CN1033645A - The gene engineering method of controlling plant virus disease - Google Patents
The gene engineering method of controlling plant virus disease Download PDFInfo
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- CN1033645A CN1033645A CN 88105596 CN88105596A CN1033645A CN 1033645 A CN1033645 A CN 1033645A CN 88105596 CN88105596 CN 88105596 CN 88105596 A CN88105596 A CN 88105596A CN 1033645 A CN1033645 A CN 1033645A
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Abstract
The present invention is a kind of gene engineering method of controlling plant virus disease.By import the energy strongly inhibited host protein synthetic toxalbumin mosaic gene of not expressing or only be expressed as strand RNA usually to plant, transcribe to translate through virus induction in the cell of virus infection and generate toxalbumin, feedback ground suppresses duplicating and the diffusion in plant of this virus.Be suitable for controlling and contain subgene group positive chain RNA virus or do not contain subgene group positive chain RNA virus and that dna virus causes is annual, perennial, the virus disease of the plant of vegetative propagation, sexual propagation.This method has no adverse effects to plant itself, people and animals and environment.
Description
The invention belongs to the genetically engineered field, is a kind of gene engineering method of controlling plant virus disease.Existing technology:
1.D.C.Baulcombe el al, Nature 321:446-449(1986) described, in host cell, express the satellite RNA that produces Causative virus and suppress propagation and the diffusion of this virus in plant.This method can delay the generation of virus disease, but only is suitable for the control of the Plant diseases that virus that only a few has the satellite RNA that can suppress this virus multiplication causes, but also may cause the environmental pollution result who is difficult to predict.A kind of satellite RNA has the hazard rating that suppresses Causative virus a certain host, and just may strengthen the hazard rating of this virus to the sort of host another plant.And RNA makes a variation easily, can control originally this virus to certain host endanger useful satellite RNA might the variation postemphasis should virus to the hazard rating of former host plant.
2.A.P.Powell et al, Science 232:738-743(1986) described, in host plant, express virus coat protein gene and suppress the propagation of this virus in the host.This method can delay the generation of this virus disease, but can only control the generation and the development of the virus disease that is caused by non-kind biography venereal disease poison in the yearly plant.For reaching and strengthen control virus disease effect, host plant need accumulate in a large number should virus coat protein, this is unfavorable to plant, as is used for directly edible plant, people are also difficult psychologically to be accepted.
3.R.N Beachy et al, In:D.Evered d S.Harnett(eds) Plant resistance to viruses, 151-158, John Wileyd Sons, (1987) described, in host plant, express virus genomic one section anti-meaning RNA fragment, control the propagation of this virus in the host with this.But this method is controlled poor effect to virus disease.
The objective of the invention is to overcome the prior art weak point.By the virus induction method, make to have imported plant originally and be not expressed as proteic toxoprotein gene and be expressed as toxalbumin, cause virus infected cell " suicide ", can reach the purpose of control virus propagation and diffusion in plant.
Content of the present invention: the present invention utilizes the toxoprotein gene reading frame that suppresses the host plant protein synthesis to replace a kind of gene reading frame of viral subgenomic because of group cDNA, this reading frame former 5 ' end keeps the base sequence that can be discerned by this rdrp virus, or change the base sequence that to be discerned by other rdrp virus, or several viruses base sequence of replicative enzyme identification separately in the series connection, or connect the homology base sequence of the replicative enzyme corecognition of several viruses, being inserted in can be in plant between expression promoter and 3 ' end, import plant, be expressed as minus strand toxalbumin RNA.In the cell of related viral infections, strand RNA is transformed into normal chain toxalbumin RNA, translation produce the corresponding virus of control in host plant propagation and the toxalbumin of diffusion, as being that positive chain RNA virus comprises and is not with the non-district's cDNA fragment of translating of subgene group positive chain RNA virus 5 ' end in toxoprotein gene reading frame 5 ' termination, according to said method form mosaic gene, import plant, this kind of plant also can resist the virus disease of not being with subgenomic positive chain RNA virus to cause.
