Nucleic acid molecules encoding fatty acid desaturase genes from plants and methods of use

A nucleic acid, coding technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, plant products, etc., can solve problems such as reducing the risk of heart disease

Inactive Publication Date: 2008-02-06
BASF PLANT SCI GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, consumption of plants with increased levels of these types of fatty acids may reduce the risk of heart disease

Method used

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  • Nucleic acid molecules encoding fatty acid desaturase genes from plants and methods of use
  • Nucleic acid molecules encoding fatty acid desaturase genes from plants and methods of use
  • Nucleic acid molecules encoding fatty acid desaturase genes from plants and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0266] Example 1: General steps

[0267] a) General cloning steps:

[0268] According to Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press: ISBN 0-87969-451-3) Cloning steps such as restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, ligation of DNA fragments, transformation of Escherichia coli and yeast cells, bacterial growth, and preparation of recombinant DNA Sequence analysis.

[0269] b) Chemical substances:

[0270] Unless otherwise stated in the text, the chemicals used were p.a. grade products from the companies Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen). Purified pyrogen-free water (hereinafter referred to as H 2 O) Prepare the solution. Restriction enzymes, DNA modifying enzymes and ...

Embodiment 2

[0285] Example 2: Isolation of total DNA from plants

[0286] The details of isolating total DNA involved the treatment of 1 g fresh weight of plant material.

[0287] CTAB buffer: 2% (w / v) N-cetyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0; 1.4M NaCl; 20 mM EDTA.

[0288] N-Lauryl Sarcosine Buffer: 10% (w / v) N-Lauryl Sarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

[0289] The plant material was ground to a fine powder in a mortar and mortar in liquid nitrogen and transferred to a 2 ml Eppendorf tube. The frozen plant material was then covered with a layer of 1 ml of lysis buffer (1 ml of CTAB buffer, 100 μl of N-lauryl sarcosine buffer, 20 μl of β-mercaptoethanol and 10 μl of 10 mg / ml proteinase K solution) and placed in Incubate with continuous shaking at 60°C for 1 hour. The resulting homogenate was divided into two Eppendorf tubes (2 ml) and extracted twice with the same volume of chloroform / isoamyl alcohol (24:1) by shaking. Centrifuge at 8000g at ...

Embodiment 3

[0290] Example 3: Isolation of total RNA and poly-(A)+RNA from plants

[0291] Arabidopsis

[0292] Total RNA and poly-(A)+ RNA were isolated to study transcripts. RNA was isolated from siliques of Arabidopsis plants according to the following method:

[0293] Preparation of RNA from Arabidopsis seeds -- "hot" extraction:

[0294] Buffers, Enzymes and Solutions:

[0295] 2M KCl

[0296] Proteinase K

[0297] Phenol (for RNA)

[0298] Chloroform: Isoamyl Alcohol

[0299] (phenol:chloroform 1:1; adjust pH for RNA)

[0300] 4M LiCl, DEPC treated

[0301] DEPC treated water

[0302] 3M NaOAc, pH 5, DEPC-treated

[0303] Isopropanol

[0304] 70% ethanol (prepared with DEPC treated water)

[0305] Resuspension buffer: 0.5% SDS, 10mM Tris pH 7.5, prepared in DEPC-treated water

[0306] 1mM EDTA (as this solution cannot be treated with DEPC)

[0307] Extraction buffer:

[0308] 0.2M sodium borate

[0309] 30mM EDTA

[0310] 30mM EGTA

[0311] 1% SDS (250 μl 10% SDS s...

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PUM

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Abstract

This invention relates generally to nucleic acid sequences encoding proteins that are related to the presence of seed storage compounds in plants. More specifically, the present invention relates to FAD2-like nucleic acid sequences encoding lipid metabolism regulator proteins and the use of these sequences in transgenic plants. In particular, the invention is directed to methods for manipulating lipid metabolism related compounds and for increasing oil level and altering the fatty acid composition in plants and seeds. The invention further relates to methods of using these novel plant polypeptides to stimulate plant growth and / or to increase yield and / or composition of seed storage compounds.

Description

[0001] This application claims priority to US Provisional Application 60 / 637,531, filed December 20, 2004, which is incorporated herein by reference in its entirety. field of invention [0002] Inventions in the field of genetic engineering of plants are described herein, including isolated nucleic acid molecules encoding fatty acid desaturase 2-like (FAD2-like) polypeptides to enhance their agronomic, horticultural and quality traits. The present invention generally relates to nucleic acid sequences encoding proteins associated with the presence of seed storage compounds in plants. More specifically, the present invention relates to FAD2-like coding nucleic acid sequences of lipid metabolism regulating proteins, and the use of these sequences in transgenic plants. In particular, the invention relates to methods of manipulating compounds involved in lipid metabolism, and methods of increasing oil levels and altering fatty acid composition in plants and seeds. The present inve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/02A01H5/00
CPCC12N15/8218C12N15/8247
Inventor H·黑特尔J·吉布森P·任
Owner BASF PLANT SCI GMBH
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