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  • 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)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

• the present invention relates to a plant promoter.
• the major structural components of the plant cell wall in terms of quantity are polysaccharides, including cellulose, hemicellulose and pectin polymers which in certain specialised cell types, e.g. vascular/epidermal cells, are complemented by further components whose presence is adapted to the functional role of the cell.
• the plant cell wall of dicotyledonous plants has also been shown to contain two types of structural protein, the hydroxyproline-rich glycoproteins (HGRPs) and more recently a glycine-rich protein (GRPs) class.
• HGRPs termed extensions have a characteristic S-P 4 repeat structure, the proline residues being hydroxylated and subsequently glycosylated.
• proline-rich proteins have now been isolated from dicotyledonous plants. Many of these have been shown to have cell wall associations although the functions remain as yet undefined. It is likely that in different cell types and in different species the PRPs present will vary.
• the present invention provides a promoter having the nucleotide sequence: (a) from -1369 to -49 upstream of the PRP140 gene, or
• the invention also provides a DNA fragment comprising such a promoter operably linked to a heterologous gene encoding a protein.
• a vector which comprises a heterologous gene encoding a protein under the. control of a promoter as above such that the gene is capable of being expressed in a plant cell transformed with the vector.
• a suitable vector is one in which the promoter is fused directly to the 5•-end of the gene.
• the vector may further contain a region which enables the gene and the promoter to be transferred to and stably integrated in a plant cell genome.
• the vector is generally a plasmid.
• Plant cells can be transformed with such a vector.
• the invention therefore further provides plant cells which harbour a promoter as above operably linked to a heterologous gene encoding a protein.
• Transgenic plants may be regenerated from such plant cells.
• a transgenic plant can be obtained which harbours in its cells a promoter as above operably linked to a heterologous gene encoding a protein. Seed may be obtained from the transgenic plants.
• the invention further provides a method of producing a desired protein in a plant cell, which method comprises:
• the invention additionally provides a method of producing a transgenic plant capable of producing a desired protein, which method comprises:
• the desired proteins can be isolated from the transformed plant cells obtained by the first method and from the plants obtained by the second method.
• the present promoter is composed of the sequence upstream of the wheat PRP140 gene from base -1369 to base -49 or of the wheat PRP378 gene from base -2316 to base -12, base 1 being A of the ATG translational start codon for PRP.
• the sequence of the PRP140 promoter can be deduced from Figure 6.
• the sequence of the PRP378 promoter can be deduced from Figure 5.
• the promoter may be obtained by preparing a genomic library of wheat DNA, screening the library for the PRP140 or PRP378 gene and digesting the sequence upstream of the wheat PRP140 or- PRP378 gene with appropriate restriction enzymes and/or exonucleases. There is a NspBII restriction site at base -49 and a Xbal restriction site at base -1369 of the upstream sequence of the PRP140 gene. There is a
• plasmid vectors which contain an upstream sequence of the wheat PRP140 or PRP378 gene comprising the promoter sequence. These vectors include pIPKH-12/2.3, pXNot/1.45, pBIXN/1.3 and pHN/2.4.
• ___ coli MC 1022 harbouring these vectors were deposited at the National Collection of Industrial and Marine Bacteria, Aberdeen, GB on 9 November 1989 under accession numbers NCIMB 40223, NCIMB 40224, NCIMB 40225 and NCIMB 40226 respectively.
• pBIXN/1.3 was deposited under the designation pIPXN/1.3.
• the PRP140 promoter may be released from pXNot/1.45 by digesting the plasmid with Xbal and NspBII.
• the PRP378 promoter may be released from pHN/2.4 by linearisation with Notl-BstXI, incubation with a nuclease, religation of the plasmid population, selecting a plasmid with a deletion at its 3'-end to base-12 of the PRP 378 gene and digesting the plasmid with Hindlll.
• the promoter sequence may be modified by one or more base substitutions, insertions and/or deletions and/or by an extension at either or both ends. However, the modified promoter sequence must still be capable of acting as a promoter. Sequences from base -769 to base -49 of the upstream sequence of the wheat PRP140 gene, and shorter, have been found not to be sufficient to direct expression of a protein at satisfactory levels. On the other hand, the sequence from base -961 to base -49 upstream of the wheat PRP140 gene does direct protein expression at a satisfactory level.
• a degree of homology of at least 60% between a modified sequence and the unmodified natural sequence from base -1369 to base -49 upstream of the wheat PRP140 gene or from base -2316 to -12 upstream of the wheat PRP378 gene.
• the degree of homology may be at least 75%, at least 85% or at least 95%.
