
Reference studies include: Wu, ed. (1979) Meth. Enzymol.68J.H.
Miller (1972)Experiments in Molecular GeneticsR. Daviset al. (1980)A
dvanced Bacterial Genetics; and R.F.Schleif and P.C.Wnesink (1982)Pra
ctical Methods in Molecular Biology.       [0054]   Except for plasmid IIc, the plasmid is a plasmid only, for example, p
Precede the display with "p" as in 3.8 or pKS4. Cells containing the plasmid were identified
Cells and plasmids shown in parentheses.
Bacterium tumefaciens (pTi15955) or K802 (pKS4-KB). The table below
1, 2, 3, 4 and 5 are useful for identifying plasmids and their relationships
Provide a good index. Table 6 below shows the deposited bacteria. FIG. 1 shows an embodiment.
5 provides an informative comparison of the constructs described in 5, 6 and 8.       [0055] [Table 1]       [0056] [Table 2]     [0057] [Table 3]     [0058] [Table 4]     [0059] [Table 5]       [0060] [Table 6]      [0061] (Example 1)   The purpose of this example is to eukaryotic non-T-DNA under the control of its own promoter.
Is to teach the expression of the gene.       [0062]1.1 Preparation of special plasmid derivatives   The restriction site isHindIII digestion, single-stranded sticky ends with DNA polymerase I
Filling, blunt end ligation, transformation to K802, tetracycline resistance
Selection, plasmid isolation from drug-resistant clones, and their unique limitations
Plasmid pBR322 was used to confirm the removal of the Removed. This plasmid was transformed into p350 (pBR322-HindIII).       [0063]1.2 Preparation of shuttle vector   p203 isBamDigested with HI and its T-DNA Bam17 fragment (see FIG. 2)
It was isolated by elution from the gel after gel electrophoresis. This fragmentBamHI
Was mixed and ligated with p350 linearized in, and the reaction was transformed into K802
Was replaced. Plasmids isolated from ampicillin-resistant transformants appear on restriction map
Characterized bySmaDigest with I and blunt endsHindLinked to III linker
.HindIII Sticky endHindExposed by cleavage with III, the linear plasmid
Transforms itself into K802 after circularizing itself by ligation
Was.       [0064]   Plasmids isolated from ampicillin-resistant transformants are characterized by restriction areas.
The structure of the plasmid, as shown in FIG. 3, is named p395.
Was. p376, discussed below, was also isolated at this point (FIG. 3). p395 isBa
mDigested with HI and the Bam17T-DNA fragmentSmaI siteHindIII
Converted but eluted following agarose gel electrophoresis. This Bam17 fragment isB
glIt was mixed and ligated with pRK290 linearized in II. The reaction was transformed into K802
After selection, the transformant prepares a plasmid characterized by a restriction map
Used to This particular plasmid was named p490-8 / 14.       [0065]   p376 has its origin as described above in this example,SmaI, now,HindIII characteristics
It was converted, but was found to have a deletion of about 0.8 Kbp to the right. P3 on
Equivalent to that Bam17, as was done for 95BamHIT-DNA fragment separated
AndBglIt was ligated to pRK290 which had been linearized in II. Transformation selection, plus
After mid isolation and characterization, the particular plasmid was named p458-1.       [0066]1.3 Kanamycin resistance and phaseolin gene insertion   Fragment carrying the phaseolin geneHindIII From digested pKS-KB3.8, Agaro
Prepared by gel electrophoresis. This fragmentHindP490- linearized with III It was mixed with 8/14 and linked. Kanamycin-resistant transformant of K802
It was used to prepare a mapped plasmid. Two components are isolated
P499 / 6/7 had a bean (base) sequence to the right of kanamycin resistance (Figure 4).
) And p499 / 6/8 was in the opposite direction (Figure 7).       [0067]   PKS-KB3.8 with purified phaseolin geneHindIII fragment alsoHind
It was mixed with p458-1 linearized in III and ligated. The plasmid is again
Restriction maps were prepared from namycin-resistant transformants. Again, both directions are isolated
P496-2 (Fig. 6) and p496-1 (Fig. 7) are each to the right of the kanamycin resistance gene.
And had Phaseolin on the left.       [0068]1.4 Double homologous recombination of Ti plasmid   The phaseolin and kanamycin genes were as described in Example 14
The plasmid is contained in the Ti plasmid maintained in C. tumefaciens cells.
I was impregnated. Two Ti plasmids were used as recipients: pTi159
55 is an octopine-type plasmid; and pTiA66 is Agrobacterium IS (insert
In) Does not work due to natural insertion of sequencetmsA6 octopine-type plastic with gene
It is a strain derived from Sumid. Defined in p499 / 6/7, p499 / 6/8, p496-2 and p496-1
The pTi15955 plasmids containing the modified structures are p529-8, p529-7, p529-11 and p529-2, respectively.
It was named. The same structures in pTiA66 are p539-6, p539-5, p539-2 and p539-1, respectively.
It was named.       [0069]1.5 Plant infection   Agrobacterium tumefacier containing p529 and p539 series Ti plasmids
The cells are stuck with a needle and infect the sunflower plant stem by injecting certain bacterial cells.
Used to make.       [0070]1.6 Phaseolin detection   Phaseolin protein chains were analyzed by ELISA as described in Example 14. A gall was detected. All humps tested include phaseolin
The amount was 20 n / g of fresh weight of tissue.
It varies between g and 0 ng, with an average of about 10 mg / g. Protein denaturing gel (SDS-PolyA
(Crylamido) by Western blot analysis
It showed a fairly small but apparently high molecular weight separated band. The exact number of bands
The size varies between hosts, which is the host's specific post-translational processing.
Is the result of       [0071]   The messenger RNA strand of phaseolin was synthesized as described in Example 12.
