Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/8223—Vegetative tissue-specific promoters
  • C12N15/8227—Root-specific
• • C—CHEMISTRY; METALLURGY
  • 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 isolated regulatory elements, in particular promoters and 3' regulatory elements, that drive root specific or root abundant expression in plants, in particular in monocotyledonous plants, to vectors containing the regulatory elements, to host cells containing the vectors, to plants containing the host cells and to methods for isolating regulatory elements that contribute to tissue-preferred gene expression in plants.
• Gene expression is considered to comprise a number of steps from the DNA to the final protein product. Initiation of transcription of a gene is generally believed to be the predominant controlling factor in determining expression of a gene.
• the tran- scriptional controls are generally located in relatively short sequence elements embedded in the 5 '-flanking and/or 3" -flanking region of the transcribed gene with which DNA-binding proteins may interact . These DNA sequence elements serve to promote the formation of transcriptional complexes and eventually initiate gene expression processes. It is furthermore known that the regulation of gene expression often depends upon the development stage and the tissue specificity of the cell concerned. Thus, certain tissues of organisms such as plants may exhibit a metabolism and a protein composition different from other tissues of the plant or different from the same tissue in a different developmental stage.
• Controlling the expression of genes in transgenic plants is considered to have high commercial value.
• the ability to control gene expression is useful for conferring resistance and immunity to certain diseases or to modify the metabolism of the tissue.
• the transfer of heterologous genes or genes of interest into a plant under control of tissue-specific genes provides a powerful means of conferring selective advantages to plants and to increase their commercial value.
• Root preferred gene expression will provide several advantages to a plant, such as alteration of the function of the root tissue; modification of the growth rate; resistance to root preferred pathogens, pests, herbicides or adverse weather conditions; as well as broadening the range of soils or environments in which said plant may grow. Root abundant or root specific gene expression would provide a mechanism according to which morphology and metabolism may be altered to improve the yield and to produce useful proteins in greater amounts .
• 097/44448 describes promoter elements conferring root preferred gene expression.
• the disclosed promoter elements were obtained from maize seedlings.
• the isolated promoter elements may not be suitable for any desired transformation problem.
• O94/02619 discloses regulatory sequences for root specific or root abundant gene expression derived from Brassica sp .
• this promoter was not shown to work in monocotyle- donous plants with an efficiency great enough to provide means for commercial use.
• the technical problem underlying the present invention is to provide novel regulatory elements for use in cloning and expressing root specific or root abundant genes, in particular for use in monocotyledonous plants which provide a high expression efficiency together with a high tissue specificity.
• the problem is solved by providing a purified nucleic acid sequence according to the above wherein the sequence is selected from the group consisting of (al) the nucleic acid sequence set out in SEQ ID No . 2 or a part or complementary strand thereof, (bl) a nucleic acid sequence which hybridizes to the nucleic acid sequence defined in SEQ ID No . 2 and (cl) alleles or derivatives of the nucleic acid sequence defined in (al) or (bl) .
• the problem is solved by providing a purified nucleic acid sequence according to the above which is selected from the group consisting of (a2) the nucleic acid sequence set out in any one of SEQ ID Nos.
• (b2) a nucleic acid sequence which hybridizes to any one of the nucleic acid sequences defined in any one of SEQ ID Nos. 3 to 11 and alleles or derivatives of the nucleic acid sequence defined in (a2) or (b2) .
• the invention also relates to purified nucleic acid sequences for use in cloning and expressing a root specific or root abundant gene in a plant that are selected from the group consisting of (a3) the nucleic acid sequence set out in any one of SEQ ID No. 12, 13, 14 or 15 or a part or complementary strand thereof, (b3) a nucleic acid sequence hybridizing to any one of the sequences defined in (a3) and (c3) alleles or derivatives of the nucleic acid sequences defined in (a3) or (b3) .
• sequences set out in SEQ ID No . 1 to 11 comprise isolated 5' regulatory elements and are considered to be promoters or parts thereof, i.e. promoter elements. These sequences are capable of modulating, initiating and/or contributing to the transcription of nucleic acid sequences operably linked to them. In a preferred embodiment of the present invention these sequences may additionally contain at their 3 ' terminus the nucleotide sequence from position 1761 to 1780 as depicted in SEQ ID No. 14, which is CCTGGACTCG CTCACTGGCA.
• sequences set out in SEQ ID No. 12, 13 and 15 comprise isolated 3 ' regulatory elements and are considered to be 3 ' transcription regulatory elements, or parts thereof, for instance polyadenylation signals .
• sequences of SEQ ID Nos . 12 and 15 linked in 5 ' to 3 ' direction to each other represent the complete 3 1 regulatory element of the present invention.
• sequences are capable of modulating, in particular contributing to, or terminating the transcription of nucleic acid sequences operably linked to them.
• sequence set out in SEQ ID No. 14 is a genomic clone comprising all of the above sequences except the 3' regulatory element of the SEQ ID No . 15.
