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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
  • C12N15/8205—Agrobacterium mediated transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression

Definitions

• This invention relates to the integration of heterologous genes into the genome of plant cells.
• heterologous gene we mean a gene which is a non-plant gene, a modified gene, a synthetic gene, a gene from a different plant strain or species, or a homologous gene from a different location in the plant's genome.
• T-DNA tumor formation
• genes for the synthesis of opines e.g., nopaline and octopine, which are produced by plant tissue containing integrated Ti-derived DNA.
• Ti plasmids are classified by the type of opine synthase they carry; nopaline synthase (NOS) or octopine synthase (OS) .
• NOS nopaline synthase
• OS octopine synthase
• Short sequences flanking the T-DNA left and right border regions are important for T-DNA integration. Lawton et al. Hort. Sci. 19:10-12 (1984) discuss Ti plasmids as potential vectors for genetic engineering. An exogenous gene and selectable marker were integrated into plant cells using a recombinant Ti plasmid. No evidence of normal transcription of the introduced DNA was found.
• Zambryski et al. EMBO 2:2143-2150 (1983) report the construction of a recombinant Ti plasmid, the tumorigenic portion of which had been deleted. Plant cells transformed with this vector grew normally and expressed NOS, which was left intact on the plasmid as a transformation marker. Genes, for tumor induction, were subsequently inserted into the recombinant Ti plasmid, and plant cells transformed with this plasmid expressed both NOS and the tumor phenotype.
• Herrera-Estrella et al. EMBO 2:987-995 (1983) report the construction of a recombinant Ti plasmid containing an antibiotic resistance marker attached to T-DNA regulatory elements. Progeny cells from tobacco protoplast transfectants expressed antibiotic resistance, but yielded only tumor callus.
• the invention allows for the development of modified plant varieties in much shorter time periods than is possible using traditional methods.
• the invention greatly broadens the gene pool from which useful genes can be selected, to include not only genes from other plants, but also genes from fungi, bacteria, and animals, as well as hybrid and synthetic genes.
• the invention features, in one aspect, a vector capable of being integrated into the -chromosome of a plant protoplast cell; the vector includes 1) a DNA region encoding a selectable marker protein, transcription of this DNA region being under the control of a regulatory DNA sequence capable of effecting transcription in plant cells; 2) a polyadenylation site; 3) a desired heterologous gene, or a site for the insertion thereof in the vector; and 4) DNA capable of causing the integration of (1) , (2) , and (3) into a chromosome of the plant cell, the vector lacking sufficient Ti-derived DNA to cause tumor formation in the plant or to cause the production of opines in the plant.
• the invention features a vector having (l)-(4), above, and in addition includes DNA sufficient to enable the vector to replicate in A. tumefaciens and to transform a plant cell when A. tumefaciens containing the vector is cocultivated with the plant cell; this vector also lacks sufficient tumorigenic DNA to cause tumor formation in the plant.
• the regulatory DNA is derived from a Ti plasmid encoding nopaline synthase or octopine synthase, in which case the vector is incapable of effecting the expression of nopaline or octopine, and the Ti border DNA is derived from a plasmid containing a gene encoding nopaline synthase or octopine synthase.
• the vectors of the invention can be used to effect the normal expression in plants of desired proteins, with selection of transformants facilitated by a selectable marker protein, also expressed normally. Normal expression of the marker protein is effected by the regulatory sequence, preferably T-DNA-derived, and the polyadenylation site. Tumor formation is prevented by the exclusion of sufficient T-DNA to cause tumor formation. Furthermore, metabolic energy of the
• Th vector of the invention can be used in any suitable plant species.
• suitable plants are dicotyledenous plants, e.g., tobacco, tomato, and petunia plants, in which Ti plasmids are known in nature to cause tumor formation.
• Other suitable plants include monocotyledenous plants capable of being transformed by Ti plasmids.
• Fig. 1 is a diagrammatic representation of two vectors of the invention, containing border regions derrived from a NOS-encoding Ti plasmid.
• Figs. 2-12 are diagramatic representations of intermediate constructions and steps leading to the vectors of Fig. 1.
• Fig. 13 is . a diagrammatic representation of a region of one of the vectors of claim 1,. and. Fig. 14 is a diagrammatic representation of the other.
• Fig. 15 is a diagrammatic representation of two vectors of the invention, containing border regions derived from an OS-encoding Ti plasmid.
• Figs. 16-23 are diagrammatic representations of intermediate constructions and steps leading to the vectors of Fig. 15.