, Was detected in the hump. All humps tested were phaseolin (RNA
) Strand was found to be contained in the poly (A) 5 + 4 RNA fraction;
About 0.005% of the total poly (A) RNA. Denatured DNA gel (methylmercury-
Agarose) Northern blot analysis shows the original phaseolin message.
A large molecular weight separated band of the same size as the RNA (1.6 Kbp).
. Phaseolin was also infected by the pTiA66 vector by ELISA
Detected in shoot-tissues from cells.       [0072]   Detection of both phaseolin protein and phaseolin messenger RNA signals
The amount to be modified is the amount of agro with unmodified pTi15955 and unmodified pTiA66.
Isolate crown gall transformed with B. tumefaciens
It is remarkable and substantial above the noise level observed when analyzing.       [0073] (Example 2)   This example demonstrates that the complete phaseolin gene can be obtained using the T-DN taught in Example 1.
Teaches insertion into T-DNA similar to A. This configuration is nopaline Ti plastic
Carries the sequence inserted into the nopaline synthesis gene region in the pmidC58, a pmid
Utilizes a shuttle vector designed to:       [0074]2.1 Structure of shuttle vector   The nopaline-type plasmid pTiC58 (FIG. 8a)SmaDigested with I, nopaline synthetic heredity
The offspring-encoding fragment was isolated by agarose gel electrophoresis. This fragment
BglA blunt end ligation to the II linker, which thenBglExposed by digestion by II
I was made to. The resulting DNA fragment isBglPRK290 linearized by II (Fig. 1
0). Selected by tetracycline after transformation into K802
The plasmid was isolated and a restriction map generated. That particular plus
The mid was named pCF44A (FIG. 8b).       [0075]   FourClaThe I site was created by recutting pCF44A twice in successionClaI feeling
Changed to a receptive site. The plasmid isXhoDigested with I, religated on its own
And transformed into K802. After selection, plasmid isolation and restriction
After creating the diagram,XhoI fragment (twoClaA suitable plasmid (with an I site)
Was digested with ClaI, religated on its own, and transformed into K802. Choice
After plasmid isolation and restriction mapping, a second deletion of the plasmid (this
Each ClaI fragment has all but the 5 'end of the nos gene) with pKS-nopIV (
9, 8, and 8c).       [0076]2.2 Insertion of Kan / bean gene   pKS-KB3.8 (Fig. 11)ClaKanamycin resistance and phaseoli digested by I
A 6.0 Kbp fragment carrying the gene was isolated by agarose gel electrophoresis. This fragment
ToClaMix with pKS-nopIV linearized with I, ligate and transform K802.
Transformation resistant to kanamycin and tetracycline and sensitive to ampicillin
Plasmids isolated from the strain were restriction mapped and had the structure shown in FIG.
One plasmid was named pKS-nopIV-KB3.8 # 5. Phaseolin gene is Kanamai
A similar clone located to the left of the syn-resistance was found and named pKS-nopKB3.8 # 3
Was. [0077]2.3 Transfer to Ti plasmid and plant infection   The three-parent crossing technique (see the prior art and Example 14) was performed using the nopaline-type Ti plasmid, pTiC
Used to transfer the above structure to 58. pKS-nopIV-KB3.8 # 3 and # 5 Each of the Ti plasmids C58-nop-KB # 3 and pC58
-nop-KB # 5 was characterized by restriction mapping and Southern blot analysis. Part 2
Bacteria containing any of the two plasmids (these plasmidsnos5 'end of gene
Sequence from the end andnosKanamycin / phase present in the sequence on the 3 'side of the gene
Have any direction of the fragment of the Olin gene) to inject the bacteria
Were used separately to infect the stems of sunflower plants.       [0078]2.4 Detection of expression   Expression of the phaseolin gene was determined by ELISA as in Example 13.5.
Detected throughout the warrior's hump organization.       [0079] (Example 3)   This example is phaseolin, which isPhaseolus vulgaris Many seeds of L.
It is a storage protein, the manipulation of its gene, the vector described in various alternative examples.
Teaches the procedure for inserting the phaseolin gene into the protein.       [0080]3.1 Subcloning of phaseolin gene   Charon 24A AG-PVPh177.4 (or 177.4; S.M.Sunet al. (1981) Nature289
: 37-41, J.L.Slightomet al. (1983) Proc. Natl. Acad. Sci. USA80; FIG. 14)
Genetic clone of phaseolinBglII andBamDigested with HI. Phaseoli
The 3.8 kbp fragment containing the gene and its flanking sequence were analyzed by agarose gel electrophoresis.
Are isolated byBamMixed with pBR322 (FIG. 12) linearized with HI,
Connected. The mixture was transformed into HB101 and resistant to ampicillin. Colonies sensitive to tetracycline were selected. Simply from these colonies
A restriction map of the released plasmid was created. Plasmid having the structure shown in FIG.
Was selected and named AG-pPVPh3.8 (or p3.8).BglII andBamHI site
Interconnection inactivates both sites.       [0081]   Another subclone of 177.4 isEcoConsisting of digestion by RI,
7.2k including all but the 3 'side sequence and the 5' end of the phaseolin gene
After isolation of the bp fragment and selection of HB101 transformants for ampicillin,
A restriction map was created. pBR322HindThe III site is the 5 'end of the phaseolin gene
Plasmid inserted in the direction adjacent to the end and in the 3 'untranslated region end
Was named AG-pPVPh7.2 (or p7.2; FIG. 15; Sunet al. And Slightom
et al., Id.).       [0082]3.2 Cloning and isolation of kanamycin resistance gene   pRZ102 (R.A. Jorgensonet al. (1979) Mol.gen.Genet.177; 65-72)
This is a colicin E1 plasmid carrying a copy of the
IsBamHI andHindDigested by III and previously linearized by the same two enzymes
It was mixed with pBR322, ligated and transformed into K802. Ampicillin and Kanamai
Plasmids isolated from transformants selected for both resistance to syn were
A diagram was created, and the plasmid having the structure shown in FIG. 16 was named pKS-4.