• the present invention also relates to nucleic acid sequences which hybridize, in particular under stringent conditions, to the sequences set out in any one of SEQ ID No. 1 to 15. In particular, these sequences have a degree of identity of 70% to the sequence of SEQ ID Nos . 1 to 15.
• nucleic acid sequences which hybridise to any one of the specifically disclosed sequences of SEQ. Id. Nos. 1 to 15 are sequences which have a degree of 60% to 70% sequence identity to the specifically disclosed sequence on nucleotide level .
• sequences which are encompassed by the present invention are sequences which have a degree of identity of more than 70% or 80% and even more preferred more than 90%, 95% or 99% to the specifically disclosed sequences on nucleotide level.
• the present invention also relates to nucleic acid sequences which encode proteins wherein the amino acid sequence of the proteins have a degree of identity of 66% to 90% on amino acid level, most preferably a degree of identity of more than 90%, 95% or 99% identity on amino acid level.
• the present invention relates to nucleic acid sequences, in particular DNA sequences which hybridise under the following conditions to the sequences specifically disclosed:
• Hybridisation buffer 1 M NaCl ; 1% SDS; 10% dextran sulphate; 100 ⁇ g/ml ssDNA
• Second wash 0.2 x SSC; 0.5% SDS at 65°C.
• the hybridisation conditions are chosen as identified above, except that a hybridisation temperature and second wash temperature of 68° C, and even more preferred, a hybridisation temperature and second wash temperature of 70° C is applied.
• the present invention also comprises functionally equivalent nucleic acid sequences to the sequences of any one of SEQ ID No. 1 to 15, in particular sequences which have at least ho- mology to the sequence of SEQ ID No. 1 to 15.
• the invention also relates to alleles and derivatives of the sequences mentioned above which are defined as sequences being essentially similar to the above sequences but comprising, for instance, nucleotide exchanges, substitutions (also by unusual nucleotides) , rearrangements, mutations, deletions, insertions, additions or nucleotide modifications and are functionally equivalent to the sequences set out in SEQ ID No. 1 to 15.
• the nucleic acid sequences of the present invention are, in a preferred embodiment, derived from maize (Zea mays), most preferably from maize roots .
• the nucleic acid sequences of the present invention are useful for cloning tissue specific, in particular root specific nucleic acid sequences, in particular regulatory elements and/or genes, in plants, in particular in monocotyledonous plants.
• the present invention provides the means for isolation of transcription regulatory elements that direct or contribute to tissue- preferred gene expression in plants, in particular in monocotyledonous plants, such as maize.
• the present invention also provides the means of isolating tissue specifically expressed genes, in particular root specifically expressed genes and their transcripts .
• the nucleic acid sequences of the present invention are also useful for expressing genes of interest in plants, in particular in the roots of plants and especially in the roots of monocotyledonous plants such as maize or of dicotyledonous plants such as sugar beets (Beta vulgaris) .
• the present invention provides the means to direct the expression of a gene of interest in a tissue-specific or tissue-abundant manner in roots, for instance in roots of seedlings.
• the proteins encoded by the gene of interest can be accumulated in roots .
• the promoter of the present invention is particularly useful in driving the transcription of heterologous structural genes that confer disease immunity or resistance to disease- susceptible roots .
• disease encompasses any adverse condition caused to a plant by a virus or an organism other than itself, such as fungi, bacteria and insects.
• promoter refers to a sequence of DNA, usually upstream (5 1 ) to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at the correct site. Promoter sequences are necessary, but not always sufficient to drive the expression of the gene.
• a “promoter element” constitutes a fraction of the DNA sequence of the promoter.
• a "3' regulatory element refers to that portion of a gene comprising a DNA segment, excluding the 5' sequence which drives the initiation of transcription and the structural portion of the gene, that determine the correct termination site and contains a polyadenylation signal and any other regulatory signals capable of effecting messenger RNA (mRNA) processing or gene expression.
• the polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
• Polyadenylation signals are commonly recognised by the presence of homology to the canonical form S'-AATAA-S', although variations are not uncommon.
• Nucleic acid refers to a large molecule which can be single or double stranded, composed of monomers (nucleotides) containing a sugar, phosphate and either a purine or pyrimidine .
• the nucleic acid may be cDNA, genomic DNA, or RNA, for instance mRNA.
• nucleic acid sequence refers to a natural or synthetic polymer of DNA or RNA which may be single or double stranded, alternatively containing synthetic, non-natural or al- tered nucleotide bases capable of incorporation into DNA or RNA polymers .
• gene refers to a DNA sequence that codes for a specific protein and regulatory elements controlling the expression of this DNA sequence.
• regulatory element refers to a sequence located upstream (5'), within and/or downstream (3') to a coding sequence whose transcription and expression is controlled by the regulatory element, potentially in conjunction with the protein bio- synthetic apparatus of the cell.
• Regulation or “regulate” refer to the modulation of the gene expression induced by DNA sequence elements located primarily, but not exclusively upstream from (5 1 ) the transcription start of the gene of interest. Regulation may result in an all or none response to a stimulation, or it may result in variations in the level of gene expression.
