JPS63119678A - Plasmid - Google Patents

Abstract

PURPOSE:To efficiently transform a higher plant, by preparing a plasmid having a sequence from a promoter, etc., derived from a cauliflower mosaic virus and capable of transforming a higher plant. CONSTITUTION:A plasmid having a border sequence of a cauliflower mosaic virus-derived promoter and terminator, neomycin phosphotransferase gene and T-DNA as well as about 6Kb molecular weight and capable of transforming a higher plant is prepared. Said plasmid can be expressed also by introducing into a cell of a higher plant such as the genus Brassica, etc., directly or via an Acrobacterium because of having a promoter and terminator derived from 19S or 35Sm RNA of CaMV and BR and BL of T-DNA.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a specific plasmid that can efficiently transform higher plants such as plants of the genus Brassica.

[Conventional technology]

Conventional plasmid vectors that can transform higher plants include T1 plasmid, OaMV (cauliflower mosaic virus), Escherichia coli vectors, and promoters that function in higher plants. j fA)" signal sequence has been developed.

The T1 plasmid is the best studied,9 with high transformation frequency and high reproducibility, but is not applicable to monocots. Furthermore, the length of the gene that can be inserted into OaMV vectors is limited, and the range of hosts that OaMV can infect is limited.

On the other hand, an E. coli vector incorporating a promoter that functions in higher plants can be applied to both monocots and dicots. However, there is a problem that the transformation rate is low.

Furthermore, a vector has been proposed that combines an E. coli-derived expression vector with the T-DNA border sequence of the T1 plasmid (ThθEMBOJ, L.

Core 7--g4t, /qrz) has yet to be reported, and it cannot be said that it has been sufficiently developed.

[Object of the invention and its solution]

The purpose of this BA is to provide a vector that is a combination of an E. coli-derived expression vector and 71 glasmid, and has improved transformation efficiency compared to conventional vectors.
Such an objective is achieved by Grasmid using specific plasmids, in particular as border sequences of the T-DNA of the T1 plasmid.

Hereinafter, to explain the present invention, the plasmid of the present invention is a plasmid having a molecular weight of about A Kl) (1,000 to 1 Oobp) and a restriction enzyme cleavage map as shown in FIG. rKb(r, 000±100bp
) is a plasmid with a restriction enzyme cleavage map as shown in Figure 1.

The promoter and terminator used in the plasmid of the present invention are, for example, 778 mRNA derived from OaMV, or ? &SmRNA-derived.

The neomycin phosphotransferase (NPT) gene is Tn? oj-derived NPT-4 gene and Tn! The derived NFT-31 gene is used.

Furthermore, as the border sequence of T-DNA, the DNA sequence shown below is preferably used in the BA of the present invention.

(Formula/) (Formula ko) The DNA sequence shown by (Formula/) has a light border (B
R) and the DNA sequence represented by (Formula, 2) is used as the left border (BL). These may be synthesized by chemical synthesis.

A method for producing a plasmid of the present invention having the promoter, terminator-1NPT gene, and T-DNA border sequence as described above will be explained based on FIG.

The KcoRl-8au JA fragment (/ffJbp) containing the promoter (P) of OaMV 778 mRNA was transferred to pBR3λco (Gone,
pOM/r by inserting into the coRl-BamHl site.
Get λ. In order to delete the OaMV-derived DNA part below the translation start codon (ATG) in the KcoRl-Sau, 7A fragment, pOM/r was cut with 5acl, and then approximately j! Digest about r bp. Next, the promoter is cut with EcoRl to obtain a promoter containing OaMV-derived ATG.

pUoza (Gene, /9.ko3)
9 (/9r2)) into the BaoRl-Sma 1 site to obtain pUOza-/-7. Next, the EcoRl-H1ndl fragment (/jJbp) of this grasmid was obtained, and the E
Insert into the coRl-H1nd1 site to obtain pOM/h.

After cutting this pOM/j,2 with Hlndll, it was smoothed (
fillin) L and further cut with Bgl II. Then, at this site, 3rBm RN of OaMV
Allul-Bgl containing the terminator (T) of A
Inserting the l fragment (j-, 2Abp, 1) yields pcMA7g.

At the BamHl-H1nc11 site of pOM&7g, T
n! r-derived 17) Bgll containing the NPT-11 gene
- insert an 8malUr piece (10/jbp), then
The BL represented by the above (formula λ) is Pvul-EcoR1
BRk inserted into the site and also represented by the above (Formula 1)
By inserting into the BglIl-Sma1 site, the plasmid (pTS&) shown in FIG. 1 is obtained.

In addition, 7 g of the pOMA was cut with Nrul to give approximately / Kb
After removing the Bam fragment of the recombined plasmid,
NPT- derived from Tn90.3 at Hl-H1nc1 site
■Introduce genes. For the N'P T-1 gene, for example, the DNA from the codon next to ATG to the Xho 1 site is chemically synthesized, an Xhol-Avai fragment is ligated to it, and a BamH1 site is added to the j' side of the DNA (
? xbp). And the above can be done.

