JPS63126486A - Hybrid dna tandem containing dna fragment of gemini virus - Google Patents

Abstract

PURPOSE:To obtain a hybrid DNA, containing DNA having sequentially >=2 linked DNA fragments derived from gemini viruses in the same direction and useful as a vector for introducing exogenotes into plant cells. CONSTITUTION:A hybrid DNA, prepared by directly linking DNA fragments which are obtained by converting single-stranded DNA of gemini viruses into double-stranded DNA by recombining operation and containing a region considered as a replication starting point of at least the gemini viruses DNA or DNA fragments of size 2,500-2,800 based pairs and containing a transcription starting region of a coat protein in addition to the replication starting region in replication intermediate DNA of the gemini viruses by treatment with a restriction enzyme or ligase reaction so as to provide at least two of the DNA fragment of the above-mentioned viruses having the same sequence and plasmid vector DNA, etc., for Escherichia coli linked to the other end. Exogenotes can be introduced into the afore-mentioned DNA and then into plant individuals.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention is directed to the use of dieminivirus DNA to be used as a vector for introducing foreign genes into plants or plant cells.
It relates to hybrid DNA containing fragments. More specifically, a hybrid D containing DNA in which two or more geminivirus-derived DNA11'i fragments are linked in the same direction.
Regarding NA.

<Prior Art> DNA genes from viruses are recently being developed and used as vectors in genetic recombination technology. In particular, the DNA of viruses such as the animal virus simian virus (SV) 40, papillomavirus, bovine virus, and adenovirus are often used. These were discovered as animal vectors and do not proliferate in plant cells. Therefore, these cannot be used as vectors for genetic modification of plants.

To date, tomatoes have been used as plant vectors that can be used to introduce foreign genes into plants.

An 11-plasmid possessed by Agrobacterium tumfaciens, which causes tumors in tobacco and other plants.

The only known DNA was cauliflower mosaic virus, which causes diseases in Chinese cabbage and cabbage. Ti
-Plasmid, cauliflower mosaic virus DNA
There have been reports of cases in which foreign genes are linked together and transferred to plants, but the plants that can be used are mostly limited to dicotyledonous plants. Rice is an important grain for humankind.

The development of vectors that can be applied to monocotyledonous plants such as wheat and corn has extremely important value and is also of industrial value.

D that has the potential to become a vector for genetic recombination
The cauliflower mosaic virus described above has been known for a long time as a plant virus having an NA gene, and research on its vectorization is also progressing.

Cauliflower mosaic virus DNA is found to be infectious to cabbage and Chinese cabbage even if it is grown in E. coli.
G◆Reported by Lebel Jrier (G) et al.
ene 12139 (1980)], and the same authors Pr0C, Natl, ^cad.

Sci, USA 792932-2938 (19B
In 2), cauliflower mosaic virus DNA
The E. coli vector DNA p
It has been reported that DNA of cauliflower mosaic virus was obtained by inoculating turnips while conjugated with BR322. The authors' research results opened the door to the use of cauliflower mosaic virus DNA as a plant vector, but 1
984 cauliflower mosaic virus is common DNA
Unlike viruses, reverse transcription occurs through RNA during replication, as reported by M Polovitzi et al.
3309 (1984)] and is considered to be a unique virus.

On the other hand, in recent years, a group of viruses called geminiviruses have become known. Geminivirus is a circular single-stranded D virus with a size of 2500 to 2700 bases.
It is a plant virus that carries NA as a gene, and is known to infect and multiply in tobacco, tomatoes, beans, wheat, corn, etc. That is, the host region where geminiviruses infect and propagate is not limited to dicotyledons but also extends to monocotyledonous plants, whereas Ti-plasmid and cauliflower mosaic virus are mostly limited to dicotyledonous plants. Therefore, vectorization of geminivirus itself is extremely significant.

