JPH04330233A - Method for preparing virus resistant plant - Google Patents

Abstract

PURPOSE:To prepare the title plant having excellent virus resistance by integrating a specific extraneous gene into a genome of a plant cell and transforming the integrated genome. CONSTITUTION:A recombinant double-stranded DNA molecule containing (i) a promoter functionating in a plant cell and causing production of RNA sequence of a plant virus, (ii) DNA sequence induced from the plant virus and causing production of PNA sequence of the plant virus and (iii) recombinant double-stranded DNA molecular containing a 3'-non-translation DNA sequence functionalizing in the plant cell and causing addition of polyadenylated nucleotide to 3' end of RNA sequence is inserted into a genome of the plant cell to provide a transformed plant cell. Then, a plant enhanced in resistance to infection by the plant virus and genetically transformed is regenerated to provide the objective plant.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to a method for incorporating a large amount of foreign genes into plants, a method for producing plants that are resistant to virus diseases using the method, and a method for producing plants that are resistant to virus diseases. Concerning matter and the products of its methods. The invention therefore includes applications in the fields of plant molecular biology, plant virology and plant genetic engineering.

0002

BACKGROUND OF THE INVENTION Viral infections in plants cause various adverse effects such as growth inhibition, morphological changes and reduced yield. Additionally, viral infections often make plants more susceptible to damage by other pests and pathogens. General knowledge about plant viruses can be found in the literature, eg, Mattews, R.; E. F. , Plant V
irology(Academic Press198
1), Laufer, M. A. , et al(e
ds. ), 26 Advances in Viru
s Research (Academic Press
(1981) and Kado, C. I. &H. O.
Agrawal, Principles and T.
echniques in Plant Virolo
gy (Van Nestrand Rheinhold
1972).

[0003] Plants do not have an immune system involving antibodies like animals. However, plants have developed several ways to resist infection by pathogens. For example, some types of plants produce lectins that bind to sugar moieties on the surface of invading fungi, immobilizing them. Additionally, certain types of plants apparently produce various proteins or phytoalexins that circulate throughout the plant in response to attack by bacteria, insects, and perhaps viruses.

Some types of plants are "attenuated" to young plants.
A certain degree of viral resistance can be induced by infecting a virus strain, ie, a virus strain that does not cause serious symptoms [for example, Rast. A. Ne
thelands J. Plant Patholo
gy, 78, pp. 110-112, (197
2) and Costa. A. J. Plant Dise
ase, 64, pages 538-541, (1
980)]. This method has several limitations, including: That is, (1) it can be conveniently performed only in certain types of crops; (2) Can only be used against certain types of viruses. (3) This can only be done if a suitable attenuated strain of the infectious virus has been identified or isolated. (4) The protection afforded by this method may be effective only against a limited number of viruses. (5) Infection with an attenuated strain can further seriously exacerbate infection with a second, unrelated virus through synergistic effects.

[0005]Therefore, there is a need for a method of protecting plants from viral infection that overcomes the above-mentioned problems and does not require the identification, isolation and use of attenuated viruses. Also, natural genetic resistance or cross-immunity (c
It is necessary to provide viral resistance when resistance (ross-protection) is not available. Furthermore, when using a conventionally known Agrobacterium tumefaciens Ti plasmid-derived vector (regardless of intermediate vector or binary vector), approximately 1 to 5 copies of a foreign gene are introduced per genome in the usual method. It is said that it is no more than . Although it is rare, it is known that several dozen copies can be introduced using this method. However, these methods have not succeeded in efficiently integrating a gene of interest at a high copy number.

[0006]Therefore, it would be desirable to provide a method by which hundreds of copies or more of a gene can be introduced into a plant genome with high efficiency and the cells can be efficiently selected. If such a method is available, it is expected that the positional effect of the introduced target gene on the chromosome can be diluted to some extent, which has not been possible with conventional methods. In addition, by integrating the target gene into many sites, it is possible to obtain plants that exhibit high expression in many tissues. The thing is,
It is particularly useful when systemic expression is required, eg, viral resistance. Moreover, such methods can confer the trait of virus resistance to all of its progeny, although the integrated gene generally tends to segregate by mating. In other words, if at least one copy of a functional gene can be integrated into every chromosome, there will be no separation due to hybridization. In particular, by integrating several hundred copies into chromosomes in a dispersed state, the target gene can be integrated into all chromosomes. Furthermore, by performing Southern analysis using this dispersed gene as a probe, it is possible to make it have the same role as the conventional fingerprint region on chromosomes using minisatellites found in humans, sparrows, etc., and to trace the ancestors of the plant. It is possible to estimate. It will also be possible in the future to protect the rights of those who bred the breeding stock using this method. That is, when a derivative is made using a certain plant, it can be determined that the derivative is derived from that plant based on the fingerprint area. In order to meet the above requirements, there has been a need to develop an efficient method for multiple gene integration.

