JP3243233B2 - Transgenic plants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

Background matter of the invention relates to is one that corresponds to a continuation application of US patent application No. 692,759, filed on January 18, 1985.

[0002] The present invention relates to the use of genetic engineering techniques in plant improvement. More specifically, Bacillus thyurin caddissis ( Bacillust) is used in plant cells.
huingiensis ) or the introduction and integration of a chimeric gene having considerable sequence homology with the toxic genes described below and the insecticide of said polypeptide venom transformed and descended by its progeny To achieve expression at an acceptable level.

[0003]

BACKGROUND OF THE INVENTION Recombinant DNA engineering is currently used on certain genetically engineered microorganisms, such as bacteria and yeast, to synthesize specific proteins. In the state of the art, genetic engineering for higher organisms requires one or several genetically engineered cells capable of forming a complete organism. Among higher organisms, the cells of certain plants exhibit excellent regenerative capacity and are therefore considered as potentially good materials for genetic manipulation of such plants. Furthermore, in higher plants, the exogenous DN is added to the plant genome.
A known system for introducing A has been obtained. This system is based on the gram-negative soil bacterium Agrobacterium tumefaciens.
A ( Agrobacterium tumefacie)
ns ). Aggro
Bacteria can genetically transform plant cells by stably integrating T-DNA, a well-known fragment of the Ti plasmid, into the genome of plant cells. Significant progress has recently been made to facilitate the use of Ti plasmids as vectors for plant genetic engineering. T
-It has been discovered that small orthotopic repeats flanking (the end sequence of) DNA play an important role in the integration of T-DNA. Non-tumorigenic Ti plasmid vector is T-DN
A was constructed from deletion of the oncogene due to an internal deletion of A. These Ti plasmids contain border sequences and therefore transfer T-DNA without tumor introduction. An example of such a Ti plasmid-derived vector for plant genetic engineering is DGV3850, where the gene inside the T-DNA is replaced by the commonly used cloning vector pBR322. pGV38
Several methods have been developed to regenerate infected plants, including 50. P having the sequence of pBR322 in T-DNA
GV3850 is an efficient receptor plasmid for gene transfer experiments in plant cells. In fact, the genes that have been cloned in pBR322 like plasmid, Agurobakute
It transferred to helium, in one step, pGV3850T-DNA
Is inserted through homologous recombination.

[0004] Another major advance in the development of plant manipulation techniques is the use of plant regulatory sequences to express chimeric genes in plants. In general, these chimeric genes include a promoter region from a gene naturally expressed in plant cells, the sequence to be expressed, and, preferentially,
It contains a 3 'untranslated region containing a site in the polyadenylation of a gene naturally expressed in plant cells. For example, dominant selectable markers for plant cells have been constructed using the nopaline synthase promoter and bacterial antibiotic resistance genes.

[0005] Certain chimeric genes are now successfully expressed in transformed plant cells;
There are twists and turns. The level of expression of foreign genes of non-plant origin is not only very different in different transformed crude weaves,
In general, various evidences have shown that they are very low. Such low levels of gene expression are due to several reasons. First,
Incomplete transcription of a gene due to an accidental transcription termination signal. Second
Inefficient mRNA processing. Third, reduced mRNA transfer from the nucleus to the cytoplasm. Fourth, cytoplasm m
RNA instability. Fifth, inefficient cytoplasmic mRNA
Translation. And sixth, protein instability due to plant sensitivity to specific proteins. Thus, successful transformation of plant cells using the vectors described above is not always predictable before attempting the desired transformation.

The manipulation of plant differentiated cells and their progeny to express a Bt2 polypeptide and / or a truncated version thereof and / or a polypeptide having substantial sequence homology to Bt2 involves the following: For one or more reasons, it is much more difficult than with other genes, such as antibiotic resistance genes, or other plant genes, such as thaumatin. (1) The size of Bt2 toxin is large even when shortened. (2) Unique properties of the Bt2 polypeptide (such as, but not limited to, the solubility of the polypeptide). (3) Bt2 against plant cells
Potential toxicity of the polypeptide. Alternatively, (4) the Bt2 polypeptide synthesized in the plant cell and its progeny must possess substantially the same properties as the crystalline protein synthesized in bacteria.

Bacillus thuringiensis (sometimes referred to herein as Bt ) comprises approximately 19 known variants that produce polypeptide toxins that form pseudospore crystals during the sporulation phase. I have. B. t. Is toxic to certain insect larvae.
Toxins produced by certain species exhibit strong insecticidal activity against certain lepidopteran, beetle, and dipteran larvae. For example, Tyrell, D.E. J. Etc., Journal of Bakute
See Rheology (1981), Vol. 145 (No. 2), pages 1052-1062 . Upon incorporation into insect larvae, the crystals solubilize and produce at least one active polypeptide toxin in the midgut of the insect that is believed to be altered and act on the cell wall of the midgut. Studies have shown that individual crystalline polypeptides exhibit insecticidal activity. Yamamoto, T. Etc., Current My
Clobiology , (1983) Vol. 9, 279-28
Page 4: Yamamoto, T. Achievements of By
Chemistry and Biophysics , (83
Year), Vol. 227 (Issue 1), pp. 233-241 ; Lily, M, et al., Journal of General
Microbiology , (80), Vol. 118, pp. 1-11; Bra, L .; A. Etc., the journal of
Biological Chemistry , (1981), Vol. 256 (No. 6), 3000-3004 pages.

The toxicity of the crystalline polypeptide produced by a variant of Bacillus thuringiensis is very specific to certain insects and recognized as safe by higher vertebrates. Formulations containing crystals have been used commercially as biological insecticides. For example, BioChem Product Ltd, Bacto Spain and Sand AG's Chiuriside, commercially available from Dipel Abbot Laboratories. The efficacy of preparations obtained from bacterial hosts, however, is limited, since proper control of pests requires repeated and precise use of time.
In addition, the prices associated with the production of such products make it difficult to compete effectively with other commercial products, such as pyrethroid derivatives.

[0009] The molecular genetics research, Bacillus healing
At least some of the polypeptide toxins produced by Ulingensis have been shown to be encoded by plasmids. D. P. Starley et al. (1978), Biochemical and Biofi
Zical Research Communication , Volume 84, 5
Pages 81 to 588; G. FIG. Debobokk etc. (197
7 years), Genenetica , Ings of National Academy of Sai
Ens of the USA , (1981), Vol. 78, 2993
From 2997 pages. A. Embo Jiana, Krie
Ru , (1982), Volume 1 (No. 7), 791-799
page. Adan et al., Gene (36), 289 pages, 1
985. Journal of Biological deer such as Sienev
Le ChemStation birds over, (20), 6264 pages, 198
5 years. Takumi, etc., Gee emissions (34), see 1985.

[0010] Given the major importance of plants with respect to both consumption and production of precious products, plant cells can be used to produce Bacillus cerevisiae without adversely affecting the plants.
Plants that have been genetically modified to synthesize polypeptide toxins that are very similar to those produced by Lingensis are desirable. Heterologous DNA fragment encoding a polypeptide toxins produced by Bacillus Chiyuu phosphorus thuringiensis stably integrated into the plant cell genome, a plant body in, by obtaining the expression of said heterologous DNA fragment level that combating insects, their The plant cells so transformed and their progeny are consequently resistant to certain pests. Genetically engineered plant cells and their progeny in this way are present by substantially eliminating the need for specific chemical or biological pesticides while retaining their normal morphological properties. It is an economically viable alternative to commercially available varieties and a more reliable means of controlling certain pests.

[0011] [SUMMARY OF THE INVENTION] polypeptide toxins produced by Bacillus Chiyuu phosphorus Gen cis or,
It is an object of the present invention to provide an unprecedented chimeric gene that encodes a polypeptide poison having significant sequence homology to the venom genes described herein. Plant regulatory sequences of the chimeric gene govern expression in transformed cells. Another object of the present invention is to provide a new hybrid plasmid vector containing a chimeric gene that allows for the introduction, integration and expression of the chimeric gene in the genome of a plant cell. Yet another object of the present invention is to provide a method for producing a genetically transformed plant cell comprising transforming a plant cell having the hybrid plasmid vector containing the chimeric gene. Other objects, features and advantages of the present invention will become apparent to one of ordinary skill in the art from the drawings and description below.

According to the Summary of the Invention The present invention [Means for Solving the Problems], (a) DNA fragment containing a promoter region derived from genes expressed in plant cells naturally and (b) Bacillus Chi Yu There is provided a chimeric gene expressed in differentiated plant cells which encodes a polypeptide toxin produced by Lingensis or which consists of at least one DNA fragment with considerable sequence homology thereto. The chimeric gene may be a gene whose DNA fragment (b) encodes a Bt2 protein or a small Bt2 protein having insecticidal activity, a DNA fragment having a considerable sequence homology with Bt2 or a small Bt2 protein, or D
NA fragment (b) is capable of expression in differentiated plant cells,
And a DNA fragment (c) encoding an enzyme capable of identifying a plant cell expressing the DNA fragment (b), wherein the DNA fragments (b) and (c) encode the fusion polypeptide. Contains the gene that is fused to

According to the present invention: (a) a DNA fragment which is substantially homologous to the portion of the Ti plasmid essential for transferring the T-region of the Ti plasmid into the plant cell genome (pathogenic region of the Ti plasmid): b) at least one DNA fragment depicting the DNA fragment for integration into the plant cell genome (T plasmid T
-A border sequence of the DNA portion; only one border sequence is present, preferably the right border sequence); and (c) (i) a DNA fragment containing a promoter region from a gene naturally expressed in plant cells; And (ii) bees
A hybrid plasmid comprising at least one DNA fragment encoding a polypeptide toxin produced by Russ thuringiensis , or at least one chimeric gene comprising at least one DNA fragment having considerable sequence homology thereto. -Vectors are provided.

The chimeric gene is a DNA fragment (b)
Is a Bt2 protein or a small Bt having insecticidal activity
A DNA fragment that has considerable sequence homology with BT2 or BT2 or a small size Bt2 that encodes a protein;
Alternatively, a plant cell in which the DNA fragment (b) is capable of expressing in a differentiated plant cell and expresses the DNA fragment (b) in which the DNA fragments (b) and (c) encode a fusion polypeptide And a gene that is fused to a DNA fragment (c) encoding an enzyme that allows the identification of E. coli. Furthermore, according to the present invention, (a) DNA fragment containing a promoter region derived from genes expressed in plant cells naturally and (b) Bacillus Chiyuuringen
An intermediate plasmid vector comprising at least one DNA fragment encoding a polypeptide toxin produced by cis or at least one chimeric gene comprising at least one DNA fragment having considerable sequence homology thereto is provided. .

The chimeric gene is a DNA fragment (b)
Is a Bt2 protein or a small B with insecticidal activity
A gene encoding the t2 protein, a DNA fragment having a considerable sequence homology with Bt2 or Bt2 having a small size, or a DNA fragment (b) can be expressed in differentiated plant cells, and the DNA fragment (b) ) and (c) an enzyme co you enable the identification of plant cells you expressing DNA fragments where encoding a fusion polypeptide (b)
Genetic being fused to over de reduction to the DNA fragment (c)
Contains children. Further, according to the present invention, there is provided a method of using a plant cell transformed with an insecticidal substance and its progeny. Further, according to the present invention, there are provided a plant containing a cell genome and expressing the chimeric gene as described above, and a plant seed capable of growing on a plant expressing the chimeric gene as described above. The transformed cells of the plant and their progeny express intracellularly a polypeptide toxin that is very similar to the polypeptide toxin produced by Bacillus thuringiensis ,
Very toxic to certain insects. The transformed plant cells and their progeny can be used for controlling the aforementioned insects.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows 7.5% SDS stained with Coomassie blue .
It is a result of polyacrylamide gel electrophoresis. Track 1: B. t. Kurstaki Crystal Protein Preparation Track 2: B. t. Berliner Crystal Protein Preparation Track 3: Molecular Weight Markers a: Phosphorylase B
(92500 daltons) b: Bovine serum albumin (66
C: ovalbumin (45,000 daltons) d: carbonic anhydrase (31,000 daltons) FIG. 2 is a schematic diagram of plasmid pEcoR251. EcoRI endonuclease gene (EndR
I) is fused to the _{P R} promoter _{(P R),} a single B
It contains a glII cloning site. Amp: β-
Lactamase gene. FIG. 3 shows the results of B. coli cloned into pEcoR251. t. Figure 5 shows a restriction map of the insertions present in the four immunologically positive Sau3A partially digested clones of Berliner 1715 plasmid DNA.

FIG. 4 shows the results of a 7.5% SDS polyacrylamide gel exercise swimming stained with Coomassie blue. Track 1: B. t. Kurustaki crystal protein preparation (FIG. 1, identical to track 1) Track 2: B. t. Berliner crystal protein preparation (same as FIG. 1, track 2) Track 3: Escherichia coli K51 containing Bt200 plasmid
Total lysate track 4: Control (total lysate of E. coli K514 without Bt200 plasmid)

FIG . 5 shows rabbit anti-B. t. Fig. 3 shows the results of an immunoblotting experiment using Krusutaki crystal serum. Track 1: B. t. Berliner Crystal Protein Preparation Track 2: B. t. Kurustaki crystal protein preparation Track 3: E. coli K5 containing pBt200 plasmid
14 total lysate tracks 4: control (E. coli K514 without pBt200)
FIG. 6 shows the results of an immunoblotting experiment using a rabbit anti-Kulstaki crystal serum (A) and a rabbit anti-berliner crystal serum (B). C shows Coomassie blue staining of 7.5% SDS polyacrylamide gel electrophoresis (same gel used for blotting shown in A) after blotting. Track 1: Bt2 protein track (purified as described in section 5-1) t. Berliner Crystal Protein Track 3: B.I. t. Kurustaki's Crystal Protein

FIG . 7 shows the results of Coomassie staining by SDS polyacrylamide gel electrophoresis. Track 1: Total lysate of E. coli K514 with pBt200. Track 2: Purified Bt2 protein prepared from E. coli K514 with pBt200 . FIG. 8 is a graph showing the results of an ELISA experiment.
Goat anti-B. t. Bt2 protein using mouse anti-Bt2 serum as primary antibody and solubilized B. t. Crystal protein binding curve. FIG. 9 shows ELISA showing the reactivity of various anti-berliner crystal monoclonal antibodies with (A) whole berliner crystal protein and (B) purified Bt2 protein.
It is a graph of the result of A experiment. FIG. 10 shows 130 Kd
3 shows a comparison of the N-terminal amino acid sequences of crystal proteins. 130K
The d protein is deduced from the DNA sequence reported by 1) Won et al. (Journal of Biological Chemistry, Vol. 258, 1960-1967, 1983). (Designated BtW) 2) Determined from bt2 protein.

FIG . 11 shows rabbit antibody B. t, Summary of immunological detection of polypeptides using Western blotting with Berliner crystal serum. The polypeptide is encoded by a pBt200 derivative containing various deletions resulting from restriction enzyme cleavage as shown. FIG. 12A shows a restriction map of the HpaI-NdeI fragment containing the complete Bt2 gene shown in boxes. B is
2 shows the sequence region of the Bt2 gene. Boxes represent ranges arranged from each chain. C indicates the sequencing method.
Restriction fragments were end-labeled with polynucleotide kinase, separated strand by strand, and sequenced using the method of Maxam and Gilbert. Arrows indicate the length of the region sequenced in each experiment. FIG. 13 shows a complete Bt2 showing the reading frame (21st to 3605th).
DNA sequence of the gene and its deduced amino acid sequence (1
155 amino acids). The experimentally determined amino acid sequence of the Bt2 protein is shown above the corresponding amino acids.

FIG . 14 shows the Bt2 gene (Berliner)
And B. cloned from the other three strains. t. 3 shows a comparison with a sequence deduced from a crystal protein gene. The other three strains are: t. Kurusutaki HD73 (Adan et al., Gene 36th
Volume 289, page 1985). t. Kurstaki HD1 (Shienefu, etc., journal,
Of Biological Chemistry , Volume 20, 62
64, 1985) t. Sot (Shibano et al., Gene , Vol. 34, 243
(Page, 1985) In the latter three sequences, only the amino acid sequence different from the Bt2 sequence is shown at the same position. In fact, a gap was introduced (marked with "-") to arrange the arrangement. FIG. 15 shows the pHD16 of the Bt2 gene cassette .
1 is a schematic overview of the construction of 0. FIG. 16 shows a different B
It is a schematic diagram of a t2 gene cassette.

FIG . 17 shows the experimental procedure used for the construction of plasmid pLB10. Here, also, The plasmid pLK5 described by Botterman et al.
4 and the structure of pLK57 are also shown. FIG.
1 shows the construction and structure of pLBKm25. FIG.
t: Shows the technique used to construct the NPTII fusion and Bt2 deletion. FIG. 20 shows the 3 ′ of the smallest encrypted fragment of the toxin .
FIG. 2 is a schematic diagram of the different Bt2 3 ′ terminal deletion mutant strains used for terminal mapping. The arrow indicates the position of the 3 'end.
FIG. 21 shows the results of an immunoblotting experiment using rabbit anti-berliner crystal serum. The sample analyzed was the entire extract of the Bt2 deletion clone identified in FIG. 20 and section 7. FIG. 22 shows Bt2 used to outline the smallest gene fragment encoding an active poison .
The 3 'ends of the deleted clones pLB834 and pLB879 on the sequence are shown. The deduced amino acid sequence and the location of the putative trypsin cleavage site are also indicated.

FIG . 23 shows the Bt2: NPTII fusion gene cassettes pLBKm23, pLBKm33 and pLB.
It is a schematic diagram of construction of Km14. Bt of different constructs
2: The 5 'upstream sequence of the NPTII fusion is also shown (the sequence corresponding to the BamHI site is underlined). FIG. 24 is a schematic diagram of the different Bt: NPTII fusion gene cassettes. FIG. 25 is described by Reis et al . ( Gene , Vol. 30, p. 217, 1984)
9 shows the results of NPTII measurement. The samples analyzed were supernatants of cell extracts of bacterial clones producing NPTII or various Bt2-NPTII fusion proteins. 2
3 means K514 (λ) (pLBKm23). 8
60 means K514 (λ) (pLBKm860). 865 means K514 (λ) (pLBKm865). NPT means HB101 (λdv).
(Gifted by Julien Davis, formerly a member of Biogen.)

FIG . 26 shows the approximate position of the 3 'end of the Bt sequence of the Bt: NPTII fusion (indicated by the arrow) with the different deletions. FIG. 27 shows the technique used to adapt Bt2 and BT2: NPTII cassettes for expression in plant cells. FIG. 28 shows a different manipulated T
3 shows the DNA sequence of the junction between the promoter region present in plasmid i and the coding sequence of the Bt gene cassette. The sequence derived from the original promoter region and the sequence of the coding sequence of the Bt2 gene are underlined. Some suitable restriction sites involved in the construction of the chimeric gene are indicated. The ATG start codon is boxed. FIG. 29 shows that p
It is a schematic diagram of construction of HD208. B: BamHI, Hp: HpaI, H: HindIII, E: EcoRI, Bg: BglII

FIG . 30 is a schematic diagram of the construction of pGV831: pGV831 was obtained from R. B. of the Genetic Virus Laboratory at the University of Brussels Free University in Belgium. Built by Debraele. This is described in European Patent Application No. 83112985.3.
As described in pGV700. Recombinant DNA technology has been developed by Mania Teis et al.
Over Cloning (1982)従Tsuta in Cold Spring Harbor Laboratory.

The HindIII fragment present in pGV700 is derived from pGV600 (Lehmans et al., Journal of
Morekular and Abride Genetics,
1 (pp. 149-164, 1981). Recombinant plasmid pGV742
Was isolated as a crude Cb ^R Cm ^S Ts ^S recombinant. B
Digestion with amHI and recircularization created an internal deletion in pGV742. This is pGV744.
To obtain pGV749, an internal deletion was made in pGV744 by EcoRI digestion and recircularization. pGV7
Forty-nine HindIII-NruI fragments were cloned into pGV710. pGV710 was cut with EcoRI, the 5 'overhang was filled in using DNA polymerase, and further cut with HindIII. The resulting plasmid pGV815 was isolated as a ^Sm R, Cb ^R recombinants. Both the EcoRI site and the HindIII site of pGV815 were removed by digestion with these and filling and recircularization with protruding DNA polymerase. Finally, a chimeric gene containing the Tn5 neomycin phosphotransferase gene in the nopaline synthase promoter was transformed into PKC71: nos
Isolated as a BclI-BamHI fragment of pGV8
It was cloned into 25 BglII sites. Sp ^R ,
Miles ^R recombinant plasmid pGV831 was obtained.

FIG . 31 shows that the Ti plasmid pGV385
FIG. 4 is a schematic diagram of the T region of the intermediate vector pHD205 and the intermediate vector pHD205. The crossed line shows the pG to create pHD1050.
The regions involved in the co-operation of V3850 with pHD205 are shown. T of hybrid Ti plasmid pHD1050
The area is revealed. H: HindIII Bt: Chimeric Bt2 gene under the control of nopaline synthase promoter nos: nopaline synthase gene Ap, Km: genes encoding resistance to ampicillin and kanamycin

FIG . 32 shows that the Ti plasmid pGV226
FIG. 2 is a schematic diagram of the T region of the 0 and the intermediate vector pHD208. The crossed line is the pGV to create pHD1076.
The region involved in the association of 2260 with pHD208 is shown. The T region of the hybrid Ti plasmid pHD1076 is represented. 1: vector fragment 2: T-DNA border region 3: Bt
2 gene cassette 4: Pssu promoter fragment
5: Pnos promoter fragment 6: Neomycin phosphotransferase gene cassette 7: TD
NA border region 8: The black triangle in the vector fragment represents the T-DNA border region. Ap. Sp, Kn: genes encoding ampicillin, spectinomycin and kanamycin resistance, respectively Pnos: nopaline synthase promoter Pssu: small subunit of ribulose 2-phosphate carboxylase promoter t. : Bt2 gene cassette Fig. 33 shows a schematic diagram of different intermediate expression vectors.

