Cabbage type rape herbicide resistance protein and application thereof to plant breeding

A herbicide and plant technology, applied in the field of plant protein, can solve problems such as complex situations, loss of herbicide resistance, and abnormal plant growth and development

Active Publication Date: 2013-07-24
SHENZHEN XINGWANG BIOLOGICAL SEED IND +2
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  • 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

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  • Cabbage type rape herbicide resistance protein and application thereof to plant breeding

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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 resistant to 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 sup...

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, the forward primer (5'-catcatctctctctcctctaaccatggc -3') and the reverse primer (5'-CCAGCTTCATCTCTCAGTACTTAG -3') were used to PCR amplify from the genomic DNA of the mutant plant of Zhongshuang9 above. After the mutated 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. After the positive Arabidopsis transformation is identified by PCR, the transgenic seeds of the T1 generation are harvested, and the imidazolinone herbicide "imazapyr" water spray is u...

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Abstract

The invention discloses an ALS (acetolactate synthase) protection sequence for enabling plants to have herbicide resistance and an application of the sequence. The sequence is derived from a mutant strain of double No.9 of a cabbage type rape variety and the amino acid sequence is represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12. In addition, the invention further provides a corresponding nucleic acid, an expression box, a carrier, a cell, a plant, an application and a method for obtaining the plants with the herbicide resistance, and a method for identifying the plants obtained by the 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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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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