[0007]

[Problems to be Solved by the Invention] As mentioned above, a system for large-scale expression of foreign genes has not been established in plant cells, except for storage proteins in seeds, and conventional methods have been used to express at most 1.5% of the total soluble protein. It was about 5%. This amount is E. coli,
The expression level is one order of magnitude lower than that of yeast, and is not necessarily a satisfactory expression level. Conventionally, promoters have been improved as a solution to this problem, but this alone has not been sufficient to achieve the above objective. Furthermore, development of multiple gene integration has not progressed to a level where it can be used sufficiently in both efficiency and multiplicity.

[0008] Therefore, the present invention provides a method for multiple gene integration that can pave the way for a system for mass expression of foreign gene products in plant cells, and that allows multiple gene integration to occur at many chromosomal sites. The purpose is to provide.

[0009]

[Means for solving the problem] The above problem is to efficiently introduce a large amount of foreign DNA into plant cells, selectively culture and redifferentiate the plant cells, and produce plants incorporating a large amount of foreign DNA. The problem is solved by a method including the following steps. (a) in the genome of a plant cell; (i) a DNA sequence that causes the production of RNA or protein that functions in the plant cell and causes some effect;
(ii) a DNA sequence that causes inactivation of a specific plant-specific gene (including changes in tissue specificity, etc.) when inserted into the plant cell genome; and (iii)
Activation of other specific genes inherent in plants by insertion into the plant cell genome (including changes in tissue specificity, etc.)
Inserting a recombinant double-stranded DNA molecule containing any of the DNA sequences giving rise to.

(b) A step of obtaining a plant cell transformed with the above recombinant double-stranded DNA molecule. (c) Regenerating genetically transformed plants with increased utility from the transformed plant cells. Also,
Using this method, we also provide a method for producing virus-resistant plants that does not require consideration of either the use of attenuated viruses, the presence of genetic determinants conferring resistance, or the availability of cross-immunity. The invention also involves inserting into the genome of a plant a recombinant DNA molecule containing, among other things, a small portion of the genome of a plant virus, so that the engineered plant becomes resistant to the plant virus. A method for genetically engineering plants is also provided.

The present invention also provides recombinant DNA molecules that can be used to produce genetically transformed virus-resistant plants. Furthermore, in the present invention, plant virus RN
DN that causes the production of A sequence (including antisense sequence)
Also provided are genetically transformed cells and differentiated plants, each characterized by the presence of the A sequence.

In one embodiment of the invention, a method for producing genetically transformed plants that are resistant to infection by plant viruses includes the following steps. (a) in the genome of a plant cell; (i) RN of a plant virus functioning in the plant cell;
(ii) a DNA sequence derived from a plant virus that causes the production of an RNA sequence of the plant virus; and (iii) a promoter that functions in a plant cell to produce an RNA sequence. 3
Inserting a recombinant double-stranded DNA molecule comprising a 3' non-translated DNA sequence resulting in the addition of a polyadenylated nucleotide to the 'end.

(b) A step of obtaining the plant cells transformed in the above steps. (c) Regenerating genetically transformed plants with increased resistance to infection by plant viruses from the plant cells obtained in the above steps. In one preferred embodiment, the plant virus RNA sequence encodes the coat protein of the virus.

[0014] In another aspect of the invention, there is provided a recombinant double-stranded DNA molecule that includes in its sequence: (a) Functioning in plant cells, RNA of plant viruses
(b) a DNA sequence derived from a plant virus that causes the production of an RNA sequence encoding the coat protein of the plant virus;
NA sequences, and (c) function in plant cells to produce RN
A DNA sequence corresponding to the 3' untranslated region that results in the addition of polyadenylated nucleotides to the 3' end of the A sequence.