.       [0083]3.3 Ligation of kanamycin resistance to phaseolin gene   p3.8 isClaI andBamPBR322 digested by HI and with phaseolin gene
The 4.2 kbp fragment containing the sequence was isolated by agarose gel electrophoresis. this
Is pKS-4 (Figure 16)ClaKanamycin from pBR322 (FIG. 12) linearized with I
Tn5 with resistance (neomycin phosphotransferase II, NPTII) geneCl
aI /BamMixed with HI fragment. The mixture was ligated and transformed into K802
Was. Plasmid isolation after selection of colonies resistant to ampicillin and kanamycin
And created a restriction map. The colony having the structure shown in FIG. 11 is pKS-KB3.8
It was named.       [0084]   p7.2 isEcoRI andBamDigested with HI and the 5 'end of the phaseolin gene
The 3.0 kbp fragment with everything but was isolated by agarose gel electrophoresis. This corresponds to the kanamycin gene derived from pKS4 (FIG. 16).HindP linearized by III
Tn5 with BR322 (Figure 12)HindIII /BamMixed with HI fragment. The mixture is
And transformed into K802. Colonies resistant to ampicillin and kanamycin
After selection, the plasmid was isolated and restriction mapped. Structure shown in FIG.
The colony with was designated as pKS4-KB3. Within pKS4-KB, phaseolin is a dead
Sequence coding for the 5'-most end of the gene and all 5 'flanking regions have been lost
(See FIG. 14).       [0085] (Example 4)   This example teaches how to remove introns from a gene. This is a gene
It is the same as placing cDNA in a genomic environment. Unprocessed (unp
restriction sites found in both exons at the 5 'and 3' ends of the transcribed
Or by specific mutations on the site. These sites are
Present in both strands and cDNAs. DNA containing interspersed introns is
CD that can be removed from the loan and has no intron equivalent over two sites
Replaced by a fragment of the NA clone. The reverse operation is also possible: int
The gene sequence containing Ron may be placed in a cDNA environment. Some are genetic claw
The internal fragment of the clone is inserted into the corresponding gap of the cDNA clone. This latter
Method is similar, but introns are chosen to make the fragment
Often, it is technically more difficult than to include sites that are sensitive to certain enzymes. this
Difficulty is careful selection of partial digestion conditions and desired fragments by agarose gel electrophoresis
Was overcome by purification. A further adaptation of this strategy is that individual genes within a gene
Operating the intron without affecting other introns and exons, and
And when undesired intervening restriction sites are present in the intron as described above.
Includes a stepwise exchange of sequences.       [0086]4.1 Replacement of Phaseolin Intron-Containing Fragment with cDNA   P3.8, a plasmid clone of phaseolin and its side sequence,EcoRI andS
acEach fragmented partially and completely with I. Then, the pBR322 vector and its Agarose gel electrophoresis of a 6.4 kbp fragment containing both the 5 'and 3' ends of the
Was isolated. pcDNA31 (p of cDNA created from phaseolin mRNA)
BR322 plasmid clone)SacI andEcoPartially and completely disassembled by RI
1.33 containing the entire phaseolin cDNA except the most 5 'and 3' terminal sequences
The kbp fragment was isolated by agarose gel electrophoresis. These two fragments are
They were ligated together and transformed into HB101. After colony selection, bacterial growth,
Isolate the plasmid, create a restriction map, and confirm that the plasmid has the desired structure.
And confirmed. This plasmid was named p3.8-cDNA (FIG. 18). All of that
The structure is shown in FIG.       [0087]4.2 Use of p3.8-cDNA   p3.8-cDNA is a substitute for the source of genetic DNA, such as p3.8 used in all other examples.
Note that you can. And the p3.8-cDNA when used in that way has an intron
It will result in a different analogous structure, such as one that is missing. Or this
The strategy can be used to remove introns from already created structures.       [0088] (Example 5)   The purpose of this example was to construct pTi15955 and other octopine Ti plasmids.tms(Bud "shoo"
ting "locus)tmrTi plasmid deleted through (root "rooting" locus)
Is to cause This derivative ensures that the transformed cells are intact.
Complete to regenerate into plantstmsWhentmrTransformed by pTi15955 with the gene
This is useful because it is simpler than open cells.       [0089]   tms-tmrThe deleted pTi15955 is finally replaced in two ways:tms-tmrInactivation
And insertion of foreign genes. These two transformations are set at different positions in the T-DNA.
Each conversion is then independently inserted by a different shuttle vector. To conversion
Each successful shuttle vector was selected separately, and
Requires the use of at least two markers that can be selected in the above. Usual kanamai
In addition to syn-resistance, this example utilizes chloramphenicol resistance from pBR325. Was.       [0090]5.1 Construction of chloramphenicol resistance gene clone   pBR325 isHincDigested by II, blunt endsHindLinked with III linker.
The result isHindIII digested and religated
Nicole resistance (cam) And named pKS-5 (FIG. 19). this is,cam
Contains genesHindIII /BclServes as the origin of the I fragment.       [0091]5.2 Construction of pBR322 clone of T-DNA with deletion and cam gene   The 9.2 kbp linearized DNA fragment wasHindIII Complete disassemblyBamSimple from HI partial decomposition
Released (FIG. 31).camThe gene-bearing fragment was isolated from pKS-5 and had a 9.2 kbp line.