FIG . 34 is a graph showing the results of an ELISA test of tobacco callus tissue transformed with C58Cl Rif ^R pHD1076 as described in Example 11 of Section 11. The coat antibody was goat anti-B. t. Crystal serum.
Rabbit anti-Bt2 was used as the primary antibody. Numbers 1 to 14 are transformed calli. The absorbance (OD) corresponding to the level of 4 ng Bt protein / g tissue determined in the reconstitution experiment is shown in the figure. FIG. 35 shows C58Cl Ri as described in Section 11, Example 1.
In f ^R pHD1076 is a graph showing the results of an ELISA testing of tobacco callus tissue transformed. The coat antibody was goat anti-B. t. Crystal serum. Rabbit anti-Bt2
Was used as the primary antibody. Numbers 1 to 21 are transformed calli. 1 g of tissue determined by reconstructive experiment
O.t. corresponds to the level of 4 ng Bt2 protein per D. The values are shown in the figure.

FIG . 36 is a graph showing the results of an ELISA test of tobacco callus tissue transformed with C58Cl Rif ^R pHD1076, as described in Section 11.2, Example 1. The coat antibody was a goat anti-B. t. Crystal serum. A different monoclonal antibody was used as the primary antibody. The reactivity of untransformed SR1 callus tissue (used as a negative control) is also shown. Third
FIG. 7 is a description of the experimental procedure used for the preparation of callus tissue extracts and the immunological detection of Bt2 expressed in this callus. FIG. 38 shows that the transformed tobacco leaves obtained in Section 10, Example 15 were fed as feed .
It is a graph which shows the growth rate of the 1st instar larva of sexta . The hollow bars represent the number of larvae that reached L2 stage 3 days after feeding (relative to all 20 larvae tested). FIG. 39 shows that M. aureus was fed on transformed tobacco leaves . FIG. 39 is a graph showing the complete growth rate curve of sexta larvae over 4 days (data are from the same experiment as represented in FIG. 38). The values shown are the number of larvae in the L2 stage at a certain time point (20 larvae per plant were tested).
C _{1 to C 4} are (transformed with only Pnos-NPTII gene) control plants. Other numbers (N20-1,
N20-46) is attached to individual plants presumably transformed with pGS1110.

FIG . 40 shows the cauliflower mosaic virus Cm4-184 (Gardner et al., 1981, Nuku
Reitsk Acid Research , Vol. 9, 2871-2
8 shows the DNA sequence of the P35S-1 and P35S-2 promoter fragments from p.888). FIG. 41 shows that pG
It is a schematic diagram of construction of SH50. FIG. 42 shows that pGV1
500 is a schematic diagram of the construction of 500. FIG. FIG. 43 shows that pGSH1
FIG. 2 is a schematic diagram of the construction of 50 and pGSH151. Forty-fourth
The figure is a schematic diagram of the construction of Pssu301 wild-type gene pAGS007.

DESCRIPTION OF THE PREFERRED EMBODIMENTS As used herein, the term "polypeptide" is understood to mean the entire protein or a fragment thereof. "Plants" refers to angiosperms (monocotyledon and dicotyledon)
And multi-cellular differentiated organisms that can photosynthesize including gymnosperms. "Plant cell" refers to one or more cells derived from a plant. "Plant cell progeny" refers to the callus;
Plant parts such as roots, fruits, leaves or flowers; plants; plant seeds; all cells or tissues derived from plant cells, including pollen and plant embryos. A "chimeric gene" is a portion of hybrid DNA that contains regulatory signals essential for transcription, called a promoter, fused to at least one structural gene sequence encoding a particular polypeptide. "Substantial (or substantial) sequence homology" refers to a DNA fragment having a nucleotide sequence sufficiently similar to another DNA fragment that produces a protein having similar properties, or another polypeptide exhibiting similar properties. Are understood to be polypeptides having an amino acid sequence sufficiently similar to "Identification" refers to selecting or evaluating cells that express the desired gene. Selectable markers allow growth (selection) under otherwise lethal conditions, such as kanamycin resistance (Km ^R ). The discriminating marker adds an identifying property (evaluation) unrelated to non-transformed cells. “Naturally expressed genes”
Means a DNA fragment originally as part of the genome of the plant or a DNA fragment introduced by factors such as bacteria or viruses that produce RNA, protein or both in plants without human intervention. The chimeric gene also contains an untranslated D at the 3 'end (downstream) of the structural gene sequence.
It contains NA fragments and also contains regulatory signals derived from genes that are naturally expressed in plants, called polyadenylation signals.

The naturally expressed gene contains a 3'-untranslated region which also contains a polyadenylation signal;
Then, both respond to the corresponding messenger RNA (m
RNA) region. Their corresponding mR
The NA region is located at the 3 'end of the stop codon of a single cistronic mRNA. The 3'-untranslated region of the mRNA is believed to be involved in mRNA processing, stability and / or transport. 3'- of mRNA
It is believed that the untranslated region also contains a base sequence called a polyadenylation signal that is recognized by enzymes in cells. This enzyme adds a number of atenosin residues to the mRNA molecule to form the poly A "tail" of the mRNA. In general, the method used to achieve the present invention was entitled "Method of introducing an expressible gene into an Agrobacterium strain having a plant cell genome and a hybrid Ti plasmid vector useful for this process". It is described in European Patent Application Publication No. 0116718. Mallet
Luz Chiyuu phosphorus plasminogen or encoding a polypeptide toxin produced cis, or, Bt2 (see Figure B) and one or more of the chimeric transgenic and integration into the plant cell genome has significant sequence homology Is executed as follows:

(1) isolation of at least one DNA fragment of Bacillus thuringiensis encoding a polypeptide poison by digestion of cellular DNA, and insertion of the resulting mixture of DNA fragments into a cloning medium of a bacterial host; (2) identification of a bacterial clone having a DNA fragment encoding the polypeptide toxin; and (3) characterization of the structure of the DNA fragment encoding the polypeptide toxin; and (4)
Removal of unwanted DNA fragments adjacent to the desired DNA fragment; or (5) synthesis of a DNA fragment having considerable sequence homology and similar structure to the Bt2-encoding DNA fragment; or (6) producing a fusion polypeptide Construction of a DNA fragment containing the DNA fragment of (4) fused to the DNA fragment encoding the isotope polypeptide; and (7) a plasmid that controls the plant regulatory sequences present in the bacterial host.
DNA of the above (4) or (5) or (6) into a vector
Insertion of the fragment; and (8) introduction of the plasmid of (7) by conjugation (or initiation) to a bacterial host with the appropriate accessory plasmid; and (9) Agrobacterium harboring the receptor Ti plasmid vector. Conjugation of the bacterial clone of (8) to Tyumehuaciens ; and (10) Agrobacterium teumefuashi containing the desired chimeric gene.
Identification of Aens ; and Agrobacterium according to (11) and (10).
Contact of Umm teyumehuaciens with plant cells; and (1
2) identification of transformed plant cells from a suitable culture medium; and (13) immunological detection of Bt2 antigen present in extracts of transformed plant cells; and (14) regeneration of differentiated plants. Growth of transformed plant cells.

A different cloth than that described in the following example
Vector and bacterial host strain may be used
Expected. Recombinant plasmid before transfer to plant cells
Ti-like vectors such as pGV3850 to be integrated
This is known as a serotype vector. Recombinant plasmid
Is not integrated into the existing Ti plasmid
Alternatively, most of the naturally occurring Ti plasmids have been deleted.
There are also existing Ti-based vector systems. this
These binary systems, (Hekema et al.,Neycha, 303
Volume, 179 pages (1983)) or mini Ti plastic
Sumid, (Flamonide, etc.Biotechnology, First
Volume, 262 pages (1983))
It has been shown to introduce NA. These programs
Rasmids are located adjacent to the gene introduced into the plant (slightly less).
At least one, preferably two) border sequences. Plant details
Markers that can be selected or identified in cells
Yes, but not required. Such a plasmid isA.
  Teyume HacienceSelf-replicating with existing Ti
No incorporation into Rasmid is required. Chimeric gene of the present invention
Of the toxicity required to transfer DNA such as
The function is supplied to the transformer. (Hechema, etc.Neyja
ー303, 179 (1983)).
T. Frehley, etc.Biotechnology, Volume 3, 62
9 pages (1985) and Cree et al.Biotechno
Gee3, Vol. 637 (1985).
I want to be.

A. Tyumehuaciens is not the only way to introduce genes into plants. DNA is introduced by physical means such as electroporation or by chemical means such as polyethylene glycol (PEG) fusion.
It is believed that all techniques for introducing DNA, such as the chimeric gene of the present invention, can be used. In addition, RNA viral vectors that introduce an RNA copy of the insecticidal chimeric gene are also used. In addition, plasmid vectors containing other plant regulatory sequences than those described in the examples below are used. For example, the enhancer may be included before, after, or in the vicinity of the enhancer in order to exert the function of the chimeric gene.

Plant cells transformed with the novel plasmid vector of the present invention are cultured in a suitable medium, preferably a selective growth medium, and plants expressing the polypeptide toxin are regenerated from the resulting calli. . The next generation of plant cells and their progeny must also express the polypeptide poison. Their descendants and plant cells transformed or polypeptide toxin was quite similar to the polypeptide toxins produced by Bacillus Chiyuu phosphorus thuringiensis expresses a DNA fragment having significant sequence homology with Bt2. It is expected in the present invention that the hybrid plasmid transformation vector will be used to develop plant cells and their progeny that exhibit insect resistance properties. Plants other than those described in the Examples below are transformed, especially dicotyledons such as cotton, sugar beet and soybeans, rapeseed and cabbage, lettuce and vegetables such as beans. The transformed plant cells and their progeny are protected from certain pests by expressing insect-controlling amounts of the polypeptide poison. Control means toxic (lethal) or aggressive (semi-lethal) amounts of polypeptide toxin. The transformed plants are morphologically normal and are cultivated in a conventional manner for consumption and production of the product. In addition, the transformed plants significantly reduce the need for chemical or biological pesticides used for combating lepidopteran and beetle larvae. The gene encoding the polypeptide toxin is stably integrated into the plant cell genome, and thus becomes genetically inheritable, and seeds obtained from the transformed plants also exhibit substantially the same level of polypeptide toxin. It produces the expressing plants and is therefore also protected from certain pests.

In addition, the transformed plant cells and their progeny may contain the pests and / or the habitat of said pests (ie, the place to be protected, for example, the growing cereal or the place where the cereal is to be grown) It is used to control certain pests by application to a more effective (controllable) amount of the transformed plant body alone or with other ingredients. By way of example, and not limitation, transformed plant cells and their progeny are used alone or as a single component in a formulation or composition. For practical applications, the plant cells and their progeny are used as active substances in conventional insecticidal compositions and preparations or as solid carriers. Such compositions and preparations also contain adjuvants such as surfactants and stabilizers. Examples of such compositions and preparations include pastes, powders, wettable powders, granules, baits, and aerosol compositions. Compositions and formulations are prepared in a known manner. The amount of transformed plants used depends on various factors, such as the type of pest,
It depends on the composition or formulation used, the condition of the crop which is infested with the pest, and the general weather conditions. Generally, transformed plant cells and progeny thereof will comprise from about 0.1 to about 100%, preferably from about 1.0 to about 99%, by weight in the composition or formulation. Compounds or compositions such as known insecticides, fungicides, biocides, herbicides and fertilizers that can be used in combination with the polypeptide poisons can be used in the above-described crude products and formulations to provide additional benefits and advantages. May be included as an ingredient in the composition.

In practice, certain lepidopteran and beetle larvae seek to feed on the transformed plants. As a result, a small amount of the transformed plant is consumed. The ingested substance is altered (processed) in the insect midgut to produce an active polypeptide toxin that acts on the midgut cell membrane to kill the pest or inhibit its growth. Also, indeed, when used alone or as an ingredient in a formulation or composition, certain lepidopteran and / or beetle larvae tend to feed on plants treated with the formulation or composition. A small amount of the treated plant (as a result) is consumed. The ingested material, including the formulation or composition, is altered (processed) in the midgut of the insect where it is possible to obtain a polypeptide toxin that acts on the midgut cell membrane to kill the pest or inhibit its growth. .

The technique of the present invention was generally performed as follows: B. t. Isolation and Preparation of Antibodies Specific for Crystalline Polypeptides Isolation of Bacillus thuringiensis (Bt) crystalline polypeptide B. t. 1. Preparation of (polyclonal and monoclonal) antibodies against crystalline polypeptide B. t. Creation of Gene Bank B. t. Preparation of total DNA or plasmid DNA, preferably plasmid DNA B. Partial digestion of purified DNA using appropriate restriction enzymes 2. Cloning of the DNA fragment into an appropriate E. coli plasmid expression vector B. t. Isolation of recombinant plasmids containing polypeptide genes Anti-B. t. Screening of Escherichia coli transformed cells using crystal protein serum Identification and isolation of bacterial clones expressing the polypeptide

4. Characterization of Bt2 protein The cloned B. t. Purification of polypeptide encoded by the gene The polypeptide expressed by the clone is immunologically B. t. Test to confirm identity with crystalline polypeptide C.I. 4. Test to confirm that the polypeptide expressed by the clone is pesticidal 5. Bt2 mapping and subcloning including restriction enzyme analysis, subcloning and DNA sequencing Undesired adjacent AT preceding the start ATG
6. Construction of a venom gene cassette including removal of the G triad and addition of an appropriate restriction enzyme cleavage site using a synthetic oligonucleotide linker. Construction of intermediate vector8. Construction of hybrid Ti plasmid

9. Manipulation of plants A. B. Identification of transformed plant tissue producing toxins using immunoassays and quantification of the level of toxin produced 10. Regeneration of plant from tissue 10. Detection of Bt2 venom in engineered plants Measurement of toxicity of engineered plants to insects Various types of chimeric genes (promoter gene fusions)
Has been used to genetically transform plant cells, and basically three different types of plant-specific promoters are identified: Promoters: 1. 1. Promoter from Ti plasmid (Pnos, PTR sometimes referred to as PTR2 in this) Plant promoters (Psuzupea, Psuzu3
01)

3. Plant virus promoter (P35S of cauliflower mosaic virus) Chimera gene type: Type I: A linear promoter gene fusion in which the complete Bt2 coding sequence is inserted after the promoter fragment. An example is Pnos-Bt2 (pHD
1050, pHD1060), Psupea-Bt
2 (pHD1076), PTR2-Bt2 (pGS11
61), Pssu301-Bt2 (pGS1181),
P35S-1-Bt2 (pGS1261), P358-
2-Bt2 (pGS1271). Some constructs do not contain the entire 5 'untranslated region of the original transcript (Pnos, Psuppea), while others do (PTnos).
R, Pssu301).

2. Type II: Bt2 gene is RuBisc
A Pssu-Tp-Bt2 chimeric gene fused to the small subunit transit peptide (Tp) sequence of o and expressed under the control of the Pssu promoter. In this case, the fusion protein is probably made from the original translation initiation signal of the ssu gene. Vuan den Brock, etc. (1
985) demonstrated the transfer of bacterial NPTII protein into plant chloroplasts using fusion of RuBisco's ssu transit peptide with a region encoding NPTII. From these results, we found that the chimeric gene P
ssu- Tp: Bt2 was constructed. Both the P ssu promoter and the transport peptide (Tp) fragment were derived from the pea gene used by Vuan den Brock et al. (1985). The DNA sequence at the junction is shown in FIG. It is noteworthy that the native 5 'untranslated region of the pea mRNA is retained in Pssu-Tp: Bt2, and as a result, the chimeric gene is translated from the true ssu translation start site. (PHD1080) 3. Type III: Only the part of the sequence encoding Bt2 is used ("Bt2 of reduced size")
However, linear promoter-gene fusion. A fragment of the Bt2 sequence that encodes an active poison is inserted behind a plant-specific promoter: toxic polypeptides produced in plant cells using these constructs are specific or It has different biological and biophysical properties than the complete Bt2 protein, such as solubility.
Example: pGS1162, pGS1163, pGS1262

4. Type IV: (sometimes Bt2: NPTII
Bt: a linear promoter-gene fusion where the NPTII fusion gene is inserted behind the promoter. A fusion gene was constructed containing a fragment of the sequence encoding Bt2 fused to the sequence encoding the NPTII enzyme (encoding a still active poison). The Bt: NPTII fusion gene used here was a complete neomycin phosphotransferase (NTPII)
A stable fusion protein containing the amino terminal portion of the Bt2 protein fused to the enzyme is designated as a condition. These fusion proteins have specific toxicity when compared to the intact Bt2 protein, and retain the neomycin phosphotransferase enzyme activity intact. Therefore, expression of the Bt: NPTII fusion protein in plant cells allows for the direct selection of production of this protein by isolating kanamycin-resistant (Km ^R ) transformed cells. further,
Level of miles ^R is correlated to the amount of protein that is synthesized directly. Therefore, selection of plants that are resistant to high levels of kanamycin reveals, among all possible transformations, plants that produce high levels of toxic fusion proteins. In addition, expression of the fusion protein by the Bt: NPTII fusion gene has other desirable properties, such as stability in plant cells; for example, mRNA is more stable. The differences in results obtained with these type IV fusion genes are due to the inherent differences in the nature of the expressed fusion protein when compared to the complete Bt2 protein. Example: pGS1110, pGS1151, pGS115
2, pGS1171, pGS1251, pGS125
3, pGS1281

Alternative construction of the desired transformation vectors described herein is also contemplated. For example, a heterologous promoter specific to another plant than that disclosed herein is used. The use of different heterologous promoter sequences serves for direct expression of the inserted heterologous DNA in a regulated manner. Examples of other types of adjustments used include:
Includes tissue-specific expression (leaves, stems or flowers) and inducible expression (temperature, light, or chemical factors). In addition, DNA encoding the polypeptide endotoxin
Given the sequence data, Bt2 described here
Artificially created DNA that is very similar to a DNA fragment
A transformation vector containing the fragment was constructed. This artificially generated DNA fragment was used to transform plants in substantially the same manner as described herein. The present invention will be described with reference to the following examples.

Experiment 1 Bacillus thuringiensis (Bt) crystal protein
Isolated on white as stated by Mahilon and Delcole ( Jia
Null of Microbiological Method , Third
Vol. 2, No. 69-76, 1984), crystals were obtained from strain B. t. Berliner 1715 (received from Dr. A. Krie, Embo Journal Vol. 1, No. 7,
791 to 799, 1982); t. Variant Kurustaki ( Journal of Bacteriology ,
145, No. 2, p. 1052 (1981)). Crystal protein was obtained by purifying the purified crystals at 37 ° C. for 2 hours with 0.2 M thioglycolate, 0.1 M
Solubilization was performed by incubating in NaHCO ₃ , pH 9.5, after which insoluble material was removed by low speed centrifugation. This method uses 80% of the protein present in the crystal.
Solubilize the above. The solubilized crystal protein was 7.5% sodium dodecyl sulfate polyacrylamide gel (SDS).
PAGE). The crystalline protein preparation of Bt bell liner was obtained by staining the gel with Coomassie brilliant blue (FIG. 1).
d and 130 Kd) with at least two major proteins and about 1
It contained a small amount of protein of 20 Kd. The crystal protein of the solubilized Krusutaki strain showed one major 130 Kd protein band and a weak 60 Kd band (FIG. 1).

The crystal protein that is these solubilized, using the toxicity test described in Section 5.2 below, Pierisu -
Brassica third (Pieris b rassicae)
It showed strong toxicity to the young larvae (LD50 value of 0.5 ng for larvae of Kurstaki and 0.65 ng for larvae of Berliner). 2. B. t. Preparation of antibody specific to crystal protein 2.1. B. Polyclonal antiserum t. Antisera to the crystal proteins (Berliner 1715 and Klustaki) were prepared individually in rabbits and mice. B. prepared in goats t. Crystal protein (Kurstaki)
Anti-serum against the University of Idaho L. Given by courtesy of Dr. Bra. As far as the applicant knows, antiserum is
Prepared by known methods substantially similar to those described for rabbits and mice.

