CA1321364C - Phosphinothricin-resistance gene active in plants, and its use - Google Patents
Phosphinothricin-resistance gene active in plants, and its useInfo
- Publication number
- CA1321364C CA1321364C CA000556972A CA556972A CA1321364C CA 1321364 C CA1321364 C CA 1321364C CA 000556972 A CA000556972 A CA 000556972A CA 556972 A CA556972 A CA 556972A CA 1321364 C CA1321364 C CA 1321364C
- Authority
- CA
- Canada
- Prior art keywords
- gene
- plants
- vector
- ptc
- resistance gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
Abstract
Abstract of the disclosure:
The phosphinothricin (PTC)-resistance gene isolated from the genome of Streptomyces viridochromogenes DSM 40736 is, after adaptation to the codon usage in plants, synthe-sized and incorporated into gene structures which make plants resistant to PTC after expression therein.
The phosphinothricin (PTC)-resistance gene isolated from the genome of Streptomyces viridochromogenes DSM 40736 is, after adaptation to the codon usage in plants, synthe-sized and incorporated into gene structures which make plants resistant to PTC after expression therein.
Description
HOECHST AKTIENGESELLSCHAFT HOE 87/F 333 J Dr. KL/mu :
Specification :i Phosphinothricin-res;stance yene acti~e ~n plant~r ar4d ~ts ~se 5 C~Adian Pat~nt Applic~tion S~rl~l No. 545,037 propo~ pbo~-phinothricin (PTC)-resistance gene which can be obtained from the total DNA of ~
DS~ 40736 tgeneral collection) or DSM 4112 tdeposition `~.
under the Budapest Treaty), which has been selected for phosphinothricyl-alanyl-alan;n~ (PTT)-resistance, by cut-tin~ with ~amH$, cloning of a fragment 4.0 kb in size, and selection ~or PTT-resistance, as ~ell as the use of this ~ene for the production of PTC-resistant pLants, as PTT-resistance marker in bacteria and PTC-resistance ~arker in plant cells. ~he aamHI fragment ~hich is 4 kb in size and on ~hich the resistance g~ne is located is defined in ; detail b~ a restrictlon map (Figur~ 1 of Canadian P~tent Appliration 5eri~1 No. 545,037).
ZO
The position of ths coding region has been located ~ore accurately by cloning part-regions of this 4 kb fragment. It emerged from this that the resistance gene is located on the 1.6 kb SstII-SstI fragment (positions 0.55 ~o 2.15 in Fig. 1 of Canadian Patent Application Serial No. 545,037).
Digestion with BglII results in a fragment 0.8 kb in size ~hich confers PTT-resistance after incorporation ~n a plasmid and transformation of S.
lividans. This resistance depends on the N-acetylation of PTC. Hence the resistance gene codes for ~in acetyltr~nsferase.
The DNA s~qu~nc~ of th~ abovem~ntion~d 0.8 kb fr&g~nt i~ reproducad i~
C~n~d~ Patent ApplicAtion Seri~l No. 545,037.
I~ is possible to deter~ine fro~ the sequence the s~art codon and the open reading frame of the ~ene sequence.
The ~ast nucleotide ii5 part of ~he stop codon ~GA~
Genes from Streptomycetes have a very high praportion of ~ :
G ~ C, the adenine SA3 ~ thymine (T) : guanine ~6) ~ ~
-`` 1321364 cytosine ~C) ratio being about 30 : 70. The proportion of GC in plant genes is far lo~er, being about 50~. For this reason, in a fureher development of the inventive idea, the DNA sequence of the resistance gene has been S optimized~ by de novo synthesis, to a codon usage favor-able for plant RNA poly~eras~ II.
The lnv~lltloll r~l~t~ to ~ mod~f ~ tlon ~f the r~al~t~nce g~ wh~ch iLB
1~ prQpos~d in C~n~diAn P~tant Appl~c~tion Serial ~o. 545,037, namely an adaptat;on eo th~ codon usage in p~ants. The corresponding amino acid sequence is depicted in the annex.
~urther embodiments of the invention are defined in the patent c~aims or are expLained hereinafter.
As is kno~n, the genetics code is degenerate, i.~. on~y 2 amino acids are coded for by a single tripletO where-as the remaining 18 genetically codable amino acids are assigned to 2 to 6 triplets. Thus, theoretically, a ~O ~;de;variety of:codon~-combinations-can be chosen for the synthesis of the gen~. Since the said rel~tive propor-~ion of the indiv;dua~ nucleotides in the total DNA se-quence exerts an influence, it was used as one of the criteria on ~hich the sequence optimi2ation ~as based.
The following ~odifi~ations ~ere ~ade tc the sequenced gene: `
1~ The Streptomycetes gene stare codon GTG (posit;on 258-260 in ~he sequence in ~he additionaL applica-tion) ~as replaced by the start codon ATG ~h;ch is used by plant RNA poly~erase II.
2. ~ithin the gene, ~he Strepto~ycetes gene codons ~ere changed in such a ~ay that they resu~ted in codons suitable in p~ant ~enes ~6/G ratio~.
Specification :i Phosphinothricin-res;stance yene acti~e ~n plant~r ar4d ~ts ~se 5 C~Adian Pat~nt Applic~tion S~rl~l No. 545,037 propo~ pbo~-phinothricin (PTC)-resistance gene which can be obtained from the total DNA of ~
DS~ 40736 tgeneral collection) or DSM 4112 tdeposition `~.
under the Budapest Treaty), which has been selected for phosphinothricyl-alanyl-alan;n~ (PTT)-resistance, by cut-tin~ with ~amH$, cloning of a fragment 4.0 kb in size, and selection ~or PTT-resistance, as ~ell as the use of this ~ene for the production of PTC-resistant pLants, as PTT-resistance marker in bacteria and PTC-resistance ~arker in plant cells. ~he aamHI fragment ~hich is 4 kb in size and on ~hich the resistance g~ne is located is defined in ; detail b~ a restrictlon map (Figur~ 1 of Canadian P~tent Appliration 5eri~1 No. 545,037).
ZO
The position of ths coding region has been located ~ore accurately by cloning part-regions of this 4 kb fragment. It emerged from this that the resistance gene is located on the 1.6 kb SstII-SstI fragment (positions 0.55 ~o 2.15 in Fig. 1 of Canadian Patent Application Serial No. 545,037).
Digestion with BglII results in a fragment 0.8 kb in size ~hich confers PTT-resistance after incorporation ~n a plasmid and transformation of S.
lividans. This resistance depends on the N-acetylation of PTC. Hence the resistance gene codes for ~in acetyltr~nsferase.
The DNA s~qu~nc~ of th~ abovem~ntion~d 0.8 kb fr&g~nt i~ reproducad i~
C~n~d~ Patent ApplicAtion Seri~l No. 545,037.
I~ is possible to deter~ine fro~ the sequence the s~art codon and the open reading frame of the ~ene sequence.
The ~ast nucleotide ii5 part of ~he stop codon ~GA~
Genes from Streptomycetes have a very high praportion of ~ :
G ~ C, the adenine SA3 ~ thymine (T) : guanine ~6) ~ ~
-`` 1321364 cytosine ~C) ratio being about 30 : 70. The proportion of GC in plant genes is far lo~er, being about 50~. For this reason, in a fureher development of the inventive idea, the DNA sequence of the resistance gene has been S optimized~ by de novo synthesis, to a codon usage favor-able for plant RNA poly~eras~ II.
