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ALS (Acetolactate Synthetase) mutant type gene of japonica rice as well as protein and application thereof

A mutant and gene technology, applied in the field of plant protein and plant herbicide resistance, can solve the problem that no one has reported herbicide resistance, it is difficult to predict the herbicide resistance of ALS protein in advance, and the mechanism of herbicide action is not determined, etc. question

Active Publication Date: 2017-06-20
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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
[0006] Moreover, no one at home and abroad has reported whether the mutation of acetolactate synthase in japonica rice will produce herbicide resistance

Method used

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  • ALS (Acetolactate Synthetase) mutant type gene of japonica rice as well as protein and application thereof
  • ALS (Acetolactate Synthetase) mutant type gene of japonica rice as well as protein and application thereof
  • ALS (Acetolactate Synthetase) mutant type gene of japonica rice as well as protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: The process of obtaining the imidazolinone herbicide-resistant mutant of japonica rice Zhendao 18 (Bailongtong)

[0037]Regular japonica rice seed Zhendao 18 (purchased from the Agricultural Germplasm Resources Protection and Utilization Platform of Jiangsu Province) (this is M0, soaked in water for 2 hours) 150kg was divided into 6 times with 0.5-1.0% (w / w) methanesulfonic acid Soak in ethyl ester (EMS) at room temperature for 6-9 hours, shake the seeds every 1 hour during this period; discard the EMS solution, soak the seeds 5 times in tap water, 5 minutes each time, then rinse the seeds with tap water overnight, and sow in the field the next day , and carry out conventional 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 rice (this is M2) seedlings grow to the 3-4 leaf stage, spray 3mL Bailongtong / L water ("Bailongtong" is a wat...

Embodiment 2

[0038] Example 2: Analysis of mutation sites of japonica rice mutants resistant to imidazolinone herbicides

[0039] The herbicide-resistant japonica rice mutant plants obtained in the above-mentioned Example 1 were taken to select the leaves of the mutant plants, and the genomic DNA was extracted respectively, and sent to Shanghai Hanyu Biotechnology Co., Ltd. for genome sequencing. Comparing the sequencing results with the reference gene sequence of japonica rice (https: / / www.ncbi.nlm.nih.gov / nuccore / XM_015770973.1), it was found that the above herbicide-resistant japonica rice mutant had one locus in the ALS gene Mutation, wherein the herbicide-resistant mutant plants have a mutation at the 406th base of the ALS gene, changing from G to T, resulting in a change from glycine to threonine at the 136th position of the corresponding encoded amino acid sequence, that is, herbicide resistance The nucleotide sequence of the ALS gene of the agent mutant plant is shown in SEQ ID NO....

Embodiment 3

[0040] Example 3 Cloning of the ALS gene of the japonica rice mutant resistant to imidazolinone herbicides

[0041] The leaves of the above-mentioned herbicide-resistant japonica rice mutant zhen9 were taken, and the genomic DNA of the mutant zhen9 was extracted respectively. According to the chromosomal sequence of the rice wild-type ALS gene (such as Genbank accession number XM_015770973.1), the specific primers for amplifying the ALS gene are: forward primer F5'-ATCCGAGCCACACATCGCCTCAC-3', reverse primer R 5'-CTCTTTATGGGTCATTCAGGTCAA-3 '.

[0042] Using OneTaq TM Quick 2×Master Mix (purchased from NEB Company, article number: M0483L) amplifies the 5' end sequence and 3' end sequence of the ALS gene, and the reaction system is as follows:

[0043]

[0044] The PCR amplification reaction program adopts a two-step method, annealing and extension are combined together, and 68 degrees are used.

[0045] The program is as follows: pre-denaturation: 98°C for 3min; 35 cycl...

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Abstract

The invention discloses an ALS mutant type gene of japonica rice. The 406th nucleotide on an ALS gene sequence of the japonica rice is mutated into a nucleotide T from G. The invention further discloses an ALS mutant protein encoded by the ALS mutant type gene of the japonica rice and application of the ALS mutant protein. The mutant protein comes from a japonica rice mutant plant capable of resisting ALS inhibitor herbicides. Compared with a wild type ALS sequence, the protein sequence in the invention is mutated at the locus Gly136. Due to green plant expression, the protein sequence can resist (tolerate) the ALS inhibitor herbicides, particularly imidazolone herbicides.

Description

technical field [0001] The invention belongs to the field of plant protein and plant herbicide resistance. Specifically, the present invention relates to an acetolactate synthase (ALS) mutein of japonica rice, which can impart herbicide resistance to plants, especially rice, against acetolactate synthase inhibitors. The invention discloses the sequence of the mutant proteins and their application in the field of plant herbicide resistance. Background technique [0002] Acetolactate synthase (ALS) (also called acetohydroxyacid synthase, AHAS; EC 4.1.3.18) is a key enzyme in the biosynthesis of three amino acids in plants and microorganisms. If the activity of this enzyme is inhibited or lost, the synthesis of valine, leucine and isoleucine will be hindered, affecting the synthesis of protein, and eventually the growth of the plant will be hindered until death. Therefore, a variety of high-efficiency herbicides have been developed with ALS as the target, mainly including sul...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/82A01H5/00C12Q1/68C12Q1/527
CPCC12N9/88C12N15/8205C12N15/8274C12Q1/527C12Q1/6895C12Q2600/158C12Y401/03
Inventor 张保龙王金彦陈天子凌溪铁
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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