JPS61257186A - Novel dna and hybrid dna - Google Patents

Abstract

PURPOSE:To provide a double-chain vector derived from the single-chain DNA of bean golden mossaic virus and having improved technological handleability in genetic recombination technique. CONSTITUTION:The DNA of formula having two or more chains was produced from the single-chain DNA of bean golden massaic virus (BGMV) (a kind of gemini virus) and having a length of 2585 bases. The double-chain DNA was incised with a restriction enzyme and integrated with other biological vector incised with the same restriction enzyme to obtain a hybrid DNA.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the use of bean golden mosaic virus (Bea
DNA related to Golden Mo5aic Virus (hereinafter sometimes abbreviated as "BGMV") and hybrid DNA incorporating it
Regarding. To explain in more detail, the single-stranded DN of BGMV
DNA obtained by double-stranding A, hybrid DNA obtained by incorporating this DNA into a vector for other organisms
, Hybrid DNA 1 obtained by propagating this hybrid DNA relates to DNA obtained by extracting only the BGMV-derived DNA fragment portion from this hybrid DNA and performing ring-closing ligation, and replicative DNA of BGMV.

The DNAM gene from the iL virus has recently been widely developed and used as a vector in genetic recombination technology. Examples of viruses that provide such vectors are Pabovaviruses such as Simian Virus (S'V) 40 and Polyomavirus.

Papillomaviruses and adenoviruses are known. However, these known vectors were discovered as animal-based vectors and do not proliferate in plant-based cells. Therefore, these known vectors cannot be used as vectors for plant genetic recombination.

To date, vectors that can be used as plant genetic modification vectors include Agrobacterium tumphaensis, which causes tumors in dicotyledonous plants such as tomatoes and tobacco.
The only known examples are the Ti plasmid, an extranuclear gene possessed by P. efaciens), and the DNA gene from cauliflower mosaic virus, which causes diseases in cabbage and Chinese cabbage. Other than these, vectors suitable for plant genetic recombination have not yet been developed. Furthermore, other than the above-mentioned cauliflower mosaic virus, there are no known plant viruses that have DNA genes that can be used as vectors for plant genetic modification.

Recently, Robert M. Goodman and his colleagues at the University of Illinois in the United States have discovered that BGMV, a tropical plant virus, has a diameter of about 18 mm.
It looks like two 0m particles paired together to form a single pillion, and analysis of its nucleic acid revealed that the gene of this virus is NA, a circular single-stranded polymer with a size of approximately 2500 bases. is reported under the heading.

[Virology 83171 (1977),
V 1roloay 97388 (1979)].

A large number of plant viruses have been discovered that have the shape of two particles with a diameter of approximately 18 nm paired together to form a pillion, and whose gene is a circular single strand. These viruses are called the ``dieminivirus group.''

As mentioned above, the only known plant DNA viruses are cauliflower mosaic virus and dieminivirus, and dieminivirus is extremely important as a goal for the development of vectors for genetic recombination in plants. I can expect it.

Conventionally, the Ti plasmid and cauliflower mosaic virus, which have been proposed as vectors for plant genetic modification, can only infect dicotyledonous plants, that is, host plants, whereas dieminivirus can be used not only for dicotyledonous plants but also for humans. Since monocots such as wheat and corn, which are important grains for this virus, can also serve as hosts, it is extremely meaningful to convert the DNA of this dieminivirus into a vector for genetically modifying plants.

On the other hand, cauliflower mosaic virus multiplies within the cytoplasm of plant cells, whereas dieminivirus multiplies both within the cytoplasm and nucleus. This indicates that when dieminivirus is used as a vector, it is highly likely that the nuclear genes of plants themselves can be modified. Therefore, it is clear that if it is possible to convert dieminivirus DNA into a vector, it would be of industrial value in itself.

Therefore, the present inventors made the heavy chain DNA of Bean Goal Ann Mosaic Virus (BGMV), which is a type of dieminivirus, into a double-stranded vector that is technically easy to handle in genetic recombination operations. The present invention was achieved as a result of research into hybrid DNA incorporating this.

That is, according to the present invention, (1) DNA obtained by at least partially double-stranding a single-stranded DNA with a length of 2585 bases and having the base sequence shown in FIG.
NA (hereinafter abbreviated as DNA-a) and Q) DNA obtained by at least partially double-stranding a single DNA with a length of 2647 bases and showing the base sequence in Figure 2.
(hereinafter abbreviated as DNA-b), according to the present invention, the double-stranded DNA-a and DNA-b are digested with restriction enzymes, and other biological vectors cleaved with the same restriction enzymes are prepared. hybrid DNA obtained by integrating this hybrid DNA into the host of the original vector and propagating it; DNA showing the base sequence of FIG. 1 or 2 is provided.

Bean gall mosaic virus (BGMV) is a virus that causes a gorten mosaic-like disease in common beans in Central and South America, and is a phytopatholoq virus.
y 67 (NQl) 37 (1977), the single-stranded DNA can be isolated and purified from infected plants as described below.

(1) Isolation of single-stranded DNA from BGM V Viruses were depleted from infected plants by the method described in the above literature, and approximately 500 μ9 of the purified virus obtained was incubated at 30 m
M Tris HCj (PH= 7.6) 1%S
Shake for 2 minutes with O8, proteinase KIO μg/a + Jl solution, and then perform protein extraction three times with 100 μg of phenol. Dissolved phenol is removed from the aqueous layer by ether extraction, and then the single-stranded DNA of the virus can be separated and purified by ethanol precipitation.

Double-stranding of f2+BDMV single-stranded DNA The single-stranded DNA obtained as described above is then double-stranded in vitro. In this double-stranded formation, it is preferable to use 0.002 to 2000 μg of brimer per 1 μ3 of single-stranded DNA. As a brimer, B 1Oc by J.M. Taylor et al.
hilica et B 1ophysicaAct
A, 442325 (1976) can be modified to use oligonucleotides obtained by decomposing regenerating thymic ON with DNASe. In the present invention, a brimer obtained by this modified method was used as described in the Examples below. When this oligonucleotide is used as a brimer, the brimer opening is
A ratio of 50 to 400 μ9 per μg of NA is suitable.

Furthermore, when using purified DNA containing at least 10 bases and having a completely complementary sequence to the single-stranded DNA of BGMV, the ratio of the primer used is 0.002 μg to 1 μg per 1 μg of single-stranded DNA. and further may be 0.004μg to 0.02μ9.

After adding and mixing the brimer to the single-stranded DNA at the above-mentioned ratio, the mixture is preferably heated at 50 to 80°C, especially at 60°C.
A temperature of 0 to 70°C is maintained for 1 to 10 minutes, preferably 3 to 7 minutes, and then, if necessary, rapidly cooled to a temperature of about 0°C or higher. By this operation, the primer DNA is attached to the complementary base sequence on the virus-heavy chain DNA.