[0015] Another aspect of the present invention is
Bacterial cells and transformed plant cells are provided which contain DNAs comprising elements (a), (b) and (c) above, respectively. Furthermore, another aspect of the invention provides a differentiated plant comprising a plant cell transformed as described above and exhibiting resistance to a plant virus. Other aspects of the invention include cultivating such plants, propagating such plants using explants, cuttings and propagules such as suds; Alternatively, methods are also provided that involve crossing the plant with other plants to produce progeny that are also resistant to the plant virus.

The present invention will be explained in more detail below. As a result of examining various conditions for causing multiple gene integration into plants, the present inventors found that they could use Safinia purple (Petunia hybrida), which is widely commercially available as a host.
cv. Surfinia purple), it was found that gene transfer occurred at a much higher copy number than usual. Until now, there have been no cases where more than a few hundred copies were introduced into tobacco, and the maximum was less than 100 copies. In Safinia, it was possible to introduce several hundred copies of the gene extremely efficiently. This indicates that Safinia has extremely excellent characteristics as a host for multiple gene integration. It is known that in tobacco, even when multiple gene integration occurs, the expression level does not necessarily increase. However, the present inventors have discovered that gene dosage effects can be obtained by increasing the amount of integration several tens of times more than is normally used. This is the first example showing that gene dosage effects can be observed in plants as well as in bacteria and yeast. Therefore, the host used in the present invention is not limited to Safinia, as it is expected that by effectively applying the present invention to other plants, the expression level will be increased in other plants based on the gene dosage effect.

The method for preparing and inserting the recombinant double-stranded DNA molecule into the plant cell genome of the present invention is not limited, but can be carried out as follows. Preparation of plant virus RNA As a method for preparing RNA from plant viruses, RN
Known methods for extracting A, such as the guanidine method,
It is possible to use a thermal phenol method, an SDS-phenol method, an SDS-protease-phenol method, and the like. As a plant virus, tobacco mosaic
virus, soybean mosaic virus, bean
Pod mottle virus, tobacco ring
spot virus, barley yellow dwarf virus, wheat streak virus, wheat spindle streak virus,
Soil Bone Mosaic Virus, Maze
Dwarf Mosaic Virus, Maize Chlorotic Dwarf Virus, Alfalfa Mosaic Virus, Potato Virus X
, Potato Virus Y, Potato Leaf Roll Virus, Tomato Golden Mosaic
Viruses, cucumber mosaic virus, etc. can be used.

[0018] cDNA synthesis A method for synthesizing cDNA is as follows: Okayama & B
The method of erg et al. [Mol. Cell. Biol. , 2
: 161 pages, (1982)], Land's method [Nucleic Acids Res. , 9 , 2
251 pages, (1981)] or Gubler &
The method of Hoffman et al. [Gene, 25, 263
~269 pages, (1983)] or methods similar thereto, as appropriate.
Double-stranded DNA can then be synthesized. Also Gu
Various synthesis kits based on the method of Bler & Hoffman et al. are commercially available. For example, Amersha
It is possible to obtain cDNA in a few hours from the RNA prepared above using m's cDNA synthesis system plus.

Cloning of double-stranded cDNA Double-stranded cD
NA is converted into pBluescr by, for example, the deoxyguanosine-deoxycytosine (dC-dG) method.
It can be cloned into ipt II, etc. This plasmid can be used to transform Escherichia coli etc., for example, by the method reported by Hanahan et al. This transformant, such as Escherichia coli, is cultured in an ampicillin-containing medium, and among the ampicillin-resistant strains that have grown, the alkaline method or boiling method, for example, according to the method reported by Maniatis, is used.
Prepare a plasmid using thod etc. and cD of the desired length.
Strains with NA can be selected.

Modification for efficient expression in plants and introduction of the plasmid into Agrobacterium tumefaciens The above cDNA was combined with a promoter that functions in plant cells (
For example, plant DNA virus promoters, cauliflower mosaic virus 35S promoter nopaline synthase or octopine synthase promoter, plant gene promoter ribulose diphosphate carboxylase small subunit promoter, promoter of genes encoding hydroxyproline-rich proteins, cauliflower mosaic virus. 35S promoter of mannopine synthase
promoters, etc.) and sequences that cause addition of polyadenylated nucleotides (e.g., polyA of nopaline synthase).
cDNA can be introduced into plant cells using the Ti plasmid system by ligating additional signals (additional signals) and then inserting between the T-regions of a binary vector or intermediate vector.

