CA2341780A1 - Regulatory sequences for root specific or root abundant gene expression in plants - Google Patents

Abstract

The present invention relates to regulatory elements for tissue specific, in particular root specific, expression of genes of interest in plants. The invention also relates to methods to obtain transgenic plants which show root specific expression of genes of interest and to methods for isolating root specific genes and/or root specific regulatory elements.

Description

2 PCT/EP99/06692 Regulatory Sequences for Root Specific or Root Abundant Gene Expression in Plants Description 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 particu-lar in monocotyledonous plants, to vectors containing the regu-latory elements, to host cells containing the vectors, to plants containing the host cells and to methods for isolating regula-tory elements that contribute to tissue-preferred gene expres-sion in plants.
Gene expression is considered to comprise a number of steps from the DNA to the final protein product. Initiation of transcrip-tion of a gene is generally believed to be the predominant con-trolling 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 me-tabolism 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 con-sidered 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 in-crease their commercial value.
It is also considered particularly important to develop tran-scriptional regulatory units that direct gene expression selec-tively to root tissue. Root preferred gene expression will pro-vide 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 ad-verse weather conditions; as well as broadening the range of soils or environments in which said plant may grow. Root abun-dant 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.
Thus, there is a need to provide regulatory elements capable of directing transcription specifically in root tissue. W097/44448 describes promoter elements conferring root preferred gene ex-pression. The disclosed promoter elements were obtained from maize seedlings. However, the isolated promoter elements may not be suitable for any desired transformation problem.
Furthermore, W094/02619 discloses regulatory sequences for root specific or root abundant gene expression derived from Brassica sp. However, this promoter was not shown to work in monocotyle-

-3-donous plants with an efficiency great enough to provide means for commercial use.
Thus, the technical problem underlying the present invention is to provide novel regulatory elements for use in cloning and ex-pressing 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 present invention solves the technical problem underlying the present invention by providing purified and isolated new nu-cleic acid sequences for use in cloning and expressing a root specific or root abundant gene in a plant which are selected from the group consisting of (a) the nucleic acid sequence set out in SEQ ID No. 1 or a part or complementary strand thereof, (b) a nucleic acid sequence which hybridizes to the nucleic acid sequence defined in SEQ ID No. 1 or a complementary strand thereof and (c) alleles or derivatives of the nucleic acid sequence defined in (a) or (b) or a complementary strand thereof.
In particular, the problem is solved by providing a purified nu-cleic 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) al-leles or derivatives of the nucleic acid sequence defined in (al) or (bl) .

-4-In a further particularly preferred embodiment, 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. 3 to 11, (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 abun-dant 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 se-quences defined in (a3) and (c3) alleles or derivatives of the nucleic acid sequences defined in (a3) or (b3).
The 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 pre-ferred embodiment of the present invention these sequences may additionally contain at their 3' terminus the nucleotide se-quence from position 1761 to 1780 as depicted in SEQ ID No. 14, which is CCTGGACTCG CTCACTGGCA.
The sequences set out in SEQ ID No. 12, 13 and 15 comprise iso-lated 3' regulatory elements and are considered to be 3' tran-scription regulatory elements, or parts thereof, for instance polyadenylation signals. The sequences of SEQ ID Nos. 12 and 15

-5-linked in 5' to 3' direction to each other represent the com-plete 3' regulatory element of the present invention.
These sequences are capable of modulating, in particular con-tributing to, or terminating the transcription of nucleic acid sequences operably linked to them.
The sequence set out in SEQ ID No. 14 is a genomic clone com-prising all of the above sequences except the 3' regulatory ele-ment 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 par-ticular, these sequences have a degree of identity of 70% to the sequence of SEQ ID Nos. 1 to 15.
In the context of the present invention, nucleic acid sequences which hybridise to any one of the specifically disclosed se-quences of SEQ. Id. Nos. 1 to 15 are sequences which have a de-gree of 60% to 70% sequence identity to the specifically dis-closed sequence on nucleotide level. In an even more preferred embodiment of the present invention, sequences which are encom-passed by the present invention are sequences which have a de-gree of identity of more than 70% or 80% and even more preferred more than 90%, 95% or 99% to the specifically disclosed se-quences 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.

-6-Thus, 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
Hybridisation temperature: 65° C
First wash: 2 x SSC; 0.5% SDS at room temperature Second wash: 0.2 x SSC; 0.5% SDS at 65°C.
More preferably, 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 hy-bridisation temperature and second wash temperature of 70° C is applied.
Thus, the present invention also comprises functionally equiva-lent 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 men-tioned above which are defined as sequences being essentially similar to the above sequences but comprising, for instance, nu-cleotide 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 pref-erably from maize roots.

-7-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. Thus, the present invention provides the means for isolation of transcrip-tion regulatory elements that direct or contribute to tissue-preferred gene expression in plants, in particular in monocoty-ledonous plants, such as maize. The present invention also pro-vides 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 monocoty-ledonous plants such as maize or of dicotyledonous plants such as sugar beets (Beta vulgaris?. Thus', the present invention provides the means to direct the expression of a gene of inter-est in a tissue-specific or tissue-abundant manner in roots, for instance in roots of seedlings. Accordingly, the proteins en-coded by the gene of interest can be accumulated in roots. For instance, 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.
In the context of the present invention, a number of terms shall be utilized as follows.
In the context of the present invention the term "disease" en-compasses any adverse condition caused to a plant by a virus or

