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Rice als mutant protein for making plants resistant to herbicides and application thereof

一种除草剂、植物的技术，应用在植物蛋白和植物抗除草剂领域，能够解决很难提前预测ALS蛋白产生除草剂抗性、除草剂作用机理未确定等问题

Active Publication Date: 2019-01-18
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 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. New herbicide-resistance locus in ALS protein just might be discovered by luck

Method used

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  • Rice als mutant protein for making plants resistant to herbicides and application thereof
  • Rice als mutant protein for making plants resistant to herbicides and application thereof
  • Rice als mutant protein for making plants resistant to herbicides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Obtaining wild-type sequences of rice Hefei male sterile line, Jiahua No. 1, and R89

[0068] According to the Nipponbare ALS gene (NCBI: XM_015770973) in NCBI, primers were designed for amplification, and TakaraPrimerSTAR Max DNA Polymerase (purchased from Takara Company) was used to amplify the ALS genes of rice Hefei male sterile line, Jiahua 1, and R89 wild type. Its reaction system is as follows:

[0069] 2×PrimerSTAR Max Premix 10.0μl

[0070] 10μM forward primer 1.0μl

[0071] 10μM reverse primer 1.0μl

[0072] 20-30ng / μL rice genomic DNA 1.0μl

[0073] Add sterile water to a total volume of 20 μl

[0074] The PCR amplification reaction program adopts a two-step method, annealing and extension are combined together, and 68 degrees are used.

[0075] The program is as follows: pre-denaturation: 98°C for 3min; 35 cycles: denaturation at 98°C for 10sec; extension at 68°C for 2min; incubation: 72°C for 10min.

[0076] Take 2 μ l of the PCR product and ...

Embodiment 2

[0083] Example 2: The process of obtaining rice mutants resistant to imidazolinone herbicides (Bailongtong)

[0084]1. Indica conventional rice seed Hefei CMS (Hua K01S, provided by Anhui Huakai Agricultural Technology Co., Ltd.) (this is M0, soaked in water for 2 hours) 150kg is divided into 6 times with 0.5-1.0% (w / w) Soak in ethyl methanesulfonate (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, the next day Carry out field sowing, and carry out 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 rice (this is M2) seedlings grow to the 3-4 leaf stage, spray 3.3mL Bailongtong / L water ("Bailongtong" is a water-based imidazolinone herbicide produced by BASF, Germany, The recommen...

Embodiment 3

[0086] Example 3: Analysis of mutation sites in rice mutants resistant to imidazolinone herbicides

[0087] The herbicide-resistant mutant rice plants obtained in the above-mentioned Example 2 were selected to select the leaves of the mutant plants, and the genomic DNA was extracted, and sent to Nanjing Yidao Biotechnology Co., Ltd. for genome sequencing. Comparing the sequencing results with the wild-type ALS gene of the Hefei male sterile line, it was found that the herbicide-resistant rice mutant had 2 mutations in the ALS gene, and the 512th and 1050th bases were mutated, respectively changing from C to A , T into A, resulting in the 171st and 350th positions of the corresponding encoded amino acid sequence changing from proline to histidine, and asparagine to glutamic acid, that is, the nucleus of the ALS gene of the herbicide-resistant mutant The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded ALS protein is shown in SEQ ID NO.2. ...

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Abstract

Disclosed are a rice ALS mutant protein for conferring herbicide resistance to plants, and a nucleic acid encoding the protein. The protein is derived from a rice mutant plant resistant to an ALS inhibitor herbicide, and compared to the ALS sequence in the genome of wild type rice, the protein sequence thereof is mutated at the Pro171 site and / or Asp350 site. The experiment results of spraying the ALS inhibitor herbicide, "Imazameth", in fields show that rice seedlings with 3-4 leaves and containing the herbicide-resistant ALS protein still grow normally and fructify after applying 3.3 mL of Imazameth / L of water, whereas wild type rice seedlings with 3-4 leaves show whole plant death after applying 1 mL of Imazameth / 3 L of water for 30 days.

Description

technical field [0001] The invention belongs to the field of plant protein and plant herbicide resistance, and relates to rice ALS mutant protein, gene and application thereof for making plants resistant to herbicides; specifically, the invention relates to rice acetolactate synthase (ALS) mutant protein , the protein can endow plants, especially rice, 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 are unfavorable factors restricting the stable and high yield of agricultural production. Compared with traditional cultivation methods, artificial weeding and mechanical weeding, the use of chemical herbicides is an efficient, simple and economical method of controlling weeds. [0003] The biosynthesis of branched-chain amino acids (valine, leucine, and isoleucine) in plants and microorga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/82A01H5/00A01H6/46A01G13/00
CPCA01G13/00C12N9/1022C12N9/88C12N15/8274C12Q1/6895C12Q2600/13C12Y202/01006C12Y401/03
Inventor 张保龙王金彦凌溪铁陈天子邓惠清吴魁
Owner JIANGSU ACAD OF AGRI SCI
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