Method for inserting genes into plants Obtained cD
As a method for inserting NA into plants, for example, when using a vector, Agrobacterium tumefaciens can be used, and several types of vectors can be used commercially, or vectors prepared in the same way as these can be used. It's okay. For example, the above cDNA can be introduced into plant cells and expressed using the binary vector pBI121, pAL4404 system manufactured by Clontech.

[0022] In this way, we were able to obtain a plant in which multiple gene integrations had occurred. This plant has superior expression levels and tissue specificity compared to conventional plants in which several copies of the gene have been integrated. That is, the expression level increased due to the gene dosage effect, and by integrating the target gene into many sites on the chromosome, strong expression of the gene was observed in many tissues without tissue specificity.

[0023] Multiple integration of the above-mentioned target genes can be carried out relatively easily in certain plants. for example,
Plants with a large number of receptors required for Agrobacterium tumefaciens infection; Plants with weak or few DNA degrading enzymes in the nucleus; Plants that are less stressed by Agrobacterium tumefaciens infection; Agrobacterium tumefaciens Multiple gene integration can be efficiently caused by selecting plants that do not contain substances that are toxic to Agrobacterium tumefaciens; plants that produce substances that induce the Vir gene of Agrobacterium tumefaciens; Plants with many DNA degrading enzymes in the nucleus; plants that contain substances toxic to Agrobacterium tumefaciens; plants that are extremely susceptible to Agrobacterium tumefaciens and easily die, etc. are not suitable for multiple gene integration. It is. From the above viewpoint, desirable plants that can be used in the present invention include plants belonging to the families selected from Leguminosae, Umbelliferae, Brassicaceae, Cucurbitaceae, Poaceae, and Solanaceae, and in particular, tobacco plants, tomato plants, Petunia plants are preferred.

Transformation method In the conventional transformation method, it was possible to increase the frequency of infection by increasing the concentration of Agrobacterium tumefaciens, but If present in excess, subsequent removal is often extremely difficult, and if anything, a relatively low concentration bacterial solution was often used. However, in order to achieve large-scale incorporation through large-scale infection, the concentration of the bacterial solution is an important point. That is, in order to cause gene integration in a large amount, it is effective to infect a large amount of Agrobacterium tumefaciens. Further, according to vacuum infiltration, a large amount of Agrobacterium tumefaciens bacterial liquid can be brought into contact with plant cells, and as a result, the frequency of multiple infections can be increased.

[0025] Next, the transformed plant cells obtained as described above can be used to produce target plants through the following steps. Selection of transformed cells The above-transformed plant cells can be selectively cultured using an antibiotic such as kanamycin. As a result, only kanamycin-resistant transformed cells can be obtained.

Redifferentiation of transformed cells The transformed cells obtained above can be redifferentiated by conventional tissue culture techniques. For example, a regenerated plant can be obtained by culturing Murashigeskoog's medium with the addition of 0.01-0.5 ppm of auxin and 0.5-2 ppm of cytokinin.

Rooting of transformed plants The shoots produced from the transformed cells obtained above can be transplanted into a rooting medium to obtain a complete rooted plant. For example, rooted individuals can be obtained by using a hormone-free Murashigeskoog medium or a medium to which auxin is added.

Acclimation of Transformed Plants Plants obtained and rooted as a result of transformation can be acclimated by conventional plant acclimation methods. As a result, complete plants can be obtained. The plants thus obtained exhibit strong expression of the foreign gene in a wide range of tissues. For example, it expresses the viral coat protein and exhibits virus resistance.

[0029]

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the scope of the present invention is not limited thereto. Example 1. Isolation of RNA contained in CMV (Y strain) CMV-Y provided by the University of Tokyo (Nippon Tobacco) (for details, see Nitta et al., Ann. Phytopath
．． Soc. Japan 54. 516-522 (1988)) inoculated into tobacco, and 7-14
The plants were grown at 25°C to 30°C for 1 day and infected leaves (100g) were harvested. Infected leaves were frozen (-20°C) and then treated with 0.5M sodium citrate buffer (pH 6.5) (0.1% (w/w).
v) Thioglycolic acid, containing 10mM EDTA)
Homogenization was performed using 100 ml of chloroform and 100 ml of chloroform. The resulting emulsion was centrifuged at 4,600xg for 20 minutes. 8% polyethylene glycol in the supernatant,
Add salt to 0.2M (final concentration) at 4°C.
I left it for 40 minutes.

