JPH04501354A - Transformation of cucumber by Agrobacterium tumefaciens and regeneration of transformed cucumber plants - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Transformation and traits of cucumber by Agrobacterium tumefaciens Background of the invention of regeneration of converted cucumber plants Dicotyledonous plants are affected by two pathogenic species, the Gram-negative soil bacterium Agrobacterium chume. 77 sciences (Agrobacterium tumefaciens) and Agrobacterium rhizogenes (Agrobaeterium rhizog) enes) [Bevan and Gilton (Bevan and Chilton).

1982, Ann, Rev, Genet, 16 : 357-384], respectively known as crown gall and hairy root. I'm curious. In each of these diseases, the etiology lies in large areas known as T-DNA regions. Results of transferring and integrating segments of pTi or pRi DNA plasmids It is considered that Chilton et al., l 9 77, Cell, l1:263-271; Chilton et al. lton et al), l 982, Nature + - (Nature).

295: 432-434°pTi and pR4T-DNA transfer mechanism and Some of the genes contained within these T-DNA regions that are responsible for the disease Identification of the normal process led to the design of avirulent Agrobacterium strains. [ Hepburn et al.

1985, Journal of General Microbiology. J, Gen Eral Microbiology), 131: 2961-2969 ;7-Hood et al., 1986, Journal of Ba Bacteriology (J, Bacteriology), l 68: 1291~ l　301　; Valiane and Cassedelbert (Valiane and Cassedelbert) Ca5se-Delbart), l 987.

Molecular and General Genetics (Mo1.Gen.

Genet, ), 206: l 7-23], this information is also smaller, replicable in both E. coli and Agrobacterium strains. , led to the construction of a broad host range plasmid. The Kagaru plasmid is the designed T- Agrobacterium-derived pTi or pRiDNA required for DNA region transfer Contains a signal. These plasmids are called binary plasmids. Bevan, Nucl, Acids Research (Nucl, Ac1d) s, Res, ), 12: 8711-8721; An et a l), l　985].

Many species belonging to the Cucurbitaceae family are susceptible to infection by these Agrobacterium pathogens. It is known that [Anderson and Moore oore), 1979, 7 Phytopathology (Phytopath).

69:320-323; Smarrelli et al. l　986　.

Plant Physiol, 82: 622-624]. Additionally, methods to reproduce many of these species are already established. [Malepszy and Nadolskáorzik] Nadolska-Orczyk), 1983 Diol (Z, Pflanzenphysiol,), l　l　l　: 273~ 276, Nadolska-O rczyk and Malepszy).

1984, Bulletin Po1.Aca d.

Sci, ), 32: 423-428; Jelaska, 1 986.

Biotechnology in Agriculture Luture & Foresteri ((Bi otechnology in agriculture and fores try) (Y, P, S, Bajai) 2: 371-386)].

When these two factors are combined, genetic information can be aggregated into the genome of Cucurbitaceae species. It is suggested that Robacterium-mediated transfer is easily accomplished. The secret of genetic material Development of systems for introduction and stable incorporation into family plant species is possible through normal plant breeding techniques. may be useful for the transfer of genetic traits that are difficult or impossible to transfer. Cucurbitaceae plant species Development of gene transfer technology: resistance to viral infection [Powellou-Habre et al. owell-Able et al), l 986, Science ce), 232 Near 38-743; Cuozzo et al. , l 988, Bio/1ech, 5: 549-55 '7], Herbicide Tolerance [Comai et al., 1985, Nature, 317: 741-744; Derashiova et al. (della-Cioppa et al), l 9870.

Bio/1ech, 5:579-584; Le et al. e etal), 1988. EMBO, J, 7:1241-1248; Stalker et al., 985, Journal of Biological Chemistry (J, Biol, Chem,), 260 : 4214-4728) or as soon as they become available. It may be useful to introduce engineered genes that transfer other useful properties.

Disclosure of information Transformation and regeneration of transformed cucumber plants expressing the NPTn gene are Trulson et al. (1986, Applied Digital (Appl, Genet, ), 73: 11-15). It has been tell. This transformation was carried out using Agrobacterium rhizogenes. 03-NPT T-DNA of binary plasmid pARC8 containing n gene The area was achieved by infecting cucumber hypocotyl sections. Obtained The transformed tissue produced roots, which were regenerated via somatic embryogenesis.

Nis Marepji Buddha Kay 21 Milobiissuzut, and Jay Beetzni force (S, Malepzy, K, Niemirowicz-Szczyt t, and J.

