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Wheat ALS mutant protein and nucleic acid and application thereof

A mutant, wheat technology, applied in the field of plant protein and plant herbicide resistance, can solve the problem that the mechanism of action of herbicide is not determined, and it is difficult to predict in advance that ALS protein will produce herbicide resistance.

Active Publication Date: 2017-11-17
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]At present, the mechanism of action of ALS inhibitor herbicides has not yet been determined, and it is difficult to predict in advance whether mutations at other amino acid sites of the ALS protein will cause herbicide resistance. Relying on the long-term and arduous practical exploration of researchers, and with some luck, it is possible to discover new herbicide resistance sites in ALS proteins

Method used

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  • Wheat ALS mutant protein and nucleic acid and application thereof
  • Wheat ALS mutant protein and nucleic acid and application thereof
  • Wheat ALS mutant protein and nucleic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: The process of obtaining wheat mutants resistant to imidazolinone herbicides (Bailongtong)

[0036] The seeds of No. Xu Mai No. 33 (wheat variety available in the market) (this is M0, soaked for 2 hours with clear water) 120 kg were soaked for 6 times at room temperature with 0.4-0.6% (w / w) ethyl methanesulfonate (EMS) for 6 times. -9 hours, during which the seeds were shaken every 1 hour; the EMS solution was discarded, and the tap water was stirred and soaked 5 times for 5 minutes each time, then the seeds were rinsed with tap water overnight, and the field was sown the next day, and routine fertilizer and water management (this is M1). After the plants are mature, the seeds are mixed, dried, and stored for the winter. Sow in the field the following year. When the wheat (this is M2) seedlings grow to the stage of 1 leaf and 1 core, spray 1mL Bailongtong / L water ("Bailongtong" is a water-based imidazolinone herbicide produced by BASF in Germany, it is reco...

Embodiment 2

[0037] Example 2: Analysis of mutation sites in herbicide-resistant wheat mutants

[0038] There are three copies of the ALS gene in the wheat genome, located in the wheat A, B and D genomes. From the herbicide-resistant wheat mutant plants obtained in the above-mentioned Example 1, the leaves of the mutant plants and wild-type plants were selected, and genomic DNA was extracted respectively, and the wheat ALS gene was amplified and sequenced. Sequencing results showed that compared with wild-type plants, the above-mentioned herbicide-resistant wheat mutants had mutations at 3 sites in each of the 3 genomic ALS genes, which were named 6AL-353 mutant, 6AL-551 mutant, 6AL-631 mutant, 6BL-352 mutant, 6BL-550 mutant, 6BL-630 mutant, 6DL-350 mutant, 6DL-548 mutant, 6DL-628 mutant. Wherein, the nucleotide sequence of the wheat A genome ALS gene of the 6AL-353 mutant is mutated from T to A at the 1059th position of SEQ ID NO: 1, resulting in the amino acid sequence encoded by it at ...

Embodiment 3

[0039] Example 3 Cloning of ALS gene of wheat mutant resistant to imidazolinone herbicides

[0040] The 6AL-353 mutant, 6AL-551 mutant, 6AL-631 mutant, 6BL-352 mutant, 6BL-550 mutant, 6BL-630 mutant, 6DL-350 mutant, 6DL Genomic DNA was extracted from leaves of -548 mutant and 6DL-628 mutant respectively. The specific primers designed to amplify the full-length ALS gene according to the sequencing sequence were: forward primer 5'-CCCATCTGAACCACACGCTCACC-3', reverse primer 5'-CTGATTGCGCATGTCACACTTG-3'. Using Takara PrimerSTAR Max DNA Polymerase polymerase (10U / ul) (purchased from Takara Company) to amplify the full-length sequence of the ALS gene, the reaction system is as follows:

[0041]

[0042] The PCR amplification reaction program adopts a two-step method, annealing and extension are combined together, and 68 ° C is used.

[0043] The procedure is as follows: pre-denaturation: 98°C for 3min; 35 cycles; denaturation at 98°C for 10sec; extension at 68°C for 3min; incub...

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Abstract

The invention discloses a wheat ALS mutant protein. The sequences of an ALS gene of a wheat A genome 6AL chromosome, an ALS gene of a B genome 6BL chromosome and an ALS gene of a wheat D genome 6DL chromosome are mutated. The invention further discloses nucleic acid of the wheat ALS mutant protein and further discloses application of the wheat ALS mutant protein. By expressing the protein sequence, green plants can resist (tolerate) acetolactate synthetase inhibitor type herbicides, especially imidazolone type herbicides. Wheat one-leaf one-core seedlings with the wheat ALS mutant protein still can normally grow, develop and fruit after a herbicide is applied according to the concentration of 1 mL of imazameth per L of water (three times of recommended use concentration).

Description

technical field [0001] The invention belongs to the field of plant protein and plant herbicide resistance, and relates to a wheat ALS mutant protein and its nucleic acid and its application; specifically, the invention relates to a wheat acetolactate synthase (ALS) mutant protein, which can endow Characterization of resistance of plants, especially wheat, to acetolactate synthase inhibitor herbicides. The invention discloses the sequence of the protein and their application in the field of plant herbicide resistance. Background technique [0002] Weeds in farmland are one of the main causes of 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 efficient, simple and economical method of controlling weeds in farmland. [0003] The biosynthesis of branched-chain amino acids (valine, leucine, and isoleucine) in plants and microorganisms r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/00C12Q1/68G01N33/573
CPCC12N9/1022C12N15/8274C12Q1/6895C12Y202/01006G01N33/573G01N2333/9104
Inventor 张保龙王金彦凌溪铁陈天子
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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