Endoglucanase gene promoter upregulated by nematodes

a technology of endoglucanase and promoter, which is applied in the field of tissue-specific gene promoters, can solve the problems of affecting the growth of endoglucanase, and affecting the growth of endoglucanase, and achieves the effect of reducing the number of nematodes and endocannase gene promoters, and reducing the number o

Inactive Publication Date: 2005-07-28
DAVIS ERIC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0041] An advantage of the present invention is that two or more promoters can be “daisychained” to a single structural gene. Where each promoter is responsive to a different pathogen, the plant is then provided with resistance to a plurality of promoters. For example, a second promoter may be positioned upstream from the structural gene and operatively associated therewith so that the structural gene is associated with a plurality of promoters, with each of the promoters activated by a different plant pathogen. Still more promoters can be included if desired. Other promoters that may be used in conjunction with the instant promoter are described in U.S. Pat. No. 5,750,386 to Conkling et al.
[0042] The various fragments comprising the various constructs, expression cassettes, markers, and the like may be introduced consecutively by restriction enzyme cleavage of an appropriate-replication system and insertion of the particular construct or fragment into the available site. After ligation and cloning, the DNA construct may be isolated for further manipulation. All of these techniques are amply exemplified in the literature. See, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). 4. Plant Transformation Vectors and Techniques.
[0043] A vector is a replicable DNA construct. Vectors which may be used to transform plant tissue with DNA constructs of the present invention include both Agrobacterium vectors and ballistic vectors, as well as vectors suitable for DNA-mediated transformation. Agrobacterium tumefaciens cells containing a DNA construct of the present invention, wherein the DNA construct comprises a Ti plasmid, are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell.
[0044] Numerous Agrobacterium vector systems useful in carrying out the present invention are known. For example, U.S. Pat. No. 4,459,355 discloses a method for transforming susceptible plants, including dicots, with an Agrobacterium strain containing the Ti plasmid. The transformation of woody plants with an Agrobacterium vector is disclosed in U.S. Pat. No. 4,795,855. Further, U.S. Pat. No. 4,940,838 to Schilperoort et al. discloses a binary Agrobacterium vector (i.e., one in which the Agrobacterium contains one plasmid having the vir region of a Ti plasmid but no T-DNA region, and a second plasmid having a T-DNA region but no vir region) useful in carrying out the present invention.
[0045] Microparticles carrying a DNA construct of the present invention, which microparticle is suitable for the ballistic transformation of a plant cell, are also useful for making transformed plants of the present invention. The microparticle is propelled into a plant cell to produce a transformed plant cell and a plant is regenerated from the transformed plant cell. Any suitable ballistic cell transformation methodology and apparatus can be used in practicing the present invention. Exemplary apparatus and procedures are disclosed in Sanford and Wolf, U.S. Pat. No. 4,945,050, and in Agracetus European Patent Application Publication No. 0 270 356, titled “Pollen-mediated Plant Transformation”. When using ballistic transformation procedures, 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 such systems include 1 to 5 μm gold-spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.
[0046] Plant species may be transformed with the DNA construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art. 5. Plants for Transformation and Propagation of Transformants.

Problems solved by technology

Plant parasitic nematodes cause approximately 100 billion dollars annually in crop loses worldwide.

Method used

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  • Endoglucanase gene promoter upregulated by nematodes
  • Endoglucanase gene promoter upregulated by nematodes
  • Endoglucanase gene promoter upregulated by nematodes

Examples

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example 1

Plant and Nematode Culture Maintenance

[0054] Plant Material. Tobacco (Nicotiana tabacum ‘NC95’) seeds were surface sterilized with 2.5% sodium hypochlorite for five minutes, followed by several rinses with sterile water, and germinated in Petri plates containing 0.8% Noble agar (Fisher Scientific, Pittsburgh, Pa.) supplemented with Murashige and Skoog ((1962) Physiol. Plant Path. 15: 473-497) minimal media, pH 5.8 and 3% sucrose. Tobacco seedlings were grown in a controlled temperature growth chamber at 25° C. with a 14-hour photoperiod.

