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ALS mutant gene and application of protein thereof to herbicide resistance

A mutant and genetic technology, applied in the fields of plant protein and plant herbicide resistance, can solve the problems of undetermined herbicide action mechanism and difficult to predict the herbicide resistance of ALS protein in advance

Inactive Publication Date: 2016-07-06
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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  • ALS mutant gene and application of protein thereof to herbicide resistance
  • ALS mutant gene and application of protein thereof to herbicide resistance
  • ALS mutant gene and application of protein thereof to herbicide resistance

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: The process of obtaining rice mutants resistant to imidazolinone herbicides (Bailongtong)

[0040] 150 kg of indica conventional rice 9311 seeds (purchased from Jiangsu Provincial Agricultural Germplasm Resources Protection and Utilization Platform) (this is M0, soaked in water for 2 hours) were divided into 6 times with 0.5-1.0% (w / w) ethyl methanesulfonate Ester (EMS) was soaked at room temperature for 6-9 hours, and the seeds were shaken every 1 hour during this period; the EMS solution was discarded, tap water was stirred and soaked 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 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 the rice (this is M2) seedlings grow to the 3-4 leaf stage, spray 3mL Bailongtong / L water ("Bailongtong" is ...

Embodiment 2

[0041] Example 2: Analysis of Mutation Sites of Herbicide-resistant Rice Mutants

[0042] The leaves of mutant plant 3 were selected from the herbicide-resistant mutant rice plants obtained in Example 1 above, and genomic DNA was extracted, and sent to Shanghai Hanyu Biotechnology Co., Ltd. for genome sequencing. Comparing the sequencing results with the 9311 reference genome sequence (http: / / rise2.genomics.org.cn / page / rice / download.jsp), it was found that the above-mentioned herbicide-resistant rice mutants had multiple sites on the ALS gene. Mutations, including the 75th base G to C, the 339th base A to C, the 1099th base C to T, and the 1880th base G to A in the ALS gene, resulting in changes in the corresponding encoded amino acid sequence The 25th amino acid changes from glutamine to histidine, the 113th amino acid changes from glutamine to histidine, the 367th amino acid changes from histidine to tyrosine, and the 627th amino acid changes from serine to asparagine. That...

Embodiment 3

[0043] Example 3 Cloning of ALS gene of rice mutant resistant to imidazolinone herbicides

[0044] Genomic DNA was extracted from the leaves of the above-mentioned herbicide-resistant rice mutant 9311M3. According to the chromosomal sequence of the 9311 rice wild-type ALS gene (BAC clone Genbank accession number AAAA02006431.1), the specific primers for amplifying the 3' end sequence of the ALS gene are: forward primer 3F5'-GGTCTTGCGTCTGGTTGGCGAGT-3', reverse primer 3R5 '-CTCTTTATGGGTCATTCAGGTCAA-3', the specific primers designed to amplify the 5' end sequence of ALS gene are: forward primer 5F5'-ATCCGAGCCACACATCGCCTCAC-3', reverse primer 5R5'-AGCAACAGGTCAGCCTTATCCAC-3'. The sequences amplified by the two pairs of specific primers have an overlapping portion of 230 bp, and can be spliced ​​into a complete ALS gene sequence.

[0045] Adopt TakaraPrimerSTARMaxDNAPolymerase polymerase (purchased from Takara Company) to amplify ALS gene 5' end sequence, 3' end sequence, and its r...

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Abstract

The invention discloses an ALS mutant gene. The nucleotide at the 75th locus of the ALS mutant gene in an ALS gene sequence of rice is changed from G to C, the nucleotide at the 339th locus is changed from A to C, the basic group at the 1099th locus is changed from C to T, and the basic group at the 1880th locus is changed from G to A. The invention further discloses ALS protein coded by the ALS mutant gene and application of the ALS protein. The protein comes from a rice mutant plant resistant to ALS inhibitor herbicide. Compared with a wild ALS sequence of rice, the sequence of the protein is subjected to mutation at the loci of Gln25, Gln113, His367 and Ser627. It is expressed by green plants that the sequence of the protein can resist acetolactate synthetase inhibitor herbicide and particularly imidazolone and sulfonylurea herbicide.

Description

technical field [0001] The invention belongs to the field of plant protein and plant herbicide resistance. Specifically, the present invention relates to rice acetolactate synthase (ALS) mutant protein, which can endow plants, especially rice, with the characteristic 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] Acetolactate synthase (ALS) (also known as acetohydroxyacid synthase, AHAS; EC4.1.3.18) inhibitor herbicides target ALS to cause weed death, mainly including sulfonylureas (Sulfonylureas, SU) , imidazolinones (Imida...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12Q1/68A01H5/00A01N47/44A01P13/00
CPCC12N9/88A01N47/44C12N15/8274C12Q1/6895
Inventor 张保龙陈天子王金彦凌溪铁
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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