CA2075209C - Virus/herbicide resistance genes process for producing same and their use - Google Patents
Virus/herbicide resistance genes process for producing same and their use Download PDFInfo
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- CA2075209C CA2075209C CA002075209A CA2075209A CA2075209C CA 2075209 C CA2075209 C CA 2075209C CA 002075209 A CA002075209 A CA 002075209A CA 2075209 A CA2075209 A CA 2075209A CA 2075209 C CA2075209 C CA 2075209C
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
- C12N15/8277—Phosphinotricin
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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- C12N2770/14011—Bromoviridae
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Abstract
Virus/herbicide-resistance genes, processes for the preparation thereof and the use thereof Virus genes, for example coat protein genes, which bring about a reduction in the signs of infection by the corresponding virus or bring about virus resistance can be combined with herbicide-resistance genes for the transformation of plants.
A combination of this type facilitates the selection of the transgenic plants. In addition, in practical field cultivation, the vitality of the plants is increased by the virus tolerance, and an unproved plant protection is possible owing to the herbicide-resistance gene.
A combination of this type facilitates the selection of the transgenic plants. In addition, in practical field cultivation, the vitality of the plants is increased by the virus tolerance, and an unproved plant protection is possible owing to the herbicide-resistance gene.
Description
~<r"ate:°~~
HOECHST AKTTENGESELLSCHAFT HOE 90/F 036 Dr. KH/rk Description Virus/herbicide-resistance genes, processes for the preparation thereof and the use thereof The synthesis of virus coat protein in plants leads to an enhanced resistance of the plant to the corresponding virus. European Patent Application 0 2~0 331, for eacam ple, describes the preparation of plant cells which contain such a coat protein.
Turner et al . [ EMBO J . 5 , 1181 ( 19 8 7 ) ] have carried out the transformation of tobacco and tomato plants with a chimeric gene which codes for the coat protein of alfalfa mosaic virus. The progeny of these is transformed plants which showed a significant reduction in the signs of infection with the corresponding virus, and in some cases even virus resistance.
It has now been found that such virus genes can be combined with a herbicide-resistance gene, which facili-rates the selection of the transger~ic plants. At the same time, in practical field cultivation, the vitality of the plants is increased by the virus tolerance, and an improved plant protection is possible owing to the herbicide-resistance gene. It has been generally observed that herbicide application exerts a stimulating effect on growth. The plant transformed according to the invention shows an enhancement of this effect, which makes it possible to achieve an improved plant yield.
Herbicide-resistance genes have already been disclosed.
German Offenlegungsschrift~ 37 16 309 describes the selection of non-fungoid bacteria which are resistant to phosphinothricin. The phosphinothricin-resistance gene can be localized to a fragment 2 kb in size on the DNA of these selectants.
HOECHST AKTTENGESELLSCHAFT HOE 90/F 036 Dr. KH/rk Description Virus/herbicide-resistance genes, processes for the preparation thereof and the use thereof The synthesis of virus coat protein in plants leads to an enhanced resistance of the plant to the corresponding virus. European Patent Application 0 2~0 331, for eacam ple, describes the preparation of plant cells which contain such a coat protein.
Turner et al . [ EMBO J . 5 , 1181 ( 19 8 7 ) ] have carried out the transformation of tobacco and tomato plants with a chimeric gene which codes for the coat protein of alfalfa mosaic virus. The progeny of these is transformed plants which showed a significant reduction in the signs of infection with the corresponding virus, and in some cases even virus resistance.
It has now been found that such virus genes can be combined with a herbicide-resistance gene, which facili-rates the selection of the transger~ic plants. At the same time, in practical field cultivation, the vitality of the plants is increased by the virus tolerance, and an improved plant protection is possible owing to the herbicide-resistance gene. It has been generally observed that herbicide application exerts a stimulating effect on growth. The plant transformed according to the invention shows an enhancement of this effect, which makes it possible to achieve an improved plant yield.
Herbicide-resistance genes have already been disclosed.
German Offenlegungsschrift~ 37 16 309 describes the selection of non-fungoid bacteria which are resistant to phosphinothricin. The phosphinothricin-resistance gene can be localized to a fragment 2 kb in size on the DNA of these selectants.
German Offenlegungsschrift 37 37 918 indicates a way of synthesizing the phosphinothricin-resistance gene from the genome of Streptomyces viridochromogenes. Incorporation in gene structures with whose aid transformed plants become resistant to the herbicide is likewise described therein.
The invention thus relates to a gene coding for a virus-resistance combined with a herbicide-resistance.
According to one aspect of the present invention, there is provided an isolated gene coding for a phosphinothricin-resistance combined with an isolated gene coding for a virus-resistance.
According to one other aspect of the present invention, there is provided a plant cell transformed with a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance.
According to another aspect of the present invention, there is provided a plant cell as described herein, wherein the nucleotide sequence encoding the virus coat protein can be obtained by cDNA cloning starting from the RNA of cucumber mosaic virus, of alfalfa mosaic virus or of brome mosaic virus.
According to still another aspect of the present invention, there is provided a plant cell as described herein, wherein the nucleotide sequence encoding a protein conferring phosphinothricin-resistance is from Streptomyces.
