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EPSP synthase gene from Klebsiella pneumoniae 342 and application of EPSP synthase gene

A technology of Klebsiella and EPSP synthase, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2013-01-16
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are only two glyphosate-resistant genes currently used in production, and both are protected by patents. Therefore, it is very necessary to discover new EPSP synthetase genes for the cultivation of glyphosate-resistant transgenic crops with independent intellectual property rights.

Method used

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  • EPSP synthase gene from Klebsiella pneumoniae 342 and application of EPSP synthase gene
  • EPSP synthase gene from Klebsiella pneumoniae 342 and application of EPSP synthase gene
  • EPSP synthase gene from Klebsiella pneumoniae 342 and application of EPSP synthase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 DNA fragment cloning of high tolerance to glyphosate

[0029] 1. Collection of soil samples in paddy fields where glyphosate is frequently used

[0030] Soil samples were collected from orchards that had been used at least four times a year and had been used continuously for more than ten years.

[0031] 2. Screening of glyphosate-resistant strains

[0032] Weigh 1g of the orchard soil sample frequently used by glyphosate, add 1ml of 0.9% (w / v) sodium chloride solution, shake and mix at 5000 rpm, centrifuge gently at 3000 rpm, pour off the supernatant, and then add 0.9% (w / v) 1ml of sodium chloride solution, shake and mix at 5000 rpm, let it stand on ice for 10 minutes, draw 150 μl of the solution, spread it and culture it in LB solid medium containing 60mM glyphosate for 24 hours. Inoculate the grown single colony into a test tube added with 1.6ml LB liquid medium, cultivate at 28°C for 48h, then draw 150μl of the culture solution and spread it again and cu...

Embodiment 2

[0041] Example 2 Sequence analysis of DNA fragments highly tolerant to glyphosate

[0042] The highly tolerant glyphosate plasmid pAroA obtained by screening in Example 1 by step-by-step sequencing method k.pneumonia Full sequence DNA sequencing. Analysis results show that the fragment is 2100bp in size, which contains a reading frame of 1284, its nucleotide sequence is shown in SEQ ID NO 1, and it encodes 428 amino acids, and its sequence is shown in SEQ ID NO 2. .

Embodiment 3

[0043] Example 3 Artificial synthesis and screening of EPSP synthase gene highly tolerant to glyphosate

[0044] Cloning the above plasmid containing pAroA by gene synthesis [Nucleic Acids Research, 2004, 32, e98] k.pneumonia 26 primers were designed, and the EPSP synthase gene of the present invention was synthesized and screened. The designed primers are as follows:

[0045] 1. KP-1:Tm=54,60mer

[0046] GGA, TCC, ATG, GAA, TCC, CTG, ACG, TTA, CAA, CCC, ATC, GCA, CGC, GTA, GAG, GGC, ACC, GTG, AAC, CTG

[0047] 2. KP-2:Tm=54,60mer

[0048] GAG,CGG,CCA,GCA,GCA,GCG,CGC,GGT,TGG,AGA,CGC,TTT,TCG,AAC,CTG,GCA,GGT,TCA,CGG,TGC

[0049] 3. KP-3:Tm=54,60mer

[0050] TGG, CCC, GCG, GCA, CGA, CGG, TGC, TGA, CCA, ACC, TGC, TGG, ACA, GCG, ACG, ATG, TGC, GCC, ATA, TGC

[0051] 4. KP-4:Tm=54,60mer

[0052] GAT, AGG, GTA, TAT, TGA, ACC, CCC, AGC, GCA, CTC, AGG, GCA, TTC, AGC, ATA, TGG, CGC, ACA, TCG, TCG

[0053] 5. KP-5:Tm=54,60mer

[0054] TGC, CGA, CCG, CAC, CCG, CTG, CGA, AGT, GAC,...

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Abstract

The invention discloses an EPSP synthase gene from Klebsiella pneumoniae 342 and application of the EPSP synthase gene. The EPSP synthase gene comprises 1284 basic groups and 428 coding amino acid, the nucleotide sequence is shown as SEQ ID NO 1, and the coding amino acid sequence is shown as SEQ ID NO 2. The EPSP synthase gene from the Klebsiella pneumoniae 342 has high glyphosate tolerance and can be used for cultivating transgenic crops.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to an EPSP synthase gene derived from Klebsiella pneumoniae 342 and application thereof. Background technique [0002] Glyphosate is an excellent broad-spectrum sterilizing and systemic herbicide, widely used in orchards, rubber gardens, non-cultivated land, and no-till land for pre-sowing or post-sowing treatment of corn, soybeans, and cotton, as well as post-emergence orientation deal with. Since it was registered in the United States in 1974, it has been registered in more than 100 countries around the world, becoming one of the most widely used herbicides in the world. But the herbicide is also a non-selective herbicide, which has a killing effect on crops. Therefore, in order to use glyphosate in agricultural production, crops with glyphosate resistance or degradability must be bred. [0003] The toxic mechanism of glyphosate (N-phosphonomethyl-glycine...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12A01H5/00
Inventor 田永生姚泉洪许晶彭日荷金晓芬韩洪娟韩静王波王丽娟
Owner SHANGHAI ACAD OF AGRI SCI
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