HK1016653B - Riit cirtex specific gene promoter - Google Patents

Description

Root cortex specific gene promoter

The invention is funded by the government under the national science foundation MCB-9206506. The government may have certain rights in the invention.

Technical Field

The present invention relates to tissue-specific gene promoters, in particular to promoters active in the root cortex of plants.

Background

A promoter is a DNA sequence that flanks a transcribed gene, and if the flanking gene is to be transcribed into messenger RNA, RNA polymerase must bind to the promoter. A promoter may consist of a number of different regulatory elements which influence the structural gene operably associated with the promoter in different ways. For example, a regulatory gene may enhance or inhibit the expression of a bound structural gene, developmentally regulate the gene, or contribute to tissue-specific regulation of the gene. Modification of the promoter using recombinant DNA methods may result in selective gene expression patterns. See, for example, Old and Primrose, principles of gene manipulation (4 th edition, 1989).

An example of a plant promoter is the promoter found flanking the gene for the small subunit ribulose-1, 5-bisphosphate carboxylase of petunia. See U.S. Pat. No. 4,962,028. Another example is a promoter containing the 5' flanking region of the wheat Em gene. See EPO application 335528. Yet another example is the stress-inducing regulatory element disclosed in EPO application 0330479.

Although the regulation of root gene expression plays an important role in plant development, relatively little work has been done in this regard. This deficiency is due in part to the lack of readily identifiable root-specific biochemical functions (these genes may be readily cloned and studied). Evans et al (molecular genetics 214, 153-. A number of nodule-specific genes have been cloned and characterized by Fuller et al (proceedings of the national academy of sciences USA 80, 2594-. Comparison of the 5' ends of the transcription initiation sites of the DNA sequences indicated the presence of 8 nucleotides in the repeats of the three genes examined. Unfortunately, the lack of an efficient transformation/regeneration system for most leguminous (Leguminaceae) prevents the functional analysis of these cis-acting sequences. Bogusz et al (Nature 331, 178-180(1988)) isolated a hemoglobin gene specifically expressed in the roots of non-tumorigenic plants by homology of the hemoglobin gene of the non-tumorigenic plant with the hemoglobin gene of a closely related tumorigenic plant species. Keller and Lamb (Gene and development 3,1639-1646(1989)) isolated genes encoding hydroxyproline-rich glycoproteins of the cell wall expressed during lateral root initiation. Cloning and characterization of barley root-specific lectins has recently been reported by Lerner and Raikhel (plant physiology 91, 124-129 (1989)).

Many plant pathogens and pests destroy the roots of plants, causing severe crop damage and loss. The most commonly damaged root tissue is the root cortex, a layer consisting primarily of storage parenchyma, which underlies the epidermis layer and surrounds the central vascular bundle of the root. The root cortex may also contain sclerenchyma, secretory cells, resin tracts, other structures and cell types. The cells of the root cortex have morphological and developmental similarities to the cortical cells of the aerial stalk.

In order to deliver useful traits to plants via expression of exogenous genes using genetic engineering techniques, various tissue-specific promoters are required to enable selective expression of the new traits in the appropriate plant tissues. The present invention is based on our continuing research relating to this problem.

Summary of The Invention

The present invention is based on the identification of the tobacco RD2(TobRD2) promoter, which directs root cortex specific expression of the associated gene. A first aspect of the invention is an isolated DNA molecule that directs root cortex specific transcription of a downstream heterologous DNA segment in a plant cell, the isolated DNA molecule having a sequence selected from the group consisting of (a) SEQ ID NOs: 1-9 and (b) under stringent conditions and SEQ ID NO: 1-9 and directing root cortex specific transcription of a downstream heterologous DNA segment in a plant cell.

Another aspect of the invention is an expression cassette comprising a tobacco RD2 promoter and a heterologous DNA segment located downstream of and operably linked to the promoter.

Another aspect of the invention is an expression cassette comprising a root cortex specific promoter and a heterologous DNA fragment derived from the nucleic acid sequence provided herein as SEQ ID NO: 1-9 and sequences of root cortex specific promoters selected under stringent conditions and under stringent conditions with SEQ ID NO: 1-9 and directs root cortex-specific transcription of the DNA sequence.

Further aspects of the invention include plant cells comprising an expression cassette as described above, methods of producing transformed plants from such plant cells, and transformed plants comprising such transformed plant cells.

Brief description of the drawings

FIG. 1A shows tobacco RD2 transcripts located in tobacco root cross sections by in situ hybridization in 7-day old seedlings.

FIG. 1B shows tobacco RD2 transcripts located in tobacco root longitudinal sections by in situ hybridization of 7-day-old seedlings.

FIG. 2 is a 2010 base pair sequence (SEQ ID NO: 1) of the 5' region of TobRD 2.

FIG. 3 is a schematic representation of the TobRD2 promoter/Glucuronidase (GUS) construct used to test the ability of the RD2 promoter to direct root cortex specific gene expression.

FIG. 4 is a bar graph summarizing the β -Glucuronidase (GUS) activity in roots (solid bars), leaves (diagonal bars), stems (stippled bars) of plants transformed with the chimeric reporter construct, and the results are shown in Table 1. This figure shows the activity of plants transformed with a gene construct using different promoters (CaMV 35S; Δ 2.00; Δ 1.50; Δ 1.40; Δ 1.25; Δ 0.80; Δ 0.70; Δ 0.60; Δ 0.30) while using only the vector pBI101.3 as a control. GUS activity was expressed in pmolMU/. mu.g protein/min.

FIG. 5A is a bar graph summarizing the relative β -Glucuronidase (GUS) activity in roots, leaves of tobacco plants transformed with the chimeric reporter construct, and the results are shown in Table 1, where different promoters (CaMV 35S; Δ 2.00; Δ 1.50; Δ 1.40; Δ l.25; Δ 0.80; Δ 0.70; Δ 0.60; Δ 0.30) were used, while using only vector pBI101.3 as a control. GUS activity was expressed in pmolMU/. mu.g protein/min, and the relative activity shown was root/leaf activity.

FIG. 5B is a bar graph summarizing the relative β -Glucuronidase (GUS) activity in the roots, stems of tobacco plants transformed with the chimeric reporter construct, and the results are shown in Table 1, where different promoters (CaMV 35S; Δ 2.00; Δ 1.50; Δ 1.40; Δ 1.25; Δ 0.80; Δ 0.70; Δ 0.60; Δ 0.30) were used, while using only vector pBI101.3 as a control. GUS activity was reported in pmolMU/. mu.g protein fraction, and the relative activity shown was root/stem activity.

FIG. 6A is an optical micrograph showing the histochemical localization of GUS activity in the cross section of tobacco plant roots transformed with a reporter Gene (GUS) driven by a.DELTA.2.0 promoter.

FIG. 6B is an optical micrograph showing the histochemical localization of GUS activity at the root tip of tobacco plant roots transformed with a reporter Gene (GUS) driven by a.DELTA.2.0 promoter.

Detailed description of the invention

The nucleotide sequences are shown herein in single stranded form only from left to right in the 5 '-3' direction. Nucleotides are represented herein in the form recommended by the IUPAC-IUB Commission on Biochemical nomenclature.

Transgenic plants expressing peptides that inhibit or kill particular pests or pathogens provide a means to reduce crop damage and loss. For example, expression of the bacillus thuringiensis protein in transgenic maize provides resistance to european maize borer. However, expression of a transgene in all plant tissues (constitutive expression) is disadvantageous because it can expose non-target organisms to the transgenic protein and also increase the selective pressure for development of pathogens and pests that are resistant to the transgenic protein. High levels of expression of the transgene throughout the plant may also have a negative impact on plant growth and yield. Another strategy is to express toxic peptides only in organs or tissues affected by a particular pest or pathogen. The implementation of such strategies against plant root-damaging pests and pathogens has been hampered by the lack of well-characterized root-specific promoters.

Transcription of a gene begins when a stable complex is formed between the RNA polymerase and the gene promoter. Promoters starting at all transcription units are typically about 100 base pairs long and are located immediately upstream of the transcription start site. See, for example, Maniatis et al, science 236: 1238(1987). Promoters can vary in "strength," i.e., their ability to accurately and efficiently initiate transcription, is variable. It is believed that RNA polymerase holoenzyme covers a region of approximately 50 base pairs immediately upstream of the transcribed region. The strength of transcription initiation is sometimes increased by accessory proteins that bind adjacent to the promoter region immediately upstream of the transcribed DNA. See, for example, Singer and Berg, genes and genomes, 140-.

Specific examples of root cortex specific promoters of the invention are those having a sequence identical to SEQ ID NO: 1-9 (discussed in more detail below). It is obvious that other fragments of the sequences longer or shorter than the aforementioned sequences or with minimal additions, deletions, or substitutions of the flanking regions of tobacco RD 25' may be prepared, which will also carry the TobRD2 root cortex specific promoter, and all such sequences are encompassed by the present invention. Another aspect of the invention includes promoters isolated from other tobacco genes or from non-tobacco plants as described below, which are homologous to the tobacco RD2 promoter and are capable of directing root cortex specific transcription of a downstream heterologous DNA segment in a cell.