• a longer sequence may be provided which extends upstream of base -1369 of the PRP140 gene or of base -2316 of the PRP378 gene, for example to another restriction site.
• An extension upstream typically comprises the natural nucleotide sequence upstream of base -1369 of the PRP140 gene or of base -2316 of the PRP378 gene. Such a longer sequence may be obtained from a genomic library of wheat DNA as above.
• the upstream sequence if the wheat PRP 140 gene from base -961 to base -49 may also be modified by one or more base substitutions, insertions and/or deletions and/or by an extension at either or both ends. Again, such a modified sequence must be capable of acting as a promoter. There may be a degree of homology of at least 60%, for example at least 75%, at least 85% or at least 95%, between the unmodified and modified longer sequences.
• a modified promoter sequence may be obtained by introducing changes into the natural promoter sequence. This may be achieved by any appropriate technique, including restriction of the natural sequence with an endonuclease, insertion of linkers, use of an exonuclease and/or a polymerase and site-directed mutagenesis.
• a shorter DNA sequence therefore may be obtained by removing nucleotides from the 5'-terminus or the 3•-terminus of the natural promoter sequence, for example using an exonuclease such as exonuclease III or BAL 31.
• an exonuclease such as exonuclease III or BAL 31.
• the modified sequence is placed upstream of a protein coding sequence, such as the bacterial reporter gene / 5-glucuronidase as in the Example. Tobacco leaf discs can then be transformed. The protein expressed by the transformed cells are then assayed, in the case of ,9-glucuronidase as described in Example 1.
• the promoter may be operably linked to a heterologous gene encoding a protein.
• the heterologous gene may encode any protein it is desired to express.
• heterologous is that the gene is not naturally operably linked to the promoter.
• the gene does not therefore encode PRP140 in the case where the promoter is derived from the upstream sequence of the PRP140 gene or PRP378 in the case where the promoter is derived from the upstream sequence of the PRP378 gene.
• the protein may additionally comprise a transit peptide sequence at its N-terminus, encoded within the heterologous gene sequence.
• the promoter is typically used to control the expression of genes in plant tissues.
• the protein whose expression is controlled by the promoter may be a protein encoded by a herbicide-resistance gene or a protein conferring biological control of pests or pathogens.
• the protein may therefore be an insecticidal protein, such as B. thurinqiensis toxin, to give resistance to leaf-eating insects.
• Other uses to which the promoter may be put are the production of viral coat proteins to protect against viral infection, the production of high value proteins such as pharmaceuticals and the production of proteins to alter taste or nutritive value of forage grasses, etc.
• the promoter sequence may be fused directly to a heterologous gene or via a linker.
• the linker sequence may comprise an intron.
• the linker may be composed of up to 45 bases, for example up to 30 or up to 15 bases.
• the linker sequence may comprise a sequence encoding a transit amino acid sequence, for example a transit sequence capable of directing a protein to a chosen subcellular locality such as the chloroplasts or mitochondria.
• the linker sequence may comprise a sequence having enhancer characteristics, to boost expression levels.
• DNA fragments and vectors can be prepared in which the promoter is operably linked to a heterologous gene.
• the fragments and vectors may be single or double stranded.
• Plant cells can be transformed by way of such fragment directly or by way of such a vector.
• the vector incorporates the heterologous gene under the control of the promoter.
• the vector contains regulatory elements capable of enabling the gene to be expressed in a plant cell transformed with the vector. Such regulatory elements include, besides the promoter, translational initiation and/or termination sequences.
• the vector typically contains too a region which enables the chimaeric gene and associated regulatory control elements to be transferred to and stably integrated in the plant cell genome.
• the vector is therefore typically provided with transcriptional regulatory sequences and/or, if not present at the 3'-end of the coding sequence of the gene, a stop codon.
• a DNA fragment may therefore also incorporate a terminator sequence and other sequences which are capable of enabling the gene to be expressed in plant cells.
• An enhancer or other element able to increase or decrease levels of expression obtained in particular parts of a plant or under certain conditions may be provided in the DNA fragment and/or vector.
• the vector is also typically provided with an antibiotic resistance gene which confers resistance on transformed plant cells, allowing transformed cells, tissues and plants to be selected by growth on appropriate media containing the antibiotic.
• Transformed cells are selected by growth in an appropriate medium.
• Plant tissue can therefore be obtained comprising a plant cell which harbours the heterologous gene under the control of the promoter , for example in the plant cell genome.
• the gene is therefore expressible in the plant cell.
• Plants can then be regenerated which include the heterologous gene and the promoter in their cells, for example integrated in the plant cell genome, such that the gene can be expressed.