Next, deoxyadenosine triphosphate (dATT), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP) were each added to a final concentration of 10 to 300 μM.
It is preferably added in a range of 50 to 150 IM.

At this time, for example, by substituting a part of dCTP with α-32p-deoxycytidine triphosphate (α-321)-dCTP), the degree of progress of double-strand formation and the double-stranded This is convenient because it makes it easy to trace the oxidized DNA. In addition, Mg" (e.g. magnesium chloride, magnesium sulfate, etc.) is added to promote the enzyme reaction.
It is preferably used in a concentration range of 5 to 20 Il1M. Note that in the water layer ill, rMJ is r unless otherwise specified.
It means the concentration unit of moJl/uJ. I in the reaction system
)H is maintained in the range 7.2-9, preferably 7.6-8.4 using a buffer such as Tris H(J (
1187,6-8.4) in a concentration of 30-300 IM, preferably 70-20011M.

The entire mixture contains 1 μg of IQ DNA per virus.
The amount is preferably in the range of 1 to 1000 μm, preferably 10 to 500 μm. It is also effective to use commonly used enzyme stabilizers, such as α-mercaptoethanol and dithiothreitol (DTT). The concentration of the stabilizer in the mixed solution is 0.1 to 5% (heavy equipment), preferably 0.5 to 2% (i.e.
It can be used within a range of (Li amount)%.

Next, a double-stranded enzyme is applied, such as Avian miniloblastosis virus (AMV) reverse transcriptase, T4-DNA, etc.
Examples include polymerase, Escherichia coli DNA polymerase (■), and DNA polymerase eI large fragment (Talenor enzyme). Among these, AMV reverse transcriptase or DNA polymerase trage fragment is preferred.

The proportion of double-stranded enzyme used depends primarily on its type. For example, when using AMV reverse transcriptase [Code 120248 manufactured by Seikagaku Corporation], single-strand D
1 to 20 units, preferably 5 to 1 units per μg of NA
It is effective to use 0 units. If the number is less than 1 unit, it may be difficult to form double strands sufficiently. After adding the double-stranded enzyme, firstly at a temperature of 10-30°C, preferably 15-25°C for 2-30 minutes, preferably 5-30°C.
React for 20 minutes and then heat at 30-45°C, preferably at 35°C.
30-180 minutes at a temperature of ~40°C, preferably 50-180 minutes
A reaction time of 120 minutes is advantageous.

The reaction can be stopped by adding a terminating agent such as water-saturated phenol or EDTA aqueous solution (pH=8.0) to the reaction mixture. At this time, in the case of water-saturated phenol, it is sufficient to add 1/10 to 2 times the volume of the reaction mixture, shake it, and then stretch to separate only the aqueous layer, and in the case of an EDTA aqueous solution, Mg present in the mixture You just need to use enough cars to supplement ``''. The double-stranded DNA may be separated from the DNA-containing aqueous solution subjected to the reaction termination treatment, and the separation operation is preferably performed by gel filtration. To explain an example of a chemical compound in that case, a double-stranded DNA-containing aqueous solution is treated with Sephadex G-1
0 [Pharmacia Fine Chemicals Co., Ltd. Biog
el T) 30 [Perform fractionation using a gel filtration agent such as Bio U111 Co., and measure the 32p intensity of each fraction. Collect the mother DNA-containing fractions and precipitate them in ethanol or isopropanol to obtain double-stranded DNA.
is obtained. The double-stranded DNA thus separated is B
It may contain a considerable amount of primer DNA other than double-stranded DNA based on the single-stranded DNA of GMV. This tendency is remarkable when oligonucleotides from the regenerated thymus DNA are used as primers.

Therefore, in such cases, it is desirable to purify the double-stranded DNA again to make it even more pure. This purification can be carried out using the gel filtration method described above. -Specific examples are given below, -Heavy chain DNA 20μ
When starting from 3, the double-stranded DNA was diluted with water 100~
Dissolve 10 mM Tris H (J
(1) Gel filtration is performed using a column with increased separation capacity packed with Sephadex Q-50 or Biogel p30 equilibrated with 100 mM NaCl aqueous solution (H=7.4). 10 mMTr as eluent
is H(J (pH = 7.4), 100mM
A fraction with a high 32p count at the bottom of the void volume is collected using Na Cl and precipitated in ethanol or isopropanol to obtain highly pure double-stranded BGMV DNA.

(3) Double-stranded DNA There are two types of dieminivirus in BGMV mentioned above.
There are also two types of DNAs that contain NA and are double-stranded as described above. These two types of double-stranded DNA can be made into individual DNA molecules, or can be cloned and separated. It is also possible to separate each DNA before making it into double strands (in other words, in the state of single stranded DNA) and make them into double strands in the same way.

In order to separate a mixture of single-stranded DNA of BGMV into two types of DNA, a method called "Basstrand separation" [for example, in the book "Mo1ec" by Maninais et al.
ular Cfoning-A Labator
[Method described on pages 180 to 185 of yManual] A method using twin gel electrophoresis, or a DNA fragment complementary to either one of the DNAs is added to a cesium chloride solution of the DNA mixture, and a single DNA fragment is separated by equilibrium density gradient centrifugation. It is possible to adopt a method of separating DNA that remains as a strand and partially double-stranded DNA by utilizing the density difference.

However, separation of two types of DNA can be easily carried out by converting a single-stranded DNA mixture into double-stranded DNA, cloning it into, for example, plasmid pBR322, and separating it.

This separation method will be explained in detail later.

In this way, the DNA obtained by double-stranding the DNA from BGMV has 2585 salt U pairs (hereinafter referred to as "base pairs") on one side.
Figure 1
The base sequence of The other one is 2647 base pairs (bp)
It has the size of , and shows the nucleotide sequence in Figure 2.

The double-stranded DNA according to the present invention is all single-stranded DNA.
It is sufficient that the protein is at least partially double-stranded, and it is not necessarily necessary that the entire region is double-stranded.

The double-stranded DNA of the present invention is used, for example, as a vector for plant genetic recombination.
It is used by cutting it with an enzyme consisting of In this case, if the site to be cut is at least double-stranded, the non-double-stranded site will be repaired by subsequent cloning or multiplication, and the DNA will become completely double-stranded. Therefore, the double-stranded DNA of the present invention only needs to be at least partially double-stranded, but preferably at least 80%, preferably at least 90% of the base pairs of the single-stranded DNA are double-stranded. It is fine as long as it is chained.

