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Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis

A technology of zinc finger protein and biosynthesis, applied in the field of engineered DNA binding domain, which can solve the problems of labor-intensive, time-consuming, inability to specifically target selected genes, etc.

Active Publication Date: 2012-11-14
DOW AGROSCIENCES LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, conventional methods of altering plants to improve fatty acid profiles rely on mutagenesis (e.g., chemical, radiation, etc.) and / or breeding and are time-consuming, labor-intensive, and cannot specifically target selected genes

Method used

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  • Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis
  • Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis
  • Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0204] Example 1: Identification of target sequences in Brassica napus

[0205] 1.1 Target sequence identification

[0206] In this example, the endogenous DNA sequence encoding the native β-ketoacyl-ACP synthase II (KAS II) of Brassica napus (Brassica napus) was identified. These genes were selected as exemplary targets to demonstrate that transcriptional regulation by engineered zinc finger protein transcription factors (ZFP TFs) produces the desired modification of fatty acid biosynthesis and concomitant altered seed oil profile. The enzyme β-ketoacyl-ACP synthase II catalyzes the conversion of 16:0-ACP to 18:0-ACP and subsequent formation of oleic acid (Ohlrogge and Browse, 1995, The Plant Cell, 7:957-970). Reported β- The Arabidopsis fab1 mutation of ketoacyl-ACP synthase II resulted in a 65% decrease in enzyme activity accompanied by a 7% and 3% increase in stearic acid content in leaves and roots, respectively (Wu et al., 1994, Plant Physiology, 106:143- 150). Stab...

Embodiment 2

[0213] Example 2: Design of a ZFP DNA-binding domain specific to the β-ketoacyl-ACP synthetase II gene

[0214] Zinc finger proteins were designed against various target positions in the promoter region and 5' untranslated and translated regions of the Brassica napus β-ketoacyl-ACP synthase II gene. see figure 2 . Recognition helices for representative ZFP designs are shown in Table 1. Target locations for zinc finger design are shown in Table 2.

[0215] Table 1: β-ketoacyl-ACP synthetase II zinc finger design

[0216]

[0217] Table 2: Target positions of β-ketoacyl-ACP synthase II zinc fingers

[0218]

[0219] β-ketoacyl-ACP synthetase II was designed to be incorporated into a zinc finger expression vector encoding a protein with at least one CCHC-binding finger. See US Patent Publication 2008 / 0182332. Specifically, the last finger in each protein has a CCHC structure (architecture).

[0220] The zinc finger coding sequences were then fused to sequences encod...

Embodiment 3

[0221] Example 3: ZFP TF mediates upregulation of the native β-ketoacyl-ACP synthase II gene in Brassica napus

[0222] To evaluate the function of the designed zinc finger proteins in plant cells, a method of expressing the proteins in living plant cells was employed. DNA encoding a zinc finger protein can be delivered to a plant cell under conditions where the DNA does not integrate into the genome of the plant cell. Thus, DNA molecules are transiently maintained in plant cells and serve as templates for gene expression. Alternatively, the DNA encoding the zinc finger protein can be delivered to the cell under conditions under which the DNA integrates into the genome of the plant cell. A transgenome of the zinc finger protein encoding gene is generated such that the DNA molecule is stably maintained in the plant cell and serves as a template for gene expression. Those skilled in the art can evaluate the function of these proteins in living plant cells by transiently or tra...

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Abstract

The present disclosure relates to engineered zinc finger proteins that target genes in plants involved in fatty acid biosynthesis. Methods of using such zinc finger proteins in modulating gene expression, gene inactivation, and targeted gene modification are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No. 61 / 279,528, filed October 22, 2010, the contents of which are incorporated herein by reference in their entirety. technical field [0003] The invention mainly relates to the fields of plant genome engineering transformation and protein expression. Specifically, the present invention relates to engineered DNA binding domains, such as zinc finger proteins targeting genes involved in fatty acid synthesis, and the ability of said zinc finger proteins to produce changes in fatty acid distribution in the regulation of gene expression, gene inactivation, and target gene modification. Plant approach. Background technique [0004] A diet rich in saturated fatty acids increases low-density lipoprotein (LDL), which in turn causes cholesterol deposition on blood vessels, a precursor condition closely associated with arteriosclerosis and coronary heart disease (C...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N5/04A01H5/00
CPCC12N15/8216C12N15/8247C07K14/415C07K2319/00C12N9/22C12Y203/01041C12Y301/00C12N15/8218C12N15/82
Inventor R·迪克勒弗M·古普塔J·C·米勒S·诺瓦克J·F·珀托林诺
Owner DOW AGROSCIENCES LLC
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