According to yet another aspect of the present invention, there is provided a plant cell as described herein and expressing the respective nucleotide sequence.
The invention thus relates to a gene coding for a virus-resistance combined with a herbicide-resistance.
According to one aspect of the present invention, there is provided an isolated gene coding for a phosphinothricin-resistance combined with an isolated gene coding for a virus-resistance.
According to one other aspect of the present invention, there is provided a plant cell transformed with a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance.
According to another aspect of the present invention, there is provided a plant cell as described herein, wherein the nucleotide sequence encoding the virus coat protein can be obtained by cDNA cloning starting from the RNA of cucumber mosaic virus, of alfalfa mosaic virus or of brome mosaic virus.
According to still another aspect of the present invention, there is provided a plant cell as described herein, wherein the nucleotide sequence encoding a protein conferring phosphinothricin-resistance is from Streptomyces.
According to yet another aspect of the present invention, there is provided a plant cell as described herein and expressing the respective nucleotide sequence.
According to a further aspect of the present invention, there is provided a process for the preparation of transformed plants with improved plant yield, which are transformed with a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance, and which comprises treating the virus- and herbicide-resistant regenerated plants with phosphinothricin, and this treatment resulting in improved plant yield.
According to yet a further aspect of the present invention, there is provided a method for increasing the yield from plants which are transformed with a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance, which comprises treating the plants with phosphinothricin in the growth period.
According to still a further aspect of the present invention, there is provided the use of a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance for increasing the yield from plants transformed therewith.
The invention is described in detail hereinafter, especially in its preferred embodiments. Furthermore, the invention is defined by the contents of the claims.
The genes for virus-resistance, especially the virus coat proteins, can be obtained starting from isolated virus RNA by cDNA cloning in host organisms. The preferred 3a starting material for this is the RNA of cucumber mosaic virus, of alfalfa mosaic virus or of brome mosaic virus.
Herbicide-resistance genes can be isolated from bacteria, for example of the genera Streptomyces or Alcaligenes. Preferably used is the phosphinothricin-resistance gene from Streptomyces viridochromogenes (Wohlleben, W. et al, Gene 80, 25-57 (1988)), which can be appropriately modified for expression in plants.
The genes are cloned and sequenced in each case using the vectors pUCl9, pUCl8 or pBluescript (Stratagene, Heidelberg, Product Information).
The gene is cloned in an intermediate vector with plant promoter. Examples of such vectors are the plasmids pPCV701 (Velten J. et al., EMBO J. 3, 2723-2730 (1984)), pNCN
(Fromm H. et al., PNAS 82, 5824-5826 (1985)), or pNOS (an, G.
et al., EMBO J. 4, 277-276 (1985)). Preferably used is the vector pDH51 (Pietrzak, M. et al., NAR 14, 5857, (1986)) with a 35S promoter, or the vector pNCN with a Nos promoter.
After subsequent transformation of E. coli, such as, for example, E. coli MC 1061, DH1, DK1, GM48 or XL-1, positive clones are identified by methods known per se (Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982), Molecular Cloning, a Laboratory Manual, 1st Edition, Cold Spring Harbor, New York, U.S.A.), such as plasmid mini-preparation and cleavage with an appropriate restriction enzyme.
These positive clones are then subcloned together into a binary plant vector. The plant vector which can be employed is pGV3850 (Zambrysk, P. et al., EMBO J. 2, 2143-2150 (1983)) or pOCAl8 (Olszewski, N., NAR 16, 10765-10782, (1988)). pOCAl8 is preferably employed.
3b The resulting binary plant vectors which contain plant promoters with the attached DNA fragment for the expression of virus coat protein and phosphinothricin-resistance in the T-DNA are used to transform plants. This can be carried out by techniques such as electroporation or microinjection. Preferably employed is cocultivation of protoplasts or transformation of leaf pieces with Agrobacteria. For this, the plant vector construct is transferred by transformation with purified DNA or, mediated by a helper strain such as E. coli SM10 (Simon R. et al., Biotechnology 1, 784-791 (1983)), into Agrobacterium tumefaciens such as A282 with a Ti plasmid via triparental mating. Direct transformation and triparental mating were carried out as described in "Plant Molecular Biology Manual"
(Kluwer Academic Publisher, Dardrech (1988)).
It is possible in principle to transform all plants with the binary plant vectors carrying the DNA constructed according to the invention. Dicotyledonous plants are preferred, especially productive plants which produce or store starch, carbohydrates, proteins or fats in utilizable amounts in their organs, or which produce fruit and vegetables or which provide spices, fibers and industrially useful products or pharmaceuticals, dyes or waxes and, moreover, fodder plants. As example mention 28976-Sl -may be made of tomato, strawberry, avocadoand plants which bear tropical fruits, mango, for example papaya, but also pear, apple, nectarine, apricot or peach.
Further examples of plants to be transfoaned are all types of cereals, rape, bird rape... The transformed cells are selected using a selection medium,cultured to a callus and regenerated to the plant on appropriate an medium (Shain M. et al., Theor. appl. Genet.~, 770-770 (1986)); Masson, J. et al., Plant Science ~,,'~,,167-176 ( 1987 ) ) , Zhan g. et al. , Plant Mol. Hiol. ,~,,~,551-559 (1988); McGranaham G. et al., Bio/Technology 800-804 ~, (1988); Novak F. J. et al., Bio/Technology7, 154-159 (1989)).
