DD273855A5 - Process for the preparation of a recombinant DNA molecule - Google Patents

Abstract

The invention relates to a method for producing a recombinant DNA molecule. DNA molecule can transfer herbicide tolerance based on detoxification of the herbicide to a plant. According to the invention, a recombined DNA molecule is prepared in such a way that by means of methods known per se various DNA sequence segments, which consist either of genomic DNA and / or of cDNA, are linked to form a complete glutathione-S-transferase-encoding DNA sequence , According to the invention, the linking of DNA sequence segments is carried out using in vivo recombination of DNA sequences which have homologous sections and / or the in vitro linkage of restriction fragments. The present invention further relates to the application of the recombinant DNA technology for the transformation of plants, which leads to a transmission of a herbicide resistance. More particularly, the present invention relates to the construction and use of a recombinant DNA molecule that includes a glutathione S-transferase (GST) gene that increases the enzymatic activity of GST upon expression in the plant.

Description

For this 15 pages drawings

Field of application of the invention

The invention relates to a process for the preparation of a recombinant DNA molecule which is capable of transmitting a herbicide tolerance based on the detoxication of the herbicide to a plant. In particular, the present invention relates to the preparation and use of a recombinant DNA molecule comprising a glutathione S-transferase (GST) gene and which, when expressed in the plant, increases the GST enzyme activity of the plant.

Characteristic of the known state of the art

Glutathione S-transferases (EC 2.5.1.18) belong to a class of enzymes that play an important role in the xenobiotics detoxification process. These enzymes are found in most living organisms, including microorganisms, plants, insects and higher animals. Each of the glutathione-S-transferase (GST) enzymes within this class differs from the other, but all of these enzymes have overlapping substrate specificity. See: Jakobi et al., "Glutathione S-transferase: Binding and Physical Properties", in Glutathione: Metabolism and Function, ed. I. Arias and W. Jakoby (Raven Press, New York, 1976), Reddy et al. , Purification and Characterization of Individual Glutathione S-Transferase from Sheep Liver,, Archives of Biochem., And Biophys., 224 / 87-101 (1983).

Of the many GST functions, catalysis of the conjugation of glutathione with electrophilic compounds is of particular interest. Rennberg, "Glutathione Metabolism and Possible Biological Roles in Higher Plants," Phytochemistry, 21: 2771-2781 (1982); See: Meister and Täte, "Glutathione and Related Gamma-Glutamyl Complementaries: Biosynthesis and Utilization," in Ann. Rev. Biochem., 45: 560-604 (1976). Numerous xenobiotics. including herbicides. Pesticides and insecticides are among the electrophilic compounds. In the course of conjugation of the glutathione and an electrophilic compound, the sulfhydryl group of the glutathione reacts with the electrophilic center of that compound, with the glutathione acting as the nucleophile. This conjugation is catalyzed by a specific GST enzyme. ! Race mountain, supra).

For plants, this reaction is of particular importance as it offers a means of detoxifying xenobiotics. The conjugated, electrophilic, xenobiotic compound becomes water-soluble and thus is no longer toxic to the plant.

Object of the invention

It is the goal of the invention to provide plants with tolerance to herbicides.

Explanation of the nature of the invention

The object of the invention is to develop plants which are tolerant of herbicides by significantly increasing the enzyme activity of glutathione-S-transferase in said plants using genetic engineering methods.

This object is achieved according to the invention by a process for the preparation of a recombinant DNA molecule which is capable of transferring a herbicidal tolerance based on the detoxification of the herbicide to a plant which is characterized by the fact that it is known per se Methods various DNA sequence sections, which consist of either genomic DNA and / or cDNA, would have been linked to a completely glutathione S-transferase dokierenden DNA sequence.

Successful linkage of DNA sequence segments using in vivo recombination of DNA sequences having homologous segments and / or in vitro linking of restriction fragments. These DNA sequence segments are composed of both genomic DNA and cDNA. In particular, these DNA sequence segments are composed of gene fragments from several organisms belonging to different genera. They are also composed of gene fragments of more than one strain, of a variety or of a species of the same organism. They are further composed of proportions of more than one gene of the organism.

According to the invention, the DNA sequence coding for a glutathione-S-transferase polypeptide is derived from a sucker, in particular from a rat.

In the DNA molecule prepared according to the invention, as well as its variants or mutants which code for polypeptides with glutathione-S-transferase activity, the DNA sequence which codes for a rat glutathione-S-transferase polypeptide has the following nucleolides Sequence on:

40 50 60

ATGGGTATGATACTGGGATACTGG MPMILGYW

70 80 90 1OC 110 120

aacgtccgcgggctgacagacccgatccgcctgctcgtggaatagacagactcaagctat nvrglthpirllleytdssy

130 140 150 160 170 180

GAGGAGAAGAGATACGCCATGGGCGACGCTCGCGACTATGACAGAAGCCAGTGGCTGAAT EEKRYAMGDAPDYDRSQWLN

190 200 210 220 230 240

GAGAAGTTCAAACTGGGCCTGGACTTCCCGAATCTGCCCTAGTTAATTGATGGATGGGGC · EKFKLGLDFPNLPYLIDGSR

250 260 270 280 290 300

AAGATTACCCAGAGCAATGCCATAATGGGCTACCTTGGGCGGAAGCACCAGCTGTGTGGA KITQSNAIMRYLARKHHLCG

310 320 330 340 350 360

GAGACAGAGGAGGAGCGGATTCGTGCAGACATTGTGGAGAACCAGGTCATGGACAACCGC ETEEERIRADIVENQVMDNR

370 380 390 400 410 420

ATGCAGCTCATGATGGTTTGTTACAACCGCGACTTTGAGAAGCAGAAGCCAGAGTTCTTG MQLIMLCYNPDFEKQKPEFL

430 440 450 460 470 480

AAGACCATCCCTGAGAiVGATGAAGCTCTACTCTGAGTTCGTGGGCAAGGGACCATGGTTT KTIPEKMKLYSEFLGKRPWF

490 500 510 520 530 540

GCAGGGGACAAGGTCAGCTATGTGGATTTCCTTGCTTAT -'- kCATTCTTGACCAGTACCAC AGDKVTYVDFLAYDILDQYH

550 560 570 580 590 600

ATTTTTGAGCCGAAGTGCCTGGACGCCTTGCGAAACCTGAAGGAGTTCCTGGCCCGCTTC IFEPKCLDAFPNLKDFLARF

610 620 630 640 650 660

GACGGCCTGAAGAAGATCTCTGCCTAÜATGAAGAGCAGCCGCTAOGTCTCAACACCTATA EGL K CLESAY M KSSRYL S TPI

670 680 690 700 710 720

TTTTCGAAGTTGGCCCAATGGAGT.iACAAGTAG FSKLAQWSNK

This DNA sequence may also have the following nucleotide sequence:

1 10 13 0

I ¹ AC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG

Tyr Ser Met GIy Asp Ala Pro Asp Tyr Asp Arg Ser GIn

150 1 70

TGG CTG AGT GAG AAG TTC AAA UTG GGC CTG GAC TTU CCC AAT CTG

Trp Leu Ser Giu Lys Phe Lys Leu Gily Leu Asp Phe Pro Asn Leu

19 0 210

CCC TAG TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC

.fro Tyr Leu lie Asp Gly Ser His Lyg lie Thr Gin Ser Asn AIa

2 30 25 0

ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGT GGG GAG ACA

He Leu Arg Tyr Leu GIy Arg Lys His Asn Leu Cys GIy GIu Thr

2 90 31 0

GAG GAG GAG AGG ATT CGT GTG GAC GTT TTG GAG AAC GAG GCT ATG

GIu GIu GIu Arg He Arg VaI Asp VaI Leu GIu Asn Gin AIa Met

330 3 50

GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT

Asp Thr Arg Leu GIn Leu Ala Met VaI Cys Tyr Ser Pro Asp Phe

37 0 390

GAG AGA AAG AAG CCA GAG TAC TTA GAG GGT CTC CCT GAG AAG ATG

GIu Arg Lys Lys Pro GIu Tyr Leu GIu GIy Leu Pro GIy Lys Met

4 10 43 0 450

AAG CTT TAC TCC GAA TTC CTG GGC AAG CAG CCA TGG TTT GCA GGG

Lys Leu Tyr Ser Giu Phe Leu Giy Lys GIn Pro Trp Phe Ala Giy

4 70 49 0

AAC AAG ATT ACG TAT GTG GAT TTT CTT GTT TAC GAT GTO CTT GAT

Asn Lys He Thr Tyr VaI Asp Phe Leu VaI Tyr Asp VaI Leu Asp

510 5 30

CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC

Gin His Arg He Phe Giu Pro Lys Cys Leu Asp Ala Phe Pro Asn

55 O 570

CTC AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Lou Lys Aap Phe VaI Ala Arg Phe GIu ο Iy Leu Lys Lys He Ser

5 90 61 O

GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA Asp Tyr Met Lys Ser Gly Arg Phe Leu Ser Lys Pro He Phe AIa

6 50

.4AG ATG GCC TTT TGG AAC CCA AAG DAY

Lys Meta Phe T.rp Asn Pro Lya End

Srfindungsgemä'i CCT can this DNA sequence too 0 ACA CTG GGT have the following nucleotide sequence: TGG GAC 30 ATC CGT GGG CTG GCT CAC ATG Per 1 ATG Thr Leu Gly TAC Trp Asp He bad Gly Leu Ala Kis mead 50 ATT mead CTG TTC CTG Tyr 7 ACT 0 ACA GAC ACA AGC DID GAG 90 GAC GCC He CGC Leu Phe Leu GAG Tyr Thr Asp Thr Ser Tyr Glu Asp Ala AAG bad AGC ATG 1 GGG Glu GCT CCC GAC DID GAC 13 AGA 0 AGC CAG AAG Lys TAC Ser mead Gly 10 GAT Ala Per Asp Tyr Asp bad Ser gin Lys CTG Thr GAG 150 AAG TTC Asp CTG GGC CTG 1 GAC 70 TTC CCC AAT CTG TGG Leu AiT Glu Lys Phe AAA Leu Gly Leu Asp Phe Per Ηβη Leu Trp TAC Ser 0 ATT GAT GGG Lys CAC AAG 210 ATC ACC CAG AGC AAT GCC CCC Tyr 19 TTA He aap Gly TCA His Lya He Thr Gin Ser Asn Ala Per 30 CTG Leu TAC CTT GGC Ser 25 AAG 0 CAC AAC CTT TGT GGG GAG 270 ACA 2 ATC Leu CGC Tyr Leu Gly CGG Lys His aan Leu Cys Gly Glu Thr He GAG bad AGG ATT 2 CGT bad GAC GTT TTG GAG AAC 31 CAG 0 GCT ATG GAG Glu GAG bad He bad 90 GTG Asp Val Leu Glu Asn Gin Ala mead Glu ACC Glu CTA 330 CAG TTG Val ATG GTC TGC 3 TAC 50 AGC CCT GAC TTT GAC Thr CGC Leu Gin Leu GCC mead Val Cys Tyr Ser Per Asp Phe Asp AGA V Arg 0 AAG CCA GAG Ala TTA GAG 390 GGT CTC CCT GAG AAG ATG GAG bad 37 Lya Per Glu TAC Leu Glu Gly Leu Per Glu Lys mead Glu 10 CTT J§-s TCC GAA TTC Tyr 43 GGC 0 AAG CAG CCA TGG TTT GCA 450 GGG 4 AAG Leu TAC Ser Glu Phe CTG Gly Lys Gin Per Trp Phe Ala Uly Lys Tyr 4 Leu 49 0 AAG ACG DID GTG 70 TTT CTT GTT TAC GAT GTC CTT GAT AAC Lys ATT Thr Tyr Val GAT Phe Leu Val Tyr Asp Val Leu Asp Asn He Asp

510 5 30

CAA OAG CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC Gin His Arg JLee Phe GIu Pro Lye Cys Leu Asp Ala Phe Pro Asn

55 0 570

CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Leu Lya Asp Phe VaI Ala Arg Phe GIu GIy Leu Lya Lyo He Ser

5 90 61 0 630

GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA Asp Tyr Met Lys Ser Gly Arg Phe Leu Ser Lys Pro lie Phe Ala

6 50 AlG ATG GCC TTT TGG AAC CCA AAG DAY

Lys Met Ala Phe Trp Asn Pro Lys End

According to the invention, said DNA sequence may also have the following nucleotide sequence:

10 30

A GAC CCC AGC ACC ATG UCC ATG ACA CTG GGT TAC TGG GAC ATC

Met Pro Met 'her Leu GIy Tyr Trp Asp Hey

5 0 70

CGT GGG CTA GCG CAT GCC ATC CGC CTG CTC CTG GAA TAC ACA GAC Arg GIy Leu Ala His Ala He Arg Leu Leu Leu GIu Tyr Thr Asp

90 11 0 130

TCG AGC TAT GAG GAG AAG AGA TAC ACC ATG GGA GAC GCT CCC GAC Ser Ser Tyr GIu GIu Lys Arg Tyr Thr Met GIy Asp Ala Pro Asp

1 50 17 0

TTT GAC AGA AGC UAG TGG CTG AAT GAG AAG TTC AAA CTG GGC CTG .the Asp Arg Ser GIn Trp Leu Asn GIu Lys Phe Lys Leu GIy Leu

190 2 10

GAC TTC CCC AAT CTG CCC TAC TTA ATT GaT GGA TCA CAC aAG A'i'C Asp Phe Pro Asn Leu Pro Tyr Leu He Asp GY about His Lys He

23 0 250 2

ACC UAG AGC AAT GCC ATC CTG UGC ACT CTT GGC CGU AAG UAC AAU Thr GIn Ser Asn Ala He Leu Arg Tyr Leu uly Arg Lys His Asn

70 29 0 310

CTG TGT GGG GAG ACA GAA GAG GAG AGG ATT CGT GTG GAC ATT CTG Leu Cye GIy GIu Thr GIu GIu GIu Arg He Arg VaI Asp He Leu

3 30 35 0

GAG AAT CAG CTC ATG GAC AAC CGC ATG GTG CTG GCG AGA CTT TGC

GIu Asn GIn Leu Met Asp Asn Arg Met VaI Leu AIa Arg Leu Cys

370 3 90

TAT AAC CCT GAC TTT GAG AAG CTG AAG CCA GGG TAC CTG GAG CAA

Tyr Asn Pro Asp Phe Giu Lys Leu Lys Pro Giy Tyr Leu Giu CIn

41 0 430 4

CTG CCT GGA ATG ATG CGG CTT TAC TCC GAG TTC CTG GGC AAG CGG Leu Pro GIy Met Met Arg Leu Tyr Ser GIu Phe Leu GIy Lys Arg

