Kit for screening glyphosate N-acetyltransferase antiserum

A technology of acetyltransferase and glyphosate, applied in the direction of transferase, immunoglobulin from serum, anti-enzyme immunoglobulin, etc., can solve the problem of glyphosate losing herbicide activity and achieve broad application prospects

Active Publication Date: 2014-04-23
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Glyphosate N-acetyltransferase (glyphosate N-acetylhransferase, GAT) can acetylate glyphosate to acetylglyphosate, thereby making glyphosate lose herbicide activity

Method used

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  • Kit for screening glyphosate N-acetyltransferase antiserum
  • Kit for screening glyphosate N-acetyltransferase antiserum
  • Kit for screening glyphosate N-acetyltransferase antiserum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, preparation of anti-glyphosate N-acetyltransferase serum

[0040] 1. Prokaryotic expression of tag-guide peptide-glyphosate N-acetyltransferase

[0041] 1. Amplification of glyphosate N-acetyltransferase (GAT) gene

[0042] According to the GAT gene sequence, design the primer upstream primer (GATF-B): 5'-GGATCCTCTAGAATGGCGCAAGTTAG-3' (sequence 3); downstream primer (GATR-X): 5'-CTCGAGGAGCTCTTATGCGATCCTCTT-3' (sequence 4), the upstream primer increases BamH I restriction site, downstream primers increase Xho I restriction site.

[0043] Using the plasmid p2301-35S-GAT as a template, GATF-B and GATR-X were used for PCR amplification to obtain a 696bp PCR product.

[0044]After sequencing, the PCR product has the 109-780 nucleotides from the 5' end of Sequence 1 in the sequence listing, and is a gene encoding glyphosate N-acetyltransferase fused with a chloroplast guide peptide at the N-terminal.

[0045] 2. Construction of recombinant vector

[0046] Do...

Embodiment 2

[0084] Example 2. Functional research of anti-glyphosate N-acetyltransferase serum

[0085] Use anti-glyphosate N-acetyltransferase serum to detect whether the target protein GAT is contained in the test sample, and use Western blot to carry out, as follows:

[0086] 1. Preparation of samples to be tested

[0087] The GAT transgenic tobacco is to transfer the GAT gene (340-780 nucleotides from the 5' end of sequence 1) into wild-type tobacco (the variety is Nicotiana tabacum va. Xanthi nc) through p2301-35S-GAT, The obtained GAT genetically modified tobacco.

[0088] Take 0.2g leaves of transgenic GAT tobacco and grind them into powder with liquid nitrogen, add 150μl 2×SDS protein loading buffer and ddH 2O shake and mix well, denature at 100°C for 10 minutes, quickly place on ice for 5 minutes, centrifuge at 12,000 rpm for 10 minutes, transfer the supernatant to a new centrifuge tube for later use, and obtain the total protein, which is the sample to be tested.

[0089] 2. ...

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Abstract

The invention discloses a kit for screening glyphosate N-acetyltransferase antiserum. The invention provides an application of glyphosate N-acetyltransferase in preparation of the kit for preparing and screening glyphosate N-acetyltransferase antiserum. The amino acid sequence of the glyphosate N-acetyltransferase is shown in the table 1), namely amino acid residue of the 114-260th locus from the N-end in the sequence 2 in the sequence table, or table 2), namely the sequence 2 in the sequence table. Experiment shows that by utilizing the method in which the glyphosate N-acetyltransferase antiserum is prepared from prokaryotic expression protein for the first time, the antiserum of GAT (Glyphosate N-acetyltransferase) is successfully obtained and the antiserum can be also applied to specific detection on target protein of transgenic plant.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for screening glyphosate N-acetyltransferase antiserum, and also to a preparation method and application of glyphosate N-acetyltransferase antiserum. Background technique [0002] Glyphosate (glyphosate) is an excellent herbicide with broad-spectrum killing and systemic conduction. It is widely used in agricultural production because of its low cost, high efficiency, easy degradation and low environmental impact. [0003] The target enzyme of glyphosate is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway (Amrhein et al, 1980; Steinrucken and Amrhein, 1980). Glyphosate is an analogue of 5-phosphoenol pyruvate (PEP) in the shikimate pathway, and the molecular structure of the two is very similar. In the shikimate pathway, glyphosate competes with PEP for EPSPS. The strong competitiveness of glyphosate makes the enol group of PEP unable to combine wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C07K16/40G01N33/573
CPCC07K16/06G01N33/573G01N2333/91051
Inventor 张宗英陈相儒韩成贵李大伟于嘉林燕永亮陆伟林敏孙倩王颖张永亮王献兵
Owner CHINA AGRI UNIV
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