• coding sequence refers to that portion of a gene encoding a protein, polypeptide, or a portion thereof, and excluding the regulatory sequences which drive the initiation or termination of transcription.
• the coding sequence or the regulatory element may be one normally found in the cell, in which case it is called “autologous” , or it may be one not normally found in a cellular location, in which case it is termed a “heterologous gene” or “heterologous nucleic acid sequence” .
• a heterologous gene may also be composed of autologous elements arranged in an order and/or orientation not normally found in the cell in which it is transferred.
• a heterologous gene may be derived in whole or in part from any source known to the art , including a bacterial or viral genome or episome, eukaryotic nuclear or plasmid DNA, cDNA or chemically synthesized DNA.
• the structural gene may constitute an uninterrupted coding region or it may include one or more introns bounded by appropriate splice junctions.
• the structural gene may be a composite of segments derived from different sources, naturally occurring or synthetic .
• vector refers to a recombinant DNA construct which may be a plasmid, virus, or autonomously replicating sequence, phage or nucleotide sequence, linear or circular, of a single or double stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell, in particular a plant cell.
• plant refers to photosynthetic organisms, such as whole plants including algae, mosses, ferns and plant-derived tissues.
• Plant derived tissues refers to differentiated and undifferentiated tissues of a plant, including roots, shoots, shoot meristems, coleoptilar nodes, tassels, leaves, cotyledon- dous petals, pollen, ovules, tubers, seeds, kernels and various forms of cells in culture such as intact cells, protoplasts, embryos and callus tissue.
• root includes various groups of primary, lateral seminal or crown roots, and root tips. Plant-derived tissues may be in planta, or in organ, tissue or cell culture.
• a “monocotyledonous plant” refers to a plant whose seeds have only one cotyledon, or organ of the embryo that stores and absorbs food.
• a “dicotyledonous plant” refers to a plant whose seeds have two cotyledons .
• transformation refers to the process by which cells, tissues or plants acquire properties encoded on a nucleic acid molecule that has been transferred to the cell, tissue or plant .
• Transformation and “transferring” refers to methods to transfer DNA into cells including, but not limited to, particle bombardment, microinjection, permeabilizing the cell membrane with various physical (e.g., electroporation) or chemical (e.g., polyethylene glycol , PEG) treatments.
• various physical e.g., electroporation
• chemical e.g., polyethylene glycol , PEG
• host cell refers to a cell which has been genetically modified by transfer of a heterologous or autologous nucleic acid sequence or its descendants still containing this sequence. These cells are also termed “transgenic cells”. In the case of an autologous nucleic acid sequence being transferred, the sequence will be present in the host cell in a higher copy number than naturally occurring.
• operably linked refers to the chemical fusion of two of more fragments of DNA in a proper orientation such that the fusion preserves or creates a proper reading frame, or makes possible the proper regulation of expression of the DNA sequences when transformed into plant tissue.
• expression is intended to describe the transcription and/or coding of the sequence for the gene product.
• a DNA chain coding for the sequence of gene product is first transcribed to a complementary RNA, which is often an mRNA, and then the thus transcribed mRNA is translated into the above mentioned gene product if the gene product is a protein.
• expression also includes the transcription of DNA inserted in antisense orientation to its regulatory elements. Expression, which is constitutive and possibly further enhanced by an externally controlled promoter fragment thereby producing multiple copies of mRNA and large quantities of the selected gene product, may also include overproduction of a gene product .
• translation start codon or "initiation codon” refers to a unit of three nucleotides (codon) in a nucleic acid sequence that specifies the initiation of protein synthesis.
• signal peptide refers to the N-terminal extension of a polypeptide which is translated in conjunction with the polypeptide, forming a precursor protein and which is required for its entry into the secretory pathway.
• the signal peptide may be recognised by the mechanisms within the same species or unrelated species plants, necessary for direction of the peptide into the secretory pathway.
• the signal peptide may be active in roots, seeds, leaves, tubers and other tissues of the plant.
• signal sequence refers to a nucleotide sequence that encodes a signal peptide.
• ER targeting signal refers to the N-terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor protein and which is required for its entrance into the Endo- plasmatic Reticulum (ER) of a cell.
• the ER targeting signal may be recognised by the mechanisms within the same species or unrelated species plants, necessary for direction of the peptide into the ER and/or the vacuole of a cell.
• ER targeting signals may be active in roots, seeds, leaves, tubers and other tissues of the plant.
• ER targeting sequence refers to a nucleotide sequence that encodes the ER targeting signal .
• tissue specific promoter refers to a sequence of DNA that provides recognition signals for RNA polymerase and/or other factors required for transcription to begin, and/or for controlling expression of the coding sequence precisely within certain tissues or within certain cells of that tissue. Expression in a tissue specific manner may be only in individual tissues, or cells within tissues, or in combinations of tissues. Examples may include tissue specific expression in roots only and no other tissues within the plant, or may be in leaves, petals, ovules and stamen, and no other tissues of the plant.
• tissue specific is also meant to describe an expression in a particular tissue or cell according to which the expression takes place mainly, but not exclusively, in the tissue. Such an expression is also termed “tissue abundant” .