• Fig. 24 is a digrammatic representation of octopine TL border-containing DNA.
• Fig. 25 is a diagrammatic representation of the synthetic 25 bp repeat sequence of the octopine TL right border.
• Figs. 26-33 are diagrammatic representations of intermediate constructions and steps leading to the vectors of Fig. 33.
• Fig. 34 is a diagrammatic representation of vectors containing a single synthetic border sequence. Plasmid Components
• the plasmids of the invention contain several essential DNA regions and sites, now discussed in greater detail.
• plasmids of the invention must contain a DNA region which encodes a selectable marker protein for identification of transformants.
• This marker protein can be any protein which can be expressed in plant cells and which enables the phenotypic identification of plant cells which express the protein.
• Preferred marker proteins are proteins which provide resistance to one or more antibiotics; currently most preferred is the protein aminogly ⁇ oside p osphotransferase, which • inactivates antibiotics such as kana ycin, neomycin, and G418; transformants are those plant cells able to grow in the presence of antibiotic.
• chloramphenicol acetyl transferase which provides resistance to chloramphenicol
• dihydrofolate reductase which provides resistance to methotrexate.
• Certain genes conferring herbicide resistance can also be used as selectable markers.
• Eukaryotic (e.g., plant) messenger RNA's must be polyadenylated for efficient translation and processing. Polyadenylation requires a recognition site for polyadenylation enzymes near the 3' end of the DNA region encoding the selectable marker.
• DNA Sequences for Integration DNA derived from or substantially identical to a border region of T-DNA is used to effect the integration of the plasmids of the invention into the chromosomes of host plant cells. Sequencing of several T-DNA border regions has revealed that each contains a direct repeat sequence 25 base pairs in length. One such sequence is sufficient to cause integration, and the vectors of the invention can include one or two such sequences, which may or may not be flanked by additional T-DNA border sequences. Regulatory Sequences
• Transcription of both the selectable marker gene and the desired heterologous gene, in order to lead to efficient expression, is preferably under the control of regulatory sequences normally expressed in plant cells, e.g., T-DNA promoters such as those for the NOS or OS genes.
• T-DNA promoters such as those for the NOS or OS genes.
• suitable promoter sequences include those of the ribulose bisphosphate carboxylase small subunit gene, the nitrate reductase gene, and the glutamine synthase gene.
• Synthetic, engineered, or altered natural promoters can also be used. In some instances, it will be desirable to use promoters which are regulated, e.g., promoters active only at certain times in the plant's development.
• the plasmid of the invention contain less than the entire structural gene normally under the control of the each promoter used, to ensure that metabolic energy of the transformed plant is not wasted producing protein encoded by the structural gene.
• Site for Desired Heterologous Gene The site for the insertion of a desired heterologous gene can be any site at which an endonuclease can act to cut the plasmid for insertion of the desired heterologous gene. Preferably the site is unique in the plasmid, so that the endonuclease cuts the plasmid only in the desired location. Desired Heterologous Gene
• the desired heterologous gene can be any gene which is capable of being expressed in the plant, and
• genes 5 which encodes a protein which enhances a beneficial feature of the plant, or provides a new beneficial feature.
• genes are those encoding resistance to herbicides, resistance to diseases such as tomato fusarium wilt, proteins which can be produced to
• one heterologous- gene can be introduced, and the expression of such multiple genes controlled in a coordinate manner so as to introduce more complex biochemical functions into plants.
• Examples include multi-enzyme pathways, e.g. energy-generating reactions
• transcription of the desired heterologous gene is preferably under the control of a regulatory sequence normally expressed in plant
• the fusion of the heterologous gene to the regulatory sequence can be carried out prior to the insertion of the heterologous gene into the vector, using conventional techniques.
• the gene can be inserted into the vector by itself, and the 30 regulatory sequence inserted upstream from the gene separately, prior to or following the insertion of the gene, using conventional methods.
• plasmids pJP25 and pJP46 contain the aminoglycoside phosphotransferase gene from Tn5, encoding resistance to the antibiotics kanamycin and G418 (the kan r gene) , under the transcriptional control of the NOS promoter; the nopaline T-DNA left and right border regions; a polyadenylation site; and a unique Bgl II site for insertion of a heterologous gene.
• These plasmids were constructed as follows (pJP25 and pJP46 differ only in that pJP25 contains a small amount of the NOS structural gene) .
