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Glyphosate-resistant 5-enolpyruvyl-shikimate-3-phosphate synthase gene and application thereof

A technology of enolpyruvyl shikig and phosphate synthase, applied in the fields of molecular biology and plant genetic engineering, can solve problems such as restricting application, and achieve the effect of high glyphosate resistance

Active Publication Date: 2010-10-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because glyphosate kills weeds and crops non-selectively, it can only be used before crop emergence or in non-crop planting areas, which restricts its application in agriculture

Method used

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  • Glyphosate-resistant 5-enolpyruvyl-shikimate-3-phosphate synthase gene and application thereof
  • Glyphosate-resistant 5-enolpyruvyl-shikimate-3-phosphate synthase gene and application thereof
  • Glyphosate-resistant 5-enolpyruvyl-shikimate-3-phosphate synthase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, acquisition and identification of glyphosate-resistant bacterial strains

[0025] 1. Obtaining the strain

[0026] Solid medium screening method: Weigh 20g of extremely polluted soil samples (from a glyphosate production factory in Hubei), in 0.1L of 0.9% sodium chloride solution, magnetically stir for 20 minutes, and smear on 100mM glyphosate M63 medium (recipe: potassium dihydrogen phosphate 3g, dipotassium hydrogen phosphate 7g, ammonium sulfate 2g, ferrous sulfate 0.0005g, magnesium sulfate 0.4928g, glycerol 40ml, VB10.2g, agar 15g, pH 6.80, extinguished Add 8g of glucose and 16.9g of glyphosate sterilized by filter after the bacteria, and adjust the volume to 1000ml with sterilized water), observe the result after 24 hours: there are different types of single bacterial colonies to grow, and each single bacterial colony is placed at 100mM Glyphosate-concentrated M63 liquid medium was cultured overnight and stored in glycerol for future use.

[0027] ...

Embodiment 2

[0031] Embodiment 2, acquisition of EPSPS gene of pseudomonas moraxellaceae No.1 bacterium

[0032] 1. Acquisition of the whole genome sequence

[0033]In view of the tolerance of pseudomonas moraxellaceae No.1 to glyphosate as high as 900mM, it must be that the bacterium has undergone a directed mutation under the long-term selection pressure of high concentration glyphosate, and this directed mutation is likely to be the result of the EPSPS gene The structure has changed, and we tried to clone the EPSPS gene of this bacterium by homologous cloning, but it has not been successful. To this end, we use the current fastest and cheapest sequencing method, solexa sequencing technology: 4 nucleoside tags can be added in one reaction at the same time, and SBS-sequencing by synthesis (SBS-sequencing by synthesis) can be used to reduce secondary structure It is caused by the deletion of a region, and has the characteristics of small amount of sample required, high throughput, high ac...

Embodiment 3

[0038] Example 3, Expression of glyphosate-resistant gene EPSPS-L1 in maize

[0039] 1. Construction of plant expression vectors

[0040] First, through the existing plant expression vector pHM102, its map is as attached image 3 As shown, pHM102 was digested with BamHI and Kpn1, and EPSPS-L1 was digested with Bgl II and KpnI respectively. Since the sticky ends produced after digestion with BamHI and Bgl II are the same, ligation can be performed, and the two fragments are connected to obtain EPSPS-L1 The plant expression vector pHM102EP-L1.

[0041] 2. Obtaining of transgenic EPSPS-L1 maize resistant to glyphosate

[0042] The method for obtaining the transgenic corn is to introduce the insertion sequence into the callus of the recipient plant by using a gene gun method, and obtain the transgenic plant after being screened by the herbicide glufosinate. The specific method is:

[0043] (1), induction of type II callus

[0044] a. Remove the bracts: cut off the top of the ...

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Abstract

The invention discloses a glyphosate-resistant 5-enolpyruvoyl-shikimate-3-phosphate synthase gene and application thereof, and relates to the field of plant molecular biology and plant genetic engineering. More specifically, the invention discloses a sequence of a bacterial 5-enolpyruvoyl-shikimate-3-phosphate synthase amino acid residue disclosed in SEQ ID NO:2. The plant expressing the 5-enolpyruvoyl shikimate-3-phosphate synthase has resistance to glyphosate.

Description

technical field [0001] The invention belongs to the fields of molecular biology and plant genetic engineering, specifically. The invention relates to a glyphosate-resistant gene and the protein coded by the gene. The gene can be expressed in the plant through the method of genetic transformation, so as to improve the plant's tolerance to glyphosate, and facilitate the removal of weeds in the farmland. The present invention can be applied in the breeding of crops and the screening of plant cell culture. Background technique [0002] Glyphosate is the most widely used broad-spectrum herbicide. It is non-toxic to humans and animals, it is difficult for weeds and crops to develop resistance to it under natural conditions, and it has low soil residue. The market potential is huge. However, because glyphosate kills weeds and crops non-selectively, it can only be used before crop emergence or in non-crop planting areas, which restricts its application in agriculture. In order to...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/63C12N5/10
Inventor 赖锦盛赵海铭宋伟彬赵海楠
Owner CHINA AGRI UNIV
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