Artificially synthesized glyphosate-tolerant gene and application thereof
A sequence listing and sequence technology, applied to artificially synthesized glyphosate-resistant genes and their application fields, can solve the problem of low expression level and achieve the effect of increased expression level and improved tolerance
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Embodiment 1
[0031] Example 1. Obtaining of codon-optimized glyphosate-resistant genes
[0032] 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 frequency of codon usage (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 the G+C content is reduced from 64.83% to 62.07%. The gene shown in Sequ...
Embodiment 2
[0036] Example 2, the acquisition of mG2-aroA transgenic maize
[0037] 1. Construction of recombinant expression vector pS3300-UMG2
[0038] 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 ce...
Embodiment 3
[0089] Example 3, Double Antibody Sandwich ELISA Detection of G2-aroA Protein Expression in Transgenic Maize Plants
[0090] Sample extract: Tris-Cl (pH8.0) 25mM, KCl 10mM, MgCl 2 ·6H 2 O 20mM, DTT 1mM, PMSF 1mM (add before use).
[0091] Coating buffer: take 1.5g Na 2 CO 3 , 2.93 g NaHCO 3 , and distilled water to 1000mL, pH9.6.
[0092] 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.
[0093] 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.
[0094] 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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