Basic feature of the present invention is to import the mosaic gene that can not be expressed as protein at ordinary times but just can be expressed as toxalbumin in by virus infected cell to plant.Have only in some dna virus and only induced in the reading frame of the gene that could express and the plant behind certain poisoning intrusion just by other gene products of this virus that the reading frame of the gene of abduction delivering replaces with the toxalbumin reading frame in cells infected, form mosaic gene, import plant, because these mosaic genes are only transcribed and translated in the host cell of these virus infectiones and produce toxalbumin, such plant also can obtain the resistance of virus disease that these viruses are caused.
Concrete technological line of the present invention is:
1. a kind of coat protein gene reading frame with subgene papova cDNA is changed a kind of toxoprotein gene reading frame of energy strongly inhibited host plant protein synthesis.
2. keep the former coat protein gene reading frame 5 of this section cDNA ' end and can be guaranteed the fragment of provirus positive chain RNA stability from fragment and virus 3 ' end that negative strand gene group RNA produces normal chain coat protein RNA by this rdrp virus identification, can in plant, be transcribed into the form of strand RNA, insert between plant promoter and 3 ' end, form mosaic gene.
3. mosaic gene is inserted in the plasmid vector T-DNA district of band plant screening mark gene such as chimeric neomycin phosphotransferase II (NPT II) gene,, this plasmid imported no T-DNA district and be with in the Agrobacterium of Ti-plasmids in Vir district by conjugal transfer.
4. the way of cultivating altogether by leaf video disc or protoplastis and Agrobacterium imports plant dyeing plasmagene group to the T-DNA district with this mosaic gene and selection markers gene, produces the engineering plant with this toxoprotein gene of anti-certain antibiotic.
5. from engineering plant, filter out the plant of anti-this virus disease, cultivate, obtain heredity and go up stable anti-this viral seed progeny by sexual propagation, subculture.Also can obtain the asexual propagation material that is used to produce from complete antiviral engineering plant.
6. before this toxoprotein gene reading frame, respectively there is a specific restriction enzyme otch these rdrp virus identification cDNA fragment both sides, available should virus other cDNA fragment for replicative enzyme identification, other rdrp virus specific recognition cDNA fragment does not even replace with the non-district's cDNA fragment of translating of 5 of subgene group positive chain RNA virus RNA ' end; The cDNA of the homology base fragment of each self-identifying cDNA fragment of the replicative enzyme of the also available several viruses that are together in series or corecognition replaces; Form and import the mosaic gene that plant can resist one or more virus diseases.
7. chimeric toxoprotein gene is imported the method for plant except being total to culture method with binary form Ti carrier used herein and Agrobacterium, also available unary form Ti carrier is cultivated or the dna direct transfer method altogether through Agrobacterium, chimeric toxoprotein gene is imported plant, obtain engineering plant.
8. be used to suppress the zymoprotein gene that plant protein synthetic toxoprotein gene has the modification elongation factor 2 of bacteriums such as diphtheria toxin Segment A gene, pseudomonas aeruginosa exotoxin A gene; The ribosome-inactivating protein gene of resource plant pokeweed antiviral protein etc. and food plant wheat, corn, balsam pear etc.The fragment that is used to change minus strand toxalbumin RNA and is normal chain toxalbumin RNA has various band subgene group positive chain RNA virus (as tobacco mosaic virus (TMV); cucumber mosaic virus; potato virus X; potato virus Y; alfalfa mosaic virus; bromovirus; barley stripe mosaic viruses etc.) replicative enzyme comprises the non-district's cDNA fragment of translating of no subgene group positive chain RNA virus 5 ' end from cDNA and each positive chain RNA virus that viral separately strand RNA is transcribed into the required base fragment of normal chain subgenomic RNA.