[0030] This suspension was centrifuged (8700xg, 1
5 minutes) and the precipitate was added to 10mM phosphate buffer (2mM EDT).
A). This suspension was centrifuged (87
00xg for 15 minutes) and the supernatant was
Centrifugation was performed at xg for 90 minutes. 1 precipitate (containing virus)
0mM phosphate buffer (pH 7.0) (2mM EDTA
0.5 ml) and subjected to 10-40% density gradient ultracentrifugation (35,000 rpm, 2 hours). The resulting viral zone was sucked up with a syringe needle and centrifuged (40,000 rpm, 2 hr). The obtained precipitate was suspended in 340 μl of 10 mM phosphate buffer (pH 7.0). The resulting purified CMV-Y
13 mg of particles were obtained. Of this, 500 μg of virus was added to 400 μl of Tris-HCl buffer (pH 7.5).
) (100mM NaCl, 10mM EDTA, 4%
(w/v) (containing sodium lauryl sulfate) was added and mixed, and then phenol/chloroform saturated with the same buffer was added and the mixture was stirred vigorously and left in ice for 10 minutes (with occasional vigorous stirring). After centrifugation at 15,000 rpm for 5 minutes, the aqueous layer was collected. Also, add a buffer solution to the phenol layer.
Add 400 μl of buffer saturated phenol/chloroform to the aqueous layer obtained by adding 00 μl and perform back extraction, and add the previously obtained aqueous layer, stir vigorously for 1 minute, and perform the same centrifugation. Ta. After repeating this phenol/chloroform extraction three more times, the separated aqueous layer was added with 1/10 volume of 3M sodium acetate (pH 5.5).
Add 2 times the volume of ethanol and incubate at -70℃ for 90 minutes, at -20℃
After standing for 30 minutes, RNA was collected by centrifugation at 15,000 rpm for 30 minutes at -10°C. This precipitate was suspended in 72 μl of water, and then mixed with 8 μl of 3M sodium acetate and 20 μl of water.
Add 0 μl of ethanol and store at -70°C for 30 minutes at -20°C.
After standing for 40 minutes, the precipitate containing RNA was collected by centrifugation at 15,000 rpm for 30 minutes at -10°C. After drying this precipitate under reduced pressure and dissolving it in 20 μl of water, -70
Stored at °C.

Example 2. c encoding the CP gene
Cloning DNA To clone cDNA RNA 1-4 3
DNA complementary to the ’ end (5’-TGGTCTCCT
TTTGGAGGCCC-3') was chemically synthesized using an Applied Biosystems synthesizer, and 580 μg of D
Got NA. 65 μl of the RNA solution obtained in Example 1
After heating at °C for 10 minutes, the mixture was placed on ice and double-stranded cDNA was synthesized using Amersham's cDNA synthesis system plus.

This cDNA was precipitated with ethanol, dried under reduced pressure, and dissolved in 15 μl of 10 mM Tris-HCl buffer (pH 8.0, containing 1 mM EDTA). This solution was fractionated by low melting point agarose electrophoresis, a 1 kb band corresponding to RNA 4 was excised, and then added to 10 mM Tris-HCl buffer (pH 8.0).
, containing 1mM EDTA) 0.6ml, pBlue
Add 1 μg of script EcoRV cleavage product (described below) and dissolve at 68°C for 20 minutes, then add 10 mM Tris-HC.
l Buffer (pH 8.0, containing 1mM EDTA)
By performing saturated phenol extraction twice, remove the agarose, add 1/10 volume of 3M sodium acetate and 2 volumes of ethanol, and leave at -20°C for 1 hour at -70°C for 90 minutes, then at 15000 rpm for 30 minutes at -10°C. A precipitate containing cDNA and pBluescript was obtained by centrifugation. This precipitate was dried under reduced pressure and dissolved in 10 μl of water. Of this, add 5 μl of ligation buffer (100 m
M Tris-HCl (pH 7.5), 10mM Mg
After adding 1 μl of Cl2) and 3 μl of water, add T4 ligase 0.9
μl and 0.1 μl of T4 kinase were added and left at 15° C. for 19 hours. 2 μl of this was mixed with 100 μl of E. coli XL-1 Blue competent cells, left in ice for 70 minutes, then 90 seconds at 42°C. After 2 minutes in ice, 1 ml of LB broth was added and cultured at 37°C for 1 hour, containing ampicillin. LB to do
Plated on agar medium. Sixteen of the colonies that appeared were cultured in a liquid medium containing ampicillin, and a plasmid was prepared by the boiling method. RNA by restriction enzyme analysis
We obtained one clone that was thought to contain almost the entire length of 4 (
pSKIICMV-Y1). This clone 2
After culturing in 00 ml, a plasmid was prepared by the alkaline method and CsCl density gradient ultracentrifugation, and 2120 μg of DNA was obtained.