TGG TTT GCA GGG GAC 47 MG 0 ATC ACC TTT GTG GAT TTC 490 ATT -11- 273 855 50 OCA Trp Phe Alu Gly Asp He Thr Phe Val Asp Phe He GCT Per GAT GTT CTT 5 GAG 10 AGG MC CM GTG TTT GAG 53 GCC 0 ACG TGC Ala TAC Asp Val Leu Glu bad Aeon Gin Val Phe Glu Ala Thr Cys CTG Tyr GCG TTC 550 CCA AAC CTG MG GAT TTC 5 ATA 70 GCG CGC TTT GAG Leu GAC Ala Phe Per Ann Leu Lys Asp Phe He Ala bad Phe Glu GGC Asp 59 MG 0 MG ATC TCC GAC TAC ATG 610 MG TCC AGC CGC TTC CTC Gly CTG Lys Lys He Ser Asp Tyr mead Lys Ser Ser bad Phe Leu 6 CCA Leu CCT CTG TTC ACA MG 1-3 CTi OVJl 0 GCT ATT TGG GGC AGC MG 670 DAYS Per 30 AGA Per Leu Phe Thr Lys mead Ala He Trp Gly Ser Lys End GAC bad GAC AGG TGG 6 GCT 90 TTA GGA GM AGA TAC CM 71 ATC 0 TCC TGG Asp CCT Asp bad Trp Ala Leu Gly Glu bad Tyr Gin He Ser Trp GTT Per CM GAG 730 CCC TM GGA GCG GGC AGG 7 ATT 50 CCT GAG CCC CAG Val TGC gin Glu Per End Gly Ala Gly bad He Per Glu Per Gin AGC Cys 77 GTT 0 TTC TTC CTT CCT TCC ATT 790 CCA GTC CCC AAG CCT TAC Ser CAT Val Phe Phe Leu Per Ser He Per Val Per Lys Per Tyr 8 CAG His TCA TTT TTT GGT CAT 83 CM 0 ATT CCT GCC AT THE CAC AGG 850 CTC Gin 10 CTC Ser Phe Phe Gly His Gin He Per Ala Lys His bad Leu TTA Leu GCC CTA GCA 8 ACT 70 CCT TTC CAT DAY CM MT 89 AGC 0 CTT CTA Leu AT THE Ala Leu Ala Thr Per Phe His End Gin Asn Ser Leu Leu MG Lys MG TGC 910 CCC GCC CCC ACC CCT CGA 9 GCT 30 CAT GTG ATT GGA Lys TTA Lys Cye Per Ala Per Thr Per bad Ala His Val He Gly DAY Leu 95 GCT 0 CCC MC ATG TGA TTA TTT 970 TGG GCA GGT CAG GCT CCC End TTG Ala Per Asn mead End Leu Phe Trp Ala Gly gXn Ala Per 9 CGG Leu ATG GGG TCT ATC TGG 101 AGA 0 CAG DAY ATT GCT AGC 1 AGC 030 TTT bad 90 CAG mead Gly Ser He Trp bad Gin End He Ala Ser Ser Phe GAC gin CGT AGC CM 10 GCC 50 CCT CTT CTT GCT GTT TCC 107 CGA 0 GAC DAY Asp CAC bad Ser Gin Ala Per Leu Leu Ala Val Ser bad Asp End CTA His GCA 1 AGG 090 TGT GCT GTG TCC CCA GCA 11 CTT 10 GTC ACT GCC TCT Leu TGA Ala Ars Cys Ala Val Ser Per Ala Leu Val Thr Ala Ser GTA End Val

113 O 1 150 11

ACC CGC TCC TAC CGC TCT T ¹ I ¹ C TTC CTG CTG CTG TGA GCT GTA CCT

Thr Arg Ser Tyr Arg 3er Phe Phe Leu Leu Leu End Ala VaI Pro

70 119 0 1 210

CCT GAC CAC AAA CCA GAA TAA ATC ATT CTC CCC TTA AAA AAA AAA

Pro Asp Hia Lya Prc GIu and He Leu Pro Leu Lya Ly Ly0 ii AA AAA AAA A _s Lya Lya Lye

The DNA sequence of the present invention, which encodes a glutathione S-transferase polypeptide, is operably linked to a plant promoter and expressed in the plant cell. According to the invention, it is of heterologous origin with respect to dun vegetable oil promoter.

The plant cells may be cells of tobacco, soybeans or cotton. The plant cells can be parts of a whole plant.

The plant promoter may be a promoter selected from the group of nos, ocs and CaMV promoters. The plant promoter may also be the promoter of the small subunit of the soybean ribulose bis-phosphate carboxylase or the promoter of the chlorophyll a / b binding protein. The herbicides may be a chloroacetamide, a sulfonylurea, a triazine, a diphenyl ether, an imidazolinone or a thiocarbamate. The invention also relates to a process for the preparation of a DNA transfer vector characterized by comprising a chimeric gene sequence consisting of a glutathione S -transferase gene operably linked to a plant-derived promoter is spliced into a suitable cloning vector containing replication and control sequences that stagnate from species compatible with the host cell. According to the invention, said host cell is a microorganism or a plant cell. According to the invention, a ver.'dhren for making a herbicide tolerant plant cell is also provided, which is characterized in that one transforms the plant cell with a recombinant DNA molecule according to any one of claims 1 to 14 using known transformation methods.

The present invention thus relates to recombinant DNA molecules which transfer tine herbicide tolerance to plants, wherein the herbicides are detoxified by production of proteinone. Known detoxification mechanisms include the conjugation of glutathione to electrophilic compounds, a reaction catalyzed by glutathione S-transferase; the conjugation of D-amino acid cn 2,4-dichlorophenoxyacetic acid (2,4-D) as well as the hydroxylation and Koh'enhydrat conjugation in sulfonylureas.

In particular, the present invention relates to herbicide tolerant plants transformed with a recombinant DNA molecule encoding an enzyme responsible for the detoxication of herbicides. Furthermore, the present invention relates primarily to recombinant DNA molecules characterized by a gene sequence encoding a glutathione S-transferase polypeptide, and to herbicide tolerant transformed plants having increased glutathione S-transferase enzyme activity , In the context of this invention, the plant cell is transformed with a glutathione-S-transferase gene which, in the course of its expression, confers herbicidal tolerance to the overexpression of the plant.

The present invention furthermore relates to from transformed plant cells regenerated plants and their seeds, furthermore au the progeny of regenerated from transgenic plant cells, plants as well as mutants and variants thereof zh.

The invention also relates to chimeric genetic constructs containing a glutathione S-transferase gene, to cloning vehicles and host organisms and to methods of transferring herbicide tolerance to plants. In the following, the enclosed figures are to be briefly characterized and explained:

Figure 1 sets forth the sequencing strategy for the pGTR200 cDNA insert from Y _b 200, a rat liver glutathione S-transferase gene.

Figure 2 shows plasmid pCIB710, an E. coli replicon comprising the promoter for the CaMV 35S DNA transcript and its terminator, and polyA accessory signal.

Figure 3 shows the construction of plasmid pCIB 12, an E. coli strain containing the chimeric gene with the CaMV 35S promoter linked to the glutathione S -transferase cDNA gene from rat liver Y _b 200, and the CaMV terminator is linked. Figure 4A illustrates the construction of plasmid pCIB 14, a wide-range replicon containing two chimeric genes within a T-DNA border sequence, where the chimeric gene consists of the CaMV 35S promoter linked to the glutathione S-transferase gene. Gen Y _b 200, the CaMV terminator and the kanamycin resistance gene, ncs-neo-nos, is linked. Figure 4B shows the completion of a Teklone using the in vivo recombination method. Figure 4C shows the completion of a Teklone using the in vitro linkage method.

Figure 5 shows fluorescence induction patterns typical of non-transgenic tobacco leaves. The upper curve is obtained after the leaves have taken up 10 " ⁶ M atrazine over a period of 48 hours, the lower curve being obtained when pure buffer solution is taken up.

Figure 6 shows the fluorescence induction patterns obtained in leaves of transgenic pCIB-14 tobacco plants after inoculating 10 " ⁵ M atrazine for 48 hours, revealing three classes of responses: (i) no detoxification of atrazine, upper curve ; (ii) significant detoxification of atrazine, lower curve; (Ni) a middle detoxification, middle curve.

Figure 7a shows the construction of plasmid pCIB5

 Figure 7b shows the construction of plasmid pCIB4

 Figure 7c shows the construction of plasmid pCIB2.

Figure 7d / e describes the construction of plasmid pCIB 10.

Figure 7f / g describes the construction of plasmid pCIB 10a.

In the following description, a number of printouts are used which are common in recombinant DNA technology as well as in plant genetics.

To ensure a clear and consistent understanding of the description and claims as well as the scope of the said printouts, the following definitions are set forth:

Heterologous gene (s) or DNA: A DNA sequence that codes for a specific product, product or biological radio- lation derived from a species other than that into which said gene is introduced; said DNA sequence is also referred to as foreign gene or foreign DNA.

Homologous gene (s) or DNA: A DNA sequence encoding a specific product, product or illogical function derived from the same species into which said gene is introduced.

Plant promoter: A DNA expression control sequence that ensures transcription of any homologous odar heteroIngen DNA gene sequence in a plant, when operably linked to such a promoter.

Overproducing Plant Promoter (OPP): A plant promoter capable of effecting, in a transgenic plant cell, the expression of any operatively linked functional gene sequence (s) to an extent (as measured in terms of RNA or polypeptide amount) which is significantly higher than is naturally observed in host cells that are not transformed with said OPP.

Glutathione S-Transferase: The definition of this enzyme is functional and includes any glutathione S-transferase (GST) capable of conjugating glutathione and an electrophilic compound in a given and desired plant to catalyze. Thus, not only does this term include the enzyme from the specific plant species involved in the genetic transformation, but also includes glutathione-S-transferases (GSTs) from other plant species, as well as from microorganisms or mammalian cells, if these GSTs are capable to fulfill their natural function in the transgenic plant cell.

The term GST includes amino acid sequences that are shorter or longer than the sequences of natural GSTs, such as

z. B. functionally hybrids or partial fragments of GSTs or their analogs.

Plant: Any photosynthetic active member from the Planta realm characterized by a membrane-coated nucleus, a chromosomally organized genetischas material, membrane-coated cytoplasmic organelles, and the ability to perform meiosis.

Plant Cells: Structural and physiological unit of the plant consisting of a protoplast and a cell wall.

Plant tissue: A group of plant cells organized in the form of a structural and functional unit.

Plant organ: A delimited and clearly visible differentiated part of a plant, such as root .. stem, leaf or embryo.

Detailed description of the present invention Herbicide-tolerant plants with increased GST enzyme activity:

The present invention relates to recombinant DNA molecules which confer on a plant a herbicidal tolerance based on the detoxication of the herbicide.

Known detoxification mechanisms include the hydroxylation and carbohydrate conjugation of sulfonylurea (Hutchison et al., Pesticide Biochem., Physiol., 22: 243-249 [1984]), D-amino acid conjugation to 2-4-D (Davidonis et al. Plant Phys., 70: 357-360 [1982]) and the conjugation of glutathione to electrophilic compounds catalyzed by glutathione S-transferase.

In particular, the present invention relates to herbicide-tolerant plants transformed with a recombinant DNA molecule encoding an enzyme responsible for the detoxification of herbicides. Furthermore, the present invention relates primarily to recombinant DNA molecules characterized by a gene sequence encoding a glutathione S-transferase polypeptide, and to herbicide tolerant transformed plants having increased glutathione S-transferase enzyme activity. The glutathione-containing plant cells and plants are transformed by a glutathione-S-transferase gon, which upon expression in said plant cell or plant enhances GST enzyme activity and thus confers herbicide tolerance to the plant These plants used gene manipulation techniques.

In the context of the present invention, a "herbicide-tolerant plant" is defined as a plant which survives in the presence of a normally effective herbicidal concentration and preferably exhibits normal growth.

Herbicide tolerance in plants according to the invention refers to a detoxification mechanism in the plant, even if the herbicide binding or target site is still sensitive

 Resistance means the maximum achievable tolerance.

Detoxification must be distinguished from other mechanisms for transmitting herbicide tolerance in which the herbicidal binding or target site is altered and no longer sensitive.

In the context of the present invention, the herbicide binding site in the plant remains unchanged sensitive, but the herbicide is not bound, since it is z. B. previously detoxified by the GST enzyme.

The term "herbicidal tolerance" used in the context of the present invention is therefore intended to encompass both tolerance and resistance to herbicides, said tolerance or resistance being due to the detoxification of herbicides, for example due to an increased GST enzyme activity survive damage in the presence of certain herbicides which are lethal to herbicidal-sensitive plants or impair their growth or vitality.

In the context of the present invention, herbicides are to be understood as meaning all herbicides which can be deioxified, for example by formation of conjugates of plant glutathione or glutathione analogues or homologs, or of any electrophilic compound. Particularly preferred in the context of the present invention are herbicides which have a chlorine radical. Examples of such herbicides, which are not limiting, are within the scope of the present invention:

Triazines, including chloratracets, acetamides, including chloroacetanilides, sulfonylureas, imidazolinones, thiocarbamates, chlorinated nitrobenzenes, diphenyl ethers, and others. Some specific examples of such herbicides include atrazine, alachlor, S-ethyl dipropyl thiocarbamate and diphenyl ether. See also: Herbicide Resistance in Plants (H. LeBaron and J. Gressel, ed., 1982).

Some of the mentioned herbicides are potent photosynthetic inhibitors in the plant.

Frearetal Phytochemistry, 9: 2123-2132 (1970) (chlorotriazines); Frear et al., Pesticide Biochem. to Physlol., 20: 299-310 (1983) (diphenyl ether); Lay et al .. Pesticide Biochem. and Physiol ,, 6: 442-456 (1976) (thiocarbamates); and Frear et al .. Pesticide Biochem. and Physiol., 23: 56-65 (1985) (metribuzin). Other herbicides lead to the inactivation of enzymes involved in the biosynthesis of amino acids.

Although the term "herbicides" is used to describe these compounds, the use of this term is not to be considered as limiting in the context of this invention, for example, there are numerous insecticides and pesticides (for the control of diseases, parasites and predators) which have harmful effects on the plant's vitality when applied to the plant The insecticidal or pesticidal agent may possibly be absorbed into the plant through the leaves, stem or soil via the root system.

In addition, there are numerous xenobiotics that are electrophilic compounds that can be conjugated to glutathione in a GST enzyme activity-catalyzed reaction. These xenobiotic compounds are also included in the present invention.

A specific embodiment of the present invention comprises herbicides which are sensitizers, i. Inhibitors of GST enzyme activity. These sensitizers inhibit the endogenous detoxification mechanism by forming a GST sensitizer conjugate.

The simultaneous application of a herbicide and a sensitizer, z. B. tridiphan, on a plant, leads to an inhibition of enzyme-catalyzed conjugation between glutathione and the herbicide. Ezra et al., "Tridiphane as a Synergist for Herbicides in Corn (Zea mays) and Proso Millet (Panicum miliaceum), Weed Science, 33: 287-290 (1985).

In the context of the present invention, therefore, the use of gene manipulative techniques to transfer GST enzyme activity or increased GST enzyme activity to a plant results in a transgenic plant having increased tolerance to sensitizers such as tridiphane.