-8-an organism other than itself, such as fungi, bacteria and in-sects.
The term "promoter" refers to a sequence of DNA, usually up-stream (5') 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 (or "3' end") refers to that portion of a gene comprising a DNA segment; excluding the 5' sequence which drives the initiation of transcription and the structural por-tion 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 charac-terized 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 5'-AATAA-3', 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.
The term "nucleic acid sequence" refers to a natural or syn-thetic polymer of DNA or RNA which may be single or double stranded, alternatively containing synthetic, non-natural or al-_g_ tered nucleotide bases capable of incorporation into DNA or RNA
polymers.
The term "gene" refers to a DNA sequence that codes for a spe-cific protein and regulatory elements controlling the expression of this DNA sequence.
The term "regulatory element" refers to a sequence located up-stream (5'), within and/or downstream (3') to a coding sequence whose transcription and expression is controlled by the regula-tory element, potentially in conjunction with the protein bio-synthetic apparatus of the cell. "Regulation" or "regulate" re-fer to the modulation of the gene expression induced by DNA se-quence elements located primarily, but not exclusively upstream from (5') the transcription start of the gene of interest.
Regulation may result in an all or none response to a stimula-tion, or it may result in variations in the level of gene ex-pression.
The term "coding sequence" refers to that portion of a gene en-coding a protein, polypeptide, or a portion thereof, and exclud-ing the regulatory sequences which drive the initiation or ter-mination of transcription. The coding sequence or the regula-tory 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 nu-clear 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 syn-thetic.
The term "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.
As used herein, "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, em-bryos and callus tissue. Within the context of the present in-vention, the term "root" includes various groups of primary, lateral seminal or crown roots, and root tips. Plant-derived tissues may be in plants, 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 ab-sorbs food. A "dicotyledonous plant" refers to a plant whose seeds have two cotyledons.
As used herein, "transformation" refers to the process by which cells, tissues or plants acquire properties encoded on a nucleic _11_ acid molecule that has been transferred to the cell, tissue or plant.
"Transformation" and "transferring" refers to methods to trans-fer DNA into cells including, but not limited to, particle bom-bardment, microinjection, permeabilizing the cell membrane with various physical (e. g., electroporation) or chemical (e. g., polyethylene glycol, PEG) treatments.
The term "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 se-quence will be present in the host cell in a higher copy number than naturally occurring.
The term "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 se-quences when transformed into plant tissue.
The term "expression" as used herein is intended to describe the transcription and/or coding of the sequence for the gene prod-uct. In the expression, 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 trans-lated into the above mentioned gene product if the gene product is a protein. However, expression also includes the transcrip-tion of DNA inserted in antisense orientation to its regulatory elements. Expression, which is constitutive and possibly fur-ther enhanced by an externally controlled promoter fragment ~CA 02341780 2001-03-06 WO 00/t5662 PGT/EP99/06692 thereby producing multiple copies of mRNA and large quantities of the selected gene product, may also include overproduction of a gene product.
The term "translation start codon" or "initiation codon" refers to a unit of three nucleotides (codon) in a nucleic acid se-quence that specifies the initiation of protein synthesis.
The term "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.
The term "signal sequence" refers to a nucleotide sequence that encodes a signal peptide. The term "ER targeting signal" refers to the N-terminal extension of a polypeptide, Which is trans-lated 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 unre-lated 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. The term "ER targeting sequence" refers to a nu-cleotide sequence that encodes the ER targeting signal.
A "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 control-ling 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. Here, "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.
The term "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. The term "root abundant nucleic acid sequence'' refers to nucleic acid se-quences, 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.
In a particularly preferred embodiment of the present invention the nucleic acid sequences, i.e. the 5' and/or 3' regulatory elements of the present invention contained in the vector, are operably linked to 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. Such a gene of interest may be a gene, in particular its coding sequence, conferring, for instance, disease resistance; drought resistance; insect resis-tance; herbicide resistance; immunity; an improved intake of nu-trients, minerals or water from the soil; or a modified metabo-lism in the plant, particularly its roots. Such a modified me-tabolism may relate to a preferred accumulation of useful sub-stances in roots, for instance sugars or, vice versa, in the de-pletion of substances undesirable in roots, for instance certain amino acids. Thus, in the context of the present invention, 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 gene of interest includes also a coding sequence cloned in antisense orientation to the regulatory sequences di-recting its expression. Such a construct may be used specifi-cally to repress the activity of undesirable genes in plant cells, in particular in roots, most preferably in primary, lat-eral seminal and/or crown roots. The gene of interest may also comprise signal sequences, in particular ER targeting sequences, _15_ directing the encoded protein in the ER and eventually for in-stance in the cell wall, vascular tissue and /or the vacuole.
In a particularly preferred embodiment the vector defined above is comprised of further regulatory elements directing or enhanc-ing 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 in-serted within or outside the gene of interest.
In a particularly preferred embodiment of the present invention 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.
Thus, the 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 inven-tion may also be useful to regulate the expression of genes of interest depending upon the developmental stage of the trans-ferred cell or tissue. Furthermore, the present invention al-lows the specific modification of the metabolism in roots.
In a particularly preferred embodiment of the present invention the vector furthermore contains T-DNA, in particular the left, the right or both T-DNA borders derived from Agrobacterium tume-faciens. Of course, 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-_..... __._...____.__.. . ~ 02341780 2001-03-06 faciens mediated transformation of cells. In a preferred em-bodiment of the present invention, 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, techni-cal 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. In particular, the cell being transformed by the method of the present invention is a plant, bacterial or yeast cell. In a particularly preferred embodiment of the present in-vention, the above method further comprises the regeneration of the transformed cell to a differentiated and, in a preferred em-bodiment, fertile plant.
The method to transform a cell with a vector according to the present invention may be any method known to effectively trans-fer nucleic acid sequences into a host cell, thereby allowing its expression therein and eventually the regeneration of the cell to a plant. In a particularly preferred embodiment of the present invention, the transfer of the nucleic acid sequence may be mediated by Agrobacterium tumefaciens or rhizogenes. How-ever, any other method known to eventually achieve the same re-sults may be used, such as direct uptake of nucleic acid se-quences, 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-WO 00/15662 PCf/EP99/06692 cotyledonous plant cells. The present invention also relates to cell cultures, tissue, calluses, etc. comprising a cell accord-ing to the above, i.e. a transgenic cell.
Furthermore, 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 tis-sues, 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. Preferably, the nucleic acid se-quences of the present invention are linked to genes of interest or antisense DNA normally not found in the transferred cell op-erably linked to the nucleic acid sequence of the present inven-tion, at least not in that order or orientation.
Thus, the present invention also relates to roots, leaves, cal-luses, 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 in-terest encoding a protein of interest or being an antisense con-struct.
Thus, the invention relates to plants, nonbiologically trans-formed, which possess, stably integrated in the genome of their cells, a heterologous or autologous nucleic acid sequence con-taining a regulatory element of the present invention recognised WO 00/15662 PCf/EP99/06692 _18_ by the polymerases of the cells of said plants and a gene of in-terest encoding a protein of interest or being an antisense con-struct.
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 po-lymerases of the cells.
Finally, 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 de-rived 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 op-erably linked to them.
Further preferred embodiments of the present invention are men-tioned in the claims.
The invention may be more fully understood from the following detailed sequence descriptions which are part of the present teaching. The SEQ ID Nos. 1 to 15 are incorporated in the pres-ent invention. The positions indicated below refer to the se-quence 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 by (base pair) sequence spans the region from and including position 1760 to-wards the 5' end, up to and including position 1.

_19_ SEQ ID No. 2 is a partial DNA sequence of the zmGRP3 gene of Zea mat's . The 1348 by 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 mat's. The 1096 by 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 mat's. The 746 by 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 mat's. The 554 by 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 mat's. The 406 by 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 mat's. The 190 by 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 mat's. The 1354 by 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 by 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 by sequence spans the region from and in-cluding 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 by sequence spans the region from and in-cluding 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 by sequence spans the region from and exclud-ing 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 by sequence spans the region from and exclud-ing 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 encom-passing the coding region of the gene, a sequence of 20 by be-tween 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 by sequence spans the region from and exclud-ing the moat 3' located base "C" at position 3416 of SEQ ID No.
14 to the sequence motif "TCC" at position 4488-4490. Thus, the complete genomic DNA sequence of zmGRP3 comprises 4490 bp.
The invention is further illustrated by way of example and the following drawings.
Figure 1 shows a restriction map of the genomic clone zmGRP3 de-scribed 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.
Example 1: Cloning of the genomic zmGRP3 sequence The 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 devel-oping young crown roots was made by choosing 7-9 days old wild-type maize seedlings. mRNA from 30-40 of the wild-type coleop-tilar 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. Af-ter elimination of cDNAs with a size less than 0.4 kb (kilobases) the cDNAs were ligated into the phage vector ~,-ZAP-II
(Stratagene) using the ligation kit "Gig-Pack-Gold" (Stratagene, Heidelberg). The phages were plated on E, coli strain XL 1B.
The obtained library had a titre of 3.7 x 106 pfu/ml. The in-serts had an average size of 1.4 kb. The phages of the cDNA li-brary 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.
As probes radioactively labelled 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 forma-tion of crown roots) (Hetz, et aI. Plant Cell, 1996. Pp. 10, 845-85?). The first strand cDNA was labelled using radioactiv-ity and, for further analysis, digoxigenin.
Radioactive Labelling of first strand cDNA:
Materials:
ca. 1-3 ug mRNA
5x RT-Buffer 250 mM Tris/HCl pH 8.5 40 mM MgClz 150 mM DTT
dNTP-Mix dTTP, dGTP, dATP, ea 5 mM
dCTP 1.3 mM
Random primer (0.8 ug/~tl) RNase-Inhibitor (30 U/~1) 32P-dCTP(10 ~ Ci/~C1) Superscript Reverse Transcriptase (Gibco, 100 U/P.l) For standard labelling 1-3 ~g of mRNA were dissolved in 25.5 ul of H20, denaturated for 10 minutes at 65°C, then cooled in ice water. The following agents were added in order: 10 P1 5 x RT-Buffer, 1 ~.l of Rnasin, 2.5 ~1 of dNTP-Mix, 5 ul of random primer, 5 P1 (=50 ~,Ci) '2P-dCTP, and 1 ~1 of Superscript Reverse Transcriptase. The preparation was mixed thoroughly and incu-bated for 2 hours at 42°C. A final purification was done using the "Nucleotide Removal Kit" (Quiagen, Hilden).
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 la-belled With digoxigenin and hybridized in a northern blot analy-sis against 10 ~g total RNA from wild-type and mutant coleopti-lar nodes.
The labelling with digoxigenin of first strand-cDNA was done as follows:
Materials:
ca . 1-3 P,g mRNA
5x RT-Buffer 250 mM Tris/HC1 pH 8.5 40 mM MgCl2 150 mM DTT
dNTP-Mix dCTP, dGTP, dATP, ea 5 mM
dTTP 1.3 mM
RNase-Inhibitor (Fermentas, 30 U/~C1) Superscript Reverse Transcriptase (Gibco, 100 U/P,1) Digoxigenin dUTP (Boehringer) Oligo dT (0.6 ~g/~1) EDTA solution (0.2 M) LiCl solution (4 M) EtOH (100%, 70%) The mRNA was denaturated for 5 minutes in 23.4 ~1 of HBO at 65°C, then rapidly cooled in ice water. The following agents were added in order: 10 ~.1 5x RT-Buffer, 1 ul of RNase-Inhibitor (ca. 20 units), 5 ul of oligo-dT (final concentration 0.06 ~Cg/ul) 3.6 ~.1 of DIG-dUTP (final concentration 0.13 mM) , 5 P.1 of dNTP-mix and 2 ~1 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 ~.1 of EDTA so-lution, 5 ~.1 of LiCl solution and 130 ~1 of 100% EtOH. Follow-ing a washing with 70% EtOH, the cDNA resuspended in 50 ~.1 of H,O
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 ab-sence 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 ex-clusively 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 par-tially 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".
The sequences depicted in SEQ ID Nos. 1 to 7 comprise the puta-tive TATA-box and are particularly useful for the purposes de-scribed in the present invention.
The 3' regulatory element of the present invention, in particu-lar the elements described in SEQ ID No. 12 and 13 comprise at position 2957 the polyadenylation signal. The 3' regulatory ele-ment 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 pro-tein exhibits a hydrophobic domain at the N-terminal region fol-lowed by repeated glycin-rich motifs. The hydrophobic domain at the N-terminal region is believed to be part of an ER-type sig-nal peptide. The obtained genomic clone is the first nucleic acid sequence encoding a glycin-rich protein expressed exclu-sively in the root system, in particular that of maize.