Mechanism of action of the present invention is:
1. the toxalbumin strand RNA that some amount is all arranged in the engineering plant that virus does not infect, these RNA can not become positive chain RNA and produce corresponding toxalbumin.After plant was by virus infection, virus utilized the host protein synthesis system to synthesize replicative enzyme; Minus strand toxalbumin RNA is discerned and transcribed to these replicative enzyme, produces mRNA; These mRNA utilize the translation of plant protein synthesis system to generate corresponding toxalbumin again; These toxalbumin suppress the protein synthesis of infected host cell again with feeding back.The character of the toxalbumin tool enzyme here has very strong arrestin matter synthetic ability.Like this, no matter the vigor of the original protein synthesis of host cell how, utilizes this Feedback mechanism, in the host cell of virus infection, protein synthesis all can be promptly by severe inhibition.
2. transfer to flanking cell or tissue far away with subgenomic positive chain RNA virus from cells infected, need the albumen relevant of the virus of synthetic some amount with transfer.These albumen are translated from subgenomic RNA often.Virus will be transcribed the geneome RNA that generates minus strand earlier after entering host cell generation replicative enzyme, changes each normal chain subgenomic RNA into by them again.In engineering plant, prestore some amount and have and to be discerned and to transcribe by rdrp virus under the condition of required segmental toxalbumin strand RNA, will preferentially generate toxalbumin mRNA behind the virus infection host cell, translate into toxalbumin.Before also failing to accumulate required each transfer protein of virus transfer, it is proteinic synthetic in the cells infected that this albumen suppresses rapidly with feeding back.Like this, virus can not be bred to be diffused in the adjacent cells and gone.The toxalbumin strand RNA that has the 3 ' end (promptly 5 of positive strand genomic rna ' hold) of this virus negative strand gene group RNA that rdrp virus can discern as the some amount that prestores in the engineered plant, behind this poisoning intrusion, also can preferentially generate poison in the virus infected cell and be limited in the cell that originally infects in vain and virus with similar mechanism.
Thereby the host cell of virus infection generate the minority toxalbumin make cell protein synthetic be suppressed substantially be suppressed with virus replication after, this cell also has RNA and protein degrading activity.RNA enzyme as it can be degraded viral RNA fully, and then toxalbumin and viral protein also will be eliminated thereupon, and this cell might return to not by the state before the virus infection.
Advantage of the present invention and effect:
Because this law is by viral auto-induction, feedback ground suppresses host protein that its own breeding and diffusion relied on and synthesizes and reach the effect of controlling virus disease, so, compare with the method in past, 1. this law can be controlled the development of virus disease fully.For example, can resist the diffusion of tobacco mosaic virus (TMV) in engineering plant with a kind of engineering plant that this law obtains, it is antiviral that 2. this law is suitable for controlling various plants such as annual and perennial, sexual propagation and vegetative propagation.3. agricultural goes up topmost virus disease major part by being with subgenomic positive chain RNA virus to cause, the present invention is suitable for controlling the Plant diseases that these viruses cause, also be suitable for controlling the virus disease of not being with subgenomic positive chain RNA virus to cause, use this law through extending, go back the purpose that causes viral diseases of plants that the most viruses of may command cause.4. the engineering plant that uses the present invention to obtain does not contain toxalbumin at ordinary times, only temporarily accumulates several toxalbumin molecules behind the virus infection in infected cell, and they can not invade people and animals' cell yet.Such engineering plant is not to plant itself, can bring detrimentally affect to people, animal with to environment.