• the first step was to insert the left and right T-DNA border regions from a NOS-encoding Ti plasmid into E. coli plasmid pBR327.
• nopaline T-DNA including the border regions, is present on the Ti plasmid carried by A-_ tumefaciens strain C58.
• piasmid pGV0369 (Fig. 3) bears the left nopaline T-DNA border
• the Hind III 23 fragment (Fig. 2) of plasmid pGV0329 bears the nopaline T-DNA right border and the NOS structural gene.
• Hind III fragment 23 was purified from a Hind ⁇ i digest of pGV0329 and subcloned into pBR327. As shown in Fig. 4, two plasmids were derived from this construction: pJPS18 carries the NOS polyadenylation site, and pJPL19 carries the nopaline T-DNA right border region and the NOS promoter. Plasmid ⁇ JPL19 (Fig. 5) was cut at the Nco I site and treated with Bal 31 to remove about 400 bp from each end. The DNA was then cut with Ba H I, the ends filled in with DNA polymerase, a Sma I linker (CCCGGG) added, and the plasmid recircularized.
• CCCGGG Sma I linker
• Plasmid pE61-8 (Fig. 6) was one of the plasmids obtained. pE61-8 contains the nopaline T-DNA right border region and the NOS promoter.
• the left border region was then inserted by cloning the Bgl II-Pst I fragment from pGV0369 into the BamH I site of pE61-8 to yield ⁇ E118 (Fig. 7), which contains the nopaline T-DNA right and left borders and NOS promoter, but still lacks the polyadenylation site and the kan r gene.
• plasmid pVW90 (Fig. 8) , carrying the kan gene, was constructed as follows. First, the
• Hind Ill-Sal I fragment from Tn5 was cloned in pBR327 to yield pVW60, as shown in Fig. 9.
• pVW60 there is an ATG upstream from the SD sequence of the kan r gene which could result in an incorrect translation start of the RNA of this gene in a eukaryotic cell.
• pVW60 was cut at Bgl II and treated with Bal 31 to remove DNA up to the SD sequence.
• a Sal I linker was inserted and the plasmid religated. Clones resistant and sensitive to kanamycin were obtained. - A number of these were sequenced to determine the position of the Sal I linker. The resulting clones, and the portions of the pre-kan DNA they contain, are shown in Fig. 10.
• pVW61, 62, 63, 64, 65, 70 were all kanamycin resistant.
• pVW80, 81, 82, 83, 84, 86, and 87 were all kanamycin sensitive.
• pVW61, 63, and 65 still have the extra ATG so are not useful.
• pVW62, 64, 70 have lost the extra ATG and have an SD sequence intact enough to give expression.
• pVW81, 87, 82, 80, 83, 86, and 84 have lost the extra ATG and the SD sequence
• pVW81, 87, and 82 have an intact kan r gene
• pVW80, 83, 86 and 84 have lost - li ⁇ the first 7 codons of kan r and can be used to make translational fusions with a gene expressed in plant cells.
• pVW86 was combined with the NOS polyadenylation site to form pVW90 (Fig. 8) , as follows.
• Plasmid pJPS18 (Fig. 11) , discussed above, carries the 3' end of the NOS gene and the NOS polyadenylation recognition site.
• the Xma I fragment was cut out of pVW77 and cloned into the Xma I (Sma I) site at the 3' end of the kan r gene of pVW86, to yield pVW90 (Fig. 8). (The pVW77 Xma I fragment was also cloned in pVW82, 84, and 87.)
• Plasmid pVW90 was then combined with pEH8 (Fig. 7, discussed above), as follows. pEll ⁇ was cut at the Sma I site and treated with Bal 31 to remove about 25-30 bp. The linearized plasmid was then blunt-end ligated with the Sal I fragment from pVW90 carrying the kan r gene and polyadenylation site, to yield constructions having the structure shown in Fig. 12. The fusion junction of several constructions was sequenced to obtain clones in which the kan gene was in frame with the ATG of the NOS gene. Two clones were obtained having in-frame gene fusions: pJP125 (Figs. 1 and 13) and pJP46 (Figs. 1 and 14).
• Plasmid pSUP104 is described in Simon et al. (1983) "Vector Plasmids for _in vivo and in vitro Manipulations of Gram-Negative Bacteria" in Molecular Genetics of the Bacterial Plant Interaction (Puhler, ed. , Berlin 1983) , and is available from Agrigenetics Corporation. Plasmid Construction; Octopine T-DNA Borders
• plasmids pVW125 and pVWl26 contain the right and left border regions of the right T-DNA ("TR DNA") of the octopine T-DNA (the octopine T-DNA also includes left T-DNA, or "TL DNA”) ; r the kan gene, under the transcriptional control of the NOS promoter; a polyadenylation site, and a unique
• the first step was to insert the left and right TR border regions into pB.R327.