[0055] Nematode Cultures and Inoculations. The tobacco cyst nematode (TCN), Globodera tabacum subspecies solanacearum (Miller and Gray (1972) Nematologica 18: 404-413), and the root-knot nematode (RKN), Meloidogyne incognita Race 4 (Hartman and Sasser (1985) Identification of Meloidogyne species on the basis of differential host test and perineal—pattern morphology. In Advanced Treatise on Meloidogyne, Vol. II (Biology and Control), ed. J. N. Sasse...

example 2

In Planta Localization of TCN EGases Tissue

[0056] Fixation and Embedding. For immunolocalizations, TCN-infected root pieces were excised from Petri plates twenty-four to ninety-six hours after inoculation and fixed in 1% paraformaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 1.4 mM KH2PO4, 2.6 mM KCl, 1.8 mM Na2HPO4, pH 7.4) for three hours at room temperature. After two 15 minute washes with PBS, the fixed root pieces were dehydrated in a graded ethanol series (30%, 60%, 70%, 85%, 95%, 100%, 15 min. each) and then incubated sequentially in ethanol: Histoclear (National Diagnostics, Atlanta, Ga.) 75:25, 50:50, 25:75 for 10 minutes each. After two 15 minutes incubations in 100% Histoclear, the root pieces were transferred to molten Paraplast plus (Fisher Scientific, Pittsburgh, Pa.) at 60° C. for two hours and embedded in blocks. For in situ mRNA localizations, nematode-infected tobacco root pieces were dissected from Petri plates 7-9 days or 12-14 days after infection and...

example 3

Isolation and Sequence Characterization of Tobacco EGases

[0060] To isolate poly A(+) RNA, 5 cm of infected or noninfected tobacco root pieces (excluding root tips) were excised from Petri plates and ground in a small glass homogenizer in 250 μl lysis-binding buffer (100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM EDTA, pH 8.0, 5 mM dithiothreitol, 1% LiDS; Dynal, Lake Success, N.Y.). After lysis, the homogenate was centrifuged for one minute at 13,000×g and the supernatant was transferred to a clean tube. Twenty-five microliters of Dynal magnetic oligo-(dT)25 beads equilibrated with lysis-binding buffer were added to the supernatant and placed on a rotator for 5 minutes to allow the mRNA to anneal to the beads. Using a magnetic stand the beads were washed twice in washing buffer with LiDS (10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA, 0.1% LiDS) and three times in washing buffer without LiDS. For first strand cDNA synthesis, the beads were washed several times in IX first strand buff...

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Abstract

The present invention provides a nucleic acid construct comprising a cyst and root knot nematode responsive promoter, preferably the Nicotiana Ntcel7 promoter or promoters that hybridize thereto, operatively associated with a heterologous nucleic acid segment that encodes a product disruptive of nematode attack. Plants and plant cells using the same and methods of use thereof are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This is a continuation application of U.S. patent application Ser. No. 09 / 970,367 currently pending, Oct. 21, 2001, the disclosure of which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION [0002] This invention relates to tissue-specific gene promoters, and particularly relates to a promoter which is responsive to the cyst and root knot nematodes. BACKGROUND OF THE INVENTION [0003] A promoter is a DNA sequence which flanks a transcribed gene, and to which RNA polymerase must bind if it is to transcribe the flanking gene into messenger RNA. A promoter may consist of a number of different regulatory elements which affect a structural gene operationally associated with the promoter in different ways. For example, a regulatory gene may enhance or repress expression of an associated structural gene, subject that gene to developmental regulation, or contribute to the tissue-specific regulation of that gene. Modificat...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/42C12N15/82
CPCC12N15/8239C12Y302/01004C12N9/2437
Inventor DAVIS, ERICGOELLNER, MELISSA
Owner DAVIS ERIC
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