Example 3. binary vector pBI
Insertion of coat protein gene into 121 pSKIICMV-Y1 DNA 10 μg (5 μl)
10μl of low buffer,
80 μl of water, 5 μl of ClaI, 0.5 μl of calf thymus alkaline phosphatase (CIP) (13.5 units)
s), left at 37°C for 2 hours, checked for cleavage by electrophoresis, extracted with phenol/chloroform, and extracted with 2-
After butanol concentration and ethanol precipitation, the residue was dried under reduced pressure and dissolved in 10 μl of water. Add Klenow buffer (
Klenow buffer) 2μl, 1mM NT
2 μl of P, 4.5 μl of water, and 1.5 μl of Klenow enzyme were added, and the mixture was left to stand at 30° C. for 20 minutes, and then fractionated by low melting point agarose gel electrophoresis. Approximately 4kb band cut out 0.5ml
of Tris-HCl (pH 8.0, 1mM EDTA
), then 2 μg of SacI linker was added, and 68
After dissolving at ℃ for 20 minutes, the agarose was removed by extraction with saturated phenol twice using 10 mM Tris-HCl buffer (pH 8.0, containing 1 mM EDTA).
/ Add 10 volumes of 3M sodium acetate and 2 volumes of ethanol, leave at -70°C for 60 minutes, then turn to 15,000 rpm for 10 minutes.
A precipitate containing linear DNA was obtained by centrifugation at -10°C for 1 minute. This precipitate was added again to 10mM Tris-HCl.
Buffer (pH 8.0, containing 1mM EDTA) 80
After dissolving in μl, saturated phenol extraction was performed again with 10mM Tris-HCl buffer (pH 8.0, containing 1mM EDTA), and after concentration with 2-butanol, 1/10
Add 1 volume of 3M sodium acetate and 2 volumes of ethanol,
-70℃, left for 60 minutes, then 15000 rpm, 10 minutes, -
A precipitate containing linear DNA was obtained by centrifugation at 10°C. This precipitate was dried under reduced pressure and dissolved in 8 μl of water. Of this, 1 μg (1 μl) of SacI linker was added to 4 μl.
Hoshuzo ligation kit solution A 40 μl, B 5 μl
was added and left for 2 hours at 16°C, followed by standard transformation (Han
ahan) was carried out. Of the transformants obtained, 16
The clone was grown in a liquid medium containing ampicillin, a plasmid was prepared by the boiling method, and whether or not the SacI site had been introduced was examined by restriction enzyme analysis by electrophoresis. The results showed that the SacI site was introduced into all clones. Approximately 10 μg of this is B
It was cut with amHI and SacI, fractionated by 1% low melting point agarose gel electrophoresis, and a coat protein gene portion of approximately 1 kb was excised. Add 2 times the volume of 10mM Tris-HCl buffer (pH 8.0, containing 1mM EDTA) (twice the volume of the weight of the gel), heat at 68°C for 20 minutes to melt the agarose, and then add 10mM Tris-HCl buffer (pH 8.0, containing 1mM EDTA). H.C.
l Buffer (pH 8.0, containing 1mM EDTA)
Saturated phenol extraction was performed to remove the agarose, and after concentration with 2-butanol, 1/10 volume of 3M sodium acetate and 2 volumes of ethanol were added to recover the DNA. The recovered DNA was dissolved in 10 μl of water. On the other hand, pBI 1
21.5 μg was added to low buffer (Low buffer).
r) and cleaved with SacI, NaCl was added to a final concentration of 100 mM, and 100 units of BamHI was added and cleaved. This reaction solution was subjected to ethanol precipitation as shown in Example 2 to recover DNA, and the vector portion was recovered by low melting point agarose electrophoresis. The recovered DNA was dissolved in 10 μl of water, and 3 μl of the DNA was dissolved in 1 kb Bam containing the coat protein gene obtained above.
Mix 3 μl of HI-SacI fragment solution and use Takara Shuzo's D
Ligation was performed using an NA ligation kit. This ligation product was transformed into Escherichia coli XL-1Blue by the method shown in Example 2 to obtain a kanamycin-resistant transformant. Of these, 16 clones were grown in a liquid medium containing kanamycin, plasmids were prepared by the method shown in Example 2, and restriction enzyme analysis was performed. As a result, DNA containing coat protein was found to be inserted into 6/16 clones. The plasmid obtained here was named pBI121CMV-Y. (Figure 1).

