Herbicide-resistant protein in Brassica napus and its application in plant breeding

A rape and plant technology, applied in the field of plant protein, can solve the problems of loss of herbicide resistance, complicated situation, and inability to apply in practice.

Active Publication Date: 2014-10-08
SHENZHEN XINGWANG BIOLOGICAL SEED IND +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are currently many reports in the literature that mutating some genes and then introducing wild-type plants into transgenic plants can bring herbicide resistance to plants, most of the experiments are aimed at model plants with clear genetic backgrounds such as Arabidopsis, but For the crops actually planted, especially the crops with good traits (and complex genetic background) obtained through a large number of previous transgenic and / or non-transgenic breeding, the situation is often not optimistic. As we found, in vitro and / or simulated The introduction of mutant genes with herbicide resistance in Arabidopsis thaliana, after the introduction into plants, a large number of cases of herbicide resistance loss occurred, although some retained herbicide resistance, the plant growth and development were not normal and it was impossible to practice This is likely due to the synergistic effect of a large number of genes in the plant
Therefore, for plants with complex genetic backgrounds (such as rapeseed, especially improved rapeseed), plants with herbicide resistance and their real decisive substances (such as proteins, genes, etc.) have not yet been theoretically designed. , can only be obtained by relying on the hard practice of scientific researchers, especially relying on long-term screening, and relying on some luck to obtain
For tetraploid plants, the situation is more complicated

Method used

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  • Herbicide-resistant protein in Brassica napus and its application in plant breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Extraction of protein mutants resistant to imidazolinone herbicides from Zhongshuang No. 9 mutant plants

[0036] Rapeseeds of various varieties / lines were sown, including Zhongshuang 9 (available from the Institute of Oil Crops, Chinese Academy of Agricultural Sciences). Before sowing, soak the rape seeds of each variety / line in water for 22 hours in an incubator at 25°C, then remove the seeds and control the water. Then, each canola seed was soaked with 0.4% (w / w) ethyl methanesulfonate (EMS) for 12 hours, during which the seeds were shaken every 1 hour. After 12 hours, discard the EMS solution, replace the tap water to soak the seeds, discard the tap water, and repeat the soaking 5 times in total, soaking for 5 minutes each time, then rinse the seeds with tap water for 2 hours, and turn the seeds during the rinsing process to wash off the EMS. Put a small amount of seeds on the surface of wet paper, put them in an incubator at 28°C, and measure the germi...

Embodiment 2

[0038] Example 2 Determination of in vitro activity of protein mutants against imidazolinone herbicides

[0039] Refer to the method of Fan ZJ et al. (Specific activity determination of acetolactate synthase from maize (Zea mays L.). See 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.), extracted the ALS enzymes of the above-mentioned Zhongshuang 9 wild-type and mutant plants, and determined the rate of inhibition of the corresponding enzyme activity by imidazolinone herbicides. Briefly speaking, 5 g of each plant seedling was taken and chopped, and 10 mL of 50 mmol / L K at pH 7 was added. 2 HPO 4 -KH 2 PO 4 Buffer (containing 1 mmol / L sodium pyruvate, 0.5 mmol / L MgCl 2 , 0.5 mmol / L TPP, 10 μmol / L FAD), ground and pulverized with quartz sand, filtered with 8 layers of gauze, and the filtrate was centrifuged at 4°C and 20,000 rpm for 30 minutes. Take the supernat...

Embodiment 3

[0043] The acquisition of the transgenic plant of embodiment 3 transprotein mutant gene

[0044] The gene encoding the above-mentioned mutant ALS enzyme of Zhongshuang9 was transformed into wild-type Arabidopsis plants by conventional Agrobacterium-mediated method. In short, use the forward primer (5'-catcatctctctctcctctaaccatggc -3') and the reverse primer (5'-CCAGCTTCATCTCTCAGTACTTAG -3') to PCR amplify from the genomic DNA of the mutant plant of Zhongshuang9 above. After the mutant ALS gene was sequenced correctly, the nucleotide sequence of the ALS gene shown in SEQ ID NO: 1, 3, 5, 7, 9 and 11 was cloned into the pCAMBIA1303 plasmid (purchased from Cambia Company), and positive clones were selected Transform Agrobacterium EHA105, culture the cells and transform Arabidopsis thaliana. The positive Arabidopsis thaliana was harvested by PCR to obtain transgenic seeds of the T1 generation, and sprayed with imidazolinone herbicide "imazapyr" water agent when planting , it was...

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Abstract

The invention discloses the ALS protein sequence and its application for making plants resistant to herbicides. It is derived from a mutant strain of Brassica napus variety Zhongshuang9, and its amino acid sequence is as SEQ ID NO: 2, 4, 6, 8, 10 or 12 shown. In addition, the present invention also provides corresponding nucleic acids, expression cassettes, vectors, cells, plants, applications and methods for obtaining plants with herbicide resistance, and methods for identifying plants of the present invention.

Description

technical field [0001] The invention belongs to the field of plant protein, in particular, the invention relates to a protein capable of imparting herbicide resistance (tolerance) to plants (especially oilseed rape) and its application in plant breeding. 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, and their use has played a major role in solving weed damage in farmland, promoting innovation in cultivation methods, and increasing production. [0003] Common herbicides, including sulfonylureas, imidazolinones, pyrimidotriazoles, pyrimidine salicylate and other herbicides, have been widely used commercially. However, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/21C12N1/19C12N5/10A01H5/00C12Q1/68C12Q1/527
Inventor 刘东风王承旭唐晓艳邓兴旺
Owner SHENZHEN XINGWANG BIOLOGICAL SEED IND
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