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DNA shuffling to produce herbicide selective crops

A herbicide and active technology, applied in the field of herbicide-selective crops produced by DNA recombination, can solve problems such as heavy workload

Inactive Publication Date: 2001-09-26
MAXYGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it is generally theoretically possible to determine which specific structural features confer herbicide tolerance on a particular form of P450 (or other protein encoded by a potential herbicide tolerance gene), and to understand how the gene can be modified to enhance tolerance, but the workload involved in this task is considerable

Method used

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  • DNA shuffling to produce herbicide selective crops
  • DNA shuffling to produce herbicide selective crops

Examples

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preparation example Construction

[0216] The present invention refers to a number of methods known in the art for producing transformable protoplasts of various plants and then transforming cultured protoplasts. See, eg, Hashmoto et al. (1990), Plant Physiol. 93:857; Plant Protoplasts, edited by Fowke LC and Constabel F, CRC Press (1994); Saunders et al. (1993), Plant In Vitro Symposium on Applications in Technology, UPM, 16-18, November 1993; and Lyznik et al. (1991), Bio Techniques 10:295, which are incorporated herein by reference.

[0217] chloroplast

[0218] The chloroplast may be the site of action for some herbicide tolerance, and, sometimes, the product of a herbicide tolerance gene is preferentially fused to a chloroplast transit sequence peptide, thereby facilitating entry of the gene product into the chloroplast. At this point, the shuffled herbicide tolerance nucleic acid is preferably transformed into the chloroplast of the plant host cell. Chloroplast transformation and expression can be perfo...

Embodiment 1

[0245] Example 1: Shuffling of plant EPSPS genes for glyphosate tolerance

[0246] Arabidopsis EPSPS cDNA was PCR amplified from reverse transcribed RNA using primers 5'GCAGT CCATG GAGAA AAGCG TCGGA GATTG TACTT CAACC C-3' and 5'-TAGAC TAAGA TCTGT GCTTT GTGAT TCTTT CAAGT ACTTG G-3'. This fragment was digested with NcoI and BglII, and then directionally cloned into the prokaryotic expression vector pQE60 (QIAGEN) and introduced into E. coli AroA AB2829 (Pittard, 1966). Likewise, tomato cDNA was amplified from purified phage DNA from a cDNA library (Stratagene) with primers 5′-ACGTC CATGG CAAAA CCCCA TGAGA TTGTG CTAG-3′ and 5′-CAGTA GATCT GTGCT TAGAG TACTT CTGGA G-3′ and cloned into pQE60 , and introduced into AB2829 cells. Growth of transformed cells on minimal medium lacking aromatic amino acids demonstrated that the function of the AroA mutation was complemented by the expression of the cloned EPSPS gene.

[0247] The Arabidopsis and tomato EPSPS genes cloned from pQE60 were...

Embodiment 2

[0249] Example 2: Glyphosate tolerance of recombinant forms of EPSP synthase

[0250] The positive activity of EPSP synthase was determined by monitoring phosphate production with the malachite green colorimetric assay (Lanzetta PA et al., Anal. Biochem. 100:95-97, 1979). Reactions were performed in assay buffer (50 mM HEPES, pH 7.0 and 0.1 mM ammonium molybdate) containing enzymes, 0.1 mM phosphoenolpyruvate, 0.1 mM shikimate-3-phosphate, and various concentrations of glyphosate, The final volume is 0.2 ml. After 20 minutes, 0.7 mL of malachite green reagent (3 parts 0.045% malachite green to 1 part 4.2% ammonium molybdate) was added to stop the reaction. After 10 minutes, the absorbance at 660 nm was measured with a Beckman DU600 spectrophotometer. The inhibition constant (150) for each enzyme against glyphosate was obtained from the plot of percent activity versus glyphosate concentration. The K for PEP was obtained from a plot of the rate of product formation versus PEP...

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Abstract

The present invention provides a method for shuffling DNA to obtain a recombinant herbicide tolerance nucleic acid, the protein encoded by the nucleic acid has new or improved herbicide tolerance activity, and the invention also provides the shuffled herbicide tolerance nucleic acid Libraries, transgenic plants and DNA shuffling mixtures.

Description

Field of Invention [0001] The present invention relates to the use of nucleic acid shuffling to achieve or enhance herbicide tolerance. Background of the Invention [0002] Herbicides are commonly used in modern agriculture to control weed growth in crop fields. Strategies for applying herbicides to kill weeds without harming species (crop plants) depend on the selective tolerance of certain species to a given herbicide. In other words, the plants survived the herbicide application without being visibly sick, while the weedy plants did not. [0003] "Crop selectivity" is defined as the ability of a crop to survive herbicide treatment with no visible damage (or at least minimal damage) compared to the herbicide's control of a target weed. The fact that herbicides are used on crops suggests that they are safe (selective) for crops, but provide overall or at least acceptable control of economically important weeds. [0004] Crop selectivity is dete...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/21C12N5/10A01H1/00C12N9/02C12N9/10C12N15/01C12N15/09C12N15/10C12N15/82C12Q1/02C12R1/19C12R1/91
CPCC12N9/0073C12N9/1092C12N9/0004C12N15/1027C12N15/8275C12N15/82
Inventor V·萨布拉马尼安W·P·C·斯特默尔L·A·卡斯尔U·S·穆赫哈D·L·西尔
Owner MAXYGEN
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