• Selective expression refers to expression mainly, preferably almost exclusively, in specific organs of the plant, including, but not limited to, roots, leaves, tubers or seed. The term may also refer to expression at specific developmental stages in an organ, such as in early or late embryogenesis or in seedlings. In addition, “selective expression” may refer to expression in specific subcellular locations within the cell, such as the cy- tosol or vacuole .
• root specific nucleic acid sequence refers to nucleic acid sequences, i.e. genes, coding sequences and/or regulatory elements which are exclusively or mainly active in roots of plants, in particular those which direct or contribute to a root abundant or root selective expression of a protein.
• root abundant nucleic acid sequence refers to nucleic acid sequences, i.e. genes, coding sequences and/or regulatory elements which are mainly active in roots of plants, in particular those which direct or contribute to a root abundant or root selective expression of a protein.
• the present invention also relates to a vector comprising the nucleic acid sequences according to the above, in particular to a bacterial vector, such as a plasmid, or a virus.
• nucleic acid sequences i.e. the 5' and/or 3' regulatory elements of the present invention contained in the vector
• a gene of interest which in this context may also be only its coding sequence, which may be a heterologous or autologous gene or coding sequence.
• a gene of interest may be a gene, in particular its coding sequence, conferring, for instance, disease resistance; drought resistance; insect resistance; herbicide resistance; immunity; an improved intake of nutrients, minerals or water from the soil; or a modified metabolism in the plant, particularly its roots.
• a gene of interest may confer resistance to infection by a virus, such as a gene encoding the capsid protein of the BWYV or the BNYW virus, a gene conferring resistance to herbicides such as Basta ® , or to an insecticide, a gene conferring resistance to the corn rootworm, a gene encoding the toxic crystal protein of Bacillus thuringiensis or a gene whose expression confers male sterility.
• a virus such as a gene encoding the capsid protein of the BWYV or the BNYW virus, a gene conferring resistance to herbicides such as Basta ® , or to an insecticide, a gene conferring resistance to the corn rootworm, a gene encoding the toxic crystal protein of Bacillus thuringiensis or a gene whose expression confers male sterility.
• a gene of interest includes also a coding sequence cloned in antisense orientation to the regulatory sequences directing its expression. Such a construct may be used specifically to repress the activity of undesirable genes in plant cells, in particular in roots, most preferably in primary, lateral seminal and/or crown roots.
• the gene of interest may also comprise signal sequences, in particular ER targeting sequences, directing the encoded protein in the ER and eventually for instance in the cell wall, vascular tissue and /or the vacuole .
• the vector defined above is comprised of further regulatory elements directing or enhancing expression of the gene of interest such as 5 ' , 3 ' or 5 ' and 3' regulatory elements known in the art, for instance enhancers such as the 35S-enhancer .
• regulatory elements considered in the present invention also encompass introns or parts of introns inserted within or outside the gene of interest.
• the 3 ' regulatory element is a transcription termination region, preferably a poly A addition or polyadenylation sequence, most preferably the poly A addition sequence of the NOS gene of Agro- bacterium tumefaciens .
• nucleic acid sequences of the present invention are useful since they enable the root specific expression of genes of interest in plants, in particular monocotyledonous plants. Accordingly, plants are enabled to produce useful products in their roots .
• the nucleic acid sequences of the present invention may also be useful to regulate the expression of genes of interest depending upon the developmental stage of the transferred cell or tissue. Furthermore, the present invention allows the specific modification of the metabolism in roots.
• the vector furthermore contains T-DNA, in particular the left, the right or both T-DNA borders derived from Agrobacterium tume- faciens .
• T-DNA sequences derived from Agrobacterium rhi- zogenes may also be used.
• the use of T-DNA sequences in the vector of the present invention enables the Agrobacterium tume- faciens mediated transformation of cells .
• the nucleic acid sequence of the present invention, together with the gene of interest and - optionally - further operably linked regulatory sequences, is inserted within the T-DNA or adjacent to it.
• the present invention also relates to a non-biological, technical method of genetically modifying a cell by transforming it with a vector or a nucleic acid sequence according to the above, whereby the gene of interest operably linked to the nucleic acid sequence or sequences of the present invention is expressible in the cell.
• the cell being transformed by the method of the present invention is a plant, bacterial or yeast cell.
• the above method further comprises the regeneration of the transformed cell to a differentiated and, in a preferred embodiment, fertile plant.
• the method to transform a cell with a vector according to the present invention may be any method known to effectively transfer nucleic acid sequences into a host cell, thereby allowing its expression therein and eventually the regeneration of the cell to a plant.
• the transfer of the nucleic acid sequence may be mediated by Agrobacterium tumefaciens or rhizogenes .
• any other method known to eventually achieve the same results may be used, such as direct uptake of nucleic acid sequences, microinjection of nucleic acid sequences, or particle bombardment .
• the present invention also relates to host cells transformed with the vector of the present invention, in particular plant, yeast or bacterial cells, in particular monocotyledonous or di- cotyledonous plant cells .
