Rice herbicide resistant ALS mutant protein, nucleic acid 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: 2018-04-27
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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 herbicide resistant ALS mutant protein, nucleic acid and application thereof
  • Rice herbicide resistant ALS mutant protein, nucleic acid and application thereof
  • Rice herbicide resistant ALS mutant protein, nucleic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Obtaining the full length of Huazhan wild-type rice ALS gene

[0061] The genomic DNA of the conventional indica rice seed Hua Zhan was extracted by conventional methods. The conventional indica rice seed Hua Zhan is a gift from the Yangzhou Academy of Agricultural Sciences. For information on the relative parents of the Hua Zhan, please refer to the China National Rice Research Institute http: / / www.ricedata.cn / variety / varis / 607962.htm? 607962 According to the Nipponbare ALS gene in NCBI (NCBI: XM_015770973), primers were designed for amplification, and the ALS gene was amplified by Takara PrimerSTAR Max DNA Polymerase (purchased from Takara). The reaction system is as follows:

[0062] 2×PrimerSTAR Max Premix 10.0μl

[0063] 10μM forward primer 1.0μl

[0064] 10μM reverse primer 1.0μl

[0065] 20-30ng / μL Huazhan wild-type rice genomic DNA 1.0μl

[0066] Add sterile water to a total volume of 20μl

[0067] The PCR amplification reaction program adopts a two-step meth...

Embodiment 2

[0071] Example 2: Obtaining process of rice mutants resistant to imidazolinone herbicides (Bailongtong)

[0072] Indica conventional rice seed Hua Zhan (a gift from the Yangzhou Academy of Agricultural Sciences), see the China National Rice Research Institute for information on the Hua Zhan related parents and other information http: / / www.ricedata.cn / variety / varis / 607962.htm? 607962 ,

[0073] (This is M0, soak in clean water for 2 hours) 150kg is soaked in 0.5-1.0% (w / w) ethyl methanesulfonate (EMS) for 6-9 hours at room temperature for 6 to 6 hours, and the seeds are shaken every 1 hour during this period; discard; To remove the EMS solution, turn the tap water to soak the seeds 5 times for 5 minutes each time, and then rinse the seeds with tap water overnight. The next day, field sowing and conventional fertilizer and water management (this is M1) are performed. After the plants mature, the seeds are mixed, dried, and preserved for winter. Sow the fields the following year. ...

Embodiment 3

[0074] Example 3: Analysis of mutation sites in rice mutants resistant to imidazolinone herbicides

[0075] The leaves of the Huazhan herbicide-resistant rice mutant-1 plant and the Huazhan herbicide-resistant rice mutant-2 plant obtained in Example 2 were respectively taken, genomic DNA was extracted, and sent to Nanjing Yidan Biotechnology Co., Ltd. for genome sequencing. Comparing the sequencing results with the Huazhan wild-type ALS gene, it was found that both the two resistant rice mutants had mutations at position 627 of the ALS gene, and the 1879th position of the ALS gene sequence in the Huazhan herbicide-resistant rice mutant-1 The nucleotide changes from A to C, and the 1880th nucleotide from G to T, which causes the 627th position of the corresponding amino acid sequence to be changed from serine to leucine, namely Hua Zhan The nucleotide sequence of the ALS gene of the herbicide-resistant rice mutant-1 is shown in SEQ ID NO. 1, and the amino acid sequence of the enco...

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Abstract

The invention discloses a rice herbicide resistant ALS mutant protein. The amino acid sequence of the ALS mutant protein has any one of the following mutations: serine which corresponds to an amino acid at a 627th position of rice ALS is changed into leucine, or serine which corresponds to an amino acid at a 627th position of rice ALS is changed into cysteine. The invention further discloses a nucleic acid and application of an expression cassette, a recombinant vector or cells in in the aspect of a weed resisting and removing agent for plants. A result of an experiment of spraying an ALS inhibitors herbicide 'imazameth' in a field shows that after 3.3 mL of imazameth/ L water is applied to rice 3-4-leaf seedlings which separately contains two ice herbicide resistant ALS mutant proteins ofthe invention, plants still grow and develop normally and are firm, but 30 days after 1 mL imazameth/3L water is applied to wild rice 3-4-leaf seedling, the whole plants are dead.

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 that make plants have herbicide resistance; specifically, the invention relates to rice acetolactate synthase (ALS) mutant protein The protein can give plants, especially rice, resistance to acetolactate synthase inhibitor herbicides. The present invention discloses the sequence of the protein and their application in the field of plant herbicide resistance. Background technique [0002] Weeds are an unfavorable factor restricting the stable and high yield of agricultural production. Compared with traditional methods such as relying on cultivation measures, artificial weeding and mechanical weeding, the use of chemical herbicides is an efficient, simple and economical method for weed control. [0003] At present, the use of acetolactate synthase (ALS) inhibitor chlorsulfuron has become a major breakthrough ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12Q1/6895A01H5/00A01H6/46A01H6/82A01H6/60A01H6/20A01N25/32A01N43/50A01P13/00
CPCA01N25/32A01N43/50C12N9/88C12N15/8274C12Q1/6895C12Q2600/13C12Y401/03
Inventor 张保龙王金彦凌溪铁陈天子
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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