Example 2: Northern analysis As the tissues to be compared, i.e. wild-type and mutant tissues differed only in the presence or absence of crown roots, the transcription of zmGRP3 mRNA could be identified to be closely connected with the formation of crown roots. However, to deter-mine whether the zmGRP3 transcription could also be detected in other tissues or other types of roots, primary roots, lateral seminal roots, and lateral roots (of maize) the following north-ern 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.
Figure 4 A to D illustrate the detection of zmGRP3 transcripts in northern analysis. After electrophoretic separation and blotting, the RNAs (10 ug) were hybridized with the DIG-labelled zmGRP3 cDNA. Total RNAs were isolated from; 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 tas-sel (lane 8). Fig. 48, C and D represent RNA samples taken from defined root segments of crown roots (B), of primary roots (C), or of lateral seminal roots (D) : root tip up to cm 1 (lane 1) , cm 2 ( lane 2 ) , cm 3 ( lane 3 ) , arid cm 4 ( lane 4 ) . 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 ~ 02341780 2001-03-06 r,.,... _._,...

_27_ level of accumulation occurring in the meristematic and elonga-tion region (cm 1) , low levels in early regions of cell matura-tion (cm2) and again increasing levels in later regions of cell maturation ( cm 3 and 4 ) ( cm sect ions as def fined in Held a t a1, Plant Physiol. (1993) pp. 102, 1001-1008). In crown roots the zmGRP3 mRNA is predominantly expressed in the root tip. A weak hybridization signal is detected in the later stage of root de-velopment (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.
Example 3: Preparation of transgenic plants Materials and Methods:
Isolation and culture of immature embryos for particle bombard-ment Immature maize embryos (crosses between inbred line A188 and H99) with a size of 1.0-1.5 mm were isolated under sterile con-ditions. They were cultured with the scutellar side facing up-ward 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. For the osmotic pre-treatment, the embryos were placed with the scutellar side facing upward on MSC-medium (thong et aI. 1992) in which a higher osmotic value was adjusted with sucrose to 670 mOsm/kg. The osmotic treatment was started 4 h before bombard-ment and was continued for 20 to 24 h after bombardment. In ex-periments where the rhizobial part of the embryo was bombarded, the embryo was cut immediately or one day after isolation.
Particle bombardment Particle acceleration was performed with the PDS 1000/He gun (BioRad, Munich, Germany). Five Pg plasmid DNA was precipitated onto gold particles with an average size of 0.4-1.2 ~m (Heraeus, Karlsruhe, Germany) following a protocol described by BioRad (Munich, Germany) and modified according to Becker et a1.
(1994). For stable transformation experiments, the particle-DNA
pellet was resuspended in 240 P1 ethanol. For each bombardment, 3.5 ~1 were spread on the macrocarrier.
Parameters used for particle bombardment Distance between A: rupture disk and macrocarrier 2.5 cm B: macrocarrier and stopping screen 0.8 cm C: stopping screen and target cells 5.5 cm Gas pressure 1350 psi Partial vacuum 28 inch Hg Particles gold (size: 0.4-1.2 Vim) Amount of particles 30 Pg per shot Constructs In experiments for stable transformation, the p35Spat gene (Brettschneider et al., 1997) was used as selection marker gene.
In co-transformation experiments 2.5 ~g DNA of the plasmid with the selection marker gene was mixed with the second plasmid con-taining 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)).
In detail, 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: Xho1 (1), G: Bgl II (1206), Y:
Pvul (120), K: Kpnl (1354), P:Pstl (588), N: BstNl (572, 1760), S: Sacl (664), B: BamHl (698, 1014), Z: BstXl (412), C:Clal (1497, 1525, 1570), D: Dra1 (906), Y: Xbal (963). Furthermore, 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 B-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 i~-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.
Expression studies For transient expression studies, different maize tissues, i.e.
roots, leaves of seedlings, germinating embryos, were bombarded with the promoter constructs identified above. For histochemi-cal GUS-detection, the bombarded tissues were incubated one day after bombardment at 37°C for 20 h in X-gluc staining solution (McCabe et aI. 1988). After staining leaf tissues with x-gluc chlorophyll was extracted in ethanol/acetic acid glacial (3:1) for 30 to 60 min at 65°C and washed once with 70% ethanol.
For quantification of the promoter strength, bombarded tissues were analysed in a protein GUS-assay (Jefferson et al. 1987).
For standardisation of the transformation frequency, the pro-moter constructs were co-transformed with a luciferase reporter gene under control of the actin promoter from maize (positive control) . As negative control, pUC 19 DNA was used in the same amount as the promoter constructs. One day after bombardment, proteins of the bombarded tissue were extracted and analysed for GUS and LUC activity. The analysed root tissue exhibited GUS
and LUC activity, while the other bombarded tissues only showed LUC activity. These results clearly evidence root specificity of the promoter element of the present invention.
The table I below shows the results of the transformation of 5' deletion constructs.

Promoter Root Leaf Embryo SEQ ID No. 1, 0 + - -SEQ ID No. 2, -412 + - -SEQ ID No. 3, -664 + - -SEQ ID No. 4, -1014 + - -SEQ ID No. 5, -1206 + - -SEQ ID No. 6, -1354 + - -SEQ ID No. 7, -1570 + - -negative control - - -positive control + ~ + +
I

Table I: Transformation of various maize tissues with 5' dele-tion promoter constructs.
All of the tested promoter constructs clearly and unambiguously show expression in roots, but not in leaves or in the embryo.
Even the smallest promoter fragment (SEQ ID No 7, 190 by length) shows significant expression exclusively in the root.
Figure 5 shows further gus-fusion constructs using the regula-tory 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, pBIl21-GRP3-Prom-3') or the GRP3-promoter + 2x35S-Enhancer (pBI212-2x35S-En-GRP3-Prom-3'). In some cases moreover the 3' region of GRP3 (pBI121-GRP3-Prom-3', pBI121-2x35S-En-GRP-Prom-3') was inserted instead of the NOS-terminator. The positions of restriction endonuclease-sites relevant to this are indicated under the relevant constructs (H:

HindIII, L: Sal/I, X: XbaI, B: BamHI, M: SmaI, S: SacI, E:
EcoRI ) .
Figure 5 thus shows further gus-fusions constructs using the regulatory elements of the present invention. PBI 121 is a posi-tive control while PBI-121-35S is a negative control. PBI-121-GRP3-Prom is a construct using the promoter of SEQ ID No. 1 be-ing functionally linked to a gus-reporter gene and the NOS-terminator. PBI-121-GRP3-Prom 3' is a DNA construct of the pres-ent invention using the promoter element of SEQ ID No. 1 func-tionally 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. Therefore 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 combi-nation 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 transcrip-tion 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).
The following table II shows the expression results in transient essays in roots and leaves. Accordingly, it can be shown that both the 5' and 3' regulatory elements of the present invention contribute to tissue specific expression in roots indicating that DNA constructs using both the 5' and 3' elements of the present invention or the elements separately may prove useful in genetic engineering of plants.