The lnv~lltloll r~l~t~ to ~ mod~f ~ tlon ~f the r~al~t~nce g~ wh~ch iLB
1~ prQpos~d in C~n~diAn P~tant Appl~c~tion Serial ~o. 545,037, namely an adaptat;on eo th~ codon usage in p~ants. The corresponding amino acid sequence is depicted in the annex.
~urther embodiments of the invention are defined in the patent c~aims or are expLained hereinafter.
As is kno~n, the genetics code is degenerate, i.~. on~y 2 amino acids are coded for by a single tripletO where-as the remaining 18 genetically codable amino acids are assigned to 2 to 6 triplets. Thus, theoretically, a ~O ~;de;variety of:codon~-combinations-can be chosen for the synthesis of the gen~. Since the said rel~tive propor-~ion of the indiv;dua~ nucleotides in the total DNA se-quence exerts an influence, it was used as one of the criteria on ~hich the sequence optimi2ation ~as based.
The following ~odifi~ations ~ere ~ade tc the sequenced gene: `
1~ The Streptomycetes gene stare codon GTG (posit;on 258-260 in ~he sequence in ~he additionaL applica-tion) ~as replaced by the start codon ATG ~h;ch is used by plant RNA poly~erase II.
2. ~ithin the gene, ~he Strepto~ycetes gene codons ~ere changed in such a ~ay that they resu~ted in codons suitable in p~ant ~enes ~6/G ratio~.
3. The TGA stop codon ~as placed at the end of the se-. quence to termin~te the translation processO
The beginning and end o~ the gene sequence ~ere provid~d ~ith protruding ends of restriction sites in order to be ~ble to amplify the gene and li~ate æ
.. . -. . ........... .............. .. ,.,,.. .. , - .... . ~ . .. , . . ; - ..
~32~
. .
~ it between plant regulation sequences. ~
5. Palindromic sequences were reduced to a minimum. ~:
Description of the Drawin~
The DNA sequence I according to the invention twith the S corresponding amino acid sequence) i~ depicted in Figure 1 . ' Three internal un;que cleavage sites for the res~riction enzymes XbaI ~position 152), BamHI (312~ and XmaI (436) make possible ehe subcloning o~ part-sequences ~hich can be incorporated in ~ell-inves~igated cloning vectors such as, for example, pUC18 or pUC19. In addition, a number of other unique recognition sequences for restr;ction en~ymes ~er~ incorporated within the gene~ and these, on the one hand, provide access to part-sequences o~ acetyl-transferase and, on the other hand, allo~ mod;f;cat;ons to b~ ~ade:
Restriction enzyme Sut after nucleotide ~o.
BspMII 11 SacII
EcoRV 74 HpaI S0 AatII 99 BstXI 139 ApaI 232 Sc~I 272 AvrII 30~
AflII 336 StuI 385 ~ssHII 449 FokI 4~7 BglI 536 BgLIl 550 The construction of p~rt-sequences by chemical synthesis and enzy~atic li~a~ion reactions is carrieid out in a ~anner kno~n per se ~EP-A Q,133,282, 0,136,472, 0,1559590, : . , -, ::
132~ 3~4 0~161~SD4, 0,163,249, 0,171,024, 0,173,149 or 0,177,827 Deta;ls, such as restriction analyses, ligation of DNA
fragments and transformation of plasmids in E. coli, are described a~ length in the textbook of Maniatis (Mole-cular Cloning, Maniatis et al., Cold Spring Harbor, 1982)~
The gene sequence which has been cloned in this way is then in~roduced ;nto plan~s, under the control of plant regulation signals, and its expression is induced.
EP-A 0~122,791 revie~s known methodsO In this way are obtained PTC-res;stan~ plant cells (i.e~ a selection feature for transformed cells is available), plants or parts of plants and seeds.
Some embodiments of the invention are explained in detail in the examples ~hich follo~. Unless other~ise indica-ted, percentage data therein relate to weight.
`- - 20 Examples -- - -The follo~ing media were used:
a) for bacteria:
25YT medium: O.5X yeast extract, 0.8% Bacto tryp-tone, 0.5% NaCl LP medium: 0.5X yeast extractr 1% Bacto tryptone, 1X NaCl as solid mediu~: addition of 1.5X agar to each b) for plants:
M+S medium: see Mur~shige and Skoog, Physiologica Plantarum 15 (19~2) 473 2MS medium: M~S medium containing 2% sucrose MSC10 medium: M~S medium containing 2% sucrose~ 500 mg/l cefotaxime, 0.1 mg/l naphthylacetic acid (NAA), 1 mg/l benzylaminopurine (~AP), 100 mg/l kanamyc;n MSC15 medium: M+S medium containing 2% sucrose, ,. : . :, . , ~ .
~3213~
500 mg/l cefotaxime~ 100 mg/l kanamycin.
1. Chemical synthesis of a single stranded oligonucleo-t;de The synthesis of fragment II, one of the four part-fragments I - IVD started from the terminal oLigonuc-leotide IIc (nucleotide No~ 219 to 31Z ;n the coding strand of DNA sequence I). For the solid-phase syn-thes;s, the nucleoside at the 3' end, that is to say guanosine (nucleotide No. 312) ;n ~he present case, is covalently bonded via ~he 3'-hydroxyl group to a support. The support material ;s CPG (controlled pore glass) funct;onalized w;th long-chain amino alkyl raclicals. Otherw;se, the syn~hesis follows the known (from the Said EP_A~) methOdS.
The plan of synthesis is ind;cated in DNA sequ~nce 2a ~.I.-~annex~7-!*wh;ch-lothe.r~i.se-^~ror~re-spor~ds-:to.:DNA se-quence I.
2~ Enzymatic linkage of the single-stranded oligonucleo-tides to give gene ~ragment II
For the phosphorylat;on of the oligonucleotides at the 5' end, 1 nmol of each Qf oligonucleotide~ IIb and IIc was treated with S nmol of adenos;ne tr;phos-phate and 4 un;ts of T4 polynucl~otide k;nase ;n 20 ~l of 50 mM tris-HCl buffer ~pH 7.6), 10 mM mag-nesium chloride and 1Q mM dithiothreitol (DTT) at 37G for 30 m;nutes~ The enzyme ;s inactivated by heating at 95C for 5 minutes. Oligonucleotides IIa and IId, which form the "protrud;ng" sequence ;n DNA fragment II, are not phosphorylated~ Th;s pre-vents the formation, dur;ng the subsequent l;ga~;on, of larger subfragments than correspond to DNA frag-ment II.
.. . .
132~36~
O~igonucleotides II (a-d) are ligated to give sub-fragment II as follows: 1 nmol of each of oligonuc-leotides IIa and IId and the 5'-phosphates of IIb and IIc are together dijsolved in 45 ~l of buffer containing 50 mM tr;s-HCl tpH 7.6), 20 mM magnesium chloride, 25 mM potassium chloride and 10 mM DTT.
For the annealing of the oligonucleotides correspond-ing to DNA fragm~nt II, the solution of the oligo-nucle3tides is heated at 95C for 2 minutes and then slowly ~ooled (2-3 hours) to 20C. Then, for the enzymaeic Linkage, 2 ~l o~ 0O1 M DTT, 8 ~l of 2.5 ~M adenosine triphosphate (pH 7) and 5 ~l o~ T4 DNA ligase (2000 un;ts~ arY added, and the mixture is incubated at 22C for 1b hsurs.