• the regenerated plants can be reproduced and, for example, seed obtained.
• a preferred way of transforming a plant cell is to use Agrobacterium tumefaciens containing a vector comprising the promoter operably linked to the heterologous gene.
• a hybrid plasmid vector may therefore be employed which comprises:
• a DNA sequence which enables this DNA to be transferred to the plant genome is delineated by the T-DNA border sequences of a Ti-plasmid. If only one border sequence is present, it is preferably the right border sequence.
• the DNA sequence which enables the DNA to be transferred to the plant cell genome is generally the virulence (vir) region of a Ti-plasmid.
• the heterologous gene and its transcriptional and translational control elements, including the promoter can therefore be provided between the T-DNA borders of a Ti-plasmid.
• the plasmid may be a disarmed Ti-plasmid from which the genes for tumorigenicity have been deleted.
• the gene and its transcriptional and control elements, including the promoter can, however, be provided between T-DNA borders in a binary vector in trans with a Ti-plasmid with a vir region.
• Such a binary vector therefore comprises:
• the heterologous gene under the control of the promoter and other regulatory elements capable of enabling the gene to be expressed when integrated in the genome of a plant cell; and (b) at least one DNA sequence which delineates the
• a ⁇ robacterium tumefaciens f therefore, containing a hybrid plasmid vector or a binary vector in trans with a Ti-plasmid possessing a vir region can be used to transform plant cells.
• Tissue explants such as stems or leaf discs may be inoculated with the bacterium.
• the bacterium may be co-cultured with regenerating plant protoplasts.
• Plant protoplasts may also be transformed by direct introduction of DNA fragments which encode the heterologous gene and in which the promoter and appropriate other transcriptional and translational control elements are present or of a vector incorporating such a fragment.
• Direct introduction may be achieved using electroporation or polyethylene glycol, microi jection or particle bombardment.
• Plant cells from monocotyledonous or dicotyledonous plants can be transformed according to the present invention.
• Monocotyledonous species include barley, wheat, maize and rice.
• Dicotyledonous species include tobacco, tomato, sunflower, petunia, cotton, sugarbeet, potato, lettuce, melon, soybean, canola (rapeseed) and poplars.
• Tissue cultures of transformed plant cells are propagated to regenerate differentiated transformed whole plants.
• the transformed plant cells may be cultured on a suitable medium, preferably a selectable growth medium. Plants may be regenerated from the resulting callus. Transgenic plants are thereby obtained whose cells harbour the promoter operably linked to the heterologous gene, for example integrated in their genome. The gene is consequently expressible in the cells. Seed from the regenerated plants can be collected for future use.
• the following Examples illustrate the present invention. In the accompanying drawings:
• Figure 1 shows the restriction map of the insert DNA of phage 3cX2
• Figure 2 shows the restriction map of the insert DNA of phage E3.2
• Figure 3 shows the sequence of the PRP 378 gene
• Figure 4 shows the sequence of the PRP 140 gene
• Figure 5 shows the regulatory sequence upstream of the PRP 378 gene
• Figure 6 shows the regulatory sequence upstream of the PRP 140 gene
• Figure 7 shows the construction of pBIHN/2.4
• Figure 8 shows the construction of pBIXN/1.3
• Figure 9 shows the construction of pBID -234, -400, -540, -769 and -961 N
• Figure 10 shows the construction of pIPKH -6, -12 and -50/2.3;
• Figure 11 shows the construction of pIPH-6/2.3
• Figure 12 shows the construction of pIP S-6/762 and pIP P-6/615
• Figure 13 shows the construction of pIPD-6/1839, 1510, 1289, 1023 and 816;
• Figure 14 shows the sequences of the fusion junctions of constructs based on BIN 19
• Figure 15 shows the sequences of the fusion junctions of constructs based on pUC19
• Figure 16 shows the results of Gus assays for the PRP140 promoter deletions
• FIG. 17 shows the results of Gus assays for the PRP 378 deletions; and Figure 18 shows the nucleotide sequence of the cDNA clone (WPRPl) .
• EXAMPLE 1 Isolation of PRP Genes
• the strategy employed to isolate wheat PRP promoters involved first constructing a genomic library of wheat DNA and then screening of this library using a cDNA probe for a PRP gene.