• the present invention also relates to cell cultures, tissue, calluses, etc. comprising a cell according to the above, i.e. a transgenic cell.
• the present invention relates to plants, harvest and propagation material, parts of plants, plant tissue, plant seeds, plant leaves, plant roots, leaves, or embryos containing a host cell of the present invention.
• These plants, plant tissues, plant seeds and plant parts are also called "transgenic".
• These plants or plant parts are characterised by, as a minimum, the presence of the transferred nucleic acid sequence of the present invention in their genome or, in cases where the nucleic acid sequences are autologous to the transferred host cell, are characterised by additional copies of the nucleic acid sequences of the present invention.
• the nucleic acid sequences of the present invention are linked to genes of interest or antisense DNA normally not found in the transferred cell operably linked to the nucleic acid sequence of the present invention, at least not in that order or orientation.
• the present invention also relates to roots, leaves, calluses, plant tissue, tassels, etc., nonbiologically transformed, which possess, stably integrated in the genome of their cells, a heterologous or autologous nucleic acid sequence containing a regulatory element of the present invention recognised by the polymerases of the cells of said plant parts and the gene of interest encoding a protein of interest or being an antisense construct .
• the invention relates to plants, nonbiologically transformed, which possess, stably integrated in the genome of their cells, a heterologous or autologous nucleic acid sequence containing a regulatory element of the present invention recognised by the polymerases of the cells of said plants and a gene of interest encoding a protein of interest or being an antisense construct .
• the teaching of the present invention is therefore applicable to any plant, plant genus, or plant species wherein the regulatory element of the present invention is recognised by the polymerases of the cells.
• the present invention relates to a method for isolating or cloning a root specific gene and/or root specific regulatory elements, such as promoters whereby a nucleic acid sequence of the present invention is used to screen nucleic acid sequences derived from any source, such as genomic or cDNA libraries derived from plants, in particular monocotyledonous plants.
• the nucleic acid sequences of the present invention thereby provide a means of isolating related regulatory sequences of other plant species which confer root specificity to genes of interest operably linked to them.
• SEQ ID Nos. 1 to 15 are incorporated in the present invention.
• the positions indicated below refer to the sequence numbering of SEQ ID No . 14.
• SEQ ID No. 1 is a partial DNA sequence of the zmGRP3 (glycine- rich protein: GRP) gene of Zea mays.
• the 1760 bp (base pair) sequence spans the region from and including position 1760 towards the 5' end, up to and including position 1.
• SEQ ID No. 2 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 1348 bp sequence spans the region from and including position 1760 towards the 5' end, up to and including position 413.
• SEQ ID No. 3 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 1096 bp sequence spans the region from and including position 1760 towards the 5' end, up to and including position 665.
• SEQ ID No . 4 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 746 bp sequence spans the region from and including position 1760 towards the 5' end, up to and including position 1015.
• SEQ ID No. 5 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 554 bp sequence spans the region from and including position 1760 towards the 5' end, up to and including position 1207.
• SEQ ID No. 6 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 406 bp sequence spans the region from and including position 1760 towards the 5' end, up to and including position 1355.
• SEQ ID No. 7 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 190 bp sequence spans the region from and including position 1760 towards the 5' end, up to and including position 1571.
• SEQ ID No. 8 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 1354 bp sequence spans the region from and including position 1354 towards the 5' end, up to and including position 1.
• SEQ ID No. 9 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 1497 bp sequence spans the region from and including position 1497 towards the 5' end, up to and including position 1.
• SEQ ID No. 10 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 1550 bp sequence spans the region from and including position 1550 towards the 5' end, up to and including position 1.
• SEQ ID No. 11 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 1710 bp sequence spans the region from and including position 1710 towards the 5' end, up to and including position 1.
• SEQ ID No. 12 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 856 bp sequence spans the region from and excluding the translation stop codon at position 2558 - 2560 towards the 3' end up to position 3416.
• SEQ ID No. 13 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 440 bp sequence spans the region from and excluding the translation stop codon towards the 3 ' end up to position 3000.
• SEQ ID No. 14 is a large part of the genomic DNA sequence of the zmGRP3 gene of Zea mays comprising 3416 nucleotides and encompassing the coding region of the gene, a sequence of 20 bp between and excluding the translation initiation codon ATG (position 1781 - 1783) and the nucleotides CCA at position 1758 - 1760 and the regulatory elements identified in SEQ ID Nos. 1 to 13.
• SEQ ID No. 15 is a partial DNA sequence of the zmGRP3 gene of Zea mays.
• the 1074 bp sequence spans the region from and excluding the most 3' located base "C” at position 3416 of SEQ ID No. 14 to the sequence motif "TCC" at position 4488-4490.
• the complete genomic DNA sequence of zmGRP3 comprises 4490 bp .
• Figure 1 shows a restriction map of the genomic clone zmGRP3 described in SEQ ID No. 14.
• Figure 2 shows restriction maps of 5 ' deletion constructs of the present promoter element corresponding to SEQ ID Nos . 1 to 7.