Wiezniewska), (1982), cucumbers in vitro (Cucumis 5ativus L,) Thin body Cucumbersomatic embryogenesis in vitro) J, Acta Biology (Acta) After identifying the coat protein gene by Reaction (polymerase chain reaction (PCR)) Any coat protein gene is modified by modifying its sequence using techniques to add specific restriction enzyme sites. It can also be concatenated to children. Using these restriction enzyme sites, various plant genomes can be Plants that contain the necessary genetic regulatory elements needed to control genes after introduction into the system The coat protein gene can be cloned into an expression vector.

Plant expression vector p18Uccp for cloning of various coat protein genes A schematic diagram describing the use of exp (described in Appendix B) is shown in Chart 2. aggro Plant coat proteins can be expressed in binary vectors to facilitate bacterium-mediated gene transfer. to facilitate microprojectile-mediated gene transfer. marker genes such as the β-glucuronidase gene (Jefferson (Jefferson et al., 1987) into the plasmid. (See Chart 2).

Plants resistant to virus diseases and their production methods are available in the "Cucumber Mosaic Virus" Cucumber Mo5aic Virus CoatP European patent ratio of the invention name rotein Gene) J! [No. 223452, and U.S. Patent No. 1 [07/234404, filed Aug. 19, 1988; and “Botivirus coat protein genes and plants transformed with them (P otyvirus Coat Protein Genes and Plan 198 of the name of the invention named tsTransformed Therewith) J The date June 19, 2009 is set forth in U.S. patent application Ser. No. 07/368,710. .

The use of viral coat protein genes to achieve resistance in transgenic plants is Powell-Abel et al. (1986).

“Viral coat protein genes are derived from plant virus classes (tobacco, tsukumo, poti, and dwarf). CaMV promoter and downstream 3' plant Constitutively expressed in plants after ligation of the rearenylation signal.”

The construction of a plant-expressible coat protein gene is described in U.S. Patent Application No. 07/07, attached as Appendix A. No. 234,404, Examples 3 and 4, and U.S. patent publications attached as Appendix B. Examples 8 to 11 of Application No. 07/368,710.

Example 6 Transfer of herbicide resistance gene This example describes a plant-expressable gene that renders a plant resistant to a particular class of herbicides. Explain how to create. Such plants are destroyed by usually lethal herbicides for several reasons: (1) (2) of previously used herbicides that are normally lethal to crops in the second category; Using different crops in close rotations in soils that may contain residues Can be done. The two genes of interest are chlorsulfuron (chlorsulfuron). ron) OJ: U sulfometuron mesil (sulfometuron m) ethyll) herbicide (7 Falco et al., 1985) of the acetolactate synthase (ALS) gene (plants or are susceptible to the mutant derivatives (derived from bacterial sources) and the herbicide gly7-osate. Enolbirubilkimate-3-phosphate synthase (EPSFS) Coded by Stalker et al., 1985) It is a mutant of the gene that causes

Important enzymes that are either resistant to or detoxify certain herbicides The gene encoding the element was isolated from CaMV35S (Pietrzuk et al. ak et al), 1986), ribulose-1,5-diphosphate carboxy Mazurase small subunit genes (Mazur and Chuwi) and Chui), 1985)) or other strong plant gene promoters. downstream from plant promoters such as (See Chart 3).

This gene is then transferred into an Agrobacterium-derived vector (binary or cysotic). This genetic material is transformed into a genome using the plant transformation method described above. (Chart 3). Additionally, this gene can be expressed in plants. cloned into a vector containing a possible marker gene (Chart 3) and transferred to cucumber plants using the Loprogenoteal-mediated gene transfer method (Example 3). can be entered.

Example 7 Insects - Transfer of resistance genes In nature, there are a number of polypeptides that are toxic to insects and nematodes. Moved to. Under diffused cool white fluorescent light (4K]X) with 16 hours of illumination time, 26° These cultures were incubated for an additional 2 weeks at C. Then grow the tissue Transferred to MS medium without regulators.

Once the plantlets develop extensive root threads on the latter medium, they can be placed under specialized cultivation. Transferred to explant mix and covered with Ziploc storage bag for curing. Then it is played The plants were potted in soil and grown in a greenhouse.

If using explants from cotyledons and hypocotyls, add 2,4-D to the induction medium. Concentrations range from 1-Omglo to 2', Omglo.

The concentration of kinetin added is approximately 0.5 mg per explant from cotyledons and hypocotyls. /(2.

Tissue transformation is described by Horsch et al., Science (S. (1985), 227:1229-1231. The method was followed. 3 day old cucumber seedlings (Tsukumis sativus e. Cucumis 5ativus L.

Cotyledons from cv, Polnsett) 76)) were cut into small pieces. Applicable A small piece of avirulent Agrobacterium strain C58Z707 (Hepburn et al. Hepburn et al., 1985) or virulent strain 058 (D. Depicker et al., 1980) liquid Soaked overnight in culture. Both Agrobacterium strains use the binary plasmid pGA4. 81 (An et al., 1985; An).