Mixed with the ligated fragments, ligated,E.coliTransformed into chloramphenicol
Selected for resistance and named pKS-oct.Cam203 (Figure 20).       [0092]   pKS-oct.Cam203 may be used to construct many TL deletion mutants of pTi15955
Plasmid clone. Contains the right arm of TL and the resistance gene to the left of the right arm
. The various left arms of TLcamTo the left of the gene (HindIII site). For example,
And once p102 is attached, the deletion is 5.2 kbp long, andtmsWhentmrAll of
Including. If p103 is attached, the deletion will be 3.2 kbp long,tmsPart oftmr
Including all of See FIG.       [0093]   pKS-oct.Cam203 isHindDigested by III. p102 or p103HindBy III
Digested and fragments of 2.2 kbp or 2.0 kbp T-DNA were isolated and linearized.
Ligation with pKS-oct.Cam203, transformation, and the resulting pKS-oct.del II (Fig.
21) or pKS-oct.del I (FIG. 22) was isolated. These structures are
To Agrobacterium tumefaciens, homologous recombination, and
And chloramphenicol resistance. Or one using established methods
The structure of the plasmidBamLinearized with HI, and pRK290BglConnect to II site
This inserted the structure into pRK290.       [0094] (Example 6)   The Ti plasmid was used in this example.tmrInHpaFrom the I sitetmlInSmaBetween the I sites
Mutated by deleting T-DNA. The Ti plasmid that can be modified is pTi1
Includes 5955, pTiB6, pTiA66 and others. This configuration is shown in FIG.       [0095]6.1 Isolation of the cam gene   pKS-5 (Fig. 19)HindIII andBclDigested by I. Example with smallest fragment
Isolated after separation on an agarose gel as taught in Section 5.       [0096]6.2 Construction of T-DNA pBR322 clone with deletion   Right hand arm of T-DNA deletion is p203SmaI siteBglBy inserting the II site
(See FIG. 23). p203 isSmaDigested by I,BglDigested in II
And religated and transformed into K802. In another configurationBamHI linkerBglII
Substitutes forBamThe HI partial isolate is isolated. As a result
The resulting plasmid is p203-BglNamed II, andBglII andHindErased by III
Be transformed into Its big containing fragmentsBglThe II / HindIII vector was described in Example 6.1.
The chloramphenicol resistant fragment isolated as described was ligated. K
Loramphenicol resistance is selected after transformation into K802. The resulting program
Rasmid is named p2f (FIG. 23).       [0097]6.3 Composition of left hand arm of T-DNA deletion clone HindIII site is p202HpaI site,HpaDigest with I andHindLink with III linker
It is inserted by doing.HindAfter exposure of sticky ends by digestion with III,HindI
2 kbp with II endHpaThe I fragment is isolated.HindIII terminalHpaI fragmentHindIII minutes
Thaw and transform into K802. After colonies containing the desired components have been isolated,
The plasmid is then named p3e (FIG. 24).       [0098]6.4 Construction of T-DNA deletion clone   The clone's left-hand arm was eluted from an agarose gel after electrophoresis.
, P3eHindObtained by purifying the 2 kbp fragment of III digestion. p2fHindIII
And treated with alkaline phosphatase, mixed with the 2 kbp fragment
Ligated, transformed into K802, and selected for chloramphenicol resistance
You. Plasmids are isolated from individual colonies and characterized by restriction mapping
Can be A plasmid having two arms in the desired vertical arrangement direction was selected, and pKS-oc
It is named t.del III (FIG. 25).       [0099]   pKS-oct.del III, Agrobacterium tumefaciens
And homologous recombinants were selected by selection with chloramphenicol.
Selected. Sunflower and tobacco roots and shoots are mentioned in other examples
And the resulting tumor is tested for orpin.       [0100] (Example 7)   This exampletmrWhentmlAnd teaches a configuration for deletion of
You.       [0101]7.1 Construction of chloramphenicol resistance with BglII site   pBR325 isHincDigested by II, blunt endsBglLinked to the II linker,Bgl
It is digested and religated by II (FIG. 26). Chloramphenicol resistance is K802
Or after transformation of GM33. The resulting plasmid, pKS-6,camRemains
Have a geneBglII /BclServes as a source of I fragments.       [0102]7.2 Construction of tmr, tml deletion clone   p203 isHpaI andSmaDigested by I. Blunt endsBglAfter linking with II linker,Bgl
Digested by II,BglThe cohesive ends were exposed and religated, and transformed into K802. Place
The desired structure is confirmed and named p2 (FIG. 27).       [0103]7.3 Construction of T-DNA deletion clone (pKS-oct.del IIIa) camHave a geneBglThe II fragment was isolated from pKS-6,BglConnect to p2 cleaved at II
Is done. Chloramphenicol resistance is selected after transformation of K802. as a result
The resulting plasmid was named pKS-oct.del IIIa (FIG. 28) and is described in Example 6.4.
Tested as stated.       [0104] (Example 8)   The purpose of this construction is to insert the chloramphenicol geneHpaPart onlyt
mrIt is to provide an example of mutation of the locus. This gene is derived from pKS-6BglII /Bcl
Isolated as an I fragment, p203BglConverted to II siteHpaConnected to the I site.       [0105]8.1 Conversion of Hpa site to BglII site   p203 isHpaDigested byBglII linked to a linker,BglCut by II
, Are reconnected. After transformation of K802, colonies were selected,BglFor insertion of II site
The search is performed by creating a restricted map (FIG. 29).       [0106]8.2 Isolation of cam gene   pKS-6 isBglII andBclDigested by I. The smallest piece is agarose
Isolated by gel electrophoresis.       [0107]8.3 Construction of Mutant T-DNA Clones   The modified p203 from Example 8.1 isBglII digested from Example 8.2
PreparedcamLinked to the gene and transformed into K802. Chloramfe
Selected for Nicole resistance, isolated from resistant transformants, and mapped with restriction enzymes
After characterization by PCR, the plasmid is named pKS-oct.tmr (FIG. 30).
