Recombinant DNA (deoxyribonucleic acid) fragment containing roundup ready gene and application thereof

Abstract

The invention discloses a recombinant DNA (deoxyribonucleic acid) fragment containing a roundup ready gene and application thereof. The recombinant DNA fragment provided by the invention comprises the following components from 1) to 3): 1) a promoter; 2) the roundup ready gene which is promoted by the promoter to be transcribed, the nucleotide sequence of the roundup ready gene is a) or b), wherein a) represents a sequence 2 in a sequence table, and b) represents a protein which has at least 98% of identity with the sequence 2 in the sequence table and is shown by a coding sequence 9; and 3) tanscription termination sequence. Compared with the transgenic corn which contains the recombinant DNA fragment in which prokaryote roundup ready gene G2-aroA is transplanted, the transgenic corn which is transplanted with the recombinant DNA fragment 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 a recombinant DNA fragment containing a glyphosate-resistant gene and its application, in particular to a recombinant DNA fragment containing a glyphosate-resistant gene 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...

Examples

Embodiment 1

[0041] Example 1. Obtaining of codon-optimized glyphosate-resistant genes

[0042] This example is based on the amino acid sequence of the G2-aroA gene (Chinese patent, application number 03826892.2, authorized announcement number CN 100429311 C) (the nucleotide sequence is shown in sequence 1 in the sequence table), under the premise of ensuring that the amino acid sequence remains unchanged , first artificially optimized the G2-aroA gene by adopting maize-biased 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 (sequence 1) is only 84%, and...

Embodiment 2

[0047] Example 2, the acquisition of mG2-aroA transgenic maize

[0048] 1. Construction of recombinant expression vector

[0049] 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

[0100] Example 3, Double Antibody Sandwich ELISA Detection of G2-aroA Protein Expression in Transgenic Maize Plants

[0101] Sample extract: Tris-Cl (pH8.0) 25mM, KCl 10mM, MgCl 2 ·6H 2 O 20mM, DTT 1mM, PMSF 1mM (add before use).

[0102] Coating buffer: Take 1.5g Na 2 CO 3 , 2.93 g NaHCO 3 , and distilled water to 1000mL, pH9.6.

[0103] 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.

[0104] 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.

[0105] 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. ...

technical field

[0001] The invention relates to a recombinant DNA fragment containing a glyphosate-resistant gene and its application, in particular to a recombinant DNA fragment containing a glyphosate-resistant gene 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...

Examples

Embodiment 1

[0041] Example 1. Obtaining of codon-optimized glyphosate-resistant genes

[0042] This example is based on the amino acid sequence of the G2-aroA gene (Chinese patent, application number 03826892.2, authorized announcement number CN 100429311 C) (the nucleotide sequence is shown in sequence 1 in the sequence table), under the premise of ensuring that the amino acid sequence remains unchanged , first artificially optimized the G2-aroA gene by adopting maize-biased 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 (sequence 1) is only 84%, and...

Embodiment 2

[0047] Example 2, the acquisition of mG2-aroA transgenic maize

[0048] 1. Construction of recombinant expression vector

[0049] 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

[0100] Example 3, Double Antibody Sandwich ELISA Detection of G2-aroA Protein Expression in Transgenic Maize Plants

[0101] Sample extract: Tris-Cl (pH8.0) 25mM, KCl 10mM, MgCl 2 ·6H 2 O 20mM, DTT 1mM, PMSF 1mM (add before use).

[0102] Coating buffer: Take 1.5g Na 2 CO 3 , 2.93 g NaHCO 3 , and distilled water to 1000mL, pH9.6.

[0103] 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.

[0104] 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.

[0105] 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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