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Protein for endowing wheat with herbicide resistance and application of protein in plant breeding

A herbicide, plant technology, applied in the field of plant protein

Active Publication Date: 2012-07-11
FRONTIER LAB OF SYST CROP DESIGN CO LTD +1
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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, and can only be obtained through the hard work of scientific researchers, especially long-term screening, and relying on some luck to get

Method used

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  • Protein for endowing wheat with herbicide resistance and application of protein in plant breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Extraction of protein mutants resistant to imidazolinone herbicides from Ningchun No. 4 mutant plants

[0037] Get 400kg of commercially available wheat varieties—Ningchun No. 4 seeds, immerse the seeds in water for 4 hours, remove the seeds, and control the water. Then, the seeds were soaked in 0.5% (w / w) ethyl methanesulfonate (EMS) for 12 hours, ensuring that the liquid surface was submerged in the seeds. Discard the EMS solution after 12 hours, change the tap water to soak the seeds, discard the tap water, repeat the soaking 5 times, soak for 5 minutes each time, then rinse the seeds with tap water for 2 hours, turn the seeds during the washing process, so that the EMS is washed, and slightly air-dried After sowing, harvest when the plants mature. The harvested seeds are planted in the field, and when they grow to the three-leaf stage, they are treated with the imidazolinone herbicide imazethapyr ((RS)5-ethyl-2-(4-isopropyl-4-methyl-5 -Oxo-3-imidazolin...

Embodiment 2

[0039] Example 2 Determination of in vitro activity of protein mutants against imidazolinone herbicides

[0040] 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 wild-type and mutant plants of Ningchun No. 4 above, and measured the rate of inhibition of the corresponding enzyme activity by imidazolinone herbicides. In brief, 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 (which contains 1mmol / L sodium pyruvate, 0.5mmol / L MgCl 2 , 0.5mmol / L TPP, 10μmol / L FAD), ground and pulverized with quartz sand, filtered with 8 layers of gauze, and the filtrate was centrifuged at 4°C and 20,000rpm for 30 minutes. Take the supernatant, add ammonium...

Embodiment 3

[0044] The acquisition of the transgenic plant of embodiment 3 transprotein mutant gene

[0045] Beijing Weiming Kaituo Agricultural Biotechnology Co., Ltd. was entrusted to use the conventional Agrobacterium-mediated method to transfer the gene encoding the mutant ALS enzyme of Ningchun 4 into wild-type Arabidopsis plants. In brief, using the forward primer (5'-AACTTCCAACCCCTCTCTCTGCCTC-3') and the reverse primer (5'-TCAGTACGAGGTCCTGCCATC-3'), PCR amplified from the genomic DNA of each mutant plant of Ningchun 4 above. The mutant ALS gene was added, and after the sequence was correct, the ALS gene whose nucleotide sequence was shown as SEQ ID NO: 1, 3 and 5 was cloned into the pCAMBIA1303 plasmid (purchased from Cambia Company), and the positive clone was selected to transform into Agrobacterium AGL0, The bacteria were cultured and transformed into Arabidopsis thaliana. After harvesting the positive Arabidopsis thaliana by PCR, the transgenic seeds of the T1 generation were...

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Abstract

The invention provides an acetolactate synthetase (ALS) protein for endowing a plant with herbicide resistance. The ALS protein is derived from a wheat variety Ningchun 4, and carries Ala179 and / or Ser627 mutation. In addition, the invention also provides corresponding nucleic acid, an expression kit, a vector, a cell, a plant, application and method of obtaining the plant with the herbicide resistance, and a method for identifying the plant.

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 wheat) 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 significant role in solving farmland weed damage, 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, sinc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/21C12N1/19C12N5/10A01H5/00C12N15/82A01H4/00A01H1/02C12Q1/68C12Q1/527
Inventor 马力耕王晓雪周宽基邓兴旺
Owner FRONTIER LAB OF SYST CROP DESIGN CO LTD
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