Herbicide-resistant corn protein and application thereof in plant breeding

A herbicide and plant technology, applied in the field of plant protein, can solve problems such as impossibility of practical application, loss of herbicide resistance, abnormal growth and development of corn, etc.

Active Publication Date: 2013-05-15
深圳洁田模式生物科技有限公司
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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 A mutant gene with herbicide resistance was introduced into Arabidopsis thaliana, and after being introduced into corn, etc., a large number of cases of herbicide resistance loss occurred. Although some of them retained herbicide resistance, the growth and development of corn were not normal, so it was impossible to practice , which is likely due to the synergistic effect of a large number of genes in maize
Therefore, for plants with complex genetic backgrounds (such as corn, especially improved corn), 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

Method used

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  • Herbicide-resistant corn protein and application thereof in plant breeding
  • Herbicide-resistant corn protein and application thereof in plant breeding
  • Herbicide-resistant corn protein and application thereof in plant breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Extracting protein mutants resistant to imidazolinone herbicides from Beijing No. 21 mutant plants

[0035] Sow various varieties / lines of corn, including corn inbred line Jing 21 (available from the Corn Research Center of the Institute of Crop Science, Chinese Academy of Agricultural Sciences). Nicotinic acid ((RS) 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-3-imidazolin-2-yl)nicotinic acid, CAS No. 81335-77-5) water (concentration of 0.5%, purchased from Shandong Xianda Chemical Co., Ltd.) was soaked for 18 hours, drained and sown in the experimental field, and the germination of corn seeds was observed after 10 days. After a lot of experimental screening, I finally discovered by chance that there are green seedlings that continue to grow ( figure 1 ), retained and propagated these green seedlings, and confirmed that they were 2 Jing 21 mutant plants resistant to imidazolinone herbicides. These plants could grow and develop normally, and the rest of the corn die...

Embodiment 2

[0037] The in vitro activity assay of the anti-imidazolinone herbicides of embodiment 2 protein mutants

[0038] 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 the18th Asia-Pacific Weed Science Society Conference.515-522.May28-June2, 2001. Beijing: Standards Press of China.), extracted the ALS enzymes of the wild-type and mutant plants of the above-mentioned Jing 21, and determined the rate of inhibition of the corresponding enzyme activity by imidazolinone herbicides. In brief, take 5g of each plant seedling and chop it up, add 10mL of pH7 50mmol / LK 2 HPO 4 -KH 2 PO 4 Buffer (which contains 1mmol / L sodium pyruvate, 0.5mmol / 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 supernatant, add ammonium sulfate to make it r...

Embodiment 3

[0042] The acquisition of the transgenic plant of embodiment 3 transprotein mutant gene

[0043] 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 the above-mentioned Jing 21 into wild-type Arabidopsis plants. In brief, use the forward primer (5'-CCGTCCGGTCTGTAGCGTGTAC-3') and the reverse primer (5'-CATAACAGATAGCTGACGGCCTAC-3') to PCR amplify from the genomic DNA of the above-mentioned Jing 21 mutant plants After the mutant ALS gene was sequenced correctly, clone the ALS gene whose nucleotide sequence is shown in SEQ ID NO: 1 and 3 into the pCAMBIA1303 plasmid (purchased from Cambia Company), select the positive clone and transform it into Agrobacterium AGL0, and culture the cells And transform Arabidopsis thaliana, PCR identified transformation-positive Arabidopsis thaliana after harvesting to obtain transgenic seeds of the T1 generation, spraye...

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Abstract

The invention provides an ALS (Acetolactate Synthase) protein capable of endowing plants with herbicide resistance. The amino acid sequence of the herbicide-resistant corn protein is as shown in SEQ ID NO: 2 or 4. Furthermore, the invention further provides corresponding nucleic acid, expression cassettes, vectors, cells, the plants, application and a method for obtaining the plants with herbicide resistance, and a method for identifying the plants in the invention.

Description

technical field [0001] The invention belongs to the field of plant proteins, in particular, the invention relates to a protein capable of imparting herbicide resistance (tolerance) to plants (especially corn) 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): C12N9/88C12N15/60C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/68A01H5/00A01H1/02G01N33/68
Inventor 黄拔严石久英
Owner 深圳洁田模式生物科技有限公司
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