Embodiments of the invention:
The mosaic disease that the present invention causes with tobacco resisting tobacco mosaic virus (TMV) is an example.The toxalbumin of using is the diphtheria toxin Segment A, and it can not enter people and animals' cell, as long as several molecule just is enough to the protein synthesis of a cell of severe inhibition promptly.The fragment that can be discerned by the TMV replicative enzyme used herein is the fragment before the coat protein gene reading frame.When recombinating, for guaranteeing that mosaic gene has high transcriptional capability and obtains behind the positive chain RNA high conversion ability being arranged, select cauliflower mosaic virus strong promoter (CaMV355 promotor) and its 3 ' end for use, kept the complete non-district's base of translating of former TMV coat protein mRNA5 ' end.In order to keep strand RNA and goodish stability to be arranged by the positive chain RNA that it transcribes generation, we are placed on 3 ' end with the long base fragment of 0.6Kb before the former coat protein reading frame, and 5 ' end has kept the desired base sequence of former TMV3 ' end generation positive chain RNA higher structure.Here mosaic gene imports plant by binary form Ti carrier through Agrobacterium.The entire operation flow process is as shown below:
1. mosaic gene reorganization flow process:
(1) band TMV3 ' end comprises the pOM5 II of coat protein gene cDNA
2Cut through Hind III enzyme, fill and lead up with the Klenow enzyme, Apa I partly enzyme is cut, and gets required Hind III-Apa I-Apa I fragment, in the Apa I between importing carrier pRT100 cauliflower mosaic virus CaMV35S promotor and its 3 ' end, the Sma I otch, gets pKC503.
(2) pKC503 cuts with Sac I and Cla I enzyme, remove former coat protein gene fragment, the fragment that goes up 61 base pairs of original Dra I-Cla I with synthetic Sac I-Nco I-Sal I-Dra I poly joint and pKC503 links to each other, and gets pKC505.
(3) the Nco I of band diphtheria toxin (DT) the Segment A gene of pTH-1 is to the EcoRI fragment, imports earlier in the Nco I, EcoRI otch of pRAJ275, connects Sac I otch before making the Nco I, pRAJ275DT.PRAJ275DT is cut with Sal I and Hpa I enzyme and the Sal I-Hpa I fragment of band DT Segment A gene is imported between the Sal I and Sac I otch of pKC505, the Sac I is used T in advance
4Archaeal dna polymerase is filled and led up, and gets pKC525.
Here all undertaken with the reorganization operation of back by people such as T.Maniatis " Molecular Cloning A Laboratory Manual " (the Cold Spring Harbor Laboratory 1982) method that book is chatted of showing.
2. mosaic gene imports Agrobacterium and imports the method that tobacco produces the resisting tobacco mosaic virus engineering plant:
(1) with the mosaic gene among the pKC525, cut out with the Hind III, import the Hind III otch of Bin19 carrier, get pKC545.Like this, chimeric toxoprotein gene just is placed on chimeric neomycin phosphotransferase II (NPT II) the gene limit that can make the anti-kantlex of plant; The two is all in the T-DNA district of Bin19 carrier.
(2) pKC545 is by the assistance of pRK2013, through conventional conjugal transfer operation, import in the Agrobacterium of band LBA4404, with the LB substratum screening that is mixed with kantlex, Streptomycin sulphate and rifomycin, inhibition to the kantlex sensitivity only with the Agrobacterium of LBA4404 with to the band pKC545 of Streptomycin sulphate, rifomycin sensitivity or the intestinal bacteria of pPK2013, just sift out required band LBA4404 and the Agrobacterium pAKC545 of pKC545.LBA4404 contains the Vir district and the Ti-plasmids that do not contain the T-DNA district, and when Agrobacterium and injured plant tissue, cells contacting, its Vir gene expression product will make the T-DNA district of pKC545 enter vegetable cell.
(3) Agrobacterium pAKC545 is inoculated in the LB liquid nutrient medium, cultivated 12 hours down at 28 ℃, with one to three times of LB liquid nutrient medium dilution, contact 5 to 10 minutes with the video disc that the aseptic tobacco leaf of anti-kantlex is not newly cut, after bacterium liquid was removed in the aseptic filter paper suction, the leaf video disc forwarded MS to
0Solid medium was placed three days in 25 ℃ of incubators of 12 hours of continuous irradiation every day, changeed in band 6-Bian Ji purine (1 μ g/ml), cephalo
On the MS solid medium of oxime sodium (300 μ g/ml) and kantlex (100 μ g/ml), under same culture conditions, placed about 20 days again, get the young shoot of a lot of transformation of tobacco; Cut these young shoots, forward to above-mentioned antibiotic but do not have on the MS solid medium of 6-benzyl purine and take root, must resist the transformation of tobacco seedling of kantlex after about 20 days, these tobacco seedlings are transplanted to the greenhouse from substratum, after waiting to survive, infect the engineering cigarette strain that to sift out anti-fully TMV with TMV.