Rabbits were dialyzed against 0.5 mg of a solubilized crystalline protein preparation (PBS pH 7.4, mixed with an equal volume of complete Freund's adjuvant (CFA).
l) was injected subcutaneously. Three months later, the rabbits received another injection of the same type of preparation, and three weeks later, a blood sample was taken. BALBc mice are CFA (1/1)
) Was injected subcutaneously with 100 μg of the crystal protein solution. Four to six weeks later, mice received a booster injection of 50 μg of crystalline protein PBS, and four days later,
A blood sample was taken. The antigen reactivity of the serum was confirmed by an immunodiffusion test (Octalony test). A strong cross-reaction was observed between Berliner 1715 and the Kurustaki crystal protein preparations, indicating that they contained antigenic and relevant components. Some mice were sacrificed and spleens were removed for cell fusion experiments. (See 2.2)

2.2 Monoclonal Antibodies Although not essential for the identification of clones expressing the toxins described herein, B.C. t. Hybridomas producing monoclonal antibodies to crystalline proteins were originally described by Kohler and Milstein ( Naturer , 25th Edition) .
6, 495-497 (1975)). Spleen cells of immunized BALBc mice (see 2.1) were fused with the SP2 / 0 myeloma cell line (M. Schulman et al., Nature , 276).
Vol., 269 pages, 1978). Cells were seeded in microtiter plates at 3 × 10 ⁵ per well, and after 10 to 14 days, goat anti-mouse immunoglobulin labeled with alkaline phosphatase as supernatant secondary antibody (Sigma, A-5153) Was used to detect the presence of anti-crystal protein antibodies (Engval and Petess, Scandinavian Journal of Immunity).
Noroji, sea squirts 7, 1978). About 4% of the wells were positive for the antigen (crystal protein). Positive clones were subcloned twice by limiting dilution. Positive subclones were selected and grown and the culture supernatant containing the monoclonal antibody was collected. B. t. A total of 1 to produce monoclonal antibodies that react with Berliner crystal protein
Seven hybridoma cell lines were generated.

3.B. t. Plastic of Berliner 1715 strain
Construction of gene bank for Smid DNA B. t. Both Berliner strain 1715 are on plasmids
Clone to contain two related toxic genes located
Studs etc. (Journal of Bacteriology,
54, pp. 419-428 (1983))
I told. The complete endotoxin gene is
Using partial digestion of S.A. t. Be
Gene Van of the total plasmid DNA of Lulina 1715 strain
Isolated from B. t. Berliner 1715 cells
LB medium (mirror,Experiments in Morekiyu
Ra Zinuteiks(1972), Cold Sp
Ring Harbor Laboratory, New York)
Growed at 7 ° C. Plasmid DNA is B. t. Berli
1715 strain, described by Kronstad et al.
Denaturation-restoration method (Journal of Bacteriology
ー54, 419-428 (1983)
Year)). 0.5% agarose gel
Analysis of the plasmid DNA
Several different pluses where products exist at different molecular concentrations
Amides. To build a gene bank
In addition, 30 μg of plasmid DNA is
Partially digested with Sau3A at 0 ° C. 100 μl
The samples were each of Incubation 10, 20, 3
Collected after 0, 45 and 60 minutes, the phenol-clo
Extracted in Roholm. DNA digested with Sau3A
Is fractionated by size on a 10 to 40% sucrose density gradient
DNA fragments of different fractions
Calculated on an 8% agarose gel. Large from 6 to 10Kd
Fractions containing DNA in the size range were collected and BglII
Ligated to pEcoR251 vector DNA digested with
Was. The pEcoR251 plasmid is shown in FIG.
As described above, plasmid pLK5 (Zebau and Stanley)
-Embo JournalVolume 1, 1217 to 1224
P (1982)_RTo the promoter fragment
EcoRI endonuclease inheritance of fused chimeras
The EcoRI-PvuII fragment is replaced by the
Is a derivative of plasmid pBR322. pEco
R251 is in the EcoRI endonuclease gene
It contains one BglII site, into which the insertion of the gene
Inactivate. The pEcoR251 vector contains
Positive selection cloning vehicle as described by Dricht
pSCC31 (Journal of Bacteriology,
Vol. 154, pp. 1005-1008, 1983
Year). Sau3A DNA
The fragment was digested with BglII and slowed to pEcoR251.
Was. Recombinant plasmids are Dajate and Ehritz
(Gene, Vol. 6, (1980) 23-28
As described by di), E. coli K514 strain
Responsive cells (Coleson, etc.GeneticsVolume 52, 10
43-1050, 1965).
Selected by transformation. Cells
Phosphorus (100 μg / ml) added LB medium (Mira
-EXPERIMENTS IN MOLECUILLA GIENETE
Ix(1972), Cold Spring Herba
-Laboratory, New York). From 600
Several recombinant clones each containing 1500 recombinant clones
Gene Bang was built. 12 random choices
Analysis of the recombinant plasmid present in the clone
At least 10 genebanks of each clone
It contains inserts ranging in size from 5 to 15 Kb.
I was confirmed

4. B. t. Recombination involving the crystal protein gene
Isolation of Bovine Plasmids Genebank colonies were isolated from purified B. t. Rabbit sera raised against the Berliner crystal protein was used to search for bacteria that produce crystal protein (see section 2.1 above). The method used is Helfmann like (Procedure
Dength of National Academy of Sai
Ens of the USA, Vol. 80, pages 31 to 35,
1983) is slightly improved. 1
Bacterial colonies grown in a 50 mm square petri dish were replicated on a nitrocellulose sheet (0.45 μm, No. 401196, manufactured by Schleicher & Schuel). The sheets were soaked in 0.1 M NaOH until the colonies dissolved. The sheet is then air dried, washed with phosphate buffered saline (PMS) pH 7.4 for 30 minutes, and
Incubation overnight at 4 ° C or 2 hours at room temperature with gentle stirring in PBS containing 1% crude ovalbumin (Sigma, A-5253). The nitrocellulose sheets were washed with PBS and incubated for 2 hours at room temperature with gentle stirring in rabbit anti-crystal serum diluted in PBS, 1% ovalbumin, 0.2% Triton X-100. After further washing the sheet, a goat anti-rabbit antibody labeled with peroxidase (Sigma,
A-6154) (room temperature, 2 hours). After extensive washing with PBS / 0.2% Triton, the sheets were reacted with a substrate solution (substrate was 4-chloro-1).
Naphthol, sigma, C-8890). Positive colonies were developed as dark blue dots. Using a series of dilutions of the purified crystalline protein solution, the detection limit for this test was calculated to be 1 to 10 ng protein / ml. After all, 4
Two different immunopositive clones were isolated from 1250 Genebank clones. Plasmid DN
A: Zabeau and Stanley, Embo Journal , No. 1
Volume, pages 1217 to 1224, prepared from each clone according to the method of 1982. An initial restriction map was constructed by performing single and simultaneous restriction digests. Enzymes EcoRI, EcoRV, B
From a comparison of the restriction maps of amHI, SacI, MluI and PstI (see FIG. 3), all four plasmids showed a fully sized DN showing a clear overlap of the regions.
It became clear that it had the A fragment. These results indicate that the Bt2 gene is encoded by a 4.2 Kb region common to four different recombinant plasmids. In a subsequent study, the 7.5 kb BamH of the B12 clone was
The I-PstI fragment (see FIG. 3) was converted to plasmid pUC.
8 (J. Viera and J. Messing, Gene , Volume 19,
259-268, 1982) and the recombinant plasmid was named pBt200.

5. Characterization of Bt2 protein 5.1 130 kd fragment encoded by pBt200
K514 strain E. coli containing the crystal protein identified pBt200 plasmid (Fourth
(See section) gave a strong positive response to the colony test.
This is further attributed to the enzyme-linked immunosorbent assay (ELISA).
(Engbal and Petse, 1978, Scandinavian Journal of Immunology, Appendix 7). The following methods are used in the ELISA search:
Flexible polyvinyl microtiter plates coated with goat anti-Bt crystal protein antibody were incubated with cell-free extracts of bacterial colonies (cell-free extracts freeze-thaw cell clumps, .1M
Obtained by incubation in NaOH for 15 minutes followed by neutralization with 0.1 M HCl). After washing, the diluted rabbit or mouse anti-B. t. Crystal protein serum was added. After 1-2 hours of incubation, the plates are washed and rabbit or mouse anti-B. t. Incubated with crystal serum. 1 to 2
After a period of incubation, the plates were washed and incubated with alkaline phosphatase labeled goat anti-rabbit or anti-mouse IgG (Sigma A-8025, A-5153). After incubation and washing, the substrate (p-nitrophenyl phosphate, Sigma 104-105) was added and the reaction was monitored by measuring the absorbance (OD) at 405 nm. The detection limit of the test for the purified solubilized crystal protein was calculated to be in the range of 0.1 to 1 ng / ml.

The whole cell protein extract of E. coli harboring pBt200 was analyzed by SDS PAGE. A strong new protein band became visible in the high molecular weight range, corresponding to a molecular weight of about 130 kd. This band was absent in K514 cells containing the pUC8 vector plasmid without the insert. This new protein is also SDSPA
GE. t. One of Berliner's main crystal proteins; t. It co-migrated with the main crystal protein of Kurusutaki (see FIG. 4). This protein, called Bt2, and B.
t. The relationship with the crystal protein was confirmed by immunoblotting. Western blotting experiments were performed using both rabbit anti-Bt Klustachy crystal serum and rabbit anti-Bt Berliner crystal serum. A strong reaction of the Bt2 protein was observed in both antisera (see FIGS. 5 and 6). These results indicate that the cloned Bt2
Gene is B. t. B. kurstaki 130 kd crystal protein is immunologically related. t. One of Berliner's crystal proteins
Indicates that the individual is coded.

[0055] The amount of positive reactants in the bacterial extract was determined by EL
Quantified by ISA test. Using purified crystal protein as a standard, the amount of crystal protein produced in E. coli with pBt200 was estimated to be in the range of 5-10% of the total cellular protein content. This estimate is in good agreement with the band intensity of the SDSAGE Bt2 protein band after staining with Coomassie Blue. PBt2 (named Bt2 protein)
To further characterize the 130 Kd protein encoded by the 00 plasmid, a rapid purification method was developed that exploited the relative insolubility of the protein. K514 (pBt20
50 g of 53 g of cells obtained from the 2l overnight culture of
1 of 50 mM TRIS pH 7.9, 50 mM EDT
A, suspended in 15% sucrose, lysozyme (100 μg /
ml), and sonication (Labsonic 1510,
The mixture was mixed with 200 ml of PBS pH7 containing 2% Triton X-100 and incubated for 30 minutes on ice. 15000g of cell-free extract
And the supernatant was removed. The precipitate containing the Bt2 protein was resuspended in the same buffer and the procedure was repeated. Thereafter, the precipitate was washed twice with 200 ml of PBS. To solubilize the Bt2 protein, precipitate the precipitate with 50 ml of extraction buffer 0.2
N-thioglycolate and 0.1 M NaHCO ₃ , pH
Resuspended at 9.5 for 2 hours at 37 ° C. In this way, Bt
Efficient (> 90%) and selective solubilization of the two proteins was obtained (FIG. 7). These semi-purified protein preparations were used in subsequent studies. Using a similar immunization procedure as described in section 2.1, antisera to Bt2 protein were generated in rabbits and mice. These antisera are based on the EL described above.
In the ISA test, Bt2 itself reacted equally well with the solubilized crystal proteins of Bt Berliner and Klustachy (FIG. 8 shows the results with mouse serum).

A similar positive reaction was obtained by affinity chromatography of Bt2 immunoadsorbent (CNBr-activated Sepharose 4B, Bt2 protein conjugated to Pharmacia) using antibody generated from anti-Bt crystal serum by affinity chromatography. Was observed. These antibodies are also described in t. The reaction was carried out by Western blotting with the 130 Kd protein present in both Berliner and Kurustaki crystals. Finally,
In ELISA, all B.I. t. Nine of the 17 monoclonal (monoclonal) antibodies raised against the berulina-crystal protein also reacted with the Bt2 protein. (Code numbers: 1F6, 167, 4D6, 4F3, 8G10, 10
E3, 1.7, 4.8, C73) (FIG. 9). t. Reacted with Kurustaki crystal protein. Generally, Bt2 protein and B. t. Both of the main 130Kd crystal proteins are alkaline pH for complete solubilization.
And the presence of a reducing agent. Both precipitate at pH 4-5. Therefore, the cloned gene product B
t2 is B.I. t. Berliner and B.A. t. It exhibits properties similar to the biochemical properties of the main 130 Kd crystal protein of Klustachy and is immunologically related to these crystal proteins.

The Bt2 protein was further purified by DEAE ion exchange chromatography and Cefacryl gel filtration. The amino-terminal sequence of this purified protein can be analyzed using a method such as Hewick ( Journal of Biological
Chemistry , 256, 7990-7997, 1981) Determined using a gas phase sequencer (Applied Biosystems). First 20 N
The terminal amino acid sequence is t. It has been found that the sequence is substantially identical to the N-terminal sequence deduced from the DNA sequence of the Kurustaki gene (Won et al., Journal of Biological Chemistry, No. 25).
Vol. 8, (No. 3) 1960 to 1967 (198
3 years)) (Fig. 10).

5.2 Insect Toxicity of Bt2 Protein t. Is known to be a significant poison for certain lepidopteran larvae. For Bt2 protein To test whether exhibit similar toxicity, toxicity tests Kiyabetsu butterfly pin
On the larva of Ellis Brassica . ppm (1p
pm = 1 μg / ml), and the protein solution of known concentration was serially diluted with water. Small discs (0.25 cm ² ) were cut from fresh cabbage leaves, and 5 μl of the test solution was added to each disc. The disks were air dried and each disk was placed in a vial containing one larva. The third instar larvae are P. bra
Obtained from synchronized cultures of deer . Ten hours before molting, these larvae were cultured in individual vials without food.
Immediately after molting, the larvae are fed one leaf daix.
When the first disk is consumed, the larva is given a fresh disk without any sample. For each sample dilution, 50 larvae were tested. 1 every 24 hours
Food intake and viability were monitored for up to 20 hours. Table 1
As can be seen in the Bt2 sample preparation, t. P.I. of comparable level to the solubilized crystals of Berliner 1715 It showed toxicity to Brassica larvae.

P. Bt2 against Brassica larva growth
To test the effect of the semi-lethal dose of the venom, the following experimental design was used: cabbage leaves were soaked in a solution containing a known concentration of Bt2 protein (0.01 to 1 ppm) and dried. A group of 100 third instar larvae that had been synchronized and fed were fed with Bt2 coated leaves. The leaves were periodically replaced with new leaves treated in the same way. Larval growth was followed for 7 days, corresponding to the time required to progress from 3 to 5 instars. As can be seen from the results presented in Table 2, Bt2 protein was found in P. cerevisiae at a semi-lethal dose. It significantly inhibited the growth of Brassica larvae.
Inhibition of growth was evident at a concentration of 0.01 ppm, corresponding to 2.67 ng protein / g leaf. First 48
At time, larvae fed leaves coated with 0.01 ppm ate 3.6 cm ² leaves (83 mg) and consequently ingested 0.22 ng of Bt2 protein. At this point, 93% of the larvae were still at the L3 stage, while only 33% of the control larvae were at this stage. Therefore, an inhibitory effect on growth is observed at toxic doses well below the LD ₅₀ value (1.65 ng / larva, see Table 1).

[0060] These results, P. Figure 2 shows how much level of Bt2 protein synthesis must be reached in transformed plant cells to develop insect resistance to Brassica . A level of 2.7 ng Bt2 protein per gram of tissue is sufficient to slow larval growth. This is already suitable by itself to stop the catastrophic spread of larvae in the field. The toxicity test using Bt2 protein was also done sphingidae moth larvae (Mandeyuka sexta). As shown in Table 3, the Bt2 protein was slightly more toxic than the whole Berliner crystal protein (12.5
ng / cm ² 100% dead). In addition, significant growth inhibition is observed at the semi-lethal dose (2.5 ng / cm ² ): 7 compared to 30.5 mg of control larvae.
4.4 mg body weight after day. Due to the fact that Mandeyuca is fed artificial feed (see: RA Bell and F.M.
G. FIG. Joachim (1976) Anyuinal Entology
Of the Society of America Vol. 69, 3
65-373), the results are somewhat different, expressed as ng toxin added per cm ^{2 of} agar medium.

6. Characterization of the Bt2 gene To determine the location of the Bt2 venom gene on the 7.2 Kb BamHI-PstI fragment of the pBt200 plasmid, a series of deletions were made in the 7.2 Kb DNA fragment using HpaI, KpnI, and IbaI, respectively. . Protein that is encrypted by these deletions Burasumido, ELISA techniques and Western blotting (also, here are cited as immunoblotting connexion queuing) (Tobin, etc., Bro Sea de Ings-of-Nashiyo
Naru Academy of Science of USA ,
76, 4350-4354, 1979; N. Barnett, An. Biochemistry , No. 1
12, 195-203 (1981)).

The results (shown in FIG. 11) are summarized as follows: (1) Deletion of the HpaI fragment results in lower levels of complete Bt2 protein synthesis. This finding indicates that the deletion only affects the regulatory region, not the structural gene. (2) Deletion of the Kpn fragment produces a ± 70 Kd protein fragment that can be detected by immunoblotting. (3) X near 5 'end
The ba deletion does not yield a protein fragment that can be detected by Western blotting. These results indicate that the complete gene encoding the 130 Kd protein is a 4.3 Kb HpaI.
-Indicates that it is located in the PstI fragment (see FIG. 11). To determine the exact structure of the Bt2 gene,
The complete nucleotide sequence of the 4060 base pair (bp) HpaI-NdeI fragment was determined by Maxam and Gilbert sequencing. The sequencing procedure used was the twelfth
It is shown in the figure.

The submitted nucleotide sequence has a complementary strand
Determined primarily by sequencing. Arrangement
Looking at the column, 3605 starting from the 141st position
1155 amino acids with a molecular weight of 127 Kd ending in
Single large follower that can encode any protein
The existence of a frame has become apparent. This is the SDS Polyac
Bt2 protein molecule determined by Lilamide gel electrophoresis
This corresponds to an amount of 130 Kd. In addition, the nucleotide sequence
The amino terminal amino acid sequence predicted from
It is identical to the amino acid sequence determined for the t2 protein (first
0 and FIG. 13). Complete amino acid distribution of Bt2 venom
The row contains the 3 cloned and sequenced genes.
Species separate B. t. : B. t. Kurusutaki HDI (Day
Pell (Shiunev, etc.Journal of Biological
·chemistry20, Volume 6264, 1985
Year), B. t. Kurusutaki HD73 (Adan, etc.Gee
N36, 289, 1985) and B.I.
t. Sot (such as Shibano,Gene34, 243 pages
Di, 1985)
Shows some homology.

These other B.I. t. A comparison of the sequence with Bt2 at the amino acid level (FIG. 14) shows that they encode similar but different proteins with considerable homology, but a region of stretched stretches. Was. 7. Construction of the "Toxin Gene" Cassette 7.1. Construction of a cassette with the complete Bt2 gene Examination of the DNA sequence of the Bt2 gene revealed that the 160 bp region immediately upstream of the ATG translation initiation codon contained a five ATG triad. Usually, translation of eukaryotic genes begins with the first AUG of the message (R
U replaces T in NA). These AUG triads act as starting AUGs, and
It is preferentially recognized as a two initiation codon and can therefore reduce the level of expression in transformed plant cells. In addition, these AUGs are in the other open reading frame, giving rise to nonsense polypeptides. These A
Bt2 to prevent the start of translation from UG triplet
The upstream of the gene sequence was deleted by endonuclease treatment before inserting the pBt2 gene into the Ti expression vector. As a result, a deletion derivative of the pBT200 plasmid in which the upstream sequence was deleted up to the start ATG was constructed. 35 μg of pBt200 DNA was digested with HpaI and 12 mM MgCl ₂ , 12 mM CaCl ₂ , 0.6 mM
MNaCl, 1 mM EDTA and 20 mM Tris-H
In 300 μl of Cl-pH 8.0, 6 units of Bal
31 exonuclease (Biolab, New England) treatment for 1, 1.30, 2, 2.30 and 3 minutes
The test was performed at 30 ° C. 1 μg of Bal31-treated molecule of each reaction was added to 0.13 μg of phosphorylated BamHI linker (Biolab, New England) at 4 ° C. for T4D.
Ligation was performed using a total of 20 μl using 2 units of NA ligase.

After inactivating the T4 ligase for 10 minutes at 68 ° C., each ligation mixture was digested with 20 units of BamHI.
Digested at 7 ° C for 1 hour. Subsequently, 50 ng DNA was added for a total of 1
Recircularization was performed at 4 ° C. for 20 hours with 0.1 unit of T4 DNA ligase in 00 μl. 1/5 of this linked mixture
Are sensitive cells of Escherichia coli K514 ( Coleson et al.
Takes , Vol. 52 (1965) 1043-105
(Page 0) as described by Tadgiate and Erich ( Gene , 6 (1980), pp. 23-28). Cells are carbenicillin (100
μg / ml) (mirror, experimental
Mens in Morekular Genetics , (19
72) Cold Spring Harbor Laboratory, New York.

