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Synthetic roundup ready gene expression vector and application thereof

An expression vector and glyphosate-resistant technology, which can be used in the application, use of vectors to introduce foreign genetic material, cells modified by introducing foreign genetic material, etc., can solve the problem of low expression

Active Publication Date: 2012-08-22
BEIJING ORIGIN SEED TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the glyphosate-resistant gene G2-aroA was discovered in 2004, it can be highly expressed in the host Pseudomonas fluorescens G2 and Escherichia coli, showing high resistance to glyphosate (Yichen Sun, Min Lin and Yiping Wang. Novel AroA with high tolerance to Glyposate, encoded by a gene of Pseudomonas putida 4G-1 isolated from a

Method used

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  • Synthetic roundup ready gene expression vector and application thereof
  • Synthetic roundup ready gene expression vector and application thereof
  • Synthetic roundup ready gene expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Obtaining of codon-optimized glyphosate-resistant genes

[0037] In this embodiment, according to the amino acid sequence (the nucleotide sequence is shown in sequence 1 in the sequence table) of the G2-aroA gene (Chinese patent, application number 03826892.2, authorized announcement number CN100429311C), under the premise of ensuring that the amino acid sequence remains unchanged, first The G2-aroA gene was artificially optimized and transformed by adopting maize preferred codons. Try to avoid using maize rare codons, and adjust the codon usage frequency (Table 1), so that the codon usage frequency of G2-aroA protein is close to the usage frequency in maize (Table 1). On this basis, the typical AT-rich sequence that causes instability of plant gene transcripts in the DNA sequence is removed, and the hairpin structure is removed, and the new nucleotide sequence obtained is sequence 2 in the sequence listing. The homology between sequence 2 and G2-aroA gene (...

Embodiment 2

[0041] Example 2, the acquisition of mG2-aroA transgenic maize

[0042] 1. Construction of recombinant expression vector

[0043] In order to improve the expression level of the mG2-aroA gene (sequence 2) in the recipient organism, the inventors added an omega sequence and a Kozak sequence to the 5' end of the mG2-aroA gene when constructing the recombinant expression vector of the mG2-aroA gene, The Ω / Kozak sequence (abbreviated as OMK) is shown as sequence 6 in the sequence listing. The Ω sequence is a translation enhancing sequence derived from the coding region of the capsid protein gene of a plant virus. It consists of 67 bp and is enriched in TTAAC sequences. There is a UAUUUUUACAACAA sequence and 4 UUAC sequences at the 5' end, which are formed during the translation process of protein synthesis. Ribosome and rRNA binding sites. Kozak sequence is a sequence encoding ribosome binding protein that promotes the translation process of foreign genes in plant cells. The co...

Embodiment 3

[0094] Example 3, Double Antibody Sandwich ELISA Detection of G2-aroA Protein Expression in Transgenic Maize Plants

[0095]Sample extract: Tris-Cl (pH8.0) 25mM, KCl 10mM, MgCl 2 ·6H 2 O 20mM, DTT 1mM, PMSF 1mM (add before use).

[0096] Coating buffer: take 1.5g Na 2 CO 3 , 2.93 g NaHCO 3 , and distilled water to 1000mL, pH9.6.

[0097] Washing solution (PBST): take 1mL Tween 20, add phosphate buffer saline (PBS) to make up to 1000mL, pH7.5; phosphate buffer saline (PBS): take 8.0g NaCl, 0.2g KH 2 PO 4 , 2.96 g Na 2 HPO 4 12H 2 O, add 1000mL distilled water, pH7.5.

[0098] Sample buffer solution (PBST): Take 1mL Tween 20, add phosphate buffer saline (PBS) to make it 1000mL, pH7.5; Phosphate buffer saline (PBS): take 8.0g NaCl, 0.2g KH 2 PO 4 , 2.96 g Na 2 HPO 4 12H 2 O, add 1000mL distilled water, pH7.5.

[0099] Substrate buffer: take 0.1g MgCl 2 or 0.2g MgCl 2 ·6H 2 O, 97.0 mL diethanolamine, dissolved in 1000 mL distilled water, pH 9.8, stored at 4°C. ...

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Abstract

The invention discloses a synthetic roundup ready gene expression vector and application thereof. The expression vector provided by the invention is the expression vector for expressing the synthetic roundup ready gene; and the synthetic roundup ready gene is the DNA (deoxyribonucleic acid) molecule of a) or b): a) a nucleotide sequence is a sequence 2 in a sequence table; and b) the nucleotide sequence has at least 98% of identity with the sequence 2 in the sequence table, and the protein is shown by a coding sequence 10. Compared with the transgenic corn which is transplanted with the expression vector containing a prokaryote roundup ready gene G2-aroA, the transgenic corn which is transplanted with the expression vector provided by the invention is obviously higher in the expression quantity of G2-aroA protein; and the transgenic corn is also obviously higher in the glyphosate tolerance.

Description

technical field [0001] The invention relates to an artificially synthesized glyphosate-resistant gene expression vector and its application, in particular to a glyphosate-resistant gene expression vector designed and synthesized according to the maize preferred codon and its application. Background technique [0002] Most of the exogenous genes used in plant transgenic breeding, such as Bt and EPSPS, are from prokaryotes. Due to the characteristics of prokaryote genes, such as 1) AT content is higher, exceeding 60%, causing the mRNA of gene expression to be in the plant. It is easily degraded in the body; 2) There are intron cut points and transcription terminator sequences similar to eukaryotic genes, resulting in incomplete transcription, abnormal splicing of mRNA, etc.; 3) There are large differences between codons and plant codons, resulting in The protein translation efficiency is reduced; 4) its structure is significantly different from that of plants and other eukaryo...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/11C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 韩庚辰刘素霞邓德芝姜付坤宋哲李雪峰
Owner BEIJING ORIGIN SEED TECH
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