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Wheat ALS mutant gene and application thereof in resisting herbicide

A mutant, wheat technology, applied in the field of plant protein and plant herbicide resistance, can solve the problems of undetermined action mechanism of herbicides, and it is difficult to predict ALS protein in advance to produce herbicide resistance, etc., and achieve the effect of good growth state.

Active Publication Date: 2017-04-26
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the mechanism of action of ALS inhibitor herbicides has not 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. New herbicide-resistance locus in ALS protein just might be discovered by luck

Method used

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  • Wheat ALS mutant gene and application thereof in resisting herbicide
  • Wheat ALS mutant gene and application thereof in resisting herbicide
  • Wheat ALS mutant gene and application thereof in resisting herbicide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: The process of obtaining wheat mutants resistant to imidazolinone herbicides (Bailongtong)

[0034] Xumai seeds (purchased from Jiangsu Provincial Agricultural Germplasm Resources Protection and Utilization Platform) (this is M0, soaked for 2 hours with clear water) 120kg were divided into 6 times with 0.4-0.6% (w / w) ethyl methanesulfonate (EMS ) at room temperature for 6-9 hours, during which the seeds were shaken every 1 hour; the EMS solution was discarded, and the seeds were stirred and soaked in tap water 5 times, each time for 5 minutes, and 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 3mL of Bailongtong / L water ("Bailongtong" is a water-based imida...

Embodiment 2

[0035] Example 2: Analysis of mutation sites in herbicide-resistant wheat mutants

[0036] Among the herbicide-resistant wheat mutant plants obtained in Example 1 above, the leaves of the mutant plant No. 38 and the leaves of the wild-type plant were selected, and genomic DNA was extracted and sent to Shanghai Hanyu Biotechnology Co., Ltd. for genome sequencing. Sequencing results revealed that, compared with the wild-type plant, the above-mentioned herbicide-resistant wheat mutant No. 38 had a single-base mutation at the 1180th nucleotide of the ALS gene sequence on chromosome 6BL of its B genome, changing from G to A , causing the 394th amino acid of its corresponding encoded amino acid sequence to be changed from alanine to threonine, that is, the nucleotide sequence of the 6BL chromosome ALS gene of the B genome of the No. 38 wheat herbicide-resistant mutant plant such as SEQ ID NO.1 As shown, the amino acid sequence of the ALS mutant protein encoded by it is shown in SEQ ...

Embodiment 3

[0037] Example 3 Cloning of ALS gene of wheat mutant resistant to imidazolinone herbicides

[0038] The leaves of the above herbicide-resistant wheat mutant No. 38 were taken to extract genomic DNA. The specific primers for amplifying the full-length ALS gene were designed according to the sequencing sequence: forward primer 5'-CCATCACCCCTCCCCAATTCC-3', reverse primer 5'-CACTTGTAGGTCTTGTAGGTCG-3'. Using Takara PrimerSTAR Max DNA Polymerase polymerase (10U / μl) (purchased from Takara Company) to amplify the full-length sequence of the ALS gene, the reaction system is as follows:

[0039]

[0040] The PCR amplification reaction program adopts a two-step method, annealing and extension are combined together, and 68 ° C is used.

[0041] 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; incubation: 72°C for 10min.

[0042] Take 2 μl of PCR products and detect by 1% agarose gel electrophoresis, a...

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Abstract

The invention discloses a wheat ALS mutant gene. According to the mutant gene, the 1180th nucleotide of the ALS gene sequence of the wheat B genome 6BL chromosome is changed from nucleotide G to nucleotide A. The present invention further provides a wheat ALS mutant protein encoded by the wheat ALS mutant gene and an application of the mutant protein. The mutant protein originates from wheat mutant plants capable of resisting ALS inhibition herbicide. Compared with the wheat wild type ALS order, the mutant protein order is mutated in the Ala 394 site. The expression of the protein order through a green plant can resist (endure) acetyl lactate synthase inhibitor herbicide, particularly imidazolidinone herbicide. The 1-leave-1-core wheat seedlings with the wheat ALS mutant protein can still grow, develop and bear fruits normally after an imazameth aqueous solution with a concentration of 3mL imazameth per liter of water (nine times of the recommended concentration) is applied to the seedlings.

Description

technical field [0001] The invention belongs to the fields of plant protein and plant herbicide resistance, and in particular relates to a wheat ALS mutant gene and its application in herbicide resistance. Background technique [0002] Weeds in farmland are one of the main causes of crop yield reduction. The use of chemical herbicides is recognized as the most efficient, simple and economical method of controlling weeds in farmland. [0003] Acetolactate synthase (ALS) (also known as acetohydroxyacid synthase, AHAS; EC 4.1.3.18) inhibitor herbicides use ALS as a target to cause weed death, mainly including sulfonylureas (Sulfonylureas, SU), Imidazolinones (Imidazolinones, IMI), triazolopyrimidines (TP), pyrimidinylthio(or oxy)–benzoates, PTB; pyrimidinyl-carboxyherbicides; PCs] and sulfonamide carbonyl Triazolones (Sulfonylamino-carbonyltriazolinones, SCT) and other 13 types of compounds. ALS inhibitor herbicides have the advantages of strong selectivity, broad herbicidal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/82A01H5/00C12Q1/68
Inventor 张保龙陈天子王金彦凌溪铁
Owner JIANGSU ACAD OF AGRI SCI
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