The end points of the plasmid deletion were first analyzed by sizing the EcoRI fragment of the newly generated recombinant plasmid on a 2% agarose gel. The nucleotide sequence at the exact end of the deletion of the plasmid with the deletion ending just before the start of the Bt2 gene was determined. The cloned pHD100 is
It has a deletion ending 8 bp before the initiating ATG and has deleted all upstream non-initiating ATGs. The cloned pBa3.3 contains a BamHI linker fused to the fourth base pair of the coding sequence, and the cloned pBa23-3 contains a Bam linker fused to -33 base pairs. In the second operational step, the non-coding sequence at the 3 'end of the venom gene was Bal31 exonuclease (Biolab, New England)
Was removed using. pHD100 plasmid DNA3
0 μg was digested with NdeI and treated with Bal31 exonuclease at 30 ° C. for 3, 4, 5, 6, and 8 minutes in buffer. At each time interval, each contains 6 μg of Bal31 treated DNA molecules.
The μl was removed. After adding a phosphorylated Bgl II linker (Biolab, New England) to the Bal31-treated DNA molecule, the DNA molecule was 0.1 units T4
Recyclized overnight at 4 ° C with ligase. The ligation mixture was used for E. coli K514 strain-sensitive cells ( Coleson et al.
Vinegar, Vol. 52 (1965), from 1043 to 1050 page), as Dajieto and Eritsuhi stated (di
Over emissions, Vol. 6, (1980) 23 from page 28),
Transformed. Cells were carbenicillin (100 μg
/ Ml) in LB medium (mirror, experimental
Two in Morecular Genetics (1973)
Year) Cold Subling Harbor Research Institute, New York. Following the determination of the size of several plasmid deletions using restriction enzyme digestion and agarose gel electrophoresis, pHD160, pHD162, pHD163 were maintained for subsequent studies. For pHD160, BglI
The I site is located about 300 bp behind the TAA stop codon of the Bt2 gene: In pHD162, BglI
I is ± 250 bp behind TAA, pHD16
In 3, BglII is 334 of the Bt2 coding sequence.
2 (bp). The construction of pHD160 is schematically shown in FIG. Thus, the BamHI inserted and deleted at the BamHI site of the Ti expression vector
A venom gene cassette having the Bt2 gene on the HI-BglII fragment was constructed. To construct pHD164,
Bam of pHD160 containing the 5 'end of the coding sequence
The HI-SacI fragment is the corresponding Bam of pBa3.3.
Replaced with the HI-SacI fragment. To construct pHD159, the BamHI-SacI of pHD163 was used.
The fragment was replaced with the BamHI-SacI fragment of pBa3.3 (FIG. 16). To make plasmid pDC3 (FIG. 16), plasmid pHD164 was
The fragment was digested with aI, ligated to a BglII linker, and the fragment containing the Bt2 gene was cloned into the BglII site of pLK57 (FIG. 17). In this way, BamHI
The BglII site of the BglII cassette is
It was placed in close proximity to the AA stop codon.

7.2. A mosquito containing the engineered Bt2 gene
Set construction 7.21 Small Bt2 gene 7.2.1.1. Reasoning B. t. Fundamental studies on the functional properties of crystalline proteins have shown that crystalline proteins with a size of ± 130 Kd are relatively insoluble and can be converted into low molecular weight active toxins that can exert toxic effects on insects. Is a protoxin that requires processing in the midgut of insects (LA Bra Giunia, DB Vectorel, KJ Kramer,
Y. I. Sietna, A. I. Aronson and P.M. C. Fittsuji Ames, 1980, Review of Ma
Microbiology , Vol. 8, pp. 147-203; A. Bra Giunia, K. J. Clamer, D.C.
J. Cox, B. L. Johns, L. I. Davidson and G.W. L. Lockhart, 1981 Journal
・ Biological Chemistry , Vol. 256,
3000 to 3004 pages: A. Angs, Cana
Dian Journal of Microbiology ,
2nd volume, 416 pages (1956): M. Recadet, " Microvial Toxins ", Volume II,
T. C. Monte E & S. Kadeis, Academic
Press Inc. New York & London, 1970,
Pages 437 to 471). The specific activity of Bt toxin when ingested by insects as part of a manipulated plant component is determined not only by the total amount of toxin present, but also by the degree of utilization of the active toxin released into the midgut. Was. An insect

B. t. It has been shown to be more effective at solubilizing and / or "processing" protoxins than others (Dr. P. Lucy in "2nd Workshop, Bacterial Protein Toxins") Announcement, Wepion, Belgium: 30 June to 4 July 1985
Date; minutes and published). Therefore, 1) construct an already processed or partially processed toxin exhibiting sufficient toxic activity and 2) a small size toxin from Bt2 that has properties that are more soluble than the native Bt2 protein. It is advantageous for the operation of insect resistant plants. Plants expressing such small polypeptides show higher specific toxicity to insects than plants expressing the same level of intact Bt2. 7.2.12. Construction of deletion mutants Position B. of poison gene behind the P _L probe motor t. The gene encoding the 130Kd crystal protein toxin of Berliner 1715 was transformed into pUt to produce pBt200.
C8 (Vieera and Messing, Jean , Vol. 1, 259
268 (1982)). This gene, called Bt2, and the resulting poison (Bt2
The properties of (protein) are described in Sections 5 and 6. To ensure high levels of expression that can be regulated in E. coli, Bt
2 gene was placed behind the P _L promoter (first
7). Bt on the 7.7 Kb BamHI-PstI fragment
The plasmid pBt200 with two genes was cut with HpaI, treated with Bal31, ligated to a BamHI linker, cut with BamHI, and self-ligated (as described in section 7.1). From the resulting clones, the deletion derivative is selected with the upstream sequence of various lengths, (view and Botsutaman like during printing 17, Gene, see 1986) expression plasmid pLK54 behind the P _L promoter of Was inserted using restriction enzymes BamHI and PstI. The resulting plasmids were evaluated for Bt2 protein production and selected for further work, followed by one of the Bt2 high producing plasmids, designated pLB10. Plasmid pLB10 starts with pBa23-3 (FIG. 17, section 7.1).

2. Construction of Deletions From the internal deletions previously made in pBt200 with XbaI and KpnI, only the KpnI deletion results in an immunologically detectable Bt-derived protein (see Section 6). Deletion is pLB
No. 10 was made using the restriction enzymes KpnI and HindIII. Western blotting analysis and ELISA
Shows that only a KpnI deletion mutant containing a large fragment extending from position 2167 to the beginning of the Bt2 gene is stable at about 80%.
It has been shown to produce Kd polypeptides. Hind
Polypeptides encoded by III deletion derivatives are probably hypersensitive to E. coli proteases. Interestingly, the KpnI deletion mutant encoding the polypeptide showed insecticidal activity equivalent to the complete Bt2 protein:
In one experiment, third-instar P. The LD ₅₀ value for Brassica larvae was determined to be 2.5 ng / larva for the Kpn deletion variant compared to 2 ng / larva for complete Bt2. This result indicates that the small Bt2 gene product resulting from the KpnI deletion contains a complete active toxin unit.

The previous data showed that B, which encodes the active toxin,
The small t2 gene fragment is contained within the KpnI deletion fragment, indicating that it is more extensive than the HindIII site. To map the exact end point of the smallest fragment encoding the active toxin, deletion mutants containing a reduced size N-terminal fragment were constructed. To achieve this, a method was used that allows the simultaneous construction of a translational fusion between the deletion mutant and the NPTII gene (see section 7.2.2). The construction of the intermediate plasmid pLBKm25 is shown in FIG.
It is outlined in As shown in FIG.
LBKm25 is composed of pLB10 (see previous section) and pLKm9.
1 (described in 7.2.2.2).
As shown, this plasmid contains one Sa
A DNA sequence with a stop codon is provided in three open reading frames after the I site. This construct was cut with KpnI, digested with Bal31, cut with SalI, treated with Klenow polymerase and religated (No. 19).
Figure). In this way, the deleted coding region is fused to a stop codon with minimal nonsense coding sequences. An overview of the deleted clone is given in FIG. Whole cell extracts were made from the clones (after induction) and analyzed in western blotting and ELSA for quantitative detection of Bt2-like polypeptides and in insect toxicity tests to search for active toxins. The results are shown in FIG. 21, where the end point of the coding region is H
This shows that the closer to the indIII site, the less the detection of stable polypeptides.

At a certain position (downstream of HindIII), the Bt2-like protein became almost undetectable. In addition, the toxicity of the extract of these clones suddenly falls when the 3 'end point passes a specific position between HindIII and KpnI. Minimal toxicity polypeptide (pLB87
Two clones characterized by 9) and the most non-toxic polypeptide were verified by DNA sequence analysis. This analysis shows that the exact endpoint of the stable active toxin is one of the Bt2 gene.
797 and 1820 (second
2). Therefore, Bt ending downstream of 1820 (bp)
All two N-terminal gene fragments include the gene fragment encoding the active toxin. Interestingly, a whole cell extract of one clone (pLB820) showed a stronger response in Western blotting with polyclonal antisera and monoclonal antibodies. In addition, the protein produced by this clone was more soluble in E. coli than the KpnI deletion gene product and showed sufficient toxic activity. 7.2.2. Fusion gene with NPTII 7.2.2.1. Amino terminal fusions with reasoning NPTII gene is known to be able to produce a fusion protein that gives kanamycin resistance to the bacteria (Reis etc., EMBO journal, Vol. 3, 33
17 pages, 1984). Such gene fusion is a very promising application because NPTII is the best selectable marker in plant engineering. Indeed, using such NPTII fusion proteins to transform plants, selection for high kanamycin resistance is a direct selection for high expression of the fusion product. Therefore, toxin gene fusions with NPTII can be used to transform plants by selecting for kanamycin resistance and to select for transformed plants that express high levels of toxin.

7.2, 2.2. Structure of fusion gene cassette
Different fragments of the construction Bt2 gene were fused to the N-terminus of NPTII. Bt: one of the fusion genes named BPT2
The individual is described in more detail below. 1. Construction of Bt: NPT2 fusion gene The construction of the Bt: NPT2 gene is shown in FIG. pLK54 is, pB the transcriptional termination region of the _{P L} promoter and two phage fd is included in tandem
It is an R322 derivative (Section 7.2.1.2). pKm1
09/90 contains the NPTII gene of Tn5 in pBR322 (Reis et al., Embo Journal , 19
1984) (Fig. 24). PKm containing NPTII gene
The 1141 bp gene fragment of 109/90 is pLK54
Into pLKm90. NPTI
To create a Bgl II site behind the I gene,
BglII linker, Klenow or Renou (Klen
ow) After the polymerase treatment, it was ligated at the XbaI and SalI sites. PLKm91 is now ready. p
HD159, after Bam31 treatment with BamHI linker at the fourth base pair and BglII linker with Bal31, 334
PBt200 fused to two base pairs (7.1.
Section). The BamHI-BglII fragment of this plasmid containing the deleted Bt2 gene was pL
Inserted into the BamHI site of Km91, pLBKm10
Generated the Bt2: NPTII fusion gene. pLBKm
To construct 13, the Klenow-treated BglII fragment of aspartic acid at position 728 was added to BLK of pLKm91.
It was inserted between the amHI site (after filling) and the BglII site.

To produce the Bt: NPTII fusion protein in E. coli, pLKm10 and 13 analogous plasmids containing the 5 'leader sequence of the Bt2 gene with a ribosome binding site were created. Therefore, the position of -33 Bam
The BamHI-SacI fragment of pLBKm13 was exchanged from another Bal31 deletion derivative of pBt200, pBa23-3, having an HI linker, and pLBKLm23
Was attached. In order to express the fusion protein Bt: NPT2 behind the Pnos promoter and 35S promoter, a BamHI-SacI fragment of pHD160 (7.1) was used.
(Described in the section below) was cloned between the same sites in p2BRm13, resulting in pLBRm33. Finally, construction using the Pettiunia ssu-promoter (see Section 8) resulted in the 3 'non-coding region being NPTI.
Improved Bt: NP with I stop codon removed
TII was used. To achieve this, NPTII
NcoI-Bg of pLBKm13 containing the 3 'end of the gene
The II fragment was NcoI-Bg derived from p2Km91.
It was replaced by the II fragment (FIG. 23). This plasmid was cut with DcbI, treated with Klenow polymerase, and ligated to a BglII linker; the resulting DNA was then cut with NcoI and BglII. 23rd
The figure also shows the 5'Bt2 sequences of the different constructs.

Briefly, the Bt: NPT2 gene includes (FIG. 24): 1) 8 marks upstream of the start ATG codon or +4 or -3.
The 5 'end of the Bt2 gene starting at position 3 and extending to nucleotide position 2173. 2) 16 bp linker fragment. 3) NPTII coding region starting from the 13th nucleotide. 2. Fusion gene Bt placed behind the _{P L} promoter of the characteristics of the fusion protein expressed in E. coli plasmid construct pLBKm23: NPT2 is
It was expressed in E. coli to study the properties of the fusion protein.

2.1. Identification of E. coli fusion protein E. coli clone transformed with pLBKm23
-Analyzed by PAGE and Western blotting. Coomassie staining of the complete bacterial extract on SDS-PAGE indicates that the expected size of the fusion protein (± 110 Kd)
The presence of a new protein band with an apparent molecular weight corresponding to. The protein may also be anti-Bt2 serum or anti-N
Western blotting using PTII serum could also be seen. In addition, a positive reaction band indicated the expected size of the fusion protein. The protein is quite stable in E. coli because only a limited amount of degradation products are detected (low molecular weight band). 2.2. Kanamycin resistance of engineered Escherichia coli clones
In addition, the E. coli clone containing the NPTII active pLBKm23 gene of the Bt: NPT2 protein was actually kanamycin resistant. Bacteria are 100 μg / ml K
m (as a comparison, the wild-type NP
The TII gene confers resistance of 100 μg / ml or more).

[0076] NPTII activity of the Bt-NPT2 fusion protein, etc. (Reis As stated in the other place, Gee
Emissions, Vol. 30, page 217, 1984) NPTII
It was evaluated using tests. A cell extract of an E. coli clone expressing the Bt: NPT2 protein was a wild-type NPTII.
Were placed on a gel under non-denaturing conditions along with the extract of an E. coli clone producing E. coli. Cell extracts were prepared as follows. E. coli clones were grown in a culture containing 20 ml of LB medium at 28 ° C. for about 4 hours, centrifuged, resuspended in 1 ml of TES buffer, and 50 W
Sonicated twice for 1 minute and centrifuged at 15000 rpm for 30 minutes; the supernatant was used for the experiments. Protein location and NPT
II specific activity was determined by phosphorylation of kanamycin in the natural state using ³² P-ATP (Reis et al.
1984, Gene , Vol. 30, pages 217-223). Gels of the same sample were run side-by-side and used for Western blotting (with anti-Bt2 and anti-NPTII antibodies). Purified Bt2 and purified N contained in this gel
A series of diluted samples of PTII were compared to determine the amount of protein present in the samples used to determine activity;
The specific activity of the t: NPT2 protein was shown to be approximately the same as the specific activity of NPTII (FIG. 25).

2.3. Bt: NPT2 fusion protein in E. coli
White behavior The results in the previous section indicate that the complete NPTII enzyme and the fusion protein NP
The TII specific activities have been shown to be substantially equivalent. This appears to be somewhat inconsistent with the behavior of the protein "in vitro". Escherichia coli producing wild-type NPTII is resistant to 1000 μg / ml Km or more, whereas Escherichia coli engineered with Bt: NPT2 that expresses comparable levels of protein is resistant to only 100 μg / ml. However, proteins (e.g. in this case, by using a strong P _L promoter) produced in E. coli at high levels is known to tend to precipitate in the cell. Given that a substantial fraction of the Bt: NPT2 protein is present in the precipitated form of E. coli (and most of the protein is enzymatically inactive), the result is expected to be a low-resistance phenotype. B using Western blotting
Analysis of bacterial extraction of the t: NPT2 clone showed that virtually all of the protein was present in an insoluble form. After lysis and centrifugation of the cells, most of the detectable Bt: NPT2 (compared to the NPTII wild-type protein present mostly in the supernatant fraction) was present in the precipitate. Only a fraction of the Bt: NPT2 protein was present in the supernatant.
To solubilize the fusion protein present in the precipitate fraction, 8M
Urea and 2% ME (mercaptoethanol) were required. After dialysis to reconstitute the solubilized protein, a concentrated Bt: NPT2 protein preparation for use in insect toxicity tests was obtained (see 2.4).

2.4. Pre-toxicity testing of the Bt: NPT2 protein showed that the polypeptide encoded by the KpnI deletion fragment of Bt2 still had 100% of the toxic activity of the complete Bt2 protein (7. 2.
1.2. section). In addition, studies on the identification of the least active toxin fragment showed that this Kpn fragment contained an active toxin (± 60 Kd) that exhibited the full toxin activity of the Bt2 molecule. Next, it was determined whether the Bt: NPT2 fusion protein still had the same degree of toxicity. To this end, the toxicity levels of concentrated Bt: NPT2 and produced Bt2 protein to insect larvae were compared. The exact amount of the fusion protein in the sample was determined by ELISA (using a monoclonal antibody specific to the N-terminal region of Bt2, 4D6) and Western blotting (comparison of the band intensities of the diluted sample of the fusion protein and purified Bt2). ). M. Sexta and P. B
The results showed that the specific activity of the toxin on the deer larvae was virtually identical to the complete Bt2 and Bt: NPT2 fusion proteins (Tables 4 and 5). Taken together, the above data indicate that the Bt: NPT2 protein has NPTII activity "in vivo" or "in vitro" and also has a potential insecticidal activity equivalent to Bt2 venom. . Therefore, this small toxin clearly represents a valuable alternative for the engineering of plants that express high levels of insect venom.

2.5. Emits a highly resistant phenotype of kanamycin
The results obtained with the Bt: NPTII gene constructed prior to the selection of additional Bt2: NPTII fusions to appear show that this fusion protein is conferred kanamycin resistance, exhibits normal levels of toxicity, and is relatively stable So it was very promising. However, the kanamycin resistance conferred by the Bt: NPT2 fusion was relatively lower than wild type cells. Separately generated Pnos-Bt: N
NPTI where PT2 protein is comparable to wild-type NPTII protein
Since it had specific activity I, the low resistance of kanamycin
It was concluded that this was due to the low solubility of the fusion protein in E. coli.
The relatively low tolerance phenotype interferes with an effective selection system in plants. Therefore, it was considered that other Bt-NPTII fusions may have different physicochemical properties leading to a higher Km resistance phenotype "in vivo". The NPTII gene
An experiment was designed to randomly fuse to the Bt2 sequence in the region between the nI site and the processing site. The experiment was as follows:

[0080] Using KpnI site Bt2 gene and _{P L} promoter fused to NPTII gene at the 1100bp downstream of the plasmid containing (19 see Figure) interesting element called PLBKm25 (Figure 18). 1
Two XhoI sites separate Bt from the NPTII gene. This plasmid was linearized by KpnI digestion and
a131 exonuclease. Ba131
Was titrated so that the deletion did not proceed beyond the HindIII site located upstream of the C-terminal processing site. The plasmid treated with Ba131 was treated with XhoI.
Linked to the linker, digested with XhoI and self-ligated. As a result, NPTII is fused to a fragment of the altered size Bt2 gene. This plasmid transformed into E. coli contains the NPTII gene in-frame with Bt
Kanamycin resistance was conferred under the condition of fusion to the two genes. Transformants were low level kanamycin (20 μg
/ Ml) and the ability to grow at higher concentrations of kanamycin was searched. 145 kanamycin-resistant transformants were searched for growth ability at higher concentrations of kanamycin. Eight transformants were improved to be more resistant and could grow at concentrations higher than 200 μg / ml kanamycin. The fusion point of all eight clones is 2
Determined with restriction enzymes that map to 0 bp accuracy.
Surprisingly, 7 out of 8 clones had a fusion point around the HindIII site at position 1680 of the Bt gene. One clone (pLBKm860) has ± 20
It was mapped to 50 positions. Although most of the deletions were fused around position 1800, none of these had been conferred the kanamycin high resistance phenotype. Seven clones with the fusion point around the HindIII site were:
Inability to encrypt too short active toxins. However, one of the clones (pLBKm860) was: A more stable, more small Bt protein was found in the supernatant as more protein was detected per amount of whole cell extract in Western blotting analysis. , It was more soluble. B of clones 860 and 865
The position of the 3 'end point of the t2 coding sequence is shown in FIG. The toxicity of the fusion protein and the small size Bt2 gene product is shown in Table 6.

7.3. Size for expression in plant cells
Cassette containing small Bt gene or Bt gene fusion
The plasmids pLBKm860 and 865 were modified as described in FIG. 27 to create plasmids pLBKm1860 and pLBKm1865, respectively. p
LBKm2860 was derived from pLBKm860 (FIG. 27). BamHI-Sac of pLB820 and 884
By replacing the I fragment with the BamHI-SacI fragment of pLBKm14, pLB1820 and 18
A new plasmid called 84 was created. pLB
2820 was derived from pLB1820. As in the example,
Final constructs pLBKm1860, pLBKm1865
And pLBKm2860 are shown in FIG.

8. Intermediate expression vector containing toxin gene
Construction 8.1. Overview A summary of the engineered plasmids constructed in Table 7 and used in plant transformation experiments is provided. Each engineered Ti plasmid contains the receptor Ti with an intermediate vector.
This is the result of plasmid integration. Each intermediate vector contains a chimeric toxin gene consisting of a plant promoter sequence from a supported expression vector and a Bt gene cassette. Chimera Gene Assembly Table 7 provides the plasmids from which the Bt gene cassette was isolated. The construction of these plasmids is described above. A detailed map of the expression vectors listed in Table 7 is provided in FIG. 33 (A to J). pHD2
The detailed construction of the two chimeric genes present at 05 and pHD208 is given as an example. The experimental method used standard recombinant DNA technology, and the experimental procedure was Mania Teis (Cold Spring Harbor, 1982).
It was done according to. The different plant promoters used were (Table 7): Pnos: TD encoding nopaline synthase gene
Constitutive and relatively weak promoter from NA. Pssu-pea: from a weakly expressed member of the small subunit group of the pea ribulose 2-phosphate carboxylase gene. Expression is slightly inducible in green tissue. PTR2: _T R -DNA of the octopine Ti plasmid
Constitutive promoters of origin (Berten et al., 1984,
Embo Journal , Vol. 3, p. 2723). In the expression vectors pGSH150 and pGSH160, the dual promoter fragment is derived from pop443 (Berten et al., 1984, Embo Journal , Vol. 3, 272).
3 pages). 5 'non-inversion of 2' promoter Was completed by adding a BamHI linker to create

Cloning into the BamHI site therefore completely leaves the 5 'untranslated region of the 2' gene,
The Bt gene cassette is fused at the start ATG of the 2 'gene. The importance of the complete 5 'untranslated region to obtain high levels of expression of the chimeric gene has been demonstrated (Jyones et al., Embo Journal , Vol. 4, 2411, 1985). PSSU301 : Ha Petitunia r
rb of pettiunia where about 50% of bs mRNA is transcribed
s (Ribulose diphosphate carboxylase small subunit) gene promoter (Dane et al., Embo Journal, 1984, 4, 12). By site-directed mutagenesis, the sequence around the initiation codon (T
AACT ATG GCTT) has NcoI on the start codon.
It was changed to TAA CCATGG CTT to create a site. Similarly, from GGCTTCTAAGTT Improvements around the stop codon created a BglII site. As a result, the coding region of the ssu-301 gene
6. Exactly removed by NcoI-BglII digestion;
It was replaced by the Bt gene cassette as described in section 2. Promoter fragment (PssuPET)
And the separation and characterization of the 3 'end (ssu-PET) is described in Dean et al., The Proceedings of National Academy of Sciences of USA, Vol. 82,
4964 (1985). pA
The construction of GS007 (FIG. 33H) is described in FIG.