WO 00/15662 PCT/EP99/06b92 Table II: Transient expression studies Construct Activity in the Activity in the Transient Assay Transient Assay in Roots in Leaves pBIl21 +++++ +++++

pBI121-35S

pBI121-GRP3-Prom +

pBI121-GRP3-Prom-3' ++

pBI121-2x355-En-GRP3-Prom-3'+++

(+: strong expression, degree indicated by number of "+", . no or hardly any expression).
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 un-der light (16 h) .
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 selec-tion 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 se-lection, 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 DNA isolation and Southern blot hybridisation For the analysis of stable transformants, total 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) . In-troduced DNA of the present invention (SEQ ID No. 1) was de-tected using a modified protocol of the non-radioactive digoxi-genin chemiluminescent method (Neuhaus-Url et al. 1993). Fil-ters were hybridised with PCR-labelled Dig probes. Stably transformed transgenic maize plants could be obtained.
Example 4 In situ hybridization experiments of various types of maize roots with zmGRP
Method:
In situ hybridisation experiments were carried out according to a modified protocol from Coen et al., 1990 (Cell 63, 1311-1322).
First the 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.
The 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 200 bp. Hybridisation was carried out in 50% formamid at a tem-_g5_ perature of 50°C. Washing and the immunological staining was carried out exactly as described by Coen et al., 1990.
Results:
Longitudinal sections of different 5 day old root types were used. These slices were derived from primary root as well as from lateral crown roots and from stem derived crown roots. The embryogenic primary root as well as lateral and stem derived crown roots showed the same hybridisation pattern. In all cases there was no signal with the sense probe, whereas With the an-tisense probe there was a strictly root specific hybridisation.
This hybridisation was cell specifically and was restricted to the epidermal cell layer and cells of the columella root cap.
In all other tissue types there was no hybridisation signal.
References Becker, D., Brettschneider, R. and Lorz, H. (1994) Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. The Plant Journal 5, 299-307.
Brettschneider et al (1997) Efficient transformation of scutella tissue of immature maize embryos. Theoretical Applied Genetics (94), 337.
Coen et al., 1990 (Cell 63, 1311-1322).
Dellaporta, S.L., Wood, J. and Hicks, 3.8. (1983) A plant DNA
minipreparation: Version II Plant Mol. Biol. Rep. 4, 419-21.
D~Halluin, K., Bonne, E., Bossut, M., De Beuckeleer, M. and Lee-mans, J. (1993) Transgenic maize plants by tissue electropora-tion. The Plant Cell 4, 1495-1505.
Fang et al., (1989) Plant Cell 1, 141-150 Franck et al., (1989) Cell 21 (1), 285-294 McCabe, D.E., Swain, W.F., Martinelli, B.J. and Christou, P.
(1988) Stable transformation of Soybean (Glycine max) by parti-cle acceleration. Bio/Technology 6, 923-926.
McElroy, D., Zhang, W., Cao, J. and Wu, R. (1990) Isolation of an efficient Actin promoter for use in rice transformation. The Plant Cell 2, 163-171.
Neuhaus-Url, G, and Neuhaus, G. (1993) The use of the non-radioactive dioxigenin chemiluminescent technology for plant genomic Southern blot hybridisation: a comparison with radioac-tivity. Transgenic Research 2, 115-120.
Zhong, H., Srinivasan, C. and Sticklen, M.B. (1992) In-vitro morphogenesis of corn (Zea mays L.) Planta 187, 483-489.

