JPS6314693A - Plant virus rna vector - Google Patents

Abstract

PURPOSE:A plant virus RNA vector useful for transformation of plant cell, obtained by replacing a coat protein gene range of tobamovirus with an adventitious gene. CONSTITUTION:Plasmid pBR325 containing chloramphenicol acetyltransferase (CAT) gene derived from Escherichia coli HB101 strain is treated with restriction enzyme to construct pCL29 (A) having a coat protein gene range of tobacco mosaic virus (TMV) of pLFW3 replaced with CAT gene. On the other hand a fragment of pt5L29 is linked to a fragment of ptC3L5-2 to construct pCLB29 (B). Then the components A and B are digested with MluI, subjected to transcription reaction with Escherichia coli RNA polymerase and prepared RNA is purified by phenol extraction and ethanol precipitation.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to plant viral RNA vectors,
This invention relates to transcription vectors for producing vectors, vectors 1 and 1, methods for producing these, and methods for introducing foreign genes into plant cells.

・Prior art Tobamovirus is a rod-shaped RNA virus isolated from plants such as tobacco, tomatoes, cowpeas, and cucumbers.Tobacco mosaic virus (TMV), which uses tobacco and tomatoes as hosts, and cucumbers, etc., as hosts A. cucumber green spot mosaic virus (CGMM)
V) etc. belong to this category.

It is known that there are several types of TMV, such as a common type isolated from tobacco, a tomato type and a cowpea type isolated from tomatoes and cowpeas.

Already, tomato T and MV-L strains and its attenuated strain TMV-
The nucleotide sequences of several types of tobamoviruses such as the LIIA strain have been determined and reported (for example, NishiN15h
i et al: Nucleic, Ac1ds
Res, 13°5585 (1985); Kaida: Cell Engineering Vol. 1.4. No, 11. P, 979-990
(19'85)").

In addition, we created full-length cDNA of TMV-L strain and TMV-L, A strain RNA, and used it with Ahlquist (Ahlquist).
After cloning into the transcription vector pPM1 developed by E.
Successfully regenerated NA.

The present inventors further conducted research for the purpose of utilizing vamovirus RNA as a plant vector for use in transforming plant cells, and completed the present invention.

Summary of the Invention The present invention provides an RN for use in introducing foreign genes into plant cells.
A vector, said 1'? Provided are transcription vectors for producing NA vectors, methods for producing them, and methods for introducing foreign genes into plant cells.

DETAILED DESCRIPTION OF THE INVENTION The RNA vector of the present invention can be produced by replacing the coat protein gene-j'pfl region of I. hamovirus with a desired foreign gene.

It is known that the tobamovirus genome encodes four types of proteins: two types of proteins, which have been identified as tll, which are involved in virus replication, a 30-year-old protein, and a coat protein. , T
Regarding MV, it is known that it consists of four types of genes: protein 130 and its read-through protein 180, protein 30, and coat protein. The RNA vector of the present invention can be produced by replacing the gene region encoding this coat protein with a foreign gene.

This RNA vector can be produced by the following steps.

1) Synthesis of full-length cDNA of viral RNA 2) Full-length recombinant cD in which the coat protein coding region of full-length cDNA of viral RNA is replaced with a foreign gene
Construction of a transcription vector containing NA 3) Linearize the above transcription vector 4) Perform a transcription reaction using RNA polymerase on the linearized transcription vector by a conventional method to obtain the desired recombinant RNA.
Manufacture the vector.

At this time, if the transcription reaction is performed with RNA polymerase in the presence of m'G ppp G and the 5' end of the RNA is blocked with a cap structure, the infectivity of the recombinant RNA to plant cells increases markedly. However, it is not essential to block the 5' end of the RNA with a camp structure. Furthermore, regarding the 3' end of RNA, it is desirable that many nucleotides not be connected beyond the 3' end, so that when transcribing RNA from cDNA using a transcription vector, the 3' end of the RNA is transferred to the positive ill. It is desirable that ゛ be completed. For this purpose, cDNA as a template is
Insert an appropriate restriction enzyme (e.g., M
It is desirable to incorporate a cleavage site by u T ) and terminate transcription by cleaving the cDNA there prior to transcription.

