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Compositions and methods for the modification of physiological responses in plants

一种组合物、植物的技术，应用在生物化学设备和方法、植物细胞、重组DNA技术等方向，能够解决“关闭”基因表达的能力慢、诱导水平低、植物非毒性诱导剂特异性不够等问题

Inactive Publication Date: 2010-09-29
ROHM & HAAS CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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  • Compositions and methods for the modification of physiological responses in plants
  • Compositions and methods for the modification of physiological responses in plants
  • Compositions and methods for the modification of physiological responses in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1: Plasmids

[0105] Gene cassette components include an activation cassette comprising: G10-90 constitutive promoter (nucleotides 1-243 of SEQ ID NO: 1), VP16 activation domain (nucleotides 249-529 of SEQ ID NO: 1) , the GAL4 DNA binding domain (nucleotides 534-983 of SEQ ID NO: 1), binds to the T52V mutant ecdysone receptor ligand bound to the NOS terminator sequence (nucleotides 2070-2364 of SEQ ID NO: 1) domain (nucleotides 990-1997 of SEQ ID NO: 1), which were cloned separately. Similarly, the target cassette assembly was cloned separately, and the target cassette included: 5 copies of the GAL4 response element (nucleotides 2391-2492 of SEQ ID NO: 1) and the minimal 35S promoter (nucleotides 2391-2492 of SEQ ID NO: 1 2499-2554), and the mutant etr1-1 gene (nucleotides 2557-4764 of SEQ ID NO: 1) and the 35S terminator sequence (nucleotides 4791-5001 of SEQ ID NO: 1 ). SEQ ID NO: 1 is shown in FIG. 4 .

[0106] These components are in SK - Expression cas...

Embodiment 2

[0128] Embodiment 2: Preparation of transgenic tobacco plants

[0129] Tobacco plants were transformed with the plasmid of Example 1, prepared by standard Agrobacterium-mediated leaf disc transformation as described in Fisher and Guiltinan 1995, Plant Molecular Biology Reporter 13:278-289. Plants were propagated on rooting medium containing kanamycin, and then the introduction and inheritance of the gene expression system was verified by PCR, as described below.

[0130] One leaf from each plant maintained in a magenta box was collected, snap-frozen in liquid nitrogen and stored at -80°C. The leaves (50-100 mg) were then transferred to KONTES tubes and ground using a drill with a KONTES disposable grinding rod for about 1 minute, followed by addition of lysis buffer for a few seconds. DNA was purified using the DNEASY Mini Kit (Qiagen) protocol. DNA was finally eluted in 100 μl.

[0131] Specific PCR primers were designed to amplify 529bp of VGE, 495bp of GVE, 463bp of mark...

Embodiment 3

[0138] Example 3: Effect of Regulating Ethylene Sensitivity on Tobacco Plant Growth

[0139] A triple reaction assay modified from Guzman and Ecker 1990 The Plant Cell, 2: 513-523 was performed to determine the role of modulation of ethylene sensitivity in the transformed tobacco plants of Example 2. Two leaves were obtained from each transformed plant designated 185-1, 184-3, 185-8, 185-11, 187-7, 187-13, 187-17 and 187-21 and cut into small pieces. These patches were plated on MSS plates containing 100 ng / L kanamycin with or without 10 μM of the inducer, 3,5-dimethyl-benzoic acid N-(1-ethyl-2, 2-Dimethyl-propyl)-N'-(3S-hydroxymethyl-5-methyl-2,3-dihydro-benzo[1,4]dioxine-6-carbonyl) Hydrazine. MSS plates without antibiotics or inducers were used as controls.

[0140] These plated plant tissues were grown at 25°C with light in the growth chamber to induce germination. The shoots were transferred to fresh plates and incubated for about 2 weeks at 25°C for continued growth....

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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 gene expression system modulates sensitivity to ethylene in transgenic plant cells, tissues, organs and entire plants in the presence of an inducing composition controlling ethylene sensitivity. Ethylene sensitivity 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 technique [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, Salinity excess and response to disease (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 signal transduction / 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. Commercially, a conventional route to modulate the effects of ethylene in plants, including fruits, vegetables and flowers, involves the application of chemicals to the plants, fruits, flowers or vegetables, such as 1-methylcyclopropene (1-MCP; Nongxin Co....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82
CPCC12N15/8291C12N15/8249C12N15/8238C12N15/8266C12N15/8217C12N15/8237
Inventor J·L·罗西尚D·R·加里
Owner ROHM & HAAS CO
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