In this way, for example, a sensitizer and a herbicide can be applied in combination to herbicide-sensitive and at the same time to herbicide-tolerant plants according to the present invention. This combination of sensitizer and herbicide will typically be toxic to the herbicide-sensitive plants, but not to transgenic plants.

Any plant containing glutathione or an analogous compound such as homo-glutathione, which is susceptible to genetic manipulation using genetic engineering techniques, may be used in the invention. The transgenic plant must also be able to express the GST gene.

In the context of the present invention, plants are to be understood as meaning plant cells, plant protoplasts, plant tissue cultures which can be cultivated and induced to form whole plants, but also plant calli, plant lumps and plant cells as intact parts of a plant and plant parts.

The term "plant" also refers to that which is transformed by means of gene manipulation techniques.

The highest glutathione concentrations in plants are usually found in the subcellular compartments of the plant tissue, such. In the plant plastids, especially in the chloroplasts (Rennenberg, H., Phytochemistry, 21: 2771-2781 [1982]). Glutathione has a gamma-L-glutamyl-L-cysieinyl-glycine structure. A homologous form of glutathione, homo-glutathione, has been found in some plants. It has the structure of gamma-L-glutainyl-L-cycteinyl-beta-alanine (Carnegie, P., Biochem J., 89: 459-471 [1963] and Carnegie., Biochem. 89: 471-478 [1963]).

Plants have different amounts of glutathione or homo-glutathione. For example, in various legumes mainly homo-glutathione is found, while other legumes preferably contain glutathione. Normally, in cases where one of the two components, either homo-glutathione or glutathione, predominates in the plant, the other component is present only in low concentration. (Race mountain, supra).

The region coding for the glutathione S-transferase (GST) gene used according to the present invention may be of homologous or heterologous origin relative to the plant cell or plant to be transformed. However, it is in any case necessary that the gene sequence encoding GST be expressed and result in the production of a functional enzyme or polypeptide in the resulting plant cell. The present invention therefore includes plants which possess either homologous or heterologous GST genes and which express the GST enzyme.

Furthermore, the GST may be derived from other plant species or from organisms belonging to a different taxonomic unit, e.g. From microbes or from mammals.

As previously described, the GST enzyme is a class of multifunctional enzymes. Therefore, it is necessary to select a GST gene which catalyzes the conjugation of glutathione and an electrophilic compound.

Since glutathione acts as a substrate of GST, prior to this invention, it was uncertain whether the GST enzyme specific for the conjugation of glutathione would accept homo-glutathione in transformed plants. Frear et al., Phytochemistry, 9: 2123-2132

(1970) (shows that glutathione-S-transferase is specific for reduced glutathione). The required GST enzyme specific for glutathione can be identified and selected by using an assay that determines substrate specificity to differentiate the different glutathione S-transferases. In a typical assay, glutathione-specific GST can be characterized by affinity chromatography (Tu et al., Biochem. And Blophys.

Research Comm., 108: 461-467 [19821; Tu et al., J. Biol. Chem., 258: 4659-4662 [1983]; and Jakoby et al., in Glutathione; Metabolism and Function, (Raven Press, New York, 1976]).

In a specific embodiment of this invention, the GST is a plant GST that is homologous to the plant to be transformed. In another embodiment of this invention, the GST is a plant GST heterologous to the plant to be transformed.

The plants that have a GST surplus include corn and sorghum. Another embodiment of the present invention includes a mammalian GST. Mammalian GSTs are known and described in Reddy et al. Archives of Biochem. and Biophys., 224: 87-101 (1983) (sheep liver); Tu et al., J. Biol. Cham., 258: 4, 659-4662 (1983) (rat tissue from heart, kidney, liver, lung, spleen and testis). The preferred GST gene is characterized by the coding region of the GST gene from rat liver, in particular by Yb200 described in Example I and the completed Yb 187 described in Example IB.

Another part of the present invention relates to the coding region of a GST gene from the rat brain, in particular the cDNA clone GIY ·, which is described in Example IE. However, other genes are also known which can be used in the context of the present invention. See: Mannervik, B., Adv. Enzymol. Relat. Areas MoI. BIoI., 57: 357-417 (1985), incorporated by reference into this invention.

The DNA sequence coding for the glutathione-S-transferase can be constructed exclusively from genomic or from cDNA. Another possibility is the construction of a hybrid DNA sequence consisting of both cDNA and genomic DNA. In this case, the cDNA may be from the same gene as the genomic DNA, or both the cDNA and the genomic DNA may be from different genes. In any case, however, both the genomic DNA and / or the cDNA, each by itself, may be made from the same or different genes.

If the DNA sequence includes portions of more than one gene, these genes may be either from one and the same organism, from several organisms belonging to more than one strain, variety or species of the same genus, or from organisms containing more than belonging to a genus of the same or another taxonomic unit (Reich).

The various DNA sequence sections can be linked to one another by means of methods known per se to form a complete glutathione S-transferase-encoding DNA sequence. Suitable methods include, for example, in vivo recombination of DNA sequences having homologous portions as well as in vitro linking of restriction fragments.

Thus, a variety of embodiments are included within the broad concept of this invention.

One of these embodiments of the invention is characterized by a chimeric gene sequence containing:

(a) a first gene sequence encoding a glutathione S-transferase polypeptide having glutathione S-transferase activity upon expression of the gene in a given plant cell; and

(b) one or more additional gene sequences operably linked to both sides of the GST coding region. These additional gene sequences contain promoter and / or terminator regions. The plant regulatory sequences may be heterologous or homologous relative to the host cell.

Any promoter and terminator capable of inducing induction of expression of a GST coding region may be used as part of the chimeric gene sequence. Examples of suitable promoters and terminators are e.g. These include the nopaline synthase genes (nos), the octopine synthase genes (ocs) and the cauliflower mosaic virus genes (CaMV).

An effective representative of a plant promoter that can be used is an overproducing plant promoter.

This type of promoter, if operably linked to the gene sequence for the GST, should be able to mediate the expression of said GST in such a way that the transformed plant is resistant to a herbicide due to its presence GST enzyme activity is tolerant.

Overproducing plant promoters that can be used in the present invention include the small subunit (ss) of soybean ribulose-1,5-bisphosphate carboxylase (Berry-Lowe et al., J. Molecular and App Gen., 1: 483-498 [1982]) and the promoter of the chlorophyll a / b binding protein. These two promoters are known to be induced by light in eukaryotic plant cells (see

z. Genetic Engineering of Plants, to Agricultural Perspective, A. Cashmore, Plenum, New York 1983, pages 29-38; Coruzzi G. et al., The Journal of Biological Chemistry, 258: 1399 [1983] and Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2:

The chimeric gene sequence consisting of a glutathione S-transferase gene operably linked to a plant promoter can be spliced into a suitable cloning vector. In general, one uses plasmid or virus (bacteriophage) vectors with replication and control sequences derived from species compatible with the host cell.

The cloning vector usually carries an origin of replication, as well as specific genes that lead to phenotypic selection characteristics in the transformed host cell, in particular to resistance to antibiotics or to certain herbicides. The transformed vectors can be selected from these phenotypic agents after transformation in a host cell.

As host cells in the context of this invention are prokaryotes in question, including bacterial hosts such. B.

A. tumefaciens, E.coli, S. typhimurium and Serratia marcescens, as well as cyanobacteria. Furthermore, eukaryotic hosts such as yeasts, mycelium-forming fungi and plant cells may also be used in the invention.

The cloning vector and the host cell transformed with this vector are used according to the invention to increase the copy number of the vector. With an increased copy number, it is possible to isolate the vector carrying the GST gene and, for. B. for the introduction of the chimeric gene sequence in the plant cell to use.

The introduction of DNA into host cells can be carried out by methods known per se. For example, bacterial host cells can be transformed after treatment with calcium chloride. In plant cells, DNA can be introduced by direct contact with the protoplasts produced from the cells.

Another way to introduce DNA into plant cells is to bring the cells into contact with viruses or with Agrobacterium. This can be accomplished by infection of sensitive plant lines or co-culturing of protoplasts derived from plant cells.

These procedures will be discussed in detail below.

For the direct introduction of DNA into plant cells, a number of methods are available. For example, the genetic material contained in a vector can be microinjected directly into the plant cells by means of micropipettes for the mechanical transfer of the recombinant DNA. The genetic material may also be introduced into the polyethylene glycol after treatment of the protoplast with it. (Paszkowski et al., EMBO J., 3: 2717-2722

Another object of the present invention relates to the introduction of the GST gene into the plant cell by means of electroporation (Shillito et al. Biotechnology, 3: 1099-1103 [1985]; Fromm et al., Proc. Natl. Acad. Be. USA, 82: 5824 [1985]).

In this technique, plant protoplasts are electroporated in the presence of plasmids containing the GST gene.

Electrical impulses of high field strength lead to a reversible increase in permeability of biomembranes and thus facilitate the introduction of the plasmids, electroporated plant protoplasts renew their cell wall, divide and form callus tissue. The selection of the transformed plant cells with the expressed GST enzyme can be carried out with the aid of the phenotypic markers described above.

The cauliflower mosaic virus (CaMV) can also be used as a vector for the introduction of the GST gene into the plant in the context of this invention (Hohn et al., In "Molecular Biology of Plant Tumors", Academic Press, New York, 1982, Page 549-560; Howell, U.S. Patent No. 4,407,956).

The entire viral DNA genome of CaMV is thereby integrated into a bacterial parent plasmid to form a recombinant DNA molecule that can be propagated in bacteria. After cloning, the recombinant plasmid is cleaved by means of restriction enzymes either randomly or at specific non-essential sites within the viral portion of the recombinant plasmid, e.g. Within the gene coding for the transmissibility of the virus by aphids in order to incorporate the GST gene sequence.

A small oligonucleotide, a so-called linker, which has a unique, specific restriction recognition site can also be integrated. The recombinant plasmid thus modified is cloned again and further modified by splicing in the GST gene sequences: a single restriction site.

The modified viral portion of the recombinant plasmid is then excised from the parent bacterial plasmid and used for inoculation of the plant cells or the whole plants.

Another method for introducing the GST genes into the cell uses the infection of the plant cell with Agrobacterium tumefaclens, which is transformed with the GST gene. The transgenic plant cells are then subjected to appropriate; culturing conditions known to those skilled in the art, so that they form shoots and roots and ultimately whole plants result.

The GST gene sequences can be converted into suitable plant cells, for example with the aid of the Ti plasmid of Agrobacterium tumefaclens (DeCleene et al. Bot. Rev., 47: 147-194 [1981]; Bot. Rev., 42: 389-466 (1976).) In the course of infection by Agrobacterium tumefaciens, the Ti-Piasmid is transferred to the plants and stably integrated into the plant genome.

(Horsch et al., Science, 233: 496-498 [1984]; Fraley et al., Proc Natl Acad ScI., USA, 80: 4803 (1983)).

For plants whose cells are not sensitive to infection with Agrobacterium, one can resort to the co-cultivation of Agrobacterium with the corresponding protoplasts.

Ti plasmids have two regions that are essential for the production of transformed cells. One of them, the transfer DNA region, is transferred to the plant and leads to the induction of tumors. The other, the virulence-conferring (vir) region, is essential only for the formation but not for the maintenance of the tumors. The transfer DNA region can be increased in size by incorporation of the GST gene sequence without impairing transmissibility.

By removing the tumor-causing genes, whereby the transgenic plant cells remain non-tumorigenic and by incorporating a selectable marker, the modified Ti plasmid can be used as a vector for the transfer of the gene construction according to the invention into a suitable plant cell.

The vir region effects the transfer of the T-DNA region of Agrobacterium to the genome of the plant cell, regardless of whether the T-DNA region and the vir region are present on the same vector or on different vectors within the same Agrobacterium cell. A vir region on a chromosome also induces transfer of T-DNA from a vector into a plant cell.

Preferred is a system for transferring a T-DNA region from an Agrobacterium to plant cells, characterized in that the vir region and the T-DNA region are on different vectors. Such a system is known by the term "binary vector system" and the vector containing the T-DNA is referred to as a "binary vector".

Any T-DNA containing vector that is transmissible in plant cells and that allows for selection of the transformed cells is suitable for use in this invention. Preferred is a vector made from a promoter, a coding sequence and pCIB10.

Any vector having a vir region that effects transfer of a T-DNA region of Agrobacterium to plant cells may be used in the invention. The preferred vector having a vir region is pCIB 542, plant cells or plants transformed according to the present invention can be selected by means of a suitable phenotypic marker which is part of the DNA in addition to the GST gene. Examples of such phenotypic markers, but are not intended to be limiting, include antibiotic resistance markers, such as kanamycin resistance and hygromycin resistance genes, or herbicide resistance markers. Other phenotypic markers are known to those skilled in the art and may also be used within the scope of this invention.

All plants whose cells can be transformed by direct introduction of DNA or by contact with Agrobacterium and subsequently regenerated to complete plants can be subjected to the process according to the invention for the production of transgenic whole plants containing the transferred GST gene. There is a growing body of evidence suggesting that, in principle, all plants can be regenerated from cultured cells or tissues including, but not limited to, all major cereals, sugarcane, sugarbeet, cotton, fruit trees and other tree species, Legumes and vegetables.

The target crops in the context of the present invention include, for example, those selected from; of the series: Fragaria, Lotus, Medlcago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapls, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersfon, Nlcotlar.a, Solanum, Petunia, Digitalis, Majorana, Cichorlum, Helianthus, Lactura, Bromus, Asparagus, Antirrhinum, Hemerocallis, Nemesia, Pelargonium, Panicum, Pennlsetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum and Sorghum. including those selected from the series: Ipomoea, Passiflora, Cyclam6ti, Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Lilium, Narcissus, Pineapple, Arachis, Phaseolus and Pisum.

The knowledge about the extent to which all these plants can be transformed with the help of Agrobacterium are still limited at present. In vitro, for example, it is also possible to transform plant species which are not natural host plants for Agrobacterium. For example, monocotyledonous plants, especially the cereals and grasses, are not natural hosts for Agrobacterium.

In the meantime, there is increasing evidence that monocotyledons can also be transformed with Agrobacterium, so that using new experimental approaches that are now becoming available, crops and grasses species are also susceptible to transformation (Grimsley, N., et al , Nature, 325: 177-179 (1987J).

Regeneration of cultured protoplasts to whole plants is described in Evans, et al., Protoplast Isolation and Culture, Handbook of Plant Cell Culture, 1: 124-176 (MacMillan Publishing Cn N ™ York 1 f) R3); MRDavey, "Recent Developments in the Culture of Regeneration of Plant Protoplasts", Protoplasts, 1983 - Lecture Proceedings, pages 19-29, (Birkhäuser, Basel 1983); PJ Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," in Protoplast's 1983 Lecture Proceedings, pages 31-41, (Birkhäuser, Basel 1983), and H. Binding, "Regeneration of Plants," in Plant Protoplasts , Pages 21-37, (CRC Press, Boca Raton 1985).