The double-stranded DNA according to the present invention, shown in FIG.
There is an oven reading frame (hereinafter abbreviated as "○RF") 1 extending from ATG of arrow (1) to TGA of arrow (2), and within this frame, ATG of arrow (1)
Starting from TG and moving toward the right in Figure 1, we can see the arrows (2
), the protein is encoded with an amino acid sequence according to the so-called "universal codon table" until the end with TGA. Note that the amino acid translation stop codon TAA, TAG, or TGA does not appear in the intermediate codon between arrow (1) and arrow (2).

Similarly, in the DNA shown in FIG. 1, another protein is encoded in 0RF2 from GTA shown by arrow (3) to AAT shown by arrow (4) in the left direction of FIG. In the double-stranded DNA according to the present invention shown in FIG. 2, the AT indicated by the arrow (5)
0 from G to TAA shown by arrow (6) in the right direction in Figure 2
The protein is encoded by RF3. Similarly arrow +
7) Click the arrow (81(7)) to the left in Figure 2 from GTA.
AATmar (7) to ORF4, arrow (9) Jl: To the left of Figure 2, arrow (2) to 0RF5 to GAT,
From ATG of arrow Ql) to 0RF6 to TGA of arrow 02) to the right in FIG. 2, to 0RF7 from GTA of arrow 03 to AAT of arrow a4 to the left of FIG.
Separate proteins are encoded in PRF8 from GTA 51 to AAT shown by arrow 08) to the left in Figure 2, and in 0RF9 from GTA 071 to AAT shown by arrow 08) to the left in Figure 2. There is. The amino acid sequences of the proteins encoded by 0RF1-9 are translated according to the universal codon table and are shown in FIGS. 3-11, respectively.

Each starts from the amino acid translation start codon ATG (the corresponding amino acid is methionine; MET) and ends at the stop codon.
The DNA base sequence is shown up to the previous codon.
The amino acids corresponding to each codon are shown below.

FIG. 3 shows 0RF1, and there is a possibility that 0RF1 encodes a protein whose translation is initiated from the ATG indicated by two arrows. 0RF2 shown in FIG. 4 may encode a protein whose translation is initiated from the two arrows ATG. In 0RF4 shown in FIG. 6, a protein is encoded from ATG indicated by an arrow.

0RF5 shown in FIG. 7 may encode a protein whose synthesis starts from the arrow ATG. Figure 8
0RF6, which is not shown, may encode a protein whose synthesis starts from the arrow ATG.

0RF7 shown in FIG. 9 may encode a protein whose synthesis starts from the arrow ATG. Figure 1
0RF8 shown at 0 may encode a protein whose synthesis starts from the arrow ATG.

In 0RF9 shown in FIG. 11, there is a possibility that the protein whose synthesis starts from the arrow ATG is heated.

The double-stranded DNAs-a and -b of the present invention can be used alone or in combination as a vector for plant genetic recombination. Further, DNA-a or -b can be cleaved at a specific restriction site with a restriction enzyme to form linear DNA. This DNA is mixed with a third DNA cut out using the same restriction enzyme at approximately the same molar ratio, and a DNA binding enzyme (e.g. T4-DNA ligase) is added using a conventionally known method.
③ A third DNA is attached to a specific site of a or Ib by
It can be a circular DNA containing.

Here, the third DNA mainly refers to DNA fragments encoding genes that can impart new traits to conventional plants. These include, for example, antibiotic resistance genes such as kanamycin resistance genes, neomycin resistance genes, methotrexate resistance genes, herbicide resistance genes (glyphosate resistance genes, atrazine resistance genes, sulfuron resistance genes, paraquat resistance genes, etc.); Examples include protein genes (eg, genes encoding phaseolin, glycinin, zein, etc.). Also, a combination of two or more of these
It is preferred as the third DNA because it can be more effective in selecting transformed plants. Such third D
When inserting NA into the DNA of the present invention, there are no restrictions on the insertion site, but it is preferable to insert using the protein coding region of 0RF1-9 described above.

Removing the protein synthesis start codon ATG and ending with the stop codon in one ORF, and introducing therein a third DNA containing ATG and ending with the stop codon in the same direction, avoids the [)NA duplication of the DNA of the present invention. This is preferable because the production capacity can be used effectively and the function of the protein encoded by the third DNA can be fully expressed.

If the third DNA encodes a gene that confers resistance to antibiotics (e.g. kanamycin or neomycin), this third DNA is
By using DNA into which a plant has been incorporated, plant cells can be transformed into cells resistant to the antibiotic (for example, kanamycin or neomycin).

If the third DNA encodes a gene resistant to a herbicide (e.g. glyphosate), plant cells are transformed by using DNA in which this resistance gene has been incorporated into DNA-a or -b, and the herbicide (e.g. glyphosate) resistant cells.

In this case, the transformation method includes, for example, a method in which the above-mentioned DNA is applied to a protoplast-formed plant cell in the presence of Ca, polyethylene glycol, or Ca+ in a high pH state, and further into the plant cell. There is a method of mechanically introducing the above-mentioned DNA (microinjection).

Further, an enzyme capable of cleaving the double-stranded DNA-a or -b of the present invention preferably at one site, such as Hind m
, CfaI, B0JITI, Xll1nI, or EC0RV, and can be inserted into other biological vectors cleaved with the same restriction enzymes as shown below. In this case, other biological vectors include conventionally known vectors, such as plasmid vectors for E. coli (e.g. p
BR322, pBR325, pBR328, pMB9.
1) KO2, etc.) cosmid vectors (e.g. 11K
Y 2662, etc.), phage vectors (e.g. Sharon 1, etc.), plasmid vectors for Bacillus subtilis (e.g. l) U B 110. pU B 112. l)S
A 0501゜pvp4. pTA1060.
pTA1020. pC194゜pC221,l)0
223 etc.), yeast vectors (e.g. YRp7.
YEp13. Y Ip32. pYCL.

pYC2, etc.) can be used.

U of hybrid DNA with these other biological vectors
A method uses double-stranded DNA-a and DNA-b.
Completely digest the mixture with, for example, HindlI, and mix this with the digested product of Hindl[[ of other biological vectors, such as plasmid 1IBR322 for E. coli.
A hybrid DNA of DNA-a and 1) BR322 can be obtained by cyclization using a DNA-binding enzyme (for example, T4-DNA ligase).

Similarly, a mixture of double-stranded DNA-a and DNA-b was completely decomposed with CJaI, and then 1) B
DNA-b and 1) BR32 by mixing with a CjaI-digested product of R322 and cyclizing it with a DNA binding enzyme.
Hybrid DNA with 2 can be obtained.

When obtaining these hybrid DNAs, the desired hybrid DNAs can be obtained more efficiently if the two DNAs to be combined are mixed in approximately equimolar proportions. Other biological vectors are cleaved with restriction enzymes and then treated with alkaline phosphatase to cleave the 5' DNA.
It is preferable to perform a terminal dephosphorylation reaction in preparing a hybrid DNA, since it is possible to largely prevent self-circularization using only vectors for other organisms.

