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Method for obtaining protein with glufosinate-ammonium resistance and glutamine synthetase mutant

A glutamine and synthetase technology, applied in the field of genetic engineering, can solve problems affecting plant growth and development, consumer resistance, and insufficient tolerance for commercial application, etc., to achieve the effect of satisfying growth and development

Pending Publication Date: 2022-07-22
SICHUAN GEVOTO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, due to the wave of anti-genetics, the acceptance of genetically modified crops in the world is still low. Even in the Americas with the largest planting area of ​​genetically modified crops, genetically modified crops are mainly limited to several crops such as corn, soybeans, and cotton.
In particular, the bar gene and pat gene are derived from microorganisms, not from the crop itself, which is more likely to cause consumers' resistance
[0007] The glufosinate-ammonium acetylase encoded by bar gene and pat gene can inactivate glufosinate-ammonium by acetylation, but before glufosinate-ammonium contacts GS, it is difficult for the enzyme to inactivate glufosinate-ammonium completely, because many GS are distributed in Therefore, when glufosinate-ammonium is applied to transgenic crops with bar gene and pat gene, it will interfere with the nitrogen metabolism of plants to varying degrees, and at the same time affect the normal growth and development of plants
Although overexpression of wild-type GS in plants can reduce the sensitivity of transgenic plants to glufosinate-ammonium, the degree of tolerance is not enough for commercial application

Method used

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  • Method for obtaining protein with glufosinate-ammonium resistance and glutamine synthetase mutant
  • Method for obtaining protein with glufosinate-ammonium resistance and glutamine synthetase mutant
  • Method for obtaining protein with glufosinate-ammonium resistance and glutamine synthetase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] The rice (Oryza sativa) glutamine synthase (GS1) mutant provided in this example is obtained from the wild-type rice glutamine synthase itself (named OsGS1-WT, and the amino acid sequence is shown in SEQ ID NO.1, The 55th amino acid residue S of the coding nucleotide sequence of SEQ ID NO. 5) was mutated to D, T, and L, and the obtained rice GS1 mutants were named R55D, R55T, and R55L, respectively.

[0117] The amino acid sequence alignment of rice GS1 mutants R55D, R55T, R55L and wild-type rice GS1 is as follows figure 2 As shown, in the figure: the position indicated by the arrow is the mutation site.

[0118] In this example, the coding sequence of each rice GS1 mutant is at the position encoding the 55th amino acid, the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.

[0119] amino acid D T L a GAT ACC ...

Embodiment 2

[0122] The soybean (Glycine max) GS1 mutant provided in this example is composed of wild-type soybean GS1 itself (named as GmGS1-WT, the amino acid sequence is shown in SEQ ID NO. 3, and the coding nucleotide sequence is SEQ ID NO. The 55th position of 7) (corresponding to the 55th position of the reference sequence (SEQ ID NO. 1)) was obtained by mutating the amino acid residue S to D. The obtained soybean GS1 mutants were named G55D, respectively.

[0123] The amino acid sequence alignment of soybean GS1 mutant G55D and wild-type soybean GS1 is as follows image 3 As shown, in the figure: the position indicated by the arrow is the mutation site.

[0124] In this example, the coding sequence of each soybean GS1 mutant is at the position encoding the 55th amino acid, and the codons used for the corresponding amino acid are shown in the following table, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.

[0125] ...

Embodiment 3

[0128] The maize (Zea mays) GS1 mutant provided in this example is composed of wild-type maize GS1 itself (named ZmGS1-WT, the amino acid sequence is shown in SEQ ID NO.2, and the coding nucleotide sequence is SEQ ID NO.6 ) at position 55 (corresponding to position 55 of the reference sequence (SEQ ID NO. 1)) is obtained by mutating the amino acid residue S to D. The resulting maize GS1 mutants were named Z55D, respectively.

[0129] The amino acid sequence alignment of maize GS1 mutant Z55D and wild-type maize GS1 is as follows Figure 4 As shown, in the figure: the position indicated by the arrow is the mutation site.

[0130] In this example, the coding sequence of each maize GS1 mutant is at the position encoding the 55th amino acid, the codons used for the corresponding amino acid are shown in the table below, and the nucleotides in the remaining positions are the same as the corresponding wild-type coding sequence.

[0131] amino acid D a GAT

[...

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Abstract

The invention discloses a method for obtaining a protein with glufosinate-ammonium resistance and a glutamine synthetase mutant, and relates to the technical field of gene engineering. The method comprises the following steps: comparing an amino acid sequence of a target protein with a reference sequence, and mutating the amino acid sequence of the target protein corresponding to a 55th amino acid residue S and / or a 64th amino acid residue P of the reference sequence; and selecting the glufosinate-ammonium resistance enhanced protein. The glutamine synthetase mutant provided by the invention is originally derived from plants and is more easily accepted by consumers. The glutamine synthetase mutant has glufosinate-ammonium resistance after mutation, and a plant converted with the glutamine synthetase mutant not only has glufosinate-ammonium resistance suitable for commercial application, but also can keep normal enzyme catalytic activity of glutamine synthetase, and can meet normal growth and development of the plant.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a method for obtaining a protein with glufosinate-ammonium resistance and a glutamine synthase mutant. Background technique [0002] Glutamine synthetase (GS) is a key enzyme in plant nitrogen metabolism, which catalyzes the conversion of glutamate (Glu) to NH in the glutamate synthetase cycle. 3 Condensed to form glutamine (Gln), which is involved in the metabolism of nitrogenous compounds in plants. According to the distribution and subcellular localization, the isoenzymes of higher plant GS (which belong to GSII) can be divided into two types: one is located in the cytoplasm called cytoplasmic GS (GS1) with a molecular weight of 38-40kDa; the other is located in the cytoplasm. The chloroplast (or plastid) is called plastid-type GS (GS2), with a molecular weight of 44-45kDa. [0003] Glufosinate (glufosinate ammonium, trade name Basta) is a glutamine synth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/70C12N15/82A01H5/00A01H6/00
CPCC12N9/93C12N15/70C12N15/8277C12Y603/01002
Inventor 陈容邓龙群卢远根付颖钊张震侯青江胥南飞
Owner SICHUAN GEVOTO BIOTECH CO LTD
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