The regeneration differs from plant species to plant species. In general, however, a suspension of transformed protoplasts, cells, or tissues containing numerous copies of the GST genes is first prepared. On the basis of these suspensions, the induction of embryo formation can subsequently take place. The development of the embryos is allowed to proceed to maturity and germination, as is the case with naturally occurring embryos. The culture media usually contain various amino acids and hormones, v.'ie z. Auxin and cytokinins. It also proves to be advantageous to add glutamic acid and proline to the medium, especially in species such as maize and alfalfa. Sprouting and rooting generally occur simultaneously. Efficient regeneration depends primarily on the medium, the genotype and the history of the culture. If these three variables are adequately controlled, the regeneration is completely reproducible and repeatable. Plants suitable for use in the invention include, for example, species of the genera Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Citrus, Linum, Mani sot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus , Lycopersicon, Nicotlana, Solanum, Petunia, Majorana, Cichorium, Helianthus, Lactuca, Asparagus, Antirrhinum, Panicum, Pennlsetum, Ranunculus, Salpiglossis, Glycine, Gossypium, Malus, Prunus, Rosa, Populus, Allium, Lilium, Narcissus, Pineapple, Arachis , Phaseolus and Pisum.

After discovering that oryza (rice) can be regenerated from protoplasts to whole plants, it should now also be possible to regenerate other plants of the Gramineae family. The following plants can therefore also be used in the context of the present invention: lolium, zea, triticum, sorghum and bromus. Particularly preferred according to this invention are plants of the genera Nlcotana spp. (e.g., tobacco), Glycine spp. (especially Glycine max, soybean) and Gossypium spp. (Cotton).

The mature plants grown from transformed plant cells are crossbred with themselves for semen production. Some of the Sarners include the Gone for increased GST enzyme activity in a ratio that obeys the established laws of inheritance. These seeds can be used to produce herbicide tolerant plants. The tolerance of these seeds can be determined, for example, by growth of the seeds in soil containing herbicide. Alternatively, the herbicidal tolerance of the transformed plant can be determined by applying the herbicide to the plant.

Homozygous lines can be obtained by repeated self-fertilization and inbred line production. These inbred lines can in turn be used to develop herbicidal tolemnt hybrids. In this method, a herbicide-tolerant inbred line is crossed with another inbred line for producing herbicide-resistant hybrids. Parts that can be obtained from regenerated plants, such. B. flowers, seeds, leaves, branches, fruits u. a. are also part of the present invention, provided that these parts include herbicide tolerant cells. Progeny, including hybrid offspring), varieties and mutants of the regenerated plants are also part of the present invention.

Use of GST gene constructions and herbicide tolerant plants

The previously described GST gene constructs can be used in vectors as intermediates in the preparation of herbicide tolerant plant cells, plant organs, plant tissues, and whole plants. The importance of herbicide tolerant plants according to the present invention is evident. Namely, these plants allow farmers to first plant a herbicide-tolerant crop and then treat the field with the herbicide to control weeds without damaging the crop.

In addition, a herbicide tolerant plant allows farmers to grow field crops on areas that have been previously treated with herbicides, for example in a crop rotation, where a naturally tolerant plant of a naturally tolerant plant is a naturally sensitive plant follows. These herbicidally treated crops may contain a "herbicide excess" in the soil such that inherently herbicidal sensitive crops may be damaged by this excess herbicide in the soil if they do not prove to be tolerant (Sheets, T., Residue Reviews, 32: 287-310 [1970]; Burnside et al., Weed Science, 19: 290-293 (1971)).

For example, farmers usually grow corn and soybeans in alternating successions. While the corn plants are inherently tolerant to certain herbicides, eh? .. as various triazines such as atrazine, the sensitive! S soybean plants can be damaged if they are planted in an area that has previously been treated with herbicides.

When using a herbicide tolerant plant according to the present invention, damage due to herbicidal excess in the soil is avoided (Fink et al., Weed Science, 17: 35-36 [1969] [Soybeans]; Khan et al., Weed Research, 21: 9-12 [1981] (Oats and Timothy plants; Brinkmann et al., Crop Science, 20: 185-189 [1980] [Oats]; Eckert et al., J.RangeMgmt., 25: 219-224 [Ί972] [ Wheatgrass]).

The present invention also includes a method of plant control, which is characterized in that a mixed population consisting of a herbicide-sensitive plant such. Thus, the method of foliar treatment of herbicide-tolerant plants and herbicide-sensitive weeds with herbicides in the field, both types of plant in the field of herbicides, is a herbicide-tolerant plant At the same time as the treatment operation is brought into contact with the herbicide, it is also an object of the present invention.

Another object of the present invention relates to a previously described method of plant control, which is characterized in that a herbicide and a sensitizer in a mixed population are applied simultaneously to both herbicide tolerant and herbicide-sensitive plants.

The term "plant controlling amount of a herbicide" functionally describes a rate of application of a herbicide capable of affecting the growth or development of a particular plant, thus either minimizing the rate of application so as to decrease it or suppression of growth or development is sufficient, or it may be so large that irreversible damage to the sensitive plant occurs.

The actual application rate depends on the one hand on the herbicide used, on the other hand on the plant to be controlled. For dicotyledonous plants and weeds e.g. As a rule, application rates between 0.5 and 1.5 kg / ha are needed.

For monocotyledonous plants the required herbicidal concentrations are generally between 0.5 and about 2.0 kg / ha.

Various herbicides, such as the sulfonylureas, are known to be effective even at very low rates.

The herbicides may be contacted with the subject plants using well-known spraying or control methods. For example, the prepublished foliar application may be used to control weeds by atrazine in the presence of atrazine-tolerant plants of the present invention.

In the general description of the present invention, reference will now be made, for purposes of brevity, to specific embodiments which are included in the specification for purposes of illustration and which are not limiting unless specifically noted.

Ausführungsbelspie '

The invention is explained in more detail below with reference to some examples.

The methods used in the following examples are generally described in Maniatis et al. Molecular Cloning, Cold Spring Harbor Laboratory, (1982). Enzymes may be purchased from New England Biolabs unless otherwise noted. They will be used in accordance with the manufacturer's instructions, unless it is separately referred to.

Example IA: Isolation of the GST cDNA clone Y "200

Antibody:

Antisera to homogeneous Lober-glutathione-S-transierasone (GSTen) (affinity chromatographic fraction) are prepared according to previously described procedures (Tu et al., Nudele Acids Res., 10: 5407-5419 [1982]). The IgG fraction is purified on a Protein A Sepharose (Pharmacia) column and concentrated by ultrafiltration (Amicon, XM-50 membrane) as described by Kraus and Rosenberg (Kraus & Rosenberg, Proc Natl Acad ScI USA , 79:

/ ini * _dni9 [1982]). It is then stored in 50% glycerol and 0.2 mg / ml heparin at -18 ° C.

Isolation of polysomes

The livers (about 26 g) of two male Sprague-Dawley rats (body weight about 300 g) are mixed with a Potter Elvehjem Komogenisator in 150 ml (final volume) 50 mM Tris-HCl, pH 7.5.25 mM MgCl ₂ , 0.25 M sucrose solution containing bentonite (1 mg / ml). Homogenized heparin (C, 2 mg / ml) and cycloheximide (1 μg / ml) in different aliquots to a 15% (w / v) homogenate. The polysome isolation is performed exactly according to the method described by Kraus and Rosenberg (see above). Yields before and after dialysis are ¹ 389 A ₂₆₀ units and 1208 A ₂ M units, respectively.

Immobilization of Polysome Antibody Complexes and Elutlon Specific mRNA 1130A ₂ 6o Polysome units are recovered for immunoabsorption with anti-GST IgG (7.1 mg). Protein A Sepharose affinity chromatography and the elution of bound RNA molecules are performed according to the instructions of Kraus and Rosenberg supra. The sliced RNA molecules are immediately adapted to ύ, 5Μ NaCl and 0.5% dodecyl sulfate and further purified on an oligo (dT) cellulose column (Aviv & Leder, Proc Natl Acad Sci USA, 69: 1408- 1412 [1972]; Bantle et al., Anal. Biochem., 72: 413-417 [1976]). The purified poly (A +) RNA molecules are detected by in vitro translation and immunoprecipitation (Tu et al., Nucleic Acids Res., 10: 5407-5419 [1982], Pelham and Jackson, Eur.

J. Biochem., 87: 247-256 (1976)) before being used for cDNA synthesis. The immunoprecipitated material is separated on a dodecyl sulfate polyacrylamide gel and visualized by fluorography (Laemmli, Nature, 227:

680-684 [1970]; Swanstrom and Shenk, Anal. Biochem., 86: 184-192 [1978]).

Isolation of GST cDNA clones

The synthesis of the cDNA is carried out analogously to the method of Okayama and Berg (Okayama and Berg, Mol. Cell BIoI., 2: 161-170 (1832)), except for minor deviations, according to a modification of Gubler and Hoffmann (Gubler and Hoffman, Gene, 25: 263-269 (1983)). Approximately 100-500ng of poly (A +) RNA from immunoprecipitated polysomes are used for cDNA synthesis. The reverse transcription of the mRNA into the corresponding cDNA is carried out in a reaction unit of 40μΙ, the 5OmMTnS-HCl pH 8,3,10OmM NaCl, 1OmM MgCl ₂ , 1OmM DTT, 4mM sodium pyrophosphate, 1.25mM of visrdNTP's, 1800U / mlRNAsin (PromegaBiotec) , 100 μg / ml Oligo (dT) u-i8 (Pharmacia / PL) and 3000 U / ml reverse transcriptase (Molecular Genetics).

The reaction mixture is incubated for a period of 25 minutes at a temperature of 43 ⁰ C. The reaction is then terminated by addition of 2μΙ 0.5 M EDTA. The reaction mixture is extracted with one part by volume of phenol / chloroform and the aqueous phase is then back-extracted with half a volume of chloroform. The organic phase is back-extracted again with TE buffer.

The aqueous phases are then combined. After adding one volume of 4M ammonium acetate, the nucleic acids are precipitated by addition of two volumes of ethanol followed by cold-rolling on dry ice for 20-30 minutes. The solution is then warmed to room temperature for 5 minutes and centrifuged for 15 minutes in an Eppendorf centrifuge ("Microfuge") at a temperature of 4 ° C. The pellet thus obtained is dissolved in 25 μM TE buffer The process of nucleic acid precipitation and recovery is repeated as described above by addition of ammonium acetate (25μΙ; 4M) and 100μΙ of ethanol The resulting pellet is then washed with 70% ethanol, dried and dissolved in 20μΙ of water.

Replacement of the mRNA tcDNA hybrid mRNA is performed in a 50 μΙ reaction unit containing 20 mM Tris-HCl pH 7.5.5 mM MgCl ₂ , 10 mM ammonium sulfate, 10 mM KCl, 0.15 mM beta-NAD, 0.04 mM four dNTP's, 20μΙ of the first strand product, 1Ο-2Ομΰϊβ ³² ΡΡαΑΤΡ, 10U / ml E. coli DNA ligase (New England Biolabs), 230U / ml DNA polymerase (Boehringer Mannheim) and 8.5U / ml E. coli RNAse H (Pharmacia / PL). The incubation period is 90 minutes at a temperature of 12 ° C to 14 ° C and another Stundo at room temperature. The double-stranded cDNA is purified by PnenoLChloroform extraction and recovered by ethanol precipitation as described for the first strand product. The last dried pellet is dissolved in 20μΙ water.

10μΙ of the double-stranded cDNA are probed in a total volume of 20μΙ containing 10μm potassium cacodylate pH 7.0mM CoCI ₂ , 0.2mM DTT, 0.1mM dCTP and 500U / ml terminal deoxynucleotide transferase (Pharmacia / PL) with a dCTP Annex provided. The incubation period is 1 to 2 minutes at a temperature of 37 ⁰ C. Then 20μΙ 4mM EDTA are added and the enzyme heat-inactivated by incubation at 65 ⁰ C for 10 minutes.

PstI-digested and dG-tagged pBR322 (Bethesda Research Labs, Inc.) is added in a molar ratio of about 1: 1 to the dC-containing double-stranded cDNA (i.e., about 5-10 fold excess in size) on the molecular weight of the vector compared to the assumed amount of cDNA). The DNA-containing solution (cDNA + vector) is liszu a total DNA concentration of 0.5-2.0 ng ^ l (0.5 ng are optimal) in the presence of 1OmM Tris-HCl pH 7.5, 1 mM ED ΓΛ and 15 mM NaCl diluted. This mixture is first incubated for 5 minutes at 65 ⁰ C, then the actual renaturation reaction ("annealing") takes place at a temperature of 55-58 ° C over a period of 90 minutes.

E. coli strain MM 294 is transformed with the renatured cDMA: vector complex using 5μΙ DNA per 200 μΙ aliquot of transformation competent cells (Hanahan, J. Mol. BIoI., 155: 557-580 [1983]). The transformation frequency of the control is 1-2 x 10 transformants per μg of covalently closed circular pBR322 DNA. The transformed cells are plated on LM-plates (without magnesium) containing 17pg / ml tetracycline (Hanahan, supra). The colonies thus obtained are assayed for ampicillin sensitivity. The tetracycline resistant and ampicillin sensitive (about 50%) colonies are picked for further analysis.

Hybrid selected in vitro translation of pGTR200

Plasmid DNA molecules from 354 ampicillin sensitive transformants are purified using an alkaline lysis method (Birnboim and Doyl, Nudele Acid Research, 7: 1513-1523 (1979)) .The purified DNA molecules are then used to determine the size of the integrated cDNA library. Of these, a total of 134 visible cDNA inserts were contained after performing agarose-gel electrophoresis, and more than 800 nucleotide inserts were subjected to further analysis by Southern blot hybridization (Southern, J. Mol.

BiOI, 98: 503-517 [1975]) using Y, (pGTR261) and Y _c (pGTR262) (as sample molecules Lai et al, J. Chem BiOI, 259: 5536-5542 [1984J;.... Tu at a!, J. Βίo !. Chem., 259: 9434-9439 [1984]).

12 clones that do not hybridize with these probes are further characterized by hybrid selected in vitro translation (Cleveland et al., Cell, 20: 95-105 (198O)) One of these negative clones is designated pGTR200 (A +) - RNA molecules from rat liver, were tldktioniert by pGTR 200 DNA that of activated aminophenylthioether cellulose (APT paper) is immobilized, 'are at 75 ⁰ C and 100 "C eluted and then used for the in vitro Translation in the rabbit reticulocyte lysate system The in vitro translation products are immunoprecipitated by antisera against the total rat liver GSTs, followed by dodecyl sulfate polyacrylamide gel electrophoresis The immunoprecipitated product has the same mobility as Y _b By pGTR 200 no other class of GST subunits selected. Previously performed hybrid selected in vitro translation experiments with Y, and Y _c no Y _b subunit products (Lai et al., supra; Tu et al., Supra).