Claims (3)
1, the invention belongs to the genetically engineered field, it is a kind of gene engineering method of controlling plant virus disease, it is characterized in that to be expressed as protein usually to the plant importing, but in by the cell of virus infection, can be expressed as the mosaic gene of toxalbumin, make infected cell " suicide ", thus the control virus disease.Concrete grammar is:
(1) in toxoprotein gene reading frame 5 ' termination the preceding paragraph or the series connection several sections be used to produce the desired base sequence of normal chain subgenomic RNA by one or several viral replicative enzyme from strand RNA, reassemble into the mosaic gene that in host plant, can be expressed as minus strand toxalbumin RNA;
(2) produced normal chain subgenomic RNA desired homology base sequence with being used to from strand RNA by the replicative enzyme corecognition of several viruses on toxoprotein gene reading frame 5 ' termination, reassemble into the mosaic gene that in host plant, can be expressed as minus strand toxalbumin RNA;
(3) the non-district that translates of 5 ' end that viral RNA is complete on toxoprotein gene reading frame 5 ' termination reassembles into the mosaic gene that can be expressed as minus strand toxalbumin RNA in host plant;
(4) must be formed the mosaic gene that in plant, can be expressed as normal chain toxalbumin RNA by this viral product recognition sequence and toxoprotein gene reading frame what this viral product was induced the gene of just expressing down with one of dna virus;
(5) with in the plant only in that just the promotor of the gene of special abduction delivering or enhanser add promotor and toxalbumin reading frame composition can be expressed as normal chain toxalbumin RNA in plant mosaic gene behind the poisoning intrusion;
(6) mosaic gene has strong promoter, makes the base sequence that normal chain toxalbumin RNA efficiently translates and makes minus strand and the stable order of positive chain RNA, and mosaic gene imports plant by Agrobacterium Ti carrier or dna direct transfer method.
2, the gene engineering method of a kind of controlling plant virus disease according to claim 1, it is characterized in that described toxoprotein gene is a diphtheria toxin Segment A gene, the zymoprotein gene of the modification elongation factor 2 of bacteriums such as pseudomonas aeruginosa exotoxin A gene; The ribosome-inactivating protein gene of resource plant pokeweed antiviral protein etc. and wheat, corn, balsam pear etc.
3, the gene engineering method of a kind of controlling plant virus disease according to claim 1 is characterized in that described virus is the positive chain RNA virus of band subunit groups such as tobacco mosaic virus (TMV), cucumber mosaic virus, potato virus X, potato virus Y, alfalfa mosaic virus, bromovirus, barley stripe mosaic virus and is not with subgenomic positive chain RNA virus and dna virus.