The following examples serve to illustrate the invention further.
8zamcples 1. Isolation of the virus coat protein gene The virus was purified by modification of the method of Lot, M. et al . , Anual Phytopath. ~, 25-32 ( 1972 ) . Alfalfa was infected with alfalfa mosaic virus and, after 14 days, disrupted in the same volume of 0.5 M sodium citrate (pH 6.5)/5 miK EDTA/0.5% thioglycolic acid. Then 1 volume of chlorofoan was added, and the mixture was centrifuged at 12,000 x g for 10 min. The supernatant was mixed with 10% PEG 6000 (~r/w) and stirred cautiously overnight. Zt was then csntrifnged at 12,000 x g for 10 min and reauspendod in 50 ml of 5 mM sodium borate, 0.5 mM EDTA (pH 9). Triton g-100 (final concentrations 2%) was added and then the mixture was stirred for 30 min and centrifuged at 19,000 x g for 15 min. The virus pellet after centrifugation at 105,000 x g for Z h was taken up in 5 mM borate buffer/0.5 mM EDTA (pH 9.0) and subjected to a sucrose centrifuqation (5-25%).
Individual fractions from the gradient were analyzed on an agarose gel in order to find the virus-containing - 5 - cC~ ~., ~' , ~; ~.,. , ...
zone. The virus RNA was purified of coat protein by phenol/SDS extraction (Peden, K.W. et al., Virology 53, 487-492 (1973). The RNA components were fractionated using 2.8~ polyacrylamide with 40 mM tris acetate buffer (pH 7.5) as described in Synous, R.H., Aust. J. Biol.
Sci. 31, 25-37 (1978). The RNA was removed from the gel by electrophoresis in dialysis tubes and precipitated.
cDNA transcripts of RNA3 or RI~A4 were prepared as des-cribed in Langenreis, K. et al., Plant Mol. Biol. 6, 281-288 (1986) using synthetic oligonucleotide primers with 3'-complementary nucleotides to the template, each of which had an Smal or PstI cleavage site at the 5' end.
The reactions for the cDNA synthesis were carried out in accordance with the "Current Protocols in Mol. ~3iol. " ed.
Ausubel, F. et al., John Wiley and Sons.
The cDNA was cloned into the SmaI/PstT-cut p~JC 19 vector.
It was possible to delete the insertion again using Smal/HindIII.
The method described above can equally be used to isolate the CMV coat protein gene.
2. Isolation of the herbicide-resistance gene A phosphinothricin-resistance gene with the following sequence was synthesized in a synthesizer using the phosphoamidite method.
y;s.~,.~y,.... ~,~;n 2. ~ t.g y '~' GTC GAC ATG TCT CCG GAG AGG AG~a CCA GTT GAO ATT AGG CCA GCT
According to yet a further aspect of the present invention, there is provided a method for increasing the yield from plants which are transformed with a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance, which comprises treating the plants with phosphinothricin in the growth period.
According to still a further aspect of the present invention, there is provided the use of a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance for increasing the yield from plants transformed therewith.
The invention is described in detail hereinafter, especially in its preferred embodiments. Furthermore, the invention is defined by the contents of the claims.
The genes for virus-resistance, especially the virus coat proteins, can be obtained starting from isolated virus RNA by cDNA cloning in host organisms. The preferred 3a starting material for this is the RNA of cucumber mosaic virus, of alfalfa mosaic virus or of brome mosaic virus.
Herbicide-resistance genes can be isolated from bacteria, for example of the genera Streptomyces or Alcaligenes. Preferably used is the phosphinothricin-resistance gene from Streptomyces viridochromogenes (Wohlleben, W. et al, Gene 80, 25-57 (1988)), which can be appropriately modified for expression in plants.
The genes are cloned and sequenced in each case using the vectors pUCl9, pUCl8 or pBluescript (Stratagene, Heidelberg, Product Information).
The gene is cloned in an intermediate vector with plant promoter. Examples of such vectors are the plasmids pPCV701 (Velten J. et al., EMBO J. 3, 2723-2730 (1984)), pNCN
(Fromm H. et al., PNAS 82, 5824-5826 (1985)), or pNOS (an, G.
et al., EMBO J. 4, 277-276 (1985)). Preferably used is the vector pDH51 (Pietrzak, M. et al., NAR 14, 5857, (1986)) with a 35S promoter, or the vector pNCN with a Nos promoter.
After subsequent transformation of E. coli, such as, for example, E. coli MC 1061, DH1, DK1, GM48 or XL-1, positive clones are identified by methods known per se (Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982), Molecular Cloning, a Laboratory Manual, 1st Edition, Cold Spring Harbor, New York, U.S.A.), such as plasmid mini-preparation and cleavage with an appropriate restriction enzyme.
These positive clones are then subcloned together into a binary plant vector. The plant vector which can be employed is pGV3850 (Zambrysk, P. et al., EMBO J. 2, 2143-2150 (1983)) or pOCAl8 (Olszewski, N., NAR 16, 10765-10782, (1988)). pOCAl8 is preferably employed.