The TobRD2 promoter as used herein refers to a DNA molecule having a sequence identical (or substantially homologous) to a contiguous segment of DNA found at the 5' end of the transcribed region of the tobacco RD2 gene. SEQ ID NO: 1 provides the sequence of the 2kb region found immediately 5' to the start of transcription in TobRD2 gene. The TobRD2 promoter includes a region of at least 100 base pairs, a 150 base pair region, or preferably a 200 base pair region immediately 5' to the TobRD2 transcribed region and directs root cortex specific expression. As used herein, a region that is "substantially homologous" is at least 75%, more preferably 80%, 85%, 90%, or even 95% homologous.

As used herein, a root cortex specific promoter is a promoter that preferentially directs expression of an operably bound gene in root cortex tissue, relative to expression in leaf or stem tissue or other tissues of the root.

Other plant root cortex specific promoter sequences include those that are at least about 75% homologous (more preferably 80%, 85%, 90%, or even 95% homologous) to a fragment of about 100 bases of the tobacco RD2 promoter immediately upstream of the transcribed DNA region and which are capable of directing root cortex specific transcription of a downstream heterologous DNA segment in a plant cell. Other plant root cortex specific promoters include those similar to SEQ ID nos: 1-9, and more preferably at least about 75% homology (more preferably 80%, 85%, 90%, or even 95% homology) to the TobRD2 promoter, and which sequence is capable of directing root cortex specific transcription of a downstream heterologous DNA segment in a plant cell.

Highly stringent hybridization conditions that allow hybridization of a heterologous DNA sequence to a DNA sequence set forth herein are well known in the art. For example, these sequences can be hybridized to the DNA disclosed herein in 25% formamide, 5 XSSC, 5 XDenhardt's solution, 100. mu.g/ml single stranded DNA, 5% dextran sulfate at 42 ℃ under 25% formamide, 5 XSSC, 0.1% SDS at 42 ℃ for 15 minutes to allow hybridization of sequences with 60% homology. More stringent conditions have more stringent wash conditions: 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS, 60 ℃ or even 70 ℃, using standard in situ hybridization assay. (see Sambrook et al, molecular cloning, A laboratory Manual (2 nd edition 1989) (Cold spring harbor laboratory)). In general, a plant DNA sequence encoding a root cortex specific promoter and hybridizing to a DNA sequence encoding a tobacco RD2 root cortex specific promoter disclosed herein has at least about 75%, 80%, 85%, 90% or even 95% or more homology to a DNA sequence encoding a tobacco RD2 root cortex specific promoter disclosed herein.

The root cortex specific promoter of the present invention is useful in directing tissue specific expression of transgenes in transformed plants. Expression of such tissue-specific transgenes is useful in providing resistance to damage caused by pests and pathogens that attack plant roots. Furthermore, when the root cortex is the major accumulating organ for photosynthetic products, the expression of transgenes intended to alter stored carbohydrates can be directed by these promoters. Exogenous genes of particular importance for root cortex specific expression include: genes encoding proteins that bind heavy metals (e.g., metallothionein); a gene encoding a protein resistant to pests and pathogens present in soil; genes encoding proteins resistant to heat, salt (salinity) and drought; a gene encoding a protein for desalting; a gene encoding a protein that metabolizes a plant-stored compound to another preferred product or form.

Tissue-specific promoters may also be used to convert the protoxins to an active form at selected tissue sites. Hsu et al (pesticide science, 44, 9(1995)) reported the use of a chimeric gene comprising the root-specific promoter TobRB7 and the β -glucuronidase gene to preferentially convert the prototroph in roots to the active form. The inactivated pesticide pro (hydroxymethyloxamyl glucuronide) is applied to the leaves and then transported through the plant phloem to the roots where it is converted by glucuronidase into the active nematicide form.

In addition, root cortex specific promoters are histologically useful and can be used to identify or stain root cortex tissue through the use of a reporter gene (e.g., β -glucuronidase).

The term "operably linked" as used herein means that the DNA sequences are contained on a single DNA molecule and the DNA sequences are linked such that the function of one DNA sequence can be affected by another DNA sequence. Such that a promoter is operably associated with a gene when it is capable of affecting the expression of that gene (i.e., the gene is under the transcriptional control of the promoter). The promoter is said to be "upstream" of the gene, and the gene may be said to be "downstream" of the promoter.

The DNA construct or "expression cassette" of the invention comprises, in the 5 'to 3' direction of transcription, the promoter of the invention, a heterologous DNA segment operably associated with said promoter, and optionally a transcriptional and translational termination region (e.g., a termination signal and polyadenylation region). All of these regulatory regions should be capable of functioning in transformed cells. The 3' termination region may be derived from the same or different gene as the transcription initiation region.

Plants can be divided into plants without chlorophyll (such as fungi) and plants with chlorophyll (such as green algae, moss); and further can be divided into plants containing chlorophyll and vascular tissue (e.g., ferns, gymnosperms, pines, monocotyledons, and dicotyledons). The latter plant group includes those that may have roots, stems and leaves. The term "plant" as used herein includes all organisms as described above. The term "native plant DNA" as used herein refers to DNA isolated from a plant that has not been genetically engineered or non-transformed (e.g., a plant variety produced by breeding).

The term heterologous gene or heterologous DNA fragment as used herein refers to a gene or DNA fragment used to transform a cell by genetic engineering techniques, and such a gene may not be naturally present in the cell. Structural genes are parts of genes that comprise a DNA segment encoding a protein, polypeptide, or portion thereof, possibly including a ribosome binding site and/or a translation initiation codon, but lacking a promoter. The term may also refer to copies of a structural gene that are naturally present in the cell, but are artificially introduced. The structural gene may encode a protein not normally found in plant cells into which the gene or a promoter operably associated with the gene is introduced. The gene operably linked to the promoter for expression in plant species of the present invention may be derived from a chromosomal gene, cDNA, synthetic gene, or a combination thereof. The term heterologous DNA fragment as used herein also encompasses DNA fragments which encode non-protein products (e.g., ribozymes or antisense RNAs). Antisense RNAs are well known (see, for example, U.S. Pat. No. 4,801,540(Calgene corporation)).

Important genes in plants for use in the present invention include those that affect a variety of phenotypic and non-phenotypic characteristics. Among these phenotypic characteristics are proteins (e.g., enzymes) that provide resistance to various environmental stresses, including but not limited to stresses caused by dehydration (due to heat, salt, drought), herbicides, toxic metals, trace elements, pests, and pathogens. Resistance may be due to changes in the target site, an increase in the mass of the target protein in the host cell, an increase in the amount of one or more enzymes associated with the product biosynthetic pathway that protects the host from stress, and the like. The structural gene may be obtained from prokaryotes or eukaryotes, bacteria, fungi (e.g., yeast, viruses, plants, and mammals), or may be synthesized in whole or in part. Specific genes include the 3-enolpyruvylphosphithokinate synthase gene having resistance to glyphosate, nitrilase, genes of the biosynthetic pathway of proline and glutamine, and metallothionein.

The structural genes to which the promoter of the present invention is operably linked may be those encoding proteins toxic to insects, such as Bacillus thuringiensis crystal proteins toxic to insects. U.S. patent 4,853,331 discloses a DNA sequence encoding a bacillus thuringiensis toxin toxic to Coleoptera (Coleoptera) and variants of this sequence, with the portion encoding toxicity retained (see also U.S. patents 4,918,006 and 4,910,136) (the entire disclosures of all U.S. patent documents cited herein are incorporated herein by reference). PCT application WO 90/02804 discloses the gene sequence of bacillus thuringiensis which makes plants toxic to Lepidoptera (Lepidoptera). PCT application WO 89/04868 discloses transgenic plants transformed with a vector that promotes the expression of a crystal protein toxin from Bacillus thuringiensis, and the sequences of this vector can be used in conjunction with the present invention. PCT application WO90/06999 discloses DNA encoding a crystal protein toxin of Bacillus thuringiensis active against Lepidoptera. U.S. patent 4,918,006 discloses another gene sequence encoding an insecticidal crystal protein. Examples of gene sequences encoding other insect toxins are the gene sequences encoding chitinases (e.g., EC-3.2.1.14) disclosed in U.S. Pat. No. 4,940,840 and PCT application WO 90/07001. In U.S. patent application 08/007,998, genes encoding a nematode porin protein useful in the production of root nematode resistant transgenic plants are disclosed. Strains of Bacillus thuringiensis capable of producing active polypeptide toxins against nematodes are disclosed in U.S. Pat. Nos. 4,948,734 and 5,093,120(Edwards et al).

When the gene expression product is localized in a non-cytoplasmic cellular compartment, a structural gene may be constructed which comprises a region encoding a specific amino acid sequence which allows the product to be transferred to a specific site (e.g., the cytoplasmic membrane) or secreted into the cytosolic or external environment of the cell. Various secretory leader sequences, membrane integration sequences and transfer sequences to direct the expression product of the peptide to a specific site are described in the literature. See, e.g., Cashmore et al, Biotechnology (1985) 3: 803-808, Wickner and Lodish, science (1985) 230: 400-407.

