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Wheat ALS mutation gene and application of protein thereof in aspect of herbicide resistance

A mutant protein and mutant technology, which is applied in the field of plant protein and plant herbicide resistance, can solve the problems that the mechanism of herbicide action is not determined, and it is difficult to predict the herbicide resistance of ALS protein in advance, so as to achieve the effect of good growth state

Active Publication Date: 2017-05-31
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 mutation gene and application of protein thereof in aspect of herbicide resistance
  • Wheat ALS mutation gene and application of protein thereof in aspect of herbicide resistance
  • Wheat ALS mutation gene and application of protein thereof in aspect of herbicide resistance

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: The process of obtaining wheat mutants resistant to imidazolinone herbicides (Bailongtong)

[0035] 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

[0036] Example 2: Analysis of mutation sites in herbicide-resistant wheat mutants

[0037] Among the herbicide-resistant wheat mutant plants obtained in Example 1 above, the leaves of the mutant plant No. 75 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. 75 had a single-base mutation at nucleotide 331 of the ALS gene sequence on chromosome 6BL of its B genome, changing from G to A , causing the 111th amino acid of its corresponding encoded amino acid sequence to be changed from valine to isoleucine, that is, the nucleotide sequence of the 6BL chromosome ALS gene of the B genome of the No. 75 wheat herbicide-resistant mutant plant such as SEQ ID NO. 1, the amino acid sequence of the encoded ALS protein is shown in SEQ ID NO.2. The No. 75 wheat m...

Embodiment 3

[0038] Example 3 Cloning of ALS gene of wheat mutant resistant to imidazolinone herbicides

[0039] The leaves of the above-mentioned herbicide-resistant wheat mutant No. 75 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:

[0040]

[0041] The PCR amplification reaction program adopts a two-step method, annealing and extension are combined together, and 68 ° C is used.

[0042] 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.

[0043] Take 2 μl of PCR products and detect by 1% agarose gel electrop...

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Abstract

The invention discloses a wheat ALS mutation gene. The 331th nucleotide of an ALS gene sequence of 6BL chromosome of a wheat B genome is changed into nucleotide A from G. The invention further discloses wheat ALS mutation protein encoded by the wheat ALS mutation gene and application of the wheat ALS mutation protein. The protein is from a wheat mutant plant of an anti-ALS inhibitor herbicide. Compared with a wheat mild ALS sequence, the protein sequence mutates at a Val111 site. A green plant expresses that the mutation protein sequence can be resistant and tolerant to an acetolactate synthase inhibitor herbicide, especially an imidazolone herbicide. After 3mL of imazameth / L water (9-fold recommended concentration) is applied to a wheat seedling with one leaf and one core of the wheat ALS mutation protein, the plant can still normally grow, develop and fruit.

Description

technical field [0001] The invention belongs to the fields of plant protein and plant herbicide resistance, and relates to the application of a wheat ALS mutant gene and its protein in 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] 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/82A01H5/00C12Q1/68C12Q1/527A01N25/32A01P13/00
CPCA01N25/32C12N9/88C12N15/8274C12Q1/527C12Q1/6895C12Q2600/13C12Y401/03G01N2333/988
Inventor 张保龙陈天子王金彦凌溪铁
Owner JIANGSU ACAD OF AGRI SCI
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