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Glyphosate-resistant gene and application thereof

A glyphosate-resistant gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of not being able to directly provide glyphosate-resistant genes

Inactive Publication Date: 2010-01-06
沈志成
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these numerous EPSPS genes cannot directly provide people with glyphosate-resistant genes

Method used

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  • Glyphosate-resistant gene and application thereof
  • Glyphosate-resistant gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1, the resistance determination of anti-glyphosate bacteria

[0035] Deinococcus radiodurans is an ancient bacterium. In order to determine whether it has resistance to glyphosate, D. radiodurans was inoculated in M ​​63 medium containing 25mM glyphosate. The formula of M 63 medium is as follows: (per liter of M63: 13.6g KH 2 PO 4 , 2g (NH 4 ) 2 SO 4 , 0.5 mg FeSO 4 -7H 2 O, 2.4 mg MgCl 2 , 10g glucose, 10mg Thiamine-HCl and 25mg L-Proline, 40g agarose, pH7.0).

[0036] Since M63 does not contain aromatic amino acids, bacteria that can survive and grow on this medium can synthesize aromatic amino acids under high concentrations of glyphosate, and should be resistant bacteria. The results show that D.radiodurans can grow in M ​​63 medium with 25mM glyphosate, while the control Escherichia coli (E.coli) cannot grow under 25mM glyphosate, but can be cultured in M ​​63 without glyphosate basal growth. Therefore D. radiodurans may contain glyphosate res...

Embodiment 2

[0037] Embodiment 2, the cloning of resistance gene

[0038] A bacterial resistance to glyphosate may have multiple mechanisms. Such as possessing glyphosate-resistant EPSPS gene; having an enzyme that can degrade glyphosate; being able to limit the absorption of glyphosate, or possessing an enzyme that modifies glyphosate to inactivate glyphosate, etc. All genome studies of D. radiodurans were determined (Whtie O, Eisen J A, Heidelberg J F, et al. Genome sequence of the radio-resistant bacterium Deinococcus radiodurans R1. Science, 1999, 286: 1571-1577). Therefore, the discovery of glyphosate-resistant genes can start from the discovery of possible resistance genes in the genome. D. radiodurans contains a possible EPSPS gene. However, whether a bacterial EPSPS is resistant to glyphosate and the level of resistance cannot be predicted by the amino acid sequence of the EPSPS. Especially the anti-glyphosate ability of this type of novel EPSPS involved in the present invention...

Embodiment 3

[0040] The determination of the anti-glyphosate performance of embodiment 3, G7 gene

[0041] The glyphosate-resistant ability of Escherichia coli containing pMD18-T-G7 (containing G7 gene) vector was determined.

[0042] Escherichia coli containing pMD18-T-G7 and pMD18-T (empty vector, control) were first cultured in LB, when OD 600 When it reaches 0.6, take 0.2ml and centrifuge at 5000rpm for 1 minute, discard the supernatant. The precipitated cells were washed with M63 culture medium and then centrifuged, and then the precipitated cells were washed with the M63 culture medium containing 60mM glyphosate (every liter of M63: 13.6gKH 2 PO 4 , 2g (NH 4 ) 2 SO 4 , 0.5 mg FeSO 4 -7H 2 O, 2.4 mg MgCl 2 , 10g glucose, 10mg Thiamine-HCl and 25mg L-Proline, pH7.0) diluted to OD 600 0.02, then cultivated at 37°C (shaking table 250 rpm), and after 24 hours, measure the OD 600 . OD of clones containing pMD18-T-G7 600 is 0.84, while the control containing pMD18-T is only 0.05...

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Abstract

The invention discloses a glyphosate-resistant gene. A nucleotide sequence of the gene can be any one of the sequences as follows: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or a sequence with sameness of not smaller than 45 percent in comparison with the SEQ ID NO:3. The invention also discloses protein peptide coded by the glyphosate-resistant gene, and an amino acid sequence of the protein peptide can be any one of the sequences as follows: SEQ ID NO:1, SEQ ID NO:2 or a sequence with sameness of not smaller than 45 percent in comparison with the SEQ ID NO:1. The use of the glyphosate-resistant gene is expressed in a plant through transgenosis so as to ensure that the plant obtains the glyphosate-resistant capacity.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular, the invention relates to a glyphosate-resistant gene and its coded protein. This gene can be used to express in plants to make plants resistant to glyphosate, so that glyphosate can be used to selectively control weeds. The present invention can also be applied in the breeding of crops and the screening of plant cell culture. Background technique [0002] Glyphosate is a very important herbicide, which inhibits an important enzyme in the biosynthesis of aromatic amino acids in plants, namely 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glyphosate is an extremely broad-spectrum herbicide that is equally lethal to crops. So in order to be able to selectively kill weeds while the crops are growing, the crops need to acquire glyphosate resistance. [0003] Glyphosate is also able to inhibit 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the biosynthesis of ar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/195C12N15/63C07K16/12C12N15/82C12R1/01
Inventor 沈志成
Owner 沈志成
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