The hybrid DNA thus obtained is also at least partially double-stranded, like the DNA shown in FIG. 1 or 2, and can be used as a vector for plant genetic recombination.

This hybrid DNA is transferred to the host of the original biological vector, for example, to E. coli in the case of a hybrid DNA with a vector for E. coli, to Bacillus subtilis in the case of a hybrid DNA with a vector for Bacillus subtilis, and to a vector for WfE bacteria. In the case of hybrid DNA with E. coli or Bacillus subtilis, it is transformed into competent cells according to known methods, and in the case of yeast, it is treated with Zymolyses to form spheroplasts. Transformants can be selected by using appropriate drug resistance or auxotrophy, or by performing colony hybridization or the like.

To explain in more detail one example of this method, for example, hybrid DNA is a mixture of the Hindl[I digested product of Figure 1 (CfaI digested product in the case of the DNA in Figure 2) and the Hindl[[digested DNA
In the case of -b, it is a hybrid DNA obtained by ligating (JaI digested product) with T4-DNA ligase.
1. Biol, 53159 (1970)] and ampicillin 50
Colonies generated on agar (L-Agar) plates containing μg/mf1 were analyzed using the method of Grunstein and Hogness [PrOc, Natl, Acad
, Sci, 723961] (in this case, 32p-labeled BGMV DNA is used as a probe),
Colonies having DNA that hybridizes with 32p-labeled BGMv DNA are selected. In this way, Escherichia coli HB 1 was transformed with the hybrid DNA in which the DNA of FIG.
01 can be obtained.

Hybrid DNA can be isolated from the transformant thus obtained by a known method. The separated hybrid DNA is completely double-stranded throughout, and as mentioned above, it can be transformed into plant cells by incorporating a third DNA, and can also be grown in E. coli etc. It is possible.

In order to prepare a large quantity of the desired hybrid DNA, a bacterial strain containing the hybrid DNA is grown, and if necessary, an operation is performed to amplify only the hybrid DNA to lyse the bacterial cells, and the DNA is extracted from the aqueous solution containing the hybrid DNA. Just separate it. When the hybrid DNA is, for example, a hybrid DNA with plasmid I) BR322 for Escherichia coli, the E. coli transformant containing the hybrid DNA is extracted from the E. coli transformant, for example, according to the experiment of Maninais et al.
lMo1ecular C1onina-A La
boratoryManual J [Co1d S
pring l-1arbor Laborator
y (1982)], pages 88 to 94, and amplification of the hybrid DNA using each lysis method.
The covalently closed circular (hereinafter referred to as "ccc")
It becomes possible to separate only hybrid DNA of the type (abbreviated as ).

In addition, by decomposing the hybrid DNA isolated in this way with the same restriction enzyme used for cloning, the DNA fragment shown in Figure 1 (or 2) and vector DNA can be obtained.
It can be divided into fragments. Preferably Figure 1 (or 2
) is separated and then ligated and circularized using 74-DNA ligase or the like. To separate only the DNA fragments shown in Figure 1 (or 2), agarose gel electrophoresis is generally performed, and the band portions of the DNA fragments shown in Figure 1 (or 2) are cut out from the gel and Nos. 164 to 17 of the experimental vehicle of Aniatides et al.
DNA fragments can be separated and purified from the gel according to various methods described on page 2. The DN thus separated
The A fragment is ligated and circularized by known methods, e.g. using T4-DNA ligase, with the DNA of FIG. 1 (or 2)
In addition to those in which only one A is self-cyclized, some are formed in which two or more A are bonded and cyclized. Only one of these self-cyclized molecules appeared on agarose gel electrophoresis as above 3.
It becomes a 3K bp band. DNA can be extracted and purified from this banded gel in the same manner as described above. This D
NA is completely double-stranded throughout the ring and is useful as a vector for plant genetic recombination.

When DNA obtained through cloning is often replicated in E. coli that has the dam methylase gene, certain base sequences, such as the N at position 6 of adenine in GATC, are methylated to prevent it from being degraded by specific restriction enzymes. I can do it. When the DNA-a of the present invention is not subjected to such methylation, C! It is cleaved at two places by a■, but when it is methylated, one of the places is not cleaved by CfaI because of the base sequence GATCGATC. This fact indicates that when the DNA-a of the present invention is used as a plant vector, C! a
■It can be advantageously used when producing recombinant DNA at the site.

Furthermore, DNA obtained by cloning has the same infectivity as a virus, as does DNA directly extracted from a virus.

In the present invention, the base sequences of DNA-a and DNA-b are determined by the so-called MaXam Gi Ibert method [M
eth.

ds in E naymology 65499-5
60 (1980)] and DNA fragments, J.M.
Cloning was performed using the M137 vector developed by Messing et al. [J, Messing et al.
Nucl, Ac1ds Res, 9309-32
1 (1981)], the so-called dideoxy method developed by F. 5anaer et al. [F. 5anaer et al.
at at, J, Mol.

Biol, 143161-178 (1980)
], the decision was made.

The base sequence of DNA-a is shown in Figure 1 of the present invention, and DNA-a
The base sequence of b is shown in Figure 2. In Figure 11 and Figure 2, the upper row of the duplex shows the sequence from the 5'-end on the left to the 3'-end, and the lower row shows the sequence complementary to the upper row from the 3'-end on the left.
The sequence is shown in the direction from the -end to the 5'-end. Furthermore, in Figure 1, the T at the 5' end of the base on the left side of the upper row is connected to the 2585th base T at the 3' end via the phosphate group, respectively.
-23'-linked to form circular DNA. At the same time, A at the 3' end of the base on the left side of the lower row is 3'-,
5'-linked to form circular DNA. In FIG. 2, similarly, the 5'-terminus of the bases on the left side of the upper row is combined with the 2647th base G at the 3'-terminus to form a circular DNA. At the same time, the 3' end A of the base on the left in the bottom row is 5
It is bonded to the 2647th base C at the 'terminus via a phosphate group to form a circular DNA. In other words, DNA-a
and -b are each circular DNAs.

The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereto.