The gene fragment II is puri~ied by geL el~ctropho-resis on a 10X Polyacrylamide gel ~ithout addition of urea, Z0 x 40 cm, 1 ~m thick), ~he marker sub-stance used b~;ng ~X 174 DNA (from ~RL) cut ~ith - ?3 ~ -Hinfl,:or p~R3~2 cut:~ with -HaelII.
6ene fragm~nts I, III and IV are prep~red analogous-ly, although the "protruding" sequences are, before th~ annealin~, converted into the 53-phosphates be-cause no l;gat;on step is necessary.
3. Preparat;on of hybrid plas~ids containin~ gene frag-m~nts I, II, III and IV.
a~ Incorporation of gene frag~nt I in pUC18 Th~ commercially available Plasmid pUC1~ is opened in a known ~anner using the restriceion endo nucLea-ses SalI and XbaI in accordance ~ith the manufact-ure~s' instructions. The digest;on mixtur~ is frac-t;onated by el~ctrophoresis ;n a kno~n manner on a 1% agaras~ gelO and the fragmen~s are visuali~ed by staining ~ith ethidium bro~ide~ The plasmid band (about 2.6 kb) is then cut out of She agarose ~32~36~ :
gel and removed from the agarose by electro-elut;on.
1 ~g of plasmid, opened with XbaI and SalI, is then ligated with 10 ng of DNA fragment I at 16C
overnight.
b) Incorporation of gene fragment II in pUC18.
In analogy to 3), pUC18 is cut open ~ith XbaI and eamHI and ligated ~ith gene fragment II which has previously heen ph~sphorylated at the protru-ding ends as described in Exa-ple 2.
c~ Incorporation of gene fragmen~ III in pU~18 In analogy to a)~ pUC18 is cut open ~;th BamHI
and XmaIII and ligated with gene fragment III.
20~ d3 :In~orpora~--ion~o~;~gen~:~ragment.-IV--in pUC18 In analogy to a), pUC18 is cut ~ith YmaIII and SalI and ligated wi~h ~ene fragmer,t IV.
Construction of the co~plete gene and cloning in a pUC plasmid a) Transformation and amplification of gene fragments I - IY
The hybrid plasmids obtained ;n this way are transformed into E. coli. For this purpose~ the . ~
strain E. coli K 12 is made competent by treat--ment with a 70 mM calciu~ chLoride soLu~ion, and the suspension of the hybrid pLasmid in 10 mM
tr;s-HCl buffer (pH 7.5), which is 70 mM in cal-cium chioride, is added~ The transformed strains are selected as is customary, utiLizing the anti-biot;c resis~ances or sensitivities conf~rred by ~2~6~
-- 8 ~
the plasmid, and the hybrid vectors are amplified.
After the cells have been killed, the hybrid plas-mids are ;solated and cut open ~ith the restric-t;on enzymes or;ginally used, and gene fragments I, II, III and IV are isolated by gel electropho-resis.
b) Linkage of gene fragments I, II, III and IV to give the total gene Subfragments I and II obtained by amplification are linked as follows. 100 ng of each of the isolated fragments I and II are dissolved togeth-er in 10 ~l of buffer containing 50 mM tris-HCl (pH 7.6), 20 mM magnesium chloride and 10 mM DTT, and this solution is heated at 57C for 5 minutes.
After the solution has cooled to room temperature, 1 ~l of 10 mM adenosine triphosphate (pH 7) and 1 ~l of T4 ligase (400 units) are added~ and the 20 ~ mixt~re is incuba~-ed ~t-room~tempe-rature for 16 hours. After subsequent cutting with the restric-~ion enzy~es SalI and BamHI, the desired 312 bp fragment (nucleotides 1-312, SalI-~amHI) is puri-fied by gel electrophoresis on an 8% polyacryl-~5 amide gel, the warker substance used being ~X 174 RF DNA tfro~ 9RL~ cut with the restriction enzyme H3eIII .
Gene frag~ents III and IV are linked together in the same way, there being obtained after purifi-cation a 246 bp fragment (nucleotides 313-558~
Ba~HI~SalI~ The marker used for the gel electro-phoresis is pBR322 cut with the restriction en~
zyme MspI.
To contruct the total gene (DNA sequence I), 15 ng of the 312 bp fragment and 12 ng of the 246 bp fragment are ligated, as described above, with 1 ~9 of the commercially available plasmid pUC18 ~3~3~
_ 9 _ which has previously been cut open with the rest-riction enzyme SalI and enzymatically dephosphory-lated at the ends. After transformation and ampli-fication (as described in Example 4a), the correct clone having the 558 bp fragment corresponding to DNA sequence I is identified by SalI digestion~
The beginning and end o~ the gene sequence ~ere provid~d ~ith protruding ends of restriction sites in order to be ~ble to amplify the gene and li~ate æ
.. . -. . ........... .............. .. ,.,,.. .. , - .... . ~ . .. , . . ; - ..
~32~
. .
~ it between plant regulation sequences. ~
5. Palindromic sequences were reduced to a minimum. ~:
Description of the Drawin~
The DNA sequence I according to the invention twith the S corresponding amino acid sequence) i~ depicted in Figure 1 . ' Three internal un;que cleavage sites for the res~riction enzymes XbaI ~position 152), BamHI (312~ and XmaI (436) make possible ehe subcloning o~ part-sequences ~hich can be incorporated in ~ell-inves~igated cloning vectors such as, for example, pUC18 or pUC19. In addition, a number of other unique recognition sequences for restr;ction en~ymes ~er~ incorporated within the gene~ and these, on the one hand, provide access to part-sequences o~ acetyl-transferase and, on the other hand, allo~ mod;f;cat;ons to b~ ~ade:
Restriction enzyme Sut after nucleotide ~o.
BspMII 11 SacII
EcoRV 74 HpaI S0 AatII 99 BstXI 139 ApaI 232 Sc~I 272 AvrII 30~
AflII 336 StuI 385 ~ssHII 449 FokI 4~7 BglI 536 BgLIl 550 The construction of p~rt-sequences by chemical synthesis and enzy~atic li~a~ion reactions is carrieid out in a ~anner kno~n per se ~EP-A Q,133,282, 0,136,472, 0,1559590, : . , -, ::
132~ 3~4 0~161~SD4, 0,163,249, 0,171,024, 0,173,149 or 0,177,827 Deta;ls, such as restriction analyses, ligation of DNA
fragments and transformation of plasmids in E. coli, are described a~ length in the textbook of Maniatis (Mole-cular Cloning, Maniatis et al., Cold Spring Harbor, 1982)~
The gene sequence which has been cloned in this way is then in~roduced ;nto plan~s, under the control of plant regulation signals, and its expression is induced.
EP-A 0~122,791 revie~s known methodsO In this way are obtained PTC-res;stan~ plant cells (i.e~ a selection feature for transformed cells is available), plants or parts of plants and seeds.
Some embodiments of the invention are explained in detail in the examples ~hich follo~. Unless other~ise indica-ted, percentage data therein relate to weight.