.       [0108] (Example 9)   Regeneration in this example is a ninth stimulus stimulated by the Ri based TIP plasmid. Gin tumors, essentially comprising M.-D. Chiltonet al. (1982) Nature295: 432
Performed as described by.       [0109]9.1 Infecting hairy roots   The discoid structure of carrots is about 10% in 0.1 ml of water.⁹Cells are inoculated. Obtained root
(root) 1 to 1.5 cm of the end is cut off and lacks hormones (1 to 1.5%
Agar) Monier medium (D.A.Tepfer and J.C.Tempe (1981) C.R.Hebd.Seanc.Acad.Sc.
i., Paris295: 153-156) and grown at 25-27 ° C in the dark. By bacteria
Uncontaminated cultures were transferred every 2-3 weeks and lacked hormone and agar Monier
It is further planted and cultured in a medium.       [0110]9.2 Regeneration of roots into plants   Root tissue cultured as described in Example 9.1 contained 0.36 μM 2,4-D.
Place on solid (0.8% agar) Monier medium supplemented with 0.72 μM kinetin
You. Four weeks later, the resulting callus tissue is a liquid Monier that lacks hormones.
Place in medium. During the one month, keep warm in a shaker (150rpm) at 22 ℃ ~ 25 ℃
Calli are separated into suspension media and embryos differentiated, containing hormone-free Monier media.
When placed in a bird dish, it grows into a seedling. These seedlings grow in the medium and gradually decrease
After "hardening" by exposure to humid air, the greenhouse or garden (
Field plot).       [0111]9.3 Use of non-hairy root vectors   FunctiontmrGene-free Ti-based vectors are described in Examples 9.1 and 9
. Used in place of Ri-based vectors as described in 2. Appropriate
The construction of the various deletions is described in Examples 6, 7 and 8.       [0112] (Example 10)   Regeneration in this example was stimulated by a Ti-based TIP plasmid
Including tobacco tumors, essentially K.A.Bartonet al. (1983) Cell32: 1033-10 Performed as described by 43.       [0113]10.1 Crown Gall Infection   Tobacco tissue was first described in A.C. Braun (1956) Canc. Res.16: 53-56
Thus, it is transformed using a method utilizing the converted stem fragments. The stem is a 7% quotient
The surface is sterilized with commercialized Chlorox and 80% ethanol, and sterilized distillation
Rinse with water, cut into 1 cm sections, and agar solid MS medium lacking hormone (T. Murashig
e and F.skoog (1962) Physiol.Plant.15: 473-497) in the Petri dish containing
Place. Inoculation is performed by injecting the bacteria into the cut basal surface with a needle.
More accomplished. Stems are cultured in the light at 25 ° C. for 16 hours per day. Callus that grew up
calli) is removed from the upper surface of the stem section and contains 0.2 mg / ml carbenicillin
Placed on a solid MS medium lacking a porcelain solution, and three times a month, sometimes with new MS
Transfer to Rubenicillin Medium and see if cells are floating in the medium
Tested. Sterile tissues were grown under medium conditions as described above (25 ° C .; 16 hr .: 8 hr.
(Dark place) in solid MS medium without supplementation.       [0114]10.2 Transformed tissue culture media   Clones were from A. Binns and F. Meins (1979) Planta145: 365-369
Obtained from sterile tissue transformed as described. Calli is 2
Or 3 days in liquid MS with 0.02mg / l naphthaleneacetic acid (NAA) at 25 ° C
Into a cell suspension, while shaking at 135 rpm, 543 and 2
Filtered through a 13 μm stainless steel screen. The filtrate passed through is concentrated to 0.5
% Dissolved agar, 2.0 mg / l NAA, 0.3 mg / l kinetin and 0.4 g / l Difco yeast
Approximately 8 × 10 5 ml of MS medium containing extract^ThreePlated at a concentration of ml of cells
. Colonies that reached a diameter of about 1 mm were picked at the tip of a scalpel, and 2.0 mg / l NAA and 0.3 m
Place on solid MS medium containing g / l kinetin and grow. Raw as a result
Calli were split individually and tested for the transformed phenotype.
You.       [0115]10.3 Plant regeneration   The transformed clone is placed on a solid MS medium containing 0.3 mg / l kinetin,
Cultured as described in Example 10.1. The forming buds are 1/10 strength M
S medium salt, containing 0.4mg / l thiamine, lacking sucrose and hormones
And root them by placing them on a solid (1.0% agar) medium at pH 7.0. root
The plantlets were grown in medium and described in Example 9.2.
As harder, in either a greenhouse or a garden (field plot)
Transferred to soil.       [0116]10.4 Vectors used   The method described in Examples 10.1, 10.2 and 10.3 works
tmrSuitable for Ti-based vectors lacking the gene. The proper construction of the deletion is actually
It is described in Examples 6, 7 and 8. These methods are also based on Ri-based vectors
Effective when used with. Infection of the stem fragment converted in Example 10.1
Are often used to establish TIP-transformed plant cell lines.
Useful.       [0117] (Example 11)   Fosseolin isPhaseolis vulgarisAbundant storage protein (about 50% of total seed protein
). Transfer of a functional foseolin gene to alfalfa plants and fosse
Translation of foseolin, which is stored in Olin Messenger RNA, is
Significant economic value because white synthetic is introduced into leaf material to be used as livestock feed
Have. Alfalfa is a valuable plant for the transfer and expression of the foseolin gene.
Things. Because it is the acceptor as livestock feed,
It grows quickly and can fix nitrogen through symbiosis with rhizobium,
Susceptible to infection with Ngol and can be converted from single cells or protoplasts to alpha
This is because regeneration into rufa plants is possible.       [0118]   This example demonstrates the introduction of an expressible foseolin gene into a complete alfalfa plant
Teach.       [0119]11.1 Construction of shuttle vector   Alfalfa plants are genetically engineered ag
Regenerated from crown gall tissue containing Robacterium plasmid. First stage
So we engineered a functional foseolin genetmr ^-Whentms ^-T-DNA transformation
Construct a "shuttle vector" containing the variant. This configuration has a functioning
Omycin phosphotransferase (NPT II) structural gene (kanamycin resistance
Recombination with a nopaline synthase promoter (M.-D.Chilton,e
t al(18 January 1983) Reported by 15th Miami Winter Symposium; J.L.
