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Wheat ALS (anti-lymphocyte serum) mutant type protein endowing plants with herbicide resistance as well as gene and application thereof

A herbicide and mutant technology, applied in the field of plant protein and plant herbicide resistance, can solve the problems of undetermined mechanism of action of herbicide, and it is difficult to predict in advance that ALS protein will produce herbicide resistance.

Active Publication Date: 2017-08-08
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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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 (anti-lymphocyte serum) mutant type protein endowing plants with herbicide resistance as well as gene and application thereof
  • Wheat ALS (anti-lymphocyte serum) mutant type protein endowing plants with herbicide resistance as well as gene and application thereof
  • Wheat ALS (anti-lymphocyte serum) mutant type protein endowing plants with herbicide resistance as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: The process of obtaining wheat mutants resistant to imidazolinone herbicides (Bailongtong)

[0032]120 kg of Yannong 19 seeds (purchased from Jiangsu Agricultural Germplasm Resources Protection and Utilization Platform) (this is M0, soaked in water for 2 hours) 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 imidazolinone he...

Embodiment 2

[0033] Example 2: Analysis of mutation sites in herbicide-resistant wheat mutants

[0034] From the herbicide-resistant mutant wheat plants obtained in Example 1 above, the leaves of the mutant plants and wild-type plants were selected to extract genomic DNA, and sent to Shanghai Hanyu Biotechnology Co., Ltd. for genome sequencing. Sequencing results found that: compared with wild-type plants, the above-mentioned herbicide-resistant wheat mutants had a single-base mutation at nucleotide 1889 of the 6AL chromosome ALS gene sequence of the wheat A genome, changing from G to A, resulting in The 630th amino acid of its corresponding encoded amino acid sequence is changed from serine to asparagine, that is, the nucleotide sequence of the 6AL chromosome ALS gene of the A genome of the wheat herbicide-resistant mutant plant is shown in SEQ ID NO.1, and its encoded The amino acid sequence of the ALS protein is shown in SEQ ID NO.2. The nucleotide sequence of the ALS gene on the 6AL c...

Embodiment 3

[0035] Example 3 Cloning of ALS gene of wheat mutant resistant to imidazolinone herbicides

[0036] The leaves of the above-mentioned herbicide-resistant wheat mutants were taken to extract genomic DNA. 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:

[0037] 2×PrimerSTAR Max Premix 10.0μl

[0038] 10μM forward primer 1.0μl

[0039] 10μM reverse primer 1.0μl

[0040] 20-30ng / μL wheat genomic DNA 1.0μl

[0041] Add sterile water to a total volume of 20 μl

[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-denatura...

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Abstract

The invention discloses a wheat ALS (anti-lymphocyte serum) mutant type protein endowing plants with herbicide resistance, and also discloses nucleic acid for coding the protein. The protein is from a wheat mutant plant with ALS-inhibitor herbicide resistance; compared with an ALS sequence in a wild type wheat A-genome, a protein sequence mutates at an Ser630 site. The plant expressed protein sequence can be resistant to an acetolactate synthetase-inhibitor herbicide, in particular to an imidazolinone herbicide. In the wheat ALS mutant type protein, after imazameth and water at the volume ratio of 3:1,000 (9 times of using concentration is recommended) is applied to a wheat seedling with one leaf and one core, the plant can still grow and develop normally and bear fruits.

Description

technical field [0001] The invention belongs to the field of plant protein and plant herbicide resistance, and relates to wheat ALS mutant protein, gene and application thereof which make plants have herbicide resistance; specifically, the invention relates to wheat acetolactate synthase (ALS) mutant protein , the protein can endow plants, especially wheat, with the characteristics of resistance 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] Acetolactate synthase (ALS) (also known as acetohydroxyacid synthase, ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/82A01H5/00C12Q1/68G01N33/573
CPCC12N9/88C12N15/8274C12Q2600/13C12Y401/03G01N33/573G01N2333/988
Inventor 张保龙王金彦凌溪铁陈天子
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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