SEQUENCE LISTING
<110> Agricultural Technology and Genetics GmbH
<120> Regulatory Sequences for Root Specific or Root Abundant Gene Expression in Plants <130> 23143 ATG
<140>
<141>
<150> EP 98117251.3 <151> 1998-09-01 <160> 15 <170> PatentIn Ver. 2.1 <210> 1 <211> 1760 <212> DNA
<213> Zea mays <400> 1 ctcgagatgt tggatagaag aggaattgcg agcaaccgag gtctccaagg cacgccaacg 60 cgcatcctgc gcagtgaggt ggtcgctgat gtcgcgaagg agcttcgatt gttcgtcgat 120 cgcgagcttc agatttggct ccatgacccc agattgtttg gcgcggcgcg tgtaatgagc 180 agacgccaga cccgagaccg gtgtctccga taccaattgt tagcaatagc agcggacaag 240 cagcgaacaa tccgagagac atagtttggg ggaggagcag atagcttgga gaggaagaag 300 aacaatagtg acagcgtaga ttcctctctg gtctcctctg ttaccctctc tcaatctaat 360 actcctatta caaggtcggc ccatcaggcg acccagtcgc ggttccacga acttgggatt 420 gggtttgtgc ggacgccgac tggttcttgg ctctgctgga gtcttgtctg tggagctgtt 480 gctcgcgctg ctaacaatct cttgttgttt gcgacgactt cgcgtggcca cgcgttaact 540 gcgctctgct caaagaaatt gcctatacag tgcctggcgt cagctgcagg cgttgaatcc 600 gaggtcgcca gtacgaggca aacgctgagc tgcatgctgt accggcttta ttaataactg 660 agctctactc gctacgttag ctataggcac cagaggcgga tccagagggg gggcaaagta 720 ggccatggcc ccccctcaat tttgtacacc cttttatacc taatgattat ttagtattaa 780 gtgtagaaaa aatcaaaatt ttagatagta taaaaatctt gttctaaatc tctaaaacta 840 atagctagtt gctaaaatta gccaaaaggt ttagaatgga tcaactaatt gttagctata 900 ccctttaaat aagtattaat tgttagatgg cccaacctag ctaaaaccag ttaacagtta 960 gctctagatg tttagaacat ggcctaaaca accatattat gagcctactc gccggatcct 1020 ctcggttcag tcctagcatc taccagtctg actctaatct tcccgacacc acctaaacca 1080 gtctgctcgc tgccctgttt ccgccaccga cccattcccg atgcgacgtc acccgacttc 1140 gccccttcgt agcgcctccg tcccttgtcc tgtttttctc gtgacgtcga tccgctcgac 1200 aaattagatc tcgctcgtcg caacgatatg acttatcccc tgcggtcggt ggacttgacg 1260 aactagaaaa ttatggctct catgtggtct ttttggcccc tcctaaattt tcgtcctgcg 1320 tccgccactc tatagctgcg ggaqtgcggg gtaccgtatc gtgaatctga agtcgaacag 1380 tgatggcgta ctatctaatg tcccgtgcag taatatcact gttgccgacg atgggaatct 1440 ctagttttga cagaaaccaa ggcaactgct atgctagcta attaattcca gagagatcga 1500 tttctacagt gctagctgca acaatcgatg caattggcat cagacgatat atgctaatgg 1560 ttttctttat cgatacgtgg tcaacagtgc gatccagatt acagagttct ctcgccctga 1620 tcggatctca tcgcacatgg acacccatct gccaacccaa cgcggggcgg ggaacccgaa 1680 SUBSTITUTE SHEET (RULE 26) acatcgcgtc catgcacgac ccccacgcag ctataaatac ccatgcaatg caatgcagcg 1740 ggtcatcatc atcgactcca 1760 <210> 2 <211> 1348 <212> DNA
<213> Zea mat's <400> 2 ttgggattgg gtttgtgcgg acgccgactg gttcttggct ctgctggagt cttgtctgtg 60 gagctgttgc tcgcgctgct aacaatctct tgttgtttgc gacgacttcg cgtggccacg 120 cgttaactgc gctctgctca aagaaattgc ctatacagtg cctggcgtca gctgcaggcg 180 ttgaatccga ggtcgccagt acgaggcaaa cgctgagctg catgctgtac cggctttatt 240 aataactgag ctctactcgc tacgttagct ataggcacca gaggcggatc cagagggggg 300 gcaaagtagg ccatggcccc ccctcaattt tgtacaccct tttataccta atgattattt 360 agtattaagt gtagaaaaaa tcaaaatttt agatagtata aaaatcttgt tctaaatctc 420 taaaactaat agctagttgc taaaattagc caaaaggttt agaatggatc aactaattgt 480 tagctatacc ctttaaataa gtattaattg ttagatggcc caacctagct aaaaccagtt 540 aacagttagc tctagatgtt tagaacatgg cctaaacaac catattatga gcctactcgc 600 cggatcctct cggttcagtc ctagcatcta ccagtctgac tctaatcttc ccgacaccac 660 ctaaaccagt ctgctcgctg ccctgtttcc gccaccgacc cattcccgat gcgacgtcac 720 ccgacttcgc cccttcgtag cgcctccgtc ccttgtcctg tttttctcgt gacgtcgatc 780 cgctcgacaa attagatctc gctcgtcgca acgatatgac ttatcccctg cqgtcggtgg 840 acttgacgaa ctagaaaatt atggctctca tgtggtcttt ttggcccctc ctaaattttc 900 gtcctgcgtc cgccactcta tagctgcggg agtgcggggt accgtatcgt gaatctgaag 960 tcgaacagtg atggcgtact atctaatgtc ccgtgcagta atatcactgt tgccgacgat 1020 gggaatctct agttttgaca gaaaccaagg caactgctat gctagctaat taattccaga 1080 gagatcgatt tctacagtgc tagctgcaac aatcgatgca attggcatca gacgatatat 1140 gctaatggtt ttctttatcg atacgtggtc aacagtgcga tccagattac agagttctct 1200 cgccctgatc ggatctcatc gcacatggac acccatctgc caacccaacg cggggcgggg 1260 aacccgaaac atcgcgtcca tgcacgaccc ccacgcagct ataaataccc atgcaatgca 1320 atgcagcggg tcatcatcat cgactcca 1348 <210> 3 <211> 1096 <212> DNA
<213> Zea mat's <400> 3 ctactcgcta cgttagctat aggcaccaga ggcggatcca gagggggggc aaagtaggcc 60 atggcccccc ctcaattttg tacacccttt tatacctaat gattatttag tattaagtgt 120 agaaaaaatc aaaattttag atagtataaa aatcttgttc taaatctcta aaactaatag 180 ctagttgcta aaattagcca aaaggtttag aatggatcaa ctaattgtta gctataccct 240 ttaaataagt attaattgtt agatggccca acctagctaa aaccagttaa cagttagctc 300 tagatgttta gaacatggcc taaacaacca tattatgagc ctactcgccg gatcctctcg 360 gttcagtcct agcatctacc agtctgactc taatcttccc gacaccacct aaaccagtct 420 gctcgctgcc ctgtttccgc caccgaccca ttcccgatgc gacgtcaccc gacttcgccc 480 cttcgtagcg cctccgtccc ttgtcctgtt tttctcgtga cgtcgatccg ctcgacaaat 540 tagatctcgc tcgtcgcaac gatatgactt atcccctgcg gtcggtggac ttgacgaact 600 agaaaattat ggctctcatg tggtcttttt ggcccctcct aaattttcgt cctgcgtccg 660 ccactctata gctgcgggag tgcggggtac cgtatcgtga atctgaagtc gaacagtgat 720 SUBSTITUTE SHEET (RULE 2B) ggcgtactat ctaatgtccc gtgcagtaat atcactgttg ccgacgatgg gaatctctag 780 ttttgacaga aaccaaggca actgctatgc tagctaatta attccagaga gatcgatttc 840 tacagtgcta gctgcaacaa tcgatgcaat tggcatcaga cgatatatgc taatggtttt 900 ctttatcgat acgtggtcaa cagtgcgatc cagattacag agttctctcg ccctgatcgg 960 atctcatcgc acatggacac ccatctgcca acccaacgcg gggcggggaa cccgaaacat 1020 cgcgtccatg cacgaccccc acgcagctat aaatacccat gcaatgcaat gcagcgggtc 1080 atcatcatcg actcca 1096 <210> 4 <211> 746 <212> DNA
<213> Zea mays <400> 4 gatcctctcg gttcagtcct agcatctacc agtctgactc taatcttccc gacaccacct 60 aaaccagtct gctcgctgcc ctgtttccgc caccgaccca ttcccgatgc gacgtcaccc 120 gacttcgccc cttcgtagcg cctccgtccc ttgtcctgtt tttctcgtga cgtcgatccg 180 ctcgacaaat tagatctcgc tcgtcgcaac gatatgactt atcccctgcg gtcggtggac 240 ttgacgaact agaaaattat ggctctcatg tggtcttttt ggcccctcct aaattttcgt 300 cctgcgtccg ccactctata gctgcgggag tgcggggtac cgtatcgtga atctgaagtc 360 gaacagtgat ggcgtactat ctaatgtccc gtgcagtaat atcactgttg ccgacgatgg 420 gaatctctag ttttgacaga aaccaaggca actgctatgc tagctaatta attccagaga 480 gatcgatttc tacagtgcta gctgcaacaa tcgatgcaat tggcatcaga cgatatatgc 540 taatggtttt ctttatcgat acgtggtcaa cagtgcgatc cagattacag agttctctcg 600 ccctgatcgg atctcatcgc acatggacac ccatctgcca acccaacgcg gggcggggaa 660 cccgaaacat cgcgtccatg cacgaccccc acgcagctat aaatacccat gcaatgcaat 720 gcagcgggtc atcatcatcg actcca 746 <210> 5 <211> 554 <212> DNA
<213> Zea mays <400> 5 gatctcgctc gtcgcaacga tatgacttat cccctgcggt cggtggactt gacgaactag 60 aaaattatgg ctctcatgtg gtctttttgg cccctcctaa attttcgtcc tgcgtccgcc 120 actctatagc tgcgggagtg cggggtaccg tatcgtgaat ctgaagtcga acagtgatgg 180 cgtactatct aatgtcccgt gcagtaatat cactgttgcc gacgatggga atctctagtt 240 ttgacagaaa ccaaggcaac tgctatgcta gctaattaat tccagagaga tcgatttcta 300 cagtgctagc tgcaacaatc gatgcaattg gcatcagacg atatatgcta atggttttct 360 ttatcgatac gtggtcaaca gtgcgatcca gattacagag ttctctcgcc ctgatcggat 420 ctcatcgcac atggacaccc atctgccaac ccaacgcggg gcggggaacc cgaaacatcg 480 cgtccatgca cgacccccac gcagctataa atacccatgc aatgcaatgc agcgggtcat 540 catcatcgac tcca 554 <210> 6 <211> 406 <212> DNA
<213> Zea mays SUBSTITUTE SHEET (RULE 26) <400> 6 cgtatcgtga atctgaagtc gaacagtgat ggcgtactat ctaatgtccc gtgcagtaat 60 atcactgttg ccgacgatgg gaatctctag ttttgacaga aaccaaggca actgctatgc 120 tagctaatta attccagaga gatcgatttc tacagtgcta gctgcaacaa tcgatgcaat 180 tggcatcaga cgatatatgc taatggtttt ctttatcgat acgtggtcaa cagtgcgatc 240 cagattacag agttctctcg ccctgatcgg atctcatcgc acatggacac ccatctgcca 300 acccaacgcg gggcggggaa cccgaaacat cgcgtccatg cacgaccccc acgcagctat 360 aaatacccat gcaatgcaat gcagcgggtc atcatcatcg actcca 406 <210> 7 <211> 190 <212> DNA
<213> Zea mays <400> 7 cgatacgtgg tcaacagtgc gatccagatt acagagttct ctcgccctga tcggatctca 60 tcgcacatgg acacccatct gccaacccaa cgcggggcgg ggaacccgaa acatcgcgtc 120 catgcacgac ccccacgcag ctataaatac ccatgcaatg caatgcagcg ggtcatcatc 180 atcgactcca 190 <210> 8 <211> 1354 <212> DNA
<213> Zea mays <400> 8 ctcgagatgt tggatagaag aggaattgcg agcaaccgag gtctccaagg cacgccaacg 60 cgcatcctgc gcagtgaggt ggtcgctgat gtcgcgaagg agcttcgatt gttcgtcgat 120 cgcgagcttc agatttggct ccatgacccc agattgtttg gcgcggcgcg tgtaatgagc 180 agacgccaga cccgagaccg gtgtctccga taccaattgt tagcaatagc agcggacaag 240 cagcgaacaa tccgagagac atagtttggg ggaggagcag atagcttgga gaggaagaag 300 aacaatagtg acagcgtaga ttcctctctg gtctcctctg ttaccctctc tcaatctaat 360 actcctatta caaggtcggc ccatcaggcg acccagtcgc ggttccacga acttgggatt 420 gggtttgtgc ggacgccgac tggttcttgg ctctgctgga gtcttgtctg tggagctgtt 480 gctcgcgctg ctaacaatct cttgttgttt gcgacgactt cgcgtggcca cgcgttaact 540 gcgctctgct caaagaaatt gcctatacag tgcctggcgt cagctgcagg cgttgaatcc 600 gaggtcgcca gtacgaggca aacgctgagc tgcatgctgt accggcttta ttaataactg 660 agctctactc gctacgttag ctataggcac cagaggcgga tccagagggg gggcaaagta 720 ggccatggcc ccccctcaat tttgtacacc cttttatacc taatgattat ttagtattaa 780 gtgtagaaaa aatcaaaatt ttagatagta taaaaatctt gttctaaatc tctaaaacta 840 atagctagtt gctaaaatta gccaaaaggt ttagaatgga tcaactaatt gttagctata 900 ccctttaaat aagtattaat tgttagatgg cccaacctag ctaaaaccag ttaacagtta 960 gctctagatg tttagaacat ggcctaaaca accatattat gagcctactc gccggatcct 1020 ctcggttcag tcctagcatc taccagtctg actctaatct tcccgacacc acctaaacca 1080 gtctgctcgc tgccctgttt ccgccaccga cccattcccg atgcgacgtc acccgacttc 1140 gccccttcgt agcgcctccg tcccttgtcc tgtttttctc gtgacgtcga tccgctcgac 1200 aaattagatc tcgctcgtcg caacgatatg acttatcccc tgcggtcggt ggacttgacg 1260 aactagaaaa ttatggctct catgtggtct ttttggcccc tcctaaattt tcgtcctgcg 1320 tccgccactc tatagctgcg ggagtgcggg gtac 1354 SUBSTf~UTE SHEET (RULE 26) -S-<210> 9 <211> 1497 <212> DNA
<213> Zea mays <400> 9 ctcgagatgt tggatagaag aggaattgcg agcaaccgag gtctccaagg cacgccaacg 60 cgcatcctgc gcagtgaggt ggtcgctgat gtcgcgaagg agcttcgatt gttcgtcgat 120 cgcgagcttc agatttggct ccatgacccc agattgtttg gcgcggcgcg tgtaatgagc 180 agacgccaga cccgagaccg gtgtctccga taccaattgt tagcaatagc agcggacaag 240 cagcgaacaa tccgagagac atagtttggg ggaggagcag atagcttgga gaggaagaag 300 aacaatagtg acagcgtaga ttcctctctg gtctcctctg ttaccctctc tcaatctaat 360 actcctatta caaggtcggc ccatcaggcg acccagtcgc ggttccacga acttgggatt 420 gggtttgtgc ggacgccgac tggttcttgg ctctgctgga gtcttgtctg tggagctgtt 480 gctcgcgctg ctaacaatct cttgttgttt gcgacgactt cgcgtggcca cgcgttaact 540 gcgctctgct caaagaaatt gcctatacag tgcctggcgt cagctgcagg cgttgaatcc 600 gaggtcgcca gtacgaggca aacgctgagc tgcatgctgt accggcttta ttaataactg 660 agctctactc gctacgttag ctataggcac cagaggcgga tccagagggg gggcaaagta 720 ggccatggcc ccccctcaat tttgtacacc cttttatacc taatgattat ttagtattaa ?80 gtgtagaaaa aatcaaaatt ttagatagta taaaaatctt gttctaaatc tctaaaacta 840 atagctagtt gctaaaatta gccaaaaggt ttagaatgga tcaactaatt gttagctata 900 ccctttaaat aagtattaat tgttagatgg cccaacctag ctaaaaccag ttaacagtta 960 gctctagatg tttagaacat ggcctaaaca accatattat gagcctactc gccggatcct 1020 ctcggttcag tcctagcatc taccagtctg actctaatct tcccgacacc acctaaacca 1080 gtctgctcgc tgccctgttt ccgccaccga cccattcccg atgcgacgtc acccgacttc 1140 gccccttcgt agcgcctccg tcccttgtcc tgtttttctc gtgacgtcga tccgctcgac 1200 aaattagatc tcgctcgtcg caacgatatg acttatcccc tgcggtcggt ggacttgacg 1260 aactagaaaa ttatggctct catgtggtct ttttggcccc tcctaaattt tcgtcctgcg 1320 tccgccactc tatagctgcg ggagtgcggg gtaccgtatc gtgaatctga agtcgaacag 1380 tgatggcgta ctatctaatg tcccgtgcag taatatcact gttgccgacg atgggaatct 1440 ctagttttga cagaaaccaa ggcaactgct atgctagcta attaattcca gagagat 1497 <210> 10 <211> 1550 <212> DNA