`- - 20 Examples -- - -The follo~ing media were used:
a) for bacteria:
25YT medium: O.5X yeast extract, 0.8% Bacto tryp-tone, 0.5% NaCl LP medium: 0.5X yeast extractr 1% Bacto tryptone, 1X NaCl as solid mediu~: addition of 1.5X agar to each b) for plants:
M+S medium: see Mur~shige and Skoog, Physiologica Plantarum 15 (19~2) 473 2MS medium: M~S medium containing 2% sucrose MSC10 medium: M~S medium containing 2% sucrose~ 500 mg/l cefotaxime, 0.1 mg/l naphthylacetic acid (NAA), 1 mg/l benzylaminopurine (~AP), 100 mg/l kanamyc;n MSC15 medium: M+S medium containing 2% sucrose, ,. : . :, . , ~ .
~3213~
500 mg/l cefotaxime~ 100 mg/l kanamycin.
1. Chemical synthesis of a single stranded oligonucleo-t;de The synthesis of fragment II, one of the four part-fragments I - IVD started from the terminal oLigonuc-leotide IIc (nucleotide No~ 219 to 31Z ;n the coding strand of DNA sequence I). For the solid-phase syn-thes;s, the nucleoside at the 3' end, that is to say guanosine (nucleotide No. 312) ;n ~he present case, is covalently bonded via ~he 3'-hydroxyl group to a support. The support material ;s CPG (controlled pore glass) funct;onalized w;th long-chain amino alkyl raclicals. Otherw;se, the syn~hesis follows the known (from the Said EP_A~) methOdS.
The plan of synthesis is ind;cated in DNA sequ~nce 2a ~.I.-~annex~7-!*wh;ch-lothe.r~i.se-^~ror~re-spor~ds-:to.:DNA se-quence I.
2~ Enzymatic linkage of the single-stranded oligonucleo-tides to give gene ~ragment II
For the phosphorylat;on of the oligonucleotides at the 5' end, 1 nmol of each Qf oligonucleotide~ IIb and IIc was treated with S nmol of adenos;ne tr;phos-phate and 4 un;ts of T4 polynucl~otide k;nase ;n 20 ~l of 50 mM tris-HCl buffer ~pH 7.6), 10 mM mag-nesium chloride and 1Q mM dithiothreitol (DTT) at 37G for 30 m;nutes~ The enzyme ;s inactivated by heating at 95C for 5 minutes. Oligonucleotides IIa and IId, which form the "protrud;ng" sequence ;n DNA fragment II, are not phosphorylated~ Th;s pre-vents the formation, dur;ng the subsequent l;ga~;on, of larger subfragments than correspond to DNA frag-ment II.
.. . .
132~36~
O~igonucleotides II (a-d) are ligated to give sub-fragment II as follows: 1 nmol of each of oligonuc-leotides IIa and IId and the 5'-phosphates of IIb and IIc are together dijsolved in 45 ~l of buffer containing 50 mM tr;s-HCl tpH 7.6), 20 mM magnesium chloride, 25 mM potassium chloride and 10 mM DTT.
For the annealing of the oligonucleotides correspond-ing to DNA fragm~nt II, the solution of the oligo-nucle3tides is heated at 95C for 2 minutes and then slowly ~ooled (2-3 hours) to 20C. Then, for the enzymaeic Linkage, 2 ~l o~ 0O1 M DTT, 8 ~l of 2.5 ~M adenosine triphosphate (pH 7) and 5 ~l o~ T4 DNA ligase (2000 un;ts~ arY added, and the mixture is incubated at 22C for 1b hsurs.
The gene fragment II is puri~ied by geL el~ctropho-resis on a 10X Polyacrylamide gel ~ithout addition of urea, Z0 x 40 cm, 1 ~m thick), ~he marker sub-stance used b~;ng ~X 174 DNA (from ~RL) cut ~ith - ?3 ~ -Hinfl,:or p~R3~2 cut:~ with -HaelII.
6ene fragm~nts I, III and IV are prep~red analogous-ly, although the "protruding" sequences are, before th~ annealin~, converted into the 53-phosphates be-cause no l;gat;on step is necessary.
3. Preparat;on of hybrid plas~ids containin~ gene frag-m~nts I, II, III and IV.
a~ Incorporation of gene frag~nt I in pUC18 Th~ commercially available Plasmid pUC1~ is opened in a known ~anner using the restriceion endo nucLea-ses SalI and XbaI in accordance ~ith the manufact-ure~s' instructions. The digest;on mixtur~ is frac-t;onated by el~ctrophoresis ;n a kno~n manner on a 1% agaras~ gelO and the fragmen~s are visuali~ed by staining ~ith ethidium bro~ide~ The plasmid band (about 2.6 kb) is then cut out of She agarose ~32~36~ :
gel and removed from the agarose by electro-elut;on.
1 ~g of plasmid, opened with XbaI and SalI, is then ligated with 10 ng of DNA fragment I at 16C
overnight.
b) Incorporation of gene fragment II in pUC18.
In analogy to 3), pUC18 is cut open ~ith XbaI and eamHI and ligated ~ith gene fragment II which has previously heen ph~sphorylated at the protru-ding ends as described in Exa-ple 2.
c~ Incorporation of gene fragmen~ III in pU~18 In analogy to a)~ pUC18 is cut open ~;th BamHI
and XmaIII and ligated with gene fragment III.
20~ d3 :In~orpora~--ion~o~;~gen~:~ragment.-IV--in pUC18 In analogy to a), pUC18 is cut ~ith YmaIII and SalI and ligated wi~h ~ene fragmer,t IV.
Construction of the co~plete gene and cloning in a pUC plasmid a) Transformation and amplification of gene fragments I - IY
The hybrid plasmids obtained ;n this way are transformed into E. coli. For this purpose~ the . ~
strain E. coli K 12 is made competent by treat--ment with a 70 mM calciu~ chLoride soLu~ion, and the suspension of the hybrid pLasmid in 10 mM
tr;s-HCl buffer (pH 7.5), which is 70 mM in cal-cium chioride, is added~ The transformed strains are selected as is customary, utiLizing the anti-biot;c resis~ances or sensitivities conf~rred by ~2~6~
-- 8 ~
the plasmid, and the hybrid vectors are amplified.
After the cells have been killed, the hybrid plas-mids are ;solated and cut open ~ith the restric-t;on enzymes or;ginally used, and gene fragments I, II, III and IV are isolated by gel electropho-resis.
b) Linkage of gene fragments I, II, III and IV to give the total gene Subfragments I and II obtained by amplification are linked as follows. 100 ng of each of the isolated fragments I and II are dissolved togeth-er in 10 ~l of buffer containing 50 mM tris-HCl (pH 7.6), 20 mM magnesium chloride and 10 mM DTT, and this solution is heated at 57C for 5 minutes.
After the solution has cooled to room temperature, 1 ~l of 10 mM adenosine triphosphate (pH 7) and 1 ~l of T4 ligase (400 units) are added~ and the 20 ~ mixt~re is incuba~-ed ~t-room~tempe-rature for 16 hours. After subsequent cutting with the restric-~ion enzy~es SalI and BamHI, the desired 312 bp fragment (nucleotides 1-312, SalI-~amHI) is puri-fied by gel electrophoresis on an 8% polyacryl-~5 amide gel, the warker substance used being ~X 174 RF DNA tfro~ 9RL~ cut with the restriction enzyme H3eIII .
Gene frag~ents III and IV are linked together in the same way, there being obtained after purifi-cation a 246 bp fragment (nucleotides 313-558~
Ba~HI~SalI~ The marker used for the gel electro-phoresis is pBR322 cut with the restriction en~
zyme MspI.