Geminivirus was first discovered in 1977 by Bart M. Gutsman et al.
(abbreviated as GMV), and its genes are approximately 2,500.
discovered that it is a circular single-stranded DNA consisting of bases [Virology 83.171 (1977)],
After that, Mung Bean Yellow Mosaic Virus (MYMV) [P.
Tong Meerkom et al. phytopatho-1ogy
Wataru 41 (1981)], Tomato Golden Mosaic Virus (TGMV) [J. C. Matthiis et al. Summa Phytophathological
2B? (1975)], Catsara tent virus (CLV) [B.D. Harrison et al., Nature
270760 (1977>), Tobacco Leaf Curl Virus (TLCV) [Ozaki et al. Japanese Society of Plant Pathology Bulletin 4
562 (1979)], Euphorbia mosaic virus (EMV) [K. Nis. Kim et al. Phytopat
hology 69980 (1979)], ]
Tomato Y Q-do IJ-7 virus Tomato Y
DV) [Ozaki et al. Japanese Society of Plant Pathology Bulletin 44167
(1978) ], ] Beat Curly Top Virus BT
CV) [De Elu? Mufuod, Phytopa
thology 64136 (1974)], maize streak virus (MSV) [B.D./'1 Rison et al. Nature 270760 (1977)], and dwarf virus WDV>.

Cloris striate virus (C8V) [R.I.B. Francesky et al. Viroloc+y
101233 (1980)1. Miss Kansas Streak Virus (HisV) [Yamashita et al. Bulletin of the Japanese Society of Plant Pathology 51582 (1985) is known.

All of these are circular double-stranded DNA whose genes are approximately 2,500 to 2,800 bases long, and the early cauliflower mosaic virus (circular double-stranded DNA).

A completely different type of DNA (approximately 8 kilobase pairs in size)
Since it is a plant virus, it is necessary to develop a unique technology to turn it into a plant vector.

Structure of the present invention> The present inventors have discovered that one of the geminiviruses, tJIDDNAe,
Hybrid DNA is a hybrid DNA in which double-stranded DNA or double-stranded DNA in an intermediate state of replication of gemini virus genes is incorporated into E. coli vector DNA, yeast vector DNA, etc. so that it is technically easy to handle in genetic recombination operations.
As a result of conducting research on gemini virus DNA, it was surprisingly discovered that DNA in which at least two DNA fragments are linked in the same direction infects plants and multiplies well, resulting in the present invention. It is.

That is, the present invention relates to a hybrid DNA containing DNA in which at least two geminivirus DNA fragments are linked in the same direction.

As mentioned above, more than 10 types of geminiviruses are known, and their genetic DNA was published in Japanese Patent Application Laid-Open No. 60-5468.
It can be made into double strands by the method described in Publication No. 3 and Japanese Patent Application Laid-Open No. 60-54684. The DNA thus double-stranded can be used as the geminivirus DNA fragment of the present invention.

In addition, in geminiviruses, the replication intermediate type DNA is background music.
V (see Japanese Patent Application Laid-Open No. 60-54684), MYMV
(Refer to Japanese Patent Application Laid-Open No. 60-54683), TGMV [
Hamilton et al Nucleic Ac1ds Re5ea
rch, 104901 (198, 2)], and it is believed that these are commonly present in other geminiviruses as well. Among these replication intermediate DNAs, those with a size of 2500 to 2800 bases'
A circular double-stranded DNA consisting of can also be used as the geminivirus DNA fragment of the present invention.

These geminivirus DNA fragments may contain the entire length of the geminivirus gene, or may be partially deleted. At least Gemini virus D
It is sufficient that the region includes a region considered to be the origin of NA replication. The area is BGMV.

MYMV, TGMV, and CLVk-tjT are two types of DNA (that is, bisegmented DNA) found in each of these regions, each consisting of about 200 bases with extremely high base sequence homology. In the geminivirus DNA, it is a region consisting of about 200 bases that has 50% or more homology with the base sequence of the replication initiation region found in the four types of geminiviruses mentioned above.

Further, when the geminivirus genetic DNA is bisegmented, the segmented DNA that encodes the viral coat protein preferably contains not only the DNA replication initiation region but also the transcription initiation region of the coat protein. The transcription initiation region of the coat protein is a region from the replication initiation region of about 200 bases of geminiviruses with high base sequence homology to the protein translation initiation signal codon (ATG> of the coat protein).

The present invention is characterized in that two or more DNA fragments of the dieminivirus are linked in the same direction (generally referred to as 'tandem'). When two or more viruses are linked in tandem, the infection and proliferation of geminivirus-derived DNA becomes better when introduced into plants, and the performance as a vector becomes correspondingly higher.

Furthermore, in the present invention, in addition to a DNA portion in which at least two digit tandem DNA fragments of dieminivirus are linked, a hybrid D having DNA of a DNA replication origin and a selection marker gene that acts in Escherichia coli, yeast, or Bacillus subtilis is used.
NA is particularly advantageous because the hybrid DNA can be amplified and prepared in E. coli, N mother bacteria, or Bacillus subtilis.

