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An oleosin 5' regulatory region for the modification of plant seed lipid

A technology of oleosin and regulatory regions, which is applied in the direction of using vectors to introduce foreign genetic material, cells and plant peptides modified by introducing foreign genetic material, and can solve the problem of lack of Δ6-desaturase activity and other problems

Inactive Publication Date: 2000-07-05
RHONE POULENC AGRO SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is due to the lack of Δ6-desaturase activity in most plants

Method used

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  • An oleosin 5' regulatory region for the modification of plant seed lipid
  • An oleosin 5' regulatory region for the modification of plant seed lipid
  • An oleosin 5' regulatory region for the modification of plant seed lipid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Isolation of membrane-bound polyribosomal RNA and construction of borage cDNA library

[0080] Membrane-bound polysomes were isolated 12 days after pollination (12DPP) from borage seeds using the method established for pea by Larkins and Davies (1975 Plant Phys. 55:749-756). RNA was extracted from polysomes as described by Mechler (1987, Methods in Enzymology 152:241-248, Academic Press). With Oligotex-dT TM Beads (Qiagen) to isolate Poly-A from membrane-bound polysomal RNA + RNA.

[0081] The corresponding cDNA was prepared with Stratagene's ZAP cDNA synthesis kit. The cDNA library was constructed into the λZAP II vector using the λZAP II kit. Primary libraries were packaged with Gigapack II Gold packaging extract (Stratagene).

Embodiment 2

[0083] Isolation of Δ6-desaturase cDNA from Borage

[0084] hybridization program

[0085] The amplified borage cDNA library was seeded at low density (500 pfu on 150 mm Petri dishes). High levels of seed storage protein cDNA were reduced (subtracted from total cDNA) by screening for corresponding cDNA.

[0086] (1994, Current Protocols in Molecular Biology, Wiley Interscience, N.Y.) using the random guided DNA synthesis technique described by Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley Interscience, N.Y.) produces the hybridization probe that is used for screening borage cDNA library, and with the seed storage protein cDNA of abundant expression that has identified before correspond. Unbound nucleotides were removed using a G-50 spin column (Boehringer Manheim). Hybridization probes were denatured by boiling in a water bath for 5 minutes, followed by rapid cooling on ice. The nitrocellulose filter carrying the immobilized recombinant p...

Embodiment 3

[0125] Generation of transgenic plants and preparation and analysis of fatty acid methyl esters (FAME)

[0126] Tobacco (Nicotiana tabacum cv. xanthi ), only the initial transformants were selected on 100 μg / ml kanamycin.

[0127] Freeze transgenic plant tissue in liquid nitrogen and lyophilize overnight. FAMEs were prepared as described in Dahmer et al., (1989) J. Amer. Oil. Chem. Soc. 66:543-548. In some cases, the solvent was evaporated again and the FAMEs were resuspended in ethyl acetate and extracted once with deionized water to remove all water-soluble contaminants. FAMEs were analyzed by Tracor-560 gas liquid chromatography as previously described (Reddy et al., 1996 Nature Biotech. 14:639-642).

[0128] As shown in Figure 3, transgenic tobacco leaves containing borage cDNA produced GLA and stearidonic acid (OTA) (18:4 Δ6, 9, 12, 15). These results thus demonstrate that the isolated cDNA encodes a borage Δ6-desaturase.

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Abstract

The present invention is directed to 5' regulatory regions of an Arabidopsis oleosin gene. The 5' regulatory regions, when operably linked to either the coding sequence of a heterologous gene or a sequence complementary to a native plant gene, direct expression of the coding sequence or complementary sequence in a plant seed. The regulatory regions are useful in expression cassettes and expression vectors for the transformation of plants. Also provided are methods of modulating the levels of a heterologous gene such as a fatty acid synthesis or lipid metabolism gene by transforming a plant with the subject expression cassettes and expression vectors.

Description

Background of the invention [0001] Seed oil content has traditionally been improved through plant breeding. Using recombinant DNA technology to alter the composition of the seed oil can speed up this process and, in some cases, alter the oil in ways that cannot be achieved by breeding alone. The oil composition of Brassica has been significantly altered by modifying the expression of a large number of lipid metabolism genes. These manipulations to improve the composition of seed oils focus on altering the proportion of endogenous constituent fatty acids. For example, antisense suppression of the Δ12-desaturase gene in transgenic rapeseed has resulted in an increase in oleic acid of up to 83%. Topfer et al., 1995 Science 268:681-686. [0002] There have been some successful attempts at modifying the seed oil fraction of transgenic plants by introducing new genes that allow the production of fatty acids that the host plant was previously unable to synthesize. Van de Loo et a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C07K14/415C12N1/21C12N5/10C12N9/02C12N15/09C12N15/29C12N15/82
CPCC07K14/415C12N15/8247C12N9/0083C12N15/8234
Inventor T·L·托马斯Z·S·李
Owner RHONE POULENC AGRO SA
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