Shake gently to ensure all edges are infected. After that, the pieces were blotted dry and prepared for regeneration.

Example 3 Use of microprojectiles for transformation Described in specific examples of the present invention Cucumber somatic embryos induced using the cultured tissue source were treated with 2-0 mg/4 2.4-D and on MS medium containing 0.5 mg/12 kinetin. to the manufacturer Therefore, as described, all plasmids containing useful genes encoding microprojectiles coated with DNA bombard these tissues with la). After shock, these tissues contain fresh medium. Transferred to a flat plate. The DNA construct used contains the NPT n gene that can be expressed in plants. If so, add the selected drug kanamycin lOO~200mg/mQ to a fresh plate. did. A cucumber tissue bombarded with microprojectail is used as a specific example of the present invention. Play it using the method described above.

In addition, microprojectile transformation does not impact somatic embryogenic callus. It should be recognized that this can also be achieved by

Example 4 Regeneration of transformed embryoid bodies Regeneration of potentially transformed cucumber callus tissues was performed as follows. : Cotyledonous pieces from 3-6 day old germinated seedlings were treated with 2.4-D and 0 m glo. and cultured on MS basal medium supplemented with 0.5 mg/Q kinetin. 4 days later, Agrobacterium-infected cucumber cotyledon pieces were treated with 500 mg/mθ carbinicillin ( carbinicil in) and 100 to 200 mg/m (2 Kanamai The cells were transferred to the same medium supplemented with syn. The cells were further cultured for 6 weeks at 26°C in the dark.

After 4 weeks of incubation, a characteristic gel-like callus forms on the surface of the explant and Formed at the site of contact with the medium. After 6 weeks, cotyledon pieces were treated with 1 mg/Q NAA and Transferred to MS basal medium supplemented with 0.5 mg/4 kinetin. Within two weeks, the embryo A similar body began to appear on the surface of the gel-like callus. Plantlets have 50 to 1 embryoid bodies. 00 mg 10 kanamycin and 500 mg/Q carbinicillin. It was obtained by transferring to MS basal medium containing.

Calcium obtained from medium supplemented with kanamycin (100-200 mg'/12) All of the tissues were transformed.

The purpose of this example was to demonstrate that, when expressed in plants, Constructs for expression of plant viral coat protein genes that reduce symptoms or confer resistance It is about rebirth. In general, Nucle Biologica), 10: 218 ~220゜BiC and Di Tricolin (P, Chee and D, T ricoli) (1988) rCucumis sativas L. Somatic embryo formation and plant regeneration ( Somaticembryogenesis and plant regen eration from cell 5uspension of Cucum is 5ativus L, ) J, Plant Cell Reports (Plant C e1lReports), 7: 274-277゜Deaf As used herein, the terms have the following meanings = "Plantlets" Fully grown plants with shoots and roots that are regenerated asexually by cell culture means.

An “explant” is a hypertrophic (of somatic or germline) cell, whether wild type or cultured. Breeding, or genetic mutants, is the cultivation of transformants or second plants. or any part of the plant left.

"Hormone" affects the growth or differentiation of plants and is used in reference to various media. means an exogenous plant growth regulator such as

"Call" and its plural "calliforms" means a plant that has been cut, bruised or injured. refers to an unorganized population of cells that forms in response to and on explant tissue during culture. Refers to the growth of uncombined cells that can form.

"Embryoid body" means a structure similar in appearance to a plant zygote.

"Medium" means a culture medium known to those skilled in the art for culturing plant tissues.

"Kinetin" is a known hormone.

2.4-D-2,4-dichlorophenoxyacetic acidNAA-α-naphthaleneacetic acid MS medium Murashige & Skoog medium .

The present invention consists of MS medium and exogenous hormones 2.4-D and kinetin. Incubate the explants in induction medium for 4-5 weeks, sufficient to induce competent embryoid bodies. Tsucumis sativa obtained from cotyledons or hypocotyls characterized by culturing for Provided is a method for producing T. sativus embryoid bodies from S. sativus explants.

Further, the embryoid body produced by the method of claim 18 is transformed into a complete form. Transgenization of Tsukumis sativus characterized by obtaining transformed plants The present invention provides a method for producing a plant.

Furthermore, Tsukumis sativus plant tissue, induction medium, 2.4-0 and kine A composition comprising chin is provided.

Specific example of the invention Generally, the process of the present invention includes the steps shown schematically in Chart 1.