250t of 57gr of BGMV-infected top crop leaves of a.cis.
ulno0.1M') Sodium chloride-1On+ME
Grind in DTA buffer (pi-1 = 7.8) (contains 1.3gr of cischain), filter through double gauze, centrifuge the filtrate at 10,000 (:, -40 minutes at 4°C), and A supernatant was obtained. To this supernatant, 2.4S of salt was added.
J, then polyethylene glycol (ly1w7800
~9000) was added and stirred at 4°C for 1 hour, followed by centrifugation at 10000G for 25 minutes at 4°C. Remove the supernatant and add 1M to the precipitated polyethylene glycol pellet.
Sodium phosphate buffer (pl-1=7.8
) Add 10- to make it uniform, and make this 10000G 30
The supernatant was centrifuged at 4°C for 4 minutes, the supernatant was removed, and the supernatant was centrifuged at 4°C for 4 hours at 30,000 rpm using a Beckman ultracentrifuge SW2O, 1O-ter to obtain the crude virus as a precipitate. . This crude virus was homogenized in 0.5 d of 0.1 M sodium phosphate buffer (pH = 7.8), and then subjected to continuous 10-40% sucrose gradient centrifugation (same 5W 40.10-ter as above). After centrifugation, centrifuge the tube for 0.6 d from the bottom of the centrifuge tube.
Fractions were collected one by one. The absorbance A260 of each fraction was measured, and a virus peak was observed in fractions Nα9 to 16.
These fractions were collected and homogenized by adding twice the amount of 0.1M sodium phosphate buffer (pH = 7.8), and the 5w40
．． A virus pellet was obtained again at 4°C for 3 hours at 35,000 rpm in a 10-meter machine, and this was added to 0.1M of 0.5IR1.
Homogenize in sodium phosphate buffer (1lH = 7.8) and repeat 10-40% sucrose continuous density gradient centrifugation (2,
The mixture was heated at 90,000 l'pm for 3 hours (4'C3w40.10-ter), and 0.6d fractions were collected from the bottom of the centrifuge tube.

Only virus peaks were obtained in fractions No. 13 to 18, and both fractions were collected and equalized by adding twice the amount of 0.1M sodium phosphate buffer (pH = 7.8).
．． The virus was precipitated at 60,000 rpm for 3 and a half hours at 4° C. at 3w40,10°C to obtain purified BGMV.

b Isis-NA-- ・Above purification B
GMV with 750 μm of sterile water, 15 μm of I M Fr
1s H(J (pi-17,6), 10 of 75μ ship
% sodium dodecyl sulfate (SDS) and 7.5 µm of proteinase K (1 µg/µρ solution), shaken at room temperature for 2 minutes, and then mixed with 10 mM Tris HCf (DH7,6
>-700μ of phenol saturated with 1mM EDTA aqueous solution was added, shaken for 3 minutes, centrifuged for 5 minutes in an Etzbendorf small centrifuge, and the aqueous layer was taken out. This aqueous layer was subjected to the same phenol extraction procedure twice more to obtain a final aqueous layer of 950 μm, and then the aqueous layer was extracted with 70 μm of chloroform.
After adding 0 µm of water and shaking for 2 minutes, the aqueous layer was taken out and 700 µm of ether was added to this aqueous layer to extract phenol. This extraction operation was performed three times.

Add 100μ of 3M sodium acetate buffer (+) 84.8) and ethanol to 1000μρ of the resulting aqueous layer.
5111i! and kept at 20°C day and night, and centrifuged at 350,000 rpm in a Beckman long centrifuge, 5w50.10-tar.
Centrifugation was performed for 0 minutes to obtain DNA as a precipitate. This DNA 1
0mM Tris HCj O, 1mM EDTA aqueous solution e
0.5 μg/μ of BGMV-heavy chain DNA dissolved in ooμu
It was made into an aqueous solution.

Ic BGMV 11 DNA in vitro 2
Chaining; BGMV-heavy chain DNA (0.5 μg/μ sentence) 2
Add 155 μJ of sterile water to μU, 1.1M Tris HCj (
pH = 8.0) At 30 μl, 6 bovine thymus DNA oligonucleotides (16.6 μg/
μm) Mix 12 μm and hold at 70℃ for 3 minutes, then 0℃
After rapidly cooling to 8011MM while keeping it at 0℃!
IICj 230μm, o, smM deoxyadenosine triphosphate, 0.8mM deoxyguanosine triphosphate and 0
．． 3 each of 8111M deoxythymidine triphosphate
0μ sentence, 0.8111M deoxycytidine triphosphorus μ
and deoxycytidine 5'-[α-32p]triphosphate (manufactured by Amasya Japan Co., Ltd. 3oooc; /11m+0
1.10iCi/ate code number pB 10205)
Add 2.4 μm and 2 μm of AMV reverse transcriptase (Seikagaku code 120248.5 units/μ day) at 20°C.
After reacting for 10 minutes at 37° C. for 1.5 hours, 50 μl of phenol was added, shaken, and centrifuged for 5 minutes in an Etzbendorf centrifuge, and the aqueous layer was gel-filtered. Gel filtration is 101MT
ris HCj (1)H7,4) -0,IMNa
Sephadex Q pre-equilibrated with Cf aqueous solution
-75 (manufactured by Pharmacia Fine Chemicals) with a diameter of 6
The layer tube was passed through a column packed with 5Id. After applying the reaction solution, 4 drops (approximately 100 μm) were taken out and the intensity of both portions was measured. The first peak appears in fraction Nα5-12, and unreacted α-32p appears after Nα15.
-A peak due to deoxycytidine triphosphate was observed. Fraction N (collect 15-12 and add 60μ of 3M Na C1
, 1.8 mfl of ethanol was added, kept at -20°C for 3 hours, and then heated at 25,000 rpm for 20 minutes (Hetsuman ultracentrifugation t
aSW50.10-ter), centrifuged and removed the supernatant, added a fresh 70% ethanol aqueous solution 2Id cooled to -20°C, centrifuged at 20,000 rpm for 2 minutes, and removed the supernatant. The precipitated DNA was dried under reduced pressure at 20 #1 #1 Hg for 1.5 minutes. Obtained D
NA was dissolved in 1 iMTris HCI and in V
Obtain double-stranded DNA with itrO.

(The intensity of 32p was 46x 10' cps) Preparation of Ikoma DNA Oligonucleotides 1 Bovine Thymus DN
A 66I115F 0. IMNa C110mMM.

C12,10+aMTris HCf (pH7,4)
DNase I [manufactured by Millibore Corporation (2182 units/g)] 460μ
0.2 M E
DTA O, 66m was added to stop the reaction. This reaction solution was subjected to protein extraction three times with 7d water-saturated phenol, and then extracted three times with 7d ether to remove phenol from the aqueous layer. Add 20% of ethanol to the resulting aqueous layer and leave at 20°C for 2 hours.
Centrifugation was performed at 0G for 4 minutes to obtain a DNA pellet. this DNA
Pellet was 0.5d. IM Na CI 10m
Dissolved in MTris H(J (pH 7,4), 0.I
MNaCjlolMTris HCf (1)l-17
, 4) pre-equilibrated S epl+adex G −
75 (manufactured by Pharmacia) (column length 42 cttr, column diameter 0.5 cIR) for approximately 1.2 d.
Aliquots were taken one by one. A large peak was seen in the horns of Nα 10 to 32, and the fractions of Nα 16 to 26 were collected (12.5 ad!).
After standing at 0°C for 30 minutes, centrifuge (4000G, 8 minutes) and dissolve the resulting DNA pellet in 11d of distilled water.
And so.