• octopine TR DNA is present in A_ ⁇ tumefaciens strain B6.
• BamH I fragment 2 (16.5 kb) from the B6 Ti plasmid wa.s cloned in pBR327 to yield pVW51 (Fig. 17).
• the TR left border on pVW51 in an Eco RI-BamH I fragment, was cloned in pBR327 to yield pVW58 (Fig. 17).
• a unique restriction site had to be created between the two borders.
• the Eco RI site between the left and right borders was converted to a Sma I site by a partial Eco RI digest, the sticky ends were filled in, and a Sma I linker was then inserted into one or the other Eco RI site, to yield pVW78 (Fig. 19) , which bears a unique Sma I site between the borders, and pVW79 (Fig. 20) , which bears a unique EcoRI site between the borders.
• a plasmid was constructed in which the kan gene was under the transc ⁇ ptional control of the NOS promoter. This was carried out generally as described above, in the construction of plasmids containing nopaline T-DNA border regions, although the resulting vectors differed slightly in structure from those constructed with the nopaline T-DNA borders.
• the first intermediate, containing the NOS promoter is plasmid pE85-21 (Fig. 21) , in which the Sma I site is 2 bp upstream from the NOS ATG.
• the kan r gene-containing intermediate pVW93 (not shown) has a Sal I linker inserted 2 bp upstream from the ATG of the kan r gene.
• pE85-21 was cut with Sma I and treated with Bal 31 to remove a. few (5-10) bases.
• the Sal I fragment from pVW93 carrying the kan gene and the polyadenylation site was blunt end ligated into pE85-21 to yield pVWl04 (Fig. 22).
• the Bel I-Bgl II fragment from pVWl04 carries the NOS promoter, the kan gene, and the polyadenylation site. This fragment was purified and cloned into the Sma I site of pVW78 between the two TR borders, to yield pVW121 (Fig. 23).
• Plasmid pVWl21 carries the right TL border, which was deleted by cutting with Xho I and BamH I and religating. This yielded plasmids pVWl25 and pVWl26, which bear the kan r gene oriented in different directions. Ba HI linkers were ligated to the EcoRI-XhoI fragment of pVW125 and it was cloned into the BamHI site of plasmid pSUPl04 to yield plasmid pVWl30 (Fig...15) . Plasmid Construction; Synthetic borders
• T-DNA border regions have been sequenced. i.e., borders of T-DNA (from a nopaline-producing Ti plasmid) and TL and TR DNA borders (from an octopine-producing Ti plasmid) . All have a similar direct repeat of 25 base pairs at the border regions. The sequences of the borders derived from different T-DNA are not identical for all 25 base pairs.
• the 25 base pair direct repeat of the octopine right TL border was synthesized, using conventional techniques, and is represented in Fig. 25.
• This synthesized DNA was cloned into the E. coli vector pUC8 (Fig. 26) to make it more easily accessible for further cloning.
• Sal I linkers were ligated on the ends of the synthetic DNA and it was cloned into the Sal I site of pUC8 to yield plasmid pVW132 (Fig. 27) .
• Sma I linkers were also ligated on the synthetic .DNA so that it could be cloned into the Sma I site of pUC8, yielding plasmids PVW133 (Fig. 28) and ⁇ VWl34 (Fig. 29).
• the orientation of the border fragments relative to each other in the 3 plasmids is indicated by the arrow.
• the orientations of the border sequences in Ti plasmids are . indicated in Figure 30, A. and B.
• the single vector was constructed such that the relative orientations of the synthetic border sequences was the same as the orientation of the border sequences in the naturally occurring Ti plasmid.
• the Hind III-BamH I fragment from pVW134 Fig. 29
• Fig. 27 containing the synthetic border sequence in the correct orientation
• was cloned into the Hind III-BamH I site of pVWl32 (Fig. 27) yielding plasmid pVWl36 (Fig. 31) .
• a selectable marker was cloned into the BamH I site between the synthetic border sequences.
• the Bel I-Bgl II fragment of plasmid pVWl04 (Fig. 32) , containing the kanamycin resistance gene and polyadenylation site attached to the nopaline synthase promoter, was cloned into the BamH I site of plasmid pVW136 (Fig. 31) to yield plasmid pVWl43 (Fig. 33A) and plasmid pVW142 (Fig. 33B) .