〔Effect of the invention〕

The plasmid of the present invention contains CaMV/S, j! rsm
RNA promoter, terminator and T-DNA
Since it has BR and BL, it can be expressed either by direct introduction into the rule cells of higher plants such as plants of the genus Brassica or by introduction via Agrobacterium. Moreover, the plasmid of the present invention is relatively small compared to conventional plasmids, and the transformation rate is improved.

〔Example〕

The present invention will be further explained below with reference to Examples.

Example/(Method for producing pTsj) OaMV (8 strains) was treated with KcoRl at 37°C for 30 minutes.
1'jkb) was extracted, treated with phenol and ethanol, treated with Elau, 7A at 37°C for 30 minutes,
A fragment of oRl-5aujA (lxbp) was obtained.

pBR32- was treated with FicoRl and BamHl at 37°C for 50 minutes, and then treated with plucali phosphatase (BAP) at 37°C for 30 minutes. Both of these D
NA was treated with 7 enol-chloroform and ethanol to remove protein, and then treated with T,-ligase overnight at 72°C to bind, yielding pOM/Zakko.

pOM/ff was treated with 5 acl at 37°C for minutes, then treated with phenol-chloroporm and ethanol, and then treated with Bal J / at 37°C for - minutes, 3 minutes, 73 minutes, and 2 minutes.
Each treatment was carried out for 0 minutes. After stopping the reaction with phenol-chloroborm, precipitating with ethanol and suspending in water, Ec
DNA treated with oRl at 37°C for 70 minutes, subjected to polyacrylamide gel electrophoresis, and treated with Ba1J/ was recovered.

This DNA is pUct's E! It was ligated to the ooR1-8ma1 site using T41J gauze to obtain pUoz-1.27.

The base sequence was determined by the dideoxy method to see how far the base was removed by Ba1.7/. Among them, a plasmid in which the promoter was deleted to immediately after the ATG was selected, and the plasmid was treated with KOORI and Bindu as described above to isolate a portion of the promoter/jJbp. This fragment was inserted between the EaoRI-Bind1 sites of pxa to obtain pOM/j-.

pOM/j was cut with Hlndll, blunted with Klenow fragment, and then cut with Bgl11. OaMV-
Bgl l to remove part of the terminator from 8.
Cut with /ff? A fragment of jbp was obtained and further treated with AluI to isolate the terminator partial S co-Abp. This fragment was inserted between the alnali (blunt) and Bgl of the above pOM/yo- to obtain pOM47g.

The NPT-1 gene was obtained from Tn& by treating with Bgl-1 and 8mal, and BamHl of pOM67ff
- Inserted into the H1r1c11 site. This plasmid is
vuJ.

After EcoR1 treatment, chemically synthesized BL (formula ^) was inserted. Similarly, BR (formula) was inserted into the Bgllll-Sma1 site to obtain pTsj.

Application example 1 Using pTsj obtained in Example/ as a vector, tobacco (SR
/) protoplasts were treated with polyethylene glycol (PE).
An example of direct introduction using G) is shown below.

Tabacum var BRl (N, tabacum var BRl) shoot apical culture (Murashige-skoog medium, 3
% sucrose (subcultured every 3 weeks) to enzymatic (cellulase 1%, pectolyase o, /%
, 0, '1 M mannitol% pHt, t). Protoblast 0. ttM Manitou/
After washing, K3 medium (4hiro-DO, 1m9/1.
NAA/, 0171&/A', EAPθ-2m9/1
1. Suspended in 1031/l of ci:I sugar.

DNA was extracted using the Potrykus method (P'1ant Mo
regularBiologyReporter
3. / /ku/sg, /qgr). i.e. 15μI pTs! ; as carrier DN
A (Calf thymus DNA) was added to the protoplast suspension (number of cells/X 10'Al)/ml and incubated with μI.

Both circular and linear DNAs were used.

The protoplast suspension was incubated for 3 min at 4°C and then kept on ice for 10 s before adding the DNA.

After incubating the DNA and protoplasts for 5 minutes at room temperature, 0.3 d of PEG4tθ polyaqueous solution was added. After incubating for 1 minute, add O, gM mannitol,
-! Bamboo cleaning solution (0,4(M-r2)-l, OaOm1
After washing the protoplasts several times with 0mM), they were suspended in 10rttl of 3 medium and cultured at -5°C. After culturing for 1 week, kanamycin was added to a final concentration of 200 m9/l, and the culture was continued. The transformed cells become resistant to kanamycin, so they are grown on solid medium (LS medium).
Physiol, Plant, output 100-7λ (/
fA&))λ, F −D O,! rln9/l, N
AI ml/l, BAPO, 1m9/73, K
Transfer to netin 001m9/l, sucrose J, Gelrite O 0.2%, kanamycin m9/JBAP, Shi, Yo゛@, 7%, Gelrite O0λchi, Kanamycin 2
m9/l). The shoots with leaves were then transferred to MS medium (MS medium, hormone free, Gelrite 0.1%, Kanamycin 2-/11) to form adventitious roots. After roots emerged and seven plants were transferred to vermiculite to acclimatize the roots, the plants were transferred to culture soil and cultivated in pots to obtain transformed plants.