The DNA of the DNA replication origin and selection marker gene can be used in E. coli plasmid vectors (e.g. 1) BR322, 1) BR
325, pBR325, pBR328, 1) NEO, p
UC8゜pKC7, pH89, etc.> Cosmid vectors (e.g. pKY2662, etc.), phage vectors (e.g. Sharon 1, etc.), yeast vectors (e.g. YR
p7゜YEI)13. YIp32. pYcl, p
YC2, etc.), plasmid vector for Bacillus subtilis (pUB
llo, pUB112.1) SAO501゜plP4
．． pTA1060. DE194. pTA1
020. pc194. pC22LpC223, etc.).

Among these, plasmid vector DNA for E. coli is preferred because it is the easiest to handle and the DNA can be easily prepared and amplified.

In the present invention, a hybrid DNA in which at least two geminivirus DNA fragments are directly linked in the same sequence direction and a plasmid vector DNA for E. coli, for example, is linked to the other end is easy and simple to create. preferred.

The hybrid DNA of the present invention can be introduced into a plant solid or a plant cell in a covalently closed circular (CCC> form [a commonly used term indicating the form of DNA]) and can be introduced into a plant body. Since the geminivirus DNA fragment portion is extremely easy to replicate and propagate, it is a hybrid DNA with an excellent structure and can be said to be an excellent plant vector.

In the hybrid DNA of the present invention, the site into which the foreign gene is introduced is the hybrid DNA into which the foreign gene has been introduced.
There are no particular restrictions as long as the site can be replicated and propagated in the plant. The foreign gene to be introduced is inserted into a geminivirus DNA fragment or bound to one part of the geminivirus DNA fragment, so that at least two units are the same, with the geminivirus DNA fragment and the foreign gene being one unit. Preferably, the hybrid DNA is formed by directional sequence linkage.

The foreign genes to be introduced are mainly DNA fragments encoding genes that can bring new traits to conventional plants. Such as, for example, antibiotics (e.g. kanamycin, neomycin, melitrexate, etc.)
Resistance genes, herbicides (e.g. glyphosate, atrazine, sulfuron).

Balaquat, etc.) resistance genes, consisting of species storage protein trade genes (eg phaseolin, glycinin).

Genes that code for Biin, etc.).

In order to introduce the hybrid DNA of the present invention into a plant solid, for example, 0.15) INaα-0 of the hybrid DNA
, 015M sodium citrate aqueous solution (DNA'ail
O ~ 200 μg/11 f) was added to Celi
te) to the plant body. Alternatively, in order to introduce Ca2 into plant cells, add Ca2 to protoplast cells.
+ and a method of acting the hybrid DNA of the present invention in the presence of polyethylene glycol, a protoplast cell or a cell with a cell wall, and the hybrid DNA of the present invention
the so-called electroporation method or electroinjection method by electric shock in the presence of
Furthermore, there is a method of mechanically directly introducing the DNA of the present invention into plant cells (microinjection).

In particular, the hybrid DNA of the present invention does not require the assistance of bacteria to be introduced into plants, unlike the Ti plasmid, and can be used to infect the entire body of a plant simply by direct choreography, making it an excellent vector. I can say it.

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

Example 1 DNApBG described in JP-A No. 60-5468, 11
fll (3μ9) and I) BGCI (3μg)
Restriction enzymes [manufactured by Takara Shuzo]) tindll, C1a
Completely decompose the decomposed product according to the supplier's instructions, and perform agarose gel electrophoresis on the decomposed product in the same manner as described in the earlier publication of JP-A-Sho, to obtain DNA fragments of Bean Goal Ann Mosaic Virus. [2.58 kilobase pair DNA fragment from pBG old (hereinafter referred to as old fragment), 1) BGC
From I, a 2.65 kilobase pair DNA fragment (hereinafter referred to as C1
fragment)] and E. coli vector plasmid DN
A pBR322 fragment [p13R32 from p8GH1
2-H fragment (referred to as pBR322-C fragment from pBGcl) was purified in the same manner as in JP-A-60-546841g, and dissolved in 20μ of sterile distilled water.