3b The resulting binary plant vectors which contain plant promoters with the attached DNA fragment for the expression of virus coat protein and phosphinothricin-resistance in the T-DNA are used to transform plants. This can be carried out by techniques such as electroporation or microinjection. Preferably employed is cocultivation of protoplasts or transformation of leaf pieces with Agrobacteria. For this, the plant vector construct is transferred by transformation with purified DNA or, mediated by a helper strain such as E. coli SM10 (Simon R. et al., Biotechnology 1, 784-791 (1983)), into Agrobacterium tumefaciens such as A282 with a Ti plasmid via triparental mating. Direct transformation and triparental mating were carried out as described in "Plant Molecular Biology Manual"
(Kluwer Academic Publisher, Dardrech (1988)).
It is possible in principle to transform all plants with the binary plant vectors carrying the DNA constructed according to the invention. Dicotyledonous plants are preferred, especially productive plants which produce or store starch, carbohydrates, proteins or fats in utilizable amounts in their organs, or which produce fruit and vegetables or which provide spices, fibers and industrially useful products or pharmaceuticals, dyes or waxes and, moreover, fodder plants. As example mention 28976-Sl -may be made of tomato, strawberry, avocadoand plants which bear tropical fruits, mango, for example papaya, but also pear, apple, nectarine, apricot or peach.
Further examples of plants to be transfoaned are all types of cereals, rape, bird rape... The transformed cells are selected using a selection medium,cultured to a callus and regenerated to the plant on appropriate an medium (Shain M. et al., Theor. appl. Genet.~, 770-770 (1986)); Masson, J. et al., Plant Science ~,,'~,,167-176 ( 1987 ) ) , Zhan g. et al. , Plant Mol. Hiol. ,~,,~,551-559 (1988); McGranaham G. et al., Bio/Technology 800-804 ~, (1988); Novak F. J. et al., Bio/Technology7, 154-159 (1989)).
The following examples serve to illustrate the invention further.
8zamcples 1. Isolation of the virus coat protein gene The virus was purified by modification of the method of Lot, M. et al . , Anual Phytopath. ~, 25-32 ( 1972 ) . Alfalfa was infected with alfalfa mosaic virus and, after 14 days, disrupted in the same volume of 0.5 M sodium citrate (pH 6.5)/5 miK EDTA/0.5% thioglycolic acid. Then 1 volume of chlorofoan was added, and the mixture was centrifuged at 12,000 x g for 10 min. The supernatant was mixed with 10% PEG 6000 (~r/w) and stirred cautiously overnight. Zt was then csntrifnged at 12,000 x g for 10 min and reauspendod in 50 ml of 5 mM sodium borate, 0.5 mM EDTA (pH 9). Triton g-100 (final concentrations 2%) was added and then the mixture was stirred for 30 min and centrifuged at 19,000 x g for 15 min. The virus pellet after centrifugation at 105,000 x g for Z h was taken up in 5 mM borate buffer/0.5 mM EDTA (pH 9.0) and subjected to a sucrose centrifuqation (5-25%).
Individual fractions from the gradient were analyzed on an agarose gel in order to find the virus-containing - 5 - cC~ ~., ~' , ~; ~.,. , ...
zone. The virus RNA was purified of coat protein by phenol/SDS extraction (Peden, K.W. et al., Virology 53, 487-492 (1973). The RNA components were fractionated using 2.8~ polyacrylamide with 40 mM tris acetate buffer (pH 7.5) as described in Synous, R.H., Aust. J. Biol.
Sci. 31, 25-37 (1978). The RNA was removed from the gel by electrophoresis in dialysis tubes and precipitated.
cDNA transcripts of RNA3 or RI~A4 were prepared as des-cribed in Langenreis, K. et al., Plant Mol. Biol. 6, 281-288 (1986) using synthetic oligonucleotide primers with 3'-complementary nucleotides to the template, each of which had an Smal or PstI cleavage site at the 5' end.
The reactions for the cDNA synthesis were carried out in accordance with the "Current Protocols in Mol. ~3iol. " ed.
Ausubel, F. et al., John Wiley and Sons.
The cDNA was cloned into the SmaI/PstT-cut p~JC 19 vector.
It was possible to delete the insertion again using Smal/HindIII.
The method described above can equally be used to isolate the CMV coat protein gene.
2. Isolation of the herbicide-resistance gene A phosphinothricin-resistance gene with the following sequence was synthesized in a synthesizer using the phosphoamidite method.