Example 6 67 g of pOM obtained in Example 9 of pTB903RL was treated with Nrul at 37°C for 30 minutes to remove the Nrul-Nrul cleavage of approximately The cleavage sites were ligated by overnight treatment.

The obtained plasmid was treated with BamHl and Hlnall at 37°C for 30 minutes, respectively. On the other hand, Tn90.7 to 9
tAbp NPT-1 was prepared and added to the BaI and H1ncl-treated plasmid at /u℃/night at T41.
It was bound with J gauze.

Thereafter, BL and BR were inserted in the same manner as in Example 1 to obtain plasmid pTs90JRL having a molecular weight of about A kl).

pTB90JRXr 7kBc-oRl obtained in Application Example
Broad host range vector pTJs? &(ThθE
tMBOJ,.

Above, after cutting the core 7-coffQ (/?zari) with alndffi and smoothing it, F! A plasmid ligated with the c oRI IJ linker was obtained and transformed into Escherichia coli 1M10.

This transformed E. M. JM/09 strain was added at 10μI/
LB liquid medium containing kanamycin of Ll and 3 μp/ml of tetracycline (Re(/), plasmid pRK containing kanamycin resistance gene, 2fl)/J was cultured in LB liquid medium containing 10 μj9/ml of kanamycin. The cells were cultured at 37°C for 72 hours. Agelobacteria LBA & IO4 strains are 7 Ambicin/! Cultures of three bacteria cultured in b x K B liquid medium (Ref/) containing μE/ml at 30°C for 72 hours were each cultured at 0.0%. Mix ll1l by 1l, diameter, 2cIrL
Bacteria were collected on a nitrocellulose filter (Oo-1 μm). This filter was placed on an NA plate medium (Rθf/) and cultured at 30°C for an hour.

The filter was soaked in /ml of λ dilution 1 (Ref/) and gently shaken to suspend the bacteria growing on the filter surface. After diluting the bacterial solution to 1000 cultures with the same diluent, 0. /at ampicillin (jOμyd), kanamycin (10μ9/In-t), ambicin (3μl)
After spreading with a glass rod, the cells were cultured at 30° C. for 3 hours to form a single colony. The single colony was spread on the same medium and 30
The cells were incubated at ℃ for an hour to form a single colony again. This operation was repeated 6 times, and the E. coli JM109 vector plasmid was transferred to Agerobacteria LBA.
The Teitei Oy strain can be colonized.

*Ref / ; DNA OIOning, Q
47. A leaf disc (LD) with a diameter of 6 mm was punched out from a mature leaf of Dej3 tobacco (SR/) using a cork poultice.
qolI strain (YEtBf@ji, Beefex!V!, yeast extract ip, g3.peptone!g/13゜sucrose, t
j9/l, Mio4.2×10-”m/
1) Mixed with pH adjustment and c). The LD is J432. The orchard was thoroughly washed with tissue vapor and placed on a feeder layer medium with tobacco suspension culture as a nurse. The feeder layer is N A A O, / m9 / A in the 3 medium.
, BAP/-OIn9/l, 0. Approximately 0.3% tobacco suspension culture per plate in A% agarose Type I. The one containing lIi was used. After 1 week, transformants were selected on the medium described above. After one week, the callus was transferred to regeneration medium (LS medium) to form adventitious buds.
Moved to. Once stems and leaves have formed, they are transferred to a hormone-free medium when they reach an appropriate size, and adventitious roots are formed to obtain transformed callus-derived plants.

[Brief explanation of the drawing]

Figures 1 and 1 show schematic diagrams of the plasmids of the invention. In the figure, Orl indicates the replication initiation site, and hK indicates the ampicillin resistance gene. FIG. 3 shows a schematic diagram showing the method for producing the plasmid of the present invention. Applicant: Mitsubishi Corporation, Mitsubishi Chemical Industries, Ltd. Agent: Patent attorney Hase - 11 or 7 people Is 13 (So/) 1) ↓δacl -JBa/31 y? J31. ffi□ (Zono 2nd 3rd Ward (So, 3)

Claims (1)

[Claims] (1) A plasmid that has a cauliflower mosaic virus-derived promoter and terminator, a neomycin phosphotransferase gene, and a T-DNA border sequence, and is capable of transforming higher plants, and the plasmid has a molecular weight of approximately 6 Kb and has a molecular weight of approximately 6 Kb. A plasmid having a restriction enzyme cleavage map as shown in FIG. 1 or a plasmid having a molecular weight of about 5 Kb and a restriction enzyme cleavage map as shown in FIG.