<213> Zea mays <400> 10 ctcgagatgt tggatagaag aggaattgcg agcaaccgag gtctccaagg cacgccaacg 60 cgcatcctgc gcagtgaggt ggtcgctgat gtcgcgaagg agcttcgatt gttcgtcgat 120 cgcgagcttc agatttggct ccatgacccc agattgtttg gcgcggcgcg tgtaatgagc 180 agacgccaga cccgagaccg gtgtctccga taccaattgt tagcaatagc agcggacaag 240 cagcgaacaa tccgagagac atagtttggg ggaggagcag atagcttgga gaggaagaag 300 aacaatagtg acagcgtaga ttcctctctg gtctcctctg ttaccctctc tcaatctaat 360 actcctatta caaggtcggc ccatcaggcg acccagtcgc ggttccacga acttgggatt 420 gggtttgtgc ggacgccgac tggttcttgg ctctgctgga gtcttgtctg tggagctgtt 980 gctcgcgctg ctaacaatct cttgttgttt gcgacgactt cgcgtggcca cgcgttaact 540 gcgctctgct caaagaaatt gcctatacag tgcctggcgt cagctgcagg cgttgaatcc 600 gaggtcgcca gtacgaggca aacgctgagc tgcatgctgt accggcttta ttaataactg 660 agctctactc gctacgttag ctataggcac cagaggcgga tccagagggg gggcaaagta 720 ggccatggcc ccccctcaat tttgtacacc cttttatacc taatgattat ttagtattaa 780 gtgtagaaaa aatcaaaatt ttagatagta taaaaatctt gttctaaatc tctaaaacta 840 SUBSTITUTE SHEET (RULE 26) WO 00/15662 PC'1'/EP99/06692 atagctagtt gctaaaatta gccaaaaggt ttagaatgga tcaactaatt gttagctata 900 ccctttaaat aagtattaat tgttagatgg cccaacctag ctaaaaccag ttaacagtta 960 gctctagatg tttagaacat ggcctaaaca accatattat gagcctactc gccggatcct 1020 ctcggttcag tcctagcatc taccagtctg actctaatct tcccgacacc acctaaacca 1080 gtctgctcgc tgccctgttt ccgccaccga cccattcccg atgcgacgtc acccgacttc 1140 gccccttcgt agcgcctccg tcccttgtcc tgtttttctc gtgacgtcga tccgctcgac 1200 aaattagatc tcgctcgtcg caacgatatg acttatcccc tgcggtcggt ggacttgacg 1260 aactagaaaa ttatggctct catgtggtct ttttggcccc tcctaaattt tcgtcctgcg 1320 tccgccactc tatagctgcg ggagtgcggg gtaccgtatc gtgaatctga agtcgaacag 1380 tgatggcgta ctatctaatg tcccgtgcag taatatcact gttgccgacg atgggaatct 1440 ctagttttga cagaaaccaa ggcaactgct atgctagcta attaattcca gagagatcga 1500 tttctacagt gctagctgca acaatcgatg caattggcat cagacgatat 1550 <210> 11 <211> 1710 <212> DNA
<213> Zea ways <400> 11 ctcgagatgt tggatagaag aggaattgcg agcaaccgag gtctccaagg cacgccaacg 60 cgcatcctgc gcagtgaggt ggtcgctgat gtcgcgaagg agcttcgatt gttcgtcgat 120 cgcgagcttc agatttggct ccatgacccc agattgtttg gcgcggcgcg tgtaatgagc 180 agacgccaga cccgagaccg gtgtctccga taccaattgt tagcaatagc agcggacaag 240 cagcgaacaa tccgagagac atagtttggg ggaggagcag atagcttgga gaggaagaag 300 aacaatagtg acagcgtaga ttcctctctg gtctcctctg ttaccctctc tcaatctaat 360 actcctatta caaggtcgqc ccatcaggcg acccagtcgc ggttccacga acttgggatt 420 gggtttgtgc ggacgccgac tggttcttgg ctctgctgga gtcttgtctg tggagctgtt 480 gctcgcgctg ctaacaatct cttgttgttt gcgacgactt cgcgtggcca cgcgttaact 540 gcgctctgct caaagaaatt gcctatacag tgcctggcgt cagctgcagg cgttgaatcc 600 gaggtcgcca gtacgaggca aacgctgagc tgcatgctgt accggcttta ttaataactg 660 agctctactc gctacgttag ctataggcac cagaggcgga tccagagggg gggcaaagta 720 ggccatggcc ccccctcaat tttgtacacc cttttatacc taatgattat ttagtattaa 780 gtgtagaaaa aatcaaaatt ttagatagta taaaaatctt gttctaaatc tctaaaacta 840 atagctagtt gctaaaatta gccaaaaggt ttagaatgga tcaactaatt gttagctata 900 ccctttaaat aagtattaat tgttagatgg cccaacctag ctaaaaccag ttaacagtta 960 gctctagatg tttagaacat ggcctaaaca accatattat gagcctactc gccggatcct 1020 ctcggttcaq tcctagcatc taccagtctg actctaatct tcccgacacc acctaaacca 1080 gtctgctcgc tgccctgttt ccgccaccga cccattcccg atgcgacgtc acccgacttc 1140 gccccttcgt agcgcctccg tcccttgtcc tgtttttctc gtgacgtcga tccgctcgac 1200 aaattagatc tcgctcgtcg caacgatatg acttatcccc tgcggtcggt ggacttgacg 1260 aactagaaaa ttatggctct catgtggtct ttttggcccc tcctaaattt tcgtcctgcg 1320 tccgccactc tatagctgcg ggagtgcggg gtaccgtatc gtgaatctga agtcgaacag 1380 tgatggcgta ctatctaatg tcccgtgcag taatatcact gttgccgacg atgggaatct 1440 ctagttttga cagaaaccaa ggcaactgct atgctagcta attaattcca gagagatcga 1500 tttctacagt gctagctgca acaatcgatg caattggcat cagacgatat atgctaatgg 1560 ttttctttat cgatacgtgg tcaacagtgc gatccagatt acagagttct ctcgccctga 1620 tcggatctca tcgcacatgg acacccatct gccaacccaa cqcggggcgg ggaacccgaa 1680 acatcgcgtc catgcacgac ccccacgcag 1710 <210> 12 <21t> 856 SUBSTITUTE SHEET (RULE 26) <212> DNA
<213> Zea mays <400> 12 gtttccctgc ggcatgcagc tggagcgctc ggcgttagtg tcagtccatt ttggagtcgg 60 ttcgtgtacc tgagttttcc ttgaggagat gctggttttc gctttctttg ttgcaagaat 120 tattagtcag gaataatggg cctcgcgtga ttgtagttcg agtaggtagg tagtccagca 180 tgcaactgac atgtaatggc tactacgagt atatgcttgc atcgccatct tttatttgct 240 gtgccccttg ttgttacagt gttcctcatc gatctttaga ggtcgttgct accggttttt 300 atcttccaaa acacaaaata tactggaacg actcttgcag tctcttgcta ctgttgaatg 360 tgactattga ctagctaatt agctatagta gcatttatac agttgttgtc aattattata 420 tatagggttg gaaaaaaaaa ctcgagctcg acgacttaka ttgggtctcg cagcagctcc 480 agctcggctt gactcggact ggctcgacgc tcggacgagc ccgagccgag cctatttttg 540 tggctcgtga aaagagcgag ccaactcggc tcgctccaac tcgcgagcta gctcgtggct 600 cgaggtaaca acaattttta catataattc taattagcat ataatattag taccgagtat 660 acaattaatt tatgttattt gagattacta tacaaattaa tctagtatta gtgatatatc 720 tattttacta atttaaaata tgttatttaa aatattttta agattttata ttataattta 780 gagagcagat ctacgtttat agctagtacc gagctgcttt ttccagctca ccagatgggt 840 gcgccgaacc gagctc B56 <210> 13 <211> 440 <212> DNA
<213> Zea mays <400> 13 gtttccctgc ggcatgcagc tggagcgctc ggcgttagtg tcagtccatt ttggagtcgg 60 ttcgtgtacc tgagttttcc ttgaggagat gctggttttc gctttctttg ttgcaagaat 120 tattagtcag gaataatggg cctcgcgtga ttgtagttcg agtaggtagg tagtccagca 180 tgcaactgac atgtaatggc tactacgagt atatgcttgc atcgccatct tttatttgct 240 gtgccccttg ttgttacagt gttcctcatc gatctttaga ggtcgttgct accggttttt 300 atcttccaaa acacaaaata tactggaacg actcttgcag tctcttgcta ctgttgaatg 360 tgactattga ctagctaatt agctatagta gcatttatac agttgttgtc aattattata 420 tatagggttg gaaaaaaaaa 440 <210> 14 <211> 3416 <212> DNA
<213> Zea mays <400> 14 ctcgagatgt tggatagaag aggaattgcg agcaaccgag gtctccaagg cacgccaacg 60 cgcatcctgc gcagtgaggt ggtcgctgat gtcgcgaagg agcttcgatt gttcgtcgat 120 cgcgagcttc agatttggct ccatgacccc agattgtttg gcgcggcgcg tgtaatgagc 180 agacgccaga cccgagaccg gtgtctccga taccaattgt tagcaatagc agcggacaag 240 cagcgaacaa tccgagagac atagtttggg ggaggagcag atagcttgga gaggaagaag 300 aacaatagtg acagcgtaga ttcctctctg gtctcctctg ttaccctctc tcaatctaat 360 actcctatta caaggtcggc ccatcaggcg acccagtcgc ggttccacga acttgggatt 420 gggtttgtgc ggacgccgac tggttcttgg ctctgctgga gtcttgtctg tggagctgtt 480 gctcgcgctg ctaacaatct cttgttgttt gcgacgactt cgcgtggcca cgcgttaact 540 gcgctctgct caaagaaatt gcctatacag tgcctggcgt cagctgcagg cgttgaatcc 600 SUBSTITUTE SHEET (RULE 26) _g_ gaggtcgcca gtacgaggca aacgctgagc tgcatgctgt accggcttta ttaataactg 660 agctctactc gctacgttag ctataggcac cagaggcgga tccagagggg gggcaaagta 720 ggccatggcc ccccctcaat tttgtacacc cttttatacc taatgattat ttagtattaa 780 gtgtagaaaa aatcaaaatt ttagatagta taaaaatctt gttctaaatc tctaaaacta 840 atagctagtt gctaaaatta gccaaaaggt ttagaatgga tcaactaatt gttagctata 900 ccctttaaat aagtattaat tgttagatgg cccaacctag ctaaaaccag ttaacagtta 960 gctctagatg tttagaacat ggcctaaaca accatattat gagcctactc gccggatcct 1020 ctcggttcag tcctagcatc taccagtctg actctaatct tcccgacacc acctaaacca 1080 gtctgctcgc tgccctgttt ccgccaccga cccattcccg atgcgacgtc acccgacttc 1140 gccccttcgt agcgcctccg tcccttgtcc tgtttttctc gtgacgtcga tccgctcgac 1200 aaattagatc tcgctcgtcg caacgatatg acttatcccc tgcggtcggt ggacttgacg 1260 aactagaaaa ttatggctct catgtggtct ttttggcccc tcctaaattt tcgtcctgcg 1320 tccgccactc tatagctgcg ggagtgcggg gtaccgtatc gtgaatctga agtcgaacag 1380 tgatggcgta ctatctaatg tcccgtgcag taatatcact gttgccgacg atgggaatct 1440 ctagttttga cagaaaccaa ggcaactgct atgctagcta attaattcca gagagatcga 1500 tttctacagt gctagctgca acaatcgatg caattggcat cagacgatat atgctaatgg 1560 ttttctttat cgatacgtgg tcaacagtgc gatccagatt acagagttct ctcgccctga 1620 tcggatctca tcgcacatgg acacccatct gccaacccaa cgcggggcgg ggaacccgaa 1680 acatcgcgtc catgcacgac ccccacgcag ctataaatac ccatgcaatg caatgcagcg 1740 ggtcatcatc atcgactcca cctggactcg ctcactggca atggctacca gcaccaagct 1800 cgtggccctt ggcttggccg tcctcgtgag cgttgggttc tccgatgcag cgagggtagc 1860 taggcttggg agctatgcgt ctgccggagg tggcggcggc ggtggaggcg gcggctccgg 1920 atcgaccggg gctgggtacg gcggtgggtc tggcggtggc ggtggctacg gcataggcaa 1980 gggtggcggg gactggtgga acaatttcgt ctccagcgta gcgggcggtg gtggtggggg 2040 gcaaggcgga ggaggtggca ccaacggtgg atccgggtcc ggcggtgggt cgggctatgg 2100 ttctggcacc agttccaccg cagcctctgg ccccagcagt ggcaactacg ccaacgctga 2160 gggcaagggc gccggtgggg gcatgggcgg tggtgccgac gcccgacggc cctacggttc 2220 tggtgccggt ggaggcgttg gcaagggcca gggcgagagt ggcgtcgcat tggcaccgtc 2280 gtccgatggc tactacaatg gcggcgccgc tgatgctacc ggtggtggtt ccggcgctgg 2340 cggtggacat ggcggtggcg cagccggagc tccaagctac gggactggag gtggccttgg 2400 cggaggcaag ggcgaggccg gcagcgacgg gtcctggggt tcaggttacg cggcgggaat 2460 cggtgccggc actggcggcg gcggtggcgg cggatcgcaa ggcggctctg gtggtggcgg 2520 cggctctggg tccggatccg gcagcggtgg aatccactga gtttccctgc ggcatgcagc 2580 tggagcgctc ggcgttagtg tcagtccatt ttggagtcgg ttcgtgtacc tgagttttcc 2640 ttgaggagat gctggttttc gctttctttg ttgcaagaat tattagtcag gaataatggg 2700 cctcgcgtga ttgtagttcg agtaggtagg tagtccagca tgcaactgac atgtaatggc 2760 tactacgagt atatgcttgc atcgccatct tttatttgct gtgccccttg ttgttacagt 2820 gttcctcatc gatctttaga ggtcgttgct accggttttt atcttccaaa acacaaaata 2880 tactggaacg actcttgcag tctcttgcta ctgttgaatg tgactattga ctagctaatt 2940 agctatagta gcatttatac agttgttgtc aattattata tatagggttg gaaaaaaaaa 3000 ctcgagctcg acgacttaga ttgggtctcg cagcagctcc agctcggctt gactcggact 3060 ggctcgacgc tcggacgagc ccgagccgag cctatttttg tggctcgtga aaagagcgag 3120 ccaactcggc tcgctccaac tcgcgagcta gctcgtggct cgaggtaaca acaattttta 3180 catataattc taattagcat ataatattag taccgagtat acaattaatt tatgttattt 3240 gagattacta tacaaattaa tctagtatta gtgatatatc tattttacta atttaaaata 3300 tgttatttaa aatattttta agattttata ttataattta gagagcagat ctacgtttat 3360 agctagtacc gagctgcttt ttccagctca ccagatgggt gcgccgaacc gagctc 3416 <210> 15 <211> 1074 <212> DNA
<213> Zea mat's SUBSTITUTE SHEET (RULE 2B)