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| CN 88105596 CN1033645A (en) | 1988-10-22 | 1988-10-22 | The gene engineering method of controlling plant virus disease |
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| CN 88105596 CN1033645A (en) | 1988-10-22 | 1988-10-22 | The gene engineering method of controlling plant virus disease |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0425004A2 (en) * | 1989-10-03 | 1991-05-02 | Aveve N.V. | Genetic manipulations with recombinant DNA comprising sequences derived from RNA virus |
| EP0479180A2 (en) * | 1990-10-05 | 1992-04-08 | Hoechst Aktiengesellschaft | Virus resistant plants, method for their production |
| WO1992021757A1 (en) * | 1991-05-30 | 1992-12-10 | Plant Genetic Systems, N.V. | Nematode-responsive plant promoters |
| EP0612208A1 (en) * | 1991-10-04 | 1994-08-31 | North Carolina State University | Pathogen-resistant transgenic plants |
| WO1994026912A1 (en) * | 1993-05-06 | 1994-11-24 | Institut für Pflanzengenetik und Kulturpflanzenforschung | Method and vector construct for increasing the expression of transgenes |
| WO1994029464A1 (en) * | 1993-06-04 | 1994-12-22 | Sandoz Ltd. | Virus resistant plants |
| EP0641384A1 (en) * | 1992-05-14 | 1995-03-08 | Ribozyme Pharmaceuticals, Inc. | VIRUS RESISTANT PLANTS CONTAINING INDUCIBLE CYTOTOXIC mRNAs |
| TR28954A (en) * | 1992-03-13 | 1997-08-04 | Cambridge Advanced Tech | Root knot nematode resistance |
| WO1998003668A1 (en) * | 1996-07-22 | 1998-01-29 | Zeneca Limited | Virus resistance in plants |
| EP0806481A3 (en) * | 1996-05-09 | 1998-02-04 | Metapontum Agrobios s.c.r.l. | Method of preparation of transgenic plants resistant to viral infections and so obtained plants |
| EP0896059A3 (en) * | 1990-12-20 | 1999-07-07 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for the production of pathogen resistant plants |
-
1988
- 1988-10-22 CN CN 88105596 patent/CN1033645A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0425004A3 (en) * | 1989-10-03 | 1991-08-07 | Aveve N.V. | Genetic manipulations with recombinant dna comprising sequences derived from rna virus |
| EP0425004A2 (en) * | 1989-10-03 | 1991-05-02 | Aveve N.V. | Genetic manipulations with recombinant DNA comprising sequences derived from RNA virus |
| EP0479180A2 (en) * | 1990-10-05 | 1992-04-08 | Hoechst Aktiengesellschaft | Virus resistant plants, method for their production |
| EP0479180A3 (en) * | 1990-10-05 | 1992-08-26 | Hoechst Aktiengesellschaft | Virus resistant plants, method for their production |
| EP0896059A3 (en) * | 1990-12-20 | 1999-07-07 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Method for the production of pathogen resistant plants |
| WO1992021757A1 (en) * | 1991-05-30 | 1992-12-10 | Plant Genetic Systems, N.V. | Nematode-responsive plant promoters |
| EP0612208A4 (en) * | 1991-10-04 | 1995-07-12 | Univ North Carolina | Pathogen-resistant transgenic plants. |
| EP0612208A1 (en) * | 1991-10-04 | 1994-08-31 | North Carolina State University | Pathogen-resistant transgenic plants |
| CN1080302C (en) * | 1992-03-13 | 2002-03-06 | 先进技术(剑桥)有限公司 | Root knot nematode resistance |
| TR28954A (en) * | 1992-03-13 | 1997-08-04 | Cambridge Advanced Tech | Root knot nematode resistance |
| EP0641384A1 (en) * | 1992-05-14 | 1995-03-08 | Ribozyme Pharmaceuticals, Inc. | VIRUS RESISTANT PLANTS CONTAINING INDUCIBLE CYTOTOXIC mRNAs |
| EP0641384A4 (en) * | 1992-05-14 | 1995-07-12 | Ribozyme Pharm Inc | VIRUS RESISTANT PLANTS CONTAINING INDUCIBLE CYTOTOXIC mRNAs. |
| WO1994026912A1 (en) * | 1993-05-06 | 1994-11-24 | Institut für Pflanzengenetik und Kulturpflanzenforschung | Method and vector construct for increasing the expression of transgenes |
| WO1994029464A1 (en) * | 1993-06-04 | 1994-12-22 | Sandoz Ltd. | Virus resistant plants |
| EP0806481A3 (en) * | 1996-05-09 | 1998-02-04 | Metapontum Agrobios s.c.r.l. | Method of preparation of transgenic plants resistant to viral infections and so obtained plants |
| WO1998003668A1 (en) * | 1996-07-22 | 1998-01-29 | Zeneca Limited | Virus resistance in plants |
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