The above-mentioned transcription vectors include already known transcription vectors that have a replication initiation region, a selection marker, a promoter, a template DNA insertion site immediately after the transcription initiation site, and a restriction enzyme cleavage site immediately downstream of the inserted DNA. can be used. For example, Ahlquist (A
pPMl etc. (this vector was developed by AgriGenetics Research Associates)
Limited filed a patent application: Published Patent Publication 1986 57
No. 79 has been filed, and if this vector is to be used commercially, the consent of the right holder is required. In the experiments leading to the present invention, pPMl provided by Mr. Ahlquist was used. ) can be used.

In pPMl, transcription starts from the very end of the inserted cDNA, and it has a restriction enzyme site at the part corresponding to the 3' end of the RNA. It cuts at this site, linearizes it, and then transcribes the virus RNA. This is a transcription vector designed to avoid connecting extra nucleotides beyond the 3'' end that would affect infectivity.

In the present invention, tobamoviruses used as vectors include tomato-based, common-based, cowpea-based TMV and CGMMV. It is possible to use wild-type strains and strains that exhibit infectivity without causing disease symptoms or attenuated strains that have been mutated by recombinant technology.

A complete 14 c I) NA to viral RNA can be easily created by a known method of inverting the RNA 7 times with reverse transcriptase. For example, the genomic RNA of the 1' MV-L strain (cDNA of 1 to (7215 No. 11),
A and 3° end 1', 6 Kb cDNA, and TM
V, L++A strain full-length c, I) Plasmid pLT-1) cloned with NA 27, p
L-1-13 and pL++A A25 are known, respectively.
m, Rifi, 1915-1923 (1984),
Takamastucl al, :Nucle
ic Ac1ds Res, 11.3767-3
778 (1983), and NishiguN15h
ial, : Nucleic Ac1ds Res,
13.5585-5590 (1985).

The DNA used as a template for producing the RNA vector of the present invention is virus T? I) The gene 1irf region of the coat protein of the full-length NA cDNA is replaced with a foreign gene DNA, A.It is produced by incorporating NA into a transcription vector, but the construction of this recombinant transcription vector itself requires genetic recombination. This can be done by using a conventional method.

In particular, a replication vector constructed by removing the coat protein gene region of the tobamovirus RNA cDNA and introducing a restriction enzyme cleavage site that enables insertion of a foreign gene into this region, such as the transcription vector pLDC329 shown in Figure 2, is constructed. It consists of a cDNA of tobamovirus RNA in which the region, selection marker, promoter, and coat protein gene region have been converted into restriction enzyme cleavage sites for foreign gene insertion, and the transcription initiation nucleotide of the promoter is the first nucleotide of the cDNA. This can be easily done by using a universal transcription vector that is connected as follows.

A desired transcription vector can be constructed by inserting a desired gene into the foreign gene insertion site of this universal transcription vector using conventional genetic recombination techniques.

Genes introduced into plant cells according to the present invention are not particularly limited, but include, for example, genes that improve resistance to high and low temperatures, genes that improve resistance to frost, pests, pathogenic microorganisms, viruses, etc. Genes that improve tolerance to herbicides, genes involved in plant growth, enzymes involved in nitrogen fixation, genes related to photosynthesis, genes related to nutritional characteristics and flavor of plants, genes for useful substances Various genes can be mentioned.

Transformation of plant cells &,l: ,1. This can be carried out by infecting plant cells with the recombinant RNAΔ vector created as described above. At this time, the virus is reconstituted using the virus coat protein, and then the plants are inoculated! By infecting plant cells with the same method, the sensitivity can be increased. Infection of plants is carried out by diluting the reconstituted reaction solution with water, buffer, etc. as necessary to an appropriate concentration, or by infecting viruses or I'? This can be easily carried out by suspending NA in a buffer solution or the like and inoculating the plant together with Carbolanda J.