-9-<400> 15 ggctcgttgc atgagcgagc cgagtcaagc tcgggttggc tcgtttccac ccctagttat 60 atacgatcaa attggaccgc acgacactga cattaacact attaaacata acactattat 120 taatcgtgtc gtgctatatt agcattagtg actagctatc ggtacagaca tgacactata 180 ttgcctaatc atgtcgtgcc tgacactata tagagggtat ctctagtagt ttttctatct 240 tagttcctat ctgtcggtac cctgaaacta gggtatccct tactattgta taaagacgca 300 gtacccacgc ggctatcttt agacgcgtgg taaacgagct gtatgtggga ccaggccatg 360 actcgcccca gcctcgggcg actactctag gccagcaaca gcgcttgacc ccaccacatg 420 ggcgggtccg cggccgccac gtgtccagag aaagtgatat actccaaggc atcaacagtg 480 agtccggaca tgggagagtg tcggacccat gccggacccc tgtatatacg gtccaggcct 540 ccaagtttgg cccaggacct taggccattg ccgctgtcgc ccggcggtgg aatgagtatc 600 atgtcgtagc tgccgtcgag ggacatgaac tcaagactcc cgaaacggag caccttcccg 660 ggttggagag gttgctggag actgcccatc tggagcttga cgggaagctg ttcgtcaaca 720 cgcagcaggc ccctacctgg cgcgccaact gtcggcgttt cgagaccggg gggtccctaa 780 gccgacgagt gaatctcgcc gcgtgcccca gcccagatgg gtcgacgcga ggccgagcgc 890 gaagggggga agtgaggtgg ccggagatag gcgtgagaga ggtgcaaatc ccgcggcctc 900 cgtgttcgtc tcgcgcccag gtcgggtgcg cttgcagtag ggggttacaa gcgtccacgc 960 gggagaggga gcaagcggcc taacacgagc gcatgtctcg tcctcgtccc cgcgcggcca 1020 accctatcta agagggccct ggtccttcct tttataggcg taaggagagg atcc 1074 suBS~ sH~Er cRU~ zs~

