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Constructs for expressing herbicide tolerance genes, related plants, and related trait combinations

A technology of transgenic plants and herbicides, applied in the fields of botanical equipment and methods, plant products, genetic engineering, etc.

Inactive Publication Date: 2011-07-06
DOW AGROSCIENCES LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The heterologous expression of this gene in other microorganisms, or the transformation of this gene into plants, has not been reported so far

Method used

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  • Constructs for expressing herbicide tolerance genes, related plants, and related trait combinations
  • Constructs for expressing herbicide tolerance genes, related plants, and related trait combinations
  • Constructs for expressing herbicide tolerance genes, related plants, and related trait combinations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1. Method for identifying genes that confer glufosinate-ammonium resistance in plants

[0111] As a method for identifying genes possessing herbicide-degrading activity in planta or cell culture, current public databases such as NCBI (National Center for Biotechnology Information) can be mined. To start the process, one must have an identified functional gene sequence encoding a protein with the desired characteristics (ie, phosphinothricin acetyltransferase). This protein sequence was then used as input to the BLAST (Basic Local Alignment Search Tool) (Altschul et al., 1997) algorithm for comparison against stored available NCBI protein sequences. Using default settings, this search returned more than 100 homologous protein sequences at different levels. These range from high identity (85-98%) to very low identity (23-32%) at the amino acid level. Traditionally, only sequences with high homology would be expected to retain similar properties to the input sequ...

Embodiment 2

[0113] Example 2. Optimization of sequences for expression in plants and bacteria

[0114] 2.1- Background

[0115] In order to obtain higher levels of expression of heterologous genes in plants, it may be preferable to reengineer the protein coding sequences of said genes so that they are expressed more efficiently in plant cells. Maize is one such plant in which the heterologous protein coding region can preferably be redesigned prior to transformation to increase the expression level of the gene and the level of the encoded protein in the plant. Therefore, an additional step in the design of genes encoding bacterial proteins is the reengineering of heterologous genes for optimal expression. See, for example, Kawabe et al. (2003), "Patterns of Codon Usage Bias in Three Dicot and Four Monocot Plant Species," Genes Genet. Syst., pp. 343-352; and Ikemura et al. (1993), "Plant Molecular Biology Labfax", Croy , ed., Bios Scientific Publishers Ltd., p.3748), and all relevant ref...

Embodiment 3

[0132] Example 3. Cloning of Transformation Vectors

[0133] 3.1 - Construction of a binary plasmid containing DSM-2 (v2)

[0134] The DSM-2 (v2) codon-optimized gene of the coding sequence (DASPICO45) was cut with restriction enzymes BbsI (New England Biolabs, Inc., Beverly MA, Cat. No. R0539s) and SacI (New England Biolabs, Inc., Cat. No. R0156s) . The resulting fragment was ligated into pDAB773 at the corresponding restriction sites NcoI (New England Biolabs, cat# R0193s) and Sad. Positive colonies were identified by restriction enzyme digestion. The resulting clone contains Rb7MAR v3 / / At Ubi10 Promoter v2 / / Gene of Interest / / Atu Orf 13'UTR v3. The plasmid containing DSM-2 (v2) as the gene of interest was labeled pDAB3774.

[0135] The Rb7MAR v3 / / AtUbi10 Promoter v2 / / Gene of Interest / / Atu Orf13'UTRv3 expression cassette was cloned as an AgeI (New England Biolabs, Inc., Cat. No. R0552s) restriction fragment into the binary vector pDAB3736. This expression cassette was cl...

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Abstract

The subject invention relates in part to constructs for expressing herbicide tolerance genes, related plants, and related trait combinations. Such constructs and plants comprise a gene referred to herein as DSM-2. This gene was identified in Streptomyces coelicolor (A3). The DSM-2 protein is distantly related to PAT and BAR. DSM-2 can be used as a transgenic trait to impart tolerance in plant cells and plants to the herbicidal molecules glufosinate, phosphinothricin, bialaphos, and / or the like. The subject invention also relates to combination of the subject herbicide tolerant crop (HTC) traits along with other traits, including other HTC traits and / or insect resistance (IR).

Description

Background of the invention [0001] A selectable marker is a detectable genetic trait or segment of DNA that can be identified and tracked. A marker gene often serves as a flag for another gene (sometimes called a target gene). Marker genes are often used together with target genes for transformation of target cells. Target cells that heritably take up a target gene can be identified by selecting for cells that also express the marker gene. The marker gene is sufficiently close to the target gene that the two genes (marker gene and target gene) are genetically linked and usually inherited together. The current standard for selectable markers is the "pat" gene, which encodes an enzyme called phosphinothricin acetyltransferase. [0002] Glutamine synthetase ("GS") is a key enzyme in many plants for the development and life of plant cells. GS converts glutamate to glutamine. GS is also involved in ammonia assimilation and nitrogen metabolism. GS is involved in the pathway us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N15/87
CPCC12N15/821C12N15/8274Y02A40/146
Inventor 贾斯廷.M.利拉特里.R.赖特蒂莫西.D.海伊托尼亚.S.莫伊纳汉利萨.W.贝克
Owner DOW AGROSCIENCES LLC
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