The expression cassette may be provided as a DNA construct which also has at least one replication system. For convenience, replication systems functional in E.coli are usually included, such as ColE1, pSC101, pACYC184, and the like. In this way, the resulting construct can be cloned and sequenced at various stages after each manipulation, and the correctness of the manipulation can be checked. In addition, a wide host range of replication systems, such as those of P-1-incompatible plasmids (e.g., pRK290), can be used, or in place of E.coli replication systems. In addition to a replication system, may contain at least one marker, which may be useful in one or more hosts, or different markers may be used for different hosts. That is, one marker may be used for selection in prokaryotic hosts and another marker for selection in eukaryotic hosts (particularly plant hosts). The markers may provide protection against the hazards of biocides (e.g., antibiotics, toxins, heavy metals, etc.), the markers may provide complementary effects by delivering prototrophy to an auxotrophic host; or the marker may provide a visible phenotype by generating a novel compound in the plant. Examples of suitable markers that may be used for selection of a plant host include, but are not limited to, β -Glucuronidase (GUS) (producing indigo), luciferase (producing visible light), NPTII (producing kanamycin resistance or G418 resistance), HPT (providing hygromycin resistance), and mutant aroA genes (providing glyphosate resistance).

Various fragments comprising various constructs, expression cassettes, markers, etc., are introduced sequentially by restriction with appropriate replication systems and insertion of specific constructs or fragments at available sites. After ligation and cloning, the DNA construct may be isolated for further manipulation. These techniques are described in detail in the literature by way of example. See, e.g., Maniatis et al, molecular cloning: a laboratory Manual, Cold spring harbor laboratory, Cold spring harbor, N.Y. (1982).

The vector is a replicable DNA construct. Vectors that may be used to transform plant tissue by the DNA constructs of the present invention include Agrobacterium vectors and ballistic vectors, as well as vectors suitable for DNA-mediated transformation. Agrobacterium tumefaciens cells containing the DNA constructs of the invention, wherein the DNA constructs comprise a Ti plasmid, are useful in a variety of methods for producing transformed plants. Infecting plant cells with Agrobacterium tumefaciens to produce transformed plant cells, and then regenerating plants from the transformed plant cells.

A large number of vector systems of the genus Agrobacterium for use in the present invention are known. For example, U.S. patent 4,459,355 discloses a method of transforming susceptible plants (including dicotyledonous plants) with an agrobacterium strain containing a Ti plasmid. Us patent 4,795,855 discloses the use of agrobacterium vectors to transform woody plants. Furthermore, U.S. Pat. No. 4,940,838(Schilperoort et al) discloses a binary Agrobacterium vector (i.e., a vector in which Agrobacterium comprises a plasmid containing the vir region but no T-DNA region of a Ti plasmid, together with a second plasmid containing the T-DNA region but no vir region) useful in the present invention.

Microparticles carrying the DNA constructs of the invention, which are suitable for impact transformation of plant cells, are also useful in producing transformed plants of the invention. Advancing said microparticles to plant cells to produce transformed plant cells, and regenerating plants from the transformed plant cells. Any suitable ballistic cell transformation method and apparatus may be used to practice the invention. Examples of apparatus and methods are disclosed in U.S. Pat. No. 4,945,050 to Sanford and Wolf and in Agracetus European patent application publication 0270356 (entitled "pollen-mediated plant transformation"). When using the ballistic transformation method, the expression cassette may be incorporated into a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in these systems include 1-5 μm gold powder spheres. The DNA construct is deposited on the microparticles by any suitable technique, such as by precipitation.

A transformed host cell is a cell that has been transformed or transfected with a construct (produced using recombinant DNA techniques) comprising a DNA sequence disclosed herein. Transformation of plants can be accomplished using the DNA constructs of the present invention by DNA-mediated transformation of plant cell protoplasts followed by regeneration of the plant from said transformed protoplasts according to methods well known in the art.

The promoter sequences disclosed herein can be used to express heterologous DNA sequences in any plant species that is capable of using such a promoter (i.e., the plant's RNA polymerase is capable of binding to the promoter sequences disclosed herein). Examples of plant species suitable for transformation with the DNA constructs of the present invention include monocots and dicots, including but not limited to tobacco, soybean, potato, cotton, sugar beet, sunflower, carrot, celery, flax, cabbage and other crucifers, pepper, tomato, citrus, soybean, strawberry, lettuce, corn, alfalfa, oat, wheat, rice, barley, sorghum, and canola. Thus, an example of a plant species that can be transformed with a DNA construct of the invention is a dicotyledonous plant, and a more specific plant species that can be transformed with a DNA construct of the invention is a member of the Solanaceae family.

Any plant tissue that can subsequently be clonally propagated (either by organ regeneration or embryo regeneration) can be transformed with the vectors of the invention. The term "organ regeneration" as used herein refers to a process by which shoots and roots can develop from a meristematic center in succession; the term "embryo regeneration" as used herein is a process by which shoots and roots can develop from somatic cells or gametes in a coordinated manner (rather than sequentially). The particular tissue selected will depend on the clonal propagation system that can be used (and is best suited) for the particular plant species being transformed. Examples of tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, macrogametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristematic tissue (e.g., cotyledon meristem and hypocotyl meristem).

The following examples are intended to illustrate various specific embodiments of the present invention and are not intended to limit the invention.

Example 1

Isolation of genome root cortex specific RD2 Gene

A tobacco (Nicotiana tabacum) genomic library of DNA isolated from tobacco seedlings was constructed in EMBL 3 SP 6/T7. lambda.vector (ClonTech, Pale Alto, Calif.). Genomic clones containing the tobacco RD2 gene were isolated from the primary library using TobRD2cDNA (Conkling et al, plant physiology 93, 1203(1990)) as a probe. A total of 1.2X 10 cells were selected on K802 bacterial cells⁷And (4) recombinant phage. The plaques were transferred to a nylon membrane (Magnagraph) and the DNA was fixed by autoclaving (10 min, gravity cycle). All hybridizations were performed in aqueous solution (5 XSSC [750mM sodium chloride, 75mM sodium citrate) at 65 ℃]5 XDenhardt [ 0.1% of various ficoll, BSA, polyvinylpyrrolidone]0.5% SDS, 100mg/ml denatured salmon sperm DNA) for 16 hours. The filtrate was washed in 0.2 XSSC and 0.1% SDS at 60 ℃.

By screening for 1.2X 10⁷The recombinant phages identified 13 genomic clones that hybridized with the TobRD2 cDNA. These clones were isolated and further characterized by restriction mapping. Restriction maps were constructed by the rapid mapping method of Rachwitz et al (Gene, 30: 195 (1984)). The entire sequence of a clone homologous to the TobRD2cDNA was determined and the promoter determined. The genomic clone region 5' to said translated region was determined by comparing the sequences of TobRD2cDNA and genomic clones. The sequence of this untranslated region was determined and the TATAA box of the putative promoter was identified. In plant promoters, the TATAA box is generally from transcriptionNucleotides-35 to-29 from the start site. The 5' end of the transcript was identified using primer extension experiments.

FIG. 2 shows the 2010 base pair region (SEQ ID NO: 1) upstream of the transcribed region of TobRD2 cDNA. This sequence comprises the putative start of the transcribed region (at nucleotide 2000) and the TATAA box of the promoter (nucleotides 1971-.

Example 2

Nucleic acid sequencing

The restriction fragments of the isolated genomic clone (example 1) were subcloned into the bluescript (pBSKS II + or pBS KS II +; Stratagene, La Jolla, Calif.) vector. A series of one-way deletions for each clone and both DNA strands was obtained by exonuclease III and S1 nuclease digestion (Henikoff, Gene 28, 351 (1984)). The DNA sequence was determined by the dideoxy chain termination method (Sanger et al, Proc. Natl. Acad. Sci. USA, 74, 5463(1977)) using a sequencing enzyme (Cleveland, OH, USA Biochemical Co.). In all cases, both DNA strands were sequenced.

Example 3

In situ hybridization

To determine the spatial distribution of TobRD2 mRNA transcripts in various tissues of roots, in situ hybridization was performed in non-transformed plants. The antisense strand of TobRD2 was hybridized in situ with TobRD2 mRNA in root tissue using techniques such as those described by Meyerowitz (plant molecular biology report 5, 242(1987)) and Smith et al (plant molecular biology report 5, 237 (1987)). The 7-day old tobacco (nicotiana tabacum) seedling roots were fixed in phosphoric acid-buffered glutaraldehyde, embedded in Paraplast Plus (Monoject, st. louis, MO) and cut into 8 mm thick sections to obtain cross-sections and longitudinal sections. An antisense TobRD2 transcript synthesized in vitro in the presence of 35S-ATP was used as a probe. The labeled RNA was hydrolyzed by alkali treatment to obtain fragments with an average length of 100-200 bases before use.

Hybridization in 50% formamide at 42 ℃ for 16 hours, containing approximately 5X 10 molecules per ml of hybridization solution⁶One/min (cpm) labeled RNA. After exposure, the films were developed and observed under bright and dark field microscopes.