Claims (29)

Claims

1. A nucleic acid sequence for use in cloning and expressing a root specific nucleic acid se-quence in a plant which is selected from the group consisting of a) the nucleic acid sequence set out in SEQ ID No_ 1 or a complementary strand thereof, b) a nucleic acid sequence which has a degree of identity of more than 70 % to the nucleic acid sequence defined in (a) and c) alleles of the nucleic acid sequence defined in (a) or (b).

2. The nucleic acid sequence of claim 1 which is selected from the group consisting of a) the nucleic acid sequence set out in SEQ ID No.
2 or a complementary strand thereof, b) a nucleic acid sequence which has a degree of identity of more than 70 % to the nucleic acid sequence defined in (a) and c) alleles of the nucleic acid sequences defined in (a) or (b).

3. The nucleic acid sequence according to claim 1 or 2, which is selected from the group consist-ing of a) the nucleic acid sequence set out in any one of SEQ ID No. 3 to 11 or a complementary strand thereof, b) a nucleic acid sequence which has a degree of identity of more than 70 % to the nucleic acid sequence defined in (a) and c) alleles of the nucleic acid sequence defined in (a) or (b).

4. A nucleic acid sequence for use in cloning and expressing a root specific gene in a plant which is selected from the group consisting of a) the nucleic acid sequence sec out in any one of SEQ ID No. 12 to 15 or a complementary strand thexeof, b) a nucleic acid sequence which has a degree of identity of more than 70 % to the nucleic acid sequences defined in (a) and c) alleles of the nucleic acid sequence defined is (a) or (b).

5. The nucleic acid sequence of any one of the preceding claims, which is derived from maize.

6. A vector comprising the nucleic acid sequence of any one of the preceding claims.

7. The vector of claim 6, which is a bacterial or viral vector.

8. The vector of any one of claims 6 or 7, wherein the nucleic acid sequence of any one of claims 1 to 5 is operably linked to a gene of inter-est.

9. The vector of claim 8, wherein the gene of in-terest is chosen from a gene which confers a character of agronomic or industrial benefit.

10. The vector of claim 9, wherein the gene which confers a character of agronomic or industrial benefit is a gene for resistance to infection by a virus, a gene conferring resistance to a herbicide or to an insecticide or, alterna-tively, a gene whose expression confers male sterility.

11. The vector of any one of claims 6 to 10, which comprises further regulatory elements directing or enhancing the expression of the gene of in-terest.

12. The vector of any one of claims 6 to 11, wherein the further regulatory elements are 5',3' or 5' and 3' elements.

13. The vector of claim 12, wherein the 3' regula-tory element is the poly A addition sequence of the NOS gene of Agrobacterium tumefaciens.

14. The vector according to any one of claims 6 to 13, which furthermore contains T-DNA.

15. The vector of claim 14, which contains the left, the right or both T-DNA borders.

16. The vector according to claim 14 or 15, wherein the nucleic acid sequence is located within the T-DNA or adjacent to it.

17. The vector according to claim 16, wherein the nucleic acid sequence in conjunction with the gene of interest is located within the T-DNA or adjacent to it.

18. A host cell containing the vector of any one of claims 6 to 17.

19. The host cell of claim 18, which is a plant, yeast or bacterial cell.

2o. The host cell of claim 19, which is a cell from a monocotyledonous or dicotyledonous plant.

21. A cell culture comprising a cell according to any one of claims 18 to 20.

22. A plant comprising a host cell according to any one of claims 18 to 20.

23. Seeds, propagation material, harvest material and plant tissue, comprising a host cell ac-cording to any one of claims 18 or 20 or de-rived from a plant according to claim 22.

24. A method of genetically modifying a cell by transforming a cell with a nucleic acid se-guence according to any one of claims 1 to 5 or a vector according to any one of claims 6 to 17, wherein the gene of interest contained in the vector is expressible in the cell.

25. The method of claim 24, wherein the cell is a plant, bacterial or yeast cell.

26. The method of claims 24 or 25, wherein the transformed cell is regenerated into a differ-entiated plant.

27. The method of any one of claims 24 to 26, wherein the cell is transformed by transfer of a vector or nucleic acid sequences from a bac-terium to the cell.

28. The method of any one of claims 24 to 27, wherein the cell is transformed by direct in-take of a nucleic acid sequence or a vector, by microinjection or particle bombardment with a nucleic acid sequence or a vector.

29. A method for isolating a root specific gene and/or root specific regulatory element from a plant, whereby a nucleic acid sequence of any one of claims 1 to 5 or a vector of any one of claims 6 to 17 is used to screen nucleic acid sequences derived from the plant.