Nucleotide sequence of the pGTR200DNA insert

The DNA sequence of the cDNA in pGTR200 is carried out according to the strategy shown in Figure 1 using the chemical method of Maxam and Gilbert (Maxam and Gilbert .. Methods Enzymol. 65: 499-F6011980]). The DNA fragments obtained due to cleavage with various restriction enzymes are labeled at the 3 'end. Each determination was repeated at least once.

The nucleotide sequence is shown below. For the 218 residues of the open reading frame, the one-letter code for amino acids was used (Old and Primrose, Principles of Gene Manipulation, [1985], Blackwell's Publications, London, p. 346). The poly (A) addition signal, AATΑΛΑ is underlined.

10 20 30 40 50 60

CTGAAGCCAAATTGAGAAGACCACAGCGCCAGAACCATGCCTATGATACTGGGATACTGG

MPMILGYW

70 80 90 100 110 120 AACGTCCGCGGGCTGACACACCCGATCCGLCTGCTCCTGGAATACACAGACTCAAGCTAT NVRGLTHPIRLLLEYTDSSY

130 140 150 160 170 180 GAGGAGAAGAGATACGCCATGGGCGACGCTCCCGACTATGACAGAAGCCAGTGGCTGAAT EEKRYAMGDAPDYDRSQWLN

190 200 210 220 230 240 GAGAAGTTCAAACTGGGCCTGGACTTCCCCAATCTGCCCTACTTAATTGATGGATCGCGC ekfklgldfpnlpylidgsr

250 260 270 280 290 300 AAGATTACCCAGAGCAATGCCATAATGCGCTACCTTGCCCGCAAGCACCACCTGTGTGGA KITQSNAIMRYLARKHHLCG

310 320 330 340 350 360 GAGACAGAGGAGGAGCGGATTCGTGCAGACATTGTGGAGAACCAGGTCATGGACAACCGC ETEEERIRADIVENQVMDNK

370 380 390 400 410 420 atgcagctcatcatgctttgttacaaccccgactttgagaagcagaagccagagttcttg mqlimlcynpdfekqkpefl

430 440 450 460 470 430 AAGACCATCCCTGAGAAGATGAAGCTCTACTCTGAGTTCCTGGGCAAGCGACCATGGTTT KTIPEKMKLYSFFLGKRPWF

490 500 510 520 530 540 GCAGGGGACAAGGTCACCTATGTGGATTTCCTTGCTTATGACATTCTTGACCAGTACCAC AGDKVTYVDFLAYDILDQYH

550 560 570 580 590 600 ATTTTTGAGCCCAAGTGCCTGGACOCCTTCCCAAACCTGAAGGACTTCCTGGCCCGCTTC IFEPKCLDAFPNLKDFLARF

610 620 630 640 650 660 GAGGGCCTGAAGAAGATCTCTGCCTACATGAAGAGCAGCCGCTACCTCTCAACACCTATA EGLKKISAYMKSSRYLSTPI

670 680 690 700 710 720 TTTTCGAAGTTGGCCCAATGGAGTAACAAGTAGGCCCTTGCTACACTGGCACTCACAGAG FSKLAQWSNK *

730,740,750,760,770,780 AGGACCTGTCCACATTGGATCCTGCAGGCACCCTGGCCTTCTGCACTGTGGTTCTCTCTC

790,800,810,820,830,840 CTTCCTGCTCCCTTCTCCAGCTTTGTCAGCCCCATCTCCTCAACCTCACCCCAGTCATGC

850 860 870 880 890 900 CCACATAGTCTTCATTCTCCCCACTTTCTTTCATAGTGGTCCCCTTCTTTATTGACACCT

910 920 930 940 950 960 TAACACAACCTCACAGTCCTTTTCTGTGATTTGAGGTCTGCCCTGAACTCAGTCTCCCTA

970 980 990 1000 1010 1020

GACTTACCCCAAATGTMCACTGTCTCAGTGCCAGCCTGTTCCTGGTGGGGGAGCTGCCC

1030 1040 1050 1060 1070

CAGGCCTGTCTCATCTTT AATAAA GCCTGAAACACAAAAAAAAAAAAAA

Example IB: Isolation of partial GST cDNA clone Y _b 187

With the help of dor in principle the same method gives the cDNA clone Y _b 187. Y _b 187 is a partial cDNA clone lacking at the 5 'end of the coding sequence 96 nucleodites. The nucleotide and corresponding amino acid sequence as well as the sequence for the 96 missing nucleotides and for Y _b 187 are given below:

1 0 30

ATG CCT ATG ACA CTG GGT TAC TGG GAC ATC CGT GGG CTG GCT CAC Met Pro Met Thr Leu GIy Tyr Trp Asp He Arg Gly Leu Ala His

50 7 0 90

GCC ATT CGC CTG TTC CTG GAG ACT ACA GAC ACA AGC ACT GAG GAC Ala He Arg Leu Phe Leu GIu Tyr Thr Asp Thr Ser Tyr GIu Asp

1 10 13 0

AAG AAG TAC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG Lys Lys Tyr Ser Met GIy Asp Ala Pro Asp Tyr Asp Arg Ser GIn

150 1 70

TGG CTG AGT GAG AAG TTC AAA CTG GGC CTG GAC TTC CCC AAT CTG Trp Leu Ser Glu Lys Phe Lys Leu Gily Leu Asp Phe Pro Asn Leu

19 0 210

CCC TAC TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC Pro Tyr Leu He Asp Gly Ser His Lys He Thr GIn Ser Aan Ala

2 30 25 0 270

ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGT GGG GAG ACA He Leu Arg Tyr Leu GIy Arg Lys His Asn Leu Cys GIy GIu Thr

2 90 31 0

GAG GAG GAG AGG ATT CGT GTG GAC GTT TTG GAG AAC CAG GCT ATG GIu GIu GIu Arg He Arg VaI Asp VaI Leu GIu Asn Gin AIa Met

330 3 50

GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT Asp Thr Arg Leu GIn Leu AIa Met VaI Cys Tyr Ser Pro Asp Phe

37 0 390

GAG AGA AAG AAG CCA GAG TAC TTA GAG GGT CTC CCT GAG AAG ATG Giu Arg Lys Lys Pro GIu Tyr Leu GIu GIy Leu Pro GIu Lys Met

4 10 43 0 450 AAG CTT TAC TCC GAA TTC CTG GGC AAG CAG CCA TGG TTT GCA GGG Lys Leu Tyr Ser GIu Phe Leu GIy Lys GIn Pro Trp Phe Ala GIy

4 70 49 0

AAC AAG ATT ACG TAT GTG GAT TTT CTT GTT TAC GAT GTC CTT GAT Asn Lys He Thr Tyr VaI Asp Phe Leu VaI Tyr Asp VaI Leu Asp

510 5 30

CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC Gin His Arg He Phe GIu Pro Lys Cys Leu Asp Ala Phe Pro Asn

55 0 570

CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Leu Lys Asp Phe VaI AIa Arg Phe GIu GIy Leu Lys Lys He Ser

5 90 61 0 630 "GAC TAC ATG AAG AGC GGC CGC ITC CTC TCC AAG CCA ATC TTT GCA Asp Tyr Met Lys Ser Gly Arg Phe Leu Ser Lys Pro He Phe AIa

6 50

AAG ATG GCC TTT TGG AAC CCA AAG TAG Lys Met Ala Phe Trp Asn Pro Lys End

Example IC: The complete clone corresponding to Y _b 187

The missing nucleotides are added either in vivo by recombination or in vitro by linking the appropriate restriction enzymes to Y _b 187. These methods are shown in Figures 4B and 4C.

Recombination can be achieved by the introduction of a genomic clone DNA clone, as a component of the plasmid colE1 (eg pUC8 or pBR32ü) and a cDNA clone, which is located on the plasmid inc PI (eg pRK290), in the E. coli strain SR43 (ATCC Accession No. 67217, filed September 22, 1986). The strain SR43 carries the mutations polA1 supF183 (Tacon, W. & Sherratt, D., Mol. Gen. Genet., 147: 331-33511976)), so that the repiikation of the colE1 plasmid at a restrictive temperature (42 ⁰ C ) is inhibited.

A conversion of SR 43 bacteria containing the two said plasmids from a permissive temperature of 28 ⁰ C to 42 ⁰ C while maintaining the selectivity to antibiotic resistance, on the plasmid colE1 (eg

Ap-resistance of pBR322) allows the selection of bacteria containing a cointegrate form of the two plasmids.

Such cointegrated plasmids come about through recombination of homologous DNA regions.

The isolation of cointegrated plasmids allows the cloning of the entire cDNA sequence and the upstream genomic DNA in the form of a single PstI or BamHI fragment.

Restriction of the upstream DNA with Ba 131, followed by the addition of a BamHI linker, allows the isolation of clones containing the entire coding region (confirmed by DNA sequence analysis).

The entire coding region is cloned in the form of a BamHI fragment into pCIB710, as described in Example IA, and used for plant cell transfer.

The same fragment is cloned into pDR540 for expression in E. coli as described in Example IV, below.

Another possibility for constructing a complete clone corresponding to Y _b 187 is to link appropriate restriction fragments of the cDNA and genomic clones (Figure I).

Plasmid pUC 19 contains the 5 'end of the genomic clone spliced in as a PstI-HindIII fragment containing approximately the first 400 bases of the coding sequence.

The 3 'end of the coding sequence of cDNA kr.nn can be isolated as a 630 base »Hinfl fragment from the partial cDNA clone in BR 325. That at his only | The linFl site cut pUC plasmid and the Hinfl fragment carrying the 3 'end of the gene are linked together to restore the entire coding sequence of the GST gene. The complete gene is isolated as a Bgll-PstI fragment.

Isolation of the entire coding rogion, without the upstream DNA sequences, can be achieved by excision of the upstream DNA using Ba 131 followed by BamHI linker addition.

This BamHI fragment is then spliced into pCIB710 or into pDR 540, respectively, according to the information given above.

Example ID: Hybrid clones

Hybrid clones derived from coding sequences for different genes are, in principle, constructed in the same manner as previously described for the combination of the partial cDNA clone Yb 187 and the missing nucleotides (example IC), using heterologous genes as starting material for the two DNA fragments we used the.

Example IE: Isolation of the GST cDNA clone GIY _b

Using the phage expression vector λ gt11 (Young, RA and Davis, RW, Proc Natl Acad., USA, 80: 1194 [1983]), a cDNA library is prepared starting from a poly (A) RNA, previously isolated from rat brain according to the methods described above (see Example IA).

The cDNA is then isolated by antibody screening using antibodies directed against GST from the rat brain.

The isolation of the RNA and the preparation and isolation of the associated cDNA is carried out according to previously known methods, as described, for. Lei Young and Davis (Young, R.A. and Davis, R.W., Science, 222: 778-782 (1983)).

The nucleotide sequence and the associated amino acid sequence of the resulting cDNA clone GIY _b are shown below:

10 30

A GAC CCC AGC ACC ATG CCC ATG ACA CTG GGT TAC TGG GAC ATC Met Pro Met Thr Leu GIy Tyr Trp Asp He

5 0 70

CGT GGG CTA GCG CAT GCC ATC CGC CTG CTC CTG GAA TAC ACA GAC Arg GIy Leu Ala His Ala He Arg Leu Leu Leu GIu Tyr Thr Asp

90 11 0 130

TCG AGC TAT GAG GAG AAG AGA TAC ACC ATG GGA GAC GCT CCC GAC Ser Ser Tyr GIu GIu Lys Arg Tyr Thr Met GIy Asp Ala Pro Asp

1 50 17 0

TTT GAC AGA AGC CAG TGG CTG AAT GAG AAG TTC AAA CTG GGC CTG Phe Asp Arg Ser GIn Trp Leu Asn GIu Lys Phe Lys Leu GIy Leu

190 2 10

GAC TTC CCC AAT CTG CCC TAC TTA ATT GAT GGA TCA CAC AAG ATC Asp Phe Pro Asn Leu Pro Tyr Leu He Asp GYy Ser His Lys He

23 O 250 2

ACC CAG AGC AAT GCC ATC CTG CGC TAT CTT GGC CGC AAG CAC AAC Thr Gin Ser Aan Ala lie Leu Arg Tyr Leu Giy Arg Lye Hie Aen

70 29 0 310

CTG TGT GGG GAG ACA GAA GAG GAG AGG ATT CGT GTG GAC ATT CTG Leu Cys GIy GIu Thr GIu GIu GIu Arg He Arg VaI Asp He Leu

AAT CAG 3 30 GAC AAC CGC ATG GTG CTG 35 0 CTT TGC GAG Asn Gin CTC ATG Asp Asn bad mead Val Leu GCG AGA Leu Cys Glu Leu mead 3 90 Ala bad AAC CCT 370 GAG AAG CTG AAG CCA GGG GAG CAA DID Asn Per GAC TTT Glu Ly s Leu Lye Per Gly TAC CTG Glu Gin Tyr 41 0 Asp Phe 430 Tyr Leu 4 CCT GGA CGG CTT TAC rpr · r \ ίυυ GAG TTC AAG CGG CTG Per Gly ATG ATG bad Leu Tyr Ser Glu Phe CTG GGC Ly s bad Leu mead mead Leu Gly

50 47 0 490

CCA TGG TTT GCA GGG GAC AAG ATC ACC TTT GTG GAT TTC ATT GCT Pro Trp Phe Ala GIy Asp Lye He Thr Phe VaI Asp Phe He AIa

5 10 53 0

TAC GAT GTT CTT GAG AGG AAC CAA GTG TTT GAG GCC ACG TGC CTG Tyr Asp VaI Leu GIu Arg Asn Gin VaI Phe GIu AIa Thr Cys Leu

550 5 70

GAC GCG TTC CCA AAC CTG AAG GAT TTC ATA GCG CGC TTT GAG GGC Asp Ala Phe Pro Asn Leu Lye Asp Phe He AIa Arg Phe GIu GIy

59 0 610 6

CTG AAG AAG ATC TCC GAC TAC ATG AAG TCC AGC CGC TTC CTC CCA Leu Lys Lya He Ser Asp Tyr Met Lys Ser Ser Arg Phe Leu Pro

30 65 0

AGA CCT CXG TTC ACA AAG ATG GCT ATT JGG Arg Pro Leu Phe Thr Lys Met Ala He Trp

6 90

CCT GAC AGG TGG GCT TTA GGA GAA AGA TAC Pro Asp Arg Trp AIa Leu GIy GIu Arg Tyr

730 7

TGC CAA GAG CCC TAA GGA GCG GGC AGG ATT Cys Gin GIu Pro End GIy Ala GIy Arg He

77 0 790

CAT GTT TTC TTC CTT CCT TCC ATT CCA GTC His VaI Phe Phe Leu Pro Ser He Pro VaI

10 83 0 850

CTC TCA TTT TTT GGT CAT CAA ATT CCT GCC AAA CAC AGG CTC TTA Leu Ser Phe 'Phe Gly His Gin He Pro Ala Lys His Arg Leu Leu

8 70 89 0

AAA GCC CTA GCA ACT CCT TTC CAT DAY CAA AAT AGC CTT CTA AAG Lys AIa Leu AIa Thr Pro Phe His End Gin Asn Ser Leu Leu Lys

