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Problems solved by technology
These deficiencies include: use of toxic or volatile inducers, low induction levels, poor translocation / mobility in plants, slow ability to "turn off" gene expression, or insufficient specificity for non-toxic inducers in plants, etc.
Induction of ethylene insensitivity at will and maintenance for a predetermined time frame has not been successfully achieved in the prior art
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Examples
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Embodiment 1
[0110] Example 1: Plasmids
[0111] Activation cassettes of individually cloned gene cassette assemblies include:
[0112] G10-90 constitutive promoter,
[0113] VP16 activation domain,
[0114] GAL4 DNA binding domain, and
[0115] Ecdysone receptor ligand binding domain linked to NOS terminator sequence.
[0116] Similarly, to clone the targeting cassette components individually, the targeting cassette includes:
[0117] an inducible promoter consisting of 5 copies of the GAL4 response element and a minimal 35S promoter, and
[0118] ETP1 gene (GENBANK accession number NM_112874) or ETP2 gene (GENBANK accession number NM_112777) and
[0119] 35S terminator sequence.
[0120] All individual sequences are identified with reference to the nucleotide number of SEQ ID NO: 1 below.
[0121] During the assembly of the activation cassette, the following components fuse in two different orders to form two different activation cassettes:
[0122] VP16 AD to GAL4 LBD to DBD EcR...
Embodiment 2
[0133] Embodiment 2: Preparation of transgenic Arabidopsis plants
[0134] Arabidopsis plants were transformed with the Agrobacterium plasmid of Example 1 using standard flower dipping procedures. Seeds were harvested and inoculated on kanamycin-containing medium. Transformed plants were selected for their ability to grow on kanamycin and screened by PCR to verify the presence of the ETP1 or ETP2 gene. Positive transformants were selfed to produce T1 seeds. Seeds were grown on media containing kanamycin to identify transgenic homozygous lines. Homozygous plants were used for the experiment of inducing ethylene insensitivity.
Embodiment 3
[0135] Example 3: Effects of Modulating Ethylene Sensitivity on Growth of Arabidopsis Plants
[0136] Triple reaction assays (modified as described below based on Guzman and Ecker, 1990 cited above) were performed to determine the modulation of ethylene sensitivity in the transformed Arabidopsis plants of Example 2. Wild-type ein2-5 (ethylene-insensitive mutant control) and p1004 or p1005 Arabidopsis transformant seedlings were assayed. The Arabidopsis seeds were sterilized on the surface and soaked in 20 μM inducer, and kept in the dark at 4°C for 4 days. Seeds were inoculated on 0.5X MS containing 1% sucrose and 20 μM inducer, with and without 20 μM ACC (ethylene precursor), and incubated at 21° C. in the dark for 4-8 days. In some experiments, 5 μM AgNO was added to the medium 3 (ethylene inhibitor that induces ethylene insensitivity) served as a control. Responses to ethylene were recorded on the last day. Transgenic plants containing an activation cassette and a targe...
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Abstract
A gene expression system for controllable expression of ethylene response in a plant cell includes an activation cassette comprising a DNA-binding domain that recognizes a response element; an ecdysone receptor ligand binding domain; and an activation domain; and a target cassette comprising an inducible promoter, which comprises, in operative association, the response element and a minimal promoter responsive to the activation domain. The inducible promoter controls the expression of a nucleic acid sequence that encodes a selected regulatory protein that modifies sensitivity to ethylene of certain signal proteins in the plant. Interaction among the components of the activation cassette and target cassette, when in a plant cell, in the presence of an inducing composition, increases expression of the selected regulatory protein, and in turn decreases expression and accumulation of the signal protein in the plant, thereby and decreasing ethylene sensitivity in the plant cell. This increase in the expression of the regulatory protein, particularly in the presence of ethylene, is controlled by the timing, the concentration and the duration of the application of the inducing composition. Transgenic plant cells, tissues, organs and entire plants are provided, which in the presence of the inducing composition control ethylene sensitivity. Ethylene sensitivity and / or ethylene production in such transgenic plants and tissues may be controlled for purposes of manipulating ripening, flower senescence and other ethylene sensitive functions of the plant.
Description
Background of the invention [0001] The plant hormone ethylene is a signaling molecule that regulates many physiological processes in the plant life cycle, including during germination, flower and fruit development, and plant responses to various environmental stressors such as drought, heat, salt Overdose and disease responses (see, eg, Chen et al., 2005, Annals of Botany, 95:901-915; Czarny et al., 2006 Biotechnol. Adv., 24:410-419). Ethylene biosynthetic pathways and signaling / regulatory pathways and networks are well known. For example, see figure 1 and 2 , Wang et al., "Ethylene Biosynthesis and Signaling Networks", "The Plant Cell", 2002 (edited by the American Society of Plant Biologists), p. Pages S131-S151. [0002] In plants, several key steps in the ethylene signaling pathway are highly regulated. As for the EIN2 (ethylene insensitive 2) and EIN3 (ethylene insensitive 3) proteins, their expression is induced by ethylene, which leads to an increased ethylene resp...
Claims
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