y;s.~,.~y,.... ~,~;n 2. ~ t.g y '~' GTC GAC ATG TCT CCG GAG AGG AG~a CCA GTT GAO ATT AGG CCA GCT
4 TAC AGA GGC CTC TCC TCT GG't CAA CTC TAA,TCC GGT CGA
~4 b~' 7~'81 fir, ACA GCA GCT GAT ATG GCC GCG GTT TGT GIST ATC GTT AAC CAT TAC
TGT CGT CGA CTA TAC CGG CGC CAA ACA CTA TAG CAA TTG GTA ATO
99 1C~~ 117 1b ATT GAG ACG TCT ACA GTG AAC TTT AGG ACA GAG CCA CAA ACA CCA
TAA CTC TGC AGA TGT CAC TTG AAA TCC TGT CfC GGT GTT TGT GGT
~. 44 1 ~._, 132 9.71 CRA GAG TGG ATT GAT GAT CTr'~ GAG AGG TTG CAA GAT ArA TAC CCT
GTT CTC ACC TAA CTA CTA GAT CTC TCC AAC GTT CTA TCT ATG GGA
189 199 d«7 ~i6 TGG TTO GTT GCT GAt3 GTT GA~3 GET GTT OTG GCT GGT ATT GCT TAC
ACC AAC CAA CGA CTC CAA CTC CCF~ CAA CAC CGA CC~1 TAA CGA ATG
"~4 '4C Wig: 261 27r,~
GCT GGG CCC TGG AAG GCT AGG ABC GCT TAC GAT TGG ACA GTT GAG
CGA CCC GGG ACC TTC CGA TCC TTG CGA ATG CTA ACC TGT CAA CTC
79 ~~18 .~~.97 Ct:W ~1~
AGT ACT OTT TAC GTG TCA CAT ArG CAT CAA AGG TTG ~3GC CTA GGA
TCA TGA CAA ATG CAC AGT GTA TCC GTA cTT TCC AAC CCG GAT CCT
4 ~ r~R ~ 4d agl ~ 6t t TCC ACA TTG TAC ACA CAT TTO CTT Af~G TCT ATG GAG GCG CAA GGT
AGG TGT AAC ATG TGT DTA AAC GAA TTC AGA TAC CTC CGC CTT CCA
X69 ~71~ X87 ~9~ 4c;y TTT AAG TCT GTG GTT GCT GTT ATA GGC CTT CCA AAr GAT CCA TCT
AAA TTC AOA CAC CAA CGA CAA TAT CCG GAA GGT T1'G CTA GGT AGA
414 4'~. 4.~ 441 4~~y GTT AGG TTG CAT CAO GCT TTO OGA TAC ACA C~CC CGG GGT ACA TTG
CAA TCC AAC GTA CTC COA AAC CCT AT13 l~aT COG GCC CCA TGT AAC
4~9 469 47'? ~lg~ 4~~
CGC GCA GCT GAGA TAC AAG CAT G3T GG~4 T~3G CAT GA'T GTT GGT TTT
GCG CGT CGA CCT ATG TTC Gl'A CCA CC'T ACC OTA CTA CAA CCA A~1A
'5114 ~1~ '~,~~,;w '5~.,1 ~54r:~
TGG CAA AGG GAT TTT GAO TTY CCA GC'T CCT CCA AGO CCA GTT AGG
ACC GTT TCC CTA AAA CTC AAC GOT COA GGA GOT TCC GGT CAA TCC
:~49 ~~8 CCA OTT ACC CAO ATC TC,A O
GDT CAA TOO C3TC TAO ACY CAI CT~ ~' This is a modification of the sequence for the acetyl-transferase r~ene published by Wohlleben in Gene 7~D, 25-37 (1988).
It is likewise possible to examine a genomic DNA bank from the Streptomyces viridochromogenes used by Wohlleben in EMBL3 in E. coli for the acetylation of phosphino-thricin. The acetylated product can be very easily fractionated by thin-layer chromatography.
The gene was cloned in pUCl9 and sequenced. Expression in plants was carried out as SalI fragment.
3. Fusion of herbicide-resistance gene with Nos pro-moter The vector pNCN was digested with Bam/SalI, and the resulting 2.5 by piece was isolated. The protruding ends were digested off with mung bean nuclease. The acetyl transferase gene was isolated as 0.5 by piece after SalI
digestion and filled in with Rlenow. After ligase, it was possible to isolate positive clones by plasmid mini preparations. The orientation was evident from a SalI/Bam digestion.
4. Fusion of coat protein gene with 35S promoter A fragment, 0.5 base-pairs long, from pAI RNA3 (the pUCl9 vector with coat protein gene insert) was isolated after digestion with SmaI/HindIII. The protruding ends were digested off by mung bean nuclease. The vector pDH 51 was cut with XbaI, and ends were filled in with Klenow polymerase. Fragment and vector were ligated and trans-formed into MC 1061 (p35/AI). The same construction was carried out with pCM RNA3 for the coat protein of CMV
(p35/CM).