Explant source and preparation Cucumber (Tsukumis sativus L. is 5at-ivus L cv, Po1nsett) 76, Asgro Cormorant seed e campa = - (Asgrow 5eed Co, )) seeds in regular water Soaked for approximately 15 minutes. The outer skin of the seeds was removed by hand. 70 seeds with the outer skin removed % ethyl alcohol for 1 minute. Next, use commercially available bleach (hypochlorous acid). 25% (v/v) solution of sodium) for 25 minutes. The seeds are then destroyed. The cells were rinsed four times with distilled water. Sterilized seeds were soaked in 0.8% water agar (Diffusion) in a dark place. Developed on Difco Laboratories at 28°C. It sprouted. Unless otherwise noted, all media were supplemented with 3% sucrose and 0.5% sucrose. Solidified with 8% vegetable agar (cibco). Turn on the pH of all media. It was adjusted to 5.8 before the autoclave treatment.

All media were autoclaved at 121°C for 20 minutes.

Example 1 Induction of embryos and regeneration of plants 3-5 day old in vitro grown seedlings was used as the tissue source. Tissue sections (5X5 mm size) from cotyledon tissue sources and Hypocotyl segments (0.6 to 1 mm long) were used as explants. 2.4-D (1,0,2,0mg/7+) decainetin (0,5,1,0,2,0mg/a Approximately 100 explants were cultured for 4 weeks at 26°C in the dark on induction medium supplemented with did. After 4 weeks, the frequency and type of histology developed was evaluated. Next, culture Add MS medium + 1. omg/Q NAA+0.5mg is present. best known ibis Bacillus thrin is a different strain of Bacillus thrin. For example, Bacillus israelenis (B. giensis); , 1sraelenis) is active against Diptera (mosquitoes, blackflies), and B. Thuringinensis is active against Lepidoptera and B, san diego is Corioptera (Co). lioptera). Toxic proteins found in each of these bacteria Whites are highly specific for insect damage; they are non-toxic to other organisms. be. Thus, the transfer and transfer of genes encoding such toxic proteins in plants is possible. This technology reduces insect damage without the use of chemical insecticides and thereby protects other organisms. It is advantageous to avoid danger to the body. encodes many of these toxic proteins The genes responsible for this have been isolated and sequenced (Schmeph et al. et al), 1985; Waalwijketal, 1985; Sekar et al., 1987), Bi Chu transfer of the toxin gene into tobacco and its ability to protect plants from insect damage. The usefulness was reported by Vaeck et al. (1987). ing. Thus, as described herein; regeneration and transformation systems and plants. The combination of the use of expressible Bacillus toxin genes (Chart 4) Genetic characteristics can be transferred to cucumbers (see Example 2 for gene transfer method). and 3).

Example 8 Transfer of antimicrobial genes This example shows that plants can be made resistant to infection by bacteria and fungi. Explain how to create genes. Several classes of polypeptides have been isolated in nature. have been found to convey broad-spectrum antimicrobial activity; e.g. zBgainin (Zasloff, 1987) , defensinX Daher et al. , 1988), lysozyme (Bomanet et al., 1985), Cercorbin (CercorpinX Bomanet al.), 19 89), engine (attacin) (Haltmarket et al. al), l 983), thionin mannet al), l 988), etc. These antimicrobial peptides or More active modified forms have been synthesized and designed for expression in plants.

The promoters for expression of these antimicrobial polypeptide genes have tissue specificity. may include other constitutive types. Tissue-specific promoter, or wound ( woand) inducible promoter (Sanchez-Serrano et al. 5errano et al), l 987), if it is required. and only in tissues vulnerable to attack by bacteria or fungi or reprints. It would be useful for the purpose of producing antimicrobial peptides. Thus, antimicrobial polypep The design of the gene encoding Tide was carried out by the transformation and When combined with regeneration procedures and regeneration procedures, it is possible to transfer useful traits to Sukhwarash plants. shall be. Chart 5 shows Agrobacterium-mediated and microprojectile We summarize the construction of plasmids that can be used in gene transfer systems.

chart 1 Germinate the seeds to obtain seedlings.

Cut out cotyledons from 3-day-old plants.

Agrobacterium immersion or DNA-coated microprojections Inoculate the plant tissue by bombarding with a tube.

2. Transfer the tissue to MS medium supplemented with 4-D and kinetin and culture for 4 days. Ru.

2.4-D, fresh supplemented with kinetin, carbenicillin and kanamycin Transfer to culture medium.

Culture in the dark for 6 weeks (callus can be observed growing on the sides).

Transfer to fresh medium supplemented with NAA and kinetin.

The embryoid bodies are transferred and placed in a regeneration medium to produce plants.

chart 2 coat protein gene vector −−−・−・−−−・・〉Plant poly(A) signal chart 3 Herbicide resistance gene vector Σ−u’ BR plant mutant plant 4.7°a motor ALS or EPSPS gene poly or other herbicide tolerance or detoxification gene chart 4 insect resistance gene vector chart 5 Antibacterial gene vector Attachment A Examples 3 and 4 of U.S. Patent Application No. 07/234,404 Example 3 CMV-WL Construction of pUA19 clone containing coat protein gene Using standard methods, the entire EcoR engineering fragment from lambda clone WL3Z8 was Bethesda Lisa, P.O. Box 6°09, Gaithersburg, Fragment Gison 20877 (available from Betbesda Research) plasmid vector The clone pWL3Z8.1 was obtained by transfecting into tar pT, Jc19. Next, Maki Sam and Gilbert (Methods in Enzymology) In Enzymology) 65: 499, l 980). Ec in pUc19 by the technique described.