Comparative Example 1 Renovating thymus DNA oligonucleotide [16.6μ/μ
1) except when using 1.5μU instead of 12μρ
When a double-stranded experiment was conducted under exactly the same conditions as experiment C, the count of 32p in the polymeric DNA part was 8 x 1.
It can be seen that only 115 or less double strands have occurred compared to the case of 1C which was 0' cpm.

Comparative Example 2 The primer was Bioch by G.M. Theiler et al.
imica et Biophysica Acta
Primer mixture (DNA 1 degree 5
μg/μ base) 40p protein was used in place of the oligonucleotide (16,6 μg/μ base) 12μ base in the 1C experiment,
Double stranding was carried out in the same manner as in the 1C experiment except that 127 μl of sterile water was used instead of 155 μl.

The strength of 32p in the polymeric DNA part is 15x 10' c
There was pm t'. Double-stranded DN obtained in Comparative Example 2
Restriction enzyme C2 was added under exactly the same conditions as in the experiment 1d using A.
a I Hind I [I, Sa u I, etc., and autoradiography was carried out in exactly the same way as in experiment 1d below, and Hind II [the decomposed product was DN
A did not move at all from its slot position in the agarose gel. In addition, some of the substances digested with other restriction enzymes remained in the slot position, and D
The NA was also quite smeared and the band was not as clean as in the case of 1d.

3μ of 32p-labeled double-stranded DNA (32p strength 46 x 10' Clam / 100μ) obtained by lc (3μ
2p strength 7300cpm) and 4μ restriction enzyme digestion buffer - [100mM Tris HCf (p
H7,9), 70mM Mgs2C12, 70mM
β-mercaptoethanol and 0.1% bovine serum albumin are used as the basic solution. In CfaI digestion, this basic solution is called buffer art for restriction enzyme digestion, Aval, Hind.
m, B(l ma■, KpnI.

In PstI, xln decomposition, further NaC is added to the above base solution.
The solution in which 1 is added until it reaches 500 IllM is called the restriction enzyme digestion buffer.
, in ECOR■ decomposition, 1500 NaCl is added to the base solution.
The solution added to give a concentration of 111M is called a restriction enzyme digestion buffer. ] 4 μ base, 4 μ distilled water, 32 μ μ base, and restriction enzyme A
vat (21 nits/μn), Bgin (6 units/, C1, X1nI (6 units/μJl)
＞Nco engineering (3,5 units/μJl), EcoRV
(4,51 nits/u1. Hind m (5 units/μN), 5aUI (6-L nits/1lfl>,
Among the restriction enzymes of Cfa I (5 units/μ cross), CjaIEOORV is based on AV manufactured by Berger Mannheim.
aI is manufactured by Bethesda Research Laboratory, Inc., NC0T
, XmnI are manufactured by New England Biolapse Co., and the others are manufactured by Takara Shuzo ■. Add 4 units of water at 37°C.
DNA degradation was performed for more than 4 hours.

When decomposing with two types of restriction enzymes, first add a low-salt concentration enzyme, and then incubate at 37°C at a salt concentration suitable for the enzyme.
Hydrolyze for a few hours, then adjust the salt concentration to be suitable for the enzyme for high salt concentration, add the enzyme for high salt concentration, and further
Hydrolyzed for hours. After hydrolysis, 8μ of a solution of 0.25% bromophenol blue 50% glycerol 10% SDS was added, heat treated at 65°C for 5 minutes, and then 1.5%
Agarose gel electrophoresis was performed. The agarose used was Sigma's type ■ for electrophoresis. Electrophoresis buffer: 40 mM l”lis acetate, 2n+M
An EDTA (1)H8,0) aqueous solution was used. Thickness 5r
11 ~ at a voltage of 1.5 V/cI11 in a horizontal gel of H
Electrophoresis was performed for 15 hours. During this electrophoresis, DNA
E
Completely digested with COR and Hindll and pBR322D N A 002ug completely digested with TaqI were used. After electrophoresis, take out the agarose gel, dry it on a gel drying plate, and use p470-4 for the experiment by Maniathes et al. described in the text.
Autoradiography was performed as described in 72. As a result, DN was degraded and observed using various restriction enzymes.
Table 1 shows the sizes of the A fragments.

Furthermore, the DNA fragments observed after autoradiography can be classified into Group A, which is a group of fragments that appear more densely compared to the size of the fragment, and groups of 8, which are a group of fragments that appear faintly compared to the size of the fragment. .

Also, 3.3 in parentheses indicates that DNA is an oven circle (
0,C,), the apparent molecular weight of the linear DNA size marker is 3.3K bp. From this table, DNA 43 that produces fragments of group A
Before being digested with restriction enzymes, it is thought to be in the shape of an open circle (0, C,) and approximately 2.58 Kbp in size. Similarly B
The DNA that produces the group fragments is O20 before being digested with restriction enzymes.
It is described as having a size of 2.65 Kbt).

Next, by classifying each restriction enzyme alone and degrading it by a combination of the two types, and analyzing these decomposition patterns, the relative positional relationships of the various restriction enzyme cleavage points were determined, and A
Figure 3 was obtained as a restriction enzyme cleavage map for the intact DNA that gave rise to DNA fragments of group B, and Figure 4 was obtained as a restriction enzyme cleavage map for the intact DNA for the DNA of group B.

Table 1 1e knee N) lindI [I and -
50 pM of the 100μ solution of the double-stranded DNA obtained in Example 1c was added in advance to 10 mM Tris HCf (
3i equilibrated with 100 m M Na CR aqueous solution (pH 7.4).

Gel filtration was performed using a column filled with gel p30 with a diameter of 6 mrtr and a length of 30 cm. 1 as eluent
0mM Tris H(J (・H7,4), 100m
Using MNa C1 aqueous solution, 4 drops (approximately 400 αU> each) were fractionated. A high count peak of 32p was seen in the 11th to 21st fractions, and when the optical density 0D260 of the fractionated liquid was measured, the OD was found in the 28th fraction. The value started to increase and showed large values in both parts.
Collect fractions 7 to 21 (2.1m>), add 3M sodium acetate aqueous solution (1) 84.8>200μ and 4ae of ethanol, hold at 20°C for 2 hours, and then perform Beckman ultracentrifugation in a 1ASW50.10-tar. 30000rpm
Centrifuged for 30 minutes. Discard the supernatant and reheat to 75% at 20°C.
Add 5 d of ethanol and centrifuge at 20,000 rpm for 2 minutes.
The supernatant was discarded again to obtain a DNA pellet. 2#1llll-
After vacuum drying for 12 minutes, this DNA pellet was dissolved in 50μ of distilled water, and 50μ of this DNA solution was added with the restriction enzyme)-1indl1 digestion buffer (Example 1).
Description) 6 μl and restriction enzyme HindI were added and reacted at 37° C. for 4 hours. Next 10tl! l yeast t
An RNA (2 μ9/μm) solution and 70 μm of distilled water were added, and 100 μl of water-saturated phenol was added, followed by shaking and centrifugation to remove only the aqueous layer. From this aqueous layer, phenol was removed by extraction with ether three times.