P35S: is a strong constitutive promoter from cauliflower mosaic virus (CAMV). The P35S-1 promoter fragment was isolated from CAMV isolated C
m4-184. This is a fragment of the CAMV genome (nt 6492 to 7454) (Gardner et al., 1992).
1981, Nukuletsk Acid Research, Vol. 9, 2
871 to 2888) and B for cloning
Including the downstream of the amHI site (FIG. 40). P35S-2 is
Includes 6492 to 7466 CAMV sequences following the ClaI site. P35S-1 and P35S-2 are therefore 35
Only the length of the 5 'untranslated sequence of the SCAMV transcript differs. Promoter · Bt gene linked Figure 28 provides different engineered as present in Ti plasmid, the DNA sequence of the junction between Corde queuing sequence of the promoter region and the Bt gene cassettes. Sequences derived from the original promoter region and the coding sequence of the Bt2 gene are underlined. Some suitable restriction enzyme sites included in the chimeric gene assembly are indicated. The ATG start codon is boxed. Ligation between the Bt gene cassette and the 3 'end All chimeric genes have a 3' end with a sequence containing a 3 'untranslated region which naturally contains the polyadenylation site of the gene expressed in plant cells. . The following array was used:

[0084]3 'OCS (pHD1060, pHD10
76,at pHD1080) 7 containing the 3 'untranslated region of the octopine synthase gene
A 06 bp PvuII fragment (119 according to Gielen et al.
39 to 11233Evo Journal, Vol. 4, 83
5, 1984).3't7 (pGS1151, pGS1161, pGS1
152, pGS1153, pGS1162, pGS11
63, pGS1251, pGS1261, pGS125
3, pGS1262, pGS1271, pGS128
1 ) Cloned into the SmaI site of pUC8, EcoR
T-DNA gene re-isolated as an I-SalI fragment
212 bp EcoRV-C containing the 3 'untranslated region of 7
The laI fragment (positions 2317 to 21 according to Gielen et al.
05,Embo Journal, Volume 4, 835 pages, 1
984).

182 containing the 3 ′ untranslated region of the 3 ′ wos (in pGS1110) nopaline synthase gene
bp TaqI-ClaI fragment (positions 1290 to 1472 according to Depicker et al ., JMAG Volume 1,
561 page, 1982) 3 ' SSU301 (pGS1171, pGS1181 )
Middle) ± 1.2 kb Bg from 3 'end of ssu301 gene
The II-BamHI fragment was constructed by site-directed mutagenesis as follows: stop codon region (which is TAA) ssu301 ... TTC TAA GTTATA encoding

[0086]

EXAMPLES Example 1 Bt2 of nopaline synthase promoter, pHD160
DN containing 3 'untranslated region of nopaline synthase gene containing toxin gene cassette and polyadenylation site
The construction of an intermediate vector, pHD205, containing a chimeric Bt2 toxin gene consisting of the A fragment is described in this example. In the chimeric gene, the Bt2 gene cassette is connected so that the expression of the Bt2 protein can be obtained from the promoter of the nopaline synthase. The Bt2 gene cassette was excised from pHD160 with BamHI and BglII, and pLGV2382 (Hellera Estrela et al.
Mombo Journal , Volume 2, pages 987-995,
1983) at the BamHI site. 2 μg of pHD160 DNA was treated with 2 units of BglII and BamHI (Boehringer Mannheim) at 37 ° C. for 1 hour, respectively, at the incubation buffer ( Molecular Cloning (1982) described by Maniateis et al.
Year) Cold Spring Harbor Laboratory, pages 133-134) in a final volume of 20 μl. Under the same conditions, 5 μg of pLGV2382 DNA
It was digested with amHI. Subsequently, the terminal 5 'phosphate is D
NA, using the conditions described by Mania Teis et al. ( Molecular Cloning , (1982) Cold Spring Harbor Laboratory, 133-134 ).
Page) Calf intestinal alkaline phosphatase (CIP)
(Boehringer Mannheim). PLGV238 digested with BamHI and treated with CIP
1/5 of the 2 DNA in a final volume of 20 μl was digested with 0.1 μg of BamHI-BglII digestion pH using 0.01 units of T4 DNA ligase (Boehringer Mannheim).
Ligation to D100 DNA. The ligation buffer and the incubation were as recommended by Boehringer Mannheim (Pamphlet "T4 Ligase", Boehringer Mannheim, August 1980, 10M.88.
0.486). The ligation mixture was prepared according to Daziate and Erich, Gene , Vol. 6, (1980) 23-28, sensitive cells of E. coli K514 ( Coleson et al., Genetics , 52, (1965) 104
3 to 1050). Cells were cultured on LB plate medium (Miller, Experiments in Molecular Equipment ) supplemented with carbenicillin (100 μg / ml).
• Genetics (1972) Cold Spring Harbor Laboratory, New York. Transformants were purified by microscale DNA preparations according to Burnboim and Dolly ( Nucleic Acid Acid.
Research , Volume 7 (1979) 1513-1523
Page) The presence of the recombinant plasmid was searched. pL
BamHI-BglI at the BamHI site of GV2382
The orientation of the I fragment was determined by double digestion of BamHI-PstI. The pattern of double digestion of the recombinant plasmid shows four fragments after agarose gel electrophoresis.
In the α direction, about 5700 bp, 3000 bp, 2300 bp
bp and 920 bp fragments, while in the β-direction approximately 6200 bp, 3000 bp, 1800 bp and 920 bp
bp fragment. A recombinant plasmid having the α-direction (toxin gene under the control of the nopaline synthase promoter) was used in the next experiment and is called pHD205.

Example 2 This example describes the construction of pHD208.
The intermediate vector pHD208 contains the promoter (Pssu) of the pea gene encoding the small subunit of ribulose diphosphate carboxylase, the Bt2 toxin gene cassette of pHD160 and the 3 'untranslated octopine synthase gene containing the polyadenylation site. Region containing a chimeric Bt2 toxin gene. The fragment of the chimeric gene was assembled in the cloning vector pGV831 as described in this example and shown in FIG. The construction of pGV831 is summarized in FIG.

Step 1 706 bp containing the 3 'untranslated region of the octopine synthase gene into pGV831 to obtain pGV858
Insertion of the PvuII fragment of 5 μg of pGV831 DNA
Is a buffer of the incubation described by Mania Teis et al. ( Molecular Cloning (1982)
Year)) at 37 ° C. for 1 hour with 5 units of HpaI. Then, Maniateisu, etc., Morekiyura-black
Using conditions, such as those described by E. Honing , 1982, the terminal 5 'phosphate was removed from the DNA by CIP treatment. 20 μg of pGV99 DNA (de
Grave et al., The journal of more and more
・ Applied Genetics , Volume 1 , 499-5
12 pages, 1982) with 20 units of PvuII
Digested at 7 ° C for 1 hour. The resulting DNA fragments were separated by electrophoresis on a horizontal agarose gel (0.8% agarose in TBE buffer). 706bp Pv
The agarose band containing the uII fragment was cut out,
DNA was recovered by electroelution. After phenol and ether extraction, the DNA was centrifuged in an Eppendorf centrifuge for 1 hour.
Precipitate with ethanol by centrifugation for 0 min.
% Ethanol, dried and resuspended in ₂₀ μl H ₂ O. 0.03 μg of HpaI digested and CIP treated pGV831 was obtained in a final volume of 10 μl and 1 unit of T4
Using ligase, 0.1 μg of purified 706 bp
Fragment. The concatenated mixture can be prepared as described by Daziate and Erich ( Gene , Vol. 6, (1)
980) pp. 23-28), E. coli K514 sensitive (competent) cells (Coleson et al.
^Genetics , 52 (1965), ^1043-1050 ). Cells were cultured in LB plate medium (mirror, Experiments in Molecular Culture ) supplemented with carbenicillin (100 μg / ml).
Enetiques (1972) Cold, Spring Harbor Laboratory, New York. The resulting recombinant plasmid was characterized by double digestion of pstI and ApaI. One of the resulting plasmids, p
From GV858, approximately 5300bp, 1700b
The desired digested fragments of p, 1500 bp and 900 bp were obtained and used further.

[0089]Stage 2: Improvement to obtain pH 503 Construction of Pssu fragment and insertion of this fragment of pGV858
In. pUC8 (VIERA and Messing,Gene, 19th
Volume, 259-266, 1982)
The polylinker of III-BamHI was replaced with pKC7 :: Ps
The BamHI site is inserted into Pssu
It is located immediately downstream of the promoter. pKC7 :: P
ssu is prepared at the Genetics Laboratory, Ghent State University, Belgium
Was done. Vector pKC7 (Lao and Logiers,Gee
N, Volume 7. 1979)
Sulfate diphosphate carboxylase (RUDP-cose)
Of peas encrypting small sub-units (ssu)
One promoter of the gene (Hellera Estrela,Ney
CharVol. 310, pp. 115-120, 1984
Year)) EcoRI-HindIII fragment. p
1 μg of KC7 :: PSSu is 1 unit of HindIII
And 1 unit of BamHI. 1 μg of pUC8
DNA is 1 unit of HindIII and 1 unit of BamHI
At 37 ° C for 1 hour. Each digested D
0.1 μg NA is mixed and 0.01 μl in a final volume of 20 μl.
Ligation was performed using units of T4 DNA ligase. Consolidation
The mixture was prepared from Escherichia coli K514 sensitive cells (daziate and
Ritsuhi,Gene, Volume 6, (1980) 23-28
Page). Cells are 100 μg / ml
LB plate medium (Miller,D
Kusperiments in Morekiura Genetics
(1972) Cold Spring Harbor Study
Place, New York). The obtained recombinant plastic
One of the Smids, pGV861. Km of pKC7^RHeredity
The HindIII-BamHI fragment containing the offspring is 20 bp.
HindIII-BamHI polylinker of pUC8
Was replaced by

[0090] Maniateisu, etc., Morekiyura-cloning
Packaging, Cold Spring Harbor Laboratory, 133
The terminal 5 'phosphate was removed from the DNA by CIP treatment using the conditions described on page 134 (1982). Maneki Teis and more
Ning , Cold Spring Harbor Laboratory, 19
In a final volume of 20 μl, 2 μg of pGV861 was digested with 2 units of BglII, BamHI and RvuI for 1 hour at 37 ° C. using the Incubation Buffer described in 1982. Digested with 0.2 μg BamHI, CI
P-treated pGV858 has 0.05 μg of BamHI
-Final material 2 added to pGV861 digested with BglII-RvuI
0 μl and ligated with 0.01 units of T4 DNA ligase (Boehringer Mannheim). The concatenated mixture comprises Daziate and Erich. Gene , Volume 6, (1980)
According to pages 23 to 28, competent cells of E. coli K514 ( Coleson et al., Genetics , No. 52).
(1965) 1043-1050). Cells were carbenicillin (100 μg /
ml of LB plate medium (mirror, experiment
Unz in Morekiura Genetics (1972)
), Cold Spring Harbor Laboratory, New York). For carbenicillin-resistant clones, a microscale technique described by Barnboim and Dolly ( Nucleitsch Acid Research , Vol. 7,
(1979) Restriction enzyme digestion of DNA prepared according to pages 1513 to 1523 searched for the presence of the recombinant plasmid. Recombinant plasmid 1
First, in pHD503, a BglII-BamHI fragment containing the peer ssu promoter is inserted in the correct orientation before the 3 'end of the octopine synthase gene. pH
D503 contains one BamHI site between the Pssu promoter and the 3 'end of the octopine synthase gene.

Step 3 : Intermediate expression vector PHD208
To the BamHI site of pHD503 to obtain
Insertion of the mHI-BglIIBt2 gene cassette. 2μ
g of pHD160 DNA in a final volume of 20 μl
° C, 1 hour, 2 units of BglII and unit of BamHI
Completely digested. 5 μg of pHD503 DNA was digested with 5 units of BamHI completely under the same conditions,
In order to remove the terminal 5 'phosphate from the DNA, molecular cloning (1982) by Mania Teis et al.
(Cold spring harbor research institute, 133)
To page 134) using the conditions described in
Treated with P. 0.1 μg of BamHI-BglII
The digested pHD160 DNA was added to 0.2 μg of BamHI digested and CIP treated pHD503 DNA to a final volume of 20 μg.
and ligation using 0.01 units of T4 DNA ligase. The ligation mixture was transformed into competent cells of E. coli K514 (Digitate and Erich, Gene , 6 (1980) 23-28). Cells were streptomycin (20 μg / ml)
And spectinomycin (50 mg / ml)
B-plate medium (Miller, Experiments in Moreki
Yulare Genetics (1972), Cold Spring Harbor Laboratory, New York. Streptomycin-spectinomycin-resistant clones were developed by Bernboim and Dolly ( Nucleic and
・ Research , Volume 7, pages 1513 to 1523, 1
979) was searched for the presence of the recombinant plasmid by restriction enzyme digestion of DNA prepared from these clones by the microscale technique described in US Pat. P
A recombinant plasmid, pHD208, containing a cassette of the Bt2 gene in the correct orientation with respect to the ssu promoter was isolated and used for the next experiment.

Example 3 This example describes the construction of pGSH151. The intermediate vector pGSH151 contains octopine T
i-plasmid TR-DNA transcript 2 promoter (PTR2) (Berten et al., 1984, Embo Journal, Vol. 3, p. 2723), pLBKm13 Bt: NPTIL fusion gene cassette and T-DNA of octopine Ti plasmid A chimeric Bt: NPTII fusion gene consisting of the 3 'untranslated region of gene 7. Fragments of the chimeric gene were collected as described in this example. All of the technology, Maniateisu, etc., Moreki
Performed as described in Yular Cloning (1982). Step 1 : Construction of pGSH50 (FIG. 41). This plasmid contains an ATG followed by a unique BamHI site.
-Includes the TR promoter PTR2 with the complete 5 'untranslated region with the initiation codon and the 3' untranslated end of transcript 7. p
OP443 (Berten et al., 1984) describes a ClaI containing the octopine Ti plasmids PTR2 and PTR1.
-A HindIII fragment. To delete the BamHI site, POP443 was totally digested with BamHI and SalI, and the cohesive ends were replaced with E. coli polymerase I.
And self-ligated with T4 ligase. After transformation, ampicillin-resistant colonies were selected and their plasmids were searched for lack of BamHI and SalI sites to yield pOP443BSF. P
To create a DlaI site before the 3 'untranslated end of transcript 7 of AP2034 (Berten et al., 1984), pA
P2034 was digested entirely with BamHI, treated with the Klenow fragment of E. coli polymerase, and ligated to a kinesized ClaI-linker. The DNA was then digested with ClaI and self-ligated with T4 ligase: Among the Amp ^R transformants, PAR2043C was selected. From pOP443BSF, a ClaI-HindIII fragment containing the TR promoter was transformed into pAP2034.
Cloned into the corresponding site of C, resulting in pGSH50.

Step 2 : Construction of pGV1500 (FIG. 42). pGV825 is manufactured by Nubrask &
・ Research , Vol. 13, p. 4777, (1985
Year); to reduce its size, p
GV825 was digested with RvuII and self-ligated.
The resulting plasmid pGV956 contains T-FDN
In A, one BamHI site and one BglII site
Includes parts. pJB63 is described by Botterman et al. (Printing, Gene , (1986)).
BamHI-B containing only one restriction site
The glII fragment was cloned into the corresponding site of PGVG56, resulting in pGV1500. Step 3 : Construction of pGSH150 (FIG. 43) pGSH50 was digested with EcoRI, treated with the Klenow fragment of E. coli polymerase I, and digested with HindI.
Digested with II. The resulting fragment containing the TR-promoter was cloned into HpaI and HindI of plasmid pGV150.
It was cloned between the II sites.

Step 4 : Construction of pGSH151 (FIG. 3) BamHI-Bg of pLBKm13 containing Bt2 gene
The II fragment was cloned into the BamHI site of pGSH150 to create an in-frame fusion of the Bt2 gene beginning at the second codon of the ATG start codon behind PTR2.

9. Toxin gene to Agrobacterium
Introduction of the intermediate expression vector containing Agrobacterium The introduction of the intermediate expression vector into the acceptor Ti plasmid is performed in two steps:
The intermediate expression vector has two auxiliary plasmids: R64drd11 containing tra function and pGJ2 containing mod function.
E. coli GJ23 strain (Huinegan et al., Molecular and General Dienete)
Ikus, Vol. 185 (1982), pages 344-351). Second, strains of E. coli harboring all three plasmids are available from Vuan Haute et al. ( Embo Journal , No. 2).
411-418, 1983), and is conjugated to a strain of Agrobacterium containing a receptor Ti plasmid having a region of homology to the intermediate expression vector. The recombinant Ti plasmid resulting from a single crossover event is isolated by selecting the antibiotic resistance marker carried by the intermediate expression vector. By way of example, co-integration of pHD205 with pGV3850 and co-integration of pHD208 with pGV2260 are described. The intermediate vector and the receptor Ti plasmid used are listed in Table 7 and are shown in FIGS.
Is shown in

Example 1 To obtain the hybrid Ti plasmid pHD1050, the intermediate expression vector PHD205 was inserted into the receptor Ti plasmid pGV3850. As shown in FIG.
pHD1050 contains a chimeric Bt2 gene under the control of the Pnos promoter as well as the nopaline synthase gene located between the T-DNA border fragments. Psmid pH
D205 is transformation by Dajieto and Eritsuhi (di
Over emissions, Vol. 6, 1981, was introduced into competent cells of E. coli strain GJ23 in from 23 page 28). pHD205
In order to select Escherichia coli GJ23 cells transformed with E. coli, cells were cultured in LB medium supplemented with carbanicillin (100 μg / ml) (Miller, Experiments in More).
Kyura Genetics , (1972) Cold
Spring Harbor Laboratory, New York). One colon gold GJ23 colony transformed with pHD205 was inoculated into liquid LB medium and cultured overnight (about 18 hours). 0.1 ml of this culture was used for pGV38
50 (Zabrysky, etc., Embo Journal , Volume 2,
C2 including pages 2143 to 2156 (1983)
8Cl Rif ^R (also known as GV3101, Vuan Larebec et al., Nayiyah , Vol. 252, 169-1
70 ml, 1974) overnight and incubated at 28 ° C overnight in solid LB medium (Miller, X-Ray).
Perments in Morekulare Genetics (1
972) Cold Spring Harbor Laboratory, New York). The Agrobacterium strain containing the hybrid Ti plasmid obtained from one crossing was a minimal A medium supplemented with neomycin (400 μg / ml) (Miller, Experiments in Morequiula).
PHD2 on the Genetics (1972), Cold Spring Harbor Laboratory, New York.
Isolation was performed by selecting the kanamycin / neomycin marker carried by the 05 plasmid.
One of the object to be bonded, the physical structure of T- region of pHD1050 is Daesu etc. (Nukureitsuku and Research, Vol. 7, (1979) 1837 from 1849 pages) follow the method is stated connexion, C58C1 Rif ^R pHP
Determined by hybridization of ³² P-labeled pHD205 to HindIII digests of all 1050 PNAs. The structure of the T region of pHD1050 is shown in FIG.

[0097]Example 2:Hybrid Ti plasmid pHD10
To obtain 76, the intermediate expression vector pHP208 is
The receptor was inserted into the Ti plasmid pGV2260. Figure
As shown in Figure 32, pHD1076 is a Pnos pro
Neomycin phosphotran under motor control
As with chimeric genes containing swelling enzymes, T-DNA
Under the control of the Pssu promoter located between the border fragments
And the chimeric Bt2 gene. Ti plasmid p
GV2260 is described in European Patent Application No. 83112985.
3 (Publication No. 0116718). Plastic
Sumid pHD208 is available from Daziate and Erich (Gee
N6 (1980) 23-28).
Transformation into E. coli GJ23-responsive cells
Was entered. Transformed with pHD208. E. coli GJ
To select 23 cells, the transformation mixture was
LB plate culture to which benicillin (100 μg / ml) was added
Earth (mirror,Experiments in Morekular
Genetics(1972), Cold Spring
・ Harbour Laboratories, New York). Trait
One of the transformed E. coli colonies inoculated into liquid LB medium
And cultured overnight. Contains all three plasmids
0.1 ml of an overnight culture of E. coli was used for C58Cl Rif
^ROvernight culture of (pGV2260) and LB at 28 ° C overnight
Medium (mirror,Experiments in Morekiurar
・ Genetics(1972), Cold Spring
(Haber Laboratories, New York).
Obtained from one crossover between pGV2260 and pHD208
Containing hybrid Ti plasmidAgrobacteriumof
The strain is streptinomycin (300 μg / ml) and strike
Leptomycin (300 μg / ml) and streptomycin
Minimum A medium supplemented with isine (1 μg / ml)
-EXPERIMENTS IMOREKIULAR GENENETY
Cousin(1972) Cold Spring Harbor Laboratory
On the pHD208 plasmid
Streptomycin-spectinomyci incorporated
Were selected by selecting the appropriate marker.