Example 4. Triparental hybrid E. coli XL-1Blue (pBI121CMV-Y)
, E. coli HB101 (pRK2013) and Agrobacterium tumefaciens LBA4404 (pAL4
404) were each cultured in liquid, spotted on LB medium, and cultured overnight at 30°C to perform ternary hybridization. After scraping the spot and diluting it with LB medium, kanamycin (final concentration 30 μg/ml), rifampicin (
plating on LB medium containing (final concentration 5 μg/ml)
did. The colonies that appeared as a result were plated again in the same manner to form single colonies.

Example 5. Transformation Virus-free Safinia pink, purple, mini, and NC purple leaf pieces (all commercially available) grown aseptically were added to the overnight culture of Agrobacterium tumefaciens obtained in Example 4. (LB medium) and the bacterial solution was infiltrated into the leaf pieces by vacuum infiltration. After that, a suspension culture solution for protection culture was placed under the filter paper in advance, and a callus induction medium (MS-B5, pH 5.7, s.
ucrose 3%, 2,4-D1ppm, agar 1%
) and cultured in the light at 25°C for one week, then kanamycin 300 μg/ml and carbenicillin 500 μg/ml.
The cells were transplanted into the same medium containing g/ml. The calli that appeared after about one month were treated with regeneration medium (MS-B5, pH 5.7,
sucrose 3%, NAA 0.1ppm, BA
1 ppm, kanamycin 300 μg/ml, carbenicillin 500 μg/ml, agar 1%) for redifferentiation. After the redifferentiated shoots became large enough, IBA rooting medium (1/2MS, IBA 0.5
ppm, gellan gum 0.2%) and allowed to root. After sufficient rooting, the gellan gum was thoroughly washed off, transplanted to vermiculite, and placed in a mist room for acclimatization. It was propagated by cuttings and buds with well-elongated branches.

Example 6. Southern analysis The kanamycin-resistant callus obtained in Example 5 was crushed in liquid nitrogen and subjected to CT.
Total DNA was extracted by AB method. Obtained DNA 2
μg was cut with restriction enzyme HindIII and 0.7
% agarose gel electrophoresis, transferred to a nitrocellulose filter, and then Southern analysis was performed using the region encoding the coat protein as a probe. As a result, the cauliflower mosaic virus 35S promoter and the region encoding the CMV coat protein were found to be 0.6 kb and 1.2 kb, indicating that the region encoding the CMV coat protein is integrated into the plant genome.
Band b was observed in all clones. This showed that all the calli obtained by transformation in Example 5 contained the coat protein gene. In addition, two of these clones have approximately 50 per haploid genome.
0 copies of the gene were integrated (Figure 2).

