EPSP synthase gene derived from ochrobactrum anthropi and application thereof
A technology of EPSP synthase and Paleobacterium hominis, applied in the field of microbial genetic engineering, can solve biological safety problems and other problems, and achieve the effect of strong affinity and high glyphosate resistance
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Embodiment 1
[0041] Cloning of DNA Fragment Highly Tolerant to Glyphosate
[0042] 1. Collection of soil samples in paddy fields where glyphosate is frequently used
[0043] Soil samples were collected from orchards that had been used at least four times a year and had been used continuously for more than ten years.
[0044] 2. Screening of glyphosate-resistant strains
[0045] Weigh 1g of the orchard soil sample frequently used by glyphosate, add 1ml of 0.9% (w / v) sodium chloride solution, shake and mix at 5000 rpm, centrifuge gently at 3000 rpm, pour off the supernatant, and then add 0.9% (w / v) 1ml of sodium chloride solution, shake and mix at 5000 rpm, let it stand on ice for 10 minutes, draw 150 μl of the solution, spread it and culture it in LB solid medium containing 60 mM glyphosate for 24 hours. Inoculate the grown single colony into a test tube with 1.6ml of LB liquid medium, culture at 28°C for 48h, then draw 150μl of the culture solution and apply it again to the LB solid medi...
Embodiment 2
[0064] Sequence analysis of DNA fragments highly tolerant to glyphosate and functional verification of EPSP synthase
[0065] 1. Sequence analysis of DNA fragments highly tolerant to glyphosate
[0066] The high-tolerance glyphosate plasmid pAroA-O.anthropi screened in Example 1, that is, the complete sequence of the EPSP synthase gene of the present invention, was subjected to DNA sequencing by step-by-step sequencing method. The analysis results show that the inserted fragment is 3100bp in size, which contains a reading frame of 1353, and its sequence is shown in SEQID NO 1. The polynucleotide sequence it contains has a full length of 1353 bases and 451 encoded amino acids. Its amino acid sequence is shown in SEQ ID NO 2.
[0067]Amino acid sequence homology analysis results show: AroA-O.anthropi amino acid sequence and II type EPSP synthase from Agrobacterium CP4 have 82.89% homology (see figure 2 ), and less than 30% homology with type I EPSP synthase from Escherichia co...
Embodiment 3
[0070] Artificial synthesis of EPSP synthase gene with high tolerance to glyphosate
[0071] The above-mentioned highly glyphosate-tolerant EPSP synthase gene was cloned by gene synthesis method [Nucleic Acids Research, 2004, 32, e98]. The designed primers are as follows:
[0072] 1. AroA-1: Tm=54, 60mer
[0073] ATG, TCC, CAT, TCT, GCA, TCC, CCG, AAA, CCA, GCA, ACC, GCC, CGC, CGG, TCG, GAG, GCA, CTT, ACG, GGC
[0074] 2. AroA-2: Tm=54, 60mer
[0075] ACG, ATG, CGA, GAT, GGA, TTT, GTC, ACC, CGG, AAT, GCG, AAT, TTC, GCC, CGT, AAG, TGC, CTC, CGA, GCG
[0076] 3. AroA-3: Tm=54, 60mer
[0077] GAC, AAA, TCC, ATC, TCG, CAT, CGT, TCC, TTC, ATG, TTC, GGC, GGC, CTT, GCA, TCG, GGT, GAA, ACC, CGC
[0078] 4. AroA-4: Tm=54, 60mer
[0079] GGT, ATT, GAT, GAC, GTC, TTC, GCC, TTC, CAG, AAG, GCC, GGT, GAT, GCG, GGT, TTC, ACC, CGA, TGC, AAG
[0080] 5. AroA-5: Tm=54, 60mer
[0081] GGC, GAA, GAC, GTC, ATC, AAT, ACC, GGT, CGC, GCC, ATG, CAG, GCC, ATG, GGC, GCG, AAA, ATC, CGC, AAG
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