[0098] The object to be conjugated was rifampicin (100 µg
/ Ml) L with the addition of spectinomycin (100 μg / ml) and streptomycin (300 μg / ml)
B medium (mirror, Experiments in Morekiura)
-Genetics (1972), Cold Spring Harbor Laboratory, New York.
One of the object to be bonded, the physical structure of pHD1076 is Daesu etc. (Nukureitsuku amide Research, Vol. 7 (19
1979), pp. 1837-1849), according to the method described by C58Cl Rif ^R pHD107.
6 Pst digested total DNA was determined by hybridizing ³² P-labeled pHD208. The physical structure of pHD1076 is shown in FIG.

Example 3 To obtain the hybrid Ti plasmid pGS1151, the intermediate expression vector pGSH151 was inserted into the receptor Ti plasmid pGV2260. The method used was described by Deitag et al. (1980) Proceedings of Na
National Academy of Sciences of US
A , tripa rental crossover according to volume 77, pages 7347 to 7351. One of E. coli K514 colonies transformed with pGSH151 was inoculated into liquid LB medium and cultured at 37 ° C. overnight. This culture solution 0.1
ml is HB101 (pRK2013) (Figursky and Herinski, (1979) Procedures )
・ National Academy of Sciences
Of USA , Vol. 76, pp. 1648-1652) with 0.1 ml of overnight culture and C58Cl Rif.
^R (Vuan Larebek et al., Naycha , Vol. 252, 1
(Pages 69 to 170) on an LB plate with 0.1 ml, and cultured at 28 ° C. overnight. Cells were collected from the LB plates and the dilution was made up with spectinomycin (30
0 μg / ml) and streptomycin (1 μg / ml)
A medium (Miller, Experiments I)
(Molecular Genetics , 1972, Cold Spring Harbor Laboratory, New York). The object to be conjugated was hafampicin (100 μm).
g / ml), spectinomycin (100 ug / ml)
And streptomycin (300 μg / ml)
Purified in B medium. One of the objects to be joined, pGS1151
The physical structure of, Daesu, etc. (Nukureitsuku-and-Li
C58ClRiP ^R (pGS1151), according to Search , Vol. 7 (1979) pp. 1837-1849).
For the PstI-BamHI digested total DNA of ³²
It was determined by hybridizing P-labeled pGSH151.

10. Chimeric toxin residue inserted into the genome
Method for Isolating Plant Cells and Plants Containing Genes : Transformation of Tobacco Plant Cells with a Transformation Vector as Described in Section 9 and Transformation of Callus Tissue and / or Differentiated Plants from the Transformed Cells Two different methods for reproduction are described here. Method 1: Agrobacterium of the proto-plus co-culture tobacco protoplasts C58Cl
Isolation of transformed tobacco cell lines by co-culturing with Rif ^R and searching for the presence of a determination marker such as nopaline or expression of a selection marker such as kanamycin resistance, and whole plants from the transformed callus line This method describes the regeneration of the body.

Step 1 : Preparation of protoplast a) Nicotiana tabacum cv. Petei Havana SR-
One sterile plant is grown in a test tube to a height of 10 to 12 cm in a test tube for 4 weeks on a medium containing half the vitamin and sucrose and half the inorganic composition of the Murashige and Skoog medium.
( Murasige and Skoog, Physiology of Plan
G , Vol. 15, pp. 473-497 (1962
Year)). b) Three well-opened leaves of young leaves were mixed with 20 ml of the following solution containing 1.4% Cellulase Onoka R-10 and 0.4% Macerozyme Onoka (both from Yakult Yakuhin Kogyo Co., Ltd., Japan). Incubate with. _{KCl2.5g / l, MgSO 4 · 7H} 2 O1g /
1, KH ₂ PO ₄ 0.136 g / l sorbitol 7
3 g / l, polyvinylpyrrolidone 100.3 g / l c) Incubate overnight in the dark at 24 ° C. d) Filter with a nylon filter having a mesh size of 50 μm. e) Centrifuge at 80 g for 10 minutes in a 15 ml tip, remove the supernatant and resuspend the pellet in 20 ml of the same buffer without enzyme. f) Centrifuge at 80g for 10 minutes to remove excess enzyme and remove supernatant. g) Precipitate was washed with 0.22% CaCl ₂ .2H ₂ O and 0.4
Resuspend in 20 ml of 1/2 Murashige-Skoog medium supplemented with M mannitol, pH 5.6. h) Centrifuge at 80g for 10 minutes and remove the supernatant. i) Resuspend the pellet in 20 ml of Medium 55 (see below). j) counting protoplasts ^are diluted to 10 5 pp / ml. Incubate in a 5 cm Petri dish (2.5 ml per Petri dish) in the dark for about 4 days.

Step 2: Co-culture with Agrobacterium C58ClRif ^R strain containing hybrid Ti plasmid (Section 9) a) Incubate Agrobacterium C58Cl Rif ^R in LB medium until it is saturated, and use an Eppendorf centrifuge for 1 minute The supernatant is removed by centrifugation.
And resuspended in Cl _2. When about 30% of the protoplasts begin initial cell division, 50 μl of bacterial suspension is added to 2.5 ml of protoplast suspension (this is approximately 100 to 500 bacterial cells per proplast). Is). b) Incubate in the dark for 48 hours. c) Transfer the cell suspension to a centrifuge tube and add
The Petri dish was washed with an equal volume of medium 55 supplemented with 00 mg / l and added to a centrifuge tube. The mixture was centrifuged at 80 g for 10 minutes, and the supernatant was removed.
Resuspended in 55 equivalents of medium supplemented with g / l. d) transferred into Petri dishes 5 cm (2.5 ml / dish), this time, the cell concentration is about 10 ⁴ cells / ml. Incubate at 400 lux, 16 hours a day, at 23 ° C. for 1 to 2 weeks until small clumps of 4 to 8 cells are formed. e) Add an equal volume of medium 56 (see below). f) After 3 to 4 weeks, harden with 0.7% agarose to reduce mannitol concentration (0.24 instead of 0.44M).
M), and colonies are spread on the medium 56 to which clafolan (250 mg / l) is added. At this stage, it must contain more than 50 cells per colony.
If Km ^R is used as a selectable marker, 50
μg / ml kanamycin is added to the medium as a selection agent. g) Incubate at 800 lux for 16 hours a day at 23 ° C. for 2 to 3 weeks. h) Transfer the separated calli to the same medium. Germinate. At this stage, when the nopaline is used as an identification marker, callus tissue, arts, etc., Plant Science
Taken to search for the presence of nopaline using a method such as that described by Tech Letters, Vol. 17, pages 43-50 (1979).

[0103]Stage 3:Regeneration of transformed tobacco plants a) Nopaline-positive or kanamycin-resistant calli with 4
Grow for a week. b) Differentiated calli with hormone-free Murashige-Sukuo
Transfer to medium. c) Grow for 3 weeks. d) The shoots are separated and transferred to the same medium so that the plants form roots
Grow for two to three weeks until they grow. e) At this stage, the small plants contain half of the hormones.
50ml of Murashige and Skoog (M & S) culture medium
Transferred to a 250 ml container for growth. f) Grow for 2 to 3 weeks. Nopaline detection or Kanamai
For the search for syn-resistance and for the immunological detection of toxins
The lower leaves are removed. Kanamycin resistance in plants
Leaf disks for testing (sometimes with leaf sections and
The test is performed as follows.
A small disk is cut from a plant grown "in vitro".
And extracted at various concentrations of kanamycin sulfate (50 to 5
Callus induction medium (MeS macro-
Anide micronutrients, vitamins, 3%
Yogurt, 500 mg / l clafolan, 1 mg / l NA
A and 0.1 mg / l BAP) in a Petri dish
Moved. Incubation for 3 weeks in plant tissue culture room
After Yong, calli growing on leaf disks are observed.
The Km resistance level of a plant is determined by removing callus tissue from leaf disks.
Determined as the highest concentration of kanamycin when it occurred
It is. Search for the presence of nopaline (nopaline test)
Is M. Arts, M.A. Jacob, JP. Hernalste
Ein, M.S. Vuan Montaguille and J.M. Ciel, (1
979)Plant Scientific Letter
Z, Vol. 17, pages 43-50
Implemented according to law.Medium 55 tissue: ―Micronutrient of Murashige and Skoog salt
Of half the strength of -Improved micronutrient heller by 1000 times
  1ml / l -Vitamin Morel & Wet More 1000x 1m
l / l -100ml / l inositol -FeSO₄5.57 g / l and Na₂EDTA7.45
10 ml / l of stock solution containing g / l -Benzylaminopurine 1ml / l, naphthaleneacetic acid
  3ml / l -Mannitol 80g / l (0.44M), sucrose
20g / l

Vitamin 1000 times per 100 ml
-Morel and Wetmore 100 mg Ca-pantothenic acid: biotin 1 mg; niacin 100 mg; pyridoxine 100 mg; thiamine 1
00mg improved micro Niyu bird entry, Heller _{(500ml) 500mg ZnSO 4 · 7H} 2 O; 50mgH 3 BO
_{3; 50mg MnSO 4 · 4H 2} O; 50mgCuS
_{O 4 · 5H 2 O; 15mgAlCl} 3; 15mgNiC
l Composition of medium 56: Medium 56 contains 0.2 m of naphthaleneacetic acid.
Identical to medium 55 except that g / l and 1 mM glutamine are added.

Method 2 : Agro with hybrid Ti plasmid
Infection of Bacterium C58Cl Rif ^R strain into leaf sections Infection of leaf sections with C58Cl Rif ^R and isolation of transformed cell lines by selection on a medium containing kanamycin are described herein. Sterile Nicotiana tabakum c
v. Petei Havana SR-1 is a test tube circle, complete M & S
Complete M & S supplemented with 1/2 of organic nutrient and sucrose
Cultured on plant nutrient agar containing 1/2 of the S salt mixture.
Twenty SR-1 leaf sections of about 1 cm ² were obtained from C58Cl R
was floated on 5ml liquid M & S medium in 9cm petri circumference comprising washing the bacterial suspension 0.1ml of an if ^R (no hormones). Incubation was carried out at 25 ° C. for 48 hours in the dark while shaking at 60 rpm. Then, the leaf section is 500m
Washed twice with M & S (without hormone) containing g / l clafolan and then placed on callus and bud forming medium. This medium contains M & S macro and micro nutrients and vitamins, 3% sucrose, 500mg
/ L clafolan, 500 mg / l kanamycin sulfate,
Contains 0.1 mg / l NAA and 1.0 mg / l BAP. The final pH of the medium is 5.8. Six leaf disks are placed per 9 cm Petri dish containing approximately 30 ml of medium and incubated for 3 hours at 23 ° C. (± 1 ° C.) with a 2000 lux light cycle for 16 hours per day. 3
Weeks later, discs with callus and small buds
Are transferred to the same medium for another 3 weeks. At this point, sprouts over 1 cm in length are stripped of hormones and kanamycin and M & S containing 500 mg / l of graf orane is transferred to the medium. Thereafter, the buds are transferred to hormone-deprived M & S approximately every three weeks, and clafolan concentrations are reduced sequentially (first shift: 25).
0 μg / ml, second time: 125 μg / l, third time: 0 μg
g / ml). During the first half M & S transition, leaf material is removed to test for kanamycin resistance. The leaf disks were prepared with different concentrations of kanamycin sulfate (5
0-500 mg / l) callus induction medium (M & S
Macro and micro nutrients and vitamins, 3%
Sucrose, 500 mg / l Craforan, 1 mg / l NA
A and 0.1 mg / l BAP). If the substance proves to be free of Agrobacterium, the plant Km resistance is again tested in a medium without clafolan.

Example 1 Transformation with pHD1050
Callus and plant T-DNA : P nos -Bt2 (Bt2 gene fused to P nos ) marker : Nopaline synthase as marker gene with additional border sequence between Bt gene and nos gene Transformation method : Protoplast infection Approximately 250 calli were examined for nopaline, and 19% showed Nos ⁺ representing high frequency transformation.
It was. In a total of 180 different callus lines, both nos ⁺ and nos ⁻ resulting from these transformation experiments were examined for the presence of Bt2 using the sensitive ELISA described above (section 5.1). . Most clones were tested immediately after transformation during the early phase of growth (only 5 mm in diameter), and some were tested after the subculture period (3 months later). Based on the results of the immunoassay, 25 callus lines were selected for plant regeneration. Several plants were regenerated from each callus, and each of the plants was given a different number (149 plants in total). 149 plants were bred "in vitro", followed by 138 being transferred to the greenhouse. All these plants all appeared normally, bloomed and seeded.
Several plants were used for insect toxicity tests. Total DNA was prepared from callus lines 161, 165 and 206, and B
The integration of the t2 gene was analyzed by Southern blotting. Integration of at least one copy of the Bt2 gene per genome was detected.

Example 2 Transformed with pHP1060
Callus and plant T-DNA : P nos-Bt2 selection marker : kanamycin resistance (Km) Transformation method : protoplast infection (method 1) and leaf disk infection (method 2). According to Method 1, kanamycin resistant protoplast clones were obtained and grown as calli. Calli were randomly selected and placed in production medium for bud formation. Buds isolated from these kanamycin shochu clones are propagated "in vitro" as plants. Subsequently, some of these plants were transferred to the Solstice. According to Method 2, kanamycin-resistant callus tissue and buds were induced. Uncloned callus tissue was maintained in an "in vitro" subculture. Kanamycin resistant shoots were isolated and expanded "in vitro" as plantlets (2-5 cm). These plantlets were retested for kanamycin resistance (50 μg / mlkm) using the leaf disk test. Apparently, shoots resistant to this concentration of kanamycin were selected for subsequent "in vitro" growth. The plants were eventually transferred to the greenhouse. Using Southern blotting analysis, NPTII
The presence of both the gene and the Bt2 gene was confirmed in the leaf tissue of these plants.

Example 3 : Salmon transformed with pHD1076
Callus and plant T-DNA: Pssu-Bt2 ( Bt2 gene fused to PSSu) selection marker: kanamycin resistance Transformation method: using the conditions stated in the leaf disk infection method 2, transformation and bud callus Induction was performed on infected leaf disks. Using the callus induction method, many calli were obtained by partial purification and maintained as individual semiclones. Based on the results of positive immunoassays, five of these lines were selected for the next expansion (1076-4,
10, 11, 12, 13). From the bud induction method used at the early stage of leaf disk infection, 72 kanamycin-resistant plants were regenerated (selection at 50 μg / ml km).
When retested by the leaf disk test, it turned out that 65% of these were truly resistant to 50 μg / ml km. Callus tissue was created from the leaves of some plants grown "in vitro" and augmented "in vitro" for further testing.

Example 4 : Transformed with pHD1080
Callus and plant T-DNA : Pssu-transport peptide (Tp) Bt2 selection marker : kanamycin resistance / (Nos) Transformation method : leaf disc infected kanamycin shochu callus and buds were induced according to method 2. About 20 kanamycin-resistant callus strains (or lines)
Were tested for nopaline expression and all were found to be positive. 86 kanamycin resistant shoots were selected, expanded "in vitro" and retested for kanamycin resistance (using leaf disc test) and nopaline expression. Fifty-two plants (60%) were kanamycin resistant and nopaline positive, and these were further expanded "in vitro". About 10% of the plants expressed only one of the two markers.

Example 5 : Transformed with pGS1110
Plant T-DNA : Pnos-Bt: NPTII (fusion) Selectable marker : Kanamycin resistance / NOS transformation method : Leaf disk infection Leaf disks of SR-1 plants maintained "in vitro" were Agrobacterium Tyumefu Assistance C58
Incubated with a suspension of Cl Rif ^R pGS1110 (method 2) for 48 hours. Complete NPTII
Similar dilutions of the different control strains containing the chimeric gene that encodes were also included. After 2 weeks, the formation of active shoots on M & S medium containing 50 mg / l kanamycin was observed in both control and pGS1110. However, after transfer to fresh selection M & S media, differences were apparent between the control and pGS1110. Some buds on the latter and the incubated disks turned yellow and grew slowly. Green sprouts with the best growth were transferred to a medium without kanamycin. They were rescued in this way and began to grow normally after a second transfer to medium without kanamycin. About 70 buds are pGS111
Rescued from 0 transformation experiments. 35 of these shoots
From a screening (search) of the individuals, 28 (85%) of these produced noperin,
It was shown to be a transformant of the vote. This important observation suggests that sprouts are not maintained for long periods of time in media containing kanamycin, but that there is a phenotypic selection for expression of the fusion protein. The resulting shoots were grown "test Kannai" as plantlets in a non-selective medium. Many of these plants were tested for kanamycin resistance using the leaf disk test. Most expressed callus resistance as they formed calli in media containing kanamycin. Various levels of resistance were recorded ranging from 50 to 500 mg kanamycin / litre. However, most plants had low levels of kanamycin resistance. Two out of a total of 61 plants were resistant to 200 μg / ml kanamycin and 500 μg / ml
It showed partial resistance to ml kanamycin (very weak callus growth). For many plants, copies were transferred to vermiculite pots. When reaching a height of 10 to 15 cm, an initial insect toxicity test was performed on the leaves of these plants (see section B).

Example 6 : Transformed with pGS1161
Plant T-DNA : PTR2-Bt2 selection marker : Kanamycin resistance Transformation method : Leaf disk infection Leaf disks of SR-1 plants maintained "in vitro" were Agrobacterium tumefaciens C58C
Incubated with 1 Rif ^R pGS1161 suspension for 48 hours. As a control, A, including NPTII under pTR domination. Tyumefu Assistance C58Cl
Rif ^R pGS1160 was included. Two weeks later, bud formation was observed on a medium containing 50 mg / l kanamycin sulfate. After three weeks, the disks were transferred to fresh selection medium, and after another three weeks, the best growing shoots were transferred to medium without kanamycin. The level of kanamycin resistance is systematically determined using a leaf disk test. Most plants have high levels of tolerance (500
Callus formation was shown at μmg / ml km.

Example 7 : Transformed with pGS1151
Plant T-DNA : pTR2-Bt: NPT2 (fusion) selection marker : Kanamycin resistance Transformation method : Leaf disk infected SR-1 plant leaf disks cultured in vitro
Agrobacterium teyumehuaciens C58Cl
Incubated with the suspension of Rif ^R pGS1151 for 48 hours. As a control, NP under pTR control
A. teumefuashiensu C58Cl Ri including TII
f ^R pGS1160 were included. Bud formation and bud growth in media containing 50 mg / l kanamycin sulfate were slightly slower on disks treated with pGS1151 than on control disks (pGS1160). After three weeks, the discs were transferred to fresh selection media and after another four weeks,
The best growing shoots were transferred to medium without kanamycin. Buds were propagated as plants in "test skannai" and the level of kanamycin resistance in these plants was systematically determined using a leaf disk test. Many plants have 500 μg / m
It was completely resistant to 1 kanamycin (normal callus growth). This data indicates that the PTR promoter governs higher levels of fusion protein expression in tobacco leaves than the Pnos promoter (pGS1110, Example 5 in this section). Copies of the plants were transferred to pots and raised in the greenhouse. A detailed insect toxicity test was performed on a selected set of plants exhibiting high kanamycin resistance (see Section 13). The level of kanamycin resistance was determined systematically using the leaf disk test.

Example 8 : pGS1162 or pGS11
Plant T-DNA transformed with 63 : PTR2-Bt2 / 820-PTR2-B
t2 / 884 selection marker : Kanamycin resistance Transformation method : Leaf disk infection Leaf disks obtained from SR-1 plants grown "in vitro" were Agrobacterium teumefaciens C58ClRif ^R pGS1162, pGS1163
Or infected with pGS1160 (as a control). The disks were prepared at different concentrations of kanamycin (50-100-2).
(00 mg / l). Buds from all three concentrations were transferred to medium without Km. Km resistance was checked by a leaf disc test in a callus induction medium containing 50 to 500 μg / ml kanamycin.

Example 9 : Transformed with pGS1152
Plant T-DNA : pTR2-Bt: NPT860 Selection marker : Kanamycin resistance Transformation method : Leaf disk infection Leaf disks obtained from SR-1 plants grown "in vitro" were Agrobacterium tumefaciens Infected with C58Cl Rif ^R pGS1152.
Agrobacterium teyumehuaciens C58Cl
Disks infected with Rif ^R pGS460 were included as controls. The discs have different concentrations (50, 10
(0, 200 μg / l) of kanamycin. Buds were obtained for all three concentrations, although less abundant than on control disks infected with C58Cl Rif ^R pGS1160.