• Figure 3 shows restriction maps of 3 ' deletion constructs of the present promoter element corresponding to SEQ ID Nos. 8 to 11.
• Figure 4 shows a northern blot analysis for various tissues of Zea mays .
• Figure 5 depicts DNA constructs used for transient expression experiments .
• genomic clone zmGRP3 was isolated by differential screening of a cDNA library made from maize coleoptile nodes, subsequent isolation of a specific root specific cDNA and isolation of the corresponding genomic clone .
• a cDNA library from maize coleoptilar nodes with emerging developing young crown roots was made by choosing 7-9 days old wild- type maize seedlings.
• mRNA from 30-40 of the wild-type coleoptilar nodes (about 15 mg per piece of tissue) was isolated with oligo (dT) -cellulose using the "Quick prep mRNA purification kit” (Pharmacia, Freiburg) .
• Double stranded cDNA was prepared using the "Time Safer" -cDNA synthesis kit of Pharmacia Freiburg.
• the cDNAs were ligated into the phage vector ⁇ -ZAP-ll (Stratagene) using the ligation kit "Gig-Pack-Gold" (Stratagene, Heidelberg) .
• the phages were plated on E. coli strain XL IB.
• the obtained library had a titre of 3.7 x 106 pfu/ml .
• the inserts had an average size of 1.4 kb.
• the phages of the cDNA library were plated out in low density (about 1000-2000 pfu per agar plate (12 x 12 cm) ) . Per plate two nitrocellulose filters as replicas were prepared. Both filters were then separately hybridized, each with a different probe.
• first strand cDNAs of mRNA were used which a) were derived from the coleoptilar nodes of wild- type seedlings (with formation of crown roots) and b) derived from coleoptilar nodes of rt-cs mutant seedlings (without formation of crown roots) (Hetz, et al . Plant Cell, 1996. Pp. 10, 845-857) .
• the first strand cDNA was labelled using radioactivity and, for further analysis, digoxigenin.
• Plaques giving a signal on the filter hybridized with the wild- type probe, but not giving a signal on the filter hybridized with rt-cs probe were excised and analysed. An in vivo excision was carried out and the cDNA insert was cloned into plasmid pBluescript . Altogether, 32 plaques were excised and analysed. For the further analysis, the isolated plasmid DNA clone was labelled with digoxigenin and hybridized in a northern blot analysis against 10 ⁇ g total RNA from wild-type and mutant coleoptilar nodes .
• the mRNA was denaturated for 5 minutes in 23.4 ⁇ l of H 2 0 at 65°C, then rapidly cooled in ice water.
• the following agents were added in order: 10 ⁇ l 5x RT-Buffer, 1 ⁇ l of RNase- Inhibitor (ca. 20 units), 5 ⁇ l of oligo-dT (final concentration 0.06 ⁇ g/ul) 3.6 ⁇ l of DIG-dUTP (final concentration 0.13 mM) , 5 ⁇ l of dNTP-mix and 2 ⁇ l of Superscript Reverse Transcriptase.
• the preparation was then incubated for 2 hours at 42°C.
• the labelled first strand cDNA was precipitated by adding of 2 ⁇ l of EDTA solution, 5 ⁇ l of LiCl solution and 130 ⁇ l of 100% EtOH. Following a washing with 70% EtOH, the cDNA resuspended in 50 ⁇ l of H 2 0 and stored at -20°C.
• the northern analysis showed that the clone isolated hybridized with wild-type total RNA, but not with the mutant total RNA. As the mutant tissue differed from the wild-type tissue in the absence of crown roots, the expression of the isolated clone could be related to the formation of crown roots in maize seedlings . According to northern blot analysis, the clone was expressed exclusively in the cells of roots of young maize seedlings, in particular in the tissue. The clone was used to isolate a full- length cDNA clone. The full-length cDNA clone (zmGPR3 cDNA) was in turn used to isolate a genomic clone zmGRP3 which is partially described in SEQ ID No. 14 as follows:
• the sequence given in SEQ ID No. 14 comprises the 5' regulatory elements of the present invention, the coding region of the maize zmGRP3 gene and the 3 ' regulatory elements of the present invention except the most distal part of the 3 ' region which, however, is given in SEQ ID No. 15.
• the coding region of zmGRP3 spans from the translation initiation codon at position 1781 to 1783 to the translation termination signal TGA at 2558 to 2560.
• the 5 ' regulatory element of the present invention comprises at position 1712 up to 1719 the putative TATA-box "tataaata" .
• sequences depicted in SEQ ID Nos. 1 to 7 comprise the putative TATA-box and are particularly useful for the purposes described in the present invention.
• the 3' regulatory element of the present invention in particular the elements described in SEQ ID No. 12 and 13 comprise at position 2957 the polyadenylation signal.
• the 3' regulatory element located 3' to SEQ ID No. 12 and 13 and depicted in SEQ ID No. 15 may prove particularly useful as expression enhancing element .