3M sodium acetate (1)] = 4.8 > 10
After adding 350 µm of water and ethanol and leaving it for 4 hours at -20°C, it was centrifuged at high speed. The resulting DNA pellet was dried and dissolved in 17 µm of distilled water.

For this DNA 17 μm solution, the following “J-pBR3
22 Hindll [/atase treatment with alkali boss (
DNA 1μg/μ, Q) solution 1μρ and DNA-binding enzyme buffer 7-[650m MTriS HCj (pH
=7.4), 65 IIIMMg CI2.10 m
MDTT, 4mM ATP, 40mM
3 Permidine aqueous solution 12μ and T4
DNA ligase (Code Kami 2010B 1 manufactured by Takara Shuzo Co., Ltd.
, 2 pieces 1-/μfi) add 0.5μ and 14℃22
The mixture is reacted for a period of time, and then treated at 65° C. for 5 minutes to obtain hybrid DNA.

Next, using this hybrid DNA, E. coli H3101
Transform competent cells.

Add 40 μl of the above HindIII restriction enzyme buffer to a 340 μl aqueous solution containing 50 μ9 of lasmid pBR322 DNA.
μ polish and add I-(indl [[20μ
After reaction time 1 MTris HCj (1) H8,0>
Add 44 μl of bacterial alkaline phosphatase (RAP) (0.4
10 μm (unit/μU) was added and treated at 65° C. for 7 hours.

This reaction dephosphorylates the 5' end of DNA.

This reaction solution was subjected to protein removal using 400 µm of water-saturated phenol. Then phenol/chloroform (4
/1 volume ratio) The mixed solution was further removed with 400 μp twice to remove tampan, and finally the phenol component was extracted from the aqueous solution three times with 600 μp of ether.

To 350 μm of the aqueous layer, 30 μm of 3M sodium acetate and 1100 μm of ethanol were added, and after standing at -20° C. for 2 hours, it was centrifuged at high speed. The resulting DNA pellet was dried and dissolved in 50 μm of distilled water. 1) BR322 l-1ind
ll/alkaline phosphatase treatment (DN/'Mlμg
/μ sentence) liquid.

A single colony of E. coli 1-lB107 was grown in culture medium L-
The mixture was transferred to 5 ml of broth and cultured with shaking at 37°C for 11 hours.

This 2me was inoculated into fresh L-broth 200d and cultured with shaking at 37°C for 2 hours and 20 minutes until the 0D600 was 0.4.
When the temperature reached 0, the culture solution was cooled to 0°C for 11 L, and 5000 rpm using Tomy's cooling high-speed stretching machine Nα90-tar.
Centrifuged for 5 minutes. Discard the supernatant and remove the precipitated E. coli
The bacteria were homogeneously dispersed in 101 M Nacj water of η and centrifuged again at high speed (Nα 40-tar, 500 Orpm for 5 minutes) to precipitate the bacteria. To this bacterial pellet, 60 M CaCf water was added and the whole was homogenized and kept at 0°C for 20 minutes.

High-speed centrifugation again (Na 40-ter, 4000 rpm 5
10 minutes) Discard the supernatant and place it in a pellet pellet for 30 m
Add ICaCfz and a 15% aqueous glycerinol solution to gently homogenize the whole, dispense 200μ aliquots into 1.5ml Eppendorf tubes, and keep frozen at -80°C. This CaC92-treated E. coli 1-IBlol (
Transformation was performed by returning the combipunto cells to 0°C, adding an aqueous hybrid DNA solution that had been subjected to a ligation reaction using T4 DNA ligase, holding at 0°C for 40 minutes, and then incubating the combipunto cells at 0°C for 40 minutes. It was heated for 2 minutes at 37°C. Next, 1.5 d of 1-broth was added and heated at 37°C.
After holding for a period of time, this culture solution was spread on eight 1-agar plates (diameter 9α) containing 50 μ9/d of ampicillin at a rate of about 200 μp per plate. After keeping at 37°C for 16 hours, 41 transformed l-lB101 colonies were transferred to f!
? Ta. From these 41 colonies, 3 oiling lysis described in Experiment 1 of Maniathes et al., p. 328 was obtained.
A mini-preparation of plasmid DNA was performed by the method, and then the plasmid DNA was digested with )lindl1. As a result 2.
58 Kbp, 1. f33 Kbp, 1.02
A plasmid containing Kbl) was obtained. 2.58 Kbp
The hybrid DNA of the fragment and 1) BR322 is transferred to pBG.
Hlo, pBGHlo DNAG, t pBR32
Within host E. coli of 2? ! Even when SI nuclease was applied to the DNA grown from the second product, there was no change at all, and the entire DNA was completely double-stranded.

In the Ie experiment, double-stranded DNA and pBR322 were
indI [change the decomposition to ClaI decomposition (change the salt concentration during decomposition to a low concentration for Cfat decomposition)
Perform almost the same procedure as Ie, and add 17μ of sterile water of CIa1-digested double-stranded DNA and CIa of pBR322.
Using 1 μl of alkaline phosphatase treatment (DNAI μg/μ) solution, 2 μl of the DNA binding enzyme buffer, and 0.5 μl of T4 DNA ligase at 14°C 22
[Hybrid DN
I got an A. Using this hybrid DNA, hybrid DNA was obtained in the same manner as in the le experiment. Using this hybrid DNA, E. coli H
B1oi combipunto cells were transformed and 41 transformants were obtained. These 41 colonies are boiled as in the case of IO.
' Perform mini DNA preparation and use this plasmid DNA
A was digested with ClaI. As a result, 2.65Kbp,
Three hybrid plasmids containing fragments of 1.33 Kbp and 1.25 Kbp were obtained. 2.65 Kb
One of the hybrid plasmid DNAs containing the p fragment was
1B G C10. tlB G C10 was replicated in Escherichia coli and underwent no changes even when treated with St nuclease (the entire DNA was completely double-stranded).