(11) Bt2 protein in engineered plant tissue
The expression of Bt2 in engineered plants (callus tissue or differentiated plants) was described in Section 5 and followed using an ELISA adapted to test plant extracts. . Conditions for preparing and testing plant extracts were established in reconstitution experiments where purified Bt2 protein was mixed with plant extracts. In reconstitution experiments, a considerable loss of antigenic activity of the Bt2 protein due to the presence of plant extracts (20
%) Were not observed. In ELISA, 0.1
As little as mg / ml of purified Bt2 protein was detectable. However, in reconstitution experiments, some variable state occurs in the background, probably caused by plant proteins present in the extract. Therefore, a reliable limit of detection under these conditions was on the order of 1 ng / g tissue, corresponding to a level of 2 ng Bt2 protein / g plant tissue. 11.1. Search for individual calli For the immunological search of individual calli, the following experimental method was established: 200 mg callus tissue from 150 to 20
It was mixed with 0 μl of extraction buffer. The extraction buffer had the following composition: 500 mM Na ₂ CO ₃ solution 5
0%, 100 mM DIT and 50% fetal calf serum. The tissue was homogenized by crushing with a spatula, and the cell debris was then centrifuged. B. as described.
t. PBS pH 7.4 + 1 in wells of a microtiter plate coated with goat antibody to crystalline protein
50 μl of supernatant was added to 50 μl of 0% fetal calf serum. Throughout the method, samples were kept in ice and micrometer plates were incubated at 4 ° C. for 1.5 to 2 hours. Then, as stated in 5.1,
For the detection of Bt2 protein, the ELISA method was continued (under formation of culture supernatant) using either rabbit anti-Bt2 serum or a mixture of anti-Bt2 monoclonal antibodies 4D6, 10E3 1.7 and 4.8. Was done.

Example 1 : Transformation with C58Cl Rif ^R pHD1050
Analysis of Callus Transformants Transformed callus clones were obtained by the method of protoplast co-culture as described in Section 10, Example 1. 19% of clones express nopaline (N
os ⁺ ), at least 19% were transformed. However, the intermediate expression vector (p2
Due to the additional border sequence of GV2382), the nos gene and the Bt2 gene are inserted linearly and independently. Therefore, both NOS ⁺ and NOS ⁻ clones were examined in an ELISA test. A total of 180 callus clones (130 nos ⁻ , 50 nos
s ⁺ ) was tested. Some clones were retested once or twice at different time intervals after initial growth from protoplast culture. No obvious positive signal was recorded. When the substrate reaction time in the assay was extended (incubation overnight at 4 ° C.), some clones (both nos and nos) had a very weak signal above background (which is a control callus without the Bt2 gene). Was produced. However, the values obtained were clearly below the reliable detection limits of the test system,
No definitive conclusions could be drawn regarding the expression of the Bt2 protein in these calli.

Example 2 : Transformed with C58Cl Rif ^R pHD1076
Detection of Bt2 protein in tobacco callus coarse weave Agrobacterium C58Cl Rif ^R (pHD10
76), the transformed callus tissue obtained from the leaf section infected with E. coli (see Section 10, Example 3) was immunologically
The presence of the t2 protein was examined. After initial growth, the calli were transferred 20 days later for the second generation. When optimal growth is reached, 200 mg of E from each callus line
Used for immunological search by LISA. In the first experiment, a signal was obtained in which 9 out of 14 transformed calli were clearly more positive than the background obtained with 4 control calli (untransformed SR-1 callus).
This was shown when the reaction was performed with a specific rabbit anti-Bt2 serum (see FIG. 34). As determined by comparison with the positive control (control SR-1 mixed with a known amount of Bt2 protein)
Three transformed calli produce about 5 ng Bt / g tissue
A signal corresponding to two proteins was generated. As a negative control, when reacted with normal rabbit serum, all samples were (SR-1)
A signal equal to the back ground level (obtained with the control callus) was produced. In a second experiment, 13 of the 21 transformed calli gave a signal well above background (FIG. 35). One in the callus
Individuals produced a signal corresponding to 24 ng Bt / g tissue. These results indicate that Bt2 protein is produced at detectable levels in the callus fraction transformed with pHD1076. Approximately 5 weeks after the first ELISA experiment, the four selected series (1076-10, 11, 12 and 13) that gave high positive values in the initial search were E
The LISA was retested. Several "subclones" of each line were tested (the original callus was split into sections that were grown independently in the next growth cycle;
Each new section is here a subclone). One subclone of 1076-10 was positive, one subclone was negative, two subclones of 1076-12 were positive, three subclones of 1076-13 were positive, and two were negative. These results were originally reported by B.S. t. When callus tissue determined to be positive grows significantly, B. t.
This indicates that a negative callus occurs.

11.2. Bt of accumulated callus extract
Detection of 2 In order to perform a detailed immunoassay search with increased detection sensitivity, a considerable amount of enriched extract of transformed callus tissue was prepared. The method developed here to obtain an extract enriched in Bt2 protein involves the precipitation of Bt2 at pH 4-5.
It is based on the nature of Example 1 : Callus transformed with pHD1076 Callus transformed with pHD1076 was 0.0
140 g of non-cloned transformed calli grown on a medium containing 5 mg / ml kanamycin sulfate were collected (callus lines 1076-4, 10, 11, 1).
2,13 and many unscreened lineage pools). The extract is made up with 70 ml of extraction buffer (500
mMa ₂ CO ₃ , pH 10, 5 mM DIT, 170 μm
g / ml PMFS) in the presence of callus. The supernatant obtained after centrifugation at 15000 rpm was diluted by adding 50 ml PBS, pH 7.5. Subsequently, the pH of the diluted extract was 1 M
HCl was added to pH 6 and incubated at 0 ° C. for 20 minutes, and the supernatant was separated by centrifugation and
(Fraction pH 6). When the pH was lowered to 4.5, a new precipitate was similarly separated (fraction pH
4.5). The precipitate is washed once with H ₂ , followed by 20 minutes at room temperature in the following buffer: Na ₂ CO ₃ 500 mM pH 1
0, DTT 50 mM, PMSF 170 μg / ml (1.5 ml for precipitation pH 6 and 2 ml for precipitation pH 4.5). The substance solubilized under these conditions is 1
Separated after 5000 rpm centrifugation, these samples were called 1076 pH6 and 1076 pH4.5, respectively. Exactly the same method was used to prepare an extract of the substance of normal SR-1 callus (used here as a negative control) and was called SR-1 pH 4.5.
Individual specimens were obtained. The total protein content of these samples was: 1076 pH6 600 μg / ml 1076 pH4.5 6560 μg / ml SR-1 pH6 380 μg / ml SR-1 pH4.5 3840 μg / ml To estimate the efficiency of the method, the initial sample homogenization A reconstitution experiment was performed in which 1 μg of purified Bt2 was added to 20 g of SR-1 control callus tissue. The presence of Bt2 protein in these extracts was determined by ELISA with goat anti-Bt crystal serum and rabbit anti-Bt2 6002. Fraction 1 compared to negative control (SR-1)
A strong reaction was recorded at 076 pH 4.5. Fraction 1
076 pH 6 gave a signal only slightly higher than SR-1 pH 6, which indicates that this fraction was Bt2
It shows that it contains only a small part of the protein content.

In ELISA, fraction 1076 pH4.
5 also shows five different monoclonal antibodies specific for the Bt2 protein: 1.7, 4D6, 4.8, 10E3.
And 1F6 (see FIG. 36). This strongly indicates that fraction 1076 pH 4.5 contains a Bt2 protein that is identical in shape to bacterial Bt2. Next, Bt2 was extracted from the extract using immunoprecipitation.
Tried to get rid of the protein. 5% rabbit anti-Bt2
Serum was added to the extract incubated at 4 ° C. for 1 hour. Subsequently, 5% goat anti-rabbit Ig serum was added and incubated for 1.5 hours at 4 ° C. The precipitate was removed by centrifugation and the supernatant was tested by ELISA. This supernatant contains at least 1/10 of the Bt2 activity of the original 1076 pH 4.5 fraction,
The substance producing an ELISAAT-positive signal is specifically anti-Bt
2 antibody, and it was confirmed that the substrate that reacted positively by ELISA was Bt2. In the next experiment, the sample was carbonate buffer (pH 10)
Dialyzed against. Quantification of the Bt2 content of the 1076 pH 4.5 extract was performed by testing a solution of purified Bt2 protein as a dilution of the extract and as a standard in ELISA. The quantitative value was 122 ng Bt2 / ml extract (total volume 2 ml). Reconstruction experiments (adding Bt2 to SR-1 control callus at the beginning of extraction) showed that only 20% of Bt2 protein was lost during extraction and 80% was contained in the pH 4.5 fraction. . Based on these results, the total amount of Bt2 protein present in the original 140 g callus can be calculated to be 305 ng, with 2.2 ng / g being a result that is in good agreement with the original estimates given in individual callus searches. is there. (See FIGS. 34 and 35). These data indicate that Bt2 protein present in the extract of transformed calli
As described above, it is specifically concentrated by the precipitation method at pH 4.5, indicating that the amount of Bt2 protein produced in these plant tissues can be more accurately quantified.

Example 2 Transformed with pHD1050
A pool of 500 g of selected callus clones (approximately 25 callus lines giving a value above background, based on the results of previous ELISA tests on individual calli) was obtained from Example 1 Homogenized in the presence of 1000 ml of extraction buffer using the same method as described in Substances that remain soluble at pH 6 but precipitate at pH 4.5 are separated by centrifugation,
Subsequently, it was redissolved in a small amount of carbonate buffer, pH 10 (see Example 1). Analysis of the substance by ELISA revealed a positive signal corresponding to 60 ng / ml or 1.2 ng / g of callus tissue Bt2 (Table 8). Example 3 : Transformation with pHD1060 and pHD1080
Transformed Callus In this example, a slightly different and broader extraction method is used for the separation of Bt2 protein from engineered plants. A technique was developed to recover the Bt2 protein, which was not solubilized in a single extraction step as used in the methods of Examples 1 and 2, but remained intact. This is the case when the Bt2 protein contains some highly hydrophobic regions capable of interacting with the plant cell membrane structure and is therefore less likely to be solubilized without detergents. The robust methods used here allow for the recovery of additional proteins on insoluble plant cell structures. A schematic diagram of the technique is shown in FIG. The first protein fraction was extracted with carbonate buffer pH10 + DTT and acid precipitated (p
H4.5) (fraction I). This fraction corresponds to a pH 4.5 extract obtained using the methods of Examples 1 and 2. The material that is not solubilized in the first extraction step and remains in the precipitate is then treated with the same extraction buffer containing 1% Triton X-100. The protein solubilized under these conditions and precipitated at pH 4.5 is contained in Fraction II. The last step involves solubilization with 2% SDS and acetone precipitation to give fraction III. Fractions I and II were analyzed by ELISA and Western blotting; Fraction III containing SDS was analyzed by Western blotting only. The results of the ELISA are listed in Table 8: 1.9 ng and 1.4 ng (fraction I) and 0.27 / g of original tissue, respectively.
Positive signals corresponding to Bt2 levels of ng and 0.29 ng (fraction II) were detected in fractions I and II of both 1060 and 1080 plasmids. Western blotting of the material solubilized with SDS (fraction III) revealed the presence of a faint 130 kd band in both 1060 and 1080 callus material (using rabbit anti-Bt2 serum). The limit of detection for Western blotting was 10 ng per lane. Therefore, these fractions contained at least 1060 at 0.39 ng / g and 1080 at 0.5 ng / 3. In Western blotting of Fraction I at 1050, 1060 and 1080,
Perhaps because the concentration of Bt2 protein was too low in these fractions, the presence of 130 kd was unknown. This result indicates that low levels of Bt2 protein were actually pHD1060
And expression in callus transformed with pHD1080. Although small-scale analysis of individual calli does not allow detection of immunopositive clones of these constructs, more rigorous methods of extraction and enrichment for pools of selected calli result in reliable quantitative detection of Bt2 protein. Is possible. The essential fraction of Bt2 is bound to strongly insoluble plant material. And Triton and SDS
It is released only with the use of a surfactant such as

11.3. Regenerated transgenic plant leaves
Detection of Bt2 protein For routine testing of leaf samples, the following method was established: green leaf tissue (200-400 mg) was "in vitro"
Plants (height of 5 to 10 cm as described in Section 10 Example 1) and [500 mM Na ₂
CO ₃ , 100 mM DTT, 480 μg / ml leupeptin (Migma, L-2884), 2 mM PMSF, 2 m
g / ml ascorbic acid (Sigma, A-7631) 2m
[MEDTA] and 50% FCS in the presence of an extraction buffer (200 μl). The tissue was homogenized by crushing with a spatula, after which the cell debris was centrifuged. A similar ELISA was then performed for callus tissue retrieval as described (see Section 11.1). Example 1 : Trait with C58Cl Rif ^R pHD1050
Detection of Bt2 protein in transformed tobacco plants Leaves of plants grown "in vitro" (70 separate plants regenerated from 25 selected callus clones)
The expression of t2 was tested in an ELISA using the conditions described above. Of the 70 plants tested, 5 showed a clear positive response above background obtained from untransformed SR-1 leaves corresponding to Bt2 levels ranging from 6 to 25 ng / g wet tissue. Was. One of the plants with the highest values (plant number 161-9: 25 ng) was studied in more detail. The leaf extract was rabbit anti-Bt.
Reacted positively with a mixture of two sera and a monoclonal antibody specific for the Bt2 molecule (undiluted culture supernatant of clones 1.7, 4.8, 4D6, 10E3). Furthermore, it did not react with a pool of monoclonal antibodies showing irrelevant specificity. Plant extracts were retested after freezing and thawing at least twice at -20 ° C with the same reagents. Identical results were obtained in each case, but the level of Bt2, although probably the result of Bt2 protein degradation,
It gradually decreased with each cycle of melting. Plant 16
1-9 were retested after being grown in greenhouse conditions at the stage of blooming now. Again this time, ± 5ngBt
At the level of 2 / g tissue a correspondingly positive signal was obtained. This result indicates that the level of Bt2 protein detected in the leaves of the manipulated plant changes significantly depending on the age, growth conditions, etc. of the plant.

Example 2 C58Cl Rif ^R pHD1
Search for Tobacco Plants Transformed in 060 Search for 76 kanamycin-resistant plants expanded in vitro using the ELISA method described in Example 1 did not yield any clear Bt2-positive plants. Example 3 : Trait with C58Cl Rif ^R pHD1076
Search for converted tobacco plants Extracts of leaves of plants grown "in vitro" obtained by the bud induction method were examined by ELISA (method as in Example 1). Seventeen plants were tested and none showed a positive signal. Subsequently, another 21 plants were examined when they were grown in pots and reached a height of 15 to 20 cm.
Again, no positive signal above background was recorded in the ELISA for examining the plants.

Example 4 Transformation with pHD1080
Search for Tobacco Plants Approximately 30 plants grown “in vitro” are ELISA
Was examined in. One plant (plant number 174) gave a positive signal corresponding to 20 ng / g Bt2 with both rabbit anti-Bt2 serum and a pool of monoclonal antibodies. The extract of this plant remained positive in the retest in the next experiment. 11.4. Bt2 of callus tissue from leaves of transformed plants
Protein Detection The data outlined in Examples 2 and 3 and Example 3 show that the Pssu promoter construct used here (pHD1076) is more differentiated than in callus. It shows that the leaves of the plant are less active. To further investigate this, new callus tissue was created from the leaves of the same plant used in the previous measurements. The leaf disks were cultured in a callus induction medium and after a few weeks the calli were collected and analyzed in a similar manner (Na ₂ CO ₃ p
Extraction of Step I instead of H10 / DTT buffer with Tri
s pH 7.5 buffer was used). Calli induced simultaneously from untransformed SR.1 leaves were used as a negative control. ELISA analysis of pHD1076 transformed calli revealed detectable amounts of Bt2
The proteins are shown (Table 10). These results indicate that the chimeric gene with the pea Pssu promoter in the construct used therein is functional and has detectable amounts of Bt2.
Bt2 in leaf-derived tobacco callus tissue that does not express protein
It indicates that it induces protein expression. Therefore, chimeric genes that govern expression in undifferentiated callus tissue are not always as active in leaves of differentiated plants.

12.Bt produced in engineered callus
2 Insecticidal activity of proteins Method : Toxicity measurements were performed on mandeyuca
It was given to the first larva of Sexta. From 3
4 ml of liquid artificial feed (RA Bell and FG Yoahi
Mu, (1976),Ann. Entomol. So
c. Ann. Vol. 69, pages 365 to 373)
Each section of a square Petri dish (4 cm²Distributed)
Was. Formaldehyde was removed from feed. Fix the feed
After shaping, 200 μl of the known diluted sample was
And dried in a stream of cold air. Four new larvae
Placed in each compartment. 3-5 days of growth and death
Tracked for a while.Example 1 :Callus mosquito transformed with pHD1076
Ruth extract Concentration of pool of callus transformed with pHD1076
The extract was prepared as described in Section 11.2, Example 1.
Was prepared. The diluted extract is added to the feed surface,
Toxicity was assessed. Extracted substance 1076 pH 4.5 is evident
Crab has toxic effects on Mandeyuca sexta larvae
Had: 12.5 μl / cm²All larvae are stunted
Show harm, 50 μl / cm²Died of 100% (Table 1)
1) The toxic activity of this substance is significantly reduced after immunoprecipitation
(12.5 and 25 μl / cm²Is 100% normal
Grown and 50μl / cm²37% of deaths)
And this means that the toxic activity is dead brown due to anti-Bt2
It indicates that it was done. Non-transformed negative control
The extract of the exchanged SR-1 callus tissue is completely non-toxic.
Was. The presence of Bt2 protein in the extract was determined immunologically
Therefore, the observed toxicity should be compatible with the determined Bt2 concentration.
Could be related. The value of the immunoassay is 107
6 indicating that pH4 contains 122 ng / ml Bt2
Was. Therefore 1cm²50 μl of extract per
6.1 ng Bt2 protein / cm²Is equivalent to Use Bt2
Toxicity measurement for Mandeyuca who had been present (Section 5.2, Table
3) is 12 ngBt2 / cm²Is 100% fatal
One, 2.5 ng / cm²Induces growth inhibition
Was. All of these results are expressed in engineered plants
Bt2 is a functional toxin that produces the bacterial Bt2 gene.
And exhibit the same range of potency of toxicity.

13. Shown by leaves of transformed tobacco plants
Insecticidal activity method : To assess the insecticidal activity expressed in the leaves of transformed tobacco plants, the growth rate and mortality of Mandeyuca sexta larvae fed on these leaves are recorded and The growth rate of larvae fed leaves of transformed SR-1 was compared. The following method was used: Method 1 The following experiments were performed on leaf sections (discs) placed in Petri dishes. Four leaf disks of 4 cm in diameter were embossed and 4 × 10 M. Along with Sexta 's first instar larvae, they were placed on a wet filter paper in a Petri dish. Preferentially the young leaves of the upper part of the plant were used. Twenty-four hours later, a second disk was added. 48 hours and 100 after the start of the experiment
During the time, at regular time intervals, the number of molts was counted. From this, it was possible to calculate the MT ₅₀ time at which 50% of the larvae molt. All experiments were performed in a growth chamber with a photoperiod of 16 hours illumination and 8 hours shading at 26 ° C., 90% relative humidity. In this experiment, a series of reconstitution experiments had to be included to assess the toxin levels required to have a significant effect on growth rate and survival of Mandeyuca sexta larvae. To this end, purified solubilized Bt2 protein (Section 5.1) was used in 0.1%.
Serially diluted in PBS containing 5% Triton X-100. A standard amount of Bt2 solution was mechanically sprayed (to obtain a very uniform coating) on tobacco leaf disks. Ten L1 (first instar) larvae were placed on each leaf disk, and three disks were used for Bt2 concentrate. Larval growth rates and mortality were tracked for over 100 hours. Method 2 A method essentially similar to the previous method was also used. This experimental design, however, was somewhat more extensive in order to be more effective in reliably detecting very small effects on larval growth rates. The set-up is different in the following respects:-care is taken that all plants are at exactly the same stage and conditions with the least effect on larval growth caused by differences in leaf tissue conditions. Was. --- (Unlike previous experiments where growth was only traced to L2) Larval growth was traced to L3 stage. -Not only the larval molting time was recorded, but also the final stage larvae weight was measured. The plants used for this set-up were grown in a greenhouse until they reached a height of 60 to 80 cm, but did not flower. The leaf disk is embossed and placed on a wet filter paper in a Petri dish,
And ten first instar larvae were placed on each disk. Five groups of 10 larvae were used per plant (5-leaf disc). Growth rate and mortality were followed for more than 7 days (at this point, almost 100% of controls were at the L3 stage).

Example 1 Plants transformed with pHD 1050, 1060, 1076 and 1080 were examined in the following insect test of Method 1. With this method, no significant effect on larval growth rate and survival was recorded. B of purified bacteria
The results of the reconstitution experiments using the t2 protein were as follows: At 25 ng / g, growth inhibition rather than death was observed,
At 50 ng / g about 50% died. A wide range of toxicity tests using Method 2 were performed on many transformed plants that were determined to be Bt + by immunoassay prior to Example 2 . These plants are 169-9 (Pnos-Bt2, nos +) (pHD1
050, Section 10 Example 1) 147 (Pnos-Bt2, nos +) (pHD105
0, Section 10 Example 1) 174 (Psu-Tp-Bt2, Km ⁺ nos ⁺ )
(PHD1080, Section 10, Example 4). Bt ^- plant (161-6) and untransformed SR-1 were used as controls. The results are shown in Table 12. A) Number of larvae still in the L2 stage or already progressed to L3 or died 150 hours after the start of the test.
The transition from L2 to L3 is apparently somewhat faster in the larvae fed SR-1 and 161-6 compared to the larvae fed the Bt ⁺ plants 161-9, 147 and 174. No significant mortality was recorded in any group (10% or less is considered background). B) The average weight of the larvae at the end of the experiment is shown in the upper row (the deviation of the larvae of the five groups of ten is calculated with the average of these groups). The bottom row is the calculated weight value of 5 large larvae in each group of 10 animals. These results are consistent with the kinetics of the L3 to L4 transition: control larvae
⁺ Somewhat larger than larvae fed on plant.

