Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Preparation method and application of paracoccus denitrificans EPSP synthase gene

A technology of EPSP synthase and denitrifying paracoccus, applied in the field of microbial genetic engineering, can solve problems such as lack and disadvantage

Inactive Publication Date: 2015-06-24
SHANGHAI ACAD OF AGRI SCI
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a major glyphosate production and export country, China currently lacks the 5-enolpyruvyl-shikimate-3-phosphate synthase gene with independent intellectual property rights that is suitable for the cultivation of glyphosate-resistant transgenic crops. at a disadvantage in

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method and application of paracoccus denitrificans EPSP synthase gene
  • Preparation method and application of paracoccus denitrificans EPSP synthase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Acquisition of Novel Glyphosate Resistance Gene EPSP Synthase Gene

[0026] 1. Collection of soil samples

[0027] 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.

[0028] 2. Screening of glyphosate-resistant strains

[0029] 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 of LB liquid medium, cultivate at 28°C for 48h, then draw 150μl of culture solution and spread it again and cultivate it in LB solid medium containin...

Embodiment 2

[0039] Example 2 Artificial synthesis of novel glyphosate resistance gene EPSP synthase gene

[0040] The gene synthesis method [Nucleic Acids Research, 2004, 32, e98] artificially synthesized obtained in Example 1

[0041] The novel EPSP synthase gene of the present invention. A total of 23 pairs of primers were designed, and the designed primers were as follows:

[0042] 1. P1: Tm=54, 60mer

[0043] GGA,TCC,ATG,TCT,CAT,TCT,GCT,GAA,CCA,CTG,CCA,ATG,ACT,GCC,AGA,AGA,AGT,GGT,CCA,CTG

[0044] 2. P2: Tm=54, 60mer

[0045] CTT,GTC,ACC,TGG,AAC,CTG,AGC,TTC,ACC,AGT,CAG,TGG,ACC,ACT,TCT,TCT,GGC,AGT,CAT,TGG

[0046] 3. P3: Tm=54, 60mer

[0047] ACT,GGT,GAA,GCT,CAG,GTT,CCA,GGT,GAC,AAG,TCC,ATC,TCT,CAC,AGA,GCA,CTG,ATT,CTT,GGT

[0048] 4. P4: Tm=54, 60mer

[0049] AGT,GAT,GTG,AGT,TTC,ACC,AAC,AGA,CAG,AGC,ACC,AAG,AAT,CAG,TGC,TCT,GTG,AGA,GAT,GGA

[0050] 5. P5: Tm=54, 60mer

[0051] GCT,CTG,TCT,GTT,GGT,GAA,ACT,CAC,ATC,ACT,GGT,CTT,CTT,GAA,GGT,CAA,GAT,GTT,CTT,GAC

[0052] 6. P6: Tm=54, 6...

Embodiment 3

[0136] Example 3 Prokaryotic expression of novel glyphosate resistance gene EPSP synthase gene

[0137] The EPSP synthase gene of the present invention artificially synthesized in Example 2 was amplified by PCR with primers PZ (5'-gagagaccatgtctcattctgct-3') and PF (5'-gtctcgag ttaatcgatctgggca-3'), and KOD Plus (Toyobo Japan) It is a DNA polymerase, and the amplification conditions are as follows: 94°C for 30s, 55°C for 30s, 72°C for 90s, and 30 cycles of amplification. After the cycle is over, add 2U of rtaq enzyme (Dalian Bao Biological Engineering Co., Ltd.), extend at 72°C for 90s, and the length of the amplified fragment is 1353bp. For PCR products Nco I and xhoAfter I digestion, connect the vector pET-28a (NEB Company) with the same restriction enzyme digestion to obtain the recombinant plasmid and transform it into Escherichia coli BL21 (DE3) (Novagen Company), spread the transformants on LB solid medium and culture them for 24 hours . The gel-protein purificatio...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an EPSP synthase gene derived from Paracoccus denitrificans and application thereof. The EPSP synthase gene comprises 1332 basic groups and 443 coded amino acids; the nucleotide sequence of the EPSP synthase gene is as shown in SEQ ID NO 1, and the coded amino acid sequence is as shown in SEQ ID NO 2. The EPSP synthase gene derived from Paracoccus denitrificans disclosed by the invention has higher glyphosate tolerance, and can be applied to culture of 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 Paracoccus denitrificans and its application. Background technique [0002] The shikimate pathway is an important pathway for the synthesis of aromatic amino acids in plants and microorganisms. EPSP synthase is a key enzyme in the shikimate pathway, catalyzing 3-phosphoshikimate (S3P) and phosphoenolpyruvate (PEP) to generate 5-enolpyruvyl-shikimate-3-phosphate synthase (5-enolpyruvyl shikimate-3-phosphate synthase, EPSPS). The herbicide glyphosate is a structural analogue of PEP, which can compete with PEP to inhibit the activity of EPSPS, thereby blocking the biosynthesis of aromatic amino acids and eventually leading to plant death. So far, only the EPSPS gene of type II Agrobacterium CP4 has been successfully used in commercial glyphosate-resistant transgenic crops. Since only this single gene is widely commercial...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/54C12N9/10
Inventor 田永生许晶姚泉洪彭日荷薛永赵伟付晓燕高建杰韩红娟王丽娟王波李振军
Owner SHANGHAI ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com