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Paddy rice ALS (Acetolactate Synthase) mutant protein for endowing plants with resistance to herbicides, gene and application thereof

A herbicide and mutant technology, applied in the field of plant protein and plant herbicide resistance, can solve the problems that it is difficult to predict the herbicide resistance of ALS protein in advance, and the mechanism of herbicide action is not determined.

Pending Publication Date: 2017-08-25
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007]At present, the mechanism of action of ALS inhibitor herbicides has not yet been determined, and it is difficult to predict in advance whether mutations at other amino acid sites of the ALS protein will cause herbicide resistance. Relying on the long-term and arduous practical exploration of researchers, and with some luck, it is possible to discover new herbicide resistance sites in ALS proteins

Method used

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  • Paddy rice ALS (Acetolactate Synthase) mutant protein for endowing plants with resistance to herbicides, gene and application thereof
  • Paddy rice ALS (Acetolactate Synthase) mutant protein for endowing plants with resistance to herbicides, gene and application thereof
  • Paddy rice ALS (Acetolactate Synthase) mutant protein for endowing plants with resistance to herbicides, gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: The process of obtaining rice mutants resistant to imidazolinone herbicides (Bailongtong)

[0041] 150 kg of indica conventional rice seed Yangzhou restorer line (purchased from Jiangsu Provincial Agricultural Germplasm Resources Protection and Utilization Platform) (this is M0, soaked in water for 2 hours) was divided into 6 times with 0.5-1.0% (w / w) methanesulfonic acid Soak in ethyl acetate (EMS) at room temperature for 6-9 hours, shake the seeds every 1 hour during this period; discard the EMS solution, soak the seeds 5 times with tap water, 5 minutes each time, then rinse the seeds with tap water overnight, and carry out field work the next day Sow seeds and perform conventional fertilizer and water management (this is M1). After the plants are mature, the seeds are mixed, dried, and stored for the winter. Sow in the field the following year. When the rice (this is M2) seedlings grow to the 3-4 leaf stage, spray 3mL Bailongtong / L water ("Bailongtong" i...

Embodiment 2

[0042] Example 2: Analysis of mutation sites in rice mutants resistant to imidazolinone herbicides

[0043] From the herbicide-resistant mutant rice plants obtained in Example 1 above, a plurality of mutant leaves were selected, and genomic DNA was extracted respectively, and sent to Nanjing Yidao Biotechnology Co., Ltd. for genome sequencing. Comparing the sequencing results with the wild-type ALS gene of the Yangzhou restorer line, it was found that there were three groups of mutant plants in the above-mentioned mutant rice plants, namely, herbicide-resistant rice mutant 1, herbicide-resistant rice mutant 2 and herbicide-resistant rice mutant 3, among which, The herbicide-resistant rice mutant 1 has 2 mutations on the ALS gene, the 1880th and 1883th bases are mutated, respectively changing from G to A, and G to A, resulting in the corresponding encoded amino acid sequence The 627th and 628th positions of the ALS gene are changed from serine to asparagine, and glycine to glut...

Embodiment 3

[0046] Embodiment 3: ALS gene cloning of rice mutant resistant to imidazolinone herbicides

[0047] The leaves of the above herbicide-resistant rice mutant 1, herbicide-resistant rice mutant 2 and herbicide-resistant rice mutant 3 were taken, and genomic DNA was extracted respectively. The specific primers for amplifying the full-length ALS gene were designed according to the wild-type ALS gene sequence of Yangzhou restorer line: forward primer F5'-TCGCCCAAACCCAGAAACCC-3', reverse primer R 5'-CTCTTTATGGGTCATTCAGGTC-3'.

[0048] Adopt Takara PrimerSTAR Max DNA Polymerase polymerase (purchased from Takara company) to amplify ALS gene, its reaction system is as follows:

[0049]

[0050] The PCR amplification reaction program adopts a two-step method, annealing and extension are combined together, and 68 degrees are used.

[0051] The program is as follows: pre-denaturation: 98°C for 3min; 35 cycles: denaturation at 98°C for 10sec; extension at 68°C for 1min; incubation: 72°C...

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Abstract

The invention discloses a paddy rice ALS (Acetolactate Synthase) mutant protein for endowing plants with resistance to herbicides. The invention further discloses a nucleic acid for coding the protein. The protein comes from a paddy rice mutant plant resisting ALS inhibitor type herbicides, and in comparison with the ALS sequence in the genome of wild paddy rice, the protein sequence of the protein is simultaneously mutated at sites Ser 627 and Gly 628 or only mutated at the site Gly 628. Plants which express the protein sequence can resist (tolerate) acetolactate synthase inhibitor type herbicides, particularly imidazolinone herbicides. The herbicide-resistant effect of the mutant mutated simultaneously at the sides Ser 627 and Gly 628 disclosed by the invention is about 30 percent higher than the herbicide-resistant capability of the mutant plant only contains mutation at the site Ser 627 or Gly 628.

Description

technical field [0001] The invention belongs to the field of plant protein and plant herbicide resistance, and relates to rice ALS mutant protein, gene and application thereof for making plants resistant to herbicides; specifically, the invention relates to rice acetolactate synthase (ALS) mutant protein , the protein can endow plants, especially rice, with the characteristics of resistance to acetolactate synthase inhibitor herbicides. The invention discloses the sequence of the protein and their application in the field of plant herbicide resistance. Background technique [0002] Weeds are unfavorable factors restricting the stable and high yield of agricultural production. Compared with traditional cultivation methods, artificial weeding and mechanical weeding, the use of chemical herbicides is an efficient, simple and economical method of controlling weeds. [0003] Acetolactate synthase (ALS) (also known as acetohydroxyacid synthase, AHAS; EC 4.1.3.18) inhibitor herbi...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12Q1/68A01H5/00
CPCC12N9/88C12N15/8274C12Q1/6895C12Q2600/13
Inventor 张保龙凌溪铁王金彦陈天子邓惠清
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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