The RI cloned fragment was sequenced. Based on this information, the CMV-WL coat protein The complete sequence of the gene was determined and is shown in Chart 1. CMV-Q RNA3 (Davis and Symons, 1988 , Pyrology 164: in press). As judged by comparison, further sequencing contains all but 5'193 bp of CMV-WL RNA 3 molecules. Indicated. The nucleotide and amino acid sequences of CMV-WL and CMV-C are: differ by 22.7% (Chart 2A) and 16% (Chart 3B), respectively.

Example 4 CaMV: CMV expressible in plants with 35S polyadenylation signal -Construction of micro T-DNA plasmid containing WL coat protein gene To link the CaMV35D promoter and polyadenylation signal, From the ApaI site (located within the intragenic region of RNA3) (cloning experiment The fragment extending to the EcoRI site (ligated into the lambda clone WL3Z) Taken out from 8, (Thomas Hahn), 7 lead Friedrich Miesch Institute er In5titut), Switzerland, Basel,' CH- 4002, P.O. Box 2543) vector pDH51 (vinitrzak (Pietrzak et al., 1986) multiple cloning site. I did. This includes complete digestion with Apal and partial digestion of WL3Z8 with EcoRI. By selective digestion, a suitable ApaI or EcoRI 1090 bp fragment (matrix) was extracted. generate a blunt-ended molecule (using DNA nuclease) and subsequently convert it to pD This was achieved by ligating to the Sma1 site of H51 (see Chart 4A).

This clone, named pDH51/CPWL, was subjected to Maxam-Gilber 1 technology. It was therefore sequenced to confirm its stability for expression in plants.

The plant-expressible coat protein gene is then amenable to Agrobacterium-mediated gene transfer. The vector was moved. Following partial digestion with EcoRI, approximately 1.9 kb of Ec The oRI or EcoRI fragment was removed from pDH51/CPWL (Robert ・Robert Kay, Department of Chemistry, Washington State University, Pullman, WA The EcoR1 site of plasmid pUc1813 (available from Co., Ltd.) was inserted into the EcoR1 site of plasmid pUc1813. The lasmid pUC1813/CPWL was obtained. Conoplant expressible CMV-WL envelope A 1.9 kb fragment containing the protein gene was removed by partial HndlI digestion. (Gynehung An), Institute of Institute of Biological Chemistry vector (available from Cal Chemistry), Washington State University ligated into the HindlI site of pGA482 (An, 1986). W.O. Plasmid pGA482 was pre-modified as described in No. 29105858. The modified plasmid is decorated with a plant-expressible β-glucuronidase gene. was called pGA482/G. After cloning the expressible cassette, the plus Mido was named pGA482/CPWL/G (see Chart 5A) Chart 2 A Comparison chart 3A of CMV-WL and CMV-C coat protein genes Comparison of CMV-WL and CMV-C coat protein genes Appendix B Examples 8.9.10, 11 and 15 of U.S. patent application Ser. No. 07/368,710 Example 8 Plant development for expression of various genes in transgenic plants Construction of current cassette In a preferred embodiment of the invention, to achieve high level expression of the insert, , gene insert (promoter, 5' untranslated region, transcription initiation codon, and provide the necessary plant regulatory signals (including the addition of polyadenylation and polyadenylation signals) The following expression cassette was constructed. The expression cassette contains any inserted genes. It can also be used to express children. Therefore, the applicability of the expression cassette is limited to coat proteins. Beyond its use in expressing genes. Rather, the expression cassette It can be used to express any desired protein in genetic plants. Applicable Expression cassettes are preferred expression methods for expressing viral coat protein genes in plants. It is a system.

Preferred embodiments of expression cassettes include: constitutive promoters; promote gene expression; 5° untranslated region: start codon consisting of Kozak element; a cloning site into which the desired gene can be inserted to create a functional expression unit; and poly(A) addition signals and untranslated flanking regions that result in high-level expression. It contains a 3' untranslated region consisting of a 3' untranslated region.

In particular, the expression cassette which is a preferred embodiment of the invention is a cauliflower mosaic. Virus (Ca MV) 35 S transcription promoter, cucumber mosaic virus (CMV) 5'-untranslated region, CMV transcription start codon, and CaMV polya It consists of a denylation signal. Correct location of plant regulatory signals as well as tegument proteins Allows easy addition of genes and excision of the completed cassette, thus allowing other In order to achieve the addition of convenient restriction enzyme sites that can be transferred into the plasmid of Construction of the expression cassette utilized polymerase chain reaction (PCR) technology.

