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Phenylurea herbicide n-demethylase gene pudma and its application

A technology of demethylase and gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems that threaten human life and health, function damage, and excessive pesticides

Inactive Publication Date: 2014-10-08
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Pesticides have played a huge role in agricultural production, crop pest control, weed removal, and control of human and animal infectious diseases, but at the same time it has also brought serious environmental problems. On the other hand, pesticide residues in the environment are enriched through various channels, mainly through the food chain, and ultimately directly threaten human life and health; in addition, pesticide residues also indirectly lead to changes in the structure and function of ecosystems.
[0003] Phenylurea (or substituted urea) herbicides (phenylurea herbicides) such as isoproturon and chlorotoluron are a series of herbicides synthesized according to the different substituents on the amino group and on the benzene ring in the urea molecule. They mainly play a herbicidal effect by inhibiting photosynthesis. Most substituted urea herbicides are mainly used as pre-emergence soil treatment agents to control annual grass weeds and broad-leaved weeds, although such herbicides are used as a low Toxic herbicides are widely used in the world, but a series of environmental problems caused by their extensive use have attracted people's attention. For example, the residues of phenylurea herbicides such as isoproturon in the environment are harmful to aquatic invertebrates, algae, etc. and microbial activity can have adverse effects, and isoproturon is a carcinogen

Method used

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  • Phenylurea herbicide n-demethylase gene pudma and its application
  • Phenylurea herbicide n-demethylase gene pudma and its application
  • Phenylurea herbicide n-demethylase gene pudma and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Cloning of phenylurea herbicide N-demethylase gene pudmA

[0035] 1.1 Library construction by transposon tagging

[0036] YBL3 (CCTCC NO: M208076) can grow on inorganic salt plates using phenylurea herbicides such as isoproturon as the sole carbon and nitrogen source, and the transposon mini-Tn5 is randomly inserted into YBL3 (CCTCC NO: M208076) ) After the genome, the colonies grown on the LB plus antibiotic plate were respectively spotted on the inorganic salt plus isoproturon plate and the inorganic salt plus glucose and ammonium sulfate plate. strains, the latter was used to exclude those auxotrophs resulting from random insertions. Mutant strains with functional gene mutations were obtained by this method.

[0037] 1.2YBL3 Whole Gene Sequencing

[0038] The transposon random insertion mutagenesis method is less random and inefficient in practical applications, but if the genome sequence is known, the efficiency of the method will be greatly improved. ...

Embodiment 2

[0043] Example 2 High expression of phenylurea herbicide N-demethylase gene pudmA in BL21 (pET-29a(+)) ( figure 2 )

[0044] 2.1 PCR amplification of phenylurea herbicide N-demethylase gene pudmA

[0045] with forward primer P1:5'- GGAATTCCATATG TTTCGCTACGATCAAGTA-3' (SEQ ID NO. 3) and reverse primer P2:5'- CCGGAATTC CTAAAGAAAGATGGCCAGGTT-3' (SEQ ID NO. 4) was used as the primer, and the phenylurea herbicide N-demethylase was amplified from the total DNA of Sphingobium sp. YBL3 (CCTCC NO: M208076) by PCR method. Gene pudmA fragment.

[0046] Amplification system:

[0047]

[0048] PCR amplification procedure:

[0049]

[0050] 2.2 Validation of PCR products.

[0051] The PCR product was recovered, a few microliters were added with A tail, and it was connected to the pMD19-T vector, and the correct transformant was selected and connected to Nanjing GenScript Biotechnology Co., Ltd. for sequencing to verify the correctness of the PCR product. After verification i...

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Abstract

The invention discloses a phenylurea herbicide N-demethylase gene pudmA and an application thereof. A nucleotide sequence of the phenylurea herbicide N-demethylase gene pudmA is shown in SEQ ID NO.1; the overall length is 1907bp; the content of G+C is 55.37%; a large subunit code includes 459 amino acids; a small subunit code includes 176 amino acids; and the amino acid sequence is shown in SEQ ID NO.2. The phenylurea herbicide N-demethylase gene pudmA provided by the invention can remove N-methyl of phenylurea herbicides such as isoproturon and chlortoluron, so that the herbicidal activity is removed. Therefore, the phenylurea herbicide N-demethylase gene pudmA can be used for building genetically modified crops of an anti-phenyl urea herbicide. In addition, the phenylurea herbicide N-demethylase gene pudmA and encoded protein PudmA also can be used for removing residual phenylurea herbicide in the environment.

Description

technical field [0001] The invention belongs to the field of application of environmental microorganisms and agriculture, and relates to a phenylurea herbicide N-demethylase gene pudmA and its application. Background technique [0002] Pesticides have played a huge role in agricultural production, crop pest control, weed removal, and control of human and animal infectious diseases, but at the same time, they also bring serious environmental problems. Pesticide pollution directly leads to excessive pesticides in agricultural products, and product quality declines, seriously affecting On the other hand, pesticide residues in the environment are enriched in various ways, mainly in the food chain, and ultimately directly threaten human life and health; in addition, pesticide residues also indirectly lead to changes in ecosystem structure and function damage. [0003] Phenylurea (or substituted urea) herbicides (phenylurea herbicides) such as isoproturon and chloromalon are a ser...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N1/21A01H5/00A62D3/02C02F3/00B09C1/10C12R1/19A62D101/04A62D101/28
Inventor 李顺鹏闫新谷涛何健洪青蒋建东黄星孙纪全
Owner NANJING AGRICULTURAL UNIVERSITY
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