To contruct the total gene (DNA sequence I), 15 ng of the 312 bp fragment and 12 ng of the 246 bp fragment are ligated, as described above, with 1 ~9 of the commercially available plasmid pUC18 ~3~3~
_ 9 _ which has previously been cut open with the rest-riction enzyme SalI and enzymatically dephosphory-lated at the ends. After transformation and ampli-fication (as described in Example 4a), the correct clone having the 558 bp fragment corresponding to DNA sequence I is identified by SalI digestion~
5. Transformation of the hybrid plasmids Cumpetent E. coli cells are transformed with 0u1 to ~ .
1 ~9 of the hybrid plasmid containing DNA sequence I~ and are plated out on amplicillin-containing agar plates. It is then possible to identify clones ~hich contain the correc~ly integrated sequences in the plasmid by rapid DNA analysis (Maniatis loc.
cit.).
1 ~9 of the hybrid plasmid containing DNA sequence I~ and are plated out on amplicillin-containing agar plates. It is then possible to identify clones ~hich contain the correc~ly integrated sequences in the plasmid by rapid DNA analysis (Maniatis loc.
cit.).
6. Fusion of the synthetic gene to regulation s;gnals ~hich are recognized in plants~
The optim;zed resistance gene ~hich had been provi-ded at the ends with SalI cleavage sites was ligated in the ~alI cLe3vage site of the polyl;nker sequence of the plasm;d pDH51 (Pietrzak et al., Nucle;c Acids Res. 14 (198b) 5857). The promoter and terminator of the 35S transcript ~rom cauliflower mosaic virus, which are recognized by the plant transcription appa-ra~us, are located on th;s plasmid. The ligat;on of the resistance gene resulted in it bein~ inserted do~nstrea~ of the promoter and upstream of the termin ator of the 35S transcript~ The correct orientation of the gene ~as sonfirmed by restriction analyses.
The promo~er of the ST-LS1 gene from s~lan~m t~bero sum (Eckes et al., Mol. 6en. Genet. 205 (1986) 14) was likewise ~sed for the expression of the optim;zed acetyLtransferase gene in plants.
. . . . . ...:
: ~ , . : - ~ . --: :, . ~. . . ....
`- ~3213~4 7. Insertion of the resistance gene having the regula-tion sequences into Agrobacterium tu~efaciens a~ Cointegrate method The entire transcription uni~ comprising promoter~
optimized resistance gene and ter~inator (ExampLe 6) ~as cut out with the restriction enzyme EcoRI
and ligated in the EcoRI cleavage site of the intermediary E. coli vector pMPK110 (Peter Eckes, ___ Thesis, Univ. Cologne, 19850 pages 91 et seqO).
This intermediary vector was necessary for the transfer of the resistance gene ~ith its regu-lation sequences into the Ti pLasmid of Agrobacterium tumefaciens. This so-called conju-gation ~as carried out by the method described by Van Haute et al. (EMB0 J. 2 (1983~ 411). This entailed the gene with its regulation signals be-ing integrated in the Ti pLas~id by homoLogous . ~''ZO . ~ tre.c~b.in-ati~on,.vi-a~the~,seqllenc.es of the standard vector p9R322 ~hich are present in the pMPK110 vector and in the Ti plasmid pGV3850kanR (Jones et al., EMBQ J~ 4 (1985) 2411 :
For this purpose~ 50 ~l of fresh liquid cultures of each of the E. coli strains DH1 (hos~ strain of the pMPK110 derivative) and GJ23 (Van Haute et al., Nucleic Acids Res. 14 (1986) 5857) were mix-ed on a dry YT-agar plate and incubated at 37C
for one hour. The bacteria ~ere resuspended in 3 ~l of 10 mM MgS04 and pLated out on antibiotic-agar plates tspectinomycin 50 ~g/ml: selection for pMPK110; tetracycline 1D ~g/ml: seLection for R64drd11; kanamycin 50 ~g/ml: selec~ion for pGJ28). The bacteria growing on the selective agar plates contained the three plasmids and were grown for the conjugation ~ith Agrobacteriu~ tume-faciens in YT liquid medium at 37C. The Agro bacteria were cultiva~ed in L~ medium at 28C.
.. . . ~ . . ::
~ ` 13 2 3 6 50 ~l of each bacterium suspension were mixed on a dry YT-agar plate and ;ncubated at 28C fsr 12 to 16 hours. The bacteria were resuspended in 3 ml o~ 10 mM MgS04 and plated out on antib;otic plates (erythromycin 0.05 g/l, chloramph~nicol 0.025 g/l: selection for the Agrobacter;um strain;
streptomycin 0.03 g/l and spectinomycin 0.1 g/L:
selection for integration of pMPK110 in the Ti plasmid). Only ~grobacteria in which the pMPK110 derivative has been integrated into the bacterial Ti plasmid by homologous recombination are able to grow on the~e selected plates.
Besides the gene for resistance to the antibiotic kanamycin, uhich is active in plants and was al-ready present from the outset, the PTC-resistance gene was no~ also located on the Ti plasmid pGV3850kanR. 8efore these Agrobacterium clones vere used for transformation, a Southern blot exper~ment ~-as c~rried-out~to~checkYwhether the desired integration had taken pLace~
~' b~ Binary vector method The binary vector system described by Koncz et al. (Mol. Gen. Genet. Z04 t1986) 383) ~as used.
The vector pPCV701 described by Koncz et al.
tPNAS 84 t1987) 131) ~as modified in the follow-ing way: the restriction enzy~es BamHI and HindIII
were us~d to delete from the vector a fragment on which are located, inter aLia, the TR1 and TR2 promoters. The resulting pla~mid ~as recir-cularized. Into the EcoRI cleavage site present on this vector was inserted a fragment from the vector pDH51 which i about 800 base-pairs in length and on which were located the promoter and terminator of the 35S transcript from cauliflower mosaic virus (Pietrzak et al., Nucleic Acids Res.
1~ (1986~ 5358). The resulting plasmid pPCV801 ` 1321~
had a unique SalI cleavage site bet~een the 35S
promoter and terminator. The optimized PTC-resistanc~ gene ~as inserted into this cleavage site. Its expression ~as now under the control of the 35S transcript regulation sequences. ~ ;
Thi~ plasmid tpPCV8~1Ac~ ~as transformed into the E. coli strain SM10 tSimon et ~ io~Technology 1 t1983) 784). For the transfer of the plasmid pPCV801Ac into ~ , 50 ~l of both the SM10 culture and a CS8 Agrobact~rium cult-ur~ tGV3101~ Van L~rebekP et al.~ Nature 252 (1974) 16~) were mixed ~ith the Ti plasmid pMP90RK ~Konc~ :
et al., Mol. Gen. Genet. 204 ~1986) 383) as helper plasmid on a dry YT-agar plate, and the mixtur~ ~as incuba~ed a~ 28C
for about 16 hoursO The bacteria ~ere then resus-pended in 3 mL of 1 mM M9504 ~nd plated out on antibiotic plates (rifampicin 0.1 ~ selection for GV3101, kanamycîn 0.025 g~l: selection for pMP90RK, c~rben-ic~t W n 0~1 g/l: selection for pPCV801Ac)~ Only Agrobact~ria ~hich contained both plas~ids SpMP90RK and pPCY801Ac) ~re abl2 to grow : on th~se pL~tes~ Be~ore these Agrobacteria ~ere used for th~ plant transformation, 50uth~rn blot- :
ting ~as carri~d out to check that the plasmid pPCV801Ac is present in its correct for~ in the Agrobacteria~
, 30 8. Transformation of Nlcotina tabacum by Agrobacterium ~!~ :
~ .