To accomplish the construction of this expression cassette, the following oligomers were synthesized: 1, 5’-GAAGCTTCCGGAAACCTCCTCGGATTCC -3' contains a HindI[[ site at its 5' end, and the CaMV 5'-fra It contains 21 bases that are identical to the 21 bases in the linking region.

2, 5’-GCCATGGTTGACTCGACTCAATTCTACC , AC-3' contains a translation initiation codon that conforms to the Kozak rule. It contains an NcoI site at its 5′ end and is an antisense CMV 5′-untranslated region. It has 21 bases that are identical to the 21 bases in the strand.

3, 5’-GCCATGGTTGCGCTGAAATCACCAGTCT C-3' is at its 5' end (containing the same translation initiation codon as oligomer 2) Contains an Nco1 site and is identical to the 20 bases in the 3″-untranslated region of CaMV. It has 20 bases.

4, 5’ -GAAGCTTGGTACCACTGGATTTTGGTT- 3' contains a Hindll[ site (on antisense steel) at its 3' end; 20 pairs with the 7-ranking DNA region 3′ of the CaMV polyadenylation site. It has a base.

Using these oligomers, pUC181 shown in Chart 6B and mentioned above A two-containing sequence was amplified within the CMV expression clone designated 3/CP19. Char As shown in Section 8, pUCl 813/CPl 9 was injected using PCR technology. We amplified the regulatory region of the gene.

5″-flanking, CMV5′-untranslated region, and (Kozak rule AAXXAT Amplification of the CMV initiation codon (modified to accommodate GG) results in a length of approximately 400 A base-paired fragment was obtained and the result of amplification of CaMV3-untranslated and flanking regions. As a result, a fragment approximately 200 base pairs in length was obtained. These fragments were digested with NcoI and Hi Digested with ndn[, isolated from polyacrylamide gel, then Hind I[[ligated into digested and phosphatase-treated pUc1'8. The resulting claw This is called p18CaMV/CMV-exp and is shown in Chart 8B.

@Identification of WMVII coat protein gene Chemical methods (Maxam and Gilbert), Methods of Enzymolo gy) 65:499.1980) and enzymatic methods (Sanger et al. *Proceedings of the National Academy of Sciences ・Sciences (Proc, Natl, Acad, Sci,) USA74: 　5463. 1977) and the clone p generated as described above using both Cloned WMVI from WMVn-41-3,2 [cDNA insert sequenced] Decided. Based on this information and comparative analysis with other potyviruses, clone The nucleotide sequence of pWMVI[-41-3,2 is the complete WMv coat protein gene. It was found to contain all copies. The N-terminus of the coat protein has a dipeptide sequence G suggested by the position of 1n-5et. WMVI [Integument protein gene coding The length of the coding region (281 amino acids) is approximately 33kD protein-encoding gene. No contradiction. This WMVI [coat protein gene and protein sequence is shown in Chart 2B. show. In addition, Eggenberger et al. A related virus described as Soybean Mosaic Virus (SMV) obtained from strain N Comparing this sequence with that of - Approximately 92.5% identity excluding differences in terminal length. See chart B. these Since the two viral coat protein genes have a wide range of amino acid identity, WM Expression of coat protein genes from Vn is predicted to result in plants resistant to WMVII infection. will result in plants resistant to SMV infection.

Example 10 CaMV 35S promoter and polyadenylation signal and a plant-expressible W with CMV gene trochanteric region and translation initiator ATG. Construction of the MVn coat protein gene cassette As shown in Chart 9B, the necessary plant regulation Binding of the signal to the WMVI [coat protein gene] requires the necessary site to be and oligomers added to the 3′ sequence of the WMVI [coat protein gene]. This was achieved by using a wide variety of PCR techniques. Following this amplification, the resulting fragment The fragment was digested with an appropriate restriction enzyme and the plasmid pl 8CaMV/CMV-exp was cloned into the Nco1 site of the expression cassette containing. The WMVn skin Clones containing protein gene inserts are correct for the CaMV promoter. All you have to do is check whether it is in the right direction or not. However, during PCR amplification To confirm that no processed material was incorporated into the Checked by leotide analysis.

Amplified WMVI with Ncol restriction enzyme sites at both ends [to obtain the coat protein gene] In order to do this, the following two types of oligomers were synthesized.

1, 5’-ACCATGGTGTCTTTTACAATCAGGAAAAG -3' This adds an Nco1 site to the 5' end of the WMVI [coat protein gene and generates The same-ATG translation start present in the current cassette pl 8CaMV/CMV-eXp Preserve codons.