• the coding sequence of zmGRP3 codes for a glycin-rich protein, whose transcripts accumulate exclusively in roots of young maize seedlings following developmentally specific patterns.
• the protein exhibits a hydrophobic domain at the N-terminal region followed by repeated glycin-rich motifs.
• the hydrophobic domain at the N-terminal region is believed to be part of an ER-type signal peptide.
• the obtained genomic clone is the first nucleic acid sequence encoding a glycin-rich protein expressed exclusively in the root system, in particular that of maize.
• Example 2 Northern analysis
• the transcription of zmGRP3 mRNA could be identified to be closely connected with the formation of crown roots.
• the following northern analysis was carried out. mRNA was isolated from various organs such as kernels, shoot meristems, leaves, coleoptilar nodes, tassels, various zones of primary, lateral, seminal and crown roots and root tips of lateral roots . The mRNA was then on a gel and hybridized with the zmGRP3 cDNA isolated above.
• FIG. 4A coelopti- lar node with emerging young crown roots (lane 1) , shoot meris- tem (lane 2) , root tips of crown roots (lane 3) , of primary roots (lane 4) , or of lateral seminal roots (lane 5) , leaves (lane 6) , kernel (12-14 days after pollination, lane 7) , or tassel (lane 8) .
• Fig. 4A coelopti- lar node with emerging young crown roots (lane 1) , shoot meris- tem (lane 2) , root tips of crown roots (lane 3) , of primary roots (lane 4) , or of lateral seminal roots (lane 5) , leaves (lane 6) , kernel (12-14 days after pollination, lane 7) , or tassel (lane 8) .
• Fig. 4A coelopti- lar node with emerging young crown roots (lane 1) , shoot meris- tem (lane 2) , root tips of crown roots (lane 3)
• Lane 5 in C represents root tips (up to 1 cm) of secondary roots.
• the lower parts of A, B, C and D show the ethidium bromide stained gels displaying the 18S and 26S rRNA bands.
• Figure 4B indicates that zmGRP3 mRNA accumulation is developmen- tally and spatially regulated within the root with the highest level of accumulation occurring in the meristematic and elongation region (cm 1) , low levels in early regions of cell maturation (cm2) and again increasing levels in later regions of cell maturation (cm 3 and 4) (cm sections as defined in Held et al , Plant Physiol. (1993) pp. 102, 1001-1008) .
• the zmGRP3 mRNA is predominantly expressed in the root tip.
• a weak hybridization signal is detected in the later stage of root development (cm 4) .
• a possible explanation for this difference in accumulation between crown roots and primary/lateral seminal roots is that the development of primary and lateral seminal roots begins 4 to 5 days before crown roots . The transcript was surprisingly not detected in tips of lateral roots .
• Immature maize embryos crosses between inbred line A188 and H99 with a size of 1.0-1.5 mm were isolated under sterile conditions. They were cultured with the scutellar side facing upward on modified N6 -Medium (D'Halluin et al . 1993) with 1 mg/1 2,4-D, solidified with 0.8% agarose or 0.3% phytagel .
• the explants were bombarded 4 to 12 days after isolation.
• the embryos were placed with the scutellar side facing upward on MSC-medium (Zhong et al . 1992) in which a higher osmotic value was adjusted with sucrose to 670 mOsm/kg.
• the osmotic treatment was started 4 h before bombardment and was continued for 20 to 24 h after bombardment.
• the embryo was cut immediately or one day after isolation.
• Particle acceleration was performed with the PDS 1000/He gun (BioRad, Kunststoff, Germany) .
• Five ⁇ g plasmid DNA was precipitated onto gold particles with an average size of 0.4-1.2 ⁇ m (Heraeus,
• Particles gold (size: 0.4-1.2 ⁇ m)
• the p35Spat gene (Brettschneider et al . , 1997) was used as selection marker gene.
• 2.5 ⁇ g DNA of the plasmid with the selection marker gene was mixed with the second plasmid containing the promoter-reporter gene and precipitated together onto the particles .
• the promoter used for transient and stable transformation experiments has the sequence depicted in SEQ ID No. 1 and was fused to a GUS reporter gene. Further deletion constructs in the promoter region were made which are depicted in Fig. 2 and 3.
• Figure 2 depicts 5' deletion constructs (Fig. 2 (b) ) of the full length promoter of SEQ ID No. 1 (Fig. 2(a)), while Figure 3 shows 3' deletion constructs (Fig. 3 (b) ) of the full length promoter of SEQ ID No. 1 (Fig. 3 (a)).
• Figure 2a is a schematic representation of the cloned promoter fragment of zmGRP3.
• the positions of the restriction sites of the following restriction enzymes are presented (the numbers denote the nucleotide positions of the restriction sites in the promoter fragment) :
• X Xhol (1)
• G Bgl II (1206)
• V Pvul (120)
• K Kpnl (1354)
• N BstNI (572, 1760)
• S Sad (664)
• B BamHl (698, 1014)
• Z BstXl (412), C.- Clal (1497, 1525, 1570)
• D Oral (906)
• the position of the putative TATA box is indicated.