The target plant "ζ" is not particularly limited as long as it is a plant that can be infected with tobamovirus, such as tobacco or tomato.

The present invention will be explained in more detail with reference to Examples below.

The present invention is not limited only to this example,
It is intended to include modifications and improvements commonly occurring in the art to which the invention pertains.

Construction of cDNA of RNA Vector A recombinant cDNA containing a cDNA in which the coat protein gene region of TMV was replaced with a foreign gene was produced as follows.

In this example, E. coli strain H8101 was used for DNA cloning.

■) Full-length chloramphenicol acetyltransferase
Plasmid pBR325 (F, Boliv) containing the tyltransferase: CAT) gene
ar, Gene 4,121 (197B)), filled in using the Klenol II fragment of E. coli DNA polymerase I, and digested with 5au3AI to create a 0.74 Kb filled-in + 13 a n I.
/ S au 3 A I fragment (entire coding region of CAT, 59 bp of 5' untranslated region and 24 b
(including the 3° untranslated region of p) was isolated.

Transcription vector pl), full-length c of 'I''MV-L into Ml
A known transcription vector pLFW3 incorporating DNA (
Ishikawa et al.: 11th Annual Meeting of the Molecular Biology Society (1985)
)) was digested with AV;
- genomic RNA was cut at a site corresponding to 61fliO-ylation +1, and filled in using a Klenow (KIcnc++u) fragment, and then r, l of i' MV
) 0,2:('I<b)2 containing the 3' terminal part of TMV and 22 bp of the I-1 protein gene by digesting with 3all in the 4bp T stream of NΔ
A f domain Avall/5all DNA fragment was obtained.

After joining these two fragments, u −=
pCAT3L-10 was constructed.

pcAT31, -IO pBI? Ec derived from 322
After cutting at the oRV site and flocculization, <0.6 Ml 1-NaCl, 20mM Tr 1s-HCj! with Ba131 exonuclease. (pH8
), 12mM MgCj! ,, 12mM C
aCj! 2.11.6μgpCAT3L-to/gc
, oRV, and 2.6'uBa131 exonuclease at 30° C. for 7.5 minutes.

A 5acI linker (1) dCGAGCTCG) was connected to this, and after digestion with 5acl and 5all, CAT
DNA containing the gene and all 3 untranslated regions of TMV-
It was cloned between the 5aCI and 5all sites of t-pUCl 8. Using the binding reaction solution, E. coli
HBLol was transformed and plasmids were isolated from colonies resistant to ampicillin and chloramphenicol.

The DNA sequence was determined by the guideoxy method, and the CAT gene containing the 1 obp 5' leader region and 3 of TMV were sequenced.
``ptC3L containing the terminal end and part of the coat protein''
5-2. was selected.

On the other hand, p, LFW 3 coat protein gene region (7) BstEIr site (TMV genomic RNA 5
Digested at the site corresponding to position 799)), Ba131
With exonuclease (0,6M Na'Cp220mM
Tri 5-HC7! (pl+8.0), 12m
M M g C7! t,l 2mMCaCj! z,
10. ljgP L F W 3 / Tl s L
l': Tl, 1.3 uBa 131 mm Reacted at 30° C. for 3 minutes in a reaction solution containing kiss crease) It was truncated to the vicinity of the start codon of the coat protein gene. To this, S, J (2[After connecting the linker, Sac' l
and K p n T' (a site corresponding to opening 4390 of the TMV genome).

A 1.25-1.35 Kb Sac I/K pn I fragment containing upstream of the coat protein was
U'C18's Sac■ and ni'pn! Cloned during the leak. After transforming E. coli in the same manner as above, isolate the plasmid, 1. ) Determine the NA sequence and T
Protein gene at 30 of MV, part of the protein gene at 180, and 4b following coat protein gene A'TG
pt5L29 containing p was selected.

Construction of pCL29: 1.32 Kb 5acI/Kpnl fragment of pt5L29 (part of the protein gene at 180, part of the protein gene at 30,
protein gene and coat protein gene (including the start codon and 4 bp downstream thereof), a 0.963 Kb 5 acl/M I u I fragment of ptc3L5-2 (including the CAT gene and 10 bp upstream thereof, and TMV
) and Kpnl/ of pLFW3
The Mlul fragment was ligated to construct pCL29.