Example 7. ELISA The callus and plants obtained in Example 5 were examined to determine whether coat protein expression was observed. The calli or plants were ground in liquid nitrogen, suspended in 5 times the volume of PBST, centrifuged at 11,000xg for 10 minutes, and the supernatant was collected. 50mM sodium carbonate (pH 9
．． 6) Transfer the supernatant to a 96-well plate coated with 400x diluted anti-CMV antibody.
Aliquots of μl were left at 4°C overnight, washed 5 times for 5 minutes with PBST, and then alkaline phosphatase-labeled anti-
After reacting with CMV antibody (400x dilution) at 25°C for 4 hours and washing with PBST 5 times for 5 minutes, the substrate solution (para-nitrophenyl phosphate disodium salt was dissolved in diethanolamine to a concentration of 1 mg/ml and adjusted to pH 9.
Add 300 μl of the solution (prepared in step 6), leave it at 25°C for 1 hour, and then read it using an ELISA plate autoreader.
Absorbance was measured at 5 nm and 600 nm. the result,
The presence of coat protein in transformed Safinia was demonstrated (Figure 3). We also found that there was little tissue specificity in expression during this process.

Example 8. Virus inoculation test (1)
500, 100, 20 purified CMV virus was added to virus-free Saffinia grown by cuttings, respectively.
, 4, 0.8, 0.16, 0.032 μg/
Inoculated at a concentration of ml, and incubated in an artificial climate chamber at 22°C under light conditions 1.
The cells were cultured for 14 hours in the dark for 0 hours. As a result, 500, 100, 20, 4, 0.8μg/ml in about 2 weeks
Symptoms of disease were observed at a concentration of (Figure 4).

(2) Transformants propagated by cuttings were inoculated at concentrations of 20, 4, and 0.8 μg/ml, and cultured under the same conditions as in (1). As a result, clear virus resistance was shown in all plots (Fig. 5).

[0040]

[Effect of the invention] Plant cells transformed by the method of the present invention contain CMV- which is not synthesized at all from the original plant cell itself.
It is possible to produce Y coat protein, and C can be produced in the plant cell itself or in the redifferentiated plant
It can confer resistance to MV infection.

[0041] We have now established a method for introducing several hundred copies of a gene into the chromosomal genome of a plant using a binary vector system, but in principle this method can also be applied to intermediate vectors. This method is also applicable to vector systems derived from Ri plasmids and direct introduction methods. By this method, several hundred copies or more of a gene can be introduced into a plant genome with high efficiency and the cells can be selected. As a result, plant cells exhibiting high expression can be obtained. By this, it is expected that positional effects on the chromosome can be diluted to some extent, which was not possible with conventional methods. Furthermore, by integrating the protein into many sites, it is possible to obtain plants that exhibit high expression in many tissues. This is especially useful when systemic expression is required, such as, but not limited to, viral resistance. Furthermore, this method allows the integrated gene, which is generally separated by mating, to impart the virus-resistant trait to all its progeny. That is, by incorporating at least one copy or more of a functional gene into every chromosome, separation due to hybridization can be prevented. Genes can be integrated into all chromosomes by integrating several hundred copies into chromosomes in a dispersed state. Furthermore, by using this dispersed gene as a probe, the present invention has the same role as the conventional fingerprint region on chromosomes using minisatellites found in humans, sparrows, etc., making it possible to infer the ancestor of the plant. It is. In addition, it will be possible in the future to protect the rights of the person who bred the breeding mother using this method. That is, when a derivative of a certain plant is produced by using it as a breeding mother, it can be determined that it is derived from that plant based on the fingerprint area.

[Brief explanation of drawings]

FIG. 1 shows a flow diagram of the construction of plasmid pBI121CMV-Y.

FIG. 2 shows Southern analysis results according to Example 6.

FIG. 3 is a graph showing the expression level of coat protein in transformed Safinia according to Example 7.

FIG. 4 is a graph showing the results of a virus inoculation test on control plants.

FIG. 5 is a graph showing the results of a virus inoculation test on plants of the present invention.

Claims (1)

[Claims] 1. A method of producing a genetically transformed plant that is resistant to infection by a plant virus, the method comprising: (a) adding to the genome of a plant cell; (i) functioning in the plant cell; a promoter that causes the production of an RNA sequence of the plant virus; (ii) a DNA sequence derived from the plant virus that causes the production of an RNA sequence encoding the coat protein of the plant virus; and (iii)
Inserting a recombinant double-stranded DNA molecule containing a DNA sequence corresponding to a 3' untranslated region that functions in a plant cell to cause the addition of a polyadenylated nucleotide to the 3' end of said RNA sequence. (b) obtaining plant cells transformed with said DNA; and (c) regenerating from said transformed plant cells genetically transformed plants that are resistant to infection by said plant virus. A method for producing a transformed plant comprising: