Chlorimuron-ethyl resistance-associated protein as well as encoding gene and application thereof

A coding gene, rimsulfuron-methyl technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of plant individual death, inability to complete mitosis, hinder DNA synthesis, etc., achieve high application value, and improve chlorosulfuron resistance The effect of dragon ability

Inactive Publication Date: 2013-07-03
NORTHEAST AGRICULTURAL UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Acetolactate synthase inhibitor herbicides inhibit the activity of acetolactate synthase in plants, thereby preventing the synthesis of branched-chain amino acids, resulting in the destruction of protein synthesis, hindering DNA synthesis during cell division, thereby stopping the mitosis of plant cells In the S phase (DNA synthesis phase) of the G1 phase and the M phase of the G2 phase, DNA synthesis is interfered, so the cells cannot complete mitosis, and the plant stops growing, eventually leading to the death of the plant individual

Method used

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  • Chlorimuron-ethyl resistance-associated protein as well as encoding gene and application thereof
  • Chlorimuron-ethyl resistance-associated protein as well as encoding gene and application thereof
  • Chlorimuron-ethyl resistance-associated protein as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, identification of chlorimuron-methyl-resistant bacterial strain TR-H

[0037] 1. Determination of acetolactate synthase (ALS) enzyme activity of chlorimuron-methyl-resistant TR-H strain

[0038] The TR-H strain was selected and preserved by the Pesticide Department of Northeast Agricultural University, and the Aspergillus niger strains Hm1, Hm2, and Hm3 were provided by the Heilongjiang Institute of Microbiology. The test agent is 98% chlorimuron-methyl original drug, produced by Dalian Ruize Pesticide Co., Ltd.

[0039] Get 1.0g thalline to be tested, the extraction of acetolactate synthase and the method for measuring activity refer to the method introduced by Chen Yifeng and Lukai, and improve (Lukai, Qian Chuanfan. 2000. Naphthalene dicarboxylic anhydride relieves ethametsulfuron The Mechanism of Phytotoxicity on Rice. Acta Plant Protection. 27(3): 268~272; Chen Yifeng, Li Yiwei, Tang Risheng, etc. 1996. Establishment of a simple assay method for the ...

Embodiment 2

[0053] Example 2, the acquisition of anti-chlorimuron-methyl protein and its coding gene

[0054] Search the NCBI database for the sequence related to the ALS inhibitor target enzyme-acetolactate synthase (ALS), download the entire sequence for bioinformatics analysis, design primers, and use RT-PCR from Aspergillus niger TR-H The cDNA sequence of the gene of the acetolactate synthase with the function of resisting chlorimuron-methyl was cloned in the mycelium.

[0055] The specific method is:

[0056] The strain Aspergillus niger (Aspergillus niger TR-H) (Zhang Hongyan, Zhang Qinghe, Zhang Libin, Taobo. 2010. Study on the enzymatic characteristics of chlorimuron-methyl-resistant Aspergillus niger acetolactate synthase (ALS). Journal of Oil Crops. 32(4 ): 563-566, the public can obtain from Northeast Agricultural University; Institute of Crop Science, Chinese Academy of Agricultural Sciences) inoculated in Cha's medium (formula: peptone 5g, glucose 20g, NaNO 3 3g, KCl0.5g, M...

Embodiment 3

[0062] Example 3, Functional Verification of Anti-Chlorsulfuron-methyl Protein and Its Encoding Gene

[0063] 1. Obtaining of anti-chlorimuron-methyl protein

[0064] Clone the ORF of AnALS1 into the pGBKT7 vector and place it under the control of the T7 promoter. The specific method is:

[0065] Primers were designed according to the above AnALS1 cDNA sequence, the sequence is as follows: AncDF1, 5'GA GGA TCC GTA TGA TGC CTA TGA GAC CTT C-3' (sequence 5, the underlined mark is the BamH I enzyme recognition site); AncDR1, 5'-TC GTC GAC TTA GAA ACC GGG AAC TTT CC-3' (Seq. 6, the Sal I enzyme recognition site is underlined).

[0066] Inoculate the strain Aspergillus niger (Aspergillus niger TR-H) in the Zaburi medium containing 500 mg / L of chlorimuron-methyl, and culture it at 30°C in a shaker at 150 rpm for 48 hours, collect the mycelium and wash it with DEPC water The RNA was extracted and reverse-transcribed into the first strand of cDNA, and the cDNA was used as a temp...

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Abstract

The invention discloses a chlorimuron-ethyl resistance-associated protein as well as an encoding gene and application thereof. The protein provided by the invention is (a) or (b) as follows: (a) a protein composed of amino acid sequences shown in sequence 2 in a sequence table; (b) a chlorimuron-ethyl resistance-associated protein derived from the sequence 2 via substitution and / or deletion and / or addition of the amino acid sequences shown in the sequence 2 in the sequence table by one or more amino acid residues. Experiments prove that the invention provides a new protein, the chlorimuron-ethyl-resisting capacity of a genetically modified strain can be improved by transferring the encoding gene of the protein into escherichia coli to express, and then, a chlorimuron-ethyl-resisting product can be obtained. Therefore, the gene of the protein disclosed by the invention can be used for culturing high-chlorimuron-ethyl-resistance genetically modified crop varieties, and the protein has an extremely high application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a protein related to resistance to chlorimuron-methyl and its coding gene and application. Background technique [0002] Modern biotechnology has caused major changes in several fields of agriculture, the most prominent of which is the expression of foreign genes introduced into plants, which has brought revolutionary changes to crop breeding and freed people from using traditional hybridization techniques to breed new varieties. The situation has entered a new era of free construction of genetic traits. Breeding transgenic herbicide-resistant crop varieties through genetic engineering is the fastest-growing, deepest-studied, and most accepted case by farmers. The most critical link is the acquisition of herbicide-resistant genes, isolation from mutant herbicide-resistant plants and microorganisms Resistance genes are the most important pathway. [0003] Acetolactate synthase inhib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N15/81C12N5/10C12N1/19C12N15/11A01H5/00C12R1/685
Inventor 陶波任洪雷张洪岩金龙国邱丽娟
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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