~4 b~' 7~'81 fir, ACA GCA GCT GAT ATG GCC GCG GTT TGT GIST ATC GTT AAC CAT TAC
TGT CGT CGA CTA TAC CGG CGC CAA ACA CTA TAG CAA TTG GTA ATO
99 1C~~ 117 1b ATT GAG ACG TCT ACA GTG AAC TTT AGG ACA GAG CCA CAA ACA CCA
TAA CTC TGC AGA TGT CAC TTG AAA TCC TGT CfC GGT GTT TGT GGT
~. 44 1 ~._, 132 9.71 CRA GAG TGG ATT GAT GAT CTr'~ GAG AGG TTG CAA GAT ArA TAC CCT
GTT CTC ACC TAA CTA CTA GAT CTC TCC AAC GTT CTA TCT ATG GGA
189 199 d«7 ~i6 TGG TTO GTT GCT GAt3 GTT GA~3 GET GTT OTG GCT GGT ATT GCT TAC
ACC AAC CAA CGA CTC CAA CTC CCF~ CAA CAC CGA CC~1 TAA CGA ATG
"~4 '4C Wig: 261 27r,~
GCT GGG CCC TGG AAG GCT AGG ABC GCT TAC GAT TGG ACA GTT GAG
CGA CCC GGG ACC TTC CGA TCC TTG CGA ATG CTA ACC TGT CAA CTC
79 ~~18 .~~.97 Ct:W ~1~
AGT ACT OTT TAC GTG TCA CAT ArG CAT CAA AGG TTG ~3GC CTA GGA
TCA TGA CAA ATG CAC AGT GTA TCC GTA cTT TCC AAC CCG GAT CCT
4 ~ r~R ~ 4d agl ~ 6t t TCC ACA TTG TAC ACA CAT TTO CTT Af~G TCT ATG GAG GCG CAA GGT
AGG TGT AAC ATG TGT DTA AAC GAA TTC AGA TAC CTC CGC CTT CCA
X69 ~71~ X87 ~9~ 4c;y TTT AAG TCT GTG GTT GCT GTT ATA GGC CTT CCA AAr GAT CCA TCT
AAA TTC AOA CAC CAA CGA CAA TAT CCG GAA GGT T1'G CTA GGT AGA
414 4'~. 4.~ 441 4~~y GTT AGG TTG CAT CAO GCT TTO OGA TAC ACA C~CC CGG GGT ACA TTG
CAA TCC AAC GTA CTC COA AAC CCT AT13 l~aT COG GCC CCA TGT AAC
4~9 469 47'? ~lg~ 4~~
CGC GCA GCT GAGA TAC AAG CAT G3T GG~4 T~3G CAT GA'T GTT GGT TTT
GCG CGT CGA CCT ATG TTC Gl'A CCA CC'T ACC OTA CTA CAA CCA A~1A
'5114 ~1~ '~,~~,;w '5~.,1 ~54r:~
TGG CAA AGG GAT TTT GAO TTY CCA GC'T CCT CCA AGO CCA GTT AGG
ACC GTT TCC CTA AAA CTC AAC GOT COA GGA GOT TCC GGT CAA TCC
:~49 ~~8 CCA OTT ACC CAO ATC TC,A O
GDT CAA TOO C3TC TAO ACY CAI CT~ ~' This is a modification of the sequence for the acetyl-transferase r~ene published by Wohlleben in Gene 7~D, 25-37 (1988).
It is likewise possible to examine a genomic DNA bank from the Streptomyces viridochromogenes used by Wohlleben in EMBL3 in E. coli for the acetylation of phosphino-thricin. The acetylated product can be very easily fractionated by thin-layer chromatography.
The gene was cloned in pUCl9 and sequenced. Expression in plants was carried out as SalI fragment.
3. Fusion of herbicide-resistance gene with Nos pro-moter The vector pNCN was digested with Bam/SalI, and the resulting 2.5 by piece was isolated. The protruding ends were digested off with mung bean nuclease. The acetyl transferase gene was isolated as 0.5 by piece after SalI
digestion and filled in with Rlenow. After ligase, it was possible to isolate positive clones by plasmid mini preparations. The orientation was evident from a SalI/Bam digestion.
4. Fusion of coat protein gene with 35S promoter A fragment, 0.5 base-pairs long, from pAI RNA3 (the pUCl9 vector with coat protein gene insert) was isolated after digestion with SmaI/HindIII. The protruding ends were digested off by mung bean nuclease. The vector pDH 51 was cut with XbaI, and ends were filled in with Klenow polymerase. Fragment and vector were ligated and trans-formed into MC 1061 (p35/AI). The same construction was carried out with pCM RNA3 for the coat protein of CMV
(p35/CM).
5. Fusion of 35S/coat protein gene and nos/acetyl-transferase gene A 1.3 kb piece from the 35S/coat protein construct (p35/AI, p35/CM) after EcoRI digestion was isolated from a low melt agarose gel. The plant vector pOCA 18 was digested with EcoRI and ligated to the 1.3 kbp DNA piece.
This pOCA/35 RNA3 vector was filled in with Klenow. A
2.5 kbp HindIII piece from nos/AC was, after Rlenow treatment of the ends, inserted into the filled-in ClaI
site.
Constructions: pOCA/AcAI3 pOCA/AcCM3 - g -6. Transformation of Agrobacteria The Agrobacterium strain pMP90RK was transformed with pOCA/AcAI3 or pOCA/AcCM3 in triparental mating with SM10.
100 ~1 portions of bacteria from overnight cultures of SM10, the MC 1061 carrying the construction, and the Agrobacteria were spun down and suspended together in 30 ~1 of LB medium. These cells were placed on a small circular filter on an LB plate without antibiotic. After incubation at 37°C for 12 h, the filter was washed in 2.5 ml of 10 mM MgCl2, and aliquots thereof were selected on LB plates containing rifampicin, tetracycline and kanamycin. Positive colonies were identified by hybridi-zation with 32P-labeled DNA of the genes.
This pOCA/35 RNA3 vector was filled in with Klenow. A
2.5 kbp HindIII piece from nos/AC was, after Rlenow treatment of the ends, inserted into the filled-in ClaI
site.