910 9 30

TTA AAG TGC CCC GCC CCC ACC CCT CGA GCT CAT GTG ATT GGA TAG Leu Lys Cys Pro-AIa Pro Thr Pro Arg Ala His VaI He GIy End

95 0 970 9

TTG GCT CCC AAC ATG TGA TTT TGG GCA GGT CAG GCT CGG CCC Leu Ala Pro Asn Met End Leu Phe Trp Ala GIy Gin AIa Pro Arg

90 101 0 1 030

CAG ATG GGG TCT ATC TGG AGA CAG TAG ATT GCT AGC AGC TTT GAC GIn Met Gly Ser He Trp Arg Gin End He Ala Ser Ser Phe Asp

AGC AAG 670 GAC GGC Ser Lys DAY Asp Gly 71 0 End ATC TCC GTT CAA He Ser TGG Val gin Trp 50 GAG CCC AGC CCT Glu Per CAG Ser Per Gin 8th AAG CCT CAG CCC Lv s Per TAC Gin Per Tyr

10 50 107 0

CAC CGT AGC CAA GCC CCT CTT CTT GCT GTT TCC CGA GAC TAG CTA His Arg Ser Gin Ala Pro Leu Leu Ala VaI Ser Arg Asp End Leu ι

1 090 11 1.0

TGA GCA AGG TGT GCT GTG TCC CCA GCA CTT GTC ACT GCC TCT GTA End Ala Arg Cys Ala VaI Ser Pro Ala Leu VaI Thr Ala Ser VaI

113 0 1 150 11 ^;

ACC CGC TCC TAC CGC TCT TTC TTC CTG CTG CTG TGA GCT GTA CCT Thr Arg Ser Tyr Arg Ser Phe Phe Leu Leu Leu End Ala VaI Pro

70 119 0 1 210

CCT GAC CAC AAA CCA GAA TAA ATC ATT CTC CCC TTA AAA AAA AAA Pro Asp His Lye Pro Giu End Lie Leu Pro Leu Lye Lys Lys

AAA AAA AAA A Lys Lys Lys

Example Ii: Construction of plasmid pCIB710

Plasmid pLW111, ATCC # 40235, consists of the three smaller EcoRI fragments of the BJI strain of Cauliflower Mosaic Virus (CaMV) (Franck et al., Cell, 21: 285-294 [1980]), which clones into pMB9 become. The plasmid pLW111 is digested with BglII and a 1149 bp fragment (base pairs # 6494-7643) isolated. This fragment is spliced into the BamHI site of pUC19. This restriction fragment encodes both the promoter of the CaMV 35S RNA and the polyA addition signal (i.e., the terminator) for this transcript.

In vitro mutagenesis

Between this promoter and terminator a single BamHI-restriction site is inserted via in vitro mutagenesis.

A 36-base oligonucleotide is synthesized which, except for a BamHI restriction site inserted into base sequence # 7464 in the sequence. Jt is identical to the corresponding sequence of the CaMV region.

The above mentioned 1149 bp Bgl II fragment is first cloned into M 13mp 19; then a single-stranded phage DNA is isolated. This single-stranded DNA is then annealed to the synthetic oligonucleotide.

Using this oligonucleotide as a primer, a new DNA strand is first synthesized and then the DNA is transfected into E. coli strain JM101 (Zöllei & Smith, DNA 3: 479-488 [19801].

Isolation of the M13 phage with the incorporated BamHI site is performed as described by Zoller & Smith, supra.

Selection of the desired mutant phage

The 36-base oligonucleotide is labeled in a kinase reaction with ³² P-ATP. A selection of transfected M13 phage plaques are transferred to a nitrocellulose filter. This filter is hybridized with the labeled 36-base oligonucleotide and then washed at increasing temperatures. The labeled oligonucleotide attached to the mutant phage is still stable at higher wash temperatures. One of these elevated temperature stable phages is isolated and sequenced to confirm the presence of the BamHI site.

Construction of pCIB710

Double-stranded DNA isolated from this phage is digested with HindIII ur.d EcoRI. pUC19 is also cleaved with HindIII and EcoRI. These two digested DNA molecules are then linked together. Transformants are selected for their ampicillin resistance. One of the transformants resulting from this linkage reaction is pCIB710, a plasmid shown in Figure 2.

Example III: Construction of plasmids pCIB 11, pCIB 12, pCIB 13 and pCIB 14

1. Plasmid pGTR 200 is digested with Haell, PstI and Pvull and the 678 bp Haell / PstI fragment containing the GST coding sequence isolated from an agarose gel.

2. This Haell / PstI fragment is blunt-ended by treatment with T4 DNA polymerase, which is linked by the T4 DNA ligase BamHI linker (dCGGATCCG-New England Biolabs).

3. The plasmid pCIB710 is cut with BamHI and then treated with calf intestinal alkaline phosphatase (Boehringer Mannheim).

4. The above-described, with a BamHI linker GST fragment is spliced into the digested with BamHI plasmid pCIB710, the Liyationsmischung transformed into E. coli strain HB101 and selected the desired transformants on the basis of their ampicillin resistance. It is possible to characterize both transformants which contain the GST-coding sequence with regard to transcription, starting from the CaMV promoter, in the correct orientation (pCIB 12), as well as those with an opposite orientation (pCIB 11).

5. The plasmid pCIB 10 (see Example IV) is digested using XbaI and EcoRI.

6. The plasmid pCIB 12 is also digested with the aid of XbaI and EcoRI and the smaller fragment isolated from an agarose gel.

7. The isolated XbaI / EcoRI fragment carrying the chimeric gene is spliced into the previously digested plasmid pCIB10; the entire ligation mixture is then transformed into E. coli HB101. The transformants are selected for their kanamycin resistance. These transformants, which contain the GST coding sequence in the appropriate orientation with respect to transcription from the CaMV promoter, are designated pCIB 14 (see Figure 3).

8. Plasmid pCIB 11 is similarly manipulated to construct pCIB13. PCIB 13 is a plasmid containing the GST coding region in an orientation that precludes a regulated transcript from the CaMV promoter.

Introduction of pCIB 13 and pCIB 14 in Agrobacterium

Purified plasmid DNA of type pCIB 13 or pCIB 14 is introduced by means of the transformation into Agrobacterium tumefaclens A136 (Watson et al., J. Bacteriol. 123: 255-264 [1975]), which contains the plasmid pCIB542 (see also Example VII , below). Transformants are selected on kanamycin (50pg / ml) and on spectinomycin (Σδμρ / ηηΙ).

Example IV: Incorporation of the Y _b 200 GST in the trp-lac Expresslons Vector

Plasmid pCIB 12 is digested with BamHI and a 708 bp Bam-linked GST gene fragment isolated. The plasmid pDR540 (Pharmacia PL Biochemicals), a trp-lac expression vector (Russel, DR and Bennett, GN, Gene, 20: 231-243 [1982]) is also digested with BamHI and the GST fragment transformed into the corresponding Spliced restriction restriction site. The resulting recombinant plasmid is transformed into E. coli strain JM103.

Cultures of the resulting strain are inducible at any time by the addition of 1, OmM isopropyl-beta-D-thiogalactoside (IPTG).

After induction by IPTG and expression of the Y _b 200 GST gene, the bacterial host is pelleted. The pellet formed is then resuspended in 59 ml of 0.2 M Tris-HCl, pH 8.0, and 1 mM EDTA. To this suspension is added 0.5 ml of 10 mM phenylmethylsulfonyl fluoride (PMSF) in ethanol and 5 ml of lysozyme (10 mg / ml) in buffer solution.

The suspension is incubated for about 15 minutes at a temperature of 37 ⁰ C until the bacterial cells are lysed. The lysed bacterial cells present in the suspension are pelleted again. The supernatant is collected and then dialyzed against 25 mM Tris-HCl, pH 8.0, with 1/100 volume PMSF.

The dialyzed supernatant is then assayed for GST activity. Subsequently, the dialyzed supernatant is packed on an S-hexylglutathione-agarose affinity column, at a flow rate of 0.5 ml / min. The column is washed with 25 mM Tris-HCl and 0.2 MKCl until the absorbance at 280 nm drops below a value of 0.005. After all unbound material has been washed off the column, the bound material is eluted with 25 mM Tris-HCl, 0.2 M KCl (pH 8.0) containing 5 mM S-hexylglutathione and 2.5 mM glutathione. The eluted fractions are monitored for their absorbance at 280nm.

The fractions suspected of containing the purified enzyme are individually dialyzed against 0.1 M NaPO 4 buffer (pH 6.5) containing PEG to concentrate the reaction and then against the same buffer without PEG.

The volumes in each of the dialysed fractions are registered. For each fraction, the concentration and the amount of the sample are calculated and then diluted so that at the end in each fraction the same proportion relative to the starting fraction is present. Each fraction is examined for enzyme activity.

The determination of enzyme activity is carried out using 1-chloro-2,4-dinitrobenzene (CIDNB) as a substrate.

For each reaction, to 5 mM reduced glutathione (30.8 mg / ml in buffer, 0.1 M sodium phosphate, pH 6.5) and GST enzyme is added 1 mM CIDNB (20.2 mg / ml in ethanol).

CIDNB and reduced glutathione are made fresh daily. The reaction unit has a final volume of 1 ml. The reaction is carried out at room temperature and started by addition of CIDNB. The course of the reaction is monitored by means of a Gilford spectrophotometer at a wavelength of 340 nm. A typical course of the reaction involves 10 to 50 units of GST, where 1 unit corresponds to the conversion of 1 mmol of substrate per minute and ml.

The protein concentration of the enzymatically active fractions in the starting material is determined (Biorad, BSA standard). Each of the enzymatically active fractions is analyzed on a 15% Lämmli gel using serial dilutions of standard enzymes (I.Opg, 0.3pg and 0.1 μg ) as quantitative standards. Detection of the purified enzyme is by immunoblotting using specific antibodies.

Example V:

Construction of pCIB 10 and pCIBIOa

The construction of plasmids pCIB 10 and pCIB 10a is described in pending U.S. Patent Application Serial No. 757,098, filed July 19, 1985, entitled "Transformation of Plastids and Mitochondria" and incorporated herein by reference ,

The following individual steps, which have been described and numbered as follows in the abovementioned reference patent application, are carried out for the construction of the plasmids (see FIG. 7a-g):

Construction of pCIB10

15. A T-DNA fragment carrying the left border sequence of plasmid pTiT37 is isolated from pBR 325 (EcoRI 29) (Yadav et al., Proc. Natl. Acad. Sci. USA, 79: 6322 [1982]). pBR325 (EcoRI29) is cut with EcoRI and the 1.1 Kb fragment linked to HindIII linkers (New England Biolabs).

16. Plasmid pBR322 (Bolivar et al., Gene, 2:75) is cut with Child III.

17. The fragment containing the left border sequence mentioned in step 15 is spliced into the HindIII digested plasmid pBR322.

18. The plasmid pBR322 with the left border sequence from step 17 is digested with CIaI and HindIII; The 1.1 kb HindIII / ClaI fragment is isolated (Hepburn et al., J. Mol. Appl. Genet. 2: 211 [1983]).

19. The plasmid pUC18 (Norrander et al., Gene, 26: 101 [1983]) is cut with Hind IM and EcoRI; a 60-bb poly-linker is end-labeled with the aid of T4 polynucleotide kinase and gamma ³² P-dATP and isolated from an acrylamide gel.

20. The plasmid pBR322 is cut with EcoRI and CIaI and the large fragment isolated.

21. The 60 bp Hind IH / Eco RI polylinker and the 1.1 Kb Hind III / ClaI fragment of Eco RI 29 are spliced into the plasmid pBR 322 previously cleaved with CIaI and Eco RI previously cleaved with CIaI and Eco RI was spliced to give PCiB5 (step 15-21, see Figure 7a).

22. From Bin 6 (Bevan, Nucleic Acids Res., 12: 8711 [1984]) a chimeric gene coding for kanamycin resistance (nos-neonos) is obtained in the form of a Sall / EcoRI fragment.

23. The plasmid pUC 18 is cut with EcoRI and Sail.

24. The Sall / EcoRI fragment containing the chimeric gene mentioned in step 22 is inserted into the EcoRI and Sail cut plasmid pUC18.

25. The BamHI recognition site in the termination sequence of this chimeric gene is disrupted by cutting with BamHI, using T4 + DNA polymerase ei. and finally linked again.

26. The resulting plasmid is cut with Sst II (Bethesda Research Laboratories) and Hind III.

27. A 1.0Kb fragment which covers the 5 'portion of the nos promoter and the right border sequence of the plasmid pTiT37 is isolated by cutting pBR326 (Hind23) with Hind III and Sstll.

28. This 1.0 Kb Hind II / Sst II fragment is spliced into plasmid pUC 18 from step 26 to give pCIB4 (step 22-28, see Figure 7 b).

29. Plasmid pCIB5, containing the left DNA border sequence, is cut with Aatll, blunted by treatment with T4 DNA polymorase, and then cut again with EcoRI.

30. pCIB4 is cut with Hind III, blunted by treatment with the Klenow fragment of E. coil DNA polymerase, and then cut with EcoRI.

31. Restricted plasmid pCIB5 from step 29 is linked to the truncated plasmid pCIB4 (step 30) to form pCIB2, a ColEI replicon containing the left and right T-DNA borderline sequence containing a chimeric kanamycin resistance gene flanked a polylinker (step 29-31, see Figure 7c).

32. Plasmid pRZ 102 (Jorgenson et al., Mol. Gen. Genet. 177: 65 [1979]) is supplemented with BamHI ven Uiut and using Klenow.

33. Partial Alu I digestion of the plasmid pA03 (Oka, J. Mol. Biol., 174: 217 (1981)) is carried out.

34. The AIu I digest of the plasmid pAO3 is spliced into the plasmid pRZ 102 from step 32, whereby the desired transformants can be selected for their kanamycin resistance.

35. The resulting plasmid has the coding sequence of Tn903 on a 1.05 Kb BamHI fragment which is isolated after BamHI digestion. This fragment is then treated with Klenow DNA polymerase.

36. The plasmid pRK252, an aomeptide of the plasmid RK 2, which has a broad host range, can be described by Dr. med. Don Helinski at the University of California in San Diego (USA). This plasmid lacks the Bgl II site, which is present in the parent plasmid pRK290 (Ditta et al., Proc. Natl. Acad. Sci. USA, 77: 7347 (198O)).

pRK252 is digested with Smal and Sail, and supplemented with the help of Klenow; the large fragment resulting from this digestion is isolated.

37. The Tn903-containing fragment isolated in step 35 is spliced into the large fragment of pRK252 to give pRK252Km (step 32-37, see Figure 7d).

38. Plasmid pRK252 Km is cut with EcoRI, blunt-ended using Klenow and linked to Bgl II linkers (New England Biolabs).

39. The plasmid pC> B2 is cut with EcoRV and the smaller fragment containing the right border sequence and the nos-neonos sequence is isolated. This fragment is supplemented with Klenow polymerase and linked to Bgl II linkers (New England Biclabs).