This pCL29 has a structure in which the TMV coat protein gene region of pLFW3 is replaced with the CAT gene.

Construction of pCLB 29: The 5acl sites of the 5acl/Kpnl fragment of pt5L29 and the 5acI/M1uI fragment of ptC3L5-2 were filled out with Klenow fragment to make blunt ends, and then ligated in the same manner as for pCL29 construction. was built.

Construction of universal transcription vector pLDC329: Universal transcription vector pLDcs29 was constructed as follows, in which the coat protein gene region was removed and a restriction enzyme cleavage site (Sacl) was introduced into this region to enable insertion of a foreign gene.

After digesting pLFW3 with A Va II and filling in with Klenow fragment, 3 a c, l linker
connected, digested with 5acl and M l u I, 0
．． 23Kb 5acl/Mlul fragment (TMV
3゛untranslated region and C-terminal 22b of coat protein
p) were isolated.

This SacI/MluI fragment was converted into pt5L
1, 32 Kb 5acl/Kpnl fragment isolated from 29 and pL, Kpn isolated from FW3
I/M, 1 u I fragment (the larger fragment was used) to construct pLDcs29 in which positions 5710-6160 of the coat protein gene were removed and the 5acl site was inserted.

Transcription: Transcription vector pCL29, pCL produced as above
After digesting B29 with M I I J I, a known method (AI+1quist et al.
al.

Proc, Natl, Acad, Sci, ll
5A 01.7066-7070 (1984). The resulting RNA was purified by phenol extraction and ethanol precipitation.

This is carried out using the known acetic acid method (Fraenkel-Conra).
t, H.

The coat protein of a common strain of TMV (4 mg/m
j! ) using 0,! M sodium phosphate buffer p
Virus particles were reconstituted by treatment in H7,0) at 20°C for 16 hours.

Infection and expression of plant cells: The reaction solution reconstituted as above was diluted 5 times with buffer solution, and N1cotiana tabacum Samsun
applied to the leaves. After 10 days of cultivation, the leaves were collected and extracted with an extract (250mM Tris-HCj! (pH 7,5
), 2.5mM EDTA, 0.1% ascorbic acid, 0.5mM leupeptin, 1mM PMsF) were added, ground, and heated at 60°C for 10 minutes. Centrifugation (1
After (4,000 x g, 5 min), the supernatant was diluted with buffer at 10.0
μn and 5 μl of 10 mM acetyl-CoA and 0.0
67 μCi of 14C-labeled chloramphenicol (5
3 mCi/mmo I; Amersham) and 37
Incubated at ℃ for 30 minutes. Chloramfluor L-221- and its derivatives were extracted with ethyl acetate, dried under reduced pressure, and then suspended in 5 μl of ethyl acetate.

This LPfi+ liquid was mixed with chloroporne/methanol (95
: 5) as a solvent and subjected to silica gel thin layer layer 11 madography, chloramsodic chloramphenicol I-) and its acetyl derivative (lAccm:l acetylchloramphenicol, 3AcCr++: 3-acetylchloramphenicol, I, 3AcCrrz 1,3-Diacetylchloramphenicol) was detected by autoradiography (room temperature, 16 hours). The results are shown in Figure 6. In this figure, lane 1 is unreacted 14C-raherylloramphenicol (Cm), lane 2 is an extract of 0.5 mg of leaves of the target plant to which the rotation reaction solution has not been applied, and lane 3 is Lane 4 is the transcript of the transcription vector pLD, C329, which does not have the coat protein gene. Lanes 5 and 6 are the extract of the plant infected with the wild-type TMV. It is an extract. Lane 7 is 0.
03 units of CAT reactant.

As is clear from this result, pCL29 and pCL
Expression of CAT by B29 was confirmed.