The o~timized resistance gene ~as transferred into ~oba co plants using the so-called leaf disk trans- -~
~o~ma~ign method~
The Agrobacteria were cultured in 30 ml of L~ medium containing the appropriate antibiotics at 28C, ~hak-ing continuously (about 5 days). The bacteria ~ere th@n sQdimented by centrifugation at 700Q rpm in a ~3~13~
Christ centr;fuge for 10 minutes, and were washed once with 20 ml of 10 mM MsS04. After a further centrifugation, the bacteria were suspended ;n 20 ml of 10 mM MgS04 and transferred into a Petri dish.
Leaves of ~isconsin 38 tobacco plants growing on 2MS
medium in sterile culture were used for the leaf disk infection All the sterile cul~ur~s were main-tained at 25 to 27C in a 16 hours light/8 hours dark rhythm under ~hite light.
Tobacco leaves were cut off, and the leaf surfaces were lacerated ~ith sandpapern After the laceration~
the leaves were cut into smaller p;eces and d;pped ;n the bacterium culture. The leaf pieces were then transferred to M+S medium and maintained under nor-mal culture conditions for two days. After the 2-day ;nfect;on with the bacteria, the leaf p;eces ~ere ~ashed ;n liqu;d M~S medium and transferred to MSC1Q-agar plates. Transformed shoots ~ere selected -~ 20~ o~the;b-as.is of~ he-resist3nce~*0 the antib;otic kanamycin which had also been transferred. The first shoots became visibl~ 3 to 6 weeks later. In-div;dual shoots ~ere further cultivated on MSC15 medium in glass jars. In ehe ~eeks which followed, some of the shoots wh;ch had been cut off developed roots at the site of the cut.
It was aLso possible to select transformed plants directly on PTC-containing plant media. The presence and the expression of the PTC-resistance gene was demonstrated by DNA analysis (Southern blotting) and RNA anaiysis ~Northern blotting) of the transformed plants.
9. Demonstration of the PTC-res;stance of the transfor-med plants To check the funct;oning of ~he resistance gene in transformed plants, leaf fragments from ~ransformed ,; : : .: ~
_ 141_ and non-transfor~ed plants were transferred to M-~S
nutrient ~edia containing 1 x 10 4 M L-PTC. The fragments from non-transformed plants died, while the fragments from transformed plants were able to S regenerate new shoots. Transformed shoots took root and grew ~ithout difficuLty on M~S nutrient media containing 1 x 10 3 M L-PTC~ Transformed plants were, from sterile conditions, potted in soil and sprayed with 2 kg/ha and S kg/ha PTC. ~hereas non-transformed plants did not survive this herbicide ~reatment, transformed plants showed no da~age brought about by the herbicide. The appearance and gro~th behavior of the sprayed transformed plants was at least as good as that of unsprayed control plants.
10. Acetyltransferase assay to demonstrate acetylation of PTC in transgenic PTC-res;stant plants About 100 mg of leaf tissue from transgenic PTC--'~ ^ ?Z~.~'-,~,.,,, ~resistant;~tobacco~.pl-ants.~ rom~.n-on-t.rans.formed tobacco plants were homogenized in a buf~er composed of: 5Q mM tris-HCl, pH 7.5; 2 mM EDTA; 0.1 ~/ml leupeptin; 0.3 mg/ml bovine seru~ album;n; 0.3 ~g/ml DTT; 0.15 mg/ml phenyL~ethyLsulfonyl fluoride ~PMSF).
After subsequent centrifugation, 2n ~l of the clear supernatant ~ere incubated with 1 ~l of 10 mM radio-labe~L~d DrL-PTC and 1 ~l of 1U0 ~M acetyl-CoA at 37C for 20 minutesu 25 ~l of 1?% trichloroacetic acid w~re then added to ~he reaction mixture, fol-lo~ed by centrifugation~ 7 ~l of the supernatant were transferred to a thin-layer chromatography pla~e and subjPcted to ascending development twice in a mixture of pyrid;ne : n-butanol : acetic acid : ~ater (50 : 75 : 15 : 60 parts by volume). PTC and acetyl-PTC were separated ~rom one another in this way~ and could be detected by autoradio~raphy. Non-transformed plants exh;bited no conversion of PTC into acetyl-PTC, where-as transgenic resistant plants were capabLe of this.
..
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.
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The optim;zed resistance gene ~hich had been provi-ded at the ends with SalI cleavage sites was ligated in the ~alI cLe3vage site of the polyl;nker sequence of the plasm;d pDH51 (Pietrzak et al., Nucle;c Acids Res. 14 (198b) 5857). The promoter and terminator of the 35S transcript ~rom cauliflower mosaic virus, which are recognized by the plant transcription appa-ra~us, are located on th;s plasmid. The ligat;on of the resistance gene resulted in it bein~ inserted do~nstrea~ of the promoter and upstream of the termin ator of the 35S transcript~ The correct orientation of the gene ~as sonfirmed by restriction analyses.
The promo~er of the ST-LS1 gene from s~lan~m t~bero sum (Eckes et al., Mol. 6en. Genet. 205 (1986) 14) was likewise ~sed for the expression of the optim;zed acetyLtransferase gene in plants.
. . . . . ...:
: ~ , . : - ~ . --: :, . ~. . . ....
`- ~3213~4 7. Insertion of the resistance gene having the regula-tion sequences into Agrobacterium tu~efaciens a~ Cointegrate method The entire transcription uni~ comprising promoter~
optimized resistance gene and ter~inator (ExampLe 6) ~as cut out with the restriction enzyme EcoRI
and ligated in the EcoRI cleavage site of the intermediary E. coli vector pMPK110 (Peter Eckes, ___ Thesis, Univ. Cologne, 19850 pages 91 et seqO).
This intermediary vector was necessary for the transfer of the resistance gene ~ith its regu-lation sequences into the Ti pLasmid of Agrobacterium tumefaciens. This so-called conju-gation ~as carried out by the method described by Van Haute et al. (EMB0 J. 2 (1983~ 411). This entailed the gene with its regulation signals be-ing integrated in the Ti pLas~id by homoLogous . ~''ZO . ~ tre.c~b.in-ati~on,.vi-a~the~,seqllenc.es of the standard vector p9R322 ~hich are present in the pMPK110 vector and in the Ti plasmid pGV3850kanR (Jones et al., EMBQ J~ 4 (1985) 2411 :
For this purpose~ 50 ~l of fresh liquid cultures of each of the E. coli strains DH1 (hos~ strain of the pMPK110 derivative) and GJ23 (Van Haute et al., Nucleic Acids Res. 14 (1986) 5857) were mix-ed on a dry YT-agar plate and incubated at 37C
for one hour. The bacteria ~ere resuspended in 3 ~l of 10 mM MgS04 and pLated out on antibiotic-agar plates tspectinomycin 50 ~g/ml: selection for pMPK110; tetracycline 1D ~g/ml: seLection for R64drd11; kanamycin 50 ~g/ml: selec~ion for pGJ28). The bacteria growing on the selective agar plates contained the three plasmids and were grown for the conjugation ~ith Agrobacteriu~ tume-faciens in YT liquid medium at 37C. The Agro bacteria were cultiva~ed in L~ medium at 28C.
