EPSP synthase with high glyphosate tolerance and application thereof
A technology of EPSP synthase and glyphosate, applied in the field of 5-enol-pyruvylshikimoyl-3-phosphate synthase and its high tolerance to glyphosate herbicides, can solve the problem of inability to synthesize aromatic amino acids, plants Death, shikimic acid accumulation, etc.
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Embodiment 1
[0081] Example 1 Cloning of EPSP synthase gene highly tolerant to glyphosate
[0082] 1. Collection of soil samples in extremely glyphosate polluted environment
[0083] In the natural environment, especially in the soil of areas where glyphosate and other herbicides have been used for a long time, there are a wide variety of bacterial strains that can tolerate glyphosate or other herbicides. Samples were therefore collected from soil contaminated with glyphosate. Take soil samples 2cm-10cm below the surface for bacterial strain screening.
[0084] 2. Screening and identification of highly glyphosate-tolerant bacterial strains
[0085] Weigh 5 grams of glyphosate-contaminated soil samples, place them in a 50mL Erlenmeyer flask containing 20mL sterile water, and shake vigorously at room temperature for 15 minutes. After standing still for 1 minute, take 200uL supernatant and spread it on R2A medium containing 100mM and 200mM glyphosate On the plate, culture at 28°C for 1-3 d...
Embodiment 2
[0102] Embodiment 2KO1-EPSPS glyphosate tolerance detection
[0103] The plasmid pET-KO1-EPSPS was transformed into Escherichia coli BL21(DE3) to obtain KO1-EPSPS protein expression strain BL21(KO1-EPSPS). Escherichia coli BL21(DE3) was used as a control and cultured at 37°C for 48 hours. Observe its growth in M9 medium with different glyphosate concentrations (20 to 250 mM).
[0104] The results are shown in Table 1.
[0105] Table 1 KO1-EPSPS glyphosate tolerance test in prokaryotes
[0106]
20mM
50mM
100mM
150mM
200mM
250mM
BL21(KO1-EPSPS)
++++
++++
++++
++++
+++
+
BL21(DE3)
+
-
-
-
-
-
[0107] The experimental results showed that Escherichia coli containing KO1-EPSPS could grow on the M9 medium containing 250 mM glyphosate.
[0108] Experimental conclusion: KO1-EPSPS has extremely high tolerance to glyphosate.
Embodiment 3
[0109] Embodiment 3KO1-EPSPS protein expression and purification
[0110] Escherichia coli BL21 (KO1-EPSPS) containing pET-KO1-EPSPS protein expression plasmid was inoculated in 10mL containing Kan r In 100μg / mL LB liquid medium, after activating overnight at 37°C, take a 2% solution and add it to 20mL containing Kan r In 100μg / mL LB liquid medium, shaking culture to OD 600 value is 0.6, add IPTG to a final concentration of 0.75mmol / L, and continue to culture at 37°C and 220rpm for 2h. Then add the culture solution to the centrifuge tube at 4°C, centrifuge at 6,000xg for 5 minutes, collect the bacteria, add 1 / 20 volume of NTA-0Buffer to suspend the cells, add 200 μL lysozyme (0.4 mg / mL), and conduct low-temperature ultrasound in an ice bath for 30 minutes Breaking, amplitude 25%, interval 2sec, ultrasonication 3sec, 10-15 cycles, or until the solution becomes clear; add 10μL DNaseI to reduce the viscosity caused by cellular DNA, ice bath for 10min; The supernatant and preci...
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