• Figure 2b gives an overview of the 5' promoter deletion clones prepared. Presented is in each case the length of the promoter fragment still present (boldface) , as well as the size of the region deleted from the 5' end (in brackets) of the promoter. The fragments were cloned in a vector that contained the ⁇ - glucuronidase gene (uid A) .
• Figure 3a is a schematic representation of the cloned promoter fragment of zmGRP3. The positions of the restriction sites of restriction enzymes are presented as in Fig. 2a.
• Figure 3b is an overview of the 3 ' promoter deletion clones prepared. Presented is in each case the length of the promoter fragment still present (boldface) , as well as the size of the region deleted from the 3' end (in brackets) of the promoter. The fragments were cloned in a vector that contained the ⁇ - Glucuronidase Gen (uid A) downstream from the -46bp-region of the CaMV 35S promoter (with TATA box). Thus, these 3' deletion constructs contain the CaMV 35S promoter initiation site.
• Figure 5 shows further gus-fusion constructs using the regulatory elements of the present invention (not to scale) .
• the boxes marked "GUS” symbolise the coding region of the uidA gene from E. coli, the position of the start and stop codons is indicated in each case.
• the 35S promoter from pBI121 was deleted in the constructs produced (pBI121-35S) or replaced by the GRP3- promoter (pBI-GRP3-Prom, pBI121-GRP3-Prom-3 ' ) or the GRP3- promoter + 2x35S-Enhancer (pBI212-2x35S-En-GRP3-Prom-3 ' ) .
• Figure 5 thus shows further gus-fusions constructs using the regulatory elements of the present invention.
• PBI 121 is a positive control while PBI-121-35S is a negative control.
• PBI-121- GRP3-Prom is a construct using the promoter of SEQ ID No . 1 being functionally linked to a gus-reporter gene and the NOS- terminator.
• PBI-121-GRP3-Prom 3 ' is a DNA construct of the present invention using the promoter element of SEQ ID No. 1 functionally linked to the gus-reporter gene and a 3 ' regulatory element of the present invention having the sequence of SEQ ID No. 12 and added 3' thereto of SEQ ID No. 15.
• the 3' regulatory element spans the region from and excluding the translation stop codon at position 2558-2560 towards the 3' end up to position 4490.
• This 3' regulatory element being the combination of SEQ ID No. 12 and SEQ ID No. 15 has also been used in DNA construct PBI-121-2 X 35S-EN-GRP3-Prom-3 ' which is similar to the above described construct but additionally has the 2 X 35S-enhancer (nucleotide positions 7089 to 7357 and -346 to -78 of the CaMV virus genome relative to the start of transcription described in Fang et al . (1989) and Franck et al . (1980); data base accession No. V00141 Y 02048 VRL 12. Sept. 1993; NID: g 58821) .
• Explants were cultured after bombardment for two weeks on N6 1- 100-25 medium at 26°C in the dark. Selection was started 14 days after bombardment when the explants were subcultured on N6 1- 100-25 medium without casaminoacids supplemented with 5 mg/1 phosphinotricin (PPT) , the active compound of the commercial herbicide Basta®. After two weeks, the calli were subcultured for another two weeks on N6 1-100-25 medium with 5 mg/ 1 Basta®. For regeneration, embryogenic calli were transferred on hormonefree MS-medium with 1 mg/1 PPT and cultured at 24 °C under light (16 h) .
• PPT phosphinotricin
• Green shoots were transferred to Magenta boxes with half- strength MS-medium and 1 mg/1 PPT solidified with 0.3 % phy- tagel. Plantlets with a height of 6-8 cm which survived selection were transferred into soil after they generated roots . Small plants were cultured in soil in the greenhouse for 1 week under a plastic or glass lid. Routinely, more than 90% of the regenerants survived the transfer into soil. To confirm the selection, surviving plants were sprayed once or twice with a Basta®-solution containing 250 mg/1 PPT and 0.1% Tween. Transformants were verified by southern analysis
• genomic DNA was isolated from primary transformants and their progeny using the protocol of Dellaporta et al (1983) . 10 or 15 ⁇ g of DNA, uncut or digested with restriction enzymes were separated by electro- phoresis and transferred to a Hybond N membrane (Amersham/England) or a BiodyneA membrane (Pall/England) .
• Introduced DNA of the present invention (SEQ ID No. 1) was detected using a modified protocol of the non-radioactive digoxigenin chemiluminescent method (Neuhaus-Url et al . 1993). Filters were hybridised with PCR-labelled Dig probes. Stably transformed transgenic maize plants could be obtained.
• tissue to be analysed was fixed in 4% paraformalde- hyd over night .
• the tissue then was dehydrated in an increasing concentration of ethanol and finally embedded in paraffin.
• tissue was cut in 8-12 ⁇ m slices. After that the slices were hybridised with DIG labelled zmGRP3 RNA sense or antisense probes which had been hydrolytically digested to a size of about
• Hybridisation was carried out in 50% formamid at a tern- perature of 50°C. Washing and the immunological staining was carried out exactly as described by Coen et al . , 1990.

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