As shown in FIGS. 1A and 1B, the hybridization signal was localized in the root cortex cells. Comparison of the images of the same section in bright and dark fields indicated that the TobRD2 transcript was localized in parenchymal cells in the root cortex. No hybridization signal was observed in the epidermis or the pericycle.

Example 4

Construction of chimeric genes

A series of promoter deletions were constructed by Polymerase Chain Reaction (PCR). The templates were various deleted fragments of the 5' flanking region of the TobRD2 genomic clone generated by exonuclease III/S1 nuclease digestion (example 2).

All templates were amplified using the same set of oligonucleotide primers. One primer is the pre-primer of modified bacteriophage M13 (see, e.g., Sanger et al, proceedings of the national academy of sciences USA 74, 5463 (1977)); the 5 'end of this oligonucleotide contains the HindIII recognition sequence and an additional 5' sequence that allows more efficient cleavage by restriction enzymes. Another primer was designed to have a BamHI site at the 5 'end (and additional nucleotides for efficient cleavage) and this primer was homologous to the 16 nucleotide sequence of TobRD2, said TobRD2 having 22 bases at the 5' end of the ATG initiation codon (i.e., this primer was homologous to bases 1973-1988 of SEQ ID NO: 1).

Said PCR amplification reaction contains template plasmid DNA (5-10 ng); reaction buffer (50 mKCl; 10mM Tris-HCl; pH9.0[25 ℃ C.)]；0.1％Triton X-100；1.5mMMgCl₂) (ii) a 0.25mM each of dATP, dGTP, dTTP and dCTP; 40ng of each primer; 1.25 units of Taq DNA polymerase (Promega, Madison, WS).

The template was denatured at 94 ℃ for 1 minute of PCR cycles, the primer was annealed at 46 ℃ for 1 minute, and the strand was extended at 72 ℃ for 5 minutes. This cycle was repeated 40 times and the final extended cycle was increased by 10 minutes. PCR amplification was performed in a programmed thermal cycler (PCT-100, m.j. research).

The amplified product was digested with Hind III and Bam HI and cloned into the Hind III and Bam HI sites of the Agrobacterium binary vector pBI101.3(R.Jefferson et al, EMBO J.6, 3901-3907 (1987)). This vector contains a β -Glucuronidase (GUS) reporter gene and an nptII selectable marker flanked by T-DNA border sequences.

Example 5

Plant transformation method

The chimeric reporter construct is introduced into An Agrobacterium host harboring the modified Ti-plasmid (4404) capable of providing (in trans) vir function essential for T-DNA transfer and integration in the plant genome, essentially as described by An et al (edited by Belvin and R.Schilperoot, handbook of plant molecular biology, Martinus Nijhoff, Dordrecht, Netherlands, ppA3-1-19 (1988)). The construct is introduced into the host by electroporation of triparental paired or electrocompetent Agrobacterium cells, a technique well known in the art. Such as An, etc.: plant physiology 81, 301-305(1986) describes the leaf disc transformation and plant regeneration of tobacco (SR 1). Plants with kanamycin resistance were selected for further analysis.

Example 6

GUS determination method in transgenic plant

The transformed plants were stained for histocompatibility on a cut surface of roots, stems and leaves. After a brief vacuum infiltration of substrate, these explant tissues were incubated overnight in 1mM 5-bromo-4-chloro-3-indolyl-B-D-glucuronide (X-Gluc), 25mM sodium phosphate buffer (pH7.0), 0.5% DMSO at 37 ℃. Tissues expressing GUA activity were able to cleave this substrate and therefore stain blue.

Fluorescent GUS assays were performed as described by Jefferson et al (EMBO J.6, 3901-3907(1987)) to quantify the level of GUS expression. The cell extracts of roots, leaves and stems were incubated at 37 ℃ in the presence of 1mM 4-methyl dispersyl-B-D-glucuronide (MUG). Samples were taken at intervals of 0, 5, 10, 15 and 20 minutes. The enzymatic reaction was stopped by the addition of 0.2M sodium carbonate. The fluorometer was calibrated with 10nM and 100nM MUG. The protein concentration in the sample was determined according to the method of Bradford (anal. biochem.72, 248 (1976)).

Example 7

Chimeric gene constructs capable of directing tissue-specific gene expression

To determine TobRD₂Whether the 2010 base pair sequence of the gene (SEQ ID No: 1) includes a promoter element that directs specific expression in parenchymal cells of the root cortex, a chimeric gene was constructed. A1988 base pair region (SEQ ID No: 2) was amplified by polymerase chain reaction and cloned at the 5' end of the GUS reporter gene (as described above). The chimeric gene was introduced into tobacco (as described above) and the transgenic plants were analyzed for the ability to express GUS (as described above).

Rows 25-33 of Table 1 (transformants 325II1-325IV5) show the results of an analysis of 9 independent transformants (i.e., each transformant is the product of an independent transformation process). The Δ 2.0 promoter (SEQ ID NO: 2) was found to be capable of directing high levels of gene expression (approximately 4-fold higher than the CaMV35S promoter, which is generally referred to as a "strong" promoter) (FIG. 4). No reporter gene expressed at higher levels than the control was detected in the leaf or stem (see fig. 4, 5A and 5B showing the average activity of table 1). GUS activity was essentially restricted to the roots and was shown in FIG. 6 to be restricted, in particular, to the cortex of the roots. The plants shown in figure 6 were transformed using the Δ 2.0 promoter driving GUS (in pbi 101.3).

Multiple independent transformation leaf discs were placed in petri dishes. The designation of the transformants in table 1 indicates the promoter/numbered culture dish/number of the independent transformants. Thus, 325II1 refers to a transformant from leaf disk 1 in Petri dish II using the Δ 2.0 promoter; while 101.I1 refers to transformation using pBI101.3 (GUS without promoter as control) and the transformant numbered 1 in dish I. In Table I, prefix 121 refers to the use of pBI121 (CaMV35S promoter with GUS); 325 refers to the Δ 2.0 promoter with GUS (SEQ ID N0: 2); 484 means a.DELTA.1.4 promoter with GUS (SEQ ID NO: 3); 421 refers to the Δ 1.3 promoter with GUS (SEQ ID NO: 4); 428 refers to the Δ 1.0 promoter with GUS (SEQ ID NO: 5); 490 refers to the Δ 0.7 promoter with GUS (SEQ ID NO: 6); 491 refers to a.DELTA.0.6 promoter with GUS (SEQ ID NO: 7); 492 refers to the Δ 0.5 promoter with GUS (SEQ ID NO: 8); 495 refers to the Δ 0.2 promoter with GUS (SEQ ID NO: 9). "R-GUS" refers to GUS activity in root tissue; "L-GUS" refers to GUS activity in leaf tissue; and "S-GUS" refers to GUS activity in stem tissues. R/L refers to the relative GUS activity of the roots/leaves; R/S refers to the relative GUS activity of the roots/stems. GUS activity was expressed in pmolMU/. mu.g protein/min.

TABLE 1

TobRD2 promoter analysis

Transformant	R-GUS Activity	Mean value of	L-GUS Activity	Mean value of	S-GUS Activity	Mean value of	R/L	R/L mean value	R/S	R/S mean value
											101.I1	0.19	0.58	0.23	0.33	0.22	0.36	0.83	1.67	0.86	1.51
101.I2	0.12		0.14		0.15		0.88		0.80
											101.I3	0.13		0.35		0.32		0.37		0.41
101.I4	0.73		0.46		0.24		1.59		3.04
											101.II1	0.44				0.31				1.42
101.II3	0.59		0.23		0.47		2.57		1.26
											101.II4	0.86		0.41		0.34		2.10		2.53
101.II5	0.64		0.36		0.33		1.78		1.94
											101.III1	0.69		0.24		0.42		2.88		1.64
101.III3	0.25		0.19		0.21		1.32		1.19
											101.III4	0.71		0.37		0.27		1.92		2.63
101.III5	0.15		0.13		0.21		1.15		0.71
											101.IV1	0.21		0.10		0.13		2.10		1.62
101.IV2	0.27		0.24		0.23		1.13		1.17
											101.IV3	0.88		0.42		0.57		2.10		1.54
101.IV4	0.75		0.35		0.67		2.14		1.12
											101.IV5	1.88		0.98		1.02		1.92		1.84
											121.I5	3.00	10.50	3.65	14.36	2.25	5.81	0.82	0.71	1.33	1.69
121.IV1	24.67		30.79		11.96		0.80		2.06
											121.IV2	9.20		11.66		5.33		0.79		1.73
121.IV4	12.13		15.61		7.42		0.78		1.63
											121.4	3.50		10.10		2.08		0.35		1.68