Reference example: Transcription vector pL containing full-length TMV-L cDNA
Construction of FW3: Synthesis of full-length cDNA of TMV-RNA 1) TMV-L
The strain or attenuated strain L++A was inoculated into tobacco plants and allowed to grow. After grinding the infected tobacco, virus particles were purified by a known method, and then RNA was extracted from the virus particles by a known method (edited by Taka II Atu et al., Nucleic Ac
1ds Res, 11.3767-3778 (19
83)). This RNA was combined with an excess of synthetic primers (having a sequence complementary to the 18 residues at the 3° end of TMV-RNA; or, if necessary, the 9th residue at the 3° end).
(using the A corresponding to the th A converted to T) and annealing was performed. Using this annealed RNA, 50 II
l: / m' 1. annealed RNA and 250
Inversion of units/mI'°Prepare a reaction solution containing the enzyme,
This was reacted at 42°C for 90 minutes to synthesize cDNA.

After recovering DNA by phenol extraction and ethanol precipitation, RNA was degraded with +1 and 1 N N'a OH. This was subjected to 5-20% alkaline-sucrose density gradient centrifugation or 2.5% polyacrylamide/8.3M urea gel electrophoresis to obtain cDNA corresponding to full-length RNA.
A was collected.

Add excess synthetic primer (having a sequence corresponding to residues 1 to 19 to TMV-4N) to the full-length cD'NA synthesized as above! OmMTr i 5-HCI
(pH 7,5), IO'mMMg
After heating at 90°C for 5 minutes in C1z and 50mM NaCρ, the sample was gradually cooled and annealed.

This cDNA was injected into 1 tlmMTri 5-HCI! ,
(1)'H7', 5), 2'5mM NaC7,1
0rr + MM g C12, 5mM dioxole, 0.2mM each dNTP at 250 medium/m7
! E. coli DNA-A polymerase 1 large fragment was added thereto and reacted at 21''C for 3 hours to convert into double-stranded cDNA.

Full-length double-stranded cD purified by low melting point agarose gel
Got NA.

Cloning of full-length cDNA Construction of pUcG'J1 In order to introduce a Mlul site into the polylinker sequence site of pUC9, pC09F 2 (Mesi et al.
l, Virology 127.54-64 (19
83); cucombergreen mottl
The 21 Kb HindllI/Fill-in Mlu I fragment of Q, which contains approximately 1.6 Kb containing the Mlu I site of E. mosaic virus, was transformed into pU C9 (Vieira et al,
ne 19.259-268 (19B2) l(i
n d [7/Fill-in 5all site insert p
UcG91 was constructed.

The structure around the M I u I site of pUCG91 is
Shown in the diagram.

Construction of plasmid pLFWl containing full-length c' DNA of TM'V=L strain 1) Construction of pLM51 and pLM31 Full-length double-stranded cDN obtained as above 2l- = 20 = A (TMV-L strain) was digested with B g I II and R
After obtaining 2,62 Kb corresponding to the 5° terminal part of NA,
Purified.

3.03 containing the Pm promoter of p'P'M1
AatTI/Smal fragment of Kb and pUC
9 and a 0.47 Kb AaLn/BamHI fragment. The combined DNA is E, c
o I iMcl 0 6 1 (Casdr
laban et al.: J9Mo1. Bio
l, 138.17'9-207 (1980))
transformed into Colony hybridization and restriction enzyme maps were created, and pLM with the structure shown in Figure 4 was created.
E. coli containing 5'l was selected.

2) Construction of pLM31 The full-length double-stranded cDNA (
No. 9 of the TMV-'L3° end (r of the mutant in which T in 1 was converted to A, 1) NA) was digested with p s t l,
After obtaining 4,54 Kb4 corresponding to the 3'' end portion of the RNA, it was purified.

This is Δatll/Pst of 2.1IIKb of pUC9.
l fragment and the 0.49 Kb A of pUcG91
The atll/fillin M I u I fragment was ligated. The combined DNA was transferred to E. coli MC1061.
transformed into. Colony hybridization and restriction enzyme maps were created and pLM3 with the structure shown in Figure 4 was created.
I chose 1.