2, 5’-ACCATGGCGACCCGAAATGCTAAC7GTG -3' This adds an Nco1 site to the 3'' end of the WMVI [coat protein gene, This site can be linked to the expression cassette p18caMV/CMV-eXp.

This PCR using these two types of oligomers p18 of the WMVn coat protein gene By cloning into CaMV/CMV-exp, (p18CaMV/CMV A plant-expressible WMVII gene (referred to as -eXp) is obtained and after transcription and translation , WMVI [identical to WMVI [derived from the coat protein gene nucleotide sequence] Outer coat proteins are produced.

See chart B. However, this coat protein adds an ATG start codon. and for the necessary genetic design for (GIu-3er protease The last four amino acids of the 54kD nuclear inclusion protein (adjacent to the cleavage site) The amino acids added are Va1-5e r-Leu-Glu- The N-terminus is WMVI. The addition of these four amino acid residues White should not affect the ability to produce plants resistant to WMV ■ infection. Why? The N-terminal region of the potyvirus coat protein has either length or amino acid identity. This is because it does not seem to have been well preserved.

However, if this proves to be a problem, the replacement is WMVI [Including the use of different oligomers to obtain N-terminal changes in the coat protein gene] Dew. The cloned construct of the plant-expressible WMVn coat protein gene is p18WMV. It is called I[-exp and is shown in Chart 9B.

Example 11 Plant expressible WMVI [MicroT containing coat protein gene construct -As shown in the DNA plasmid construction chart 1OB, WMVI [Coat protein gene] Plant expression cassettes for genes (mediated transfer from Agrobacterium to plant genome) Agrobacterium T-DNA import signals and and applications containing broad host range origins of replication (for replication in Agrobacterium). Plasmid pGA482/G/CPWMVI was transferred into the appropriate micro-T-DNA. [-exp obtained. To construct this plasmid, plasmid pl 8W MVI [-exp (well external to the expression cassette, polyclonin of pUc18) Digest with HindI[I, which cuts within the plant-expressible cassette. Agrobacterium-derived microvector obtained by extracting and modifying a 1.8 kb fragment Hin of pGA482 (modifications include addition of β-glucuronidase gene) dn [tied to site; The micro T-DNA vector pGA482 was transferred to Chart 7B. This is shown in G., An, Pullman, Washington. lman), Washington State University, Institute of Biologica Institute of Biological Che available from mistry). The obtained plasmid was transformed into pGA482/G/CP Named WMVI[-exp and shown in chart IOB. This plasmid (or its derivatives) A208, C58, LBA4404, C58Z207, A4R5 Agrobacterium chume7a such as 5A4RS (pRiB287b) etc. virulent or avirulent strains of S. ciennes or Rhizogenes. Stock A 208, C58, LBA4404, and A4R5 are American type cal Char Collexicon (ATCC), Berkeley, Rockville, Maryland Available from Parklawndrive 12301. bacteria A4R3 (pRiB278b) was derived from F. Cassedervaal (F, Ca5se- Dr. Delbart, France, Versailles, L'Tideux Safe Cyr Routede 5aint Cyr,).

Available from C,N,R,A. Bacteria C58Z207 is A.C. Hepvar 7 Dr. A.G. Hepburn, Urbana, Illinois ), available from Illinois State University, College of Agriculture.

The above listed agrochemicals of the designed plasmid pGA482/G/CPWMII-exp After transfer to any of the Agrobacterium strains, using these Agrobacterium strains and the plant-expressible WMVI [coat protein gene] contained within its T-DNA region. can be transferred and integrated into the plant genome. This transfer is known to those skilled in the art. This transfer can be accomplished using standard methods for DNA transfer or Agrobacterium-mediated transformation of plant seeds (AgrobacteriumMe Diated Transformation of Germinating Plant 5eeds) J filed on December 21, 1987 This can be achieved by the method described in the patent application and No. 07/135655. addition Recently, such Agrobacterium has transferred its T-DNA region to the genome of soybean plants. It turns out that it can be imported and incorporated into. Thus, these strains Possible WMVI [Soybean mosaic virus by transferring the hull protein gene into the soybean genome] It can be used to develop soybean plant lines that are resistant to infection from this strain.

(Examples 12 to 14 are omitted) Example 15 Micro T-containing plant-expressible ZYMV coat protein gene construct Construction of a DNA plasmid According to the teachings of Example 11 with appropriate modifications, plant expression is possible. A micro T-DNA plasmid construct containing the Z YMV coat protein was constructed. . Plasmid pUc18cpZYMV (Chart 12B) (10 of the expression cassettes) with HindIII (which cuts within the multiple cloning site of pUc18 outside the division). After digestion, the 1,6kb fragment containing the plant-expressible cassette was removed and mixed. It was ligated into the HindI[I site of Clovector pGA482 (Chart 7B).