.. . . ~ . . ::
~ ` 13 2 3 6 50 ~l of each bacterium suspension were mixed on a dry YT-agar plate and ;ncubated at 28C fsr 12 to 16 hours. The bacteria were resuspended in 3 ml o~ 10 mM MgS04 and plated out on antib;otic plates (erythromycin 0.05 g/l, chloramph~nicol 0.025 g/l: selection for the Agrobacter;um strain;
streptomycin 0.03 g/l and spectinomycin 0.1 g/L:
selection for integration of pMPK110 in the Ti plasmid). Only ~grobacteria in which the pMPK110 derivative has been integrated into the bacterial Ti plasmid by homologous recombination are able to grow on the~e selected plates.
Besides the gene for resistance to the antibiotic kanamycin, uhich is active in plants and was al-ready present from the outset, the PTC-resistance gene was no~ also located on the Ti plasmid pGV3850kanR. 8efore these Agrobacterium clones vere used for transformation, a Southern blot exper~ment ~-as c~rried-out~to~checkYwhether the desired integration had taken pLace~
~' b~ Binary vector method The binary vector system described by Koncz et al. (Mol. Gen. Genet. Z04 t1986) 383) ~as used.
The vector pPCV701 described by Koncz et al.
tPNAS 84 t1987) 131) ~as modified in the follow-ing way: the restriction enzy~es BamHI and HindIII
were us~d to delete from the vector a fragment on which are located, inter aLia, the TR1 and TR2 promoters. The resulting pla~mid ~as recir-cularized. Into the EcoRI cleavage site present on this vector was inserted a fragment from the vector pDH51 which i about 800 base-pairs in length and on which were located the promoter and terminator of the 35S transcript from cauliflower mosaic virus (Pietrzak et al., Nucleic Acids Res.
1~ (1986~ 5358). The resulting plasmid pPCV801 ` 1321~
had a unique SalI cleavage site bet~een the 35S
promoter and terminator. The optimized PTC-resistanc~ gene ~as inserted into this cleavage site. Its expression ~as now under the control of the 35S transcript regulation sequences. ~ ;
Thi~ plasmid tpPCV8~1Ac~ ~as transformed into the E. coli strain SM10 tSimon et ~ io~Technology 1 t1983) 784). For the transfer of the plasmid pPCV801Ac into ~ , 50 ~l of both the SM10 culture and a CS8 Agrobact~rium cult-ur~ tGV3101~ Van L~rebekP et al.~ Nature 252 (1974) 16~) were mixed ~ith the Ti plasmid pMP90RK ~Konc~ :
et al., Mol. Gen. Genet. 204 ~1986) 383) as helper plasmid on a dry YT-agar plate, and the mixtur~ ~as incuba~ed a~ 28C
for about 16 hoursO The bacteria ~ere then resus-pended in 3 mL of 1 mM M9504 ~nd plated out on antibiotic plates (rifampicin 0.1 ~ selection for GV3101, kanamycîn 0.025 g~l: selection for pMP90RK, c~rben-ic~t W n 0~1 g/l: selection for pPCV801Ac)~ Only Agrobact~ria ~hich contained both plas~ids SpMP90RK and pPCY801Ac) ~re abl2 to grow : on th~se pL~tes~ Be~ore these Agrobacteria ~ere used for th~ plant transformation, 50uth~rn blot- :
ting ~as carri~d out to check that the plasmid pPCV801Ac is present in its correct for~ in the Agrobacteria~
, 30 8. Transformation of Nlcotina tabacum by Agrobacterium ~!~ :
~ .
The o~timized resistance gene ~as transferred into ~oba co plants using the so-called leaf disk trans- -~
~o~ma~ign method~
The Agrobacteria were cultured in 30 ml of L~ medium containing the appropriate antibiotics at 28C, ~hak-ing continuously (about 5 days). The bacteria ~ere th@n sQdimented by centrifugation at 700Q rpm in a ~3~13~
Christ centr;fuge for 10 minutes, and were washed once with 20 ml of 10 mM MsS04. After a further centrifugation, the bacteria were suspended ;n 20 ml of 10 mM MgS04 and transferred into a Petri dish.
Leaves of ~isconsin 38 tobacco plants growing on 2MS
medium in sterile culture were used for the leaf disk infection All the sterile cul~ur~s were main-tained at 25 to 27C in a 16 hours light/8 hours dark rhythm under ~hite light.
Tobacco leaves were cut off, and the leaf surfaces were lacerated ~ith sandpapern After the laceration~
the leaves were cut into smaller p;eces and d;pped ;n the bacterium culture. The leaf pieces were then transferred to M+S medium and maintained under nor-mal culture conditions for two days. After the 2-day ;nfect;on with the bacteria, the leaf p;eces ~ere ~ashed ;n liqu;d M~S medium and transferred to MSC1Q-agar plates. Transformed shoots ~ere selected -~ 20~ o~the;b-as.is of~ he-resist3nce~*0 the antib;otic kanamycin which had also been transferred. The first shoots became visibl~ 3 to 6 weeks later. In-div;dual shoots ~ere further cultivated on MSC15 medium in glass jars. In ehe ~eeks which followed, some of the shoots wh;ch had been cut off developed roots at the site of the cut.
It was aLso possible to select transformed plants directly on PTC-containing plant media. The presence and the expression of the PTC-resistance gene was demonstrated by DNA analysis (Southern blotting) and RNA anaiysis ~Northern blotting) of the transformed plants.
9. Demonstration of the PTC-res;stance of the transfor-med plants To check the funct;oning of ~he resistance gene in transformed plants, leaf fragments from ~ransformed ,; : : .: ~
_ 141_ and non-transfor~ed plants were transferred to M-~S
nutrient ~edia containing 1 x 10 4 M L-PTC. The fragments from non-transformed plants died, while the fragments from transformed plants were able to S regenerate new shoots. Transformed shoots took root and grew ~ithout difficuLty on M~S nutrient media containing 1 x 10 3 M L-PTC~ Transformed plants were, from sterile conditions, potted in soil and sprayed with 2 kg/ha and S kg/ha PTC. ~hereas non-transformed plants did not survive this herbicide ~reatment, transformed plants showed no da~age brought about by the herbicide. The appearance and gro~th behavior of the sprayed transformed plants was at least as good as that of unsprayed control plants.
10. Acetyltransferase assay to demonstrate acetylation of PTC in transgenic PTC-res;stant plants About 100 mg of leaf tissue from transgenic PTC--'~ ^ ?Z~.~'-,~,.,,, ~resistant;~tobacco~.pl-ants.~ rom~.n-on-t.rans.formed tobacco plants were homogenized in a buf~er composed of: 5Q mM tris-HCl, pH 7.5; 2 mM EDTA; 0.1 ~/ml leupeptin; 0.3 mg/ml bovine seru~ album;n; 0.3 ~g/ml DTT; 0.15 mg/ml phenyL~ethyLsulfonyl fluoride ~PMSF).
After subsequent centrifugation, 2n ~l of the clear supernatant ~ere incubated with 1 ~l of 10 mM radio-labe~L~d DrL-PTC and 1 ~l of 1U0 ~M acetyl-CoA at 37C for 20 minutesu 25 ~l of 1?% trichloroacetic acid w~re then added to ~he reaction mixture, fol-lo~ed by centrifugation~ 7 ~l of the supernatant were transferred to a thin-layer chromatography pla~e and subjPcted to ascending development twice in a mixture of pyrid;ne : n-butanol : acetic acid : ~ater (50 : 75 : 15 : 60 parts by volume). PTC and acetyl-PTC were separated ~rom one another in this way~ and could be detected by autoradio~raphy. Non-transformed plants exh;bited no conversion of PTC into acetyl-PTC, where-as transgenic resistant plants were capabLe of this.
