Acetolactate synthase mutant protein with herbicide resistance and application thereof

A technology of mutant proteins and proteins, applied in the application, enzyme, transferase and other directions, can solve the problems of complex situation, difficult to predict herbicide resistance in advance, etc., and achieve the effect of improving tolerance

Active Publication Date: 2017-10-13
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, for crops with complex genetic backgrounds (such as rapeseed), mutants with high herbicide resistance and substances that can indeed play a decisive role (such as proteins, genes, etc.) have not yet been theoretically designed, and can only rely on Obtained through the hard work of scientific researchers, especially relying on long-term screening and identification, and relying on luc

Method used

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  • Acetolactate synthase mutant protein with herbicide resistance and application thereof
  • Acetolactate synthase mutant protein with herbicide resistance and application thereof
  • Acetolactate synthase mutant protein with herbicide resistance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: The creation process of the Brassica napus acetolactate synthase mutant with high herbicide resistance

[0037] Before sowing rapeseed, carry out ethyl methanesulfonate (EMS) mutagenesis treatment to wild-type rapeseed line N131 (commonly known and public, see Pu Huiming et al., Jiangsu Agricultural Science Journal, 2010, 26(6): 1432-1434). The process is as follows: 1 day before sowing, put 0.5kg of N131 seeds into a nylon mesh bag, soak in a container filled with clean water for 12 hours, hang the nylon mesh bag on a high place until the water is basically drained, and place it in a 0.4 Treat in %EMS solution (v / v) for 8h, wash the treated seeds with running water for 4h, hang the nylon mesh bag on a high place again and drain the water, immediately sow the seeds (Mo) in a 6× 50m isolation greenhouse. In the spring of that year, after the rapeseed bloomed, it was isolated with a nylon net to keep it pure, and the mature M. 1 seed. At the beginning of Oct...

Embodiment 2

[0039] Example 2: Herbicide Resistance Identification

[0040] The resistance identification test adopts two methods of field identification and greenhouse pot test to identify and evaluate the resistance effect of the resistant mutants EM28 and DS3. The field identification test of rapeseed was carried out in the isolated breeding area of ​​rapeseed of Jiangsu Academy of Agricultural Sciences, and the pot experiment in the greenhouse was carried out in a constant temperature cultivation room. The control material wild type N131 to be treated (commonly known and public, see Pu Huiming etc., Jiangsu Agricultural Science Journal, 2010, 26 (6): 1432-1434), EM28 of the present invention, DS3 seedling emergence grow to 3-4 leaf seedling age, spray respectively Apply different concentrations of SU herbicide tribenuron-methyl (2-[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-N-methylcarbamate Sulfonyl] methyl benzoate) methylsulfuron-methyl (methyl-2 [3- (4, 6- = methoxypyrimidin-2-yl) ...

Embodiment 3

[0044] Embodiment 3: ALS enzyme activity measures in vitro

[0045] Phenotypically, DS3 is highly resistant to SU herbicides, so this herbicide was selected for in vitro enzyme activity assays to compare the differences in herbicide resistance between DS3, EM28 and the original wild-type N131. The determination refers to the method of Singh et al. (Singh BK, et al., Analytical Biochemistry, 1988, 171:173-179). Specifically, take 0.2g of leaf samples respectively, grind and pulverize them with liquid nitrogen in a mortar, add the ground samples into 4.5ml of primary enzyme extract [100mM K 2 HPO 4 , 0.5mM MgCl 2 , 0.5mM Thiamine Pyrophosphate (TPP), 10μM Flavin Adenine Dinucleotide (FAD), 10mM Sodium Pyruvate, 10% (v / v) Glycerol, 1mM Dithiothreitol, 1mM Benzene Sulfonyl fluoride (PMSF), 0.5% (w / v) polyvinylpyrrolidone], centrifuged at 4°C, 12000rpm for 20min. Take the supernatant and add an equal volume of saturated (NH 4 ) 2 SO 4 , placed on ice for 30 minutes, centrifu...

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Abstract

The invention discloses ALS (Acetolactate Synthase) mutant protein. An amino acid sequence of ALS1 is shown as SEQ ID NO: 2 and an amino acid sequence of ALS3 is shown as SEQ ID NO: 4. The invention further discloses nucleic acid for coding the mutant protein, an expression box containing the nucleic acid and a recombinant vector or cell. The invention further discloses application of the mutant protein, the nucleic acid, the expression box and the recombinant vector or cell to the aspect of herbicide resistance of plants. The invention further discloses a method for obtaining plants with the herbicide resistance and a resistant plant identification method. The ALS mutant protein disclosed by the invention is a dual-gene and dual-mutation-site ALS inhibitor type herbicide acetolactate synthase mutant which is found from rape for the first time in China; a result of an experiment of applying ALS inhibitor type herbicide tribenuron and mesosulfuron-methyl in the field shows that after the herbicide with 16 times of weed prevention and control recommended utilization concentration is applied to the rape DS3 containing the ALS mutant protein provided by the invention during a 3-leaf to 4-leaf period, the plants still grow and develop, and bear fruits normally.

Description

technical field [0001] The invention relates to the field of plant herbicide resistance and molecular breeding, in particular to acetolactate synthase mutant protein with herbicide resistance and application thereof. More specifically, it relates to a method for isolating acetolactate synthase encoding gene of high herbicide resistance in Brassica napus, constructing a vector, and cultivating highly herbicide resistant plants. Background technique [0002] Rapeseed (Brassica napus L.) is one of the largest oil crops in my country, providing edible oil sources for more than half of the population in my country. In recent years, with the development of rapeseed planting methods in my country from traditional labor-intensive to large-scale and mechanized production, effective weed control has become an important link in the whole process of mechanized production and simplified cultivation of rapeseed. Chemical weeding is an economical and effective means of controlling weeds i...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/00
CPCC12N9/1022C12N15/8274C12Y202/01006C12Q1/6895C12Q1/48C12Q2600/13C12Q2600/158G01N2333/9104
Inventor 胡茂龙浦惠明龙卫华高建芹张洁夫陈松彭琦陈锋付三雄周晓婴张维王晓东
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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