3) Construction of pLFWl Pstl of pLM51 is inserted into the Pstl site of pLM31.
pLF containing the full-length cDNA by inserting the fragment (including the Pm promoter and 5” end)
Wl was constructed.

Construction of pLFW3 The Ncol/Apal fragment of pLFWl was pL
-1-13 (Takamatsu et al, Nu
Cleic Acids Res, 11.376-37
78 (1983): approximately 1,600bp TMV
-Ncol/of the 3° end (containing DNA) of the L strain
The EcoR1/Ncol fragment was transformed into pLT7D27 (Ohno et al.:
J. Biochem, 96. 1915-1923
(1984): Eco RI of TMV-L strain RNA (containing cDNA from 5” end to position 6215)
, / N c , o I fragments resulted in pLFW3 having the structure shown in FIG.

Construction of pLFAl As above, l-CcoRI of plFW1
/ A p a I fragment pLzΔ-8250
) of the TMV L++A strain by substitution with the EcoRT/Apal fragment (: DNA
A transcription vector pLFAI containing 3.11 was constructed horizontally.

[Brief explanation of drawings]

FIG. 1 is a diagram schematically showing the construction of the transcription vector pCL29 of the present invention. In the figure, Pm is λ derived from pPM1 developed by Ahlquist.
Represents the phage promoter. 130/180K, 30 and cp 1 of TMV
30/180 represents a protein gene, 30 represents a protein gene and a coat protein gene. 5 and 3 enclosed in squares before and after represent the 5° terminal untranslated region and the 3'' terminal untranslated region, respectively. Figure 2 shows the universal transcription vector pLDC329.
It is a diagram showing an outline of construction. Figure 3 shows transcription vector pCL29 containing the CAT gene.
(pCL, B29), transcription vector pLDC32
FIG. 9 is a diagram showing the DNA structure of transcription vector pLFW3 incorporating TMV (7) RNA and +7) cDNA. FIG. 4 shows plasmid pLM31. pLM51゜pLW
DNA of F 1, pLWF3, and p-L-1-13
FIG. Figure 5 shows the M of pUcG91, pLFWl, pLFW3.
FIG. 2 is a diagram showing the DNA sequence around the l u I site. FIG. 6 is a diagram showing the results of autoradiography in an experiment to confirm the expression of CAT. Kanichi 25 = special fL'JR, VG3-14693 (9) l - -H d φ! ω ■ Σ1 "÷ 1 → ->Straw; Old 6th scale j Fig. 5 DNA sequence around the Mlu I site of pUcG91 DNA sequence around the Mlu I site of pLFW1 ApaI
DNA sequence A around the M 1 u I site of Mlul pLFW3
paI MluI Figure 6 1.3AcCm 0 0, IAcCm lAcCm000 0DO4567

Claims (7)

[Claims] (1) Plant RN produced by replacing the coat protein gene region of tobamovirus RNA with a foreign gene
A vector (2) A transcription vector whose transcription product is tobamovirus RNA, which has a sequence in which the coat protein gene region of tobamovirus RNA is replaced with a foreign gene. (3) Consists of a tobamovirus RNA cDNA in which the replication initiation region, selection marker, promoter, and coat protein gene region are replaced with foreign genes, and connected so that the transcription initiation nucleotide of the promoter is the first nucleotide of the cDNA. The transcription vector according to claim 2 (4) The transcription vector according to claim 2 or 3, wherein the promoter is a λ phage promoter. (5) Tobamovirus R with a sequence in which the replication initiation region, selection marker, promoter, and coat protein gene region have been converted into restriction enzyme cleavage sites for foreign gene insertion
A universal transcription vector consisting of a cDNA of NA, connected such that the transcription initiation nucleotide of the promoter is the first nucleotide of the cDNA. (6) Universal transcription vector according to claim 5 specified as pLDCS29 (7) Plant RN produced by replacing the coat protein gene region of tobamovirus RNA with a foreign gene
A method for integrating a foreign gene into a plant cell, which comprises inoculating a virus reconstituted using the A vector or the plant RNA vector.