..
., .: :
: :: : . ~: : ~ -: -:
- ~ ~ - :- ,. .
.
2~36~
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Claims (17)
1. A resistance gene coding for the protein of amino acid sequence I as shown in Figure 1, which gene is adapted to the codon usage in plants.
2. The resistance gene as claimed in claim 1, having DNA
sequence I as shown in Figure 1, nucleotide positions 9-554.
sequence I as shown in Figure 1, nucleotide positions 9-554.
3. A gene structure having DNA sequence I as shown in Figure 1, coupled to regulation and expression signals active in plants.
4. A vector containing the resistance gene as claimed in claim 1.
5. A vector containing the resistance gene as claimed in claim 2.
6. A vector containing a gene structure as claimed in claim 3.
7. A vector containing one or more of gene fragments I - IV.
8. A host cell containing a vector as claimed in claim 4.
9. A host cell containing a vector as claimed in claim 5.
10. A host cell containing a vector as claimed in claim 6.
11. A host cell containing a vector as claimed in claim 7.
12. A plant cell containing a gene as claimed in claim 1.
13. A plant cell containing a gene as claimed in claim 2.
14. A plant cell containing a gene as claimed in claim 3.
15. A process for generating phosphinothricin-resistant plant cells, parts of plants, plants and seeds which comprises transforming plant cells with the gene as claimed in claim 1.
16. A process for generating phosphinothricin-resistant plant cells, parts of plants, plants and seeds which comprises transforming plant cells with the gene as claimed in claim 2.
17. A process for generating phosphinothricin-resistant plant cells, parts of plants, plants and seeds which comprises transforming plant cells with the gene structure as claimed in claim 3.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3701624 | 1987-01-21 | ||
| DEP3701624.5 | 1987-01-21 | ||
| DE19873737918 DE3737918A1 (en) | 1986-08-23 | 1987-11-07 | Phosphinothricin resistance gene to which is active in plants, and its use |
| DEP3737918.6 | 1987-11-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1321364C true CA1321364C (en) | 1993-08-17 |
Family
ID=25851724
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000556972A Expired - Lifetime CA1321364C (en) | 1987-01-21 | 1988-01-20 | Phosphinothricin-resistance gene active in plants, and its use |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0275957B1 (en) |
| JP (3) | JP2993964B2 (en) |
| CN (2) | CN87100603A (en) |
| AU (1) | AU609082B2 (en) |
| CA (1) | CA1321364C (en) |
| DE (1) | DE3878699D1 (en) |
| DK (1) | DK175800B1 (en) |
| ES (1) | ES2054708T3 (en) |
| FI (1) | FI116067B (en) |
| GR (1) | GR3007859T3 (en) |
| HU (1) | HU215079B (en) |
| IL (1) | IL85143A0 (en) |
| NZ (1) | NZ223227A (en) |
Cited By (1)
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|---|---|---|---|---|
| US8912124B2 (en) | 2010-04-09 | 2014-12-16 | Bayer Cropscience Ag | Use of derivatives of (1-cyanocyclopropyl)phenylphosphinic acid, esters thereof and/or salts thereof for enhancing tolerance in plants to abiotic stress |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2018274T5 (en) * | 1986-03-11 | 1996-12-16 | Plant Genetic Systems Nv | VEGETABLE CELLS RESISTANT TO GLUTAMINE SYNTHETASE INHIBITORS, PREPARED BY GENETIC ENGINEERING. |
| EP0257542B1 (en) * | 1986-08-23 | 1992-05-06 | Hoechst Aktiengesellschaft | Phosphinotricine resistance gene and its use |
| DE3716309A1 (en) * | 1987-05-15 | 1988-11-24 | Hoechst Ag | RESISTANCE TO PHOSPHINOTHRICIN |
| DE3732972A1 (en) * | 1987-07-02 | 1989-01-12 | Hoechst Ag | RESISTANCE GENES TO PHOSPHINOTHRICIN AND ITS USE |
-
1987
- 1987-02-10 CN CN198787100603A patent/CN87100603A/en active Pending
-
1988
- 1988-01-19 FI FI880216A patent/FI116067B/en not_active IP Right Cessation
- 1988-01-19 ES ES88100631T patent/ES2054708T3/en not_active Expired - Lifetime
- 1988-01-19 EP EP88100631A patent/EP0275957B1/en not_active Expired - Lifetime
- 1988-01-19 NZ NZ223227A patent/NZ223227A/en unknown
- 1988-01-19 DE DE8888100631T patent/DE3878699D1/en not_active Expired - Lifetime
- 1988-01-20 CN CN88100322A patent/CN1057120C/en not_active Expired - Lifetime
- 1988-01-20 IL IL85143A patent/IL85143A0/en active IP Right Grant
- 1988-01-20 HU HU88217A patent/HU215079B/en unknown
- 1988-01-20 AU AU10619/88A patent/AU609082B2/en not_active Expired
- 1988-01-20 CA CA000556972A patent/CA1321364C/en not_active Expired - Lifetime
- 1988-01-20 DK DK198800239A patent/DK175800B1/en not_active IP Right Cessation
- 1988-01-21 JP JP63011851A patent/JP2993964B2/en not_active Expired - Lifetime
-
1993
- 1993-05-14 GR GR930400667T patent/GR3007859T3/el unknown
-
1997
- 1997-07-22 JP JP9196218A patent/JP3062125B2/en not_active Expired - Lifetime
- 1997-07-22 JP JP09196219A patent/JP3093686B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8912124B2 (en) | 2010-04-09 | 2014-12-16 | Bayer Cropscience Ag | Use of derivatives of (1-cyanocyclopropyl)phenylphosphinic acid, esters thereof and/or salts thereof for enhancing tolerance in plants to abiotic stress |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1057120C (en) | 2000-10-04 |
| JP3062125B2 (en) | 2000-07-10 |
| FI880216A (en) | 1988-07-22 |
| DK175800B1 (en) | 2005-02-28 |
| FI116067B (en) | 2005-09-15 |
| GR3007859T3 (en) | 1993-08-31 |
| DK23988D0 (en) | 1988-01-20 |
| IL85143A0 (en) | 1988-06-30 |
| CN88100322A (en) | 1988-08-10 |
| JP3093686B2 (en) | 2000-10-03 |
| NZ223227A (en) | 1989-06-28 |
| DK23988A (en) | 1988-07-22 |
| JPH1080289A (en) | 1998-03-31 |
| HUT47636A (en) | 1989-03-28 |
| EP0275957B1 (en) | 1993-03-03 |
| EP0275957A3 (en) | 1990-02-28 |
| AU609082B2 (en) | 1991-04-26 |
| ES2054708T3 (en) | 1994-08-16 |
| CN87100603A (en) | 1988-08-10 |
| JP2993964B2 (en) | 1999-12-27 |
| DE3878699D1 (en) | 1993-04-08 |
| EP0275957A2 (en) | 1988-07-27 |
| FI880216A0 (en) | 1988-01-19 |
| JPS63273479A (en) | 1988-11-10 |
| AU1061988A (en) | 1988-07-28 |
| HU215079B (en) | 1998-09-28 |
| JPH1080278A (en) | 1998-03-31 |
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