40. The Bgl II fragment from step 39 is linked to the pRK 252 Km plasmid from step 38 which carries a linker to form pCIB 10 (step 38-40, see Figure 7e).

Construction of pCIB 10a:

41. Plasmid pRZ102 (Jorgensen, et al., Mol. Gen. Genet., 177: [1979]) is digested with BamHI and filled in using Klenow.

42. Partial Digestion of the Plasmid ρAO3 (Oka et al., Nature 276: 845 [1978]).

43. The material digested with the aid of AIuI is spliced into the restriction-restricted piasmid pRZ 102 from step 32, the desired transformants being selected for their kanamycin resistance.

44. The resulting plasmid has the coding sequence of Tn903 on a 1.05Kb BamHI fragment; this fragment is isolated after completion of BamHI digestion and after filling with the aid of Klenow.

45. The plasmid pRK290, a derivative of the plasmid RK2, which comprises a broad host range, can be obtained from Dr. med. Don Helinski at the University of California, San Diego (USA). pRK290 is digested with Smal and Sail, supplemented with Klenow, and the large fragment resulting from this digestion isolated.

46. The Tn 903-containing fragment isolated in step 35 is spliced into the large fragment of pRK2 to give pRK290Km.

47. The plasmid from step 46 is digested with Bgl II, supplemented with the aid of Klenow, and recombined to destroy its BglI site. The result is the plasmid pKR290 Km (step 41-47, see Figure 7 f).

48. Plasmid pRK290Km is cut with EcoRI, blunt-ended using Klenow and fitted with Bgl II linkers (New England Biolabs).

49. The plasmid pCIB2 is cut with EcoRV and also provided with Bgl II linkers (New England Biolabs).

50. The plasmid pCIB2 from step 39, equipped with Bgl I linkers, is spliced into the vector from step 47 to give pCIB 10a (step 48-50, see Figure 7g!

The process steps described in item 41-50 can be carried out in the same way using the plasmid pRK252 instead of pRK290.

Example VI Construction of pCIB23 and pCIB24, vectors that transformed the GST enzyme to the chloroplasts of Align plants

The plasmid pSRS2.1, which contains the 5 'sequence of the small subunit (SSU) of ribulose-bis-phosphate-carboxylase (fUiPBC) of soybeans (Berry-Lowe et al., J. Mol. Appl. Genet., 1: 483-498 [1982]) is with Dr. med. Richard Meagher of the Department of Genetics of the University of Georgia at Athens, Georgia 30602 (USA).

This plasmid is digested with EcoRI. The 2.1 Kb fragment containing the SSU 5 'soybean region is isolated from an agarose gel. The 2.1 Kb EcoRI fragment wii 'is then digested with Ddel and a 471 bp Ddel fragment is isolated. This fragment contains the transit peptide and part of the second exon. The 471 bb Ddel fragment is treated with Klenow (New England Biolabs DNA Polymerase). A kinased Bgl II linker [d (CAGATCTG) New England Biolabs] is linked to this fragment. This Bgl II fragment is then digested with TaqI and the resulting TaqI fragments are treated with Klenow (Bethesda Research Labs DNA Polymerase). The resulting smooth-ending fragments are linked to BamHI linkers previously treated with kinase (d (CGCGGATCCGCG) New England Biolabs] and then purified, these BamHI fragments are then digested with BglII and BamHI. Approximately 400 bp fragment containing the SSU 5 'region is purified.

The plasmid pCIB710 is cut with BamHI and then treated with calf intestinal alkaline phosphatase. The 400 bp BamHI / BglII fragment (see above) is spliced into this plasmid (pCIB710). The linker product is transferred to E. coli HB101 and the transformants are selected for ampicillin.

In this way one finds transformants carrying the BamHI / BglII fragment in both orientations. In plasmid pSCR 2, the 5 'region of the transit polypeptide is immediately adjacent to the 35S promoter. In contrast, the DamHI / Bgl II fragment in the plasmid pSCR1 is in the opposite orientation.

The plasmid pCIB 12 is digested with BamHI and the 708 bp BamHI fragment carrying the GST gene is isolated.

The plasmid pSCR 2 is also cut with BamHI and then treated with calf intestinal alkaline phosphatase. The 708 bp BamHI fragment from the plasmid pCIB 12 is then spliced into the BamHI-treated plasmid pSCR2. The linkage product is transferred to HB101 and the transformants selected for ampicillin.

One finds transformants having the GST gene in both orientations with respect to the control region at the 5 'end. The clone pCIB 22 has the GST gene in a direction suitable for transcription, starting from the CaMV promoter. In clone pCIB21, on the other hand, the GST gene has the opposite orientation. pCIB22 is digested with XbaI / EcoRI. The fragment carrying the chimeric gene is isolated from a gel and purified.

The plasmid pCIB 10 is also digested with XbaI and EcoRI. The XbaI / EcoRI fragment carrying the chimeric gene is spliced into the previously digested plasmid pCIB10 and the transformants are selected for their kanamycin resistance. The resulting plasmid pCIB24 is a host-wide plasmid containing the chimeric gene in direct association with a chlo-plast transit peptide sequence.

The plasmid pCIB23 is constructed starting from the clone pCIB21 instead of pCIB22, and subjecting similar manipulatory cleavage. This plasmid contains the GST gene in the opposite orientation compared to pCIB24.

These plasmids are transferred into Agrobacterium strains in a manner similar to that previously described for pCIB13 and pCIB14.

Example VII: Construction of the plasmid pCIB542, an Agropin VIr helper plasmid, which treats a spectinomycin resistance gene in the T-DNA region

The Ti plasmid pTiBo542 (Sciaky, D., Montoya, AL & Chilton, MD, Plasmid, 1: 238-253 [1978]) is of particular interest because agtobacteria containing this plasmid are capable of producing the agronomically important ones Legumes, alfalfa and soybeans (Hood, EE, et al., Bio / Tuchnology, 2: 702-703 [1984]).

The construction of a pTiBo542 derivative having a deietion in the region of its T-DNA is described by Hood, Elizabeth E., (1985) Ph. D. thesis; Washingto University, St. Louis, Mo. (USA). In this construction, designated EHA101, the T-DNA is replaced with a kanamycin resistance gene. The starting plasmid of EHA101, A201, is ATCC under ATCC no. 53487 behind.

Starting from EHA101, a derivative is constructed in which the kanamycin resistance gene is replaced by a spectinomycin resistance gene. Plasmid pi delta 307 (E.Hood, Washington University, Ph. D. thesis [1985]) has a 1.7 Kb region homologous to the left side of Barn a of plasmid pTiBo542 (Hood, et al. , [1984]), as well as an 8Kb comprehensive? Region homologous to the right side of Barn 2 a of pTiBo 542 separated by a single EcoRI site. Plasmid pMON 30, ATCC No. 67113, contains the spectinomycin / streptomycin resistance gene (spc / str) of Tn 7 (Hollingshead, S. and Vapnek, D "Plasmid, 13: 17-30 [1985]). The plasmid pMON 30 is digested with EcoRI; the 5.5 kb fragment containing the spc / str genes is isolated from an agarose gel and spliced into the EcoRI truncated plasmid pi delta 307. The desired recombination product is selected in the form of a spectinomycin-resistant (50 pm / ml) and a tetracycline-resistant (10pg / ml) transformant. This plasmid is introduced into Agrobacterium strain AI36 / EHA101 and selected for its streptomycin resistance, tetracycline resistance and kanamycin resistance phenotype. Merodiploid cells ("homogenotes") (Matzke, AJM & Chilton, MD, J. Mol. Appl. Geriet., 1: 39-49 [1981]) with the EHA101 plasmid and the spectinomycin plasmid are after introduction of the expulsion plasmid R751-pMG2 (Jacoby, G. et al., J. Bacteriol 127: 1 278-1 285 [1976]) and selection for gentamycin (50 μg / ml) and spectinomycin The preferred merodiploid cell is characterized by a gentamycin resistant, spectinomycin-resistant, tetracycline-sensitive, and kanamycin-sensitive phenotype The structure of the resulting plasmid is confirmed by Southern blot studies.

Example VIII: Test of plants for atrazine tolerance Based on numerous studies, such as

1) the fluorescence induction; and

2) the growth ability of seedlings at atrazine concentrations,

which are toxic to control or wild-type plants, it has been shown that regenerated tobacco plants bearing the GST gene construction pCIB 14 are tolerant to atrazine.

The fluorescence induction gives information about the state of the photochemical apparatus of the plant (Voss et al .. Weed Science, 32: 675-680 (1984)). In the course of this assay, leaf tissue is irradiated with light of a particular wavelength and the resulting fluoroenzyme is registered at a second wavelength. Figure 5 shows a fluorescence induction pattern typical of a punched out (untransformed) tobacco leaf infiltrated with buffer solution taken over the truncated petiolus (under petiole). One first recognizes a large increase in fluorescence when the light is turned on, which then reaches a peak, followed by a gradual decrease in the signal over time.

This pattern makes it clear that the chloroplasts are excited to fluoresce by the incident light at a wavelength characteristic of the system. At this point, energy is released from the photosystem in the form of electrons, which flow away via the electron transport pathways of photosystems II and I - this is reflected by the gradual decrease in the fluorescence signal curve.

However, when the leaf is infiltrated with a 10 " ^b M atrazine solution, the result is an image as shown in the upper curve of Figure 5. In this part, the light energy is also initially absorbed, causing the chloroparatures to fluoresce However, there is no energy efflux because the electron flow is blocked at the quinone-binding level of photosystem II, and it looks as if the photosystem is solidified in the excited state, so that no drop is seen after the initial increase in fluorescence.

If these measurements are performed in accordance with the present invention genetically engineered tobacco plants in the presence of 10 ^"b M atrazine, sozeigen all control plants, the same fluorescence induction pattern as the non-transgenic tobacco plants.

On the other hand, when the measurements are made on the test plants, three classes of fluorescence induction patterns can be distinguished (Figure 6).

Some of the transgenic plants show no evidence of atrazine detoxification (the topmost curve), some show middle detoxification (middle curve) and some show significant (lower curve) atrazine detoxification, which is evident due to normal electron trajectory through the photosystem becomes. Out of 27 plants characterized in this way, 5 showed clear evidence of the ability to detoxify atrazine.

Example IX: Agrobacterium infection of plant material

The various genotypes of Agrobacterium tumefaclens are grown on an AB minimal medium (Watson, B. et al.

J. Bacteriol. 123: 255-264 [1975J), is added to the mannitol, cultured for 48 hours at a temperature of 28 ⁰ C. The bacteria are pelleted, resuspended in MSBN medium with a two-fold dilution and incubated for three hours at äiner temperature of 25 ⁰ C. For the preparation of the MSBN medium, a prefabricated powdery mixture is used which can be obtained from KC Biological and which contains the macro and microelements of the Murashige and Skoog medium in the form of their salts. The final concentration of the constituents of the MSBN medium corresponds to that of the medium according to Murashige and Skoog. In addition, the MSBN medium (final concentrations) contains: Benzyladenine (1ng / L); Naphthylacetic acid (0.1 mg / L); myo-inositol (1 mg / L); nicotinic acid (1 mg / L); pyridoxine [1 mg / L; thiamine HCl [10 mg]; and sucrose [30 mg / L)]. The pH is adjusted to a pH of 5.7 to 5.8.

Leaf discs are allowed to grow in Nicotians tabacumcv cultured in vitro. petite Havana SR 1 plants float for 10 minutes on a bacterial suspension in a modification of a method of Horsch, R. et al., Science, 227: 1229-1231 (1985).

The leaf discs are then transferred to filter paper on MSBN medium without antibiotics. After 48 hours, the leaf pieces are dipped in liquid MSBN medium containing 500 mg / l carbenicillin and then transferred to a solid selection medium containing 100 mg / l kanamycin and 500 mg / l carbenicillin.

Plant maturation and self-pollination

Saplings that develop on the selection medium from the kaIIi are removed and transferred to OMS medium containing 100 mg / l kanamycin and 250 mg / l carbenicillin. The development is then allowed to proceed for three more weeks.

The plantlets are then planted in soil and transferred to the greenhouse. Flowering begins 4 to 8 weeks after the plants have been transferred to the greenhouse. By the time the flower opens and its anthereri separate, they are removed with the help of tweezers and rubbed on the scars to self-pollinate each individual flower. The seed capsules mature within 40 days.

Semen progeny testing of control plants, transgenic control plants and transgenic test plants The seeds are first removed from the mature seed capsule under aseptic conditions and stored in sterile petri dishes. The seeds are then transferred to a medium-soft seed germination medium (SKM) composed of all macro- and microelements in the form of their salts and in a concentration corresponding to the medium of Murashige & Skoog (KC Biological), from 1 mg / l thiamine hydrochloride as well 0.6% purified agar (Difco *).

The medium is atrazine ^"7 M, 8 χ 10" ⁷ M, and 10 ^"6 M was added in analytical quality, in a concentration of 10" ⁷ M, 3 x 10 ^"7 M, 5 x 10 '.

Seedlings' growth and survival is determined at said concentrations and at atrazine content of zero over a period of 14 days; the results are shown in Table 1, below. Although all control plants and transgenic control plants germinate, but reach their growth with atrazine concentrations of 5 χ 10 ^"7 M or at higher concentrations, only the Kotyledonenstadium. Among the seedlings develop from seed from self-pollination of pCIB 14 plants has emerged, "approximately 75% green ⁷ M and form primary leaves, whereas these seedlings with atrazine concentrations of 8 χ 10" remain at an atrazine concentration of 5 x 10 ⁷ M or higher concentrations only form cotyledons before bleach and die.

Although the tolerant seedlings on atrazine-containing medium do not show the same good growth as on atrazine-free medium, they can still be easily distinguished from the sensitive controls on the same medium.

Atrazine concentrations (M)

Genotype of the seedlings 10 " ⁷ M 3 χ 10" ⁷ M 5 χ 10 " ⁷ M 8 κ 10" ⁷ M 10 " ⁶ M

Control + + 0 0 0

LBA4404 ++ 00 0

Βίη6 + + 0 0 0

pCIB13 ++ 00 0

pCIB 14 + + + 0 0

The table clearly shows the difference in growth and survival between the progeny of transgenic pCIB 14 plants and the controls. Positive growth is indicated by "+", negative growth by a "0".

Although the present invention has been described in detail hereinbefore by way of illustration and example for the purposes of clarity and understanding, it is to be understood that within the scope of this invention certain changes and modifications can be practiced whose limitations are solely dictated by the scope of the appended claims are.

In the context of the present invention, not only those DNA molecules which are specified with specific nucleotide sequence and encode a glutathione S-transferase polypeptide form part of the invention, but likewise their variants or mutants which are suitable for polypeptides with glutathione Encode -S-transferase activity.