Marx (1983) Science 219: 830 and R. Horsch et al. (18 January 1983) 15th
 Miami Winter Symposium). This type of configuration is described in Example 1.
I have.       [0120]11.2 Transfer to Agrobacterium and plant cells   The "shuttle vector" was then used in the prior art (Example 14) such as pTi15955.
It is transformed into a strain of Agrobacterium containing the Ti plasmid. Recombination
Bacteria containing the plasmid have been selected and the cell wall regenerated
Co-cultured with Alfa (Martonet al. (1979) Nature277: 129-131;
 G.J.Wullemset al. (1981) Proc. Nat. Acad. Sci. USA78: 4344-4348; and R.B.
Horsch and R.T.Fraley (18 January 1983) 15th Miami Winter Symposium).       [0121]   Cells are grown in culture and the resulting callus tissue is
Of specific m-RNA by ELISA (Example 12) and ELISA test
Test for the presence of certain proteins (J.L.Marx (1983) Science219: 830; R.B.Horsc
h andR.T. Fraley (18 January 1983) 15th Miami Winter Symposium).       [0122]11.3 Plant regeneration   The alfalfa plant was then A.V.P. Dos Santoset al. (1980) Z. Pfla
nzenphysiol.99: 261-270; T.J.McCoy and E.T.Bingham (1977) Plant Sci. Lette
rsTen: 59-66; and K.A.Walkeret al. (1979) Plant Sci. Letters16: 23-30
Is regenerated from callus tissue in a manner similar to that previously used. This
These regenerated plants are then converted to conventional plants, which form the basis for new commercial varieties
Propagated by growth techniques.       [0123] (Example 12)   In all examples, RNA was extracted, fractionated, and probed by the following processing.
Be informed.       [0124]12.1 RNA extraction   This process, Silflowet al.(1981) Biochemis-try13: Modification of 2725-2731
Was. An alternative to LiCL precipitation for CsCl centrifugation is Murrayet al. (1981) J. Mol. Evol.
17: 31-42. 2M rechloride with 2M urea for precipitation
The use of titanium is described in Rhodes (1975) J. Biol. Chem.twenty five: 8088-8097.       [0125]   Tissues were prepared using a Polytron or ground glass homogenizer at 4% para.
Minosalicylic acid, 1% tri-isopropylnaphthalenesulfonic acid, 10 mM dithios
Reitor (freshly made) and 10 mM Na-metabisulfite (freshly made)
Homogenized in 4-5 volumes of cooled 50 mM Tris-HCl (pH 8.0) containing
Was Octanol is used as needed to control foaming
You. Equivalent of tris-saturated phenol containing 1% of 8-hydroxyquinoline
Is added to the homogenate, then shaken, emulsified, and in 20000-30000g
Centrifuged at 4 ° C. for 15 minutes. The upper layer of water is chloroform / octanol (24: 1
) Once and centrifuged as above. Concentrated lithium chloride
The urea solutions are then each added to a final concentration of 2M and the mixture Was left at 20 ° C. for several hours. The RNA precipitate was then removed by centrifugation.
And washed with 2M lithium chloride to disperse the pellets. The precipitate is
Then it is washed with 70% ethanol-0.3M sodium acetate to make a clear solution
Dissolved in sufficient sterile water. Add 1/2 volume of ethanol and mix
The mixture is placed on water for 1/2 hour and then centrifuged to remove miscellaneous polysaccharides.
Was. The RNA precipitate is then regenerated and removed from water or sterile salt-free po
Redissolved in U buffer.       [0126]12.2 Poly (U) / Sephadex chromatography   Two poly (U) Sephadex (trademarks: Pharmacia, Inc., Uppsala, Sweden)
) A buffer solution was used. One is salt-free and contains 20 mM Tris, 1 mM EDTA and 0.1% SDS
I have. The other is the first buffer plus 0.1 M sodium chloride
. In A426, 2X storage buffer needs to be made for good association to occur
. And it is necessary to add some salt. Adjust to final concentration, then buffer
Autoclave the solution.       [0127]   Poly (U) Sephadex was obtained from Bethesda Research Laboratories. Ten
Use 1 g of poly (U) Sephadex for 0 μg of expected poly (A) RNA
Was. Hydrate poly (U) Sephadex in salt-free poly (U) buffer
Pour into the column with the cut. Raise the temperature to 60 ° C and wash the column with salt-free buffer.
Washed until the baseline in mm was smooth. Ultimately, the column is
Equilibrate at 40 ° C. with poly (U) buffer.       [0128]   RNA having a concentration of 500 μg / ml or less was heated in a salt-free buffer at 65 ° C. for 5 minutes. afterwards
After cooling, sodium chloride was added to a concentration of 0.1M. Then Kono
Until the temperature drops to a stable baseline, apply R to the column at a flow rate of 1 ml / min.
Transfer NA. Then, raise the column temperature to 60 ° C and remove the RNA from salt-free poly (U) buffer.
Eluted with buffer. RNA is usually washed out in three column volumes. R eluted
Concentrate with secondary butanol to a volume where NA can be easily used, and add sodium chloride to 10 mM.
Re Then, 2 volumes of ethanol are added for precipitation. Ethanol precipitate
Dissolve in water, NH_Four-Add acetate to 0.1M. Then re-sediment with ethanol.
