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Acetolactic acid synthetase mutants and application thereof

A kind of technology of acetolactate and synthase, applied in the field of plant genetics and breeding

Inactive Publication Date: 2013-02-20
BEIJING XINGBOYA BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, plants with herbicide resistance and their decisive substances (such as proteins, genes, etc.) have not yet been theoretically designed
[0003] Therefore, the current methods of finding and applying proteins that make plants resistant to herbicides still need to be improved

Method used

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  • Acetolactic acid synthetase mutants and application thereof
  • Acetolactic acid synthetase mutants and application thereof
  • Acetolactic acid synthetase mutants and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Genetic segregation analysis of imidazolinone herbicide-resistant mutants

[0043]The maize inbred line Jing 21 was sown on 5 mu of land, and the sowing rate was 5000 kg / mu. During the pollination season, male and female flowers were bagged separately to collect fresh male flower pollen, and the pollen was treated with paraffin oil containing 0.067% (w / w) ethyl methanesulfonate (EMS) concentration for 45 minutes, wherein the volume ratio of pollen to paraffin oil About 1:10, and then within 30 minutes after the treatment, the mixture of pollen and paraffin oil was applied to the female flower filaments and bagged. Harvest when the plants are mature. Then, the harvested seeds were treated with the imidazolinone herbicide imazethapyr ((RS) 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-3-imidazoline-2 - base) nicotinic acid, CAS No. 81335-77-5) water solution (concentration: 0.5%, purchased from Shandong Xianda Chemical Co., Ltd.) soaked for 18 hours, drained and sowe...

Embodiment 2

[0045] Example 2 Determination of in vitro activity of acetolactate synthase mutants against imidazolinone herbicides

[0046] Refer to the method of Fan ZJ et al. (Specific activity determination of acetolactate synthase from maize, Zea mays L., which is hereby incorporated by reference in its entirety), and see the book edited by Han WN et al. (The Proceedings of the 18 th Asia-Pacific Weed Science Society Conference.515-522.May 28-June 2, 2001.Beijing: Standards Press of China., which is incorporated herein by reference in its entirety), extract the wild type and each mutant plant of Jing 21 in Example 1 ALS enzyme (ie, acetolactate synthase), and the rate of inhibition of the corresponding enzyme activity by imidazolinone herbicides was determined.

[0047] Specific steps are as follows:

[0048] After taking 5g of each plant seedling and chopping them up, add 10mL of 50mmol / L K 2 HPO 4 -KH 2 PO 4 Buffer (which contains 1mmol / L sodium pyruvate, 0.5mmol / L MgCl 2 , 0.5...

Embodiment 3

[0052] Example 3 Obtaining of Transgenic Plants of Transprotein Mutant Genes

[0053] The inventor commissioned Beijing Weiming Kaituo Agricultural Biotechnology Co., Ltd. to use the conventional Agrobacterium-mediated method to convert the mutant ALS enzyme coding gene (SEQ ID NO: 1 and 3) were transformed into wild-type Arabidopsis plants, the specific steps are as follows:

[0054] Using the forward primer (5'-CCGTCCGGTCTGTAGCGTGTAC-3': SEQ ID NO: 5) and the reverse primer (5'-CATAACAGATAGCTGACGGCCTAC-3': SEQ ID NO: 6), respectively, from the mutant plants of Jing 21 above The mutant ALS gene was amplified by PCR in the genomic DNA. After the sequencing was correct, the ALS gene whose nucleotide sequence was shown as SEQ ID NO: 1 and 3 was cloned into the pCAMBIA1303 plasmid (purchased from Cambia Company), and the positive clone was selected to transform into Agrobacterium AGL0, cultivate the cells and transform Arabidopsis thaliana, and the transgenic seeds of the T1 gen...

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Abstract

The invention discloses acetolactic acid synthetase mutants and application thereof. Compared with the wild type acetolactic acid synthetase, at least one of the acetolactic acid synthetase mutants Ala173 and Trp542 is mutated. When the acetolactic acid synthetase mutants are expressed in a plant, the plant can have weedicide resistance.

Description

technical field [0001] The invention relates to the field of plant genetics and breeding. Specifically, the present invention relates to mutants of acetolactate synthase and applications thereof. More specifically, the present invention relates to acetolactate synthase mutants, isolated nucleic acids, expression cassettes, vectors or cells, methods for obtaining herbicide-resistant plants and the identification of plants obtained by the method of claim 8 or 9 method. Background technique [0002] Weeds in farmland are one of the most important factors affecting crop yields, but manual weeding consumes high human resources and costs, and it is not convenient for intensive agricultural production, which seriously restricts crop planting in the direction of high yield, high quality and low cost development process. Therefore, herbicides have emerged as the times require. However, herbicides often also kill the crop itself, so the use of herbicides is extremely limited in ti...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/68C12Q1/527A01H5/00
Inventor 黄拔严石久英
Owner BEIJING XINGBOYA BIOLOGICAL TECH
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