TABLE 1

TobRD2 promoter analysis

325II1	35.30	32.15	0.54	0.46	0.61	0.78	65.37	67.19	57.87	50.17
											325II2	24.94		0.24		0.35		103.92		71.26
325II4	13.64		0.17		0.23		80.24		59.30
											325II5	38.09				0.64				59.52
325III1	45.31		0.38
											325III2	34 05		0 44
325III5	55.81		0.76		0.77		73.43		72.48
											325IV1	16.51		0.68		0.94		24.28		17.56
325IV5	25.71		0.46		1.95		55.89		13.18
484I1	61.75	36.68		0.46		0.67		74.41		53.68
											484I3	59.72
484I4	72.35
											484I5	56.58
484V2	38.32		0.78		0.86		49.13		44.56
											484V3	23.66		0.31		2.29		76.32		10.33
484III3	63.28
											484III4	42.91		0.87		0.98		49.32		43.79
484II4	15.80		0.43		0.27		36.74		58.52
											484V4	58.25		0.46		0.48		126.63		121.35
484V1	26.86		0 81		1.27		33.16		21.15
											484V5	8.53		0.42		0.34		20.31		25.09
484IV5	17.83		0.51		0.29		34.96		61.48
											484IV3	14.05		0.35		0.34		40.14		41.32
484IV2	32.33		0.32		0.51		101.03		63.39

TABLE 1

TobRD2 promoter analysis

484II3	10.18		0.13		0.16		78.31		63.63
											484II5	33.51		0.55		0.63		60.93		53.19
484II2	52.54		0.43		0.79		122.19		66.51
											484II1	8.50		0.04		0.11		212.50		77.27
											421IV4	25.04	31.87	0.82	0.81	2.27	1.01	30.54	40.54	11.03	36.78
421V4	46.31		0.82				56.48
											421II4	79.23		0.96		1.89		82.53		41.92
421III3	17.00		0.45		1.09		37.78		15.60
											421II3	19.07		0.42		0.37		45.40		51.54
421I1	27.67		0.72		0.64		38.43		43.23
											421I3	74.45		2.27		1.44		32.80		51.70
421II2	43.36		0.88		0.56		49.27		77.43
											421I4	8.41
421V1	32.32		0.94		1.34		34.38		24.12
											421V2	5.07		0.43		0.13		11.79		39.00
421IV3	4.52		0.17		0.37		26.59		12.22
428I5	20.62	38.64	0.98	0.66	0.83	0.65	21.04	72.65	24.84	47.43
											428I2	15.05		0.97		0.25		15.52		60.20
428III3	69.87		1.10				63.52
											428III1	30.97		0.52		0.36		59.56		86.03
428V2	54.66		0.24				227.75
											428V1	85.71		0.98		1.25		87.46		68.57
428IV4	4.15				0.29				14.31

TABLE 1

TobRD2 promoter analysis

428IV5	26.42		0.43		1.10		61.44			24.02
												428V3	1.58		0.16		0.17		9.88			9.29
428V2	25.60		0.34				75.29
												428III5	90.36		0.86		0.98		105.07			92.20
												490II4	9.38	22.77		0.54		0.75			41.65		36.11
490II5	9.67		0.35		0.65		27.63			14.88
												490I1	33.62		0.93		2.02		36.15			16.84
490I2	34.66		0.98		1.13		35.37			30.67
												490I3	4.58
490III2	76.74
												490III4	58.75		1.07		1.21		54.91			48.55
490III5	6.65		0.21		0.09		31.67			73.89
												490IV2	12.24
490II1	8.09		0.22		0.21		36.77			38.52
												490IV4	20.19		0.35		0.52		57.69			38.83
490IV5	17.57		0.34		0.57		51.68			30.82
												490IV3	18.11
490I5	23.03		0.78		0.93		29.53			24.76
												490V5	8.27		0.15		0.19		55.13			43.53
												491I2	8.31	39.76		0.50		0.63			53.70		45.85
491II3	6.73
												491II4	13.01		0.23		0.19		56.57			68.47
491V5	87.40

TABLE 1

TobRD2 promoter analysis

491IV1	77.12		1.02		1.34		75.61		57.55
											491IV3	49.20		0.98		1.23		50.20		40.00
491III1	18.84		0.32		0.34		58.88		55.41
											491III2	30.82		0.47		0.58		65.57		53.14
491III5	8.46		0.28		.045		30.21		18.80
											491IV5	2.88
491II5	8.55		0.22		0.31		28.86		27.58
											491IV4	165.77
											492V2	2.40	9.89	0.21	0.57	0.24	0.54	11.43	15.59	10.00	16.72
492V4	3.17		0.27		0.48		11.74		6.60
											492I3	4.40		0.87		0.35		5.06		12.57
492I4	6.58		0.50		0.37		13.16		17.78
											492I5	10.26
492III2	11.87		0.78		1.06		15.22		11.20
											492IV4	7.38
492IV5	21.63
											492III5	11.39		0.61		0.32		18.67		35.59
492IV1	20.38		0.81		0.94		25.16		21.68
											492II3	12.15		0.42		0.53		28.93		22.92
492III1	7.03		0.64		0.58		10.98		12.12
495I1	3.58	5.83	0.37	0.41	0.43	0.54	9.68	17.98	8.33	13.35
											495I3	16.41		0.59		0.74		27.81		22.18
495I4	3.20		0.17		0.17		18.82		18.82

TABLE 1

TobRD2 promoter analysis

495I5	5.96		0.32		0.34		18.63		17.53
											495II2	8.49		0.54		0.52		15.72		16.33
495III2	5.12		0.40		0.77		12.80		6.65
											495IV1	5.57		0.21		0.45		26.52		12.38
495IV2	9.74		0.75		1.03		12.99		9.46
											495IV3	2.64		0.14		0.31		18.66		8.52
495IV4	1.20
											495V1	3.67
495V2	2.38
											495V3	7.60
495V4	6.10		0.56		0.62		10.89		9.84

Example 8

Effect of 5' promoter-deletion on expression of reporter Gene Activity

The following experiment was performed in substantially the same manner as described in example 7 above, except that the length of the TobRD2 flanking region used as a promoter was varied to investigate how portions of the flanking region influence GUS expression.

A series of 7-nested 5' -deletion mutations were generated in the region upstream of the 2010 base pair TobRD2 sequence (SEQ ID NO: 1) to serve as a promoter sequence. FIG. 3 shows these deletion mutants, designated Δ 2.O (SEQ ID NO: 2), respectively, in a schematic manner; Δ 1.4(SEQ ID NO: 3); Δ 1.3(SEQ ID NO: 4); Δ 1.0(SEQ ID NO: 5); Δ 0.7(SEQ ID NO: 6); Δ 0.6(SEQ ID NO: 7); Δ 0.5(SEQ ID NO: 8) and Δ 0.2(SEQ ID NO: 9).

The chimeric gene construct described in example 3 and containing the Δ 2.00 promoter (SEQ ID NO: 2) or truncated promoter (SEQ ID NOs: 3-9) was introduced into tobacco by the Agrobacterium-mediated leaf disc transformation method (as described in example 4). The agrobacterium vector pbi101.3 alone was used as a control, and the CaMV35S promoter was used to provide a reference standard. Regenerated plants were analyzed for GUS activity in roots, leaves and stems (Table 1: FIG. 4).

FIG. 4 gives a graphical illustration of GUS activity in roots, leaves and stems using the full-length TobRD2 promoter, a deletion series of promoters, the cauliflower mosaic virus 35S [ CaMV35S) promoter, and the vector pBI101.3 as a control. As shown in fig. 4, the 6 promoters tested were found to confer high levels of root cortex specific expression: Δ 2.00(SEQ ID NO: 2); Δ 1.4(SEQ ID NO: 3); Δ 1.3(SEQ ID NO: 4); Δ 1.0(SEQ ID NO: 5); Δ 0.7(SEQ ID NO: 6); Δ 0.6(SEQ ID NO: 7). Fig. 4 shows the average values of table 1.

FIG. 4 also shows that deletion of a region approximately 50 base pairs in length (compare. DELTA.0.6 (SEQ ID NO: 7) and. DELTA.0.5 (SEQ ID NO: 8)) greatly reduced the level of GUS expression. However, this result showed that the level of GUS expression in root tissues provided by the Δ 0.5 promoter (SEQ ID NO: 8) was the same as that caused by the CaMV35S promoter. GUS expression in the root cortex provided by the Δ 0.2 promoter (SEQ ID NO: 9) was approximately half that provided by the CaMV35S promoter.

FIGS. 5A and 5B also illustrate the organ-specific characteristics of the expression of the reporter gene using the TobRD2 promoter. In all experimental examples GUS activity was strictly expressed in the roots, whereas very little, if any, activity was detected in the stems or leaves of the same transformed tobacco plants. When the level of GUS activity detected in roots transformed with the Δ 0.60 and Δ 0.30 promoters was equal to or less than the level of GUS provided by the CaMV35S promoter (fig. 4), fig. 5A and 5B illustrate that the expression directed by the Δ 0.60 and Δ 0.30 promoters was root-specific, whereas the expression in stems and leaves was minimal, unlike the expression directed by the CaMV35S promoter.

The foregoing examples are intended to illustrate the invention specifically and are not intended to limit the invention. The following claims define the invention, with equivalents of those claims to be included therein.

Sequence listing

(1) General information:

(i) the applicant: mark a.