• High molecular weight DNA was isolated from dark grown shoots of Triticum aestivum cv. Chinese Spring (Lazarus et al. Plant Mol. Biol. .5, 8-24, 1985). Conditions for partial digestion with Sau3A were established. DNA fragments of 18-25 kb were purified by size fractionation on sucrose density gradients. Lambda Charon 35 (Loenen and Blattner, Gene ___, 171-179, 1983) vector DNA was prepared by digestion with BamHI and purification on sucrose density gradients. Vector DNA (1.5 ⁇ g) and wheat DNA (3.5 ⁇ g) were ligated at high concentration (500 ng/ ⁇ l) using T4 DNA ligase and subsequently packaged in vitro using commercially available extracts (Stratagene) .
• the regulatory sequence upstream of the PRP 378 and PRP 140 genes are given in Figs 5 and 6 respectively. Restriction enzyme sites used during subsequent cloning procedures are indicated and underlined. It was proposed that these sequences would contain all of the promoter and other regulatory elements necessary to direct correct expression of protein coding sequences placed downstream under their control.
• a series of constructs were prepared. In each case part of the PRP 378 or PRP 140 upstream regions was placed in front of the bacterial reporter gene 9-glucuronidase and the nopaline synthase terminator sequences in a suitable vector.
• PIPPKGT and PBIPKGT pIPPKGT is the EcoRI/Sall insert of pRAJ275 (Jefferson, Plant. Mol. Biol. Reporter 5., 387-405, 1987) blunted with Klenow fragment of E.
• coli DNA polymerase I coli DNA polymerase I, and ligated into the Klenow blunted Asp718 site of pUCPolyTer (the nopaline synthetase terminator fragment cloned as a Sstl/EcoRI fragment into pUC19) .
• the corresponding BIN19 based plasmid is pBIPKGT.
• pIPPKGT and pBIPKGT can be obtained from each other, (b) BIN19 Based Constructs fpBI series) (i) Construction of the plasmid pBIHN/2.4 containing 2318 bp of sequence 5' of the PRP 378 translation initiation codon and 24 amino acids of PRP 378 coding sequence in a translational fusion to fl-glucuronidase (Fig. 7)
• a 2.4 Kb Hindlll-NotI fragment of the PRP 378 gene (Fig. 1) was subcloned into the plasmid vector pUBSl to form the plasmid pHN/2.4.
• pHN/2.4 was linearised with NotI and the site filled in using Klenow fragment of E. coli DNA polymerase I.
• the insert was excised by digestion with Hindlll and inserted into Hindlll-Smal digested pBI101.3 (Jefferson, Plant. Mol. Biol. Reporter 5, 387-405, 1987) to form pBIHN/2.4.
• the fusion junction was checked by nucleotide sequencing and its sequence along with the predicted amino acid sequence is shown in Fig. 14.
• a 1.45 kb Xbal-NotI fragment of the PRP 140 gene (Fig. 2) was subcloned into the plasmid vector pUBSl to form pXNot/1.45.
• a 1.3 kb fragment was excised by digestion with Xbal and NspBII (Fig. 8) and inserted into Xbal-Smal digested pBIPKGT to form pBIXN/1.3.
• the sequence of the fusion junction is shown in Fig. 14.
• the plasmid pHN/2.4 (Fig. 7) was linearised by digestion with Notl-BstXI. Incubation with Exonuclease III for various periods resulted in deletion of varied lengths of sequence from the NotI end. Treatment with Nuclease SI and the Klenow fragment of E. coli DNA polymerase enabled the plasmid population to be religated as closed circles. Nucleotide sequencing was employed to determine the extent of deletion from the NotI end of a representative portion of the population.
• plasmids pHD -6, -12, -50/2.3 with inserts which extended approximately 2.3 kb downstream from the Malawi sites to within 6, 12 and 50 nucleotides 5' of the translation initation codon of PRP 378 were chosen for evaluation of promoter activity (Fig. 5) .
• the 3 respective plasmids were linearised by a partial SstI digestion and the overhang blunted with Mung Bean Nuclease.
• the 3 inserts were excised by digestion with Hindlll and inserted into Hindlll-Smal digested pIPPKGT to form pIPKH -6, -12, -50/2.3.
• the sequences of the fusion junctions are shown in Fig. 15.
• the plasmid pIPH-6/2.3 (Fig. 11) was linearised by PstI or partial SstI digestion and the overhang blunted with Mung Bean Nuclease. Inserts with respectively 762 and 615 nucleotides of the PRP 378 promoter in a cassette with the GUS gene and the NOS terminator were excised by digestion with EcoRI and inserted into EcoRI/Smal digested pUC19. In both constructs the sequence of the fusion junction is identical to that of pIPH-6/2.3 (Fig. 15). (iv) Construction of PlPD-6/1839. 1510. 1289. 1023. 816 representing longer forms of the PRP 378 promoter deleted from its 5' end (Fig. 13)
• the plasmid pHN/2.4 (Fig. 7) was linearised by digestion with Hindlll-Apal. Incubation with Exonuclease III for various periods resulted in deletion of varied lengths of sequence from the Hindlll end. Treatment with Nuclease SI and the Klenow fragment of E. coli DNA polymerase enabled the plasmid population to be religated as closed circles. Nucleotide sequencing was employed to determine the extent of deletion from the Hindlll end of a representative portion of the population.