Let it settle. Finally, the RNA is redissolved in sterile water and stored at -70 ° C.       [0129]12.3 Formaldehyde RNA gels and "Northern" blots   The method used is Thomas (1980) Proc. Nat. Acad. Sci. USA77: 5201 and Hoffman,
et al. (1981) J. Biol. Chem.256: 2597.       [0130]   0.75 to 1.5% agarose gel containing 20 mM sodium phosphate (pH 6.8 to 7.0)
Hardened. If an aggregated band of macromolecules appears, 6% or 2.2 M
The experiment was repeated with the addition of aldehyde (using a 36% stock solution). Holmual
The dehydration was added to the agarose after cooling to 65 ° C. Add formaldehyde
And ethidium bromide make discovery difficult. Running buffer is 10 mM sodium phosphate
(PH 6.8-7.0).       [0131]   Prior to electrophoresis, RNA was finalized at 6% formaldehyde, 50% formaldehyde.
Treatment with denaturing buffer of aldehyde, 20 mM sodium phosphate buffer and 5 mM EDTA.
RNA was incubated in buffer at 60 ° C. for 10-20 minutes. Insulation stopped by adding stop buffer
did. For 20 μl sample, 4 μl 50% glycerol, 10 mM EDTA, 5 mM phosphorus
Sodium acid and bromophenol blue were added.       [0132]   Use submerged electrophoresis. Before soaking the gel, the RNA was loaded. And 12
The gel was placed at 5 mA for 5 minutes.       [0133]   Then soak the gel in water and reduce the current to 30mA (overnight) or 50mA (6-8 hours)
Lower. The buffer was circulated and electrophoresis was performed in a cold room.       [0134]12.4 "Northern" blots   No staining for gels blotted to detect specific RNA Was. However, the separated marker lane was used for staining. Staining is 0.1M sodium acetate
Perform the staining with 5 μg / ml bromide bromide and destain in 0.1 M sodium acetate for several hours.
went.       [0135]   Prior to staining, treatment with water at 60-70 ° C. for 5-10 minutes facilitated visual recognition. B
Run lots of gel for 15 minutes with 10x standard salicynoic acid (SSC) -3% formaldehyde
Dipped in If large RNA fractions do not elute from the gel,
Treat in 50 mM sodium hydroxide for 10-30 minutes to cut the RNA before processing.
I understood. If a basal treatment was used, neutralize the gel before blotting and
Should be soaked in formaldehyde. Transfer of RNA to nitrocellulose is standard
Performed by the method.       [0136]   Prehybridization is performed at 42 ° C for a minimum of 4 hours in 50% formaldehyde, 10
% Dextran sulfate, 5XSSC, 5X Denhardt, 100 μg / ml denatured carrier DNA
In 20 μg / ml poly (A), 40 mM sodium phosphate (pH 6.8-7.0), 0.2% SDS
went. For hybridization, add the probe to the same buffer and incubate overnight.
went. Probe is about 5 × 10^FiveUsed at concentrations above cpm / ml.       [0137]   After hybridization, nitrocellulose was reconstituted at 42 ° C in 2XSSC, 25 mM phosphoric acid.
Washing was performed many times using sodium, 5 mM EDTA, and 2 mM sodium pyrophosphate solutions.
Finally, it was washed with 1 × SSC at 64 ° C. for 20 minutes.       [0138]   If the filter is not dry during autoradiography,
If the probe has been removed by extensive washing with 64 mM at 1 mM EDTA,
The best results were obtained.       [0139] (Example 13)   "Western" blot (antigen found after SDS polyacrylamide gel electrophoresis)
To do) is essentially R.P.Legocki and D.P.S.Verma (1981) Analyt
. Biochem.111: 385-392.       [0140]   Micro-ELISA (enzyme-linkedimmuno-sorbantassay) for 96 wells
The following steps were performed using an Immulon-2 type plate having a (well).       [0141]13.1 Binding of antibodies to plates   On day one, the wells were diluted 1: 1000 in coating buffer (antibody rabbit).
(Seolin IgG). Incubation was carried out at 37 ° C. for 2 to 4 hours at 200 μl / well. plate
Covered with Saran wrap. Then, place the plate in phosphate buffer-tween (PB
(S-Tween) three times. Each washing step was open for 5 minutes. Then 1%
Of bovine serum albumin (BSA) was added for washing and left for 20 minutes before discarding.
Washing was performed 5 times or more using PBS-Tween.       [0142]13.2 Homogenization of tissue   After cutting the tissue into small pieces, 1 gm of tissue / ml phosphate buffer-tube was added using a polytron.
E-2% polyvinyl, pyrrolidone-40 (PBS-Tween-2% PVP-40)
Mogenized. All samples are before and after crushing and on the phaseolin standard curve.
Before and after preparation, they were stored on ice. A standard curve was generated with the tissue homogenate. So
Standard buffer with buffer to check tissue-dependent phaseolin recovery
I made a line. After centrifuging the homogenized sample,
Then, 100 μl was put into a well and left at 4 ° C. overnight. Each Sun to avoid failure
I did the same thing for two of the pulls. During the incubation, the plate was sealed.       [0143]13.3 Enzyme binding   After incubation overnight, discard the antigen and wash the wells 5 times with PBS-Tween, 5 minutes between each wash
There was an interval between.       [0144]   The conjugate (rabbit anti-phaseolin IgG alkaline phosphatase conjugate) was added to PBS-Twe
Dilute 1: 3000 with en-2% PVP (containing 0.2% BSA) and add 150 μl to each well
. Then, it was kept at 37 ° C. for 3 to 6 hours. After incubation, discard the conjugate and wash the wells with PBS-Tween.
Wash twice. There is a 5 minute interval between each wash.       [0145]13.4 Analysis   Immediately before starting the analysis, a 5 mg tablet of p-nitrophenyl phosphate (Sigma
I got more. And freeze in the dark) and add to 10 ml of substrate until the tablets are dissolved.