Mendu，Nandini

Song，Wen

(ii) The invention provides a subject: root cortex specific gene promoter

(iii) Sequence number: 9

(iv) Communication address:

(A) address: sibley, Kenneth d; seltzer, Park and Gibson

(B) Street: post Office Drawer 34009

(C) City: charlotte

(D) State: north Carolina

(E) The state is as follows: united states of America

(F) Code of the zip code: 28234

(v) A computer-readable form:

(A) type of medium: flexible disk

(B) A computer: IBM PC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: PatentIn Release #1.0.Version #1.30

(vi) Data of the current application:

(A) application No.:

(B) application date:

(C) and (4) classification:

(viii) lawyer/attorney information:

(A) name: sibley, Kenneth D.

(B) Registration number: 31, 665

(C) Certificate number: 5051-294

(ix) And (3) telecommunication information:

(A) telephone: 919-420-2200

(B) Faxing: 919-881-3175

(2) SEQ ID NO: 1, information:

(i) sequence characteristics:

(A) length: 2010 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEQ ID NO: 1

CTCGAGGATC TAAATTGTGA GTTCAATCTC TTCCCTATTG GATTGATTAT CCTTTCTTTT 60

CTTCCAATTT GTGTTTCTTT TTGCCTAATT TATTGTGTTA TCCCCTTTAT CCTATTTTGT 120

TTCTTTACTT ATTTATTTGC TTCTATGTCT TTGTACAAAG ATTTAAACTC TATGGCACAT 180

ATTTTAAAGT TGTTAGAAAA TAAATTCTTT CAAGATTGAT GAAAGAACTT TTTAATTGTA 240

GATATTTCGT AGATTTTATT CTCTTACTAC CAATATAACG CTTGAATTGA CGAAAATTTG 300

TGTCCAAATA TCTAGCAAAA AGGTATCCAA TGAAAATATA TCATATGTGA TCTTCAAATC 360

TTGTGTCTTA TGCAAGATTG ATACTTTGTT CAATGGAAGA GATTGTGTGC ATATTTTTAA 420

AATTTTTATT AGTAATAAAG ATTCTATATA GCTGTTATAG AGGGATAATT TTACAAAGAA 480

CACTATAAAT ATGATTGTTG TTGTTAGGGT GTCAATGGTT CGGTTCGACT GGTTATTTTA 540

TAAAATTTGT ACCATACCAT TTTTTTCGAT ATTCTATTTT GTATAACCAA AATTAGACTT 600

TTCGAAATCG TCCCAATCAT GTCGGTTTCA CTTCGGTATC GGTACCGTTC GGTTAATTTT 660

CATTTTTTTT TAAATGTCAT TAAAATTCAC TAGTAAAAAT AGAATGCAAT AACATACGTT 720

CTTTTATAGG ACTTAGCAAA AGCTCTCTAG ACATTTTTAC TGTTTAAAGG ATAATGAATT 780

AAAAAACATG AAAGATGGCT AGAGTATAGA TACACAACTA TTCGACAGCA ACGTAAAAGA 840

AACCAAGTAA AAGCAAAGAA AATATAAATC ACACGAGTGG AAAGATATTA ACCAAGTTGG 900

GATTCAAGAA TAAAGTCTAT ATTAAATATT CAAAAAGATA AATTTAAATA ATATGAAAGG 960

AAACATATTC AATACATTGT AGTTTGCTAC TCATAATCGC TAGAATACTT TGTGCCTTGC 1020

TAATAAAGAT ACTTGAAATA GCTTAGTTTA AATATAAATA GCATAATAGA TTTTAGGAAT 1080

TAGTATTTTG AGTTTAATTA CTTATTGACT TGTAACAGTT TTTATAATTC CAAGGCCCAT 1140

GAAAAATTTA ATGCTTTATT AGTTTTAAAC TTACTATATA AATTTTTCAT ATGTAAAATT 1200

TAATCGGTAT AGTTCGATAT TTTTTCAATT TATTTTTATA AAATAAAAAA CTTACCCTAA 1260

TTATCGGTAC AGTTATAGAT TTATATAAAA ATCTACGGTT CTTCAGAAGA AACCTAAAAA 1320

TCGGTTCGGT GCGGACGGTT CGATCGGTTT AGTCGATTTT CAAATATTCA TTGACACTCC 1380

TAGTTGTTGT TATAGGTAAA AAGCAGTTAC AGAGAGGTAA AATATAACTT AAAAAATCAG 1440

TTCTAAGGAA AAATTGACTT TTATAGTAAA TGACTGTTAT ATAAGGATGT TGTTACAGAG 1500

AGGTATGAGT GTAGTTGGTA AATTATGTTC TTGACGGTGT ATGTCACATA TTATTTATTA 1560

AAACTAGAAA AAACAGCGTC AAAACTAGCA AAAATCCAAC GGACAAAAAA ATCGGCTGAA 1620

TTTGATTTGG TTCCAACATT TAAAAAAGTT TCAGTGAGAA AGAATCGGTG ACTGTTGATG 1680

ATATAAACAA AGGGCACATT GGTCAATAAC CATAAAAAAT TATATGACAG CTACAGTTGG 1740

TAGCATGTGC TCAGCTATTG AACAAATCTA AAGAAGGTAC ATCTGTAACC GGAACACCAC 1800

TTAAATGACT AAATTACCCT CATCAGAAAG CAGATGGAGT GCTACAAATA ACACACTATT 1860

CAACAACCAT AAATAAAACG TGTTCAGCTA CTAAAACAAA TATAAATAAA TCTATGTTTG 1920

TAAGCACTCC AGCCATGTTA ATGGAGTGCT ATTGCCTGTT AACTCTCACT TATAAAATAG 1980

TAGTAGAAAA AATATGAACC AAAACACAAC 2010

(2) SEQ ID NO: 2, information:

(i) sequence characteristics:

(A) length: 1988 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEQ ID NO: 2

CTCGAGGATC TAAATTGTGA GTTCAATCTC TTCCCTATTG GATTGATTAT CCTTTCTTTT 60

CTTCCAATTT GTGTTTCTTT TTGCCTAATT TATTGTGTTA TCCCCTTTAT CCTATTTTGT 120

TTCTTTACTT ATTTATTTGC TTCTATGTCT TTGTACAAAG ATTTAAACTC TATGGCACAT 180

ATTTTAAAGT TGTTAGAAAA TAAATTCTTT CAAGATTGAT GAAAGAACTT TTTAATTGTA 240

GATATTTCGT AGATTTTATT CTCTTACTAC CAATATAACG CTTGAATTGA CGAAAATTTG 300

TGTCCAAATA TCTAGCAAAA AGGTATCCAA TGAAAATATA TCATATGTGA TCTTCAAATC 360

TTGTGTCTTA TGCAAGATTG ATACTTTGTT CAATGGAAGA GATTGTGTGC ATATTTTTAA 420

AATTTTTATT AGTAATAAAG ATTCTATATA GCTGTTATAG AGGGATAATT TTACAAAGAA 480

CACTATAAAT ATGATTGTTG TTGTTAGGGT GTCAATGGTT CGGTTCGACT GGTTATTTTA 540

TAAAATTTGT ACCATACCAT TTTTTTCGAT ATTCTATTTT GTATAACCAA AATTAGACTT 600

TTCGAAATCG TCCCAATCAT GTCGGTTTCA CTTCGGTATC GGTACCGTTC GGTTAATTTT 660

CATTTTTTTT TAAATGTCAT TAAAATTCAC TAGTAAAAAT AGAATGCAAT AACATACGTT 720

CTTTTATAGG ACTTAGCAAA AGCTCTCTAG ACATTTTTAC TGTTTAAAGG ATAATGAATT 780

AAAAAACATG AAAGATGGCT AGAGTATAGA TACACAACTA TTCGACAGCA ACGTAAAAGA 840

AACCAAGTAA AAGCAAAGAA AATATAAATC ACACGAGTGG AAAGATATTA ACCAAGTTGG 900

GATTCAAGAA TAAAGTCTAT ATTAAATATT CAAAAAGATA AATTTAAATA ATATGAAAGG 960

AAACATATTC AATACATTGT AGTTTGCTAC TCATAATCGC TAGAATACTT TGTGCCTTGC 1020

TAATAAAGAT ACTTGAAATA GCTTAGTTTA AATATAAATA GCATAATAGA TTTTAGGAAT 1080

TAGTATTTTG AGTTTAATTA CTTATTGACT TGTAACAGTT TTTATAATTC CAAGGCCCAT 1140

GAAAAATTTA ATGCTTTATT AGTTTTAAAC TTACTATATA AATTTTTCAT ATGTAAAATT 1200

TAATCGGTAT AGTTCGATAT TTTTTCAATT TATTTTTATA AAATAAAAAA CTTACCCTAA 1260

TTATCGGTAC AGTTATAGAT TTATATAAAA ATCTACGGTT CTTCAGAAGA AACCTAAAAA 1320

TCGGTTCGGT GCGGACGGTT CGATCGGTTT AGTCGATTTT CAAATATTCA TTGACACTCC 1380

TAGTTGTTGT TATAGGTAAA AAGCAGTTAC AGAGAGGTAA AATATAACTT AAAAAATCAG 1440

TTCTAAGGAA AAATTGACTT TTATAGTAAA TGACTGTTAT ATAAGGATGT TGTTACAGAG 1500

AGGTATGAGT GTAGTTGGTA AATTATGTTC TTGACGGTGT ATGTCACATA TTATTTATTA 1560

AAACTAGAAA AAACAGCGTC AAAACTAGCA AAAATCCAAC GGACAAAAAA ATCGGCTGAA 1620

TTTGATTTGG TTCCAACATT TAAAAAAGTT TCAGTGAGAA AGAATCGGTG ACTGTTGATG 1680

ATATAAACAA AGGGCACATT GGTCAATAAC CATAAAAAAT TATATGACAG CTACAGTTGG 1740

TAGCATGTGC TCAGCTATTG AACAAATCTA AAGAAGGTAC ATCTGTAACC GGAACACCAC 1800

TTAAATGACT AAATTACCCT CATCAGAAAG CAGATGGAGT GCTACAAATA ACACACTATT 1860

CAACAACCAT AAATAAAACG TGTTCAGCTA CTAAAACAAA TATAAATAAA TCTATGTTTG 1920

TAAGCACTCC AGCCATGTTA ATGGAGTGCT ATTGCCTGTT AACTCTCACT TATAAAATAG 1980

TAGTAGAA 1988

(2) SEQ ID NO: 3, information:

(i) sequence characteristics:

(A) length: 1372 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEQ ID NO: 3

TCATGTCGGT TTCACTTCGG TATCGGTACC GTTCGGTTAA TTTTCATTTT TTTTTAAATG 60

TCATTAAAAT TCACTAGTAA AAATAGAATG CAATAACATA CGTTCTTTTA TAGGACTTAG 120

CAAAAGCTCT CTAGACATTT TTACTGTTTA AAGGATAATG AATTAAAAAA CATGAAAGAT 180

GGCTAGAGTA TAGATACACA ACTATTCGAC AGCAACGTAA AAGAAACCAA GTAAAAGCAA 240

AGAAAATATA AATCACACGA GTGGAAAGAT ATTAACCAAG TTGGGATTCA AGAATAAAGT 300

CTATATTAAA TATTCAAAAA GATAAATTTA AATAATATGA AAGGAAACAT ATTCAATACA 360

TTGTAGTTTG CTACTCATAA TCGCTAGAAT ACTTTGTGCC TTGCTAATAA AGATACTTGA 420

AATAGCTTAG TTTAAATATA AATAGCATAA TAGATTTTAG GAATTAGTAT TTTGAGTTTA 480

ATTACTTATT GACTTGTAAC AGTTTTTATA ATTCCAAGGC CCATGAAAAA TTTAATGCTT 540

TATTAGTTTT AAACTTACTA TATAAATTTT TCATATGTAA AATTTAATCG GTATAGTTCG 600

ATATTTTTTC AATTTATTTT TATAAAATAA AAAACTTACC CTAATTATCG GTACAGTTAT 660

AGATTTATAT AAAAATCTAC GGTTCTTCAG AAGAAACCTA AAAATCGGTT CGGTGCGGAC 720

GGTTCGATCG GTTTAGTCGA TTTTCAAATA TTCATTGACA CTCCTAGTTG TTGTTATAGG 780

TAAAAAGCAG TTACAGAGAG GTAAAATATA ACTTAAAAAA TCAGTTCTAA GGAAAAATTG 840

ACTTTTATAG TAAATGACTG TTATATAAGG ATGTTGTTAC AGAGAGGTAT GAGTGTAGTT 900

GGTAAATTAT GTTCTTGACG GTGTATGTCA CATATTATTT ATTAAAACTA GAAAAAACAG 960

CGTCAAAACT AGCAAAAATC CAACGGACAA AAAAATCGGC TGAATTTGAT TTGGTTCCAA 1020

CATTTAAAAA AGTTTCAGTG AGAAAGAATC GGTGACTGTT GATGATATAA ACAAAGGGCA 1080

CATTGGTCAA TAACCATAAA AAATTATATG ACAGCTACAG TTGGTAGCAT GTGCTCAGCT 1140

ATTGAACAAA TCTAAAGAAG GTACATCTGT AACCGGAACA CCACTTAAAT GACTAAATTA 1200

CCCTCATCAG AAAGCAGATG GAGTGCTACA AATAACACAC TATTCAACAA CCATAAATAA 1260

AACGTGTTCA GCTACTAAAA CAAATATAAA TAAATCTATG TTTGTAAGCA CTCCAGCCAT 1320

GTTAATGGAG TGCTATTGCC TGTTAACTCT CACTTATAAA ATAGTAGTAG AA 1372

(2) SEQ ID NO: 4, information:

(i) sequence characteristics:

(A) length: 1294 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEQ ID NO: 4

AAAAATAGAA TGCAATAACA TACGTTCTTT TATAGGACTT AGCAAAAGCT CTCTAGACAT 60

TTTTACTGTT TAAAGGATAA TGAATTAAAA AACATGAAAG ATGGCTAGAG TATAGATACA 120

CAACTATTCG ACAGCAACGT AAAAGAAACC AAGTAAAAGC AAAGAAAATA TAAATCACAC 180

GAGTGGAAAG ATATTAACCA AGTTGGGATT CAAGAATAAA GTCTATATTA AATATTCAAA 240

AAGATAAATT TAAATAATAT GAAAGGAAAC ATATTCAATA CATTGTAGTT TGCTACTCAT 300

AATCGCTAGA ATACTTTGTG CCTTGCTAAT AAAGATACTT GAAATAGCTT AGTTTAAATA 360

TAAATAGCAT AATAGATTTT AGGAATTAGT ATTTTGAGTT TAATTACTTA TTGACTTGTA 420

ACAGTTTTTA TAATTCCAAG GCCCATGAAA AATTTAATGC TTTATTAGTT TTAAACTTAC 480

TATATAAATT TTTCATATGT AAAATTTAAT CGGTATAGTT CGATATTTTT TCAATTTATT 540

TTTATAAAAT AAAAAACTTA CCCTAATTAT CGGTACAGTT ATAGATTTAT ATAAAAATCT 600

ACGGTTCTTC AGAAGAAACC TAAAAATCGG TTCGGTGCGG ACGGTTCGAT CGGTTTAGTC 660

GATTTTCAAA TATTCATTGA CACTCCTAGT TGTTGTTATA GGTAAAAAGC AGTTACAGAG 720

AGGTAAAATA TAACTTAAAA AATCAGTTCT AAGGAAAAAT TGACTTTTAT AGTAAATGAC 780

TGTTATATAA GGATGTTGTT ACAGAGAGGT ATGAGTGTAG TTGGTAAATT ATGTTCTTGA 840

CGGTGTATGT CACATATTAT TTATTAAAAC TAGAAAAAAC AGCGTCAAAA CTAGCAAAAA 900

TCCAACGGAC AAAAAAATCG GCTGAATTTG ATTTGGTTCC AACATTTAAA AAAGTTTCAG 960

TGAGAAAGAA TCGGTGACTG TTGATGATAT AAACAAAGGG CACATTGGTC AATAACCATA 1020

AAAAATTATA TGACAGCTAC AGTTGGTAGC ATGTGCTCAG CTATTGAACA AATCTAAAGA 1080

AGGTACATCT GTAACCGGAA CACCACTTAA ATGACTAAAT TACCCTCATC AGAAAGCAGA 1140

TGGAGTGCTA CAAATAACAC ACTATTCAAC AACCATAAAT AAAACGTGTT CAGCTACTAA 1200

AACAAATATA AATAAATCTA TGTTTGTAAG CACTCCAGCC ATGTTAATGG AGTGCTATTG 1260

CCTGTTAACT CTCACTTATA AAATAGTAGT AGAA 1294

(2) SEQ ID NO: 5, information:

(i) sequence characteristics:

(A) length: 1030 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEQ ID NO: 5

GGAAACATAT TCAATACATT GTAGTTTGCT ACTCATAATC GCTAGAATAC TTTGTGCCTT 60

GCTAATAAAG ATACTTGAAA TAGCTTAGTT TAAATATAAA TAGCATAATA GATTTTAGGA 120

ATTAGTATTT TGAGTTTAAT TACTTATTGA CTTGTAACAG TTTTTATAAT TCCAAGGCCC 180

ATGAAAAATT TAATGCTTTA TTAGTTTTAA ACTTACTATA TAAATTTTTC ATATGTAAAA 240

TTTAATCGGT ATAGTTCGAT ATTTTTTCAA TTTATTTTTA TAAAATAAAA AACTTACCCT 300

AATTATCGGT ACAGTTATAG ATTTATATAA AAATCTACGG TTCTTCAGAA GAAACCTAAA 360

AATCGGTTCG GTGCGGACGG TTCGATCGGT TTAGTCGATT TTCAAATATT CATTGACACT 420

CCTAGTTGTT GTTATAGGTA AAAAGCAGTT ACAGAGAGGT AAAATATAAC TTAAAAAATC 480

AGTTCTAAGG AAAAATTGAC TTTTATAGTA AATGACTGTT ATATAAGGAT GTTGTTACAG 540

AGAGGTATGA GTGTAGTTGG TAAATTATGT TCTTGACGGT GTATGTCACA TATTATTTAT 600

TAAAACTAGA AAAAACAGCG TCAAAACTAG CAAAAATCCA ACGGACAAAA AAATCGGCTG 660

AATTTGATTT GGTTCCAACA TTTAAAAAAG TTTCAGTGAG AAAGAATCGG TGACTGTTGA 720

TGATATAAAC AAAGGGCACA TTGGTCAATA ACCATAAAAA ATTATATGAC AGCTACAGTT 780

GGTAGCATGT GCTCAGCTAT TGAACAAATC TAAAGAAGGT ACATCTGTAA CCGGAACACC 840

ACTTAAATGA CTAAATTACC CTCATCAGAA AGCAGATGGA GTGCTACAAA TAACACACTA 900

TTCAACAACC ATAAATAAAA CGTGTTCAGC TACTAAAACA AATATAAATA AATCTATGTT 960

TGTAAGCACT CCAGCCATGT TAATGGAGTG CTATTGCCTG TTAACTCTCA CTTATAAAAT 1020

AGTAGTAGAA 1030

(2) SEQ ID NO: 6, information:

(i) sequence characteristics:

(A) length: 722 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEO ID NO: 6

GTACAGTTAT AGATTTATAT AAAAATCTAC GGTTCTTCAG AAGAAACCTA AAAATCGGTT 60

CGGTGCGGAC GGTTCGATCG GTTTAGTCGA TTTTCAAATA TTCATTGACA CTCCTAGTTG 120

TTGTTATAGG TAAAAAGCAG TTACAGAGAG GTAAAATATA ACTTAAAAAA TCAGTTCTAA 180

GGAAAAATTG ACTTTTATAG TAAATGACTG TTATATAAGG ATGTTGTTAC AGAGAGGTAT 240

GAGTGTAGTT GGTAAATTAT GTTCTTGACG GTGTATGTCA CATATTATTT ATTAAAACTA 300

GAAAAAACAG CGTCAAAACT AGCAAAAATC CAACGGACAA AAAAATCGGC TGAATTTGAT 360

TTGGTTCCAA CATTTAAAAA AGTTTCAGTG AGAAAGAATC GGTGACTGTT GATGATATAA 420

ACAAAGGGCA CATTGGTCAA TAACCATAAA AAATTATATG ACAGCTACAG TTGGTAGCAT 480

GTGCTCAGCT ATTGAACAAA TCTAAAGAAG GTACATCTGT AACCGGAACA CCACTTAAAT 540

GACTAAATTA CCCTCATCAG AAAGCAGATG GAGTGCTACA AATAACACAC TATTCAACAA 600

CCATAAATAA AACGTGTTCA GCTACTAAAA CAAATATAAA TAAATCTATG TTTGTAAGCA 660

CTCCAGCCAT GTTAATGGAG TGCTATTGCC TGTTAACTCT CACTTATAAA ATAGTAGTAG 720

AA 722

(2) SEQ ID NO: and 7, information:

(i) sequence characteristics:

(A) length: 574 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEQ ID NO: 7

AGGTAAAATA TAACTTAAAA AATCAGTTCT AAGGAAAAAT TGACTTTTAT AGTAAATGAC 60

TGTTATATAA GGATGTTGTT ACAGAGAGGT ATGAGTGTAG TTGGTAAATT ATGTTCTTGA 120

CGGTGTATGT CACATATTAT TTATTAAAAC TAGAAAAAAC AGCGTCAAAA CTAGCAAAAA 180

TCCAACGGAC AAAAAAATCG GCTGAATTTG ATTTGGTTCC AACATTTAAA AAAGTTTCAG 240

TGAGAAAGAA TCGGTGACTG TTGATGATAT AAACAAAGGG CACATTGGTC AATAACCATA 300

AAAAATTATA TGACAGCTAC AGTTGGTAGC ATGTGCTCAG CTATTGAACA AATCTAAAGA 360

AGGTACATCT GTAACCGGAA CACCACTTAA ATGACTAAAT TACCCTCATC AGAAAGCAGA 420

TGGAGTGCTA CAAATAACAC ACTATTCAAC AACCATAAAT AAAACGTGTT CAGCTACTAA 480

AACAAATATA AATAAATCTA TGTTTGTAAG CACTCCAGCC ATGTTAATGG AGTGCTATTG 540

CCTGTTAACT CTCACTTATA AAATAGTAGT AGAA 574

(2) SEQ ID NO: and 8, information:

(i) sequence characteristics:

(A) length: 523 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEQ ID NO: 8

GTAAATGACT GTTATATAAG GATGTTGTTA CAGAGAGGTA TGAGTGTAGT TGGTAAATTA 60

TGTTCTTGAC GGTGTATGTC ACATATTATT TATTAAAACT AGAAAAAACA GCGTCAAAAC 120

TAGCAAAAAT CCAACGGACA AAAAAATCGG CTGAATTTGA TTTGGTTCCA ACATTTAAAA 180

AAGTTTCAGT GAGAAAGAAT CGGTGACTGT TGATGATATA AACAAAGGGC ACATTGGTCA 240

ATAACCATAA AAAATTATAT GACAGCTACA GTTGGTAGCA TGTGCTCAGC TATTGAACAA 300

ATCTAAAGAA GGTACATCTG TAACCGGAAC ACCACTTAAA TGACTAAATT ACCCTCATCA 360

GAAAGCAGAT GGAGTGCTAC AAATAACACA CTATTCAACA ACCATAAATA AAACGTGTTC 420

AGCTACTAAA ACAAATATAA ATAAATCTAT GTTTGTAAGC ACTCCAGCCA TGTTAATGGA 480

GTGCTATTGC CTGTTAACTC TCACTTATAA AATAGTAGTA GAA 523

(2) SEQ ID NO: 9, information:

(i) sequence characteristics:

(A) length: 220 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: DNA (genome)

(xi) Description of the sequence: SEQ ID NO: 9

TAAAGAAGGT ACATCTGTAA CCGGAACACC ACTTAAATGA CTAAATTACC CTCATCAGAA 60

AGCAGATGGA GTGCTACAAA TAACACACTA TTCAACAACC ATAAATAAAA CGTGTTCAGC 120

TACTAAAACA AATATAAATA AATCTATGTT TGTAAGCACT CCAGCCATGT TAATGGAGTG 180

CTATTGCCTG TTAACTCTCA CTTATAAAAT AGTAGTAGAA 220

Claims (15)

1. An isolated DNA molecule which directs root cortex specific transcription of a downstream heterologous DNA segment in a plant cell, said isolated DNA molecule having a sequence selected from any one of the following group of sequences:

(a) provided herein are SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 and SEQ ID NO: 9; and

(b) a DNA sequence capable of hybridizing under stringent washing conditions, 0.3M NaCl, 0.03M sodium citrate, 0.1% SDS, at 60 ℃ with an isolated DNA molecule having the sequence of (a) above, which DNA sequence is capable of directing root cortex specific transcription of a downstream heterologous DNA segment in a plant cell.

2. A DNA construct comprising an expression cassette comprising, in the 5 'to 3' direction, a root cortex specific promoter having a sequence selected from any one of the following group of sequences and a heterologous DNA segment positioned downstream of and operably linked to the promoter:

(a) provided herein are SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 and SEQ ID NO: 9; and

(b) a DNA sequence capable of hybridizing under stringent washing conditions, 0.3M NaCl, 0.03M sodium citrate 0.1% SDS, at 60 ℃ with an isolated DNA molecule having the sequence of (a) above, which DNA sequence is capable of directing root cortex specific transcription of a downstream heterologous DNA segment in a plant cell.

3. A DNA construct according to claim 2 wherein said DNA construct further comprises a plasmid.

4. A DNA construct according to claim 2 wherein said heterologous DNA segment is a gene encoding a pesticidal protein.

5. A DNA construct according to claim 3, wherein said heterologous DNA segment encodes a gene for a crystal protein of Bacillus thuringiensis (Bacillus thuringiensis) which is toxic to insects.

6. A plant cell comprising the DNA construct of claim 2.

7. A method of producing a transformed plant, the method comprising regenerating a plant from the plant cell of claim 7.

8. An agrobacterium tumefaciens (agrobacterium tumefaciens) cell comprising the DNA construct of claim 2, wherein said DNA construct further comprises a Ti plasmid.

9. A method for producing a transformed plant, which comprises infecting plant cells with Agrobacterium tumefaciens according to claim 8 to produce transformed plant cells, and regenerating a plant from said transformed plant cells.

10. A microparticle carrying a DNA construct according to claim 2, wherein said microparticle is suitable for use in shock transformation of plant cells.

11. A method of producing a transformed plant, which method comprises advancing a microparticle according to claim 10 into a plant cell to produce a transformed plant cell, and regenerating a plant from said transformed plant cell.

12. A plant cell protoplast comprising a DNA construct according to claim 2.

13. A method for producing a transformed plant, which method comprises regenerating a plant from a plant cell protoplast according to claim 12.

14. A transformed plant cell comprising the DNA construct of claim 1.

15. A transformed plant cell comprising the DNA construct of claim 3.