• the two plasmids obtained differ only in the orientation of the Kan r gene and its regulatory elements relative to the synthetic border fragments.
• BamHI linkers were ligated to the Hind III-Eco RI fragments of ⁇ VW143 and pVW142 (Fig 33 A-B) which were cloned into the BamHI site of plasmid pSUP104 to yield, respectively, plasmids pVWl49 and pVW148, for mobilization to Agrobacterium tumefaciens strain LBA4404 for cocultivation experiments with plant cells.
• plasmids In addition to the above-described plasmids (pVW142 and pVW143) containing two synthetic border sequences flanking the kanamycin resistance gene, plasmids have been constructed which contain only one such synthetic sequence; this single synthetic sequence is capable of effecting integration of the plasmid DNA into the host plant chromosome.
• One such plasmid, pVWl44 (Fig. 34), was constructed as follows. The Bcll-Bglll fragment of plasmid pVW104 (Fig. 32), containing the kan r gerie and polyadenylation site attached to the nopaline synthase promoter, was cloned into the BamHI site of plasmid pVWl33 (Fig.
• Plasmid pVWl45 (not shown) was derived from plasmids pVW104 (Fig. 32) and pVW134 (Fig. 29) in analogous fashion, and it differs from pVWl44 only in the orientation of the single border sequence relative to the kanamycin resistance gene. Plasmid Construction; Insertion of Heterologous Genes
• Plasmids pJP25, pJP46, pVW125, and pVW126 all have a unique Bgl II site into which a desired heterologous gene can be inserted, using conventional techniques.
• the plasmids of the invention can be used to directly transform plant protoplasts, e.g., by packaging the plasmids in liposomes, using conventional techniques.
• the plasmids can be used to transform plant protoplasts or non-protoplast cells, using a "binary" cocultivation technique with A ⁇ tumefaciens.
• the pSUP104-derived plasmids (pJP60, pVW144, pVW130, pVWl48, and pVW145) were transferred to an A ⁇ _ tumefaciens strain, e.g., LBA4404, which carries a Ti plasmid, pLBA4404, deleted of the T-DNA region, so that cocultivation does not result in plant tumor formation.
• LBA4404 binary cocultivation, are described in Hoekema et al.
• pLBA4404 does, however, retain the native Ti "vir” (for virulence) functions which are essential for the transfer of the hybrid plasmid of the invention from A. tumefaciens LBA4404 to the host plant cell.
• the A ⁇ tumefaciens containing the hybrid plasmid was then cocultivated with plant cells to insert the hybrid plasmid into the plant cells, where the plasmid DNA, by virtue of the natural or synthetic border DNA, integrated into the plant cell chromosome.
• Transformants were selected by means of the selectable marker.
• Another plant transformation method known as the "quick-dip” method, involves the dipping of plant explants in the A. tumefaciens bacterial culture and then incubating the infected plant parts on medium that selects for kanamycin resistance and promotes shoot regeneration; the method is described in Horsch et al. (1985) Science 227_, 1229.
• Plant Regeneration Following selection of transformants, the plant cells (protoplasts or other cells) are cultured under conditions effecting the regeneration of mature plants. Such methods are known, e.g., for the regeneration of tobacco plants from callus culture.
• the resulting mature plant the cells of which contain integrated DNA of the vector of the invention, express the desired heterologous gene, fail to express the OS or NOS genes, and do not form tumors.
• Plasmids pJP46 and pVW125 have been deposited in the American Type Culture Collection, Rockville, MD, and given ATCC Accession Nos. , respectively, of 39930 and 39929. Applicants' assignee, Biotechnica
• the OS TL border regions (contained in A ⁇ _ tumefaciens strain ACH5) , rather than the TR border regions, can be used, and the OS rather than NOS promoter used.
• a plasmid, pOTY8 (described in Hirsch et al. (1984) Mol Gen. Genet. 195, 209; Fig. 24) contains the TL border regions.
• pOTY8 contains the left TL border (pVW57) and right TL border (pVW59) have been made.
• pVW57 contains Hind III fragment 18 carrying the left border of TL cloned in pBR327
• pVW59 contains EcoRI fragment 24 carrying the right border of TL and the promoter of the OS gene cloned in pACYC184. Any of these border regions can be used to construct vectors in the same manner as described above.

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