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Enterobacter glyphosate-tolerant gene argF, and coding protein and application thereof

A glyphosate-resistant and Enterobacteriaceae-resistant technology, applied in the field of genetic engineering, can solve the problems of weak gene resistance and few genes to be excavated

Inactive Publication Date: 2015-12-23
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since glyphosate-resistant transgenes have strong glyphosate-resistant ability in addition to the target gene, most of the genes mined at present have weak tolerance, and relatively few genes have been mined

Method used

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  • Enterobacter glyphosate-tolerant gene argF, and coding protein and application thereof
  • Enterobacter glyphosate-tolerant gene argF, and coding protein and application thereof
  • Enterobacter glyphosate-tolerant gene argF, and coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Cloning of Enterobacteriaceae glyphosate-resistant gene argF

[0040] (1) Design primers to extract microbial Enterobacteriaceae DNA:

[0041] The bacterial total DNA extraction kit (purchased from Tiangen Company) was used to extract the DNA of the microorganism Enterobacteriaceae, and the primers were designed according to the existing E. coli argF gene sequence in the NCBI database for homologous cloning. The primer sequences for amplification are:

[0042]SeqID NO.3: Upstream primer 5'-ATTTTCACACACGGACGGG-3';

[0043] Seq ID NO.4: Downstream primer 5'-ATCGGCATAATGCTGGCA-3'.

[0044] (2) PCR amplification, the specific steps are as follows:

[0045] Step 1: Perform PCR amplification using Enterobacteriaceae genomic DNA as a template to obtain specific fragments, and prepare a PCR reaction solution (50uL system, Toyobo, KOD-Plus) according to the following components: 10×PCRBuffer (5uL), 2mMdNTPs ( 5uL), 2mM MgSO 4 (2uL), primers (1.5uL each), DNA (2uL...

Embodiment 2

[0048] Example 2: Expression analysis of gene argF in Escherichia coli BL21

[0049] (1) Design specific primers, add restriction sites NdeI and XbaI and protective bases, use the cloned argF gene as a template to amplify the argF gene, and the specific primers are:

[0050] SeqID NO.5: Upstream primer 5'-CGCGGATCCATGTCTGATCTGTACAAG-3';

[0051] Seq ID NO.6: Downstream primer 5'-CTCCCCAAGCGTCGCCACCTCGAGCGG-3'.

[0052] (2) PCR amplification, product recovery, and TA cloning to PMD19-Tsimplevector, sequencing, the specific operation is the same as in Example 1), select the bacterium liquid that contains the correct amplified sequence, and utilize a plasmid extraction kit (purchased from Axygen Company) Recover the plasmid. The T vector containing the gene and the prokaryotic expression vector pCold were double digested with restriction endonucleases NdeI and XbaI, the recovered fragments were detected on agarose gel, and the cleaved vector and gene were connected with T4DNA l...

Embodiment 3

[0058] Example 3: Expression analysis of gene argF in Arabidopsis

[0059] (1) Select pMDC83 (provided by the National Soybean Improvement Center of Nanjing Agricultural University) as the plant overexpression vector. The resistance of the vector is selected for hygromycin, which is convenient for the screening of transgenic seedlings; the plant expression vector of argF gene is constructed according to the instructions of the Gateway kit operate.

[0060] (2) According to the sequence of the glyphosate-resistant gene argF of Enterobacteriaceae, design primers for amplifying the complete reading frame, and introduce the gateway linker sequence into the upstream and downstream primers:

[0061] SeqIDNO.7: The upstream primer is as follows

[0062] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGATCTGTACAAG-3';

[0063] SeqIDNO.8: Downstream primers are as follows

[0064] 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTCCCCAAGCGTCGCCAC-3';

[0065] (3) Using the PCR amplification product obtai...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses an Enterobacter glyphosate-tolerant gene argF, and a coding protein and application thereof. The gene is cloned from Enterobacter evolved from glyphosate tolerance, and the nucleotide sequence is disclosed as SEQ ID NO.1. The invention detects that any plant expression vector converter plant cell capable of transforming an exogenous gene argF can be utilized to obtain the argF-expressed transgenic plant of which the glyphosate tolerance is enhanced as compared with the non-transgenic plant. The gene disclosed by the invention can be transformed into a plant as a target gene, thereby enhancing the glyphosate tolerance of the transgenic plant and having important meanings for culturing glyphosate-tolerant crops.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an enterobacteria glyphosate-resistant gene argF, encoded protein and application thereof. Background technique [0002] The acquisition of genetic resources is the key to glyphosate-resistant transgenic breeding, and glyphosate-resistant genes can be divided into target genes and non-target genes. The target of glyphosate in cells is the key enzyme 5-enolpyruvate-shikimate-3-phosphate synthase (EPSPS) in the biosynthesis of aromatic amino acids. EPSPS only exists in bacteria and plants (Steinrücken & Amrhein, 1980), Participating in the shikimate pathway to synthesize aromatic amino acids necessary for organisms plays an important role in cellular tolerance to glyphosate. In addition to target genes, research on non-target genes in glyphosate-resistant organisms has also received some attention. Non-target genes discovered so far include C-N bond oxidat...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/82C12N15/11A01H5/00
Inventor 赵团结费云燕盖钧镒
Owner NANJING AGRICULTURAL UNIVERSITY
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