Hybrid DNA 11BGH10 and ps G
Using 20 μ9 each of C10, 20 units of HindII, and 120 units of CIa, the salt concentration was set using the restriction enzyme buffer described above, and a total of 400 M
Hydrolysis was carried out at 37°C for 10 hours. Next, 0.8% agarose gel (slot width 1.5111111. length 6C)
IIi, depth 7m, gel ring 13cm) using 1V/c
After 10 hours of electrophoresis with II+, 2.58 Kbp,
Cut out the gel in the 2.65 Kbp band and extract DNA from the gel using the method described in the experimental book by Maniais et al., p. 164-5, and then extract the DNA according to the method described in p. 166 of the same book.
A was purified. 50 μU of sterile water (approximately 0.0 μU of DNA) for both.
1μg/μs). Add the DNA ligating enzyme buffer 5 to 20μ of each linear DNA aqueous solution.
μ-mon, sterile water 25 μ-mon.

After adding 0.5μ of T4-DNA ligase and reacting for 19 hours at 12°C, 0.8% agarose gel electrophoresis was performed again in the same manner as above.
The NA band portion was cut out along with the gel, and only the DNA was extracted and purified in the same manner as described above. It is thought that this DNA is circular as shown in FIG. 1 and as shown in FIG. 2, respectively. Next, add 10μ of sterile water to each of these DNAs, and
The μ sentence was partially decomposed using Hind m and CIaI, respectively. For partial decomposition, add 5μ of the restriction enzyme buffer to a 2μ solution of DNA, add 43μ of sterilized water,
Furthermore, 0.5 HindIII was added to the DNA in Figure 1 (7).
Add 5 units of ClaI O to the DNA shown in Figure 2, react at 37°C for 5 minutes, 10 minutes, and 30 minutes. Each reaction product was subjected to 0.8% agarose gel electrophoresis to observe changes in size. Ta. As the reaction slowed down for 5, 10, and 30 minutes, the 3.3 Kbp band gradually decreased, as shown in Fig. 1D.
Linear DNA of NA and Figure 2 DNA 2.58 Kb
p, 2.65 Kbp.

bands increased, and no other bands were recognized. From this, it is thought that the above DNAs are both monolithic and circular, and are the DNAs shown in FIGS. 11 and 2, respectively.

Infectivity with Ih cloned DNA; hybrid DNA obtained with le 11BGH10 (2.3μg/μρ)
50B, 211MMCl C1210μ barley, Hi
ndI [restriction enzyme buffer 26 μp.

Add Hindn [8μu, sterile water 156μ, (37℃
After treatment for 10 hours and further treatment at 65°C for 5 minutes, NaCl and sodium citrate were added at 0.15 M and 0.01, respectively.
Add until it reaches 5M. Pour this liquid into O-ning A-DN.
It was made into liquid A. ■Hybrid DNA pBGC obtained with r
10 (0,68μg/u fl') 200μi
to 2mMM.

(J220 μΩ, the above (JaI restriction enzyme buffer 2
Add 6 μfl, (Ja 1101i, Q, 4 μl of sterilized water, treat at 37°C for 10 hours, and further treat at 65°C for 5 minutes.)
M, was added until it became 0.015M. This solution was designated as Cloning B-DNΔ solution. Cloning A-DN
160 µm of each of A solution and B-DNA solution were mixed, and this mixed solution was placed in a biotron (Koito Manufacturing Co., Ltd. Control Co., Ltd.) set at 30 °C for 14 hours during the day, 27 °C for 10 hours at night, and a humidity of 80% or higher. A total of 10 plants were inoculated by rubbing the mixture with Celite on the surface of young leaves of top-cropped kidney beans, 6 days after germination, using sterile gloves in Tron). . The conditions in the biotron were maintained as described above even after inoculation, and the onset of bean gall antrum mosa ita disease was observed on the true leaves of top-cropped kidney beans. On the 10th day after inoculation, bean gall mosaic disease was clearly observed in all 10 individuals.

As a control, the DNA solution was used as a control.
Instead of 10 1) BR322 approximately 20μ9 to 1ind
lI [digested solution and pB G C1 Or I
) Using a solution obtained by decomposing approximately 20 μ7 of BR322 to cpa
A similar infectivity test was carried out on 4 specimens, but all 41,171 specimens showed normal growth, although mosaic disease does not occur.

The above shows that the DNA in Figure 1 and the DNA in Figure 2 are useful for genetic recombination of plants.

[Brief explanation of drawings]

Figure 1 - (11 to (4+) indicate the base sequence of DNA-a of the present invention. In Figure 1, arrows (1) A T G and arrow (2) T'
GA indicates the start and end of 0RF1. Further, arrow (3) GTA and arrow (/1) AAT indicate the start and stop codons of ORF2. Figure 2-(1) to (4) show the base sequence of DNA-b of the present invention. In FIG. 2, arrow (5) ATG and arrow (61T
AA indicates the start and stop codons of ORF3. Arrows (Katana GTA and arrow (8) AAT indicate the start and stop codons of 0RF4. Arrow (9) G T A and arrow 00) GAT do not indicate the start and stop codons of 0RF5. Arrows [It) ATG and arrows 02) TGA indicate the start and stop codons of ORF6. Arrow Gel G T A
and arrow 04) AAT indicates the start and end IL codons of ORF7. Arrow (15) G T A and arrow 06)
AAT indicates the start and stop codons of 0RF8. Arrow 07) GTA and arrow □□□△AT indicate the start and stop codons of ORF9. FIG. 3 does not show the base sequence of 0RFI and the amino acid sequence corresponding to each codon. The arrow ATG (MET) indicates the starting point for the synthesis of multiple proteins. 4 to 11 show the base sequences of 0RF2 to 0RF9 and the amino acid sequences corresponding to their codons, respectively. Arrow ATG (M
ET) indicates the starting point during multiple protein syntheses. Figure 1-(1) Lfflljl, 1L+1Lal/AIaA L;Li
UAljL; Itj rTGT＾GTT＾GATT＾
TTCA13^TT^CTT 2nd - (e)

Claims (1)

[Claims] 1. Circular double-stranded DNA having the base sequence shown in FIG. 1
A. 2. Add the vector DNA for other organisms to the DNA described in Section 1.
Hybrid DNA obtained by integrating into. 3. The hybrid DNA according to item 2, wherein the vector DNA for other organisms is selected from DNAs capable of self-replication within the cells of Escherichia coli, yeast, or Bacillus subtilis. 4. Circular double-stranded DNA having the base sequence shown in Figure 2
A. 5. Add the vector DNA for other organisms to the DNA described in Section 4.
Hybrid DNA obtained by integrating into. 6. The hybrid DNA according to item 5, wherein the vector DNA for other organisms is selected from DNAs capable of self-replication within the cells of Escherichia coli, yeast, or Bacillus subtilis.