Constructions: pOCA/AcAI3 pOCA/AcCM3 - g -6. Transformation of Agrobacteria The Agrobacterium strain pMP90RK was transformed with pOCA/AcAI3 or pOCA/AcCM3 in triparental mating with SM10.
100 ~1 portions of bacteria from overnight cultures of SM10, the MC 1061 carrying the construction, and the Agrobacteria were spun down and suspended together in 30 ~1 of LB medium. These cells were placed on a small circular filter on an LB plate without antibiotic. After incubation at 37°C for 12 h, the filter was washed in 2.5 ml of 10 mM MgCl2, and aliquots thereof were selected on LB plates containing rifampicin, tetracycline and kanamycin. Positive colonies were identified by hybridi-zation with 32P-labeled DNA of the genes.
7. Transformation of alfalfa A modified version of the cocultivation method of Marton S. et al., Nature 277, 129-130 (1979) was employed for the transformation of alfalfa. Stalk sections about 1 cm long from sterile plants were placed in 40 ml of sterile MS medium in Erlenmeyer flasks, and 11 ml of a diluted overnight culture of the Agrobacteria (5 x 10' cells/ml) were added. Incubation was continued at 25°C
for 3 days. The stalk segments were then washed three times with sterile water and placed on MS medium contain-ing 300 mg/1 carbamicillin and 100 mg/1 kanamycin. A
callus from which it was possible to regenerate whole plants formed after 3 weeks.
for 3 days. The stalk segments were then washed three times with sterile water and placed on MS medium contain-ing 300 mg/1 carbamicillin and 100 mg/1 kanamycin. A
callus from which it was possible to regenerate whole plants formed after 3 weeks.
8. Testing of the plants The plants showed, after working up of RNA and hybridiza tion with radiolabeled DNA of the genes, expression of AC
gene with alfalfa mosaic virus coat protein gene.
The plants grew on phosphinothricin-containing medium and showed distinct tolerance after infection with alfalfa mosaic virus.
gene with alfalfa mosaic virus coat protein gene.
The plants grew on phosphinothricin-containing medium and showed distinct tolerance after infection with alfalfa mosaic virus.
Claims (7)
1. A plant cell transformed with a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance.
2. A plant cell as claimed in claim 1, wherein the nucleotide sequence encoding the virus coat protein can be obtained by cDNA cloning starting from the RNA of cucumber mosaic virus, of alfalfa mosaic virus or of brome mosaic virus.
3. A plant cell as claimed in claim 1 or 2, wherein the nucleotide sequence encoding a protein conferring phosphinothricin-resistance is from Streptomyces.
4. A plant cell as claimed in any one of claims 1 to 3 and expressing the respective nucleotide sequence.
5. A process for the preparation of transformed plants with improved plant yield, which are transformed with a DNA
molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance, and which comprises treating the virus- and herbicide-resistant regenerated plants with phosphinothricin, and this treatment resulting in improved plant yield.
molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance, and which comprises treating the virus- and herbicide-resistant regenerated plants with phosphinothricin, and this treatment resulting in improved plant yield.
6. A method for increasing the yield from plants which are transformed with a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance, which comprises treating the plants with phosphinothricin in the growth period.
7. The use of a DNA molecule consisting of a nucleotide sequence encoding a protein conferring phosphinothricin-resistance and a nucleotide sequence encoding a virus coat protein conferring virus-resistance for increasing the yield from plants transformed therewith.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4003045.8 | 1990-02-02 | ||
| DE4003045A DE4003045A1 (en) | 1990-02-02 | 1990-02-02 | VIRUS / HERBICIDE RESISTANCE GENES, METHOD FOR THEIR PRODUCTION AND THEIR USE |
| PCT/EP1991/000130 WO1991011517A2 (en) | 1990-02-02 | 1991-01-24 | Virus/herbicide resistance genes process for producing same and their use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2075209A1 CA2075209A1 (en) | 1991-08-03 |
| CA2075209C true CA2075209C (en) | 2002-10-01 |
Family
ID=6399257
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002075209A Expired - Lifetime CA2075209C (en) | 1990-02-02 | 1991-01-24 | Virus/herbicide resistance genes process for producing same and their use |
Country Status (11)
| Country | Link |
|---|---|
| EP (2) | EP0513054B1 (en) |
| JP (1) | JPH05506568A (en) |
| KR (1) | KR927003822A (en) |
| AU (1) | AU654662B2 (en) |
| CA (1) | CA2075209C (en) |
| DE (2) | DE4003045A1 (en) |