10 µl of H1 fragment aqueous solution, 1 µl of p8R322-H fragment aqueous solution, 3 µi' of 10x DNA ligating enzyme buffer described in JP-A-60-54684, T4-DNA ligase [
Takara Shuzo Co., Ltd.] 0.5 μl and 14.5 μl of sterilized distilled water were added and reacted for 16 hours at 3°C. Using 5 μl of the reaction solution, ``Preparation of competent cells of Escherichia coli II 8101'' in JP-A-60-54684 was carried out. Competent cells of Escherichia coli 88101 were transformed by the same method as described in the section ``And Transformation thereof'', and several hundred colonies were obtained on L-agar plates containing 50 μg/- of ampicillin.

Plasmid DNA was extracted from 10 of these colonies using the BOilinfj Lysis method similar to that used in JP-A No. 60-54684.
A mini-preparation of A was performed and the plasmid DNA was analyzed.

This plasmid DNA was digested with restriction enzyme 1tindll[,8
By decomposing and analyzing each of g[11, three types of
Place the two old fragments in the same sequence direction as Hin of pBR322.
It was found that it binds to the du site. One of these hybrid DNAs is designated as 8H[)101. Another type is to insert three tiI fragments into pBR32 in the same sequence direction.
It was found that it binds to the tlind11 site of 2. One of these hybrid DNAs is designated as p811T101.

Using 10 μl of CI fragment aqueous solution and 1 μl of 1) BR322-C fragment aqueous solution, the same reaction as that used to obtain early pBHDIOI was performed. Several hundred transformed colonies were obtained, and the plasmid DNA of 15 of them was analyzed. I did it. Digested with restriction enzymes C1a I and Bgl II.
Through analysis, it was found that five types of fragments bind two CI fragments in the same sequence direction to the C1a I site of pBR322. One piece of this hybrid DNA is p[5cI)101.

In yet another type, three CI fragments were ligated to the C1a 1 site of pBR322 in the same sequence direction. This plasmid is designated pBcTlol.

Plasmid DNADBHDIOI, I) B11T10
1, pBcDlol.

pBclol by Maniatis et al. [)1o1ecul
arCloning-A Laboratory Ma
A large volume of each purified DNA was prepared from approximately 800 m of culture medium according to the method described in pages 86 to 96 of Nual J (edited by Cold Spring Heber), and 1 to 2 mCl of each purified DNA was obtained.

These DNA1.15HNaCf2-0.015)1
A mixture of the combinations shown in Table 1 dissolved in a sodium citrate (hereinafter referred to as "1 . In all? O individuals were inoculated and grown in a Biotron (Koitoron manufactured by Koito Seisakusho) at 30°C for 14 hours during the day, 27°C for 10 hours at night, and a humidity of 980% or higher, and the onset of Bean Goldan mosaic disease was observed. The number of individuals observed to develop the disease 100 days after inoculation and the incidence of disease symptoms are as shown in Table 1 below.

Comparative Example 1 For comparison, pBR322DNA fleas were inoculated in the same manner as in Examples 1 to 3 and disease symptoms were observed, but the disease symptoms of Bean Goal Anne Mosaic were observed even 10 to 20 days after inoculation. There wasn't. The results are also shown in Table 1.

Examples 1 to 3 show that the hybrid DNA of the present invention can be inoculated into plants even when it contains the E. coli vector plasmid pBR322, and that BGM-derived DNA infects and proliferates throughout the plant. Therefore, it can be said that the hybrid DNA of the present invention is useful as a plant vector.

Furthermore, collect the liquid medicine from the DNA-inoculated individual, and add 1g of it to 0.1
M Sodium Phosphate 50mHE DTA-10%S
[)S aqueous solution (1)H8,4)3rn! and 3d phenol/chloroform (4/1 volume mixture) in a mortar, centrifuged at 4000 rpm for 5 minutes, and the aqueous layer was further extracted three times with a phenol/chloroform mixture. Aqueous solution (pH
5,4) and ethanol 4rf11, and heated to -20'C.
After 2 hours, centrifuge and add the precipitated DNA pellet to 1 liter of distilled water.
3 μm of this solution was dotted onto a nitrocellulose filter and 0.5HNaOH/1.5HHa
After treatment with Cal solution for 30 seconds, 1. OHTrisHCu
After neutralization with a solution (pH 7,0), the nitrocellulose filter was treated at 80°C for 2 hours.

