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Herbicide-resistant ACCase mutant gene and protein and application thereof

A mutant, herbicide technology, applied in the field of plant protein and plant herbicide resistance, can solve the problems that it is difficult to predict in advance the ACCase protein to produce herbicide resistance, and the mechanism of action of the herbicide has not yet been determined.

Active Publication Date: 2018-12-25
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Unfortunately, the mechanism of action of ACCase inhibitor herbicides has not yet been determined, and it is difficult to predict in advance whether mutations in other amino acid sites of the ACCase protein will cause herbicide resistance. With some luck, new herbicide resistance loci in ACCase proteins may be discovered

Method used

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  • Herbicide-resistant ACCase mutant gene and protein and application thereof
  • Herbicide-resistant ACCase mutant gene and protein and application thereof
  • Herbicide-resistant ACCase mutant gene and protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Determination of rice ACCase gene mutation site

[0062] We used the ACCase sequence (Genbank AJ310767.1) of the plastid of Alopecurus myosuroides (black grass) as a reference sequence, and performed homology alignment with the rice genome, and found that it was identical to the ACCase gene (XM_015783727.1) of the rice genome. ) has the highest homology. According to the amino acid mutation sites mentioned in the patent (CN 102946714 B), the black grass ACCase can be resistant to herbicides, which are 1785 (mutation of alanine to glycine), 1786 (mutation of alanine to proline), 1824 ( Glutamine to proline), 1864 (valine to phenylalanine), 1999 (tryptophan to glycine), 1999 (tryptophan to cysteine), 2039 (glutamine acid to glycine), 2049 (valine to leucine), 2059 (alanine to valine), 2095 (lysine to glutamic acid) as reference, through homologous alignment Methods, the amino acid sites of ACCase gene mutation in rice were identified as 1796 (alanine to glyci...

Embodiment 2

[0063] Example 2: Obtaining the full-length gene of wild-type ACCase of rice Jiahua No. 1

[0064] The leaves of the wild-type plant of Jiahua No. 1 were selected, the genomic DNA was extracted, and the primers were designed according to the ACCase gene of NCBI Nipponbare (NCBI: XM_015783727) for amplification, and KOD DNA polymerase (purchased from Toyobo) was used to amplify the ACCase gene. The reaction system is as follows:

[0065]

[0066] Add sterile water to a total volume of 50 μl

[0067] The PCR amplification reaction procedure adopts a two-step method, annealing and extension are combined together at 68 degrees.

[0068] The procedure was as follows: pre-denaturation: 94°C for 3 min; 35 cycles: denaturation at 94°C for 10 sec; extension at 68°C for 10 min; incubation: 72°C for 10 min.

[0069] Take 2 μl of PCR product and test it by 1% agarose gel electrophoresis, after finding fragments of expected size ( figure 2 ), the remaining PCR products were cleaned ...

Embodiment 3

[0070] Example 3: Obtaining the Gene Sequence of Rice ACCase Gene Mutation Site

[0071] The mutation site sequence of rice ACCase gene was amplified by point mutation kit. First, using the primers ACC-CF and ACC-CR, and using Jiahua No. 1 cDNA as a template, the full-length ACCase cDNA was cloned, and ligated into the TK007 vector (Nanjing Qingke Biotechnology Co., Ltd.) to transform E. coli TOP10 strain competent cells. The positive transformants were selected and verified by sequencing, and the correct single clone was obtained, and the plasmid was named TK007-ACCase; secondly, the mutant primers ACC-MutF and ACC-MutR were used, and TK007-ACCase was used as the template for PCR amplification, and the amplification product was restricted. After digestion with endonuclease DpnI, it was transferred into competent cells of Escherichia coli DMT strain, and positive transformants were selected for sequencing verification to obtain a plasmid with ACCase target site mutation.

[0...

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Abstract

The invention discloses rice ACCase mutant protein. An amino acid sequence of the ACCase mutant protein has one or more types of mutations corresponding to mutations of the 1796th locus, the 1797th locus, the 1835th locus, the 1875th locus, the 2010th locus, the 2050th locus, the 2060th locus, the 2070th locus and the 2106th locus of a wild-type rice ACCase amino acid sequence. The invention further discloses a nucleic acid or a gene which codes the protein. By a transgenic method, the mutant gene is expressed in herbicide-sensitive rice, and plants containing the mutant protein are resistantto acetyl coenzyme A carboxylase herbicides. After application of 3mL quizalofop-ethyl / L water (twice of recommended concentration) to three-leaf rice seedlings containing the rice ACCase mutant protein, the plants still grow, develop and fruit normally.

Description

technical field [0001] The invention belongs to the field of plant protein and plant herbicide resistance, and relates to ACCase mutant gene and protein with herbicide resistance and application thereof; The mutated ACCase protein it encodes can endow plants, especially rice, with the property of resisting acetyl-CoA carboxylase inhibitor herbicides. The invention discloses the nucleotide sequence and amino acid sequence of the mutant protein and their application in the field of plant herbicide resistance. Background technique [0002] Farmland weeds are one of the main reasons for crop yield reduction. Compared with the traditional methods of relying on cultivation measures, manual weeding and mechanical weeding, the use of chemical herbicides is recognized as the most reliable and economical means of farmland weeding technology. Since 2,4-D began to be used in the 1940s, the herbicide industry has been developing for more than 60 years, and a large number of selective h...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/82A01H5/00A01H6/00C12Q1/6895
CPCC12N9/93C12N15/8274C12Q1/6895C12Y604/01002
Inventor 张保龙王金彦凌溪铁郭冬姝
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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