• Nicotiana Constructs were mobilised from Escherichia coli MC1022 into Agrobacterium tumefaciens LBA4404 as described (Bevan, Nucl. Acids. Res. .12, 8711-8721, 1984).
• Leaf discs of Nicotiana tabacum var. Sa sun were transformed as described (Horsch et __l, Science 223. 496-498, 1984) and selected on shooting medium containing 100 ⁇ g/ml kanamycin. Typically between 5 and 15 plants containing each construct were regenerated and assayed.
• the activity of the PRP promoters in individual transformants was determined by measuring ,9-glucuronidase activity in leaf extracts. Tissue extracts were prepared and analysed for fluorescence of the reaction product 4-methyl umbel1iferone as described (Jefferson, 1987) . Reactions were usually incubated at 37°C for 4 hours with aliquot ⁇ sampled at 2 hour intervals. The protein concentration in each extract was measured to allow direct comparisons to be made between them (using a Bio-Rad kit) . Results of assays on PRP140 promoter derived constructs expression in nmol 4-methyl umbel1iferone produced/min/mg protein are shown in Fig. 16.
• the pUC19 based constructs were assayed for transient expression by Ca - PEG mediated transformation of Zea mays anther derived cell suspension (cell line Ba Tan Bai) protoplasts based on the method of Chen et al. (Chen, D.F., Tassie, A., Dale, P.J., Goldsbrough, A.P., Liang, W. , Bevan, M.W. , Flavell, R.B. and Xia, Z.A. (1988). In: Puite, K.J., Dons, J.J.M., Huizing, H.J. , Kool, A.J. , Koornneef, M. and Krens, F.A.
• Lysates of transformed protoplasts were assayed for 0-glucuronidase in the same manner as that for transformed plants (Jefferson, 1987) . Expressing activity per 1 x 10 4 protoplasts allowed comparisons to be made between constructs tested in the same experiment.
• the 2.3 kbp form of the PRP 378 promoter is significantly stronger than the 850 bp form of the CaMV35S promoter in maize protoplasts (Table 1) . In addition it is sensitive to relatively short deletions from its 3' end.
• pIPKH-12/2.3 the construct that places the promoter in almost its native context (Fig. 15) with respect to the initiating "ATG" of the GUS gene, exhibits highest levels of expression; whilst activity declines if, in effect, the promoter is moved (Fig. 15) further or closer to the ATG.
• Figure 17 illustrates the effect of deletions from 5* end of the PRP 378 promoter.
• a deletion of 800 bp reduces activity to less than 60% of the full length form, and activity continues to decline with extent of deletion.
• the full length form of the PRP 378 promoter in this experiment does not have the same level of activity with respect to CaMV 35S as those in Table 1, its fusion junction is different and the initiating ATG of the ,9-glucuronidase gene does not have the "Kozak" consensus (Fig. 15) .
• a cDNA, designated WPRP1, encoding a wheat PRP has been isolated and sequenced.
• the WPRP1 clone was detected whilst a wheat cDNA library was being screened using antibodies raised against chloroplastic fructose-1,5- bisphosphatase. Why this antibody should cross-react with the WPRP1 clone is not known.
• the cDNA isolated initially was not full length (1.3 Kb). The complete sequence came from a longer cDNA obtained by rescreening the cDNA library using the shorter clone.
• the complete nucleotide sequence of the cDNA clone (WPRP1) from wheat with its deduced amino acid sequence is shown in Figure 18. This 1548 bp cDNA contains the complete coding sequence of 1137 base pairs with 101 bp of 5' non-coding sequence and 310 bp of 3 1 non-coding sequence terminating in a poly-adenylation tail.
• Example 1.4 The localisation of Gus expression in tissues of the transformed tobacco plants of Example 1.4 was investigated. Assays were conducted as described in Example 1.5 except that tissues other than leaf were also assayed. The results are shown in Table 2 below. The values shown are pmol/min/ug protein. The numbers in brackets refer to ranking of tissue expression level within each individual transformed plant. There are significant levels of expression in all tissues. The levels of expression in petioles and stems compare favourably with CaMV 35S.

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