Example 3 Insect toxicity was measured on leaves of plants obtained by the transformation method using pGS1110 (Section 10, Example 5). The plants were 10 to 20 cm in length and the test was performed according to method 1. The transition from L1 to L2 is tracked and 1
Two groups of 0 larvae were used per plant. A remarkable growth inhibitory effect exhibited by some plants on M. sexta larvae was observed. L1- about 3 days after feeding
The data of the ratio of L2 is shown in Table 13. The control plants included in these experiments were Pnos-NPTI
It was transformed with a vector containing only I. Experiment 1 pG
Eight plants (N
20-38, N20-22, N20-18) caused growth inhibition as compared to the three control plants (C1, C2, C3). In experiment 2, four control plants (C4, C5, C6,
C7), one of the six (N20-3)
7) caused growth inhibition. The difference in growth rates is apparent when comparing the complete growth curves (FIGS. 38 and 39).
See figure). To determine if it was a true transformant, the same plants were also examined for the presence of nopaline and resistance to kanamycin. The results of the search data on the plants used in this insect test are compiled in Table 14. All four plants that showed an effect on larval growth are kanamycin resistant (Km ^R ) and nopaline positive (nos ⁺ ), and thus are positive transformants.

Example 4 Insect toxicity was measured on leaves of plants transformed with pGS1151 (Section 10, Example 7). Plants were grown at a height of 15 to 30 cm at the time of testing and in greenhouse conditions. Two independent experiments are described below: Some of the plants tested in the first experiment were retested in Experiment II to confirm the observed toxic effects. Except that only two groups of experiment I 10 larvae per plant was used (neonate larvae of Mandeyuka sexta), tested as described in Method 2 (this section) was made. Larval growth rates and mortality were tracked for more than 7 days, and larval weight at the end of this period was determined. The detailed results of Experiment I are shown in Table 15, in which larvae fed several plants transformed with PGS1151 compared to larvae fed control plants at an early stage of the experiment. It suggests that it shows significant growth inhibition. For example, 71 hours later,
60% of the larvae fed the control plant N21-107 were L2
Had progressed to the stage (2nd stage), while the plant N21-1
The number of L2 larvae at 8, 43, 53, 50 and 11 is only 15% or less. When followed for quite some time, significant mortality was recorded in larvae fed pGS1151 transformed plants. In some plants (N21-11), mortality reached 100% in less than 7 days. Mortality in control plants is only 15% in 7 days
45% of the larvae had already progressed to the L3 stage (in contrast, virtually no L3 larvae were found in other plants at 7 days). Experiment II: The results of the second insect test (II), including the newest larvae of Sexta, were performed according to Method 1 on some of the plants that were also used in Experiment I. The results are shown in Table 16. High mortality (75 to 100% mortality) was recorded in plants transformed with pGS1151, while control plants N21-102, 104 and 10
Almost all of the larvae fed 7 were still alive after 4 days. A complete list of all plants used in insect tests I and II is given in Table 17. Also, pGS1151
Shows the level of kanamycin resistance determined in plants transformed with S .; percentage mortality of larvae fed on these plants after several days: and Experiment I shows the average weight of larvae that survived after 7 days. Conclusion : Tobacco plants transformed with pGS1151 and selected for high kanamycin resistance clearly induce a severe toxic effect on larvae fed these plants. The effect on insect larvae observed here is due to B. aureus of bacterial origin.
t. Identical to that induced by the toxin (Section 5.2,
(See Tables 2 and 3); that is, growth is inhibited at an early stage (inhibition of transition from one age to the next), followed by death. Plants showing the highest level of Km tolerance (500 μg / ml Km) are also
Inducing the highest mortality is evident from Table 17. Therefore, effective expression of toxicity can be selected by selecting Km resistance using the fusion protein construction. As described therein, the use of the fusion protein not only allows the direct selection of the transformant of interest, but also has a unique advantage in that the fusion protein itself has inherently effective properties. That should be noted. For example, the Bt2: NPTII fusion protein is more stable in plant cells than the complete Bt2 protein, and messenger RNA derived from the fusion gene is more stable than Bt2 RNA.

14. New tables obtained by transformation
Stable genetic traits of the present type A substantial fraction of plants transformed with the transformation vectors described herein comprises a chimeric Bt toxin gene and a marker gene (nos, NP) stably inserted into its genome.
TII), including newly obtained DNA fragments. This was confirmed by the results of the Southern blotting experiment. The novel phenotypic traits obtained by this transformation method (Bt toxin expression, antibiotic resistance, nopaline production) are inherited according to classic Mendelian genetics. To identify stable genetic traits of new traits,
The F1 generation of the transformed plants was analyzed for Bt toxin expression and nopaline synthesis. The transformed tobacco plants were able to flower and produce seeds. Care was taken to avoid crossover. Previously identified as Bt ⁺ a 4 plants ^(161-9,10 -1, 147-8,17
From 4), seeds germinated on the agar medium, and F1 plants were analyzed for the presence of nopaline (the parent plant has nopaline synthase as a marker gene). Plants are 3 weeks after germination (approximately 1 cm in height) or 6 to 7 weeks (2
From 4 cm). The results are shown in Table 18. In plants 10-1 and 147-8, about 3/4 of F1 is
Expected from a single locus Mendelian trait (1: 2:
1) nos ⁺ Atsuta in. In the 161-9 F1 plants, nopaline signals were very weak when small plants were tested approximately 3 weeks after germination. Due to this weak expression, the nopaline signal was clearly not visible, and therefore the number of positives at this stage is underestimated. However, at 7 weeks, a positive positive signal was detected in 3/4 plants. The reason for the emergence of the plant at early age in plants is unknown. In F1 of plant 174, 4 out of 45 analyzed
The high percentage (95%) of nos ^{+ in} 3 indicates that the nos gene has been inserted into the genome at one or more loci. F1 plants were also analyzed for the expression of Bt2 toxin using an ELISA. The data of the ELISA measurement on the leaf tissue showed that the Bt2 ⁺ phenotype
os ⁺ . Therefore, Bt2
It is inherited by ⁺ characteristic stability.

The culture of the cells containing the intermediate cloning vector and the hybrid plasmid vector was carried out according to 8D-340, Glissbach, Göttingen, Germany.
0, Biotechnology Research Co., Ltd., has been deposited with the Microorganisms Collection, Germany and has been assigned the following deposit numbers:

[Table 1]

A culture of Escherichia coli K514 is commercially available. Changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims, and are understood to be within the scope of the invention.

[Table 2]

[0132]

[Table 3]

[0133]

[Table 4]

[0134]

[Table 5]

[0135]

[Table 6]

[0136]

[Table 7]

[0137]

[Table 8]

[0138]

[Table 9]

[0139]

[Table 10]

[0140]

[Table 11]

[0141]

[Table 12]

[0142]

[Table 13]

[0143]

[Table 14]

[0144]

[Table 15]

[0145]

[Table 16]

[0146]

[Table 17]

[0147]

[Table 18]

[0148]

[Table 19]

[0149]

[Table 20]

[0150]

[Table 21]

[Brief description of the drawings]

FIG. 1 shows SDS-polyacrylamide gel electrophoresis of a crystalline protein.

FIG. 2 shows the plasmid pEcoR251.

FIG. t. berliner 1715
3 shows a restriction map of a plasmid insert fragment.

FIG. 4 shows SDS-PAGE of the lysate of E. coli K514 (Bt200).

FIG. 5 shows the results of immunoblotting.

FIG. 6 shows the results of immunoblotting using different sera.

FIG. 7 shows SDS-PAGE of Bt2 protein.

FIG. 8 shows the results of ELISA.

FIG. 9 shows the results of ELISA with different antigenic proteins.

FIG. 10 shows the comparison of the N-terminal amino acid sequence of the 130 Kd protein.

FIG. 11 shows a method for immunologically detecting a polypeptide.

FIG. 12 shows a restriction map of a Bt2 gene fragment and a sequence region.

FIG. 13 shows the DNA sequence and amino acid sequence of the Bt2 gene.

FIG. 14 shows a comparison between the amino acid sequences of Bt2 and other Bt proteins.

FIG. 15 shows the construction of pHD160.

FIG. 16 shows another Bt2 gene cassette.

FIG. 17 shows the construction of plasmid pLB10.

FIG. 18 shows the construction and structure of p2BKm25.

FIG. 19 shows a BtiNPTII fusion and Bt2 deletion.

FIG. 20 shows a Bt2 3′-terminal deletion strain.

FIG. 21 shows the results of immunoblotting.

FIG. 22 shows the 3 ′ ends of pLB834 and pLB879.

FIG. 23 shows plasmids pLBKm23, 33
14 and 14 are shown.

FIG. 24 shows a Bt: NPTII cassette.

FIG. 25 shows the results of NPTII assays.

FIG. 26 shows the 3 ′ end of Bt of Bt: NPTII.

FIG. 27 shows an adaptation method of Bt2 and Bt2: NPTII.

FIG. 28 shows the DNA sequence of the junction between the promoter and cassette.

FIG. 29 shows the construction of pHD208.

FIG. 30 shows the construction of pGV831.

FIG. 31 shows the T region of pGV3850 and pHD205.

FIG. 32 shows the T region of pGV2260 and pHD208.

FIGS. 33A-33J show other intermediate expression vectors.

FIG. 34 shows the results of an ELISA of tobacco calli.

FIG. 35 shows the results of ELISA for tobacco callus.

FIG. 36 shows the results of ELISA for tobacco calli.

FIGS. 37A and 37B show a method for immunological detection of Bt2.

FIG. 38 is a section 38A view and a second 38B Figure M. sexta
The growth rate of the first instar larva is shown.

[39] FIG. 39 M. 1 shows a growth curve of sexta larvae.

FIG. 40 shows the DNA sequences of the P35S-1 and P35S-2 promoters.

FIG. 41 shows the construction of pGSH50.

FIG. 42 shows the construction of pGV1500.

FIG. 43 shows pGSH150 and pGSH151
The construction of

FIG. 44 shows the construction of pAGS007.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Marc Charles Ernesto van Montag Britssel, Belgium 120 de 72 Strastorto Strato 120 (72) Inventor Marc Albert Baetsk Zemst, Belgium 4 Aarskostosebern 4 (72) Inventor Marx Florent Oscar the Bow El Gent, Belgium Zent, El Persteg 70 (72) Inventor Jan Joseph August Lehmanns Heusden, Belgium, Elebohoten 38 (72) Inventor Hermanus Franciscus Pauls Hofte Gent, Belgium, Rosemariyunstraat 34 (58) Fields studied (Int. Cl. ^7, DB name) A01N 65/00 A01H 5/00 C12N 5/10 C12N 15/09 BIOSIS (DIALOG) WPI (D ALOG)

Claims (1)

(57) [Claims] A transformed plant, wherein the cells contain a chimeric gene, wherein the chimeric gene is stably integrated into the genome of the cell, is capable of being expressed in the cells of the plant, and The chimeric gene is a) a promoter region derived from a gene naturally expressed in plant cells; and b) Bacillus bacillus
(b) is a DNA fragment which can be obtained by cutting DNA encoding a crystal protein produced by T. thuringensis or DNA having substantial sequence homology therewith, wherein the fragment (b) encodes a polypeptide toxin of about 60 to about 80 KD. And providing the polypeptide toxin in an amount capable of controlling insects in the cells as a result of intracellular expression of the fragment (b), wherein the plant is capable of being transformed by Agrobacterium. The above plant, which is or is a dicotyledon. 2. The plant according to claim 1, wherein the DNA fragment (b) is adjacent to or fused to a DNA marker fragment (c) that can be selected or identified in the chimeric gene. 3. Said marker fragment (c) is fused to a said DNA fragment (b), resulting fragment (b) and (c) fusion Poripepu
3. The plant according to claim 2, which encodes a tide . 4. The plant according to any one of claims 1 to 3, wherein the DNA fragment (b) encodes a polypeptide toxin that is active at least against Lepidoptera. 5. The plant according to any one of claims 1 to 4, wherein the DNA fragment (b) can be obtained from a DNA encoding a Bt2 crystal protein or a protein having substantial sequence homology to the protein. 6. The DNA fragment (b) is Bacillus kurstaki (B
t. The plant according to any one of claims 1 to 4, which can be obtained by cleaving DNA encoding a crystal protein produced by K. kurosaki. 7. The Bacillus kurstaki is HD1 strain or HD73.
The plant according to claim 6, which is one of strains. 8. The DNA fragment (b) is Bacillus Berliner (B
t. The plant according to any one of claims 1 to 5, which can be obtained by cutting DNA encoding a crystal protein produced by B. berliner 1715. 9. The DNA fragment (b) is Bacillus Sotto (Bt.
The plant according to any one of claims 1 to 4, which can be obtained by cutting DNA encoding a crystal protein produced by S.toto. 10. The plant according to any one of claims 1 to 3, wherein the DNA fragment (b) encodes at least a polypeptide toxin active against Coleoptera. 11. The plant according to any one of claims 1 to 3, wherein the DNA fragment (b) can be obtained by cleaving a gene encoding a crystal protein of about 130 kilodaltons produced by Bacillus thuringiensis. . 12. The plant according to claim 11, wherein the DNA fragment (b) comprises the first 1820 nucleotides starting from the translation initiation codon of the Bt gene. 13. The plant according to any one of claims 1 to 12, wherein the DNA fragment (b) is a KpnI deletion fragment of a DNA encoding a Bt2 crystal protein. 14． The plant according to any one of claims 1 to 13, wherein the chimeric gene also contains a 3'-untranslated region derived from a gene naturally expressed in plant cells. 15. The plant according to claim 2, wherein the marker fragment (c) provides kanamycin resistance or encodes neomycin phosphotransferase. 16. The promoter region is a nopaline synthesis promoter region, a small subunit of a ribulose phosphate carboxylase promoter region, a Psu301 promoter region, a Ti-plasmid-derived promoter region,
The plant according to any one of claims 1 to 15, wherein the plant is selected from the group consisting of a TR promoter region, a promoter region derived from a cauliflower mosaic virus, a 35S-1 promoter region, and a 35S-2 promoter region. 17． A method for producing a plant capable of protecting itself against a specific insect, comprising stably incorporating the chimeric gene into the genome of the plant, wherein the chimeric gene is naturally expressed in a plant cell. B) DNA encoding a crystal protein produced by Bacillus thuringiensis or DNA obtainable by cleavage of DNA having substantial sequence homology thereto
A fragment, wherein the fragment (b) encodes a polypeptide toxin of about 60 to about 80 KD, and as a result of intracellular expression of the fragment (b), the polypeptide toxin is in an amount capable of controlling insects in the cell. The above method, wherein the plant is one that can be transformed by Agrobacterium or is a dicotyledonous plant. 18. The method according to claim 17, wherein the DNA fragment (b) is adjacent to or fused to a marker fragment (c) that can be selected or identified in the chimeric gene. 19. 19. The method according to claim 17, wherein the DNA fragment (b) encodes a polypeptide toxin active at least against Lepidoptera. 20. A DNA encoding the Bt2 crystal protein or a protein having substantial sequence homology to the Bt2 crystal protein.
20. Any one of claims 17 to 19, which can be obtained from
The method described in the section. 21. The DNA fragment (b) is Bt. Berliner 1715
21. It can be obtained by cutting DNA encoding a crystal protein produced by the method described in claim 17.
The method according to any one of claims 1 to 4. 22. A transformed plant, wherein the cells contain a chimeric gene, wherein the chimeric gene is stably integrated into the genome of the cell, is capable of being expressed in cells of the plant, and The gene encodes a) a promoter region derived from a gene which is naturally expressed in plant cells, and b) a part of a crystal protein produced by Bacillus thuringiensis, or a protein having substantial sequence homology thereto. A DNA fragment, wherein the fragment (b) encodes a polypeptide toxin of about 60 to about 80 KD, and as a result of intracellular expression of the fragment (b), the polypeptide is controlled in the cell by the polypeptide toxin; it is intended to provide an amount, or the plant is capable transformed by Agrobacterium or a dicot, said plant. 23. The plant according to claim 22, wherein the DNA fragment (b) encodes a part of a protein having the following amino acid sequence: 24. 23. The plant according to claim 22, wherein the DNA fragment (b) encodes a part of a polypeptide toxin active against beetles. 25. 23. The plant according to claim 22, wherein the DNA fragment (b) encodes a part of a polypeptide toxin active against Lepidoptera. 26. 23. The plant according to claim 22, wherein said DNA fragment (b) encodes a Bt2 crystal protein or a part of a protein having substantial sequence homology to said protein. 27. The above DNA fragment (b) is Bacillus cruschtaki (B
t Kurstaki) or Bacillus Sotto (Bt)
23. The plant according to claim 22, which encodes a part of a crystal protein produced by S. toto. 28. 23. The plant according to claim 22, wherein said DNA fragment (b) encodes a part of a crystal protein of about 130 KD produced by Bacillus thuringiensis. 29. The promoter region is a nopaline synthase promoter region, a small subunit of the ribulose phosphate carboxylase promoter region, Psu301.
The promoter region, a promoter region derived from a Ti-plasmid, a TR promoter region, a promoter region derived from a cauliflower mosaic virus, a 35S-1 promoter region, and a 35S-2 promoter region. plant. 30. A method for producing a plant capable of protecting itself against insects, comprising stably integrating the chimeric gene into the genome of the plant, wherein the chimeric gene can be naturally expressed in a) plant cells A) a promoter region derived from the gene; and b) a DNA fragment encoding a part of a crystal protein produced by Bacillus thuringiensis or a protein having substantial sequence homology thereto, wherein the fragment (b) The polypeptide toxin encodes a polypeptide toxin of up to about 80 KD, and as a result of intracellular expression of the fragment (b) thereof, provides the polypeptide toxin in an amount capable of controlling insects in the cell; or is capable transformed by um, or dicot, the method described above. 31. The plant according to claim 1 , wherein the plant is cotton, sugar beet, soybean, rapeseed, cabbage, lettuce or legumes. 32. The plant according to claim 1 , wherein the DNA fragment (b) is artificially created. 33. Plants are cotton, sugar beets, soy, rape, cabbage
18. The method according to claim 17, wherein the method is root, lettuce or legumes. 34. Plants are cotton, sugar beets, soy, rape, cabbage
23. The plant according to claim 22, which is vine, lettuce or legumes. 35. The DNA fragment (b) is artificially created
23. The plant according to claim 22, which is 36. Plants are cotton, sugar beets, soy, rape, cabbage
31. The method according to claim 30, wherein the method is root, lettuce or legumes. 37. A plant that is capable of being transformed by Agrobacterium or is a dicotyledonous plant and contains an insecticidal amount of a truncated Bacillus thuringiensis protein, wherein the truncated Bacillus thuringiensis protein is as follows: Amino acid sequence of Bacillus thuringiensis protein with 60KDa
The above plant, which comprises at least the amino acid sequence of the insecticidal fragment. 38. The cut Bacillus thuringiensis protein
White is from amino acid position 1 to 725 of the amino acid sequence
38. The plant of claim 37, comprising: 39. The cut Bacillus thuringiensis protein
White indicates the amino acid sequence of the selectable or identifiable marker protein.
39. The plant according to claim 37 or claim 38. 40. A method for protecting plant cells from Lepidopteran insects, comprising producing an insecticidal amount of a truncated Bacillus tubulingensis protein in a plant cell, wherein the truncated Bacillus tubulingensis protein comprises the following: Amino acid sequence of Bacillus thuringiensis protein with 60KDa
The above method, which comprises at least the amino acid sequence of the insecticidal fragment. 41. The cells are the cut Bacillus chuuri
Operatively linked to DNA encoding the gengensis protein
41. A DNA molecule comprising a promoter region.
The method described in. 42. A promoter from which the above promoter region can be induced
Region, tissue-specific promoter region, nopaline sinter
Ze promoter region, ribulose bisphosphate cal
Promoter region of boxylase small subunit gene
Region, the promoter region of the TR-DNA gene, or potassium.
In the flower mozaic virus 35S promoter region
42. The method of claim 41. 43. Including the insecticidal amount of the cleaved Bacillus thuringiensis protein, the cleaved Bacillus thuringiensis protein has the following amino acid sequence: Bacillus thuringiensis protein with 60KDa
The above plant cell, which comprises at least the amino acid sequence of the insecticidal fragment. 44. The cut Bacillus thuringiensis protein
White is from amino acid position 1 to 725 of the amino acid sequence
The plant cell according to claim 43, comprising: 45. The cut Bacillus thuringiensis protein
White indicates the amino acid sequence of the selectable or identifiable marker protein.
45. The plant cell according to claim 43 or claim 44 comprising: 46. The cells are the cut Bacillus chuuri
Operatively linked to DNA encoding the gengensis protein
44. A DNA molecule comprising a promoter region.
The cell according to 1. 47. A promoter from which the above promoter region can be induced
Area, organization-specific promoter region, nopaline Sinter
Ze promoter region, ribulose bisphosphate cal
Promoter region of boxylase small subunit gene
Region, the promoter region of the TR-DNA gene, or potassium.
In the flower mozaic virus 35S promoter region
47. The cell of claim 46.