Stir. Quickly add 200 μl of room temperature solution to each well. The reaction is carried out for various times (
For example, at t = 0, 10, 20, 40, 60, 90, 120 minutes), Dynatech Micro-ELISA r
It was measured using an eader.       [0146]   p-Nitrophenyl phosphate (colorless) is caused by alkaline phosphatase
Hydrolyzed to inorganic phosphoric acid and p-nitrophenol
Gave the solution a yellow color. It could be measured spectrophotometrically at 410 nm. On detection
The minimum amount that could be cut was less than 0.1 ng.       [0147] (Example 14)   Triparental crosses were generally performed as follows; other modifications known to those skilled in the art may also be used.
Can be.E. coli K802 (shuttle vector based on pRK290)E.
coli(PRK2013) and Agrobacterium tuberculosis resistant to streptomycin
-Crossed with the Mefaciens strain. pRK2013 moves to strains with shuttle vectors
Created a shuttle vector for transfer into Agrobacterium. Strept
Drugs to which mycin and shuttle vectors are resistant (kanamycin or chloram
(Phenicol is often used)
Agrobacterium cells having the shuttle vector sequence were selected. these
With cellsE.coli(PPH1J1) and transfer of pPH1J1 to Agrobacterium cells
Was. Shuttle vectors based on pPH1J1 and pRK290 can coexist for a long time in the same cell.
I can't be there. Media containing gentamicin (pPH1J1 has a resistance gene)
When grown in the ground, cells lacking the pRK290 sequence could be selected. Stre
Puttomycin and gentamicin and kanamycin or chlorampheni
Ko Only cells that are resistant to T.I.T.
It has a plasmid and the desired configuration.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram comparing plasmids used in Examples 11, 12 and 14 of the present invention. FIG. 2 is an explanatory diagram showing a T-DNA region of Ti plasmid pTi15955. FIG. 3 is an explanatory diagram showing creation of p395 and p376 used in the first embodiment. FIG. 4 is an explanatory diagram showing the structure of p499 / 6/7 used in Example 1. FIG. 5 is an explanatory diagram showing a structure of p499 / 6/8 used in the first embodiment. FIG. 6 is an explanatory diagram showing the structure of p496-2 used in Example 1. FIG. 7 is an explanatory diagram showing the structure of p496-1 used in Example 1. FIG. 8 is an explanatory diagram showing the structure of each plasmid used in Example 2. FIG. 9 is an explanatory diagram showing the structure of pKS-nopIV used in Example 2. FIG. 10 is an explanatory diagram showing the structure of pRK290 used in Example 2. FIG. 11 is an explanatory diagram showing the structure of pKS-KB3.8 used in Example 2. FIG. 12 is an explanatory diagram showing the structure of pBR322. FIG. 13 is an explanatory diagram showing the structure of p3.8 used in Example 3. FIG. 14 is an explanatory diagram showing the structure of the phaseolin gene used in Example 3. FIG. 15 is an explanatory diagram showing the structure of AG-pPVph7.2 (p7.2) used in Example 3. FIG. 16 is an explanatory diagram showing the structure of pKS-4 used in Example 3. FIG. 17 is an explanatory diagram showing the structure of pKS4-KB of Example 3. FIG. 18 is an explanatory diagram showing the preparation and structure of p3.8-cDNA of Example 4. FIG. 19 is an explanatory diagram showing creation of pKS-5 used in Example 5. FIG. 20 is an explanatory diagram showing creation of pKS-oct.Cam 203 used in the fifth embodiment. FIG. 21 is an explanatory diagram showing the structure of pKS-oct.delII used in Example 5. FIG. 22 is an explanatory diagram showing the structure of pKS-oct.delI used in Example 5. FIG. 23 is an explanatory diagram showing a method for creating p2f used in the sixth embodiment. FIG. 24 is an explanatory diagram showing a method for creating p3e used in the sixth embodiment. FIG. 25 is an explanatory diagram showing a method for creating pKS-oct.delIII used in Example 6. FIG. 26 is an explanatory diagram showing a method for producing pKS-6 used in Example 7. FIG. 27 is an explanatory diagram showing a method of creating p2 used in the seventh embodiment. FIG. 28 is an explanatory diagram showing a method for creating pKS-oct.delIIIa used in Example 7. FIG. 29 is an explanatory diagram showing a method for converting an HpaI site of p203 into a BglII site in Example 8. FIG. 30 is an explanatory diagram showing the structure of pKS-oct.tmr in Example 8. FIG. 31 is an explanatory diagram showing the structure of p203 used for creating a shuttle vector in Example 1.

Claims (1)

Claims: 1. (a) a step of inserting a plant gene having a plant-derived promoter and a plant structural gene into T-DNA, whereby a T-DNA / plant gene conjugate is obtained. Forming, the plant-derived promoter is adjacent to the 5 ′ end of the plant structural gene, and the plant structural gene is transcriptionally downstream from the plant-derived promoter; and (b) the T- Transferring the DNA / plant gene conjugate to dicotyledonous plant cells, the method comprising genetically modifying the dicotyledonous plant cells. 2. After the step (b) is carried out, the method further comprises the step of (c) detecting the expression of the structural gene of the plant in a plant cell containing the T-DNA / plant gene combination. The method of claim 1. 3. The method of claim 1, wherein said structural gene has one or more introns. 4. The method of claim 1, wherein said plant gene encodes a seed storage protein. 5. The method of claim 1, wherein the plant gene encodes phaseolin. 6. The method of claim 1, wherein the conjugate of the T-DNA and plant gene is maintained and replicated prior to step (b) as part of a shuttle vector. 7. The method according to claim 1, wherein said plant gene is inserted into octopine-type T-DNA in tml or "1.6" region. Wherein said dicotyledonous plant is a member of Konpojite or Reguminose The method of claim 1.