The obtained plasmid was named pGA482GG/cpZYMV and chart 13 Shown in B.

Designed Boo y Sumido p GA 482 CG/cp Z YMV (7) 7 After transfer to the Agrobacterium strain, the T-D Introducing the plant-expressible ZYMV coat protein gene contained within the NA region into the plant genome. Can be imported and incorporated.

Chart 12B Pπ beat -15 ★☆ 1 Recommended coat protein 1 gene BaMV coat protein gene NcoI NcoI pU(:18cpZYMV HlndIII NcoI NcoI HlndIII BamHI Swam l Pea l IClzyMv coat protein 115C11 gene Chart 13B HlndIII NcoX NcoX HlndIII BrI I No- I Pc, lqcI ZYMV envelope II Sc, II C ode 11BLI protein White gene Gus gene Submission of translation of written amendment (Article 184-8 of the Patent Act) April 2, 1991

Claims (20)

[Claims] 1. Induction consisting of MS medium and exogenous hormones 2,4-D and kinetin Culture the explants in culture for a period of approximately 4-6 weeks to induce competent embryoid bodies. Cucumis obtained from cotyledon or hypocotyl tissue characterized by A method for producing Cucumis seed embryoid bodies from seed explants. 2. Claims that the embryoid body is transgenic The method described in paragraph 1. 3. The transformed embryoid body is transformed into (a) Agrobacterium virulent or avirure. foreign DNA is introduced into the explant using the patient strain as a vector, and then (b) The inoculated tissue was placed on MS medium and induction medium consisting of 2,4-D and kinetin. The method according to claim 2, which is produced by culturing in. 4. The transformed embryoid bodies were prepared by (a) explants in MS medium, 2,4-D and kinetics; (b) culture on an induction medium consisting of micropropylene to form embryoid bodies; By introducing foreign DNA into the embryoid body by projectile bombardment The method according to claim 2 for producing. 5. 4. The method of claim 3, wherein the explant is obtained from cotyledons. 6. The concentration of 2,4-D in the medium is about 1.0 mg/l to 2.0 mg/l. The method according to claim 5, wherein the concentration of kinetin is about 0.5 mg/l. . 7. The concentration of 2,4-D is 2.0 mg/l and the concentration of kinetin is 0.5 m 7. The method according to claim 6, wherein the g/l. 8. 5. The method of claim 4, wherein the explant is obtained from cotyledons. 9. The concentration of 2,4-D in the medium is about 1.0 mg/l to 2.0 mg/l. Therefore, the method according to claim 8, wherein the concentration of kinetin is about 0.5 mg/l. . 10. The concentration of 2,4-D is 2.0 mg/l, and the concentration is 0.5 mg/l. 11. The method of claim 10. 11. 4. The method of claim 3, wherein the explant is obtained from a hypocotyl. 12. The concentration of 2,4-D in the medium is approximately 1.0 mg/l to 2.0 mg/l. Claim 11, wherein the concentration of kinetin is about 0.5 mg/l. Method. 13. The concentration of 2,4-D is 2.0 mg/l and the concentration of kinetin is about 0.0 mg/l. 13. The method according to claim 12, wherein the concentration is 5 mg/l. 14. 5. The method of claim 4, wherein the explant is obtained from a hypocotyl. 15. The concentration of 2,4-D in the medium is about 1.0 mg/l to 2.0 mg/l. Claim 14, wherein the concentration of kinetin is about 0.5 mg/l. Method. 16. The concentration of 2,4-D is 2.0 mg/l, and the concentration is 0.5 mg/l. 16. The method of claim 15. 17. (1) In induction medium containing exogenous hormone 2,4-D and kinetin Culture explants of Cucumissativus tissue in and (2) exogenous hormones supplemented with carbinicillin and kanamycin2,4 - Transfer the explants to medium containing D and kinetin for additional time in the dark. (3) incubated for 4-6 weeks, then (4) NAA and kinematics. MS basal medium supplemented with Chin for a sufficient time to induce competent embryoid bodies. , the Cucumis satay according to claim 1, characterized in that the explant is transferred thereto. Cucumissativus (Cucumissativus) from explants of Cucumissativus seeds. A method for producing a seed embryoid body (Cucumissativus). 18. The embryoid body produced by the method according to claim 17 is subjected to embryo regeneration. Cucumis sativus (Cucu) is characterized by obtaining completely transformed plants. A method for producing a transgenic plant (E. missativus). 19. Cucumissativus plant tissue, MS A composition consisting of a medium, 2,4-D and kinetin. 20. The concentration of 2,4-D in the medium is about 1.0 mg/l to 2.0 mg/l. Claim 19, wherein the concentration of kinetin is about 0.5 mg/l. Composition.