Next, using the same 32D-labeled BGMV DNA as described in JP-A-60-54684 as a probe,
)tolecular Cloning-A Labo
DNA-DNA hybridization was performed in the same manner as in Ratory Manual J, pp. 387-389, and autoradiography was performed using a commonly used method to detect BGMV in the collected leaves of Examples 1 to 3 and Comparative Example 1. We investigated whether Yamaki's DNA could be detected.

The results are also shown in Table 1 above.

Example 4 DNA pBGHlo and pBGclo described in Japanese Patent Application No. 60-98108 were treated in the same manner as in Examples 1 to 3,
DNA fragment of Bean Goal Ann mosaic virus [1]
A 2.58 kilobase pair DNA fragment (
HIO fragment), 2.65 from pBGclo
A kilobase pair DNA fragment (called a C10 fragment)] was obtained,
The old O fragment and 1) BR322-11 fragment were ligated in the same manner as in Examples 1 to 3, E. coli t18101 competent cells were transformed, and the same treatment analysis revealed that the two old O fragments were aligned in the same direction. The resulting hybrid DNA pBflD102 was ligated to the old ndln site of pBR322.

Similar processing and operations are performed on the CIO fragment, and the CI
Two O fragments are linked in the same direction to form the C of p8R322.
(Hybrid DNA pBChi1 bound to 2aI site)
I got 1q of 02.

Using pBllD102 and DBCDIQ2 DNA, predetermined lengths of DNA shown in Table 1 were inoculated into young kidney bean leaves in the same manner as in Examples 1 to 3, and disease symptoms were observed and geminivirus-derived DNA was detected. The results are shown in Table 1 above.

It can be confirmed that the hybrid DNA of this example also infects the entire kidney bean and that the gemini virus DNA is proliferating, indicating that it is useful as a plant vector.

Example 5 DNA pBGRF described in JP-A-60-54684
tll and pBGRFcl were treated in the same manner as in Examples 1 to 3 to obtain Bean Goal Ann mosaic virus DNA.
Fragment [RFI11 fragment from pBGRFHl, 1)B
RFCI fragment] from GRFCl, RFHI fragment and DBR322-H fragment, RFC1 fragment and pBR322
-C fragments were ligated in the same manner as in Examples 1 to 3, and treated and analyzed in the same manner. The RE old fragment is the old nd of pBR322.
Hybrid DNA bound to I11 site) BHRF
DIOI, RFCI fragment is cf2aI of pBR322
Hybrid DNA pBcRFD bound to site
Got lol.

DNA of pBHRF[)101 and pBcRFDlol
As in Examples 1 to 3, a predetermined amount of DNA from Cloth 1 was inoculated into young green bean leaves, and the appearance of mildew resistance and detection of geminivirus-derived DNA were performed. The results are also shown in Table 1.

The hybrid DNA of this example is similar to Examples 1 to 4,
It was confirmed that the entire kidney bean was infected and the geminivirus DNA was propagated, indicating that it is useful as a plant vector.

Claims (1)

[Claims] 1. Hybrid DNA containing DNA in which at least two geminivirus DNA fragments are aligned in the same direction.
. 2. The gemini virus is selected from the group consisting of bean golden mosaic virus, mung bean yellow mosaic virus, tomato golden mosaic virus, cassava tent virus, tobacco leaf curl virus, maize streak virus, wheat dwarf virus, and cloris striate virus. The hybrid DNA according to claim 1, which is a hybrid DNA. 3. The hybrid DNA according to claim 1, wherein the gemini virus is bean golden mosaic virus or mung bean yellow mosaic virus. 4. The hybrid DNA according to claim 1, wherein the DNA fragment is a double-stranded DNA of a geminivirus gene. 5. The DNA fragment is a replication intermediate DNA of gemini virus.
The hybrid DN according to claim 1 which is A
A. 6. The hybrid DNA according to claim 1, wherein the DNA fragment contains at least a geminivirus DNA replication origin and a virus-encoded protein transcription initiation site. 7. In addition to DNA in which at least two DNA fragments of the geminivirus are linked in the same direction, the virus has a DNA replication origin and a selection marker gene DNA that acts in E. coli. hybrid dna
. 8. Claims that include, in addition to DNA in which at least two DNA fragments of the geminivirus are linked in the same direction, a DNA replication origin and a selection marker gene that act in yeast or Bacillus subtilis. Hybrid DNA according to item 1. 9. The hybrid DNA according to claim 1, wherein a foreign gene is bound to the geminivirus DNA fragment or to one end of the fragment.