Claims (25)

claims: Anspruch [en] A process for producing a recombinant DNA molecule capable of conferring herbicidal tolerance based on the herbicide's detoxication to a plant, characterized by a) isolated using a method known per se, a glutathione S-transferase-encoding DNA sequence from a geeioneten source and b) said coding region on both sides with an odei. several additional DNA sequences operably linked. 2. The method according to claim 1, characterized in that linked by means of methods known per se different DNA sequence sections, which consist of either genomic DNA and / or cDNA, to a complete glutathione S-transferase-encoding DNA sequence. 3. The method according to claim 1, characterized in that the linking of DNA sequence sections using the in vivo recombination of DNA sequences having homologous sections and / or the in vitro linking of restriction fragments takes place. 4. The method according to claim 2, characterized in that said DNA sequence sections are composed both of genomic DNAaIs also cDNA. 5. The method according to claim 2, characterized in that said DNA sequence segments from gene fragments of several organisms belonging to different genera are composed. 6. The method according to claim 2, characterized in that said DNA sequence segments are composed of gene fragments of more than one strain, a variety or a species of the same organism. 7. The method according to claim 2, characterized in that said DNA sequence sections are composed of portions of more than one gene of the same organism. 8. The method according to any one of claims 1 to 7, characterized in that said DNA sequence encoding a glutathione S-transferase polypeptide, is derived from a sucker. 9. The method according to any one of claims 1 to 7, characterized in that said DNA sequence encoding a glutathione S-transferase P ^ lypeptid derived from a rat. 10. Verfiere'i for producing a recombinant l) NA molecule according to claim 9 and its variants or mutants which code for polypeptides with glutathione-S-Transfoijse activity, characterized in that said DNA sequence which is suitable for a glutathione-S The rat transferase polypeptide encodes the following nucleotide sequence: 40 50 60 ATGCCTATGATACTGGGATACTGG MPMILGYW 70 80 90 100 110 120 AACGTCCGCGGGCTGACACACCCGATCCGCCTGCTCCTGGAATACACAGACTCAAGCTAT NVRGLTHPIRLLLEYTDSSY 130 140 150 160 170 180 GAGGAGAAGAGATACGCCATGGGCGACGCTCCCGACTATGACAGAAGCCAGTGGCTGAAT EEKRYAMGDAPDYDRSQWLN 190 200 210 220 230 240 GAGAAGrTCAMCTGGGCCTGGACTTCCCCAATCTGCCCTACTTAATTGATGGATCGCGC EKFKLGLOFPNLPYLIDGSR 250 260 270 280 290 300 AAGATTACCCAGAGCAATGCCATAATGCGCTACCTTGCCCGCAAGCACCACCTGTGTGGA KITQSNAIMRYLARKHHLCG 310 320 330 340 350 360 GAGACAGAGGAGGAGCGGATTCGTGCAGACATTGTGGAGAACCAGGTCATGGACAACCGC ETEEERIRADIVENQVMDNR 370 380 390 400 410 420 ATGCAGCTCATCATGCTTTGTTACAACCCCGACTTTGAGAAGCAGAAGCCAGAGTTCTTG MQLIMLCYNPDFEKQKPEFL 430 440 450 460 470 480 AAG ^ CCATCCCTGAGAAGATGAAGCTCTACTCTGAGTTCCTGGGCAAGCGACCATGGTTT KTIPEKMKLYSEFLGKRPWF 490 500 510 520 530 540 GCAGGGGACAAGGTCACCTATGTGGATTTCCTTGCTTATGACATTCTTGACCAGTACCAC AGDKVTYVDFLAYD ILDQYH 550 560 570 580 590 600 ATTTTTGAGCCCAAGTGCCTGGACGCCTTCCCAAACCTGAAGGACTTCCTGGCCCGCTTC IFEPKCLDAFPNLKDFLARF 610 620 630 640 650 660 · GAGGGCCTGAAGAAGATCTCTGCCTACATGAAGAGCAGCCGCTACCTCTCAACACCTATA EGLKKISAYMKSSR YLSTPI 670 680 690 700 710 720 TTTTCGAAGTTGGCCCAATGGAGTAACAAGTAG
FSKLAQWSNK 11. A method for producing a recombinant DNA molecule according to claim 9 and its variants or mutants which code for polypeptides with glutathione S-transferase activity, characterized in that said DNA sequence encoding a glutathione S-transferase polypeptide of Rat coded, having the following nucleotide sequence: 1 10 13 0 TAC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG Tyr Ser Met GIy Asp Ala Pro Asp Tyt Asp Arg Ser Gin 150 1 70 TGG CTG AGT GAG AAG TTC AAA CTG GGC CTG GAC TTC CCC AAT CTG Trp Leu Ser GIu Lys Phe Lys Lou GIy Leu Asp Phe Pro Asn Leu 19 0 210 CCC TAC TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC Pro Tyr Leu Ue Aep Gly Ser His Lys He Thr Gin S ^ r Asn AIa 2 30 25 0 270 ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGT GGG GAG ACA He Leu Arg Tyr Leu GIy Arg Lys His Αβη Leu Cye GIy GIu Thr 2 90 31 0 GAG GAG GAG AGG ATT CGT GTG GAC GTT TTG GAG AAC CAu GCT ATG GIu GIu GIu Arg He Arg VaI Asp VaI Leu GIu Asn Gin AIa Met 330 3 50 GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT Asp Thr Arg Leu GIn Leu AIa Met VaI Cys Tyr Ser Pro Asp Phe 37 O 390 GAG AGA AAG AAG CCA GAG TAC TTA GAG GGT CTC CCT GAG AAG ATG GIu Arg Lys Lys Pro GIu Tyr Leu GIu GIy Leu Pro Giy Lys Met 4 10 43 O 450 AAG CTT TAC TCC GAA TTC CTG GGC AAG CAG CCA ^T GG TTT GCA GGG Lys Leu Tyr Ser GIu Phe Leu GIy Lys GIn Pro Trp Phe Ala GIy 4 70 49 O AAC AAG ATT ACG TAT GTG GAT TTT CTT GTT TAC GAT GTC CTT GAT Asn Lys He Thr Tyr VaI Asp Phe Leu VaI Tyr Asp VaI Leu Asp 510 5 30 CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC Gin His Arg He Phe GIu Pro Lys Cys Leu Asp Ala Phe Pro Asn 55 O 570 CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Leu Lys Asp Phe VaI AIa Arg Phe GIu GIy Leu Lys Lys He Ser 5 90 61 O 630 GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA Asp Tyr Met Lys Ser Gly Arg Phe Leu Ser Lys Pro lie Phe AIa 6 50 AAG ATG GCC TTT JGG AAC CCA AAG DAY
Lys Meta Phe Trp Aan Pro Lys End 12. A method for producing a recombinant DNA molecule according to claim 9 and its variants or mutants, di? for polypeptides with glutathione S-transferase activity, characterized in that said DNA sequence which codes for a rat glutathione S-transferase polypeptide having the following nucleotide sequence: 10 30 ATG CCT ATG ACA CTG GGT TAC TGG GAC ATC CGT GGG CTG GCT CAC Met Pro Met Thr Leu GIy Tyr Trp Asp He Arg Gly Leu Ala His 50 7 0 90 GCC ATT CGC CTG TTC CTG GAG ACT ACA GAC ACA AGC ACT GAG GAC Ala He Arg Leu Phe Leu GIu Tyr Thr Asp Thr Ser Tyr GIu Asp 1 10 13 0 AAG AAG TAC AGC ATG GGG GAT GCT CCC GAC TAT GAC AGA AGC CAG Lya Lys Tyr Ser Mat GIy Asp Ala Pro Asp Tyr Asp Arg Ser Gin 150 1 70 TGG CTG AGT GAG AAG TTC AAA CTG GGC CTG GAC TTC CCC AAT CTG Trp Leu Ser Glu Lys Phe Lys Leu Gily Leu Asp Phe Pro Asn Leu 19 0 210 CCC TAC TTA ATT GAT GGG TCA CAC AAG ATC ACC CAG AGC AAT GCC Pro Tyr Lou lie Asp Gly Ser His Lys He Thr GIn Ser Asn AIa 2 30 25 0 270 ATC CTG CGC TAC CTT GGC CGG AAG CAC AAC CTT TGT GGG GAG ACA He Leu Arg Tyr Leu GIy Arg Lys His Asn Leu Cys GIy CIu Thr 2 90 31 0 GAG GAG GAG AGG ATT CGT GTG GAC GTT TTG GAG AAC CAG GCT ATG GIu GIu GIu Arg He Arg VaI Asp VaI Leu GIu Asn Gin AIa Met 330 3 50 GAC ACC CGC CTA CAG TTG GCC ATG GTC TGC TAC AGC CCT GAC TTT Asp Thr Arg Leu GIn Leu AIa Met VaI Cys Tyr Ser Pro Asp Phe 37 ü 390 GAG AGA AAG AAG CCA GAG TAC TTA GAG GGT CTC CCT GAG AAb ATG GIu Arg Lys Lys Pro GIu Tyr Leu GIu GIy Leu Pro GIu Lys Met 4 10 43 O 450 AAG CTT TAC TCC BAA TTC CTG GGC AAG CAG CCA TGG TTT GCA GGG Lys Leu Tyr Ser GIu Phe Leu GIy Lys GIn Pro Trp Phe Ala GIy 4 70 49 O AAO AAG ATT ACG TAT GTG GAT TTT CTT GTT TAC GAT GTC CTT GAT Asn Lys He Thr Tyr VaI Asp Phe Leu VaI Tyr Asp VaI Leu Asp 510 5 30 CAA CAC CGT ATA TTT GAA CCC AAG TGC CTG GAC GCC TTC CCA AAC Gin His Arg He Phe GIu Pro Lys Cys Leu Asp Ala Phe Pro Asn 55 O 570 CTG AAG GAC TTC GTG GCT CGG TTT GAG GGC CTG AAG AAG ATA TCT Leu Lys Asp Phe VaI AIa Arg Phe GIu GIy Leu Lys Lys He Ser 5 90 61 O 630 GAC TAC ATG AAG AGC GGC CGC TTC CTC TCC AAG CCA ATC TTT GCA Aep Tyr Met Lys Ser GIy Arg Phe Leu Ser Lys Pro Ils "he AIa 6 50 AAG ATG GCC TTT TGG AAC CCA AAG DAY
Lys Met AI Phe Trp Asn Pro Lys End 13. A process for the preparation of a recombinant DNA molecule according to claim 9 and its variants or mutants which code for polypeptides with glutathione-S-transferase activity, characterized in that said DNA sequence encoding a glutathione S-transferase polypeptide of Rat coded, having the following nucleotide sequence: 10 30 A GAC CCC AGC ACC ATG CCC ATG ACA CTG GGT TAC TGG GAC ATC Met Pro Met Thr Lei GIy Tyr Trp Aap He 5 0 70 CGT GGG CTA GCG CAT GCC ATC CGC CTG CTC CTG GAA TAC ACA GAC Arg Glu Leu Ala His Ala lie Arg Leu Leu Leu Glu Tyr Thr Asp 90 11 0 130 TCG AGC TAT GAG GAG AAG AGA TAC ACC ATG GGA GAC GCT CCC GAC Ser Ser Tyr GIu GIu Lys Arg Tyr Thr Met GIy Asp Ala Pro Asp 1 50 17 0 TTT GAC AGA AGC CAG TGG CTG AAT GAG AAG TTC AAA CTG GGC CTG Phe A3p Arg Ser Gin Trp Leu Asn Giu Lys Phe Lys Leu Gily Leu 190 2 10 GAC TTC CCC AAT CTG CCC TAC TTA ATT GAT GGA TCA CAC AAG ATC Asp Phe Pro Asn Leu Pro Tyr Leu Lai Asp GIy Ser His Lys He 23 0 250 2 ACC CAG AGC AAT GCC ATC CTG CGC TAT CTT GGC CGC AAG CiAC AAC Thr GIn Ser Asn Ala He Leu Arg Tyr Leu GIy Arg Lys His Asn 70 29 0 310 CTG TGT GGG GAG ACA GAA GAG GAG AGG ATT CGT GTG GAC ATT CTG Leu Cys GIy Glu Thr GIu GIu GIu Arg He Arg VaI Asp He Leu 10 50 107 0 CAC CGT AGC CAA GCC CCT CTT CTT GCT GTT TCC CGA GAC TAG CTA His Arg Ser Gin AIa Pro Leu Leu Ala VaI Ser Arg Asp End Leu 1 090 11 10 TGA GCA AGG TGT GCT GTG TCC CCA GCA CTT GTC ACT GCC TCT GTA End Ala Arg Cys Ala VaI Ser Pro Ala Leu VaI Thr Ala Ser VaI 113 0 1 150 11 ACC CGC TCC TAC CGC TCT TTC TTC CTG CTG CTG TGA GCT GTA CCT Thr Arg Ser Tyr Arg Ser Phe Phe Leu Leu Leu End Ala VaI Pro 70 119 0 1 210 CCT GAC CAC AAA CCA GAA TAA ATC ATT CTC CCC TTA AAA AAA AAA Pro Asp His Lys Pro Glu End He He Leu Pro Leu Lys Lye Lys AAA AAA AAA A
Lys Lys Lys 14. The method according to any one of claims ι to 13, characterized in that said DNA sequence encoding a glutathione S-transferase polypeptide is operably linked to a plant promoter and / or a terminator region and in the plant cell is expressed. 15. The method according to claim 14, characterized in that said DNA sequence is of heterologous origin with respect to the plant promoter. 16. The method according to claim 14, characterized in that it is the cells of cells of tobacco, soybeans or cotton in the plant cells. 17. The method according to claim 14, characterized in that the plant cells are parts of a whole plant. 18. Process according to claim 14, characterized in that the plant promoter is a promoter selected from the group of nos, ocs and CaMV promoters. 19. The method according to claim 14, characterized in that said plant promoter is the promoter of the small subunit of the ribulose ois-phosphate carboxylase of soybean or the promoter of the chlorophyll-c, b-binding proteins. 20. The method according to claim 1, d ldurch characterized in that said herbicides are a chloroacotamide, a Sulfonyiharnsioff, a triazine, a dipnenyl ether, an imidazolinone or a thiocarbamate. 21. The method according to claim 1, characterized in that said recombinant DNA molecule in addition to the glutathione S-transferase polypeptide-encoding DNA sequence contains further sequence sections which makes said recombinant DNA molecule suitable for use as a transfer vector , 22. The method according to claim 1, characterized in that said recombinant DNA molecule in addition to the DNA sequence coding for a glutathione S-transferase polypeptide contains further sequence sections which make said recombinant DNA molecule suitable for use as an expression vector , 23. The method according to any one of claims 21 or 22, characterized in that said, a glutathione S-transferase-encoding DNA sequence is operably linked to an active promoter in plant cells and with other replication and control sequences of species which are compatible with the host cell and which are essential for the specific function of the expression and / or transvectors. 24. The method according to any one of claims 21 and 22, characterized in that said host cell is a microorganism. 25. The method according to any one of claims 21 and 22, characterized in that it is said to be a plant cell in said host cell.