| ES (1) | ES2131050T3 (en) |
| HU (1) | HU219059B (en) |
| NZ (1) | NZ236979A (en) |
| OA (1) | OA09664A (en) |
| WO (1) | WO1991011517A2 (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4442179C2 (en) * | 1994-11-26 | 1996-12-12 | Inst Pflanzengenetik & Kultur | Pathogen resistant plants and processes for their production |
| WO1996021018A1 (en) * | 1994-12-30 | 1996-07-11 | Asgrow Seed Company | Plants resistant to c strains of cucumber mosaic virus |
| EP1728868A2 (en) * | 1996-11-07 | 2006-12-06 | Syngenta Limited | Herbicide resistant plants |
| DE19836684A1 (en) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Use of a synergistic herbicidal combination including a glufosinate- or glyphosate-type, imidazolinone or protoporphyrinogen oxidase to control weeds in rice |
| CA2340013C (en) | 1998-08-13 | 2012-10-02 | Aventis Cropscience Gmbh | Herbicidal compositions for tolerant or resistant maize crops |
| DE19836700A1 (en) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Use of a synergistic herbicide combination including a glufosinate- or glyphosate-type, imidazolinone or protoporphyrinogen oxidase inhibitory azole herbicide to control weeds in cereals |
| DE19836660A1 (en) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Use of a synergistic herbicide combination including a glufosinate- or glyphosate-type, imidazolinone or protoporphyrinogen oxidase inhibitory azole herbicide to control weeds in soya |
| DE19836659A1 (en) | 1998-08-13 | 2000-02-17 | Hoechst Schering Agrevo Gmbh | Use of synergistic herbicide combination including glufosinate- or glyphosate-type, imidazolinone, protoporphyrinogen oxidase inhibitory azole or hydroxybenzonitrile herbicide, to control weeds in cotton |
| BR112012029616A2 (en) | 2010-05-21 | 2015-10-20 | Bayer Ip Gmbh | herbicidal agents for tolerant or resistant cereal crops |
| CN103002743A (en) | 2010-05-21 | 2013-03-27 | 拜耳知识产权有限责任公司 | Herbicidal agents for tolerant or resistant rape cultures |
| US20110287934A1 (en) | 2010-05-21 | 2011-11-24 | Bayer Cropscience Ag | Herbicidal composition for tolerant or resistant rice crops |
| EP2571366A1 (en) | 2010-05-21 | 2013-03-27 | Bayer Intellectual Property GmbH | Herbicidal agents for tolerant or resistant corn cultures |
| EP2524602A1 (en) | 2011-05-20 | 2012-11-21 | Bayer CropScience AG | Herbicide agent for tolerant or resistant soya cultures |
| UA117816C2 (en) | 2012-11-06 | 2018-10-10 | Байєр Кропсайєнс Акцієнгезелльшафт | Herbicidal combinations for tolerant soybean cultures |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU616635B2 (en) * | 1986-04-02 | 1991-11-07 | Pioneer Hi-Bred International, Inc. | Virus resistant plants having coat protein |
| CN87100603A (en) * | 1987-01-21 | 1988-08-10 | 昂科公司 | Vaccines against melanoma |
| DE3716309A1 (en) * | 1987-05-15 | 1988-11-24 | Hoechst Ag | RESISTANCE TO PHOSPHINOTHRICIN |
| EP0298918B1 (en) * | 1987-07-10 | 2001-09-05 | Syngenta Participations AG | Inducible virus resistance in plants |
-
1990
- 1990-02-02 DE DE4003045A patent/DE4003045A1/en not_active Withdrawn
-
1991
- 1991-01-24 DE DE59109123T patent/DE59109123D1/en not_active Expired - Fee Related
- 1991-01-24 HU HU9202500A patent/HU219059B/en unknown
- 1991-01-24 AU AU71403/91A patent/AU654662B2/en not_active Ceased
- 1991-01-24 KR KR1019920701841A patent/KR927003822A/en not_active Application Discontinuation
- 1991-01-24 JP JP91503084A patent/JPH05506568A/en active Pending
- 1991-01-24 ES ES91902693T patent/ES2131050T3/en not_active Expired - Lifetime
- 1991-01-24 CA CA002075209A patent/CA2075209C/en not_active Expired - Lifetime
- 1991-01-24 EP EP91902693A patent/EP0513054B1/en not_active Expired - Lifetime
- 1991-01-24 WO PCT/EP1991/000130 patent/WO1991011517A2/en active IP Right Grant
- 1991-01-24 EP EP98108424A patent/EP0899340A3/en not_active Withdrawn
- 1991-01-31 NZ NZ236979A patent/NZ236979A/en unknown
-
1992
- 1992-07-31 OA OA60256A patent/OA09664A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| KR927003822A (en) | 1992-12-18 |
| HU9202500D0 (en) | 1993-07-28 |
| WO1991011517A2 (en) | 1991-08-08 |
| EP0899340A2 (en) | 1999-03-03 |
| WO1991011517A3 (en) | 1992-09-03 |
| EP0899340A3 (en) | 1999-04-28 |
| OA09664A (en) | 1993-05-15 |
| AU654662B2 (en) | 1994-11-17 |
| HU219059B (en) | 2001-02-28 |
| AU7140391A (en) | 1991-08-21 |
| NZ236979A (en) | 1992-12-23 |
| CA2075209A1 (en) | 1991-08-03 |
| EP0513054A1 (en) | 1992-11-19 |
| DE4003045A1 (en) | 1991-08-08 |
| EP0513054B1 (en) | 1999-04-21 |
| ES2131050T3 (en) | 1999-07-16 |
| JPH05506568A (en) | 1993-09-30 |
| DE59109